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Synthesis of 4,7-dimethoxy-5-(5-aryl-1H- pyrazolin-3-yl)benzofuran-6-ol (4a-c): ...... combretastatin A4 analogous chalcone and its Pt-complex on cancer cells: A ...
Australian Journal of Basic and Applied Sciences, 6(3): 852-863, 2012 ISSN 1991-8178

Synthesis and Antibacterial Activity of Some Novel Chalcones, Pyrazoline and 3Cyanopyridine Derivatives Based on Khellinone as well as Ni(II), Co(II) and Zn(II) Complexes 1

Souad A. Osman, 1Hisham Abdallah A. Yosef, 1Taghrid S. Hafez, 2Abdallah A. El-Sawy, 3Hanan A. Mousa, 1Ashraf S. Hassan 1

Department of Organometallic and Organometalloid Chemistry, National Research Centre, ElBehoos Street, Dokki, 12622 Cairo, Egypt 2 Chemistry Department, Faculty of Science, Benha University, Benha, Egypt 3 Department of inorganic Chemistry, National Research Centre, El-Behoos Street, Dokki, 12622 Cairo, Egypt Abstract: Chalcones 3a-d were synthesized by condensing khellinone 1 with aryl aldehydes 2a-d. Chalcones 3a-c reacted with hydrazine hydrate in ethanol or in glacial acetic acid to afford 1Hpyrazolines 4a-c or N-acetylpyrazolines 5a-c, respectively. N-phenylpyrazolines 6a,b were synthesized by reaction of chalcones 3a,b with phenyl hydrazine in ethanol. When 3b was refluxed in ethanol containing piperidine, it yielded 4,9-dimethoxy-7-(naphth-1-yl)-6,7-dihydro-5H-furo[3,2-g]chromen-5one 7. Moreover, Chalcone 3c reacted with 2-cyanoacetamide or 2-cyanothioacetamide in ethanol to afford 3-cyanopyridines 8a and 8b respectively. The structure of the synthesized compounds was established based on elemental analysis and spectral data. The metal complexes 9, 10 and 11 of chalcone 3c; (L) with (Ni(II), Co(II) and Zn(II)) respectively, were synthesized and characterized by IR, 1H NMR, electronic absorption, magnetic susceptibility, molar conductivity and elemental analysis. The prepared complexes 9-11 had the general structural formula: [M(L)Cl2(H2O)2] where M=Ni(II), Co(II) or Zn(II) and L=3c. In order to study the structure-activity relationship, representative compounds of the synthesized products beside the metal complexes were screened for their antibacterial activities. The results indicated that some synthesized compounds had antibacterial activity as well as the metal complexes 9, 10 and 11 exhibited moderate antibacterial activities while the free ligand 3c; (L) had no activity. Key words: Khellinone; chalcones; pyrazolines; 3-cyanopyridines; metal complexes; antibacterial activity. INTRODUCTION Many benzofuran derivatives were known to have a wide variety of pharmacological activities such as antiarrhythmic (Bourgery et al., 1981), antimicrobial (Kirilmis et al., 2008; Jiang et al., 2011) and antitumor (Galal et al., 2009). Some chalcones, either natural or synthetic, were known to exhibit various biological activities, including antioxidant (Anto et al., 1995; Mukherjee et al., 2001; Arty et al., 2000), antimalarial (Li et al., 1995; Chen et al., 1997; Tomar et al., 2010), antileishmanial (Nielsen et al., 1998), anti-inflammatory (Ballesteros et al., 1995; Bandgar et al., 2010), antitumor (Pouget et al., 2001; Kumar et al., 2010), anticancer (Juvale et al., 2012; Singh et al., 2012), antibacterial (Osório et al., 2012), antifungal (Lόpez et al., 2001; Lahtchev et al., 2008) and antihyperglycemic (Damazio et al., 2010) activities. Also, Chalcones are useful synthons in the synthesis of a large number of bioactive molecules such as 1Hpyrazolines, N-acetylpyrazolines, N-phenylpyrazolines and 3-cyanopyridines etc. Some pyrazoline derivatives had been reported to show a wide range of biological activity including anti-inflammatory (Sharma et al., 2010), antibacterial (Solankee et al., 2010; Holla et al., 2000), antifungal (Siddiqui et al., 2011; Karthikeyan et al., 2007), antioxidant (Taj et al., 2011; Biradar and Sasidhar, 2011), anticancer (Shaharyar et al., 2010) and antitumor (Insuasty et al., 2011) activities. On the other hand, it had been demonstrated that molecules containing 3-cyanopyridine moiety may be able to work as a new drugs (Murata et al., 2004) which had a wide variety of pharmacological activities such as antibacterial (Khidre et al., 2011), antifungal (Gholap et al., 2007), anti-inflammatory (Hamdya and GamalEldeen, 2009; Kumar et al., 2011), anticancer (Brandt et al., 2011) and antitumor (Zhang et al., 2011) activities. Furthermore, some metal (II) complexes of chalcones had a variety of biological activities such as antiHIV, cytotoxic (Mishra et al., 2001; Prajapati et al., 2010), anticancer (Zoldakova et al., 2010), antifungal (Muthukumar and Viswanathamurthi, 2010) and antibacterial (Sumathi et al., 2011) activities. Corresponding Author: Ashraf S. Hassan, Department of Organometallic and Organometalloid Chemistry, National Research Centre, El-Behoos Street, Dokki, 12622 Cairo, Egypt E-mail: [email protected]

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In view of these facts, we here reported the synthesis of some novel chalcones, 1H-pyrazolines, Nacetylpyrazolines, N-phenylpyrazolines and 3-cyanopyridines based on khellinone 1. The structure of the synthesized compounds was established based on elemental analysis and spectral data (IR, MS, 1H NMR and 13 C NMR). The study has been extended to synthesize nickel(II), cobalt(II) and zinc(II) complexes 9, 10 and 11 with the prepared chalcone (3c). The prepared complexes had been characterized by IR, 1H NMR and UV-Vis, in addition to elemental analysis, molar conductivity and magnetic susceptibility. Some synthesized compounds, Ni(II), Co(II) and Zn(II) complexes were screened against two Gram-negative and two Gram-positive bacteria. MATERIAL AND METHODS All melting points were measured on a Gallenkamp melting point apparatus and are uncorrected. The IR spectra were recorded (KBr disk) on a Perkin Elmer 1650 FT-IR instrument. The 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were recorded on a Varian 500 MHz spectrometer in DMSO-d6 using TMS as an internal standard. 13C NMR (125 MHz) spectra using technique DEPT-135. Mass spectra were recorded on a Varian MAT 112 spectrometer at 70 ev. Elemental analyses were obtained from the Micro analytical Data Center at Cairo University, Egypt. Magnetic susceptibilities were measured at 20°C by the Gouy method at the Faculty of Science, Cairo University. Electronic absorptions were recorded on (PG Instruments ltd., +80+ UVVis) automatic spectrophotometer in DMSO. The molar conductance measurements were measured in solution of the metal complexes in DMF (10-3) using Metrohem 660 conductivity meter. Progress of the reactions was monitored by thin-layer chromatography (TLC) using aluminum sheets coated with silica gel F254 (Merck), Viewing under a short-wavelength UV lamp effected detection. All evaporations were carried out under reduced pressure at 40 oC. Preparation of Khellinone (1): 4,7-Dimethoxy-5-acetyl-6-hydroxybenzofuran (1) was synthesized according to the literature as dark yellow needles, m.p. 99-100 °C [lit. 99-101 ºC (Schöberg and Sina, 1950; Gammill, 1984)] yield about 90%. Synthesis of 1-(6-hydroxy-4,7-dimethoxybenzofuran-5-yl)-3-(aryl)prop-2-en-1-one (3a-d): General Procedure: Khellinone 1 (0.01 mole) was dissolved with aryl aldehydes 2 (0.01 mole) in 30 ml of ethanol then the solution was treated with 5 ml of 50% sodium hydroxide solution and left overnight. The reaction mixture was neutralized with dilute acetic acid (10%), the separated product was collected, washed with water and recrystallized. 1-(6-Hydroxy-4,7-dimethoxybenzofuran-5-yl)-3-(4-methoxyphenyl) prop-2-en-1-one (3a): Orange prisms, m.p. 124-125 ºC [lit. 124 ºC (Nada et al., 2002)]. 1-(6-Hydroxy-4,7-dimethoxybenzofuran-5-yl)-3-(naphth-1-yl)prop-2-en-1-one (3b): -1 3424 (OH), 1617 (C=O, Orange prisms, m.p. 136-137 ºC (EtOH), yield (60%). IR (KBr) ν max/cm 1 conjugated; hydrogen-bonded). H NMR (DMSO-d6, δ ppm) 3.90 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 7.15 (d, 1H, J=2.3 Hz, benzofuran H-3), 7.27 (d, 1H, J=16.03 Hz, α-olefinic proton), 7.53-7.59 (m, 3H, aromatic), 7.89 (d, 1H, J=2.3 Hz, benzofuran H-2), 7.95-8.01 (m, 4H, aromatic), 8.15 (d, 1H, J=16.03 Hz, β-olefinic proton), 10.13 (s, 1H, OH, D2O exchangeable). MS m/z (%): 373(69.21) [M+-H]. Anal. Calcd for C23H18O5 (374.39): C, 73.79; H, 4.85. Found: C, 73.65; H, 4.95. 1-(6-Hydroxy-4,7-dimethoxybenzofuran-5-yl)-3-(5-methylfuran-2-yl) prop-2-en-1-one (3c): Reddish-brown prisms, m.p. 128-130 ºC (EtOH), yield (76%). IR (KBr)ν max/cm-1 3423 (OH), 1620 (C=O, conjugated; hydrogen-bonded). 1 H NMR (DMSO-d6, δ ppm) 2.30 (s, 3H, CH3), 3.86 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 6.26 (d, 1H, J=3 Hz, furan H-4), 6.73 (d, 1H, J=15.3 Hz, α-olefinic proton), 6.86 (d, 1H, J=3 Hz, furan H-3), 7.07 (d, 1H, J=15.3 Hz, β-olefinic proton), 7.09 (d, 1H, J=2.3 Hz, benzofuran H-3), 7.85 (d, 1H, J=2.3 Hz, benzofuran H-2), 10.05 (s, 1H, OH, D2O exchangeable). MS m/z (%): 330 (41.17) [M++2H]. Anal. Calcd for C18H16O6 (328.32): C, 65.85; H, 4.91. Found: C, 65.65; H, 5.05. 3-(Anthracen-9-yl)-1-(6-hydroxy-4,7-dimethoxybenzofuran-5-yl)prop-2-en-1-one (3d): -1 3426 (OH), 1624 (C=O, Orange prisms, m.p. 184-185 ºC (EtOH), yield (52%). IR (KBr) ν max/cm + conjugated; hydrogen-bonded). MS m/z (%): 424(1.53) [M ]. Anal. Calcd for C27H20O5 (424.44): C, 76.40; H, 4.75. Found: C, 76.55; H, 4.63.

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Synthesis of 4,7-dimethoxy-5-(5-aryl-1H- pyrazolin-3-yl)benzofuran-6-ol (4a-c): General Procedure: A mixture of compounds 3a-c (0.01mol) and hydrazine hydrate (0.01mol) in ethanol (30ml) containing 2-3 drops of glacial acetic acid was refluxed for 4-6 hours. After cooling, the resulting solid formed was collected by filtration and recrystallized. 4,7-Dimethoxy-5-[5-(4-methoxyphenyl)-1H-pyrazolin-3-yl]benzofuran-6-ol (4a): Pale yellow crystals, m.p. 150-151 ºC (EtOH), yield (80%). IR (KBr)ν max/cm-1 3479 (OH), 3331 (NH), 1615 (C=N). 1 H NMR (DMSO-d6, δ ppm) 3.06 (dd, 1H, axial-H of CH2-pyrazoline), 3.31 (dd, 1H, equatorialH of CH2-pyrazoline), 3.70 (s, 3H, OCH3 of phenyl), 3.85 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.72 (dd, 1H, CHpyrazoline), 6.88 (d, 2H, J=6.1 Hz, ortho to OCH3), 7.05 (d, 1H, benzofuran H-3), 7.30 (d, 2H, J=6.1 Hz, meta to OCH3), 7.65 (s, br, 1H, NH, exchangeable with D2O), 7.80 (d, 1H, benzofuran H-2), 12.32 (s, 1H, OH, D2O exchangeable). MS m/z (%): 368(100) [M+]. Anal. Calcd for C20H20N2O5 (368.38): C, 65.21; H, 5.47; N, 7.60. Found: C, 65.33; H, 5.30; N, 7.80. 4,7-Dimethoxy-5-[5-(naphth-1-yl)-1H-pyrazolin-3-yl]benzofuran-6-ol (4b): Light gray crystals, m.p. 154-156 ºC (EtOH), yield (74%). IR (KBr) ν max/cm-1 3424 (OH), 3313 (NH), 1611 (C=N). 1 H NMR (DMSO-d6, δ ppm) 3.10 (dd, 1H, axial-H of CH2-pyrazoline), 3.82 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 4.00 (dd, 1H, equatorial-H of CH2-pyrazoline), 5.53 (dd, 1H, CH-pyrazoline), 7.03 (d, 1H, J=2.3 Hz, benzofuran H-3), 7.48-7.84 (m, 9H, 1H of benzofuran H-2, 7H of aromatic & 1H of NH), 12.33 (s, 1H, OH, D2O exchangeable). MS m/z (%): 388 (80.86) [M+]. Anal. Calcd for C23H20N2O4 (388.42): C, 71.12; H, 5.19; N, 7.21. Found: C, 70.95; H, 5.30; N, 7.10. 4,7-Dimethoxy-5-[5-(5-methylfuran-2-yl)-1H-pyrazolin-3-yl]benzofuran-6-ol (4c): Light gray crystals, m.p. 111-113 ºC (EtOH), yield (78%). IR (KBr) νmax/cm-1 3434 (OH), 3325 (NH), 1617 (C=N). 1 H NMR (DMSO-d6, δ ppm) 2.20 (s, 3H, CH3), 3.31 (dd, 1H, axial-H of CH2-pyrazoline), 3.55 (dd, 1H, equatorial-H of CH2-pyrazoline), 3.84 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 4.75 (dd, 1H, CH-pyrazoline), 5.97 (d, 1H, furan H-4), 6.20 (d, 1H, furan H-3), 7.07 (d, 1H, benzofuran H-3), 7.60 (s, 1H, NH, D2O exchangeable), 7.81 (d, 1H, J=2.3 Hz, benzofuran H-2), 12.20 (s, 1H, OH, D2O exchangeable). 13 C NMR (DMSO-d6, δ ppm) 13.87 (CH3-furan), 41.38 (CH2-pyrazoline), 55.46 (CH-pyrazoline), 60.89 & 61.36 (2C, 2OCH3), 105.55 (1C, furan C-4), 106.76 (1C, benzofuran C-3), 107.89 (1C, furan C-3), 144.66 (1C, benzofuran C-2). MS m/z (%): 342(100) [M+]. Anal. Calcd for C18H18N2O5 (342.35): C, 63.15; H, 5.30; N, 8.18. Found: C, 63.25; H, 5.15; N, 8.00. Synthesis of 4,7-dimethoxy-5-(5-aryl-N-acetylpyrazolin-3-yl)benzofuran-6-ol (5a-c): General Procedure: A mixture of compounds 3a-c (0.01 mol) and hydrazine hydrate (0.01 mol) in glacial acetic acid (20 ml) was refluxed for 4-6 hours. After cooling, the resulting solid formed was collected by filtration and recrystallized. 4,7-Dimethoxy-5-[5-(4-methoxyphenyl)-N-acetylpyrazolin-3-yl]benzofuran-6-ol (5a): Pale yellow crystals, m.p. 150-152 ºC (EtOH), yield (75%). IR (KBr)ν max/cm-1 3435 (OH), 1659 (C=O), 1617 (C=N). 1 H NMR (DMSO-d6, δ ppm) 2.18 (s, 3H, CH3), 3.10 (dd, 1H, axial-H of CH2-pyrazoline), 3.40 (dd, 1H, equatorial-H of CH2-pyrazoline), 3.68 (s, 3H, OCH3 of phenyl), 3.85 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 5.37 (dd, 1H, CH-pyrazoline), 6.86 (d, 2H, J=8.4 Hz, ortho to OCH3), 7.11 (d, 1H, J=2.3 Hz, benzofuran H-3), 7.16 (d, 2H, J=8.4 Hz, meta to OCH3), 7.88 (d, 1H, J=2.3 Hz, benzofuran H-2), 10.42 (s, 1H, OH, D2O exchangeable). MS m/z (%): 410(100) [M+]. Anal. Calcd for C22H22N2O6 (410.42): C, 64.38; H, 5.40; N, 6.83. Found: C, 64.15; H, 5.20; N, 7.00. 4,7-Dimethoxy-5-[5-(naphth-1-yl)-N-acetylpyrazolin-3-yl]benzofuran-6-ol (5b): Pale yellow crystals, m.p. 139-140 ºC (EtOH), yield (72%). IR (KBr) νmax/cm-1 3425 (OH), 1656 (C=O), 1619 (C=N). 1H NMR (DMSO-d6, δ ppm) 2.31 (s, 3H, CH3), 3.03 (dd, 1H, axial-H of CH2-pyrazoline), 3.81 (s, 3H, OCH3), 3.83 (s, 3H, OCH3), 4.14 (dd, 1H, equatorial-H of CH2-pyrazoline), 6.15 (dd, 1H, CH-pyrazoline), 7.01-8.10 (m, 9H, 2H of benzofuran H-2, H-3 & 7H of aromatic), 10.43 (s, 1H, OH, D2O exchangeable). MS m/z (%): 430(81.50) [M+]. Anal. Calcd for C25H22N2O5 (430.45): C, 69.76; H, 5.15; N, 6.51. Found: C, 69.55; H, 5.32; N, 6.35. 4,7-Dimethoxy-5-[5-(5-methylfuran-2-yl)-N-acetylpyrazolin-3-yl]benzofuran-6-ol (5c): Brown crystals, m.p. 120-122 ºC (EtOH), yield (71%). IR (KBr)ν max/cm-1 3430 (OH), 1657 (C=O), 1620 (C=N). 1 H NMR (DMSO-d6, δ ppm) 2.17 (s, 3H, CH3), 2.18 (s, 3H, CH3), 3.40 (dd, 1H, axial-H of CH2854

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pyrazoline), 3.78 (dd, 1H, equatorial-H of CH2-pyrazoline), 3.87 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 5.45 (dd, 1H, CH-pyrazoline), 5.95 (d, 1H, furan H-4), 6.15 (d, 1H, J=2.3 Hz, furan H-3), 7.12 (d, 1H, J=2.3 Hz, benzofuran H-3), 7.87 (d, 1H, J=1.5 Hz, benzofuran H-2), 10.53 (s, 1H, OH, D2O exchangeable). MS m/z (%): 384(100) [M+]. Anal. Calcd for C20H20N2O6 (384.38): C, 62.49; H, 5.24; N, 7.29. Found: C, 62.25; H, 5.35; N, 7.14. Synthesis of 4,7-dimethoxy-5-(5-aryl-N-phenylpyrazolin-3-yl)benzofuran-6-ol (6a,b): General Procedure: A mixture of 3a or 3b (0.01mol) and phenyl hydrazine (0.01mol) in ethanol (30ml) containing 2-3 drops of glacial acetic acid was refluxed for 5-6 hours. After cooling, the resulting solid formed was collected by filtration and recrystallized. 4,7-Dimethoxy-5-[5-(4-methoxyphenyl)-N-phenylpyrazolin-3-yl]benzofuran-6-ol (6a): Brown crystals, m.p. 176-178 ºC (EtOH), yield (65%). IR (KBr)ν max/cm-1 3426 (OH). 1 H NMR (DMSOd6, δ ppm) 3.25 (dd, 1H, axial-H of CH2-pyrazoline), 3.69 (s, 3H, OCH3 of phenyl), 3.88 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 4.10 (dd, 1H, equatorial-H of CH2-pyrazoline), 5.30 (dd, 1H, CH-pyrazoline), 6.73 (dd, 1H, aromatic), 6.87-6.88 (m, 4H, aromatic), 7.01 (d, 1H, benzofuran H-3), 7.16 (d, 2H, J=6.9 Hz, aromatic), 7.23 (d, 2H, J=8.4 Hz, aromatic), 7.83 (d, 1H, benzofuran H-2), 11.47 (s, 1H, OH, D2O exchangeable). MS m/z (%): 444(44.14) [M+]. Anal. Calcd for C26H24N2O5 (444.48): C, 70.26; H, 5.44; N, 6.30. Found: C, 70.39; H, 5.29; N, 6.17. 4,7-Dimethoxy-5-[5-(naphth-1-yl)-N-phenylpyrazolin-3-yl]benzofuran-6-ol (6b): Pale yellow crystals, m.p. 156-157 ºC (EtOH), yield (85%). IR (KBr) νmax/cm-1 3435 (OH). 1 H NMR (DMSO-d6, δ ppm) 3.27 (dd, 1H, axial-H of CH2-pyrazoline), 3.81 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 4.37 (dd, 1H, equatorial-H of CH2-pyrazoline), 6.02 (dd, 1H, CH-pyrazoline), 6.70-8.26 (m, 14H, 1H of benzofuran H-3, 1H of benzofuran H-2 & 12H of aromatic), 11.54 (s, 1H, OH, D2O exchangeable). MS m/z (%): 464(100) [M+]. Anal. Calcd for C29H24N2O4 (464.51): C, 74.98; H, 5.21; N, 6.03. Found: C, 74.85; H, 5.35; N, 5.90. Synthesis of 4,9-dimethoxy-7-(naphth-1-yl)-6,7-dihydro-furo[3,2-g]chromen-5-one (7): A solution of compounds 3b (0.01mol) in ethanol (30ml) containing 2-3 drops of piperidine was refluxed for 5 hours. After cooling, the resulting solid formed was collected by filtration and recrystallized. Yellow-orange crystals, m.p. 230-232 ºC, yield (71%). IR (KBr) νmax/cm-1 1739 (C=O, γ-lactone). 1 H NMR (DMSO-d6, δ ppm) 2.91 (dd, 1H, JHHgem=16 Hz, axial-H of CH2-pyrane), 3.31 (dd, 1H, equatorial-H of CH2pyrane), 3.81 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 6.36 (dd, 1H, CH-pyrane), 7.16 (d, 1H, J=2.3 Hz, benzofuran H-3), 7.54-8.23 (m, 8H, 1H of benzofuran H-2 & 7H of aromatic). MS m/z (%): 374(24.99) [M+]. Anal. Calcd for C23H18O5 (374.39): C, 73.79; H, 4.85. Found: C, 73.60; H, 5.00. Synthesis of 6-(6-hydroxy-4,7-dimethoxybenzofuran-5-yl)-4-(5-methylfuran-2-yl)-3-cyanopyridine derivatives (8a,b): General Procedure: A mixture of 3c (0.01 mol) with 2-cyanoacetamide or 2-cyanothioacetamide (0.01 mol) was dissolved in ethanol (30 ml) containing piperidine (three drops). The mixture was refluxed for 6 hours and then allowed to cool to room temperature. The resulting solid product was collected by filtration and recrystallized. 2-Hydroxy-6-(6-hydroxy-4,7-dimethoxybenzofuran-5-yl)-4-(5-methylfuran-2-yl)-3-cyanopyridine (8a): Light brown crystals, m.p.>300 ºC, yield (69%). IR (KBr) ν max/cm-1 3365 (OH), 2210 (C≡N), 1622 (C=N). 1 H NMR (DMSO-d6, δ ppm) 2.35 (s, 3H, CH3), 3.87 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 6.43 (d, 1H, furan H4), 6.58 (d, 1H, furan H-3), 7.15 (d, 1H, benzofuran H-3), 7.51 (s, 1H, pyridine H-5), 7.88 (d, 1H, benzofuran H2), 9.50 (s, 1H, OH of pyridine, D2O exchangeable), 12.29 (s, 1H, OH of benzofuran, D2O exchangeable). 13 C NMR (DMSO-d6, δ ppm) 14.16 (CH3-furan), 60.66 & 61.36 (2C, 2OCH3), 105.95 (1C, furan C-3), 106.62 (1C, benzofuran C-3), 110.89 (1C, furan C-4), 118.15 (1C, pyridine C-5), 144.83 (1C, benzofuran C-2). MS m/z (%): 392(87.84) [M+]. Anal. Calcd for C21H16N2O6 (392.36): C, 64.28; H, 4.11; N, 7.14. Found: C, 64.16; H, 4.25; N, 7.00. 6-(6-Hydroxy-4,7-dimethoxybenzofuran-5-yl)-2-mercapto-4-(5-methylfuran-2-yl)-3-cyanopyridine (8b): Creamy crystals, m.p. 216-218 ºC, yield (74%). IR (KBr) νmax/cm-1 3416 (OH), 2216 (C≡N), 1620 (C=N). 1 H NMR (DMSO-d6, δ ppm) 2.29 (s, 3H, CH3), 3.25 (s, 1H, SH of cyanopyridine, D2O exchangeable), 3.72 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.26 (d, 1H, furan H-4), 7.04 (d, 1H, furan H-3), 7.18 (d, 1H, benzofuran H-3), 7.66 (s, 1H, pyridine H-5), 7.88 (d, 1H, benzofuran H-2), 10.21 (s, 1H, OH, D2O exchangeable). MS m/z (%):

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408(4.00) [M+]. Anal. Calcd for C21H16N2O5S (408.43): C, 61.76; H, 3.95; N, 6.86. Found: C, 62.00; H, 3.79; N, 6.71. Synthesis of the metal complexes (9, 10 and 11): General Procedure: A solution of the metal salts (NiCl2.6H2O, CoCl2.6H2O and ZnCl2) (0.01 mol) in a minimum amount of water was added to a hot solution of the ligand 3c (0.01 mol) in methanol to form 1:1 M:L complexes. The pH of the solution was maintained around 9 by adding ammonium solution. The pH was measured with the help of pH paper. The reaction mixture was refluxed with stirring for 6 hours. The resulting precipitates was filtered off, washed several times with hot water and dried under reduced pressure. Ni–L Complex [Ni(L)Cl2(H2O)2] (9): Brown solid, m.p. >300 ºC, yield (60%). IR (KBr) νmax/cm-1 3425 (OH, water molecules), 1605 (C=O), 1262 (C-O), 513 (Ni-O). UV-vis (DMF), λmax: 409, 522 and 861 nm. Anal. Calcd for C18H20Cl2NiO8 (493.95): C, 43.77; H, 4.08. Found: C, 43.55; H, 3.95. µeff = 3.2 B.M; Λm (Ω-1 cm2 mol-1) =12.3. Co–L Complex [Co(L)Cl2(H2O)2] (10): Brown solid, m.p. >300 ºC, yield (65%). IR (KBr) νmax/cm-1 3426 (OH, water molecules), 1602 (C=O), 1265 (C-O), 513 (Co-O). UV-vis (DMF), λmax: 464 and 625 nm. Anal. Calcd for C18H20Cl2CoO8 (494.19): C, 43.75; H, 4.08. Found: C, 44.00; H, 3.90. µeff = 4.3 B.M; Λm (Ω-1 cm2 mol-1) =15.9. Zn–L Complex [Zn(L)Cl2(H2O)2] (11): Light brown solid, m.p. >300 ºC, yield (62%). IR (KBr) νmax/cm-1 3421 (OH, water molecules), 1600 (C=O), 1255 (C-O), 511 (Zn-O). 1 H NMR (DMSO-d6, δ ppm) 2.28 (s, 3H, CH3), 3.84 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 4.94 (s, 1H, OH of H2O), 6.25 (d, 1H, furan H-4), 6.75 (d, 1H, α-olefinic proton), 6.85 (d, 1H, furan H-3), 7.07 (d, 1H, β-olefinic proton), 7.10 (d, 1H, benzofuran H-3), 7.84 (d, 1H, benzofuran H-2), 10.11 (s, 1H, OH). Anal. Calcd for C18H20Cl2ZnO8 (500.66): C, 43.18; H, 4.03. Found: C, 43.35; H, 3.81. µeff = dimagnatic; Λm (Ω-1 cm2 mol-1) =11.6. RESULTS AND DISCUSSION Chemistry: The synthetic procedures adopted obtaining the target compounds which are depicted in Schemes 1 and 2. The starting materials khellinone, namely 4,7-dimethoxy-5-acetyl-6-hydroxybenzofuran 1 was prepared via the alkaline hydrolysis of the naturally occurring khellin. Condensation of khellinone 1 with pmethoxybenzaldehyde 2a, 1-naphthaldehyde 2b, 5-methylfuran-2-carbaldehyde 2c and anthracene-9carbaldehyde 2d produced the corresponding chalcones 3a-d respectively. The structures of the new chalcones 3b-d were established by their elemental analysis and spectral measurements (see Experimental and Scheme 1). The condensation of chalcones 3a-c with hydrazine hydrate in refluxing ethanol gave the corresponding 4,7-dimethoxy-5-(5-aryl-1H-pyrazolin-3-yl)benzofuran-6-ol 4a-c, while, the condensation with hydrazine hydrate in refluxing glacial acetic acid gave the corresponding 4,7-dimethoxy-5-(5-aryl-N-acetylpyrazolin-3yl)benzofuran-6-ol 5a-c. The structures of the compounds 4 and 5 were established and confirmed on the basis of their elemental analysis and spectral data (IR, MS, 1H NMR and 13C NMR). Thus, the mass spectrum of compound 4c as an example revealed molecular formula C18H18N2O5 {m/z (%): 342(100) [M+]}; its IR spectrum (KBr/cm-1) revealed the presence of OH absorption at 3434, NH absorption at 3325 and C=N absorption at 1617; the 1H NMR spectrum (DMSO-d6, δ ppm) showed two protons of CH2-pyrazoline as a doublet-doublet at 3.31 for axial-H, equatorial-H appeared at 3.55 as a doublet-doublet, CH-pyrazoline proton as a doublet-doublet at 4.75, NH and OH protons appeared as singlets at 7.60 and 12.19 respectively, which were D2O exchangeable; and the 13C NMR spectrum (DMSO-d6, δ ppm) using technique DEPT-135 for 4c showed a signal at 41.38 in the region of secondary carbon atom for CH2-pyrazoline and disappearance of a signal at the olefinic carbon region confirmed the structure 4c, the relatively high chemical shift indicating that the CH2 group was in a cyclic structure. The mass spectrum of 5c as an example revealed molecular formula C20H20N2O6 {m/z (%): 384(100) [M+]}; its IR spectrum (KBr/cm-1) revealed the presence of OH absorption at 3430, C=O absorption at 1657 and C=N absorption at 1620; the 1H NMR spectrum (DMSO-d6, δ ppm) showed two protons of CH2pyrazoline as a doublet-doublet for axial-H at 3.40, equatorial-H appeared as a doublet-doublet at 3.78, CHpyrazoline proton as a doublet-doublet at 5.45 and OH proton appeared as a singlet at 10.53, which was D2O exchangeable.

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OCH3 O H CH3 +

O

O Ar

OH OCH3

2 a-d

1 EtOH 5 ml 50% NaOH

OCH3 O Ar O

OH OCH3 3 a-d

NH2 NH2 EtOH reflux

2,3

NH2 NH2 glacial AcOH

Ar

a; 4-CH3 O-C6 H4 b; 1-naphthyl c; 5-methyl-2-f uryl d; anthracen-9-yl

reflux

O CH3

H OCH3

N

N

OCH3

N

Ar

Ar

O

N

O

OH

OH OCH3

OCH3 4 a-c

5 a-c 4,5

Ar

a; 4-CH3 O-C6 H4 b; 1-naphthyl c; 5-methyl-2-furyl

Scheme 1: The Condensation of chalcones 3a or 3b with phenyl hydrazine in refluxing ethanol gave 4,7-dimethoxy-5(5-(aryl)-N-phenylpyrazolin-3-yl)benzofuran-6-ol (6a,b) (Scheme 2). The structure of the compounds 6a,b was established and confirmed on the basis of their elemental analysis and spectral data (IR, MS and 1H NMR). Thus, as an example, the mass spectrum of 6a revealed molecular formula C26H24N2O5 {m/z (%): 444 (44.14) [M+]}. When compound 3b was refluxed in ethanol, it gave 4,9-dimethoxy-7-(naphth-1-yl)-6,7-dihydro-furo[3,2g]chromen-5-one 7, which was established and confirmed on the basis of its elemental analysis and spectral data (IR, MS and 1H NMR) Thus, the mass spectrum of 7 revealed the molecular formula C23H18O5 {m/z (%): 374 (24.99) [M+]}; its IR spectrum (KBr/cm-1) revealed the absence of OH band due to the cyclization and the C=O 857

Aust. J. Basic & Appl. Sci., 6(3): 852-863, 2012

group of 3b absorption at 1617 was shifted to 1739 due to γ-lactone carbonyl; the 1H NMR spectrum (DMSOd6, δ ppm) showed the axial-H of CH2-pyrane as a doublet-doublet at 2.91 (JHHgem=16 Hz), while equatorial-H appeared as a doublet-doublet at 3.31, a proton of CH-pyrane appeared as a doublet-doublet at 6.36 and OH proton disappeared in 1H NMR spectrum. Ph OCH 3

N

N

Ar PhNHNH2/ EtOH reflux

3 a,b

O

OH OCH 3 6 a,b

OCH3 O

EtOH/Pip.

3b

ref lux

O

O

Ar

OCH3 7

Ar CN OCH3

X

N

NC

XH

NH2

3c

EtOH/Pip. reflux

O

OH OCH 3 8 a,b

6

Ar

a; 4-CH O-C6 H4 3 b; 1-naphthyl

8 7; Ar= 1-naphthyl

X

a; O b; S

Ar 5-methyl-2-furyl 5-methyl-2-furyl

Scheme 2: Refluxing a mixture of chalcone 3c with 2-cyanoacetamide or 2-cyanothioacetamide in ethanol containing piperidine as a catalyst afforded compounds 8a and 8b respectively (Scheme 2), The structures of the compounds 8a,b were established and confirmed on the basis of their elemental analysis and spectral data (IR, MS, 1H NMR and 13C NMR). Thus, the mass spectrum of 8a revealed the molecular formula C21H16N2O6 {m/z (%): 392 (87.84) [M+]}; its IR spectrum (KBr/cm-1) revealed the presence of OH absorption at 3365, C≡ N absorption at 2210 and C=N absorption at 1622; the 1H NMR spectrum (DMSO-d6, δ ppm) showed H-5 pyridine proton as a singlet at 7.51, OH proton of pyridine and OH proton of benzofuran appeared as singlets at 9.50 and 12.29 respectively, which were D2O exchangeable, and the13C NMR spectrum (DMSO-d6, δ ppm) showed a signal at 118.15 due to C-5 of pyridine moiety. Ni(II), Co(II) and Zn(II) complexes of compound 3c; (L) were prepared in good yields from the equimolar ratio of the ligand (3c) and the corresponding metal salt (NiCl2.6H2O, CoCl2.6H2O and ZnCl2) in methanol

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(Scheme 3). All the complexes are stable in air and light. The elemental analysis of the complexes was consistent with the calculated results from the empirical formula of each compound. OCH3

OCH3

O

O

O

O CH3 O

MCl2/MeOH ref lux

OH

Cl M

O OCH 3

OCH3

OH2

CH3

O Cl H OH 2

Complex 9, M= Ni(II) Complex 10, M= Co(II) Complex 11, M= Zn(II)

3c

Scheme 3: The IR spectra of the metal(II) complexes were recorded using KBr/cm-1, it was found that the characteristic band of the hydroxyl group in the free ligand at 3423 was shifted to a lower frequency by 35-40. This shift indicated coordination of hydroxyl oxygen to the metal ions in the complexes (Keypour et al., 2002). The band of carbonyl group in the free ligand was shifted to lower frequency by 15-18, suggesting the coordination of carbonyl oxygen to metal. The band at 1272 was characteristic for ν (C-O), this band was shifted to a lower frequency by 7-17 in the complexes due to chelation of oxygen atom to metal. The new band at 513 was attributed to ν (M-O) (Raman et al., 2009). Most of the other bands appeared in the spectra of ligand were observed at nearly the same position in the IR spectra of metal complexes. In comparison with 1 H NMR spectrum of 3c, Zn(II) complex 11 confirmed that OH proton (δ 10.05 ppm in ligand) was present in the complex, shifted downfield (δ 10.11 ppm), suggesting deshielding of the hydroxyl group due to coordination, the signal at δ 4.94 ppm showed the presence of water (Silverstein and Webster, 2005). There was no appreciable change in other signals of this complex. 1H NMR of the Ni(II) 9 and Co(II) 10 complexes were recorded. Unfortunately, however due to the presence of a metal ion, proton resonance was not affected and one could observe only broad peaks indicating the formation of the complex. The molar conductivities of 10-3 M of the complexes (dissolved in DMF) at room temperature were measured. The results were in the range 15.9-11.6 Ω-1 cm2 mol-1, indicating that all the metal complexes had conductivity values in the range characteristic for non-electrolytic nature suggesting that these complexes were neutral (Domopoulou et al., 1997). The electronic spectra and the magnetic moments of metal complexes were measured in DMF. The magnetic moments were calculated from the corrected magnetic susceptibilities. The electronic spectra of the ligand 3c showed two bands at 385 and 342 nm due to charge transfer. The electronic absorption of Ni(II) complex 9 showed three bands at 409, 522 and 861 nm. These bands were assigned to the transitions 3 A2g(F)→3T1g(F), 3A2g→3T1g (p) and 3A2g→3T2g respectively, the magnetic moment value of Ni(II) complex was 3.2 B.M. The electronic spectra and the magnetic moments suggested octahedral geometry for Ni(II) complex (Cotton et al., 1999). On the other hand, the electronic spectrum of Co(II) complex 10 showed two bands at 464 and 625 nm. These bands were assigned to the transitions 4T1g(F)→4A2g(F) and 4T1g(F)→4T2g(P) respectively, for octahedral Co(II) complex, transition (4T1g→4T2g) band expected around 1050 nm was out of device range. Co(II) complex showed also magnetic moment at 4.3 B.M., which higher than the spin only value (3.87 B.M.) due to the orbital angular momentum contribution in d7 system. The electronic spectra and the magnetic moments value were consistent with octahedral geometry for the Co(II) complex (Omar and Mohamed, 2005). Zn(II) complex was diamagnetic according to the spectral data and the empirical formula of this complex, we proposed an octahedral geometry for Zn(II) complex. From the above spectral data, The prepared complexes had the general structural formula: [M(L)Cl2(H2O)2] (9, 10 and 11) where M=Ni(II), Co(II) or Zn(II) and L=3c. Antibacterial Activities: Fifteen compounds were screened in vitro for their antibacterial activities against two Gram‐positive bacteria species (Bacillus subtilis, Staphylococcus aureus) and two Gram negative bacteria species (Escherichia coli, Pseudomonas aeruginosa) using a modified Kirby‐Bauer disc diffusion method (Bauer et al., 1966). The bacteria were maintained on Meuller‐Hinton agar. DMSO showed no inhibition zone. The agar media were incubated at 35‐37 oC for 24‐48 hours for bacteria such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeuroginosa. The diameter of inhibition zone (mm) was measured. Tetracycline is used as a reference for antibacterial activities. The antibacterial screening results are compiled in Table 1. Meanwhile, Table 2 arranges the investigated compounds according to their descending order of activity. From Table 1 and 2, it could be seen that some of the investigated compounds showed marked activity with inhibition zone 859

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diameter values ranging from 9 to 15 reaching nearly one half that of the reference (Tetracycline) in case of compounds 4c, 5a and 5c against Escherichia coli. The investigation showed also that there is a structureactivity relationship. Thus, while compound 3c has no activity; its metal complexes 9, 10 and 11 showed activities against the four examined bacterial strains which reflect the effect of presence of metal atoms in this structure. The results of the metal complexes 9-11 are in agreement with the well known antifungal (Muthukumar and Viswanathamurthi, 2010) and antibacterial (Elzahany et al., 2008; Sumathi et al., 2011) activities of many metal complexes. Similarly, replacement of the 3-(aryl)prop-2-en-1-one system at position-5 in the benzofuran derivatives 3b and 3c with a 5-aryl-1H-pyrazolin-3-yl ring render the benzofuran derivatives 4b and 4c having moderate to significant activities. The activity of 4b,c are in agreement with the well known antibacterial (Holla et al., 2000) and antifungal (Siddiqui et al., 2011) activities of the benzofuran derivatives. On the other hand, when the 3-(5-methylfuran-2-yl) prop-2-en-1-one system in 3c was replaced with a 3cyanopyridine derivatives ring instead of 5-aryl-1H-pyrazolin-3-yl ring, compounds 8a and 8b were found to show no activity. Moreover, compounds 5a (Ar=4-methoxyphenyl) and 5c (Ar=5-methyl-2-furyl) showed antibacterial activities similar to their free H-analogous 4a and 4c, while 5b (Ar=1-naphthyl) was found to be inactive against the bacterial strains under investigation. However, when the acetyl group in 5a was replaced by a phenyl group, compound 6a showed no activity. Based on these results, there is a marked difference between the investigated compounds in their antibacterial activity depending upon the nature of the substitutions and/or the presence of metal complexes which confirm the structure-activity relationship (SAR) principle (Elzahany et al., 2008; Holla et al., 2000). Table 1: Antibacterial activities data of some selected compounds and the metal complexes. Compound no. Inhibition zone diameter in millimeters Gram-negative Gram-positive E. coli P.aeruginosa B. subtilis S. aureus 3b 0 0 0 0 3c 0 0 0 0 4a 13 13 12 12 4b 13 12 14 12 4c 15 14 14 14 5a 15 12 13 13 5b 0 0 0 0 5c 15 13 12 14 6a 0 0 0 0 6b 0 0 0 0 8a 0 0 0 0 8b 0 0 0 0 Complex 9 10 9 12 12 Complex 10 11 11 10 10 Complex 11 9 10 10 9 Tetracycline 32 34 32 30 Compound: > 14 mm, significant activity; 7-13 mm, moderate activity;