2.1 General methods
All manipulations were performed in a nitrogen atmosphere that was dry and deoxygenated. Freshly distilled ethanol was dehydrated in the presence of sodium metal. Analytical grade sodium borohydride (Qualigens) and sulfur (Aldrich) were used. All other reagents were AR grade. Melting points were uncorrected. TMS was used as an internal standard to record the NMR spectra of the ligands. Chemical shifts (δ) are expressed relative to TMS in units of parts per million. The Perkin–Elmer model-1430 spectrometer was used to measure infrared spectra. Elemental analysis was carried out using a Perkin–Elmer 2400 CHN analyzer. The mass spectra were obtained using a VG-70S 11-250J mass spectrometer. A Bruker Smart Apex CCD diffractometer was used to collect X-ray diffraction data. The compounds were separated and purified using column chromatography on activated silica gel (230–400) with chloroform : methanol medium.
2.2 Synthesis of chrysin derivatives of organosulfur ligands
Compounds HL2a-HL2b synthesis was prepared at 110 °C by refluxing chrysin with n-propyl amine / n-pentyl amine into toluene. For new derivatives of chrysin containing amines HL3a-HL3n, have also been prepared through Mannich reactions. HL4a-HL4n was prepared via refluxing equimolar quantities of chrysin-containing nitrogen derivatives (HL3a-HL3n) with 2-(phenylthio)aniline in ethanol (40mL). Acetic acid (1 mL) was added to this solution (Scheme 1). The solution was stirred for 3 hrs and precipitates formed. The precipitate was filtered and washed with water and ethanol. In different yields the ligands were obtained as solids (Scheme 2). The synthesized compounds were confirmed using TLC and elemental analysis and compouds purified by using chloroform and methanol (7:3).
Synthesis of (4E)-4-(2-(phenylthio)phenylimino)-8-((ethylamino)methyl)-1,4-dihydro-2-phenyl-1-propylquinoline-5,7-diol (HL4a): Yield: 73%. Anal. calcd for C33H33N3O2S: C, 73.99, H, 6.21, N, 7.84, S, 5.99. Found: C, 73.78, H, 6.08, N, 7.72, S, 5.82. Fast atom bombardment mass spectrometry (FAB-MS), m/z 536 [M+1]. 1H NMR (400 MHz, CDCl3, δ, ppm): 4.58 (1H, s, -CH), 5.43 (1H, s, -CH), 7.12-7.36 (5H, m, Ar-H, -CH), 3.08 (2H, t, -CH2), 1.57 (2H, m, -CH2), 0.96 (3H, t, -CH3), 3.83 (2H, s, -CH2), 2.05 (1H, -NH), 2.61 (2H, m, -CH2), 1.05 (3H, t, -CH3), 12.23 (1H, s, D2O exchange, -OH), 10.23 (1H, s, D2O exchange, -OH), 6.98-7.22 (9H, m, Ar-H, -CH). 13C NMR (400 MHz, CDCl3, ppm): 155.2 (C2), 102.2 (C3), 164.6 (C4), 161.4 (C5), 91.4 (C6), 159.6 (C7), 101.1 (C8), 144.5 (C9), 94.7 (C10), 134.3 (C11), 126.4 (C12, 16), 128.7 (C13, 15), 128.0 (C14), 46.7 (C17), 21.8 (C18), 11.8 (C19), 35.7 (C20), 44.2 (C22), 15.8 (C23), 160.2 (C25), 122.9 (C26), 128.6 (C27), 127.9 (C28), 132.5 (C29), 134.8 (C30), 135.7 (C32), 131.2 (C33, 37), 129.4 (C34, 36), 127.3 (C35).
Synthesis of (4E)-4-(2-(phenylthio)phenylimino)-1,4-dihydro-2-phenyl-1-propyl-8-((propylamino)methyl)quinoline-5,7-diol (HL4b): Yield: 81%. Anal. calcd for C34H35N3O2S: C, 74.29, H, 6.42, N, 7.64, S, 5.83. Found: C, 74.18, H, 6.32, N, 7.51, S, 5.74. Fast atom bombardment mass spectrometry (FAB-MS), m/z 550 [M+1]. 1H NMR (400 MHz, CDCl3, δ, ppm): 4.59 (1H, s, -CH), 5.43 (1H, s, -CH), 7.12-7.36 (5H, m, Ar-H, -CH), 3.07 (2H, t, -CH2), 1.58 (2H, m, -CH2), 0.95 (3H, t, -CH3), 3.83 (2H, s, -CH2), 2.05 (1H, -NH), 2.58 (2H, m, -CH), 1.48 (2H, m, -CH2), 0.97 (3H, t, -CH3), 10.71 (1H, s, D2O exchange, -OH), 10.13 (1H, s, D2O exchange, -OH), 6.98-7.22 (9H, m, Ar-H, -CH). 13C NMR (400 MHz, CDCl3, ppm): 152.3 (C2), 102.1 (C3), 164.6 (C4), 161.4 (C5), 91.7 (C6), 159.6 (C7), 100.8 (C8), 144.5 (C9), 94.7 (C10), 134.3 (C11), 126.4 (C12, 16), 128.7 (C13, 15), 128.0 (C14), 46.7 (C17), 21.8 (C18), 11.8 (C19), 36.4 (C20), 51.9 (C22), 23.8 (C23), 11.7 (C24), 160.2 (C26), 122.7 (C27), 128.4 (C28), 127.7 (C29), 132.4 (C30), 134.8 (C31), 135.4 (C33), 131.2 (C34, 38), 129.4 (C35, 37), 127.3 (C36).
Synthesis of (4E)-4-(2-(phenylthio)phenylimino)-8-((dimethylamino)methyl)-1,4-dihydro-2-phenyl-1-propylquinoline-5,7-diol (HL4c): Yield: 79%. Anal. calcd for C33H33N3O2S: C, 73.99, H, 6.21, N, 7.84, S, 5.99. Found: C, 73.88, H, 6.12, N, 7.71, S, 5.81. Fast atom bombardment mass spectrometry (FAB-MS), m/z 536 [M+1]. 1H NMR (400 MHz, CDCl3, δ, ppm): 4.61 (1H, s, -CH), 5.42 (1H, s, -CH), 7.12-7.36 (5H, m, Ar-H, -CH), 3.06 (2H, t, -CH2), 1.57 (2H, m, -CH2), 0.97 (3H, t, -CH3), 3.63 (2H, s, -CH2), 2.28 (6H, s, -CH3), 12.73 (1H, s, D2O exchange, -OH), 10.28 (1H, s, D2O exchange, -OH), 6.98-722 (9H, m, Ar-H, -CH). 13C NMR (400 MHz, CDCl3, ppm): 155.2 (C2), 101.9 (C3), 164.6 (C4), 161.4 (C5), 91.3 (C6), 159.6 (C7), 101.3 (C8), 144.5 (C9), 94.7 (C10), 134.3 (C11), 126.4 (C12, 16), 128.7 (C13, 15), 128.0 (C14), 46.7 (C17), 21.8 (C18), 11.8 (C19), 46.2 (C20), 45.3 (C22, C23), 160.3 (C25), 122.6 (C26), 128.4 (C27), 127.8 (C28), 132.4 (C29), 134.5 (C30), 135.5 (C32), 131.2 (C33, 37), 129.4 (C34, 36), 127.3 (C35).
Synthesis of (4E)-8-((diethylamino)methyl)-4-(2-(phenylthio)phenylimino)-1,4-dihydro-2-phenyl-1-propylquinoline-5,7-diol (HL4d): Yield: 76%. Anal. calcd for C35H37N3O2S: C, 74.57, H, 6.62, N, 7.45, S, 5.69. Found: C, 74.48, H, 6.52, N, 7.31, S, 5.61. Fast atom bombardment mass spectrometry (FAB-MS), m/z 564 [M+1]. 1H NMR (400 MHz, CDCl3, δ, ppm): 4.57 (1H, s, -CH), 5.43 (1H, s, -CH), 7.12-7.36 (5H, m, Ar-H, -CH), 3.07 (2H, t, -CH2), 1.58 (2H, m, -CH2), 0.95 (3H, t, -CH3), 3.61 (2H, s, -CH2), 2.38 (4H, m, -CH2), 1.02 (6H, t, -CH3), 10.88 (1H, s, D2O exchange, -OH), 10.33 (1H, s, D2O exchange, -OH), 6.98-7.22 (9H, m, Ar-H, -CH). 13C NMR (400 MHz, CDCl3, ppm): 155.2 (C2), 101.9 (C3), 164.6 (C4), 161.4 (C5), 91.3 (C6), 159.6 (C7), 101.3 (C8), 144.5 (C9), 94.7 (C10), 134.3 (C11), 126.4 (C12, 16), 128.7 (C13, 15), 128.0 (C14), 46.7 (C17), 21.8 (C18), 11.8 (C19), 41.9 (C20), 49.4 (C22, C24), 13.6 (C23, C25), 160.2 (C27), 122.3 (C28), 128.4 (C29), 127.3 (C30), 132.9 (C31), 134.8 (C32), 135.5 (C34), 131.2 (C35, 39), 129.4 (C36, 38), 127.3 (C37).
Synthesis of (4E)-8-((1H-pyrrol-1-yl)methyl)-4-(2-(phenylthio)phenylimino)-1,4-dihydro-2-phenyl-1-propylquinoline-5,7-diol (HL4e): Yield: 89%. Anal. calcd for C35H31N3O2S: C, 75.38, H, 5.60, N, 7.53, S, 5.75. Found: C, 75.28, H, 5.51, N, 7.45, S, 5.63. Fast atom bombardment mass spectrometry (FAB-MS), m/z 558 [M+1]. 1H NMR (400 MHz, CDCl3, δ, ppm): 4.59 (1H, s, -CH), 5.42 (1H, s, -CH), 7.12-7.36 (5H, m, Ar-H, -CH), 3.08 (2H, t, -CH2), 1.57 (2H, m, -CH2), 0.93 (3H, t, -CH3), 5.11 (2H, s, -CH2), 6.00-6.53 (4H, m, -CH), 10.94 (1H, s, D2O exchange, -OH), 10.19 (1H, s, D2O exchange, -OH), 6.98-7.22 (9H, m, Ar-H, -CH). 13C NMR (400 MHz, CDCl3, ppm): 155.2 (C2), 102.2 (C3), 164.6 (C4), 161.4 (C5), 91.3 (C6), 159.6 (C7), 101.3 (C8), 144.5 (C9), 94.7 (C10), 134.3 (C11), 126.4 (C12, 16), 128.7 (C13, 15), 128.0 (C14), 46.7 (C17), 21.8 (C18), 11.8 (C19), 38.5 (C20), 122.8 (C22, 24), 108.9 (C23, 25), 159.8 (C27), 122.7 (C28), 128.1 (C29), 127.7 (C30), 132.5 (C31), 134.8 (C32), 135.5 (C34), 131.2 (C35, 39), 129.4 (C36, 38), 127.4 (C37).
Synthesis of (4E)-8-((N-methyl-N-phenylamino)methyl)-4-(2-(phenylthio)phenylimino)-1, 4-dihydro-2-phenyl-1-propylquinoline-5,7-diol (HL4f): Yield: 72%. Anal. calcd for C38H35N3O2S: C, 76.35, H, 5.90, N, 7.03, S, 5.36. Found: C, 76.28, H, 5.81, N, 6.97, S, 5.21. Fast atom bombardment mass spectrometry (FAB-MS), m/z 598 [M+1]. 1H NMR (400 MHz, CDCl3, δ, ppm): 4.59 (1H, s, -CH), 5.45 (1H, s, -CH), 7.12-7.36 (5H, m, Ar-H, -CH), 3.06 (2H, t, -CH2), 1.54 (2H, m, -CH2), 0.94 (3H, t, -CH3), 4.64 (2H, s, -CH2), 6.55-7.10 (5H, m, -CH), 2.87 (3H, s, -CH3), 12.92 (1H, s, D2O exchange, -OH), 10.24 (1H, s, D2O exchange, -OH), 6.98-7.22 (9H, m, Ar-H, -CH). 13C NMR (400 MHz, CDCl3, ppm): 155.2 (C2), 102.2 (C3), 164.6 (C4), 161.4 (C5), 91.3 (C6), 159.6 (C7), 101.3 (C8), 144.5 (C9), 94.7 (C10), 134.3 (C11), 126.4 (C12, 16), 128.7 (C13, 15), 128.0 (C14), 46.7 (C17), 21.8 (C18), 11.8 (C19), 37.5 (C20), 149.4 (C22), 114.6 (C23, 27), 129.9 (C24, 26), 118.6 (C25), 37.6 (C28), 160.1 (C30), 122.3 (C31), 128.2 (C32), 127.2 (C33), 132.9 (C34), 134.5 (C35), 135.5 (C37), 131.2 (C38, 42), 129.4 (C39, 41), 127.4 (C40).
Synthesis of (4E)-8-((N-ethyl-N-phenylamino)methyl)-4-(2-(phenylthio)phenylimino)-1, 4-dihydro-2-phenyl-1-propylquinoline-5,7-diol (HL4g): Yield: 69%. Anal. calcd for C39H37N3O2S: C, 76.56, H, 6.10, N, 6.87, S, 5.24. Found: C, 76.48, H, 6.01, N, 6.77, S, 5.15. Fast atom bombardment mass spectrometry (FAB-MS), m/z 612 [M+1]. 1H NMR (400 MHz, CDCl3, δ, ppm): 4.63 (1H, s, -CH), 5.45 (1H, s, -CH), 7.12-7.36 (5H, m, Ar-H, -CH), 3.08 (2H, t, -CH2), 1.57 (2H, m, -CH2), 0.96 (3H, t, -CH3), 4.60 (2H, s, -CH2), 6.55-7.10 (5H, m, -CH), 3.37 (2H, t, -CH2), 1.13 (3H, s, -CH3), 10.84 (1H, s, D2O exchange, -OH), 10.13 (1H, s, D2O exchange, -OH), 6.98-7.22 (9H, m, Ar-H, -CH). 13C NMR (400 MHz, CDCl3, ppm): 155.2 (C2), 102.2 (C3), 164.6 (C4), 161.4 (C5), 91.3 (C6), 159.6 (C7), 101.3 (C8), 144.5 (C9), 94.7 (C10), 134.3 (C11), 126.4 (C12, 16), 128.7 (C13, 15), 128.0 (C14), 46.7 (C17), 21.8 (C18), 11.8 (C19), 37.5 (C20), 149.4 (C22), 114.6 (C23, 27), 129.9 (C24, 26), 118.6 (C25), 37.6 (C28), 13.2 (C29), 160.1 (C31), 122.3 (C32), 129.1 (C33), 127.3 (C34), 132.9 (C35), 134.3 (C36), 135.5 (C38), 131.2 (C39, 43), 129.4 (C40, 42), 127.4 (C41).
Synthesis of (4E)-4-(2-(phenylthio)phenylimino)-8-((ethylamino)methyl)-1,4-dihydro-1-pentyl-2-phenylquinoline-5,7-diol (HL4g): Yield: 74%. Anal. calcd for C35H37N3O2S: C, 74.57, H, 6.62, N, 7.45, S, 5.69. Found: C, 74.48, H, 6.51, N, 7.37, S, 5.61. Fast atom bombardment mass spectrometry (FAB-MS), m/z 564 [M+1]. 1H NMR (400 MHz, CDCl3, δ, ppm): 4.58 (1H, s, -CH), 5.43 (1H, s, -CH), 7.12-7.36 (5H, m, Ar-H, -CH), 3.06 (2H, t, -CH2), 1.56 (2H, m, -CH2), 1.27 (2H, m, -CH2), 1.34 (2H, m, -CH2), 0.97 (3H, t, -CH3), 3.83 (2H, s, -CH2), 2.03 (1H, -NH), 2.61 (2H, m, -CH2), 1.05 (3H, t, -CH3), 10.61 (1H, s, D2o exchange, -OH), 10.29 (1H, s, D2O exchange, -OH), 6.98-7.22 (9H, m, Ar-H, -CH). 13C NMR (400 MHz, CDCl3, ppm): 155.2 (C2), 102.2 (C3), 164.6 (C4), 161.4 (C5), 91.3 (C6), 159.6 (C7), 101.3 (C8), 144.5 (C9), 94.7 (C10), 134.3 (C11), 126.4 (C12, 16), 128.7 (C13, 15), 128.1 (C14), 44.3 (C17), 28.1 (C18), 29.9 (C19), 22.7 (C20), 14.1 (C21), 35.7 (C22), 44.1 (C24), 15.9 (C25), 160.2 (C27), 122.9 (C28), 128.6 (C29), 127.9 (C30), 132.5 (C31), 134.8 (C32), 135.7 (C34), 131.2 (C35, 39), 129.4 (C36, 38), 127.3 (C37).
Synthesis of (4E)-4-(2-(phenylthio)phenylimino)-1,4-dihydro-1-pentyl-2-phenyl-8-((propylamino)methyl)quinoline-5,7-diol (HL4i): Yield: 82%. Anal. calcd for C36H39N3O2S: C, 74.84, H, 6.80, N, 7.27, S, 5.55. Found: C, 74.68, H, 6.72, N, 7.11, S, 5.41. Fast atom bombardment mass spectrometry (FAB-MS), m/z 578 [M+1]. 1H NMR (400 MHz, CDCl3, δ, ppm): 4.59 (1H, s, -CH), 5.42 (1H, s, -CH), 7.12-7.36 (5H, m, Ar-H, -CH), 3.05 (2H, t, -CH2), 1.59 (2H, m, -CH2), 1.27 (2H, m, -CH2), 1.34 (2H, m, -CH2), 0.98 (3H, t, -CH3), 3.85 (2H, s, -CH2), 2.01 (1H, -NH), 2.56 (2H, m, -CH), 1.48 (2H, m, -CH2), 0.96 (3H, t, -CH3), 10.94 (1H, s, D2O exchange, -OH), 10.67 (1H, s, D2O exchange, -OH), 6.98-7.22 (9H, m, Ar-H, -CH). 13C NMR (400 MHz, CDCl3, ppm): 149.2 (C2), 102.2 (C3), 164.7 (C4), 161.3 (C5), 91.4 (C6), 159.5 (C7), 101.3 (C8), 144.3 (C9), 94.5 (C10), 134.5 (C11), 126.3 (C12, 16), 128.9 (C13, 15), 128.0 (C14), 44.3 (C17), 28.2 (C18), 29.8 (C19), 22.6 (C20), 14.1 (C21), 36.4 (C22), 51.9 (C24), 23.8 (C25), 11.7 (C26), 160.2 (C28), 122.7 (C29), 128.4 (C30), 127.7 (C31), 132.4 (C32), 134.8 (C33), 135.4 (C35), 131.2 (C36, 40), 129.4 (C37, 39), 127.3 (C38).
Synthesis of (4E)-4-(2-(phenylthio)phenylimino)-8-((dimethylamino)methyl)-1,4-dihydro-1-pentyl-2-phenylquinoline-5,7-diol (HL4j): Yield: 68 %. Anal. calcd for C35H37N3O2S: C, 74.57, H, 6.62, N, 7.45, S, 5.69. Found: C, 74.48, H, 6.48, N, 7.33, S, 5.61. Fast atom bombardment mass spectrometry (FAB-MS), m/z 564 [M+1]. 1H NMR (400 MHz, CDCl3, δ, ppm): 4.58 (1H, s, -CH), 5.42 (1H, s, -CH), 7.12-7.36 (5H, m, Ar-H, -CH), 3.03 (2H, t, -CH2), 1.57 (2H, m, -CH2), 1.27 (2H, m, -CH2), 1.33 (2H, m, -CH2), 0.96 (3H, t, -CH3), 3.65 (2H, s, -CH2), 2.29 (6H, s, -CH3), 11.46 (1H, s, D2O exchange, -OH), 10.21 (1H, s, D2O exchange, -OH), 6.98-722 (9H, m, Ar-H, -CH). 13C NMR (400 MHz, CDCl3, ppm): 155.2 (C2), 101.9 (C3), 164.6 (C4), 161.4 (C5), 91.3 (C6), 159.6 (C7), 101.3 (C8), 144.5 (C9), 94.6 (C10), 134.3 (C11), 126.5 (C12, 16), 128.6 (C13, 15), 128.1 (C14), 44.2 (C17), 28.2 (C18), 29.9 (C19), 22.6 (C20), 14.2 (C21), 46.3 (C22), 45.4 (C24, C25), 160.3 (C27), 122.6 (C28), 128.4 (C29), 127.8 (C30), 132.4 (C31), 134.5 (C32), 135.5 (C34), 131.2 (C35, 39), 129.4 (C36, 38), 127.3 (C37).
Synthesis of (4E)-8-((diethylamino)methyl)-4-(2-(phenylthio)phenylimino)-1,4-dihydro-1-pentyl-2-phenylquinoline-5,7-diol (HL4k): Yield: 77%. Anal. calcd for C37H41N3O2S: C, 75.09, H, 6.98, N, 7.10, S, 5.42. Found: C, 74.98, H, 6.82, N, 6.96, S, 5.31. Fast atom bombardment mass spectrometry (FAB-MS), m/z 592 [M+1]. 1H NMR (400 MHz, CDCl3, δ, ppm): 4.57 (1H, s, -CH), 5.40 (1H, s, -CH), 7.12-7.36 (5H, m, Ar-H, -CH), 3.05 (2H, t, -CH2), 1.59 (2H, m, -CH2), 1.26 (2H, m, -CH2), 1.34 (2H, m, -CH2), 0.99 (3H, t, -CH3), 3.63 (2H, s, -CH2), 2.38 (4H, m, -CH2), 1.02 (6H, t, -CH3), 10.94 (1H, s, D2O exchange, -OH), 10.26 (1H, s, D2O exchange, -OH), 6.98-7.22 (9H, m, Ar-H, -CH). 13C NMR (400 MHz, CDCl3, ppm): 155.1 (C2), 101.8 (C3), 164.5 (C4), 161.3 (C5), 91.2 (C6), 159.5 (C7), 101.2 (C8), 144.4 (C9), 94.6 (C10), 134.2 (C11), 126.3 (C12, 16), 128.6 (C13, 15), 128.1 (C14), 44.4 (C17), 28.1 (C18), 29.7 (C19), 22.5 (C20), 14.1 (C21), 41.8 (C22), 49.3 (C24, C26), 13.5 (C25, C27), 160.2 (C29), 122.3 (C30), 128.4 (C31), 127.3 (C32), 132.9 (C33), 134.8 (C34), 135.5 (C36), 131.2 (C37, 41), 129.4 (C38, 40), 127.3 (C39).
Synthesis of (4E)-8-((1H-pyrrol-1-yl)methyl)-4-(2-(phenylthio)phenylimino)-1,4-dihydro-1-pentyl-2-phenylquinoline-5,7-diol (HL4l): Yield: 86%. Anal. calcd for C37H35N3O2S: C, 75.87, H, 6.02, N, 7.17, S, 5.47. Found: C, 75.78, H, 5.91, N, 7.05, S, 5.33. Fast atom bombardment mass spectrometry (FAB-MS), m/z 586 [M+1]. 1H NMR (400 MHz, CDCl3, δ, ppm): 4.58 (1H, s, -CH), 5.44 (1H, s, -CH), 7.12-7.36 (5H, m, Ar-H, -CH), 3.04 (2H, t, -CH2), 1.58 (2H, m, -CH2), 1.27 (2H, m, -CH2), 1.33 (2H, m, -CH2), 0.97 (3H, t, -CH3), 5.12 (2H, s, -CH2), 6.00-6.53 (4H, m, -CH), 12.23 (1H, s, D2O exchange, -OH), 10.18 (1H, s, D2O exchange, -OH), 6.98-7.22 (9H, m, Ar-H, -CH). 13C NMR (400 MHz, CDCl3, ppm): 155.1 (C2), 102.3 (C3), 164.7 (C4), 161.5 (C5), 91.4 (C6), 159.7 (C7), 101.4 (C8), 144.6 (C9), 94.9 (C10), 134.4 (C11), 126.5 (C12, 16), 128.8 (C13, 15), 128.1 (C14), 44.5 (C17), 28.3 (C18), 29.9 (C19), 22.7 (C20), 14.2 (C21), 38.6 (C22), 122.9 (C24, 26), 108.9 (C25, 27), 159.8 (C29), 122.7 (C30), 128.1 (C31), 127.7 (C32), 132.5 (C33), 134.8 (C34), 135.5 (C36), 131.2 (C37, 41), 129.4 (C38, 40), 127.4 (C39).
Synthesis of (4E)-8-((N-methyl-N-phenylamino)methyl)-4-(2-(phenylthio)phenylimino)- 1,4-dihydro-1-pentyl-2-phenylquinoline-5,7-diol (HL4m): Yield: 68%. Anal. calcd for C40H39N3O2S: C, 76.77, H, 6.28, N, 6.71, S, 5.12. Found: C, 76.68, H, 6.11, N, 6.57, S, 5.04. Fast atom bombardment mass spectrometry (FAB-MS), m/z 626 [M+1]. 1H NMR (400 MHz, CDCl3, δ, ppm): 4.57 (1H, s, -CH), 5.42 (1H, s, -CH), 7.12-7.36 (5H, m, Ar-H, -CH), 3.04 (2H, t, -CH2), 1.58 (2H, m, -CH2), 1.26 (2H, m, -CH2), 1.31 (2H, m, -CH2), 0.96 (3H, t, -CH3), 4.65 (2H, s, -CH2), 6.55-7.10 (5H, m, -CH), 2.88 (3H, s, -CH3), 12.91 (1H, s, D2O exchange, -OH), 10.23 (1H, s, D2O exchange, -OH), 6.98-7.22 (9H, m, Ar-H, -CH). 13C NMR (400 MHz, CDCl3, ppm): 155.3 (C2), 102.5 (C3), 164.7 (C4), 161.2 (C5), 91.6 (C6), 159.5 (C7), 101.4 (C8), 144.3 (C9), 94.7 (C10), 134.2 (C11), 126.5 (C12, 16), 128.6 (C13, 15), 128.1 (C14), 44.3 (C17), 28.3 (C18), 29.7 (C19), 22.6 (C20), 14.2 (C21), 37.4 (C22), 149.3 (C24), 114.7 (C25, 29), 129.9 (C26, 28), 118.7 (C27), 37.8 (C30), 160.1 (C32), 122.3 (C33), 128.2 (C34), 127.2 (C35), 132.9 (C36), 134.5 (C37), 135.5 (C39), 131.2 (C40, 44), 129.4 (C41, 43), 127.4 (C42).
Synthesis of (4E)-8-((N-ethyl-N-phenylamino)methyl)-4-(2-(phenylthio)phenylimino)-1, 4-dihydro-1-pentyl-2-phenylquinoline-5,7-diol (HL4n): Yield: 66%. Anal. calcd for C41H41N3O2S: C, 76.96, H, 6.46, N, 6.57, S, 5.01. Found: C, 76.88, H, 6.31, N, 6.43, S, 4.95. Fast atom bombardment mass spectrometry (FAB-MS), m/z 640 [M+1]. 1H NMR (400 MHz, CDCl3, δ, ppm): 4.60 (1H, s, -CH), 5.43 (1H, s, -CH), 7.12-7.36 (5H, m, Ar-H, -CH), 3.05 (2H, t, -CH2), 1.57 (2H, m, -CH2), 1.27 (2H, m, -CH2), 1.33 (2H, m, -CH2), 0.96 (3H, t, -CH3), 4.60 (2H, s, -CH2), 6.55-7.10 (5H, m, -CH), 3.38 (2H, t, -CH2), 1.12 (3H, s, -CH3), 12.73 (1H, s, D2O exchange, -OH), 10.33 (1H, s, D2O exchange, -OH), 6.98-7.22 (9H, m, Ar-H, -CH). 13C NMR (400 MHz, CDCl3, ppm): 155.4 (C2), 102.1 (C3), 164.5 (C4), 161.3 (C5), 91.2 (C6), 159.5 (C7), 101.2 (C8), 144.6 (C9), 94.6 (C10), 134.2 (C11), 126.6 (C12, 16), 128.6 (C13, 15), 128.1 (C14), 44.4 (C17), 28.1 (C18), 29.7 (C19), 22.5 (C20), 14.2 (C21), 37.4 (C22), 149.4 (C24), 114.6 (C25, 29), 129.9 (C26, 28), 118.6 (C27), 37.7 (C30), 13.1 (C31), 160.1 (C33), 122.3 (C34), 129.1 (C35), 127.3 (C36), 132.9 (C37), 134.3 (C38), 135.5 (C40), 131.2 (C41, 45), 129.4 (C42, 44), 127.4 (C43).
2.3 Synthesis of Organosulfur-Copper complexes of ligands
Organosulfur ligands (0.10 mM) and copper acetate (0.10 mM) were dissolved in hot ethanol (40 mL). The triethylamine (0.15 mM) was then gradually added to the mixture while it was being stirred. The solution was heated for 4 hrs in a stirred condition before being poured into the water. The resulting precipitate (Scheme 3) was dissolved in DMC (dichloromethane), washed and dried over sodium sulphate. In different yields the copper complexes were obtained as solids (Scheme 4). TLC was used confirmed the synthesized compounds. The copper complexes are purified by the column chromatography using chloroform: methanol (1:1).
Organosulfur-Copper Complex of [CuHL4a(OAc)]: Yield: 78%. Anal. Calcd for C35H35CuN3O4S: C, 64.96, H, 5.37, Cu, 9.67, N, 6.39, S, 4.88. Found: C, 64.82, H, 5.23, Cu, 9.52, N, 6.27, S, 4.77. FAB mass spectrometry (FAB-MS): m/z 658 [M+1]. µeff (BM) = 1.96, Λm (mhocm2 mol-1) = 18.
Organosulfur-Copper Complex of [CuHL4b(OAc)]: Yield: 74%. Anal. Calcd for C36H37CuN3O4S: C, 64.41, H, 5.56, Cu, 9.47, N, 6.26, S, 4.78. Found: C, 64.32, H, 5.45, Cu, 9.32, N, 6.12, S, 4.62. FAB mass spectrometry (FAB-MS): m/z 672 [M+1]. µeff (BM) = 1.92, Λm (mhocm2 mol-1) = 20.
Organosulfur-Copper Complex of [CuHL4c(OAc)]: Yield: 76%. Anal. Calcd for C35H35CuN3O4S: C, 64.96, H, 5.37, Cu, 9.67, N, 6.39, S, 4.88. Found: C, 64.85, H, 5.26, Cu, 9.58, N, 6.24, S, 4.76. FAB mass spectrometry (FAB-MS): m/z 658 [M+1]. µeff (BM) = 1.85, Λm (mhocm2 mol-1) = 18.
Organosulfur-Copper Complex of [CuHL4d(OAc)]: Yield: 78%. Anal. Calcd for C37H39CuN3O4S: C, 64.84, H, 5.74, Cu, 9.27, N, 6.13, S, 4.68. Found: C, 64.75, H, 5.62, Cu, 9.18, N, 6.02, S, 4.55. FAB mass spectrometry (FAB-MS): m/z 686 [M+1]. µeff (BM) = 1.72, Λm (mhocm2 mol-1) = 22.
Organosulfur-Copper Complex of [CuHL4e(OAc)]: Yield: 81%. Anal. Calcd for C37H33CuN3O4S: C, 65.42, H, 4.90, Cu, 9.35, N, 6.19, S, 4.72. Found: C, 65.31, H, 4.83, Cu, 9.26, N, 6.11, S, 4.63. FAB mass spectrometry (FAB-MS): m/z 680 [M+1]. µeff (BM) = 1.94, Λm (mhocm2 mol-1) = 19.
Organosulfur-Copper Complex of [CuHL4f(OAc)]: Yield: 71%. Anal. Calcd for C40H37CuN3O4S: C, 66.79, H, 5.18, Cu, 8.83, N, 5.84, S, 4.46. Found: C, 66.64, H, 5.08, Cu, 8.74, N, 5.38, S, 4.34. FAB mass spectrometry (FAB-MS): m/z 720 [M+1]. µeff (BM) = 1.90, Λm (mhocm2 mol-1) = 12.
Organosulfur-Copper Complex of [CuHL4g(OAc)]: Yield: 75%. Anal. Calcd for C41H39CuN3O4S: C, 67.15, H, 5.36, Cu, 8.66, N, 5.73, S, 4.37. Found: C, 67.03, H, 5.20, Cu, 8.44, N, 5.66, S, 4.22. FAB mass spectrometry (FAB-MS): m/z 734 [M+1]. µeff (BM) = 1.92, Λm (mhocm2 mol-1) = 18.
Organosulfur-Copper Complex of [CuHL4h(OAc)]: Yield: 78%. Anal. Calcd for C37H39CuN3O4S: C, 64.84, H, 5.74, Cu, 9.27, N, 6.13, S, 4.68. Found: C, 64.72, H, 5.63, Cu, 9.12, N, 6.05, S, 4.52. FAB mass spectrometry (FAB-MS): m/z 686 [M+1]. µeff (BM) = 1.96, Λm (mhocm2 mol-1) = 18.
Organosulfur-Copper Complex of [CuHL4i(OAc)]: Yield: 74%. Anal. Calcd for C38H41CuN3O4S: C, 65.26, H, 5.91, Cu, 9.09, N, 6.01, S, 4.58. Found: C, 65.12, H, 5.83, Cu, 8.96, N, 5.91, S, 4.46. FAB mass spectrometry (FAB-MS): m/z 700 [M+1]. µeff (BM) = 1.83, Λm (mhocm2 mol-1) = 20.
Organosulfur-Copper Complex of [CuHL4j(OAc)]: Yield: 76%. Anal. Calcd for C37H39CuN3O4S: C, 64.84, H, 5.74, Cu, 9.27, N, 6.13, S, 4.68. Found: C, 64.73, H, 5.61, Cu, 9.18, N, 5.99, S, 4.52. FAB mass spectrometry (FAB-MS): m/z 686 [M+1]. µeff (BM) = 1.81, Λm (mhocm2 mol-1) = 18.
Organosulfur-Copper Complex of [CuHL4k(OAc)]: Yield: 78%. Anal. Calcd for C39H43CuN3O4S: C, 65.66, H, 6.08, Cu, 8.91, N, 5.89, S, 4.49. Found: C, 65.52, H, 5.97, Cu, 8.79, N, 5.75, S, 4.39. FAB mass spectrometry (FAB-MS): m/z 714 [M+1]. µeff (BM) = 1.80, Λm (mhocm2 mol-1) = 22.
Organosulfur-Copper Complex of [CuHL4l(OAc)]: Yield: 81%. Anal. Calcd for C39H37CuN3O4S: C, 66.22, H, 5.27, Cu, 8.98, N, 5.94, S, 4.53. Found: C, 66.12, H, 5.13, Cu, 8.86, N, 5.81, S, 4.45. FAB mass spectrometry (FAB-MS): m/z 708 [M+1]. µeff (BM) = 1.94, Λm (mhocm2 mol-1) = 19.
Organosulfur-Copper Complex of [CuHL4m(OAc)]: Yield: 71%. Anal. Calcd for C42H41CuN3O4S: C, 67.49, H, 5.53, Cu, 8.50, N, 5.62, S, 4.29. Found: C, 67.32, H, 5.48, Cu, 8.44, N, 5.58, S, 4.19. FAB mass spectrometry (FAB-MS): m/z 748 [M+1]. µeff (BM) = 1.90, Λm (mhocm2 mol-1) = 12.
Organosulfur-Copper Complex of [CuHL4n(OAc)]: Yield: 75%. Anal. Calcd for C43H43CuN3O4S: C, 67.83, H, 5.69, Cu, 8.85, N, 5.52, S, 4.21. Found: C, 67.73, H, 5.57, Cu, 8.74, N, 5.46, S, 4.12. FAB mass spectrometry (FAB-MS): m/z 762 [M+1]. µeff (BM) = 1.92, Λm (mhocm2 mol-1) = 18.
2.4 Biological Studies
2.4.1 Antimicrobial Activities
In vitro testing of the ligands and their complexes was performed against the bacterial species Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus epidermidis and Klebsiella pneumoniae, including the fungal species Aspergillus niger, Fusarium solani, Culvularia lunata, Rhizoctonia bataicola and Candida albicans using the Kirby Bayer disc diffusion process. As standards, Amikacin and nystatin were used. The minimum inhibitory concentration (MIC) was calculated by the process of disc diffusion [41]. The test tubes containing 5 ml of sterile nutrient/Sabouraud broth were inoculated with bacteria and fungi from 0.02 ml of 24 hrs old culture. With the aid of sterile pipettes, different amounts of compounds in DMSO were applied aseptically to the stock solution from 200 μg/ml to 5 ml of the respective media to achieve concentrations from 1 μg/ml to 50 μg/ml. Both test tubes for bacteria and fungi were inoculated at 37 °C and at room temperature, respectively. The presence of turbidity after 24 hrs and 48 hrs respectively was observed in test tubes inoculated with species. As a MIC value, the lowest concentrations of compounds inhibiting organism growth were calculated.
2.4.2 Free radical scavenging activity
The radical scavenging behaviour of DPPH (2,2-diphenyl-1-picrylhydrazyl) was evaluated according to the standard method [42,43]. In these processes, 100 ppm of DPPH solution in methanol was prepared and 1.0 mL of this solution was applied to water compound solution dilutions at various concentrations (10 ppm, 20 ppm, 40 ppm, 60 ppm, 80 ppm, 100 ppm). The absorbance was estimated at 517 nm thirty minutes later. The use of Rutin, Vitamin C and BHT was standard. In order to predict 50 % reduction in its original value, scavenging activity versus concentration (ppm) was plotted (IC50). Higher free radical scavenging behaviour suggests lower absorption of the reaction mixture. The ability to scavenge the radical DPPH was evaluated using the following equation:
Absorbance (control) : Absorbance of DPPH radical + methanol.
Absorbance (standard) : Absorbance of DPPH radical + extract / standard.
2.4.3 SOD activity
Alkaline DMSO, a source of superoxide radical (O-2) and nitrobluetetrazolium (NBT) as a scavenger (O-2) were used to test in vitro SOD activity [44]. In general, a solution containing 2.1 mL of 0.2 M potassium phosphate buffer (pH 8.6) and 1 mL of 56 mM NBT was applied to the 400 mL sample to be analyzed. The tubes were held for 15 minutes in ice and 1.5 mL of alkaline DMSO solution was added during the stirring process. Except DMSO without NaOH, absorbance was then controlled at 540 nm against a sample prepared in a similar condition. A superoxide dismutase (SOD) activity unit is the concentration of a complex or enzyme that induces a 50 percent reduction of NBT induced by alkaline DMSO inhibition.
2.4.4 Glutathione peroxidase like activit
According to Paglia et al., [45] the GPx kit (Biodiagnostic, Egypt) was used for GPx determination. 1 mL assay buffer (50 mM phosphate buffer containing 0.1 % triton X-100) and 0.1 mL NADPH reagent (24 mmol glutathione, 12 units glutathione reductase and 4.8 mmol NADPH) and 0.01 mL (41 mM) tested compounds were used in the reaction mixture and the reaction was initiated by adding H2O2 to the test compounds (0.8 mM). The contents were mixed well and the absorbance were recorded against deionized water at 340 nm over a period of 3 min. Ebselen (41 mM) was used to estimate the shift in absorption per minute (A340 nm/min) as the positive power. The values shown in Figure 4 are for the H2O2 and GSH response after context correction. Their behaviour were calculated in the case of coloured compounds after subtraction of their absorbance at the wavelength used.
2.4.5 Nuclease activity
The DNA cleavage test was performed using supercoiled pUC19 plasmid DNA by gel electrophoresis method after isolation from culture. The loading dye of 1% agarose gel and the buffer solution of 50 mmol Tris–HCl/50 mmol NaCl in water (pH 7.2) was used for cleavage. DMF was used to make test samples. The 50 μM of the different test samples and 60 μM of H2O2 were added to the isolated plasmid DNA (40 μM) and incubated for 1 hrs at 37 0C. Following the incubation period, 30 µL of plasmid DNA sample was mixed with bromophenol blue dye and loaded into the electrophoresis chamber wells. Finally, it was loaded onto the agarose gel and electrophoresed at a constant voltage of 50V for 30 min. The gel was removed after the run and stained with 10.01 µg/ml EB (ethidium bromide), and the extent of cleavage was calculated from the intensities of the bands using the Alpha Innotech Gel documentation system (AlphaImager 2200) [46, 47].
2.4.6 BSA binding experiment
The fluorescence spectra were detected using a Varian CaryEclipse spectrofluorimeter with the use of a 1 cm quartz cell. Prepare a BSA stock solution (1×10-3 M) by dissolving solid BSA in 0.01 M phosphate buffer (pH 7.4). Copper complexes of [CuHL4e(OAc)], [CuHL4g(OAc)], [CuHL4l(OAc)] and [CuHL4n(OAc)] were prepared in DMSO at a concentration of 5 × 10-3 M. The fluorescence spectra were estimated at 280 nm for the BSA excitation wavelength and 290-450 nm for the emission wavelength. Fluorescence titrations were performed by gradually increasing the concentrations of the complexes (0-2 × 10-5 M) in the BSA-cell solution (2 × 10-5 M).
The spectra of synchronous fluorescence were evaluated at two different Δλ values, (i.e the difference in BSA excitation and emission wavelengths) 15 and 60 nm. The wavelengths of synchronous emission and excitation can be expressed as λem = λex + Δλ. The concentration of BSA was kept constant at 2.0 × 10-5 M (Δλ = 15)/4.0 × 10-5 M (Δλ = 60) and different concentrations of complexes [CuHL4e(OAc)], [CuHL4g(OAc)], [CuHL4l(OAc)] and [CuHL4n(OAc)] (0-1.875 × 10-5 M) were added.
2.4.6 In vitro cytotoxic studies
The target compounds, which were chrysin derivatives of copper complexes, were put through anticancer activity tests in vitro. The growth inhibitory effects on the cell lines were determined using the MTT test (Hela and MCF-7).
MTT assay : MTT is [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide)] yellow water-soluble tetrazolium salt. Succinate dehydrogenase, a mitochondrial enzyme that cleaves the tetrazolium ring in living cells, transforms MTT to an insoluble purple formazane. The amount of formazan produced is therefore directly proportional to the number of cells that are viable. 15 μL of MTT (5 mg/mL) was applied to each well after 48 hrs of incubation in phosphate buffered saline (PBS) and incubated for 4 hrs at 37 °C. The MTT medium was then flicked off, and the obtained formazan crystals were solubilized into 100 μL of DMSO. Using a micro plate reader, the absorbance at 570 nm was measured [48]. The inhibition of the percent cell was provided by
Nonlinear regression graph was plotted between % cell inhibition and log10 concentration and IC50 (half maximal inhibitory concentration) was determined using Graph-Origin software.
2.2.8 Anti-inflammatory activity
The carrageenan-induced paw edema method was used to assess the anti-inflammatory efficacy of copper complexes. Four groups of six rats each were formed from the rat population. As a control, group I received saline as usual. 100 and 200 mg/kg bw of the copper complexes were administered to Groups II and III, respectively. Group IV received Indomethacin 10 mg/kg bw, the recommended medication. After one hour of medication administration, 0.1 ml of carrageenan (1 % w/v, Sigma, USA) in normal saline was injected into the subplantar region of the left hind paw. Following the administration of the medicines, the paw volume was measured using a mercury replacement plethysmometer (Model 7140, UGO Basile, Italy) at 0, 1, 2, 3 and 4 hrs. Anti-inflammatory activity was determined by the percentage reduction in edema as compared to the control. The formula was used to compute the % inhibition of edema,
Where, A represents the paw volume of the control at 3 hrs and B represents the paw volume of the test drug treated at 3 hrs.