【病毒外文文献】2012 Synthesis and antiviral evaluation of 7-O-arylmethylquercetin derivatives against SARS-associated coronavirus (SCV)

Arch Pharm Res Vol 35 No 1 77 85 2012 DOI 10 1007 s12272 012 0108 9 77 Synthesis and Antiviral Evaluation of 7 O Arylmethylquercetin Deri vatives against SARS associated Coronavirus SCV and Hepatitis C Virus HCV Hye Ri Park 1 Hyunjun Yoon 1 Mi Kyoung Kim 1 Sung Dae Lee 2 and Youhoon Chong 1 1 Department of Bioscience and Biotechnology Bio Molecular Informatics Center Konkuk University Seoul 143 701 Korea and 2 Swine Science Division National Institute of Animal Science RDA Cheonan 330 801 Korea Received June 18 2011 Revised July 29 2011 Accepted August 9 2011 Aryl diketoacid ADK is well known for antiviral activity which can be enhanced by introduc tion of an aromatic arylmethyl substituent A natural flavonoid quercetin has a 3 5 dihydrox ychromone pharmacophore which is in bioisosteric relationship with the 1 3 diketoacid moiety of the ADK Thus it was of our interest to test the antiviral activity of the quercetin deriva tives with an arylmethyl group attached In this study we prepared a series of the 7 O aryl methylquercetin derivatives with various aromatic substituents and evaluated their antiviral activity against the SARS associated coronavirus SARS CoV SCV as well as hepatitis C virus HCV Single difference in the aromatic substituent fine tuned the biological activity of the 7 O arylmethylquercetin derivatives to result in two different classes of derivatives selec tively active against SCV and HCV Key words Quercetin Arylmethyl Hepatitis C Severe acute respiratory syndrome SARS INTRODUCTION Quercetin 1 Fig 1 is widely distributed in the plant kingdom and is the most abundant of the flavonoid family It is often a major component of the medicinal activity of the plant and has been shown in experimental studies to have numerous effects on the body Quercetin appears to have many beneficial effects on human health including cardiovascular protection anticancer activity antiulcer effects antiallergy activity cataract prevention and anti inflammatory effects In addition quercetin exerts antiviral activity against reverse transcriptase of HIV Harada et al 1999 and other retroviruses and was shown to reduce the infec tivity and cellular replication of herpes simplex virus type 1 Amoros et al 1992 polio virus type 1 Vrijsen et al 1988 parainfluenza virus type 3 and respira tory syncytial virus RSV Kaul et al 1985 Recently we have also shown inhibition of the helicase activity of the SARS associated coronavirus SARS CoV SCV by quercetin IC 50 8 1 M Lee et al 2009a More interestingly introduction of arylmethyl substituent such as 4 ClPhCH 2 3 ClPhCH 2 and 3 CNPhCH 2 at the 7 OH position of quercetin provided the resulting quercetin derivatives 2 Fig 1 with significantly increased inhibitory activity against the SCV helicase IC 50 4 1 5 2 2 7 M respectively At this point it is worth to note that the core 3 5 dihydroxychromone moiety of the quercetin bold lines in 1 Fig 1 is in match with the 1 3 diketoacid bold lines in 3 Fig 1 the key pharmacophoric element of the antiviral aryl diketoacid ADK 3 Fig 1 in atom by atom fashion Also in line with the substitut ed quercetins the arylmethyloxy substituent dotted box in 3 Fig 1 increased the antiviral activity of the ADKs and the substituted ADKs showed potent inhibition of the RNA dependent RNA polymerase RdRp of hepatitis C virus HCV Kim et al 2008 as well as the SCV NTPase helicase Lee et al 2009b These authors contributed equally to this work Correspondence to Youhoon Chong Department of Bioscience 1 H NMR 500 MHz CD 3 COCD 3 7 86 s 1H 7 71 d J 8 0 Hz 1H 7 52 d J 7 5 Hz 2H 7 42 t J 7 5 Hz 2H 7 39 7 34 m 1H 7 00 d J 8 5 Hz 1H 6 77 s 1H 6 40 s 1H 5 26 s 2H 13 C NMR 100 MHz DMSO d 6 176 3 164 2 160 8 156 3 148 2 147 7 145 5 143 9 136 6 136 4 129 0 128 3 122 2 116 0 115 9 115 6 104 5 98 4 98 3 93 2 93 0 70 3 LC MS ESI m z Found 393 40 M H Calcd for C 22 H 17 O 7 393 10 2 3 4 Dihydroxyphenyl 7 2 fluorobenzyloxy 3 5 dihydroxy 4H chromen 4 one 2b The desired product 2b was obtained as a yellow powder in 29 yield m p 255 257 o C dec 1 H NMR 400 MHz CD 3 COCD 3 7 87 s 1H 7 73 d J 8 3 Hz 1H 7 63 t J 7 1 Hz 1H 7 46 d J 7 3 Hz 1H 7 29 7 20 m 2H 7 00 d J 8 4 Hz 1H 6 83 s 1H 6 42 d J 1 3 Hz 1H 5 33 s 2H 13 C NMR 100 MHz DMSO d 6 176 3 164 0 162 1 160 8 159 6 156 4 148 3 147 8 145 5 143 9 136 5 131 4 124 9 123 5 123 3 122 2 120 2 116 0 104 6 98 3 93 0 64 7 LC MS ESI m z Found 411 30 M H Calcd for C 22 H 16 FO 7 411 09 7 2 Chlorobenzyloxy 2 3 4 dihydroxyphenyl 3 5 dihydroxy 4H chromen 4 one 2c The desired product 2c was obtained as a yellow powder in 22 yield m p 252 255 o C dec 1 H NMR 500 MHz CD 3 COCD 3 7 86 s 1H 7 73 d J 8 5 Hz 1H 7 67 7 65 m 1H 7 52 7 5 m 1H 7 42 7 40 m 2H 7 00 d J 8 5 Hz 1H 6 82 s 1H 6 43 s 1H 5 34 s 2H 13 C NMR 100 MHz DMSO d 6 176 3 164 0 160 8 156 4 148 3 147 8 145 5 136 5 133 9 133 3 131 0 130 8 130 1 129 7 128 0 127 7 122 2 120 6 116 0 104 7 93 0 68 0 LC MS ESI m z Found 427 40 M H Calcd for C 22 H 16 ClO 7 427 06 7 2 Bromobenzyloxy 2 3 4 dihydroxyphenyl 3 5 dihydroxy 4H chromen 4 one 2d The desired product 2d was obtained as a yellow powder in 20 yield m p 248 250 o C dec 1 H NMR 400 MHz CD 3 COCD 3 7 86 s 1H 7 74 7 64 m 3H 7 45 t J 7 5 Hz 1H 7 33 t J 7 5 Hz 1H 7 00 d J 8 5 Hz 1H 6 79 d J 1 4 Hz 1H 6 42 d J 1 6 Hz 1H 5 29 s 2H 13 C NMR 100 MHz DMSO d 6 176 3 164 0 160 8 156 4 148 3 147 8 145 5 136 5 135 4 133 1 131 0 130 8 128 4 123 5 122 2 120 4 115 9 115 6 104 7 98 3 93 0 70 2 LC MS ESI m z Found 471 80 M H Calcd for C 22 H 16 BrO 7 471 01 2 3 4 Dihydroxyphenyl 3 5 dihydroxy 7 2 iodo benzyloxy 4H chromen 4 one 2e The desired product 2e was obtained as a yellow powder in 18 yield m p 252 255 o C dec 1 H NMR 400 MHz CD 3 COCD 3 7 97 dd J 7 9 0 7 Hz 1H 7 86 d J 1 8 Hz 1H 7 73 dd J 8 5 1 3 Hz 1H 7 62 dd J 7 6 1 3 Hz 1H 7 49 t J 7 0 Hz 1H 7 16 t J 7 6 Hz 1H 7 01 d J 8 6 Hz 1H 6 81 d J 2 1 Hz 1H 6 43 d J 2 1 Hz 1H 5 24 s 2H 13 C NMR 125 MHz DMSO d 6 176 4 164 2 160 9 156 5 148 3 147 9 145 6 139 8 138 6 136 6 131 0 130 7 129 0 122 3 120 5 116 1 115 8 104 8 100 1 98 4 93 2 74 5 LC MS ESI m z Found 519 30 M H Calcd for C 22 H 16 IO 7 518 99 2 2 3 4 Dihydroxyphenyl 3 5 dihydroxy 4 oxo 4H chromen 7 yloxy methyl benzo nitrile 2f The desired product 2f was obtained as a yellow powder in 28 yield m p 244 246 o C dec 1 H NMR 500 MHz CD 3 COCD 3 7 88 t J 8 0 Hz 2H 7 83 7 77 m 3H 7 73 d J 8 5 Hz 1H 7 62 t J 7 3 Hz 1H 7 00 d J 8 0 Hz 1H 6 88 s 1H 6 47 s 1H 5 46 s 2H 13 C NMR 100 MHz DMSO d 6 176 3 163 8 160 8 156 3 148 3 147 8 145 5 139 5 136 5 134 0 129 8 122 2 120 4 117 5 116 0 115 9 111 8 104 8 98 3 98 2 93 2 93 1 68 6 LC MS ESI m z Found 418 30 M H Calcd for C 23 H 16 NO 7 418 09 2 3 4 Dihydroxyphenyl 3 5 dihydroxy 7 2 nitro benzyloxy 4H chromen 4 one 2g The desired product 2g was obtained as a yellow powder in 22 yield m p 240 243 o C dec 1 H NMR 400 MHz CD 3 COCD 3 8 20 d J 8 1 Hz 1H 7 91 7 80 m 3H 7 71 d J 8 3 Hz 1H 7 66 t J 7 6 Hz 1H 6 99 d J 8 39 Hz 1H 6 82 s 1H 6 45 s 1H 5 67 s 2H 13 C NMR 100 MHz DMSO d 6 176 3 163 7 160 9 156 3 148 3 147 8 147 7 145 5 136 5 134 6 132 1 129 7 129 5 125 6 125 1 122 2 115 9 115 6 104 8 98 4 93 2 67 4 LC MS ESI m z Found 438 40 M H Calcd for C 22 H 16 NO 9 438 08 2 3 4 Dihydroxyphenyl 3 5 dihydroxy 7 2 methyl benzyloxy 4H chromen 4 one 2h The desired product 2h was obtained as a yellow powder in 23 yield m p 200 203 o C dec 1 H NMR 400 MHz CD 3 COCD 3 7 86 s 1H 7 72 d J 8 3 80 H R Park et al Hz 1H 7 47 d J 7 0 Hz 1H 7 28 7 23 m 3H 7 00 d J 8 4 Hz 1H 6 82 s 1H 6 42 d J 0 7 Hz 1H 5 26 s 2H 2 40 s 3H 13 C NMR 100 MHz DMSO d 6 176 3 171 8 164 4 160 8 156 4 148 3 147 7 145 5 137 2 136 5 134 5 130 7 130 5 129 2 128 9 128 6 122 2 115 9 104 5 98 3 93 1 69 1 19 0 LC MS ESI m z Found 407 40 M H Calcd for C 23 H 19 O 7 407 11 2 3 4 Dihydroxyphenyl 7 3 fluorobenzyloxy 3 5 dihydroxy 4H chromen 4 one 2i The desired product 2i was obtained as a yellow powder in 31 yield m p 233 235 o C dec 1 H NMR 500 MHz CD 3 COCD 3 7 85 s 1H 7 12 d J 7 5 Hz 1H 7 48 q J 8 0 Hz 1H 7 37 d J 8 0 Hz 1H 7 32 d J 10 0 Hz 1H 7 14 t J 9 0 Hz 1H 7 00 d J 8 5 Hz 1H 6 81 d J 1 5 Hz 1H 6 44 d J 2 0 Hz 1H 5 32 s 2H 13 C NMR 100 MHz DMSO d 6 176 3 164 0 163 8 161 4 160 8 156 3 148 3 147 8 145 5 139 5 139 4 136 5 131 2 124 2 122 2 120 5 115 9 114 8 104 6 98 4 93 2 69 4 LC MS ESI m z Found 411 30 M H Calcd for C 22 H 16 FO 7 411 09 7 3 Chlorobenzyloxy 2 3 4 dihydroxyphenyl 3 5 dihydroxy 4H chromen 4 one 2j The desired product 2j was obtained as a yellow powder in 28 yield m p 266 268 o C dec 1 H NMR 400 MHz DMSO d 6 7 72 d J 1 9 Hz 1H 7 57 d J 1 9 Hz 1H 7 55 s 1H 7 45 m 3H 6 90 d J 8 5 Hz 1H 6 81 d J 2 0 Hz 1H 6 50 d J 2 0 Hz 1H 5 26 s 2H 13 C NMR 100 MHz CD 3 COCD 3 178 7 165 1 161 6 157 4 148 2 147 3 145 6 139 7 134 6 130 9 128 7 128 1 126 6 123 4 121 2 115 9 115 6 104 9 98 7 98 6 70 0 LC MS ESI m z Found 427 10 M H Calcd for C 22 H 16 ClO 7 427 06 7 3 Bromobenzyloxy 2 3 4 dihydroxyphenyl 3 5 dihydroxy 4H chromen 4 one 2k The desired product 2k was obtained as a yellow powder in 26 yield m p 200 203 o C dec 1 H NMR 400 MHz CD 3 COCD 3 7 86 d J 1 6 Hz 1H 7 73 s 1H 7 71 d J 1 8 Hz 1H 7 56 7 53 m 2H 7 39 t J 7 8 Hz 1H 7 01 d J 4 5 Hz 1H 6 80 d J 2 1 Hz 1H 6 43 d J 2 0 Hz 1H 5 30 s 2H 13 C NMR 100 MHz DMSO d 6 176 3 171 9 164 0 160 8 156 3 148 3 147 8 145 5 139 4 136 5 131 5 131 0 127 3 127 0 122 3 120 5 120 2 116 0 104 6 98 3 93 2 69 3 LC MS ESI m z Found 471 20 M H Calcd for C 22 H 16 BrO 7 471 01 2 3 4 Dihydroxyphenyl 3 5 dihydroxy 7 3 iodo benzyloxy 4H chromen 4 one 2l The desired product 2l was obtained as a yellow powder in 14 yield m p 230 233 o C dec 1 H NMR 400 MHz DMSO d 6 7 86 s 1H 7 73 d J 2 2 Hz 2H 7 57 dd J 8 5 2 2 Hz 1H 7 52 d J 10 2 Hz 1H 7 23 t J 7 8 Hz 1H 6 91 d J 8 5 Hz 1H 6 80 d J 2 2 Hz 1H 6 45 d J 2 2 Hz 1H 5 22 s 2H 13 C NMR 100 MHz DMSO d 6 176 8 164 4 161 3 156 8 148 7 148 2 145 9 139 7 137 6 137 0 136 9 131 5 127 9 122 6 120 8 116 4 116 1 105 0 98 8 95 7 93 6 69 7 LC MS ESI m z Found 519 40 M H Calcd for C 22 H 16 IO 7 518 99 3 2 3 4 Dihydroxyphenyl 3 5 dihydroxy 4 oxo 4H chromen 7 yloxy methyl benzo nitrile 2m The desired product 2m was obtained as a yellow powder in 29 yield m p 236 239 o C dec 1 H NMR 400 MHz DMSO d 6 7 96 s 1H 7 84 t J 8 0 Hz 2H 7 75 d J 2 0 Hz 1H 7 65 t J 7 9 Hz 1H 7 56 dd J 8 4 2 0 Hz 1H 6 90 d J 8 4 Hz 1H 6 82 d J 2 0 Hz 1H 6 47 d J 2 1 Hz 1H 5 31 s 2H 13 C NMR 100 MHz CD 3 COCD 3 176 6 165 2 157 6 148 3 147 4 145 7 139 3 137 0 132 9 132 6 131 8 130 7 123 7 121 5 121 4 119 1 116 2 115 8 113 5 105 1 98 9 93 7 69 9 LC MS ESI m z Found 418 20 M H Calcd for C 23 H 16 NO 7 418 09 2 3 4 Dihydroxyphenyl 3 5 dihydroxy 7 3 nitro benzyloxy 4H chromen 4 one 2n The desired product 2n was obtained as a yellow powder in 30 yield m p 223 225 o C dec 1 H NMR 500 MHz CD 3 COCD 3 8 41 s 1H 8 25 d J 7 0 Hz 1H 8 00 d J 7 5 Hz 1H 7 86 s 1H 7 76 t J 8 0 Hz 1H 7 72 d J 8 5 Hz 1H 7 00 d J 8 5 Hz 1H 6 86 s 1H 6 48 s 1H 5 48 s 2H 13 C NMR 100 MHz DMSO d 6 176 3 171 9 163 8 160 9 156 3 148 3 148 2 147 8 145 5 138 9 136 5 134 5 123 1 122 8 122 2 120 5 115 9 104 7 98 4 98 3 93 2 68 9 LC MS ESI m z Found 438 40 M H Calcd for C 22 H 16 NO 9 438 08 2 3 4 Dihydroxyphenyl 3 5 dihydroxy 7 3 methyl benzyloxy 4H chromen 4 one 2o The desired product 2o was obtained as a yellow powder in 22 yield m p 206 209 o C dec 1 H NMR 500 MHz CD 3 COCD 3 7 86 s 1H 7 74 s 1H 7 72 dd J 8 5 1 5 Hz 1H 7 31 d J 5 0 Hz 2H 7 19 t J 3 5 Hz 1H 7 01 d J 8 5 Hz 1H 6 80 d J 2 0 Hz 1H 6 41 d J 2 0 Hz 1H 5 24 s 2H 2 36 s 3H 13 C NMR 100 MHz DMSO d 6 176 3 164 3 160 8 156 4 148 2 147 7 145 5 138 1 136 5 136 4 128 9 128 7 125 3 125 2 122 2 120 6 120 2 116 0 104 5 98 3 93 1 70 4 21 5 LC MS ESI m z Found 407 40 M H Calcd for C 23 H 19 O 7 407 11 Antiviral Quercetin Derivatives 81 2 3 4 Dihydroxyphenyl 3 5 dihydroxy 7 3 meth oxybenzyloxy 4H chromen 4 one 2p The desired product 2p was obtained as a yellow powder in 22 yield m p 214 218 o C dec 1 H NMR 500 MHz CD 3 COCD 3 7 86 s 1H 7 72 d J 8 5 Hz 1H 7 34 t J 8 0 Hz 1H 7 09 d J 8 0 Hz 1H 7 00 d J 8 5 Hz 1H 6 93 d J 8 0 Hz 1H 6 80 d J 2 0 Hz 1H 6 42 d J 2 0 Hz 1H 5 26 s 2H 3 82 s 3H 13 C NMR 100 MHz DMSO d 6 176 3 164 2 160 8 159 8 156 3 148 2 147 7 145 5 138 1 136 4 130 2 129 9 122 2 120 5 120 3 116 0 113 8 113 6 104 5 98 4 93 2 70 1 55 5 LC MS ESI m z Found 423 30 M H Calcd for C 23 H 19 O 7 423 11 2 3 4 Dihydroxyphenyl 7 4 fluorobenzyloxy 3 5 dihydroxy 4H chromen 4 one 2q The desired product 2q was obtained as a yellow powder in 30 yield m p 212 215 o C dec 1 H NMR 500 MHz CD 3 COCD 3 7 86 s 1H 7 72 d J 8 0 Hz 1H 7 59 t J 8 0 Hz 2H 7 20 t J 8 5 Hz 2H 7 01 d J 8 5 Hz 1H 6 79 s 1H 6 41 s 1H 5 27 s 2H 13 C NMR 100 MHz DMSO d 6 176 4 164 2 161 4 160 9 156 5 148 3 147 8 145 6 136 5 132 9 130 7 130 6 122 3 120 5 116 0 115 8 104 6 98 5 93 2 69 7 LC MS ESI m z Found 411 40 M H Calcd for C 22 H 16 FO 7 411 09 7 4 Chlorobenzyloxy 2 3 4 dihydroxyphenyl 3 5 dihydroxy 4H chromen 4 one 2r The desired product 2r was obtained as a yellow powder in 25 yield m p 228 230 o C dec 1 H NMR 400 MHz DMSO d 6 7 72 d J 1 9 Hz 1H 7 56 dd J 8 4 1 9 Hz 1H 7 50 m 4H 6 90 d J 8 5 Hz 1H 6 80 d J 1 8 Hz 1H 6 44 d J 1 9 Hz 1H 5 25 s 2H 13 C NMR 100 MHz CD 3 COCD 3 177 1 165 7 162 1 157 9 148 9 148 0 146 3 136 7 134 7 130 6 129 8 123 9 121 8 116 4 116 1 105 5 100 6 99 2 94 0 70 0 LC MS ESI m z Found 427 20 M H Calcd for C 22 H 16 ClO 7 427 06 7 4 Bromobenzyloxy 2 3 4 dihydroxyphenyl 3 5 dihydroxy 4H chromen 4 one 2s The desired product 2s was obtained as a yellow powder in 22 yield m p 255 258 o C dec 1 H NMR 400 MHz CD 3 COCD 3 7 85 s 1H 7 71 d J 8 4 Hz 1H 7 62 d J 8 4 Hz 2H 7 50 d J 8 4 Hz 2H 7 00 d J 8 5 Hz 1H 6 79 d J 1 4 Hz 1H 6 41 d J 1 6 Hz 1H 5 27 s 2H 13 C NMR 100 MHz DMSO d 6 174 4 162 1 159 0 154 4 146 3 145 8 143 6 134 5 134 2 129 9 128 4 12 3 119 7 118 5 114 0 113 7 102 7 96 5 91 3 67 5 LC MS ESI m z Found 471 30 M H Calcd for C 22 H 16 BrO 7 471 01 2 3 4 Dihydroxyphenyl 3 5 dihydroxy 7 4 iodo benzyloxy 4H chromen 4 one 2t The desired product 2t was obtained as a yellow powder in 20 yield m p 259 262 o C dec 1 H NMR 400 MHz DMSO d 6 7 79 d J 8 2 Hz 2H 7 73 d J 2 0 Hz 1H 7 57 dd J 8 5 2 1 Hz 1H 7 29 d J 8 2 Hz 2H 6 90 d J 8 5 Hz 1H 6 78 d J 2 1 Hz 1H 5 21 s 2H 13 C NMR 100 MHz DMSO d 6 176 3 164 0 160 8 156 3 148 3 147 7 145 5 137 7 136 5 136 4 130 3 122 2 120 4 115 9 115 6 104 6 98 3 94 6 93 2 69 6 LC MS ESI m z Found 519 30 M H Calcd for C 22 H 16 IO 7 518 99 4 2 3 4 Dihydroxyphenyl 3 5 dihydroxy 4 oxo 4H chromen 7 yloxy methyl benzo nitrile 2u The desired product 2u was obtained as a yellow powder in 29 yield m p 212 215 o C dec 1 H NMR 500 MHz CD 3 COCD 3 7 86 d J 8 0 Hz 3H 7 76 d J 8 0 Hz 2H 7 71 d J 8 0 Hz 1H 7 01 d J 8 5 Hz 1H 6 82 s 1H 6 45 d J 1 5 Hz 1H 5 43 s 2H 13 C NMR 125 MHz CD 3 COCD 3 176 4 163 9 161 0 156 4 148 4 147 9 145 6 142 5 136 6 133 0 128 6 122 3 120 5 119 2 116 0 115 8 111 2 104 8 98 5 93 3 69 4 LC MS ESI m z Found 418 30 M H Calcd for C 23 H 16 NO 7 418 09 2 3 4 Dihydroxyphenyl 3 5 dihydroxy 7 4 nitro benzyloxy 4H chromen 4 one 2v The desired product 2v was obtained as a yellow powder in 24 yield m p 225 227 o C dec 1 H NMR 500 MHz CD 3 COCD 3 8 31 d J 8 5 Hz 2H 7 83 d J 8 5 Hz 3H 7 71 d J 8 5 Hz 1H 7 00 d J 8 5 Hz 1H 6 83 s 1H 6 46 s 1H 5 48 s 2H 13 C NMR 100 MHz DMSO d 6 174 5 161 9 159 0 154 5 146 5 146 0 145 7 143 6 142 6 134 6 126 8 122 2 120 3 118 5 114 1 113 8 102 9 96 1 91 4 67 1 LC MS ESI m z Found 438 40 M H Calcd for C 22 H 16 NO 9 438 08 2 3 4 Dihydroxyphenyl 3 5 dihydroxy 7 4 methyl benzyloxy 4H chromen 4 one 2w The desired product 2w was obtained as a yellow powder in 21 yield m p 262 264 o C dec 1 H NMR 500 MHz CD 3 COCD 3 7 86 d J 2 0 Hz 1H 7 72 dd J 8 5 2 5 Hz 1H 7 41 d J 7 5 Hz 2H 7 24 d J 7 5 Hz 2H 7 01 d J 8 5 Hz 1H 6 79 d J 2 0 Hz 1H 6 41 d J 2 0 Hz 1H 5 23 s 2H 2 35 s 3H 13 C NMR 100 MHz DMSO d 6 175 8 163 8 160 3 155 9 147 8 147 2 145 0 137 3 135 9 1331 129 0 127 8 121 7 119 9 115 5 115 2 104 0 97 9 92 7 69 8 20 7 LC MS ESI m z Found 423 30 M H Calcd for C 23 H 19 O 8 423 11 82 H R Park et al 2 3 4 Dihydroxyphenyl 3 5 dihydroxy 7 4 meth oxybenzyloxy 4H chromen 4 one 2x The desired product 2x was obtained as a yellow powder in 19 yield m p 217 219 o C dec 1 H NMR 500 MHz CD 3 COCD 3 7 86 s 1H 7 72 d J 8 0 Hz 1H 7 45 d J 8 5 Hz 2H 7 01 6 95 m 3H 6 78 d J 2 0 Hz 1H 6 39 d J 1 5 Hz 1H 5 19 s 2H 3 82 s 3H 13 C NMR 100 MHz DMSO d 6 176 3 164 3 160 7 156 4 156 3 147 7 145 4 136 4 130 9 129 3 128 4 122 3 122 2 121 2 116 7 115 1 113 5 110 0 104 4 56 2 54 8 LC MS ESI m z Found 407 30 M H Calcd for C 23 H 19 O 7 407 11 Anti SCV assay Carboxytetramethylrhodamine TAMRA modified 45 base oligomer and fluorescein modified 25 base oligomer were purchased from Integrated DNA Tech nologies 5 20T25Tam 5 TTTTTTTTTTTTTTTTTT TTGAGCGGATTACTATACTACATTAGA TAMRA 3 and 3 0T25Flu 5 Fluorescein TCTAATGTAGTA TAGTAATCCGCTC 3 The helicase substrate was pre pared by annealing the two oligomers which resulted in 25 base pairs of dsDNA with single stranded 20 dT of 5 overhang A 80 L solution of SCV helicase 150 nM in 20 mM HEPES pH 7 4 buffer was added to each well of the 96 well assay plate which already contained 1 L of various concentrations of chemical compounds After 5 min incubation at rt the FRET based dsDNA unwinding assay was started by addi tion of 20 L 5X reaction solution 5 mM MgCl 2 45 mM ATP 25 mM DTT and 100 nM dsDNA substrate in 20 mM HEPES pH 7 4 The reaction mixture was further incubated for 2 min at 37 o C and stopped with 100 L of termination solution 0 1 M EDTA and 0 4 M trap DNA unmodified 25 bases 3 0T25 oligomer in 20 mM HEPES pH 7 4 The sample was excited at 485 nm and the fluorescence was measured at 535 nm Anti HCV assay The human hepatoma cell line Huh 7 carrying the subgenomic HCV genotype 1 replicon with the luc ubi neo reporter selective fusion gene was kindly provid ed by Dr Ralf Bartenschlager University of Heidelberg Huh 5 2 cells were seeded at a density of 5 10 3 per well in a tissue culture treated white 96 well view plate in complete DMEM supplemented with 500 g mL G418 After incubation for 24 h at 37 o C 5 CO 2 medium was refreshed with G418 and DMSO stock of test compounds were added After 4 days of incuba tion at 37 o C cell culture medium was removed and luciferase activity was determined using the Steady Glo luciferase assay system Promega Cytostatic effect Huh 5 2 cells were seeded at a density of 5 10 3 per well of 96 well plate in complete DMEM with the appropriate concentrations of G418 Serial dilutions of the test compounds in complete DMEM without G418 were added 24 h after seeding Cells were allowed to proliferate for 3 days at 37 o C after which the cell number was determined by WST 1 assay RESULTS AND DISCUSSION Chemistry Regioselective alkylation of quercetin requires selec tive protection of the five phenolic hydroxyl groups For preparation of the 7 O alkylated quercetin deri vatives we adapted a general synthetic protocol which includes peracetylation followed by regioselective deacetylation Scheme 1 Thus acetylation of quercetin 1 with acetic anhy dride Ac 2 O in pyridine afforded the peracetylated quercetin derivative 4 which underwent regioselec tive deacethylation with PhSH in NMP Li et al 2003 Sabui and Venkateswaran 2003 Lee C et al 2009a Lee HS et al 2010 to provide the corresponding quercetin 3 5 3 4 tetraacetates 5 in 74 yield Alkyla tion of 5 with variously substituted benzyl bromides followed by deacetylation by treatment with methanolic ammonia afforded a series of 7 O arylmethylquerce tins 2a 2x in moderate yields Biological activity All synthesized quercetin arylmethyl ethers 2a 2x were evaluated for their inhibitory activity against both NTPase and helicase activity of the SCV NTPase helicase Lee C et al 2009a 2009b Lee HS et al 2010 which was summarized in Table I as IC 50 values The anti HCV activity of the 7 O arylmethylquercetin deri vatives in the human hepatoma cell line Huh 7 carry ing the subgenomic HCV genotype 1 replicon with the luc ubi neo fusion gene Lohmann et al 1999 Vroljk