SYNTHESIS AND BIOLOGICAL EVALUATION OF COUMARIN CLUBBED OXAZINESHTML Full Text
SYNTHESIS AND BIOLOGICAL EVALUATION OF COUMARIN CLUBBED OXAZINES
Nilesh B. Chauhan 1, Navin B. Patel * 1 and Bhupendra M. Mistry 2
Research Laboratory 1, Department of Chemistry, Veer Narmad South Gujarat University, Udhana-Magdalla Road, Surat - 395007, Gujarat, India.
Department of Food Science and Biotechnology 2, College of Life and Biotechnology, Dongguk University, Biomedical Campus, 32 Dongguk-ro, Ilsandong-gu, Goyang-si, Gyeonggi-do, Republic of Korea.
ABSTRACT: Coumarin segment have been reported broad spectrum of different biological activities with the oxazine derivatives. In view that we have synthesized newer coumarin clubbed 4-(4-fluorophenyl)-6-substituted phenyl-2H-1,3-oxazin-2-amine and screened for their biological studies. 4-(4-fluorophenyl)-6-substituted phenyl-2H-1,3-oxazin-2-amine IIa-j condensed with 4-Methyl-6-nitro-2-oxo-2H-chromen-7-yl chloro acetate 5 to afford 4-Methyl-6-nitro-2-oxo-2H-chromen-7-yl-2-(4-(4-sustitutedphenyl) oxazine - 2- yl - amino) acetates 2a-j. The newer compounds were characterized by IR, NMR and mass spectral studies and were screened for their antimicrobial, antitubercular and antioxidant activities. Compound 2a (-C6H5) 62.5 µg/ml, 2d (2-OH) 100 µg/ml against S. aureus, 2a (-C6H5) 50 µg/ml, 2f (4-F) and 2j (3-OPh) 100 µg/ml against S. pyogenes as compared to chloramphenicol and the other compound 2c (4-Cl) showed excellent potency 25 µg/ml against E. coli and 12.5 µg/ml against P. aeruginosa while compound 2e (4-OH) exhibited comparable activity of 62.5 µg/ml and 100 µg/ml against E. coli and P. areuginosa respectively. Furthermore 2i (3-Br) also showed 100 µg/ml moderate potency against E. coli compared to the standard drugs chloramphenicol and ciprofloxacin. The study of antifungal activity indicates that 2i (3-Br) showed remarkable potency of 250 µg/ml against C. albicans and compounds 2a (-C6H5), 2b (2-Cl), 2c (4-Cl), 2f (4-F) and 2j (3-OPh) exhibited comparable activity of 500 µg/ml against C. albicans when compared to the standard drug Griseofulvin.
Coumarin, Oxazine, Antimicrobial, Antitubercular, Antioxidant activity
INTRODUCTION: Coumarin is a versatile compound which exhibits excellent activity as antibacterial 1, as a continuous work on the synthesis of bioactive coumarin-containing analogs 2 herein this article we have reported the synthesis of coumarin based heterocyclic compounds having a wide range of pharmacological activities such as antimalarial 3, antioxidant 4, antiplatelet and antithrombotic 5, antifungal 6, herbicidal 7, antiviral 8, anticoagulant 9, anti-inflammatory 10, antitumor 11, anti-oxidant activity 12 and anti cancer 13.
In addition, 4 & 7 - hydroxy and nitro-coumarins are also potent antimicrobial 14 and antioxidant and very important for the synthesis of other coumarin derivatives 15. Antimicrobial activities of 4-methyl-7-hydroxycoumarin are enhanced by nitration. It was then reacted with chloro acetyl chloride to give chloro acetate derivative of 4-methyl-6-nitro-7-hydroxycoumarin.
Oxazines as well as 2-amino oxazines are well known moiety of heterocycles having a wide variety of biological activities and their uses as medicines are well established. Oxazines derivatives are reported to exhibit diverse biological activities as antitubercular 16, anticoagulant 17, antimicrobial 18, antioxidant-anticancer 19 and antifungal agents 20. In view of these finding and in continuation of our work on the synthesis of novel heterocyclic systems exhibiting good biological activity, we have coupled chloro acetate derivative of 4-methyl-6-nitro-7-hydroxycoumarin with a series of substituted amino oxazines to synthesize newer coumarin analogous and evaluated their antimicrobial, anti tuberculosis and anti oxidant activities.
MATERIALS AND METHODS: All solvents, chemicals and reagents were purchased from Sigma-Aldrich with the highest purity and used without further purification. Melting points were determined with open capillary method on ‘Equiptronics’ digital melting point apparatus, model no. EQ-730 and melting points are uncorrected. IR spectra were recorded on a Perkin Elmer spectrophotometer (KBr pellets) instrument. 1H and 13C NMR spectra were recorded on Bruker Avance II 400MHz NMR Spectrometer using DMSO- d6 as solvent and TMS as internal standard. All chemical shifts were reported as δ values (ppm). Mass spectra were recorded using water, Q-TOF Micromass (ESI – MS). Analytical thin-layer chromatography (TLC) was performed with Merck silica gel plates and visualized with UV irradiation (254 nm) or iodine.
RESULTS AND DISCUSSION:
Chemistry: The synthetic protocol for the lead molecule 4-methyl-6-nitro-2-oxo-2H-chromen-7-yl 2-chloroacetate 5 and final compounds 2a-j is depicted in scheme. Synthesis of 4-methyl-7-hydroxy-coumarin 3 was carried out by Pechmann condensation 21 followed by nitration with cerric ammonium nitrate (CAN), water, hydrogen peroxide 22 and then reacting with chloroacetyl chloride to get 4-methyl-6-nitro-2-oxo-2H-chromen-7-yl 2-chloroacetate 5 23. 2-amino oxazines IIIa-j were prepared by cyclo-addition reaction between substituted chalcones and urea 24. These substituted amino oxazines are condensed with compound (5) to synthesize desired analogs 2a-j.
Synthesis of 4-methyl -7-hydroxy-coumarin. (3): To a cooled conc. H2SO4 (75 ml), previously prepared mixture of resorcinol 1 (0.01 mol) in ethylacetoacetate 2 (0.01 mol) was added drop wise with maintaining the temperature below 10 0C. After completion of addition stirring was continued for 1 h. The reaction mixture was poured on to crushed ice. The product was filtered and washed with distilled water to remove access of acid. The product was dissolved in cold 10% NaOH solution and re-precipitated by 10% aqueous HCl till the solution becomes acidic. The product was washed with cold water till filtrate become neutral, dried and crystallized from ethanol using activated charcoal to get white solid 3, Yield: 85%, mp: 183°C to 185°C.
Synthesis of 7-hydroxy-4-methyl-6-nitro-2H-chromen - 2-one (4): The compound 3 (0.01 mol) was charged in two portions to a solution of cerric ammonium nitrate(CAN) (0.01 mol) containing 30% hydrogen peroxide (1 ml) in water (5ml). The resulting mixture was heated at 50 - 60 0C with stirring for 1h. The completion of the reaction was monitored by TLC (ethylacetate:n-hexane 7:3), then reaction mixture was cooled, diluted with water (20 ml) and extracted with chloroform (3×30 ml). Solvent was removed under reduced pressure to offer crude product which was further purified by column chromatography using silica gel 60-120 mesh and ethyl acetate in hexane as eluent to get desired pure product 4. Yield: 72%, mp: 193 -195°C (as reported).
Synthesis of 4-methyl-6-nitro-2-oxo-2H-chromen -7-yl 2-chloroacetate. (5): To a solution of 7-hydroxy-4-methyl-6-nitro - 2H- chromen-2-one 4 (0.01mol) and α-chloro acetyl chloride (0.012 mol) in dichloromethylene (30 ml), triethyl amine (0.0102 mol) was added drop wise and the mixture was stirred for 1h. The completion of the reaction was monitored by TLC (dichloro methylene: methanol, 9:1). The reaction mixture was then washed with 1 M HCl solution (100 ml) followed by 1 M NaOH solution (100 ml × 3). The dichloromethylene layer was separated, dried over MgSO4 and evaporated. The residue was crystallized from ethyl acetate-hexane as brownish yellow crystals 5. Yield: 69%, mp: 109oC -111oC.
Synthesis of (E)-1-(4-fluorophenyl)-3-phenyl prop-2-en-1-one (IIa): p-Fluoro acetophenone 6 (0.01 mol) and benzaldehyde Ia (0.01 mol) were dissolved in 15 ml ethanol. NaOH solution (0.02 mol) in ethanol was added slowly and the mixture stirred at 20 oC for 2h until the entire mixture becomes very thick so the stirring is no longer effective. The progress of the reaction was monitored by TLC (toluene: acetone, 8:2). Then the reaction mixture was poured slowly on to 400 ml of water with stirring and kept in refrigerator for 24 h. The precipitate obtained was filtered, washed and recrystallized from ethanol. The other compounds IIb-j were prepared by the same method using substituted benzaldehydes I b-j.
Synthesis of 4-(4-fluorophenyl)-6-phenyl-2H-1,3-oxazin-2-amine (IIIa): A mixture of (E)-1-(4-fluorophenyl)-3-phenylprop-2-en-1-one IIa (0.01 mol), urea (0.01 mol) were dissolved in ethanolic NaOH (10 mL) was refluxed about 2-3h. The completion of the reaction was monitored by TLC using a mixture of ethylacetate:n-hexane (3:7). This was then poured on to 400 ml of cold water with continuous stirring for 1h and then kept in refrigerator for 24h. The precipitate 4-(4-fluorophenyl)-6-phenyl-2H-1,3-oxazin - 2 - amine IIIa obtained was filtered, washed and recrystallized.
The other compounds IIIb-j were prepared by the same method using substituted (E)-1-(4-fluoro phenyl)-3-phenylprop-2-en-1-one IIb-j.
Synthesis of 4-methyl-6-nitro-2-oxo-2H-chromen -7yl-2-(4-(4-fluorophenyl) - 6 - phenyl - 2H-1, 3-oxazin-2-yl amino)acetate (2a): Compound (5) (0.01mol), 4-(4-fluorophenyl)-6-phenyl - 2H - 1, 3-thiazin-2-amine IIIa (0.01mol) and K2CO3 (0.024 mol) in DMF was stirred for 10-15 min and refluxed for 8-9h under nitrogen.
TABLE 1: CHARACTERIZATION DATA OF COMPOUNDS 2 a-j
|Com. No.||R||Mol. Formula||M.P.||Yield|
The completion of the reaction was monitored by TLC on silica gel using a mixture of ethylacetate:toluene (3:7). After the completion of the reaction, mixture was poured on to crushed ice and precipitate was filtered, washed with saturated solution of NaHCO3, dried and recrystallized from EtOH. The other compounds 2b-j were prepared by the same method using IV b-j.
FIG. 1: GENERAL STRUCTURE OF COUMARIN CLUBBED OXAZINE
(i) Cooled (5°C - 10°C), conc. H2SO4
(ii) CAN, 30% H2O2 + 5ml H2O, stirred
(iii) α-chloroacetyl chloride, CH2Cl2, triethyl amine 1h stirred
(iv) NaOH, EtOH, 2 -3 h stirred
(v) Urea, ethanolic NaOH, 2-3h refluxed
(vi) 4-methyl-6-nitro-2-oxo-2H-chromen-7-yl 2-chloroacetate, DMF, K2CO3, 8-9 h refluxed
Spectral Characterization of the Compounds:
4-Methyl-6-nitro-2-oxo-2H - chromen-7yl- 2 - (4-(4-fluorophenyl)-6-phenyl - 2H - 1, 3-oxazin - 2-yl-amino)acetates(2a): Light brown solid, yield: 68%, mp: 125-127°C, M.F.: C28H20FN3O7. IR (KBr) ν cm−1: 3243 (-NH), 1747, 1664 (-C=O), 1594 (-C=N), 3059, 2845 (-CH3), 15348, 1349 (-NO2), 1263, 1047 (-C-O-C-), 1165 (C-F); 1H NMR (400 MHz, DMSO-d6, TMS) δ: 8.58 (s, 1H, -CH), 6.36-7.78 (m, 12H, aromatic), 5.34 (s, 1H, -CH), 3.73 (s, 2H, -CH2NH), 3.24 (s, 1H, -CH2NH), 2.42 (s, 3H, -CH3); 13C NMR (100MHz, DMSO-d6 TMS) δ: 160.12 (C-2),113.92 (C-3), 152.51 (C-4), 120.74 (C-5), 139.46 (C-6), 135.63 (C-7), 115.75 (C-8), 159.14 (C-9), 118.66 (C-10), 19.43(C-12), 168.17 (C-14), 40.96 (C-16), 111.14 (C-18),157.34 (C-20), 74.14 (C-21), 160.04 (C-22), 134.11 (C-24), 130.12 (C-25, C-29), 115.54 (C-26, C-28), 165.16 (C-27), 131.24 (C-32), 127.95 (C-33, C-37), 128.83 (C-34, C-36), 126.31 (C-35),; m/z: 529.13 (M+).
4-Methyl-6-nitro-2-oxo-2H-chromen - 7yl - 2-(4-(4-fluorophenyl) - 6 - 2- chlorophenyl - 2H - 1, 3-oxazin-2-yl-amino)acetates(2b): Cream yellow solid, yield: 69%, mp: 116-118°C, M.F.: C28H19Cl FN3O7 IR (KBr) ν cm−1: 3238 (-NH), 1740, 1669 (-C=O), 1599 (-C=N-), 3067, 2840 (-CH3), 1538, 1358 (-NO2), 1258, 1057 (-C-O-C-), 1170 (C-F), 758 (C-Cl); 1H NMR (400 MHz, DMSO-d6, TMS) δ: 8.54 (s, 1H, -CH), 6.30-7.77 (m, 11H, aromatic), 5.34 (s, 1H, -CH), 3.70 (s, 2H, -CH2NH), 3.23 (s, 1H, -CH2NH), 2.41 (s, 3H, -CH3); 13C NMR (100MHz, DMSO-d6 TMS) δ: 160.12 (C-2),113.94 (C-3), 152.51 (C-4), 120.72 (C-5), 139.44 (C-6), 135.64 (C-7), 115.77 (C-8), 159.12 (C-9), 118.67 (C-10), 19.41 (C-12), 168.17 (C-14), 40.96 (C-16), 111.13 (C-18),157.37 (C-20), 74.16 (C-21), 160.14 (C-22), 134.13 (C-24), 130.16 (C-25, C-29), 115.52 (C-26, C-28), 165.12 (C-27), 131.51 (C-32), 133.10 (C-33), 131.56 (C-37), 127.80 (C-34), 131.25 (C-36), 128.35 (C-35),; m/z: 563.09 (M+), 565.09 (M+2).
4-Methyl-6-nitro-2-oxo-2H-chromen -7yl – 2 -(4-(4-fluorophenyl) - 6 - 4 -chlorophenyl - 2H - 1, 3- oxazin-2-yl-amino)acetates (2c): Cream yellow solid, yield: 69%, mp: 116-118°C, M.F.: C28H19ClFN3O7 IR (KBr) ν cm−1: 3245 (-NH), 1736, 1663 (-C=O), 1585 (-C=N-), 3059, 2836 (-CH3), 1545, 1351 (-NO2), 1249, 1061 (-C-O-C-), 1168(C-F), 769 (C-Cl); 1H NMR (400 MHz, DMSO-d6, TMS) δ: 8.58 (s, 1H, -CH), 6.38-7.78 (m, 11H, aromatic), 5.36 (s, 1H, -CH), 3.70 (s, 2H, -CH2NH), 3.20 (s, 1H, -CH2NH), 2.40 (s, 3H, -CH3); 13C NMR (100MHz, DMSO-d6, TMS) δ: 160.11 (C-2),113.94 (C-3), 152.52 (C-4), 120.71 (C-5), 139.42 (C-6), 135.63 (C-7), 115.77 (C-8), 159.12 (C-9), 118.65 (C-10), 19.40 (C-12), 168.12 (C-14), 40.90 (C-16), 111.10 (C-18),157.34 (C-20), 74.11 (C-21), 160.02 (C-22), 134.11 (C-24), 130.11 (C-25, C-29), 115.55 (C-26, C-28), 165.07 (C-27), 128.42 (C-32), 129.34 (C-33, C-37), 128.84 (C-34, C-36), 133.62 (C-35); m/z: 563.09 (M+), 565.09(M+2).
4-Methyl-6-nitro-2-oxo-2H-chromen - 7yl- 2- (4-(4-fluorophenyl)- 6 - 2 -hydroxyphenyl - 2H-1, 3-oxazin - 2-yl-amino) acetates (2d): Dark yellow solid, yield: 65%, mp: 135-137°C, M.F.: C28H20 FN3O8 IR (KBr) ν cm−1: 3378 (-OH), 3238 (-NH), 1740, 1661(-C=O), 1597 (-C=N-), 3064, 2830 (-CH3), 1531, 1357 (-NO2), 1251, 1053(-C-O-C-), 1174 (C-F); 1H NMR (400 MHz, DMSO-d6, TMS) δ : 9.34 (s, 1H, -OH), 8.57 (s, 1H, -CH), 6.32-7.77 (m, 11H, aromatic), 5.35 (s, 1H, -CH), 3.70 (s, 2H, -CH2NH), 3.21 (s, 1H, -CH2NH), 2.44 (s, 3H, -CH3); 13C NMR (100MHz, DMSO-d6, TMS) δ: 160.13 (C-2),113.94 (C-3), 152.54 (C-4), 120.72 (C-5), 139.42 (C-6), 135.65 (C-7), 115.78 (C-8), 159.14 (C-9), 118.65 (C-10), 19.44 (C-12), 168.15 (C-14), 40.98 (C-16), 111.15 (C-18),157.36 (C-20), 74.12 (C-21), 160.05 (C-22), 134.15 (C-24), 130.16 (C-25, C-29), 115.54 (C-26, C-28), 165.11 (C-27), 110.24 (C-32), 155.82 (C-33), 123.91 (C-37), 118.03 (C-34), 121.52 (C-36), 129.36 (C-35); m/z: 545.47 (M+).
4-Methyl-6-nitro-2-oxo-2H- chromen -7yl - 2 -(4-(4-fluorophenyl)-6-4-hydroxy phenyl - 2H -1, 3 -oxazin-2-yl-amino)acetates (2e): Yellow solid, yield: 69%, mp: 141-143°C, M.F.: C28H20FN3O8 IR (KBr) ν cm−1: 3375 (-OH), 3236 (-NH), 1740, 1664 (-C=O), 1595 (-C=N-), 3064, 2833 (-CH3), 1530, 1357 (-NO2), 1251, 1055 (-C-O-C-), 1172 (C-F); 1H NMR (400 MHz, DMSO-d6, TMS) δ : 9.37 (s,1H, -OH), 8.56 (s, 1H, -CH), 6.37-7.78 (m, 11H, aromatic), 5.38 (s, 1H, -CH), 3.73 (s, 2H, -CH2NH), 3.24 (s, 1H, -CH2NH), 2.42 (s, 3H, -CH3); 13C NMR (100MHz, DMSO-d6, TMS) δ: 160.13 (C-2),113.91 (C-3), 152.55 (C-4), 120.70 (C-5), 139.41 (C-6), 135.67 (C-7), 115.73 (C-8), 159.10 (C-9), 118.54 (C-10), 19.38 (C-12), 168.13 (C-14), 40.91 (C-16), 111.06 (C-18),157.31 (C-20), 74.16 (C-21), 160.01 (C-22), 134.12 (C-24), 130.11 (C-25, C-29), 115.52 (C-26, C-28), 165.06 (C-27), 122.64 (C-32), 129.71 (C-33, C-37), 115.54 (C-34, C-36), 156.73 (C-35).; m/z: 545.47 (M+)
4-Methyl-6-nitro-2-oxo-2H-chromen - 7yl - 2- (4-(4-fluorophenyl)- 6- 4 -fluoro phenyl -2H - 1, 3-oxazin-2-yl-amino)acetates (2f): Pale yellow solid, yield: 61%, mp: 102-104°C., M.F.: C28H19F2 N3O7; IR (KBr) ν cm−1: 3238 (-NH), 1742, 1663 (-C=O), 1598 (-C=N), 3064, 2832 (-CH3), 1535, 1353 (-NO2), 1251, 1055 (-C-O-C), 1176 (C-F); 1H NMR (DMSO-d6) δ (ppm): 8.57 (s, 1H, -CH), 6.37-7.76 (m, 11H, aromatic), 5.38 (s, 1H, -CH), 3.73 (s, 2H, -CH2NH), 3.22 (s, 1H, -CH2NH), 2.43 (s, 3H, -CH3); 13C NMR (100MHz, DMSO-d6, TMS) δ: 160.12 (C-2),113.91 (C-3), 152.54 (C-4), 120.71 (C-5), 139.44 (C-6), 135.65 (C-7), 115.75 (C-8), 159.13 (C-9), 118.64 (C-10), 19.44 (C-12), 168.19 (C-14), 40.94 (C-16), 111.15 (C-18),157.32 (C-20), 74.13 (C-21), 160.08 (C-22), 134.17 (C-24), 130.14 (C-25, C-29), 115.54 (C-26, C-28), 165.11 (C-27), 123.64 (C-32), 131.71 (C-33, C-37), 116.04 (C-34, C-36), 161.54 (C-35); m/z: 547.12 (M+).
4-Methyl-6-nitro-2-oxo-2H-chromen - 7yl - 2 -(4-(4-fluorophenyl)-6-4-tolyl- 2 H- 1, 3- oxazin-2-yl-amino)acetates (2g): Pale yellow solid, yield: 65%, mp: 110-112°C., M.F.: C29H22FN3O7; IR (KBr) ν cm−1: 3234 (-NH), 1745, 1661(-C=O), 1596 (-C=N-), 3074, 2834 (-CH3), 1534, 1357 (-NO2), 1251, 1058 (-C-O-C-), 1179 (C-F); 1H NMR (DMSO-d6) δ (ppm): 8.59 (s, 1H, -CH), 6.38-7.79 (m, 11H, aromatic), 5.35 (s, 1H, -CH), 3.71 (s, 2H, -CH2NH),3.21 (s, 1H, -CH2NH),2.42(s, 3H, -CH3), 2.38 (s, 3H, -CH3); 13C NMR (100MHz, DMSO-d6, TMS) δ: 160.10 (C-2),113.93 (C-3), 152.52 (C-4), 120.76 (C-5), 139.48 (C-6), 135.63 (C-7), 115.74 (C-8), 159.14 (C-9), 118.66 (C-10), 19.43 (C-12), 168.15 (C-14), 40.96 (C-16), 111.13 (C-18),157.36 (C-20), 74.15 (C-21), 160.05 (C-22), 134.12 (C-24), 130.14 (C-25, C-29), 115.54 (C-26, C-28), 165.13 (C-27), 127.02 (C-32), 127.44 (C-33, C-37), 128.93 (C-34, C-36), 136.35 (C-35), 24.44 (C-38).; m/z: 543.14(M+), 544.15(M+1).
4-Methyl-6-nitro-2-oxo-2H-chromen - 7yl - 2- (4-(4-fluorophenyl)-6-4-propyl - 2 H- 1, 3-oxazin-2-yl-amino)acetates (2h): Light yellow solid, yield: 63%, mp: 129-131°C., M.F.: C31H26FN3O7; IR (KBr) ν cm−1: 3228 (-NH), 1735, 1654 (-C=O), 1598 (-C=N), 3071, 2832 (-CH3), 1521, 1352 (-NO2), 1256, 1049 (-C-O-C-), 1176 (C-F); 1H NMR (DMSO-d6) δ (ppm): 8.59 (s, 1H, -CH), 6.36-7.78 (m, 11H, aromatic), 5.37 (s, 1H, -CH), 3.69 (s, 2H, -CH2NH), 3.24 (s, 1H, -CH2NH),2.41(s, 3H, -CH3), 2.97 (t, 2H, -CH2CH2CH3), 1.67 (m, 2H, -CH2CH2CH3), 0.96 (t, 3H, -CH2CH2CH3); 13C NMR (100MHz, DMSO-d6, TMS) δ: 160.13 (C-2),113.94 (C-3), 152.57 (C-4), 120.73 (C-5), 139.51 (C-6), 135.59 (C-7), 115.73 (C-8), 159.23 (C-9), 118.54 (C-10), 19.42 (C-12), 168.25 (C-14), 40.96 (C-16), 111.13 (C-18),157.36 (C-20), 74.15 (C-21), 160.05 (C-22), 134.12 (C-24), 130.14 (C-25, C-29), 115.54 (C-26, C-28), 165.13 (C-27), 127.02 (C-32), 127.44 (C-33, C-37), 128.93 (C-34, C-36), 139.35 (C-35), 34.34 (C-38), 24.12 (C-39), 13.34 (C-40); m/z: 571.55(M+).
4-Methyl-6-nitro-2-oxo-2H-chromen - 7yl -2 -(4-(4-fluorophenyl) - 6 - 3 -bromo phenyl - 2H - 1, 3-oxazin-2-yl-amino)acetates (2i): Dark yellow solid, yield: 61%, mp: 132-134°C., M.F.: C28H19Br FN3O7; IR (KBr) ν cm−1: 3225 (-NH), 1733, 1652 (-C=O), 1599 (-C=N), 3071, 2831 (-CH3), 1522, 1352 (-NO2), 1257, 1048 (-C-O-C), 1177 (C-F); 1H NMR (DMSO-d6) δ (ppm): 8.58 (s, 1H, -CH), 6.37-7.72 (m, 11H, aromatic), 5.36 (s, 1H, -CH), 3.70 (s, 2H, -CH2NH), 3.23 (s, 1H, -CH2NH), 2.41 (s, 3H, -CH3); 13C NMR (100MHz, DMSO-d6, TMS) δ: 160.12 (C-2),113.91 (C-3), 152.51 (C-4), 120.74 (C-5), 139.44 (C-6), 135.63 (C-7), 115.74 (C-8), 159.15 (C-9), 118.64 (C-10), 19.41 (C-12), 168.16 (C-14), 40.97 (C-16), 111.14 (C-18),157.34 (C-20), 74.17 (C-21), 160.05 (C-22), 134.18 (C-24), 130.18 (C-25, C-29), 115.51 (C-26, C-28), 165.15 (C-27), 131.37 (C-32), 130.05 (C-33), 124.16 (C-34), 129.88 (C-35), 129.12 (C-36), 127.52 (C-37).; m/z: 607.04 (M+),
4-Methyl-6-nitro-2-oxo-2H-chromen - 7 yl- 2- (4- (4-fluorophenyl)-6-3-phenoxy phenyl - 2H - 1, 3-oxazin-2-yl-amino) acetates (2j): Yellow solid, yield: 69%, mp: 109-111°C., M.F.: C34H24FN3O8; IR (KBr) ν cm−1: 3232 (-NH), 1734, 1655 (-C=O), 1597 (-C=N-), 307, 2833 (-CH3), 1525, 1354 (-NO2), 1256, 1045 (-C-O-C-), 1173 (C-F); 1H NMR (DMSO-d6) δ (ppm): 8.56 (s, 1H, -CH), 6.38-7.77 (m, 16H, aromatic), 5.36 (s, 1H, -CH), 3.70 (s, 2H, -CH2NH), 3.24 (s, 1H, -CH2NH), 2.40 (s, 3H, -CH3); 13C NMR (100MHz, DMSO-d6, TMS) δ: 160.11 (C-2),113.93 (C-3), 152.51 (C-4), 120.73 (C-5), 139.45 (C-6), 135.64 (C-7), 115.77 (C-8), 159.13 (C-9), 118.68 (C-10), 19.42(C-12), 168.18 (C-14), 40.95 (C-16), 111.12 (C-18),157.35 (C-20), 74.13 (C-21), 160.04 (C-22), 134.14 (C-24), 130.15 (C-25, C-29), 115.57 (C-26, C-28), 165.10 (C-27), 130.64 (C-32), 127.01 (C-33), 142.01 (C-34), 127.42 (C-35), 127.03 (C-36), 126.21 (C-37), 160.51 (C-39), 118.21 (C-40, C-44), 130.02 (C-41, C-43) 122.86 (C-42); m/z: 621.15 (M+)
Antituberculer Activity: We have used the MIC to evaluate the anti-tuberculosis activity 25. Each synthesized compound was diluted obtaining 2000 mg/ml concentration, as a stock solution and then many dilution made from it i.e. 500 mg/ml, 250 mg/ml, 125 mg/ml, 100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml, 6.250 mg/ml, 3.125 mg/ml, 1.5625 mg/ml.
Reading Result: The highest dilution showing at least 99 % inhibition of colonies is taken as MIC. The result of this is much affected by the size of the inoculum. The test mixture should contain 108 organism / ml.
TABLE 2: ANTIBACTERIAL AND ANTIFUNGAL DATA OF COMPOUNDS 2 a-j
|Number||Antibacterial activity||Antifungal activity|
|S. aureus||S. pyogenes||E.coli||P. aeruginosa||C.albicans||A.niger||A.clavatus|
|MTCC 96||MTCC 442||MTCC 443||MTCC 1688||MTCC227||MTCC282||MTCC1323|
TABLE 3: MYCOBACTERIUM TUBERCULOSIS SCREENING RESULTS OF COUMARIN DERIVATIVES 2 a-j
|Compound||H37Rv MTCC 200||% Inhibition|
Antioxidant activity: In vitro antioxidant activity of all compounds 2 a-j were carried out by the 2,-diplenyl1-1-picrylhydrazyl (DPPH) and 2,2′-Azinobis (3-ethylbenzthiazoline - 6 - sulfonate) (ABTS) cation radical assay according to the literature 26. DPPH radical scavenging activity evaluation is a rapid and the best technique for screening antioxidant activities of the antioxidants. The values of IC50 of all compounds are higher than the IC50 value of ascorbic acid, which is shown in the following table.
TABLE 4: SCREENING RESULTS OF DPPH AND ABTS RADICAL SCAVENGING ACTIVITY OF 2 a–j
|Number||IC50 μg/mL ± SD||IC50 μg/mL ± SD|
|2a||64.08 ± 1.313||67.82 ± 0.94|
|2b||137.58 ± 1.538||563.02 ± 0.590|
|2c||63.66 ± 1.829||268.55 ± 0.362|
|2d||95.79 ± 0.717||384.04 ± 4.025|
|2e||64.68 ± 0.752||87.63 ± 0.149|
|2f||64.35 ± 0.966||59.74 ± 0.355|
|2g||99.94 ± 0.582||373.98 ± 5.869|
|2h||59.78 ± 1.153||265.47 ± 3.576|
|2i||56.19 ± 0.509||61.64 ± 0.757|
|2j||124.70 ± 1.436||548.83 ± 0.801|
|Ascorbic Acid||36.22 ± 0.469||22.64 ± 0.260|
RESULTS AND DISCUSSION:
Chemistry: 4-Methyl-6-nitro-2-oxo-2H-chromen-7-yl 2-chloroacetate 5 was synthesized from 7-hydroxy-4-methyl-6-nitro-2H-chromen - 2 - one 4. Compound 5 on coupling with 4-(4-Fluorophenyl)-6-substitutedphenyl-2H-1,3-oxazin-2-amine IV a-j to yield 4-methyl-6-nitro-2-oxo-2H-chroman-7yl-2-(4-(4-fluorophenyl)-6-substitutedphenyl - 2H - 1, 3-oxazin-2-ylamino) acetates 2a-j were obtained form 4-(4-fluorophenyl)-6-substitutedphenyl - 2H- 1, 3-oxazin-2-amine IV a-j Scheme.
Characterizations of intermediate and final compounds have been described in Table 1. The structures of compounds were confirmed by their spectral analysis. The characteristic band of 3242 cm-1 of -NH, while two bands for -C=O appeared at 1715 and 1671 cm-1 and 1523, 1351 cm-1 for -NO2 in IR spectrum confirmed the structure of compounds. 1H NMR spectrum of compound showed singlet at 8.55 of -CH confirmed the neighboring -NO2 group while -CH2NH showed two singlet at 3.71 and 3.23 respectively. 13C NMR spectrum of compounds showed at 160.10 and 168.20 for two different C=O and one peak at 19.40 for CH3 of coumarin. Peaks at 43.90, 81.10 and 111.30 were obtained for -CHNH, -CH of oxazine (C-18) respectively, while peaks observed at 150.20 and 164.70 for oxazine ring which confirming the structure of compounds.
FIG. 1: MINIMUM INHIBITORY CONCENTRATIONS FOR ANTIBACTERIAL ACTIVITY OF 2a-j
FIG. 2: MINIMUM INHIBITORY CONCENTRATIONS FORANTIFUNGAL ACTIVITY OF 2a-j
The minimum inhibitory concentrations (MIC) of 2a-j were evaluated against various bacterial and fungal species. The results of this activity are mentioned in Table 2.
Compound 2c with –Cl substitution at position 4 had excellent activity with 25 µg/ml while compound 2c, it showed excellent activity 12.5 µg/ml against P. aeruginosa which is comparable to chloramphenicol and ciprofloxacin while other compounds showed poor activity against S. aureus and S. pyogenes when compared with chloramphenicol, ciprofloxacin and norfloxacin. Other compounds 2i and 2j having substituent 3-Br and 3-OC6H5 respectively exhibited significant activity with MIC value 250 µg/ml. Compounds 2a (-C6H5), 2b (2-Cl), 2c (4-Cl), 2f (4-F) and 2j (3-OPh) exhibited comparable activity of 500 µg/ml against C. albicans as compared to the standard drug griseofulvin.
The minimum inhibitory concentrations (MIC) of 2a-j were tested for antituberculer activities. The results of this activity are mentioned in Table 3. Antituberculer activity results showed that, compound 2b and 2g containing 2-chloro and 3-bromo group demonstrated better activity 100 µg/ml with 99% inhibition against M. tuberculosis H37Rv.
FIG. 3: ANTITUBERCULER ACTIVITIES OF 2a-j
FIG. 4: ANTIOXIDANT POWER IC50 μg/mL ± SD of 2a-j
From the antioxidant inspections molecules 2i and 2h bearing 3-Br and 4-C3H7 respectively were appeared to have high radical scavenging efficacies as 56.19 ± 0.509 μg/ml ± SD and 59.78 ± 1.153 μg/ml ± SD of IC50 values in DPPH and 2f and 2i containing 59.74 ± 0.355 and 65.64 ± 0.757 μg/ml ± SD of IC50 values in ABTS bioassay, respectively and can be comparable to that of control ascorbic acid while other compounds have moderate to poor antioxidant power against scavenging DPPH and ABTS, results were summarized in Table 4.
CONCLUSION: The novel compound 2c had excellent activity with 25 µg/ml against E. coli and 12.5µg/ml against P. aeruginosa which is comparable to the standard drugs chloramphenicol and ciprofloxacin while other compounds showed poor activity against S. aureus and S. pyogenes when compared with chloramphenicol, ciprofloxacin and norfloxacin. Candidate compound 2b and 2g demonstrated better antiTB activity 100 µg/ml with 99% inhibition and 2i (56.19 ± 0.509) against DPPH and 2f (59.74 ± 0.355) against ABTS radical scavenging activity are highly potent amongst all compounds. While other compounds exhibited very less reactivity.
ACKNOWLEDGEMENT: The authors are highly obliged to University Grants Commission, New Delhi for awarding ‘Teacher Fellowship Award’ under ‘Faculty Improvement Program’; Principal Dr. N. M. Patel, Shree Jayendrapuri Arts and Science College, Bharuch; Dr. K. C. Patel, Dean of science faculty and Head, Department of Chemistry for Laboratory facilities and Dr. Dhanji Rajani from Microcare Laboratory. We are also thankful to S.A.I.F., Chandigarh for 1H and 13C NMR spectral analysis.
CONFLICT OF INTEREST: The authors declare that there is no conflict of interest.
- Behrami A and Dobroshi F: Antibacterial activity of coumarin derivatives synthesized from 4,7-dihydroxy–chromen-2-one and comparison with standard drug. Chem. & Pharma. Res. 2015; 7(8): 1087-1091.
- Shokhan JH and Kubba AARM: Synthesis and characterization of new coumarin derivatives containing various moieties with antibacterial activities. I J Pharmacy & Pharma Sci. 2015; 7(8): 70-74.
- Ratchanok P, Amporn S, Prasit M, Chanin N, Supaluk P, Somsak R and Virapong P: Synthesis, biological evaluation and molecular docking of novel chalcone coumarin hybrids as anticancer and antimalarial agents. J Med. Chem. 2014; 85(6): 65-76.
- Shaikh MH, Subhedar DD, Shingate BB, Khan FAK, Sangshetti JN, Khedkar VM, Nawale L, Sarkar D, Navale GR and Shinde SS: Synthesis, biological evaluation and molecular docking of novel coumarin incorporated triazoles as antitubercular, antioxidant and antimicrobial agents. Med Che Res. 2016; 25(4): 790-804.
- KontogiorgisC, Orazio N, Mangiatordi GF, Massimiliano, Tognolini, Karalaki F, Giorgio C, Patsilinakos A, Carotti A, Litina DH and Barocelli: Studies on the antiplatelet and antithrombotic profile of anti-inflammatory coumarin derivatives. J Enz Inh Med Chem. 2015; 30(6): 925-933.
- Ahmed A, Al-Amiery, Kadhum AAH and Mohhamad AB: Antifungal activities of new coumarins. Molecules, 2012; 17: 5713-5723.
- Fabrizio A, Macuso R, Lupini A, Giofre SV, Sunseri F, Gabriele B and Abenavoli MR: Phytotoxic potential and biological activity of three synthetic coumarin derivatives as new natural-like herbicides. Molecules. 2015; 20: 17883-17902.
- Hassan M,Osman H, Ali MA and Ahsan MJ: Therapeutic potential of coumarins as antiviral agents. J Med. Chem. 2016; 123:236–255.
- Jain PK and Joshi H: Coumarin: Chemical and Pharmacological Profile. J. App. Pharma. Sci. 2012; 2(6): 236-240.
- Kayal G, Kayal S, Gokhale N and Jain S: Synthesis and biological evaluation of coumarin derivatives as anti-inflammatory agents. I J Pharmaceutical Sci Res. 2016; 7(6): 2394-2401.
- Chougala BM, Shastri SL, Holiyachi M, Shastri LA, More SS and Ramesh KV: Synthesis, anti-microbial and anti-cancer evaluation study of 3-(3-benzo furanyl)-coumarin derivatives, Med Che Res. 2015; 24(12): 4128–4138.
- Kotali A, Nasiopoulou DA, Tsoleridis CA, Harris PA, Kontogiorgis CA and Hadjipavlou-Litina DJ: Antioxidant activity of 3-[N-(Acylhydrazono)ethyl]-4-hydroxy-coumarins. Molecules, 2016; 21: 138.
- Emami S and Dadashpour S: Current developments of coumarin-based anti-cancer agents in medicinal chemistry. J Med. Chem. 2015; 102: 611-630.
- Zhang R, Xu Z, Yin W, Liu P and Zhang W: Microwave-assisted synthesis and antifungal activities of polysubstituted furo[3,2-c]chromen-4-ones and 7,8,9,10-tetrahydro-6H-benzofuro[3,2-c]chromen-6-ones, Synth. Comm. 2014; 44: 3257–3263.
- Dekic V, Radulovic N, Vukicevic R, Dekic B, Stojanovic-Radic Z and Palic R: Influence of the aryl substituent identity in 4-arylamino-3-nitrocoumarins on their antimicrobial activity, J Pharm. Pharmacology. 2011; 5(3): 371-375.
- Kalra S, Kumar A and Gupta MK: Modeling of antitubercular activity of biphenyl analogs of 2-nitroimidazo[2,1-b][1,3]oxazine to rationalize their activity profile. Chem. Res. 2013; 22: 3444- 3451.
- Sawant RL, Mhaske MS and Wadekar JB: A series of Schiff bases of 1, 3-oxazines were synthesized via reaction of 1, 3-oxazine-2-amine with substituted benzaldehyde and they exhibited significant anticoagulant activity. Int. J Pharma & Pharmaceutical Sci. 2012; 4(4): 320-323.
- Sayaji SD and Pravina BP: Novel synthesis and antimicrobial activity of bis-oxazine derivatives, J Chem. & Pharm. Res. 2013; 5(5): 271- 274.
- Kalirajan R, Kulshrestha V, Sankar S and Jubie S: Docking studies, synthesis, characterization of some novel oxazine substituted 9-anilinoacridine derivatives and evaluation for their antioxidant and anticancer activities as topoisomerase II inhibitors. J Med. Chem. 2012; 56: 217-224.
- Dhanya S, Upadhya SH, Savitha and Rama M: Synthesis, characterization and QSAR studies of some New 1, 3-oxazines as Potent Antimicrobial agents. Res. J. Pharma Sci. 2013; 2(2): 15-19.
- Vogel AI, Furniss BS, Hannaford AJ, Smith PWG and Tatchel AR: Vogel’s textbook of practical organic chemistry - 5th edition, 1989; 1193.
- Ganguli N, Sukai AK and De S: Cerium(IV) ammonium nitrate mediated nitration of coumarins. Comm. 2011; 31(2): 301-309.
- Qandil AM and Fakhouri LI: α-Anilino ketones, Esters and Amides: A Chemical Study. 2012; 5: 591-612.
- Fekria MA, Soliman N, Dawoud TA and Hamza RM: Synthesis, biological and Anti-tumor: Evaluation of Some New Nucleosides Incorporating Heterocyclic Moieties. Ame J O Che 2015; 5(5): 137-148.
- Rattan A and Churchill BI: Antimicrobials in laboratory medicine, Livingstone, New Delhi, 2000; 85.
- Brand-Williams W, Cuvelier ME and Berset C: Use of a free radical method to evaluate antioxidant activity. Lebensm Wiss u -Technol 1995; 28: 25-30.
How to cite this article:
Chauhan NB, Patel NB and Mistry BM: Synthesis and biological evaluation of coumarin clubbed oxazines. Int J Pharm Sci Res 2018; 9(6): 2595-03. doi: 10.13040/IJPSR.0975-8232.9(6).2595-03.
All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.
N. B. Chauhan, N. B. Patel * and B. M. Mistry
Department of Chemistry, Veer Narmad South Gujarat University, Surat, Gujarat, India.
07 July, 2017
31 October, 2017
21 May, 2018
01 June, 2018