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Supporting information
Design, synthesis, Anti-inflammatory evaluation and Docking of novel series of Benzofuran
based Derivatives
Pratima Yadav, Praveen Singh, Ashish Kumar Tewari*
Department of Chemistry, Centre of Advance Studies, Faculty of Science, Banaras Hindu
University Varanasi 221 005, India
*Corresponding author. Tel.: +919935343986; fax: +915422368127;
E-mail address: [email protected]
1. Experimental procedures for the preparation of compounds (2a, 2b, 3a-3g and 4a-4e)
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2. 1H NMR spectra of compound (2a, 2b, 3a-3g and 4a-4e)………..
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3. 13C NMR spectra of compound (3a, 3b, 3c, 3e, 4a, 4b, 4c and 4d)………………...….…
S20-S27
4. Biological Activity
1) Assays for cyclooxygenase 1 and 2………………………………………………………..S28
2) Anti-inflammatory activity…………………………………………………………………
General procedure for the synthesis of Ethyl 5-hydroxy-2-methyl/phenylbenzofuran-3-carboxylate (2a-2b)
In a 100 ml double neck round bottom flask fitted with reflux condenser, appropriately substituted ethyl 3-aryl-3-oxopropionate (1 equiv), p-benzoquinone (1 eqiv) and anhydrous zinc chloride (1.2 eqiv), was taken in toluene and stirred at 70 °C for 15 min. Dean-stark apparatus was assembled and the reaction mixture was furthure heated to 140 °C for 12 h and cooled to ambient temperature. The completion of reaction was judged by TLC. EtOAc was added to the reaction mass and filtered through the filter paper, to remove the inorganic material. Brine was added to the reaction mixture (filtrate) and extracted, dried over sodium sulphate and concentrated to get the crude material. Further it was purified by column chromatography using 10 % EtOAc/Hexane system as eluent.
Ethyl 5-hydroxy-2-methylbenzofuran-3-carboxylate (2a)
Yield 43 %; 1H NMR (300 MHz, CDCl3): δ: 1.43 (t, J= 6.9 Hz, 3H, CH3), 2.73 (s, 3H, CH3), 4.39 (q, J= 6.9Hz, 2H, OCH2), 5.13 (br s, 1H, OH), 6.79 (d, J=8.7Hz, 2H, Ar), 7.26 (d, J=8.7Hz, 2H, Ar); FAB MS: m/z 220 (M+1).Elemental analysis forC12H12O4: Calcd: C, 65.45; H, 5.49; Found: C, 65.39; H, 5.40.
Ethyl 5-hydroxy-2-phenylbenzofuran-3-carboxylate (2b):
Yield 43 %; 1.39 (t, 3H, J= 7.2 Hz), 4.39 (q, 2H, J= 7.2Hz), 5.1 (br s, 1H), 6.8-7.9 (m, 8H, Ar); FAB MS: m/z 283 (M+1).Elemental analysis for C17H14O4: Calcd: C, 72.33; H, 5.00; Found: C, 72.30; H, 4.90.
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General procedure for the synthesis of diaryl ethers (3a-3g and 4a-4e):
A Schlenk tube equipped with a magnetic stir bar was charged with K2CO3 (1 equiv.), 8-hydroxyquinoline (0.02 equiv.), CuI (0.1 equiv.) and 2-substituted ethyl 5-hydroxybenzofuran-3-carboxylate (1 mmol) and substituted aryl/heteroarylbromide were added under a stream of nitrogen by syringe at room temperature, followed by dry DMF. The reaction mixture was heated in an oil bath at 1350C for 12h. After completion of reaction DMF was removed under reduced pressure and the reaction mixture was extracted with CHCl3. Chloroform was removed and the product was purified via SiO2 (60 × 120 mesh) column chromatography.
Ethyl 2-methyl-5-phenoxybenzofuran-3-carboxylate (3a)
Yield 0.96 g, 76 %; Rf = 0.52 Hexane/EtOAc (9:1); 1H NMR (300MHz, CDCl3): δ:1.35 (t, J= 7.0Hz, 3H, CH3), 2.77 (s, 3H, CH3), 4.35 (q, J= 6.9Hz, 2H, OCH2), 6.98-7.59 (m, 8H, Ar); 13C NMR (CDCl3, 75 MHz) δ14.4, 29.6, 60.2, 109.2, 111.4, 112.1, 116.6, 118.0, 122.6, 127.3, 129.6,149.9, 153.3, 158.3, 164.1,164.7; FAB MS: m/z 296 (M+1);Elemental analysis for C18H16O4: Calcd: C, 72.96; H, 5.44; Found: C, 72.60; H, 5.39.
Ethyl 2-methyl-5-(o-tolyloxy)benzofuran-3-carboxylate (3b)
Yield 1.03 g, 78 %; Rf = 0.52 Hexane/EtOAc (9:1);1H NMR (300MHz, CDCl3): δ: 1.31 (t, J= 7.2Hz, 3H, CH3), 2.28 (s, 3H, CH3), 2.73 (s, 3H, CH3), 4.32 (q, J= 6.9Hz, 2H, OCH2), 6.83-7.45 (m, 7H, Ar);13C NMR (CDCl3, 75 MHz) δ14.4, 22.6, 29.6, 60.1, 109.1, 110.2, 111.3, 115.1, 118.7, 123.4, 127.2, 129.3, 149.4, 152.7, 154.2, 155.4, 164.1, 164.7; FAB MS: m/z 310 (M+1); Elemental analysis for C19H18O4: Calcd: C, 73.53; H, 5.85; Found: C, 73.40; H, 5.69.
Ethyl 2-methyl-5-(p-tolyloxy)benzofuran-3-carboxylate (3c)
Yield 0.96g, 73 %; Rf = 0.52 Hexane/EtOAc (9:1);1H NMR (300MHz, CDCl3): δ: 1.35 (t, J= 7.2Hz, 3H, CH3), 2.28 (s, 3H, CH3), 2.73 (s, 3H, CH3), 4.32 (q, J= 6.9Hz, 2H, OCH2), 6.89-7.57(m, 7H, Ar); 13C NMR (CDCl3, 75 MHz) δ14.3, 22.6, 29.6, 60.1, 109.1, 111.4, 111.2, 111.4,
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116.0, 118.2, 127.1, 130.0, 132.1, 149.7, 153.9, 155.7, 163.9, 164.5; FAB MS: m/z 310 (M+1); Elemental analysis for C19H18O4: Calcd: C, 73.53; H, 5.85; Found: C, 73.49; H, 5.79.
Ethyl 5-(4-methoxyphenoxy)-2-methylbenzofuran-3-carboxylate (3d)
Yield 0.90 g, 65%; Rf = 0.52 Hexane/EtOAc (9:1); 1H NMR (300MHz, CDCl3 δ: 1.35 (t, J= 7.2Hz, 3H, CH3), 2.75 (s, 3H, CH3), 3.79 (s, 3H, OCH3), 4.34 (q, J= 6.9Hz, 2H, OCH2), 6.85-7.50 (m, 7H, ArH);13C NMR (CDCl3, 75 MHz) δ14.4, 29.6, 60.2, 109.2, 111.4, 112.1, 116.6, 118.0, 122.6, 127.3, 129.6, 149.9, 153.3, 158.3, 164.1, 164.7; FAB MS: m/z 326 (M+1); Elemental analysis for C19H18O5: Calcd: C, 69.93; H, 5.65; Found: C, 69.09; H, 5.39.
Ethyl 2-methyl-5-(pyridin-2-yloxy)benzofuran-3-carboxylate (3e)
Yield 0.94 g, 74 %; Rf = 0.52 Hexane/EtOAc (9:1);1H NMR (300MHz, CDCl3): δ: 1.38 (t, J= 7.1Hz, 3H, CH3), 2.77 (s, 3H, CH3), 4.37 (q, J= 7.1Hz, 2H, OCH2), 6.89-8.18 (m, 7H, ArH); 13C NMR (CDCl3, 75 MHz) δ 14.4, 29.6, 60.2, 109.3,111.2, 111.4, 114.2, 118.2, 127.3, 139.2, 150.4, 150.6, 158.3, 164.1, 164.7; FAB MS: m/z 297 (M+1); Elemental analysis for C17H15NO4: Calcd: C, 68.68; H, 5.09;N, 4.71; Found: C, 68.01; H, 5.04; N, 4.65.
Ethyl 2-methyl-5-(naphthalen-2-yloxy)benzofuran-3-carboxylate (3f)
Yield 1.022 g, 65 %; Rf = 0.52 Hexane/EtOAc (9:1);1H NMR (300MHz, CDCl3): δ: 1.37 (t, J= 7.2Hz, 3H, CH3), 2.78 (s, 3H, CH3), 4.42 (q, J= 7.2Hz, 2H, OCH2), 7.141-8.32 (m, 10H, Ar-H); FAB MS: m/z 347 (M+1); Elemental analysis for C22H18O4: Calcd: C, 65.45; H, 5.49; Found: C, 65.01; H, 5.04.
Ethyl 2-methyl-5-(naphthalen-1-yloxy)benzofuran-3-carboxylate (3g)
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Yield 1.022 g, 65 %; Rf = 0.52 Hexane/EtOAc (9:1);1H NMR (300MHz, CDCl3): δ: δ: 1.37 (t, J= 7.2Hz, 3H, CH3), 2.78 (s, 3H, CH3), 4.42 (q, J= 7.2Hz, 2H, OCH2), 7.16-8.31 (m, 10H, Ar-H); FAB MS: m/z 347 (M+1); Elemental analysis for C22H18O4: Calcd: C, 65.45; H, 5.49; Found: C, 65.01; H, 5.04.
Ethyl 5-phenoxy-2-phenylbenzofuran-3-carboxylate (4a)
Yield 0.931g, 69 %; Rf = 0.52 Hexane/EtOAc (9:1);1H NMR (300MHz, CDCl3): δ: 1.30 (t, J= 7.1Hz, 3H, CH3), 4.34 (q, J= 7.1Hz, 2H, OCH2), 7.01-8.02 (m, 7H, ArH); 13C NMR (CDCl3, 75 MHz) δ14.4, 29.6, 60.2, 109.2, 111.4, 112.1, 116.6, 118.0, 122.6, 127.3, 129.6,149.9, 153.3, 158.3, 164.1,164.7; FAB MS: m/z 358 (M+1); Elemental analysis for C23H18O4: Calcd: C, 77.08; H, 5.06; Found: C, 77.01; H, 5.04.
Ethyl 2-phenyl-5-(o-tolyloxy)benzofuran-3-carboxylate (4b)
Yield 0.93g, 73 %; Rf = 0.52 Hexane/EtOAc (9:1); 1H NMR (300MHz, CDCl3): δ: 1.26 (t, J= 7.2Hz, 3H, CH3), 2.33 (s, 3H, CH3), 4.32 (q, J= 6.9Hz, 2H, OCH2), 6.88-8.03 (m, 7H, Ar); 13C NMR (CDCl3, 75 MHz) δ14.0, 60.6, 110.6, 111.8, 116.2, 119.1, 123.7, 127.1, 128.0, 129.4, 129.5, 129.6, 131.3, 154.6, 155.2, 161.7, 163.7; FAB MS: m/z 372 (M+1); Elemental analysis for C24H20O4: Calcd: C, 77.40; H, 5.41; Found: C, 77.01; H, 5.04.
Ethyl 2-phenyl-5-(p-tolyloxy)benzofuran-3-carboxylate (4c)
Yield 0.93g, 73 %; Rf = 0.52 Hexane/EtOAc (9:1);1H NMR (300MHz, CDCl3): δ: 1.31 (t, J= 7.2Hz, 3H, CH3), 2.32 (s, 3H, CH3), 2.73 (s, 3H, CH3), 4.33 (q, J= 7.1Hz, 2H, OCH2), 6.91-8.02 (m, 12H, ArH); 13C NMR (CDCl3, 75 MHz) δ14.0, 29.6, 60.6, 109.1, 111.8, 112.1, 117.2, 118.4, 128.0, 128.1, 129.4, 130.1, 130.3, 149.9 154.3, 155.7, 161.7, 163.7; FAB MS: m/z 372 (M+1); Elemental analysis for C24H20O4: Calcd: C, 77.40; H, 5.06; Found: C, 77.01; H, 5.04.
Ethyl 2-phenyl-5-(pyridin-2-yloxy)benzofuran-3-carboxylate (4d)
S4
Yield 0.58 g, 74 %; Rf = 0.52 Hexane/EtOAc (9:1); 1H NMR (300MHz, CDCl3): δ: 1.30 (t, J= 7.0Hz, 3H, CH3), 4.32 (q, J= 7.1Hz, 2H, OCH2), 6.89-8.169 (m, 12H, ArH); 13C NMR (CDCl3, 75 MHz) δ13.9, 60.3, 109.0, 111.0, 111.6, 114.8, 118.0, 119.1, 127.7, 128.0,129.2, 130.0, 139.1, 147.4,150.4, 1616.5, 163.3,163.9; FAB MS: m/z 359 (M+1); Elemental analysis for C22H17NO4: Calcd: C, 73.53; H, 4.77; N, 3.90; Found: C, 73.01; H, 4.54N, 3.87.
Ethyl 5-(naphthalen-2-yloxy)-2-phenylbenzofuran-3-carboxylate (4e)
Yield 0.86 g, 60%; Rf = 0.52 Hexane/EtOAc (9:1);1H NMR (300MHz, CDCl3): δ: 0.854 (t, J= 7.5Hz, 3H, CH3), 4.32 (q, J= 7.2Hz, 2H, OCH2), 7.11-8.04 (m, 14H, ArH); FAB MS: m/z 409 (M+1); Elemental analysis for C27H20O4: Calcd: C, 79.40; H, 4.94; Found: C, 79.01; H, 4.84.
1H NMR spectra of ethyl 5-hydroxy-2-methylbenzofuran-3-carboxylate (2a.)
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1H NMR spectra of ethyl 5-hydroxy-2-phenylbenzofuran-3-carboxylate (2b)
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1H NMR spectra of ethyl 2-methyl-5-phenoxybenzofuran-3-carboxylate (3a)
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1H NMR spectra of ethyl 2-methyl-5-(o-tolyloxy)benzofuran-3-carboxylate (3b)
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1H NMR spectra of ethyl 2-methyl-5-(p-tolyloxy)benzofuran-3-carboxylate (3c)
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1H NMR spectra of ethyl 5-(4-methoxyphenoxy)-2-methylbenzofuran-3-carboxylate (3d)
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1H NMR spectra of ethyl 2-methyl-5-(pyridin-2-yloxy)benzofuran-3-carboxylate (3e)
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1H NMR spectra of ethyl 2-methyl-5-(naphthalen-2-yloxy)benzofuran-3-carboxylate (3f)
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1H NMR spectra of ethyl 2-methyl-5-(naphthalen-1-yloxy)benzofuran-3-carboxylate (3g)
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1H NMR spectra of ethyl 5-phenoxy-2-phenylbenzofuran-3-carboxylate (4a)
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1H NMR spectra of ethyl 2-phenyl-5-(o-tolyloxy)benzofuran-3-carboxylate (4b)
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1H NMR spectra of ethyl 2-phenyl-5-(p-tolyloxy)benzofuran-3-carboxylate (4c)
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1H NMR spectra of ethyl 2-phenyl-5-(pyridin-2-yloxy)benzofuran-3-carboxylate (4d)
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1H NMR spectra of ethyl 5-(naphthalen-2-yloxy)-2-phenylbenzofuran-3-carboxylate (4e)
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13C NMR spectra of ethyl 2-methyl-5-phenoxybenzofuran-3-carboxylate (3a.)
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13C NMR spectra of ethyl 2-methyl-5-(o-tolyloxy)benzofuran-3-carboxylate (3b)
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13C NMR spectra of ethyl 2-methyl-5-(p-tolyloxy)benzofuran-3-carboxylate (3c)
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13C NMR spectra of ethyl 2-methyl-5-(pyridin-2-yloxy)benzofuran-3-carboxylate (3e)
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13C NMR spectra of ethyl 5-phenoxy-2-phenylbenzofuran-3-carboxylate (4a)
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13C NMR spectra of ethyl 2-phenyl-5-(o-tolyloxy)benzofuran-3-carboxylate (4b)
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13C NMR spectra of ethyl 2-phenyl-5-(p-tolyloxy)benzofuran-3-carboxylate (4c)
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13C NMR spectra of ethyl 2-phenyl-5-(pyridin-2-yloxy)benzofuran-3-carboxylate (4d)
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Biological Activity of Compound
Anti-inflammatory activity
In vivo anti-inflammatory activity
All experiments have been conducted on adult Wistar strain albino rats of either sex, weighing between 150-200 g. The animals were obtained from the Central Animal House of the Institute of Medical Sciences, B.H.U. They were kept in colony cages under identical housing conditions at an ambient temperature of 25±2 °C and 45—55% relative humidity with a 12 h light– dark cycle in the departmental animal room and fed on standard diet. Animals were acclimatized for a week before use.
Experimental Model of Inflammation
Carrageenin Induced Paw Edema in Rats1: Target compounds were suspended in aqueous solution of carboxymethyl cellulose (CMC, 1% w/v) and administered orally at a dose level of 10 mg/kg of NSAID drugs nimusilide and their equivalent doses of the prodrugs. After 15 min control animals were similarly treated with aqueous solution of carboxymethyl cellulose (CMC, 1% w/v). One percent carrageenin suspension was prepared as a homogeneous solution in distilled water. A volume of 0.1 ml of carrageenin solution was injected through a 26 gauge needle into the plantar surface of the left hind paw below the plantar aponeurosis. The volume of paw was measured before and at different intervals for 2 h after injection of carrageenin. The difference in paw volume before and after administration of the phlogistic agent was taken as the measure of pedal edema.
Measurement of Paw Volume
The volume of hind paw of the rats up to the ankle joint was measured plathysmographically by the mercury displacement method. The ankle joint of the rats was marked with a skin marking pencil and the paw was dipped in the mercury, so that the mark on the paw coincides with a prefixed line kept constant on the syringe. The level of the mercury was every time brought to the level of this line by adjusting the height of the displaced mercury. The difference in the paw volume before and after injection of the phlogistic agents was taken as a measure of pedal edema. The change in paw volume was expressed in “ml” of mercury displaced.
Statistical Analysis: The difference in the means between each experimental group was first analyzed by Analysis of Variance (ANOVA). When the overall ANOVA was significant (p<0.05), the data was further subjected to statistical analysis by the student’s t-test.
Assays for cyclooxygenase 1 and 2
COX-1 has been isolated from Rat seminal vesicles. Recombinant human COX-2 has been expressed in insect cell expression system. These enzymes have been purified by employing conventional chromatographic techniques. Enzymatic activities of COX-1 and COX-2 were measured according to the method of Copeland et al. (1994) 2, with slight modifications using a chromogenic assay based on the oxidation of N,N,N,N,-tetra methyl-p-phenylene diamine (TMPD)
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during the reduction of PGG2 to PGH2 3,4. Briefly, the assay mixture contained TriseHCl buffer
(100 mM, Ph 8.0), hematin (15 mM), EDTA (3 mM), enzyme (100 mg COX-1 or COX-2) and the test compound. The mixture was pre-incubated at 25°C for 1 min and then the reaction was initiated by the addition of arachidonic acid and TMPD, in total volume of 1 ml. The enzyme activity was determined by estimating the velocity of TMPD oxidation for the first 25 s of the reaction by following the increase in absorbance at 603 nm. A low rate of non enzymatic oxidation observed in the absence of COX-1 and COX-2 was substracted form the experimental value while calculating the percent inhibition.
References
1. Winter, C. A.; Risely, E. A.; Nuss, G. W. Proc. Soc. Exp. Biol. 1962, 111, 544.2. Copeland, R.A., Williams J.M., J. Giannaras, J., Nurnberg, S., Covington, M., Pinto, D.,
Pick, S., Trzaskos. J.M. Proc. Natl. Acad. Sci. USA. 1994, 23, 11202.3. Pagels, W.R., Sachs, R.J., Marnett, L.J., Dewitt, D.L., Smith. W.L. J. Biol. Chem. 1983, 258,
6517.4. Egan, R.W., Paxton J., Kuehl. F.A. J. Biol. Chem. 1976, 251, 7329.
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