Abstract
Quinoxaline-incorporated Schiff bases (4a–j) were synthesized by the condensation of 2-[(3-methylquinoxalin-2-yl)oxy]acetohydrazide (3) with indole-3-carbaldehyde, furfuraldehyde, 5-(4-nitrophenyl)-2-furfuraldehyde, and substituted benzaldehydes under conventional and microwave irradiation methods. The microwave method was found to be remarkably successful with higher yields, less reaction time, and environmentally friendly compared to conventional heating method. The chemical structures of the synthesized compounds have been confirmed by analytical and spectral data. All the compounds have been evaluated for antitubercular and anti-inflammatory activities.
1. Introduction
Quinoxaline derivatives have been reported to possess a wide range of biological properties such as being anticancer [1, 2], antihistaminic [3], antiviral [4], antimicrobial [5, 6], antifungal [7], antitubercular [8, 9], and anti-inflammatory [10]. Schiff bases possess antimicrobial [11], antitubercular [12], anti-inflammatory [13], and analgesic [14] properties. In view of these facts, we herein report the facile synthesis of a new series of quinoxaline-incorporated schiff bases and screen for antitubercular and anti-inflammatory activities. In recent times, microwave-assisted organic reactions have become very popular and gain special attention due to their ecofriendly nature, safety, less reaction time, and higher yields. In continuation of our research on quinoxaline derivatives, in the present study, we made an attempt to synthesize the title compounds using conventional and microwave irradiation methods and compared the reaction time and percentage yields.
2. Experimental
Melting points were determined using open capillary tubes on ANALAB melting point apparatus and are uncorrected. IR spectra were recorded on FTIR spectrophotometer (SHIMADZU 8400 series) using KBr pellets. 1H NMR spectra were recorded in CDCl3/DMSO-d6 solvents unless otherwise mentioned. Chemical shifts are reported on AVANCE 300 MHz and INNOVA 400 MHz relative to TMS internal standard on the δ-scale. Mass spectra of the compounds were recorded on Mass spectrometer (Agilent 1100 series; EI/ES-MS) at Indian Institute of Chemical Technology, Hyderabad. The syntheses were carried out in scientific microwave oven supplied by Catalyst, India. All the reactions were monitored by thin layer chromatography on 60 F254 0.25 mm precoated aluminum plates (Merck) and visualized with UV light.
3. Results and Discussion
3-methylquinoxalin-2-ol (1) was synthesized by condensing o-phenylenediamine with ethyl pyruvate. Compound (1) was converted into ethyl [(3-methylquinoxalin-2-yl)oxy]acetate (2) by heating under reflux with ethyl chloroacetate in dry acetone and anhydrous potassium carbonate. The structure of compound (2) was confirmed by the carbonyl peak of ester at 1740 cm−1 in IR, and a mass ion peak at m/z 247 (100%) in mass spectroscopy. The reaction of the compound (2) with hydrazine hydrate (99%) in ethanol under reflux for 5-6 hr gave 2-[(3-methylquinoxaline-2-yl)oxy]acetohydrazide (3). The carbonyl peak of amide at 1680 cm−1 and NH, NH2 stretching at 3330 cm−1 and 3226 cm−1 in IR and a mass ion peak at m/z 233 (100%) corresponding to its molecular weight in mass spectrum confirmed the structure of the compound (3). From compound (3), ten different Schiff bases have been synthesized (Scheme 1) by condensing with indole-3-carbaldehyde, furfuraldehyde, 5-(4-nitrophenyl)-2-furfuraldehyde, and substituted benzaldehydes using conventional and microwave irradiation methods. The physical data of Schiff bases, time and yields in both the methods, were compared and mentioned in the Table 1.
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3.1. Antitubercular Activity
All the newly synthesized compounds were evaluated for their possible in vitro antitubercular activity at a concentration of 6.25 μg/mL against Mycobacterium tuberculosis H37RV (ATCC 27294) in BACTEC 12B medium using both micro dilution assay and the microplate alamar blue assay (MABA). Compounds exhibiting fluorescence were tested in the BACTEC 460 radiometric system. The antitubercular activity data were compared with standard drug, Rifampin at a concentration of 0.25 μg/mL. The compounds 4c, 4d, 4i, and 4j exhibited substantial antitubercular activity, particularly 4h showed 75% inhibition (MIC > 6.25 μg/mL) and emerged as the most active compound in the present series. The MIC and percentage inhibition data of the compounds that have shown activity against Mycobacterium tuberculosis are presented in Table 2.
3.2. Anti-Inflammatory Activity
Male Wistar rats (140–200 g) of both sexes were used for the studies. The rats were obtained from Mahaveer enterprises, Hyderabad. The animals were divided into groups of six each and fasted for 12 hr before the experiment. The ethical guidelines prescribed for the investigation of animals used in experiments were followed in all tests.
Carrageenan (0.1 mL, 1%) was administered into the plantar surface of the right hind paw of each rat, 1 hr after the administration of the test compounds and standard drug ibuprofen (100 mg/kg). One group was kept as control, received only 0.5% CMC solution. Before injection of carrageenan, the average volume of the right hind paw of each rat was calculated. After injection, the paw volume was measured after 3rd hr with the aid of a plethysmograph. The edema was expressed as an increase in the volume of paw, and percentage inhibition of acute edema was obtained as follows: where Among all the compounds, compounds 4a, 4b, 4d, 4g, 4h and 4i exhibited significant activity at 3rd hr (Table 3). Results are presented as Mean ± SEM (standard error of mean) of six rats. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisons, using Graph-pad Software.
3.3. Toxicity Study
Acute toxicity of schiff bases was determined in albino mice with the staircase method. Each group of 5 animals was fasted for 24 hr prior to the administration of the test compounds. The test compounds, 4a, 4b, 4d, 4g, 4h and 4i were administered orally in doses up to 2000 mg/kg and mice were kept under observation for period of 24 hrs.
4. General Procedure for the Synthesis of Schiff Bases
4.1. Microwave Irradiation Method
2-[(3-Methylquinoxalin-2-yl)oxy]acetohydrazide (3) (0.01 mole, 2.32 g) was dissolved in 10 mL of DMF and to this was added heterocyclic/aromatic aldehyde (0.01 mole) and few drops of glacial acetic acid. The mixture was transferred to a vessel and kept in the microwave oven. The oven was run at 400–480 W for different time for different reaction mixtures. The completion of the reaction was monitored continuously by TLC after every minute. The product obtained was poured into water, filtered, dried, and recrystallized from a mixture of DMF and water.
4.2. Conventional Method
Equimolar mixture of 2-[(3-methylquinoxalin-2-yl)oxy]acetohydrazide (3) and appropriate heterocyclic/aromatic aldehyde was dissolved in 90% ethanol and to this was added catalytic amount of glacial acetic acid and refluxed for 5–7 hr. Excess ethanol was removed under reduced pressure, and the residue was purified by washing with cold ethanol and recrystallized from DMF and water mixture.
2-[(3-Methylquinoxalin-2-yl)oxy]-N'-[(1E)-phenylmethylene]acetohydrazide 4a. Colorless solid; IR (KBr): 1656.74 (C=N str), 1679.88 (C=O str), 2958.6 (C–H str of CH3); 1H NMR (CDCl3): δ 11.84 (s, 1H, NH), 8.08 (s, 1H,CH=N), 7.77–7.36 (m, 9H, Ar–H), 5.46 (s, 2H, OCH2), 2.48 (s, 3H, CH3); Mass : m/z 321 (100%). Anal. Calcd for C18H16N4O2: C, 67.49; H, 5.03; N, 17.49. Found: C, 67.33; H, 5.01; N, 17.32%.
N'-[(1E)-(4-Chlorophenyl)methylene]-2-[(3-methylquinoxalin-2-l)oxy]acetohydrazide 4b. Colorless solid; IR (KBr): 1654.81 (C=N str), 1685.67 (C=O str), 2958.6 (C–H str of CH3), 754.12 (C–Cl); 1H NMR (CDCl3): δ 11.92 (s, 1H, NH), 8.23 (s, 1H, CH=N), 7.89–7.47 (m, 8H, Ar–H), 5.54 (s, 2H, OCH2), 2.81 (s, 3H, CH3); Mass : m/z 355 (100%). Anal. Calcd for C18H15Cl N4O2: C, 60.94; H, 4.26; N, 15.79. Found: C, 60.73; H, 4.15; N, 15.74%.
2-[(3-Methylquinoxalin-2-yl)oxy]-N'-[(1E)-(3-nitrophenyl)methylene]acetohydrazide 4c. Pale yellow solid; IR (KBr): 1654.81 (C=N str), 1689.53 (C=O str), 2948.96 (C–H str of CH3), 1320, 1480 (NO2 str); 1H NMR (CDCl3): δ 11.96 (s, 1H, NH), 8.42 (s, 1H, CH=N),7.98–7.53 (m, 8H, Ar–H), 5.67 (s, 2H, OCH2), 2.92 (s, 3H, CH3); Mass : m/z 366 (100%). Anal. Calcd for C18H15N5O4: C, 59.18; H, 4.14; N, 19.17. Found: C, 59.13; H, 4.11; N, 19.12%.
N'-[(1E)-(4-Hydroxyphenyl)methylene]-2-[(3-methylquinoxalin-2-yl)oxy]acetohydrazide 4d. Colorless solid; IR (KBr): 1658.67 (C=N str), 1681.81 (C=O str), 2958.6 (C–H str of CH3), 3352.05 (O–H str); 1H NMR (CDCl3): δ 11.64 (d, J = 10.2, NH), 9.94 (s, 1H, OH), 7.97 (s, 1H, N=CH), 7.78–6.83 (m, 8H, Ar–H), 5.41 (s, 2H, OCH2), 2.47 (s, 3H, CH3); Mass : m/z 337 (100%). Anal. Calcd for C18H16N4O3: C, 64.28; H, 4.79; N, 16.66. Found: C, 64.13; H, 4.71; N, 16.56%.
N'-[(1E)-(3,4-Dimethoxyphenyl)methylene]-2-[(3-methylquinoxalin-2-yl)oxy]acetohydrazide 4e. Pale brown solid; IR (KBr): 1647.1 (C=N str), 1677.95 (C=O str), 2948.96 (C–H str of CH3), 1267.14 (C–O–C str); 1H NMR (CDCl3): δ 11.23 (s, 1H, NH), 8.13 (s, 1H, CH=N), 7.83–7.47 (m, 7H, Ar–H), 5.48 (s, 2H, OCH2), 3.82 (s, 6H, OCH3), 2.43 (s, 3H, CH3); Mass : m/z 381 (100%). Anal. Calcd for C20H20N4O4: C, 63.15; H, 5.30; N, 14.73. Found: C, 63.07; H, 5.28; N, 14.62%.
N'-[(1E)-(4-Methoxyphenyl)methylene]-2-[(3-methylquinoxalin-2-yl)oxy]acetohydrazide 4f. Colorless solid; IR (KBr): 1654.81 (C=N str), 1679.88 (C=O str), 2962.46 (C–H str of CH3), 1257.50 (C–O–C str); 1H NMR (CDCl3): δ 11.53 (s, 1H, NH), 8.24 (s, 1H, CH=N), 7.82–7.58 (m, 8H, Ar–H), 5.53 (s, 2H, OCH2), 3.88 (s, 3H, OCH3), 2.51 (s, 3H, CH3); Mass : m/z 351 (100%). Anal. Calcd for C19H18N4O3: C, 65.13; H, 5.18; N, 15.99. Found: C, 65.10; H, 5.06; N, 15.63%.
N'-{(1E)-[4-(Dimethylamino)phenyl]methylene}-2-[(3-methylquinoxalin-2-yl)oxy]acetohydrazide 4g. Colorless solid; IR (KBr): 1654.81 (C=N str), 1677.95 (C=O str), 2920 (C–H str of CH3), 1365.51 (C–N str); 1H NMR (CDCl3): δ 11.16 (s, 1H, NH), 8.15 (s, 1H, CH=N), 7.75–7.38 (m, 7H, Ar–H), 5.32 (s, 2H, OCH2), 3.19 (s, 6H, N(CH3)2), 2.32 (s, 3H, CH3); Mass : m/z 364 (100%). Anal. Calcd for C20H21N5O2: C, 66.10; H, 5.82; N, 19.27. Found: C, 66.05; H, 5.66; N, 19.23%.
N'-[(1E)-1H-Indol-3-ylmethylene]-2-[(3-methylquinoxalin-2-yl)oxy]acetohydrazide 4h. Pale brown solid; IR (KBr): 1650.95 (C=N str), 1677.95 (C=O str), 2991.39 (C–H str of CH3), 3263.33 (N–H str of Ind-NH); 1H NMR (CDCl3): δ 11.58 (d, J = 19.8, 2H, Ind-H, NH), 8.268 (s, 1H, CH=N), 8.17–7.15 (m, 9H, Ar–H), 5.5 (s, 2H, OCH2), 2.47 (s, 3H, CH3); Mass : m/z 360 (100%). Anal. Calcd for C20H17N5O7: C, 66.84; H, 4.77; N, 19.49. Found: C, 66.75; H, 4.66; N, 19.39%.
N'-[(1E)-2-Furylmethylene]-2-[(3-methylquinoxalin-2-yl)oxy]acetohydrazide 4i. Pale yellow solid; IR (KBr): 1660.6 (C=N str), 1683.74 (C=O str), 2954.74 (C–H str of CH3), 1288.36 (C–O str); 1H NMR (CDCl3): δ 11.77 (d, J = 7.6, 1H, NH), 7.96 (s, 1H, CH=N), 7.84–6.64 (m, 7H, Ar–H), 5.36 (s, 2H, OCH2), 2.46 (s, 3H, CH3); Mass : m/z 311 (100%). Anal. Calcd for C16H14N4O3: C, 61.93; H, 4.55; N, 18.06. Found: C, 61.85; H, 4.66; N, 18.35%.
2-[(3-Methylquinoxalin-2-yl)oxy]-N'-{(1E)-[5-(4-nitrophenyl)-2-furfuryl]methylene}acetohydrazide 4j. Yellow solid; IR (KBr): 1654.61 (C=N str), 1689.4 (C=O str), 2956.24 (C–H str of CH3), 1286.53 (C–O str); 1H NMR (CDCl3): δ 11.69 (s, 1H, NH), 7.84 (s, 1H, CH=N), 7.78–6.73 (m, 10H, Ar–H), 5.24 (s, 2H, OCH2), 2.53 (s, 3H, CH3); Mass : m/z 432 (100%). Anal. Calcd for C22H17N5O5: C, 61.25; H, 3.97; N, 16.23. Found: C, 61.18; H, 3.68; N, 16.15%.
5. Conclusion
A novel series of schiff bases were synthesized by using conventional and microwave irradiation methods and all the compounds were characterized by physical and spectral data. Compounds 4c, 4d, 4h, 4i, and 4j exhibited potent antitubercular activity. Compounds 4a, 4b, 4d, 4g, 4h, and 4i have shown significant protection against the edema formation. With further molecular modification and manipulation of these compounds, several other promising bioactive molecules can be developed in future.
Acknowledgments
The authors are thankful to the Management of G. Pulla Reddy College of Pharmacy, Hyderabad, for providing facilities. Thanks are also due to IICT and NATCO Pharma Ltd., Hyderabad, for providing Mass and NMR spectral data.