New arylthioindoles and related bioisosteres at the sulfur bridging group. 4. Synthesis, tubulin polymerization, cell growth inhibition, and molecular modeling studies.

Chemistry MW-assisted reactions were performed on Discover LabMate (CEM), setting temperature, irradiation power, maximum pressure (Pmax), PowerMAX (in situ cooling during the MW irradiation), ramp and hold times, and open and closed vessel modes as indicated. Melting points (mp) were determined on a Buchi 510 apparatus and are uncorrected. Infrared spectra (IR) were run on a SpectrumOne FT-ATR spectrophotometer. Band position and absorption ranges are given in cm −1 . Proton nuclear magnetic resonance ( 1 H NMR) spectra were recorded on Bruker 200 and 400 MHz FT spectrometers in the indicated solvent. Chemical shifts are expressed in δ units (ppm) from tetramethylsilane. Column chromatography was performed on columns packed with alumina from Merck (70–230 mesh) or silica gel from Macherey-Nagel (70–230 mesh). Aluminum oxide TLC cards from Fluka (aluminum oxide precoated aluminum cards with fluorescent indicator visualizable at 254 nm) and silica gel TLC cards from Macherey-Nagel (silica gel pre-coated aluminum cards with fluorescent indicator visualizable at 254 nm) were used for thin layer chromatography (TLC). Developed plates were visualized by a Spectroline ENF 260C/F UV apparatus. Organic solutions were dried over anhydrous sodium sulfate. Evaporation of the solvents was carried out on a Buchi Rotavapor R-210 equipped with a Buchi V-850 vacuum controller and Buchi V-700 and V-710 vacuum pumps. Elemental analyses of the biologically tested compounds were found within ±0.4% of the theoretical values. Purity of tested compounds was >95%. Compounds 3 , 12 6 , 10 9 , 10 12 , 12 15 , 10 18 , 11 24 , 12 33 , 12 36 , 10 and 42 11 were prepared as previously reported. General Procedure for the Synthesis of Compounds 4, 7, 10, 13, 16, 19, 21, 25, 28, 31, 34, 39, and 43. Example: (1 H -Indol-3-yl)-(3,4,5-trimethoxyphenyl)methanone (4) A mixture of AlCl 3 (0.57 g; 0.0043 mol), the appropriate 1 H -indole (0.5 g, 0.0043 mol) and 3,4,5-trimethoxybenzoyl chloride (0.99 g, 0.0043 mol) in 1,2-dichloroethane (2 mL) was placed into the MW cavity (closed vessel mode, P max = 250 PSI). MW irradiation of 150 W was used, the temperature being ramped from 25 to 110 °C while stirring. Once 110 °C was reached, taking about 1 min, the reaction mixture was held at this temperature for 2 min, then cooled, diluted cautiously with water, and extracted with chloroform; the organic layer was washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by silica gel column chromatography (ethyl acetate: n -hexane = 1:1 as eluent) to furnish 4 as a white solid (0.71 g, 53%), mp 210–213 °C (from ethanol), differing from lit. 22 174–176 °C and lit. 23 132–133 °C. 1 H NMR (DMSO- d 6 ): δ 3.73 (s, 3H), 3.83 (s, 6H), 7.06 (s, 2H), 7.20–7.24 (m, 2H), 7.48 (d, J = 9.1 Hz, 1H), 8.06 (d, J = 3.0 Hz, 1H), 8.21 (d, J = 8.5 Hz, 1H), 11.99 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1571, 3179 cm −1 . Methyl 3-(3,4,5-trimethoxybenzoyl)-1 H -indole-2-carboxylate (7) Synthesized as 4 , starting from methyl 1 H -indole-2-carboxylate, yield 55%, yellow solid, mp 163–168 °C (from ethanol/water). 1 H NMR (DMSO- d 6 ): δ 3.52 (s, 3H), 3.73 (s, 6H), 3.74 (s, 3H), 7.03 (s, 2H), 7.17 (t, J = 7.2 Hz, 1H), 7.34 (t, J = 7.2 Hz, 1H), 7.54 (d, J = 7.5 Hz, 1H), 7.60 (d, J =7.3 Hz, 1H), 12.56 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1634, 1710, 3279 cm −1 . Anal. (C 20 H 19 NO 6 (369.37)) C, H, N. Ethyl 3-(3,4,5-Trimethoxybenzoyl)-1 H -indole-2-carboxylate (10) Synthesized as 4 , starting from ethyl 5-methoxy-1 H -indole-2-carboxylate, yield 39%, white solid, mp 105–110 °C (ethanol/ n -hexane). 1 H NMR (CDCl 3 ): δ 1.01 (t, J = 7.1 Hz, 3H), 3.82 (s, 6H), 3.93 (s, 3H), 4.14 (q, J = 7.1 Hz, 2H), 7.18 (s, 2H), 7.24 (t, J = 7.1 Hz, 1H), 7.41 (t, J = 7.1 Hz, 1H), 7.5 (d, J = 8.3 Hz, 1H), 7.73 (d, J = 8.1 Hz, 1H), 9.26 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1648, 1686, 3303 cm −1 . Anal. (C 21 H 21 NO 6 (383.39)) C, H, N. (5-Chloro-1 H -indol-3-yl)-(3,4,5-trimethoxyphenyl)methanone (13) Synthesized as 4 , starting from 5-chloro-1 H -indole, yield 68%, white solid, mp 240–245 °C (from ethanol). 1 H NMR (DMSO- d 6 ): δ 3.76 (s, 3H), 3.86 (s, 6H), 7.09 (s, 2H), 7.28 (dd, J = 8.7 Hz, 1H), 7.54 (t, J = 8.1 Hz, 1H), 8.19 (s, 1H), 8.23 (d, J = 2.2 Hz, 1H), 12.21 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1600, 3261 cm −1 . Anal. (C 18 H 16 ClNO 4 (345.78)) C, H, Cl, N. Methyl 5-Chloro-3-(3,4,5-trimethoxybenzoyl)-1 H -indole-2-carboxylate (16) Synthesized as 4 , starting from methyl 5-chloro-1 H -indole-2-carboxylate, yield 68%, yellow solid, mp 188–193 °C (from ethanol/water). 1 H NMR (CDCl 3 ): δ 3.69 (s, 3H), 3.84 (s, 6H), 3.96 (s, 3H), 7.16 (s, 2H), 7.36 (dd, J = 8.8 Hz, 1H), 7.44 (t, J = 8.8 Hz, 1H), 7.66 (d, J = 1.9 Hz, 1H), 9.50 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1645, 1711, 3259 cm −1 . Anal. (C 20 H 18 ClNO 6 (403.81)) C, H, Cl, N. Ethyl 5-Chloro-3-(3,4,5-trimethoxybenzoyl)-1 H -indole-2-carboxylate (19) Synthesized as 4 , starting from ethyl 5-chloro-1 H -indole-2-carboxylate, yield 38%, yellow solid, mp 160−163 °C (from ethanol). 1 H NMR (DMSO- d 6 ): δ 0.86 (t, J = 7.1 Hz, 3H), 3.73 (s, 3H), 3.75 (s, 6H), 3.96 (q, J = 7.1 Hz, 2H), 7.04 (s, 2H), 7.38 (dd, J = 10.1 Hz, 1H), 7.58 (d, J = 7.4 Hz 1H), 7.69 (d, J = 2.1 Hz, 1H), 12.78 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1636, 1684, 3294 cm −1 . Anal. (C 21 H 20 ClNO 6 (417.84)) C, H, Cl, N. Ethyl 5-Chloro-3-(2-(3,4,5-trimethoxyphenyl)acetyl)-1 H -indole-2-carboxylate (21) Synthesized as 4 , starting from ethyl 5-chloro-1 H -indole-2-carboxylate and 2-(3,4,5-trimethoxyphenyl)acetyl chloride, yield 32%, yellow solid, mp 129–134 °C (from ethanol). 1 H NMR (CDCl 3 ): δ 1.46 (t, J = 7.1 Hz, 3H), 3.80 (s, 6H), 3.83 (s, 3H), 4.40 (s, 2H), 4.50 (q, J = 7.1 Hz, 2H), 6.46 (s, 2H), 7.32 (dd, J = 8.8 Hz, 1H), 7.33 (dd, J = 9.5 Hz, 1H), 7.96 (d, J = 1.8 Hz, 1H), 9.22 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1641, 1721, 3169 cm −1 . Anal. (C 22 H 22 ClNO 6 (431.87)) C, H, Cl, N. (5-Bromo-1 H -indol-3-yl)(3,4,5-trimethoxyphenyl)methanone (25) Synthesized as 4 , starting from 5-bromo-1 H -indole, yield 53%, yellow solid, mp 235–240 °C (from ethanol). 1 H NMR (DMSO- d 6 ): δ 3.76 (s, 3H), 3.86 (s, 6H), 7.09 (s, 2H), 7.40 (dd, J = 6.6 Hz, 1H), 7.50 (d, J = 8.1 Hz, 1H), 8.18 (s, 1H), 8.39 (d, J = 2.5 Hz, 1H), 12.19 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1599, 3279 cm −1 . Anal. (C 18 H 16 BrNO 4 (390.23)) C, H, Br, N. Methyl 5-Bromo-3-(3,4,5-trimethoxybenzoyl)-1 H -indole-2-carboxylate (28) Synthesized as 4 , starting from 49 , yield 40%, white solid, mp 207–210 °C (from ethanol/ n -hexane). 1 H NMR (CDCl 3 ): δ 3.68 (s, 3H), 3.83 (s, 6H), 3.95 (s, 3H), 7.14 (s, 2H), 7.38 (d, J = 8.2 Hz, 1H), 7.48 (dd, J = 6.9 Hz, 1H), 7.81 (d, J = 1.8 Hz, 1H), 9.45 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1642, 1711, 3248 cm −1 . Anal. (C 20 H 18 BrNO 6 (448.26)) C, H, Br, N. Ethyl 5-Bromo-3-(3,4,5-trimethoxybenzoyl)-1 H -indole-2-carboxylate (31) Synthesized as 4 , starting from 48 , yield 46%, yellow solid, mp 135–140 °C (from ethanol/ n -hexane). 1 H NMR (DMSO- d 6 ): δ 0.85 (t, J = 7.1 Hz, 3H), 3.73 (s, 3H), 3.75 (s, 6H), 3.98 (q, J = 7.1 Hz, 2H), 7.03 (s, 2H), 7.33 (d, J = 1.2 Hz, 1H), 7.48 (dd, J = 6.9 Hz, 1H), 7.53 (dd, J = 8.8 Hz, 1H), 12.74 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1638, 1702, 3292 cm −1 . Anal. (C 21 H 20 BrNO 6 (462.29)) C, H, Br, N. (5-Methoxy-1 H -indol-3-yl)-(3,4,5-trimethoxyphenyl)methanone (34) Synthesized as 4 , starting from 5-methoxy-1 H -indole, yield 42%, yellow solid, mp 202–207 °C (from ethanol), lit. 14 194–195 °C. Methyl 5-Methoxy-3-(3,4,5-trimethoxybenzoyl)-1 H -indole-2-carboxylate (39) Synthesized as 4 , starting from methyl 5-methoxy-1 H -indole-2-carboxylate, yield 20%, yellow solid, mp 168–173 °C (from ethanol/water). 1 H NMR (DMSO- d 6 ): δ 3.47 (s, 3H), 3.71 (s, 3H), 3.73 (s, 3H), 3.74 (s, 6H), 6.99–7.02 (m, 3H), 7.07 (d, J = 2.3 Hz, 1H), 7.44 (d, J = 8.95 Hz, 1H), 12.49 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1635, 1710, 3274 cm −1 . Anal. (C 21 H 21 NO 7 (399.39)) C, H, N. Ethyl 5-Methoxy-3-(3,4,5-trimethoxybenzoyl)-1 H -indole-2-carboxylate (43) Synthesized as 4 , starting from ethyl 5-methoxy-1 H -indole-2-carboxylate, yield 60%, yellow solid, mp 150–155 °C (from ethanol). 1 H NMR (CDCl 3 ): δ 0.97 (t, J = 7.1 Hz, 3H), 3.82 (s, 3H), 3.84 (s, 6H), 3.94 (s, 3H), 4.09 (q, J = 7.1 Hz, 2H), 7.07 (dd, J = 6.5 Hz, 1H), 7.18 (s, 2H), 7.26 (d, J = 1.9 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 9.21 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1647, 1694, 3353 cm −1 . Anal. (C 22 H 23 NO 7 (413.42)) C, H, N. General Procedure for the Synthesis of Compounds 5, 14, 26, and 35. Example: 3-(3,4,5-trimethoxybenzyl)-1 H -indole (5) Sodium borohydride (0.80 g, 0.021 mol) was added to a solution of 4 (0.66 g, 0.0021 mol) in ethanol (85 mL). The reaction mixture was refluxed for 3 h, then cooled, cautiously diluted with water, and extracted with ethyl acetate; the organic layer was washed with brine, dried, and filtered. Evaporation of the solvent gave a residue that was purified by silica gel column chromatography (ethyl acetate: n -hexane = 1:1 as eluent) to furnish 5 as a white solid (0.26 g, 42%), mp 133–136 °C (from ethanol), lit. 22 125–126 °C and lit. 24 128–130 °C. 5-Chloro-3-(3,4,5-trimethoxybenzyl)-1 H -indole (14) Synthesized as 5 starting from 13 , yield 47%, orange solid, mp 145–150 °C (from ethanol). 1 H NMR (CDCl 3 ): δ 3.80 (s, 6H), 3.83 (s, 3H), 4.00 (s, 2H), 6.49 (s, 2H), 6.95 (d, J = 2.3 Hz, 1H), 7.14 (dd, J = 8.6 Hz, 1H), 7.28 (d, J = 8.1 Hz, 1H), 7.51 (d, J = 2.0 Hz, 1H), 8.40 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 3372 cm −1 . Anal. (C 18 H 18 ClNO 3 (331.79)) C, H, Cl, N. 5-Bromo-3-(3,4,5-trimethoxybenzyl)-1 H -indole (26) Synthesized as 5 , starting from 25 , yield 58%, white solid, mp 145–150 °C (from ethanol). 1 H NMR (CDCl 3 ): 3.81 (s, 6H), 3.84 (s, 3H), 4.01 (s, 2H), 6.50 (s, 2H), 6.94 (d, J = 2.3 Hz, 1H), 7.24–7.28 (m, 2H), 7.69 (d, J = 1.7 Hz, 1H), 8.05 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 3353 cm −1 . Anal. (C 18 H 18 BrNO 3 )) C, H, Br, N. 5-Methoxy-3-(3,4,5-trimethoxybenzyl)-1 H -indole (35) Synthesized as 5 , starting from 34 , yield 68%, white solid, mp 110–113 °C (from ethanol). 1 H NMR (CDCl 3 ): δ 3.78 (s, 6H), 3.80 (s, 3H), 3.81 (s, 3H), 4.01 (s, 2H), 6.51 (s, 2H), 6.84 (d, J = 8.8 Hz, 1H), 6.88 (t, J = 2.4 Hz, 1H), 6.97 (d, J = 2.4 Hz, 1H), 7.24 (t, J = 4.7 Hz, 1H), 7.90 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 3365 cm −1 . Anal. (C 19 H 21 NO 4 (327.37)) C, H, N. General Procedure for the Synthesis of Compounds 8, 11, 17, 20, 23, 29, 32, 41, and 45. Example: Methyl 3-(3,4,5-trimethoxybenzyl)-1 H -indole-2-carboxylate (8) To a cold solution of 7 (0.31 g, 0.00084 mol) in trifluoroacetic acid (0.96 g, 0.65 mL, 0.0084 mol) was added dropwise triethylsilane (0.22 g, 0.30 mL, 0.0019 mol). The reaction mixture was stirred at 25 °C for 24 h, then neutralized with a saturated solution of sodium hydrogen carbonate and extracted with ethyl acetate; the organic layer was washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by silica gel column chromatography (ethyl acetate: n -hexane = 3:2 as eluent) to furnish 8 as a yellow solid (0.15 g, 50%), mp 156–161 °C (from ethanol/ n -hexane). 1 H NMR (CDCl 3 ): δ 3.69 (s, 6H), 3.72 (s, 3H), 3.89 (s, 3H), 4.38 (s, 2H), 6.46 (s, 2H), 7.05 (t, J = 8.1 Hz, 1H), 7.25 (t, J = 8.3 Hz, 1H), 7.33 (d, J = 7.4 Hz, 1H), 7.57 (d, J = 8.2 Hz, 1H), 8.75 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1690, 3318 cm −1 . Anal. (C 20 H 21 NO 5 (355.38)) C, H, N. Ethyl 3-(3,4,5-Trimethoxybenzyl)-1 H -indole-2-carboxylate (11) Synthesized as 8 , starting from 10 , yield 73%, white solid, mp 115–118 °C (from ethanol/ n -hexane). 1 H NMR (CDCl 3 ): δ 1.42 (t, J = 7.1 Hz, 3H), 3.77 (s, 6H), 3.80 (s, 3H), 4.44 (q, J = 7.1 Hz, 2H), 4.47 (s, 2H), 6.55 (s, 2H), 7.13 (t, J = 7.0 Hz, 1H), 7.33 (t, J = 6.9 Hz, 1H), 7.39 (d, J = 7.5 Hz, 1H), 7.64 (d, J = 7.4 Hz, 1H), 8.82 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1672, 3337 cm −1 . Anal. (C 21 H 23 NO 5 ) (369.41)) C, H, N. Methyl 5-Chloro-3-(3,4,5-trimethoxybenzyl)-1 H -indole-2-carboxylate (17) Synthesized as 8 , starting from 16 , yield 69%, white solid, mp 172–175 °C (from ethanol/ n -hexane). 1 H NMR (CDCl 3 ): δ 3.70 (s, 6H), 3.73 (s, 3H), 3.89 (s, 3H), 4.33 (s, 2H), 6.42 (s, 2H), 7.21 (d, J = 1.9 Hz, 1H), 7.24 (t, J = 8.7 Hz, 1H), 7.51 (d, J = 1.9 Hz, 1H), 8.74 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1682, 3322 cm −1 . Anal. (C 20 H 20 ClNO 5 (389.83)) C, H, Cl, N. Ethyl 5-Chloro-3-(3,4,5-trimethoxybenzyl)-1 H -indole-2-carboxylate (20) Synthesized as 8 , starting from 19 , yield 95%, yellow solid, mp 147–150 °C (from ethanol/ n -hexane). 1 H NMR (CDCl 3 ): δ 1.41 (t, J = 7.1 Hz, 3H), 3.79 (s, 6H), 3.81 (s, 3H), 4.41 (s, 2H), 4.43 (q, J = 6.4 Hz, 2H), 6.51 (s, 2H), 7.25–7.28 (m, 1H), 7.33 (dd, J = 8.1 Hz, 1H), 7.59–7.60 (m, 1H), 8.91 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1666, 3315 cm −1 . Anal. (C 21 H 22 ClNO 5 )) C, H, Cl, N. Ethyl 5-Chloro-3-(3,4,5-trimethoxyphenethyl)-1 H -indole-2-carboxylate (23) Synthesized as 8 , startingfrom 22 , yield 40%, white solid, mp 163–165 °C (from ethanol). 1 H NMR (CDCl 3 ): δ 1.44 (t, J = 7.5 Hz, 3H), 2.88 (t, J = 7.4 Hz, 2H), 3.35 (t, J = 7.3 Hz, 2H), 3.79 (s, 6H), 3.83 (s, 3H), 4.42 (t, J = 7.1 Hz, 2H), 6.38 (s, 2H), 7.24 (dd, J = 9.2 Hz, 1H), 7.31 (d, J = 8.8 Hz, 1H), 7.49 (d, J = 1.9 Hz, 1H), 9.01 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1669, 3318 cm −1 . Anal. (C 22 H 24 ClNO 5 (417.88)) C, H, Cl, N. Methyl 5-Bromo-3-(3,4,5-trimethoxybenzyl)-1 H -indole-2-carboxylate (29) Synthesized as 8 , starting from 28 , yield 97%, yellow solid, mp 177-182 °C (from ethanol/ n -hexane). 1 H NMR (DMSO- d 6 ): δ 3.57 (s, 3H), 3.67 (s, 6H), 3.92 (s, 3H), 4.34 (s, 2H), 6.62 (s, 2H), 7.36–7.36 (m, 2H), 7.90 (d, J = 2.3 Hz, 1H), 11.88 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1675, 3322 cm −1 . Anal. (C 20 H 20 BrNO 5 (434.28)) C, H, Br, N. Ethyl 5-Bromo-3-(3,4,5-trimethoxybenzyl)-1 H -indole-2-carboxylate (32) Synthesized as 8 , starting from 31 , yield 46%, white solid, mp 150–155 °C (from ethanol/ n -hexane). 1 H NMR (CDCl 3 ): δ 1.41 (t, J = 7.1 Hz, 3H), 3.79 (s, 6H), 3.82 (s, 3H), 4.43 (q, J = 7.1 Hz, 2H), 6.51 (s, 2H), 7.28 (d, J = 9.4 Hz, 1H), 7.29 (s, 2H), 7.40 (dd, J = 6.9 Hz, 1H), 7.77 (d, J = 1.8 Hz, 1H), 8.90 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1665, 3317 cm −1 . Anal. (C 21 H 22 BrNO 6 (448.31)) C, H, Br, N. Methyl 5-Methoxy-3-(3,4,5-trimethoxybenzyl)-1 H -indole-2-carboxylate (41) Synthesized as 8 , starting from 39 , yield 58%, white solid, mp 125–128 °C (from ethanol/ n -hexane). 1 H NMR (CDCl 3 ): δ 3.77 (s, 6H), 3.79 (s, 3H), 3.80 (s, 3H), 3.94 (s, 3H), 4.42 (s, 2H), 6.53 (s, 1H), 6.96 (d, J = 2.3 Hz, 1H), 7.00 (dd, J = 6.4 Hz, 1H), 7.28 (d, J = 8.9 Hz, 1H), 8.67 (s, 1H), 11.91 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1687, 3331 cm −1 . Anal. (C 21 H 23 NO 6 (385.41)) C, H, N. Ethyl 5-Methoxy-3-(3,4,5-trimethoxybenzyl)-1 H -indole-2-carboxylate (45) Synthesized as 8 , starting from 43 , yield 90%, white solid, mp 132–135 °C (from ethanol). 1 H NMR (CDCl 3 ): δ 1.42 (t, J = 7.1 Hz, 3H), 3.78 (s, 9H), 3.81 (s, 3H), 4.44 (q, J = 7.2 Hz, 2H), 4.45 (s, 2H), 6.56 (s, 2H), 6.98–7.03 (m, 2H), 7.28–7.32 (m, 1H), 8.76 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1672, 3330 cm −1 . Anal. (C 22 H 25 NO 6 (399.44)) C, H, N. General Procedure for the Synthesis of Compounds 40 and 44. Example: Methyl 3-(Hydroxy(3,4,5-trimethoxyphenyl)methyl)-5-methoxy-1 H -indole-2-carboxylate (40) A mixture of 39 (0.34 g, 0.00085 mol) and sodium borohydride (0.03 g, 0.00085 mol) in THF (2.1 mL) and water (0.12 mL) was refluxed for 2 h. After cooling, water and ethyl acetate were added. The organic layer was removed and washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by silica gel column chromatography (ethyl acetate: n -hexane=1:1 as eluent) to furnish 40 as a brown solid (0.1 g, 29%), mp 100–103 °C (from ethanol). 1 H NMR (CDCl 3 ): δ 3.76 (s, 3H), 3.79 (s, 6H), 3.82 (s, 3H), 3.97 (s, 3H), 4.53 (d, J = 1.2 Hz, 1H), 6.50 (d, J = 7.4 Hz, 1H), 6.77 (s, 2H), 6.95 (d, J = 2.4 Hz, 1H), 7.02 (dd, J = 6.5 Hz, 1H), 7.28 (d, J = 8.4 Hz, 1H), 8.78 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1707, 2937, 3322 cm −1 . Anal. (C 21 H 23 NO 7 (401.41)) C, H, N. Ethyl 3-(Hydroxy(3,4,5-trimethoxyphenyl)methyl)-5-methoxy-1 H -indole-2-carboxylate (44) Synthesized as 40 , starting from 43 , yield 53%, white solid, mp 115–120 °C(from ethanol). 1 H NMR (CDCl 3 ): δ 1.42 (t, J = 7.2 Hz, 3H), 3.76 (s, 3H), 3.79 (s, 3H), 3.81 (s, 3H), 3.83 (s, 3H), 4.43 (q, J = 6.0 Hz, 2H), 4.44 (d, J = 1.2 Hz, 1H), 6.49 (d, J = 7.2 Hz, 1H), 6.77 (s, 2H), 6.95 (d, J = 2.3 Hz, 1H), 7.01 (dd, J = 6.6 Hz, 1H), 7.29 (d, J = 7.3 Hz, 1H), 8.78 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1676, 3209, 3402 cm −1 .Anal. (C 22 H 25 NO 7 (415.44)) C, H, N. Ethyl 5-Chloro-3-(2-oxo-2-(3,4,5-trimethoxyphenyl)acetyl)-1 H -indole-2-carboxylate (22) A mixture of 21 (0.05 g, 0.00012 mol) and selenium(IV) oxide (0.05 g; 0.00045 mol) in DMSO (2 mL) was placed into the MW cavity (closed vessel mode, P max = 250 PSI). MW irradiation of 150 W was used, the temperature being ramped from 25 to 150 °C. Once 150 °C was reached, taking about 1 min, the reaction mixture was held at this temperature for 2 min, while stirring, then cooled, diluted with water, and extracted with ethyl acetate; organic layer was washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by silica gel column chromatography (ethyl acetate: n -hexane=1:2 as eluent) to furnish 22 as a yellow solid (0.03 g, 56%), mp 177–181 °C (from ethanol). 1 H NMR (CDCl 3 ): δ 1.07(t, J = 7.2 Hz, 3H), 3.93 (s, 6H), 3.98 (s, 3H), 4.12 (q, J = 7.1 Hz, 2H), 7.35 (s, 2H), 7.42–7.43 (m, 2H), 8.40 (s, 1H), 9.50 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1650, 1672, 1724, 3275 cm −1 . Anal. (C 22 H 20 ClNO 7 (445.85)). Methyl 5-Bromo-3-(3,4,5-trimethoxyphenylthio)-1 H -indole-2-carboxylate (27) A mixture of 59 (0.25 g, 0.001 mol), 3,4,5-bis(3,4,5-trimethoxyphenyl)disulfide 10 (0.55 g, 0.0014 mol), and sodium hydride (0.071 g, 0.003 mol, 60% in mineral oil) in anhydrous DMF (2 mL) was placed into the MW cavity (closed vessel mode, P max = 250 PSI). MW irradiation of 150 W was used, the temperature being ramped from 25 to 110 °C. Once 110 °C was reached, taking about 1 min, the reaction mixture was held at this temperature for 2 min, while stirring, then cooled and quenched on crushed ice and extracted with ethyl acetate. The organic layer was washed with brine, dried, and filtered. Removal of the solvent gave crude 5-bromo-3-(3,4,5-trimethoxyphenylthio)-1 H -indole-2-carboxylic acid, which was used without further purification. The crude acid was dissolved in methanol (2.5 mL) and dichloromethane (10 mL) and treated with TMSDM (0.78 mL, 0.0015 mol, 2.0 M in hexane) while stirring at 25 °C for 30 min. Removal of the solvent gave a crude product that was purified by silica gel column chromatography (ethyl acetate: n -hexane=1:1) to furnish 27 as a white solid (0.36 g, overall yield 79%), mp 160–162 °C (from ethanol). 1 H NMR (CDCl 3 ): δ 3.72 (s, 6H), 3.81 (s, 3H), 3.98 (s, 3H), 6.48 (s, 2H), 7.33 (d, J = 9.3 Hz, 1H), 7.42 (dd, J = 1.9 and 9.3 Hz, 1H), 7.72 (d, J = 1.9 Hz, 1H), 9.20 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1680, 3296 cm −1 . Anal. (C 19 H 18 BrNO 5 S(452.32)) Ethyl 5-Bromo-3-(3,4,5-trimethoxyphenylthio)-1 H -indole-2-carboxylate (30) Synthesized as 27 , starting from 59 and 3,4,5-bis(3,4,5-trimethoxyphenyl)disulfide. 10 The crude acid (0.5 g) was dissolved in absolute ethanol (1.7 mL) and treated with thionyl chloride (0.13 mL) at 0 °C under an Ar stream. The reaction mixture was stirred at 0 °C for 20 min, heated at 65 °C for 2 h, then cooled and diluted with water and ethyl acetate. The organic layer was removed and washed with a saturated solution of sodium hydrogen carbonate and brine, dried, and filtered. Removal of the solvent gave a crude product that was purified by silica gel column chromatography (ethyl acetate: n -hexane = 1:1) to give 30 as a yellow solid (0.33 g, overall yield 70%), mp 150–152 °C (from ethanol). 1 H NMR (CDCl 3 ): δ 1.37 (t, J = 7.1 Hz, 3H), 3.72 (s, 6H), 3.80 (s, 3H), 4.43 (q, J = 7.8 Hz, 2H), 6.48 (s, 2H), 7.33 (d, J = 8.7 Hz, 1H), 7.43–7.46 (m, 1H) 7.74–7.75 (m, 1H), 9.23 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1672, 3299 cm −1 . Anal. (C 20 H 20 BrNO 5 S (452.32)). Methyl 5-Methoxy-3-(3,4,5-trimethoxyphenylsulfinyl)-1 H -indole-2-carboxylate (37) To a cold solution of 36 10 (0.05 g, 0.00012 mol) in chloroform (5 mL) was added 3-chloroperoxybenzoic acid (0.021 g, 0.00012 mol). The reaction mixture was stirred at 25 °C for 1.5 h, then diluted with water and extracted with chloroform. The organic layer was washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by silica gel column chromatography (ethyl acetate as eluent) to furnish 37 as a brown solid (0.05 g, yield 99%), mp 161–163 °C (from ethanol). 1 H NMR (CDCl 3 ): δ 3.74 (s, 3H), 3.84 (s, 9H), 3.85 (s, 3H), 6.96 (d, J = 9.1 Hz, 1H), 7.10 (s, 2H), 7.31 (dd, J = 1.9 and 9.0 Hz, 1H), 7.42 (d, J = 2.2 Hz, 1H), 9.37 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1708, 2831, 2933 cm −1 . Anal. (C 20 H 21 NO 7 S (419.45)) C, H, N, S. Methyl 5-Methoxy-3-(3,4,5-trimethoxyphenylsulfonyl)-1 H -indole-2-carboxylate (38) Was synthesized as 37 , treating 36 10 with 3-chloroperoxybenzoic acid (2 equiv), 96%, yellow solid, mp 176–180 °C (from ethanol). 1 H NMR (DMSO- d 6 ): δ 3.33 (s, 3H), 3.82 (s, 6H), 3.83 (s, 3H), 3.93 (s, 3H), 7.04 (dd, J = 6.5 Hz, 1H), 7.40 (s, 2H), 7.45 (d, J = 9.0 Hz, 1H), 7.59 (d, J = 2.4 Hz, 1H), 12.99 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1701, 3298 cm −1 . Anal. (C 20 H 21 NO 8 S (435.45)) C, H, N, S. Ethyl 1-(3,4,5-Trimethoxybenzyl)-1 H -pyrrole-2-carboxylate (46) 3,4,5-Trimethoxybenzyl chloride 15 (1.083 g, 0.005 mol) was added to a well stirred mixture of ethyl 1 H -pyrrole-2-carboxylate (0.63 g, 0.0045 mol), tetrabutylammonium hydrogen sulfate (1.53 g, 0.0045 mol), a 50% potassium hydroxide aqueous solution (15 mL) and dichloromethane (22 mL). The reaction mixture was stirred at 25 °C overnight, neutralized with 6 N hydrochloric acid and extracted with chloroform. The organic layer was washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by silica gel column chromatography (dichloromethane as eluent) to furnish 46 as a yellow oil (0.64 g, 45%). 1 H NMR (CDCl 3 ): δ 1.21 (t, J = 7.1 Hz, 3H), 3.68 (s, 6H), 3.71 (s, 3H), 4.15 (q, J = 7.1 Hz, 2H), 5.39 (s, 2H), 6.07 (dd, J = 3.9 and 2.6 Hz, 1H), 6.24 (s, 2H), 6.78 (t, J = 2.1 Hz, 1H), 6.91 ppm (dd, J = 3.9 and 1.8 Hz, 1H). IR: ν 1701 cm −1 . Anal. (C 17 H 21 NO 5 (319.35)) C, H, N. Ethyl 3,5-Dimethyl-1-(3,4,5-trimethoxybenzyl)-1 H -pyrrole-2-carboxylate (47) Was synthesized as 46 , starting from ethyl 3,5-dimethyl-1 H -pyrrole-2-carboxylate, yield 56%, pink solid, mp 68–70 °C (from toluene). 1 H NMR (CDCl 3 ): δ 1.16 (t, J = 7.1 Hz, 3H), 2.02 (s, 3H), 2.22 (s, 3H), 3.61 (s, 6H), 3.66 (s, 3H), 4.10 (q, J = 7.1 Hz, 2H), 5.37 (s, 2H), 5.71 (s, 1H), 6.03 ppm (s, 2H). IR: ν 1703 cm −1 . Anal. (C 19 H 25 NO 5 (347.41)) C, H, N. Ethyl 3,4,5-Trimethyl-1-(3,4,5-trimethoxybenzyl)-1 H -pyrrole-2-carboxylate (48) Was synthesized as 46 , starting from ethyl 3,4,5-trimethyl-1 H -pyrrole-2-carboxylate, yield 45%, yellow solid, mp 82–84 °C (from ethanol). 1 H NMR (CDCl 3 ): δ 0.36 (t, J = 7.1 Hz, 3H), 1.03 (s, 3H), 1.17 (s, 3H), 1.36 (s, 3H), 2.81 (s, 6H), 2.86 (s, 3H), 3.30 (q, J = 7.1 Hz, 2H), 4.56 (s, 2H), 5.22 ppm (s, 2H). IR: ν 1710 cm −1 . Anal. (C 20 H 27 NO 5 (361.44)) C, H, N. Ethyl 3,4-Diethyl-5-methyl-1-(3,4,5-trimethoxybenzyl)-1 H -pyr-role-2-carboxylate (49) Was synthesized as 46 , starting from ethyl 3,4-diethyl-5-methyl-1 H -pyrrole-2-carboxylate, yield 57%, yellow oil. 1 H NMR (CDCl 3 ): δ 0.99 (t, J = 7.5 Hz, 3H), 1.07 (t, J = 7.3 Hz, 3H), 1.21 (t, J = 7.1 Hz, 3H), 2.02 (s, 3H), 2.35 (q, J = 7.5 Hz, 2H), 2.68 (q, J = 7.4 Hz, 2H), 3.64 (s, 6H), 3.71 (s, 3H), 4.15 (q, J = 7.1 Hz, 2H), 5.44 (s, 2H), 5.96 ppm (s, 2H). IR: ν 1708 cm −1 . Anal. (C 22 H 31 NO 5 (389.49)) C, H, N. Ethyl 1-(3,4,5-Trimethoxybenzyl)-1 H -imidazole-4-carboxylate (50) A solution of DIPAD (1.29 g, 1.25 mL, 0.0064 mol) in anhydrous THF (18 mL) was added dropwise to a mixture of 3,4,5-trimethoxybenzyl alcohol (1.26 g, 0.0064 mol), ethyl 1- H -imidazole-4-carboxylate 26 (1.0 g, 0.0071 mol), and anhydrous triphenylphosphine (1.86 g, 0.0071 mol) in the same solvent (35 mL). The reaction was stirred at 25 °C overnight under an Ar stream. The reaction mixture was concentranted in vacuo, diluted with water, and extracted with ethyl acetate. The organic layer was washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by silica gel column chromatography (ethyl acetate:methanol = 45:5 as eluent) to furnish 50 as a colorless oil (0.2 g, 10%). 1 H NMR (CDCl 3 ): δ 1.26 (t, J = 7.4 Hz, 3H), 3.72 (s, 6H), 3.75 (s, 3H), 4.22 (q, J = 7.1 Hz, 2H), 5.36 (s, 2H), 6.33 (s, 2H), 7.54 (s, 1H), 7.69 ppm (s, 1H). IR: ν 1695 cm −1 . Anal. (C 16 H 20 N 2 O 5 (320.34)) C, H, N. Ethyl 1-(3,4,5-Trimethoxybenzoyl)-1 H -pyrrole-2-carboxylate (51) To a stirred mixture of potassium tert -butoxide (0.26 g, 0.0023 mol) and 18-crown-6 (0.47 g, 0.0018 mol) in anhydrous THF (25 mL) was added dropwise a solution of ethyl 1 H -pyrrole-2-carboxylate (0.25 g, 0.0018 mol) in the same solvent (25 mL). After cooling to 0 °C for 15 min, a solution of 3,4,5-trimethoxybenzoyl chloride (0.41 g, 0.0018 mol) in the same solvent (25 mL) was added dropwise. The reaction mixture was stirred at 25 °C for 4 h and then concentranted to a small volume and extracted with ethyl acetate. The organic layer was washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by silica gel column chromatography (dichloromethane as eluent) to furnish 51 as an orange oil (0.05 g, 8%). 1 H NMR (CDCl 3 ): δ 1.09 (t, J = 7.1 Hz, 3H), 3.78 (s, 6H), 3.82 (s, 3H), 4.02 (q, J = 7.1 Hz, 2H), 6.24 (m, 1H), 6.94 (s, 2H), 6.99 (m, 1H), 7.18 ppm (m, 1H). IR: ν 1670, 1703 cm −1 . Anal. (C 17 H 19 NO 6 (333.34)) C, H, N. Ethyl 1-(3,4,5-Trimethoxybenzoyl)-1 H -imidazole-4-carboxy-late (52) and Ethyl 1-(3,4,5-Trimethoxybenzoyl)-1 H -imidazole-5-carboxylate (53) To a solution of ethyl imidazole 4-carboxylate 26 (0.5 g, 0.0035 mol) in anhydrous dichloromethane (10 mL) was added dropwise a solution of 3,4,5-trimethoxybenzoyl chloride (0.41 g, 0.0018 mol) in the same solvent (10 mL). The reaction mixture was refluxed for 2.5 h and, after cooling, quenched on crushed ice. The organic layer was removed and washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by silica gel column chromatography (ethyl acetate as eluent). The initial eluate furnished 52 as a white solid (0.17 g, 15%), mp 130–133 °C (from ethanol). 1 H NMR (CDCl 3 ): δ 1.34 (t, J = 7.1 Hz, 3H), 3.84 (s, 6H), 3.90 (s, 3H), 4.34 (q, J = 7.1 Hz, 2H), 6.96 (s, 2H), 8.06 (s, 1H), 8.09 ppm (s, 1H). IR: ν 1672, 1702 cm −1 . Anal. (C 16 H 18 N 2 O 6 (334.32)) C, H, N. Further elution with the same solvent afforded 53 as a white solid (0.12 g, 13%), mp 140–143 °C (from ethanol). 1 H NMR (CDCl 3 ): δ 1.40 (t, J = 7.1 Hz, 3H), 3.90 (s, 6H), 3.97 (s, 3H), 4.40 (q, J = 7.1 Hz, 2H), 7.02 (s, 2H), 8.16 ppm (s, 2H). IR: ν 1674, 1707 cm −1 . Anal. (C 16 H 18 N 2 O 6 (334.32)) C, H, N. 1-(3,4,5-Trimethoxybenzyl)-1 H -pyrrole-2-carboxylic acid (54) A mixture of 46 (0.23 g, 0.00072 mol) and potassium hydroxide (0.12 g, 0.0022 mol) in methanol:water (1:1) (10 mL) was refluxed for 3 h. After cooling, the reaction mixture was washed with dichloromethane; the aqueous layer was made acidic with 2 N hydrochloric acid (pH = 2) and extracted with dichloromethane. The organic layer was washed with water, dried, filtered, and evaporated to give 54 as a brown solid (0.15 g, 74%), mp 80–81 °C (from ethanol). 1 H NMR (CDCl 3 ): δ 3.68 (s, 6H), 3.71 (s, 3H), 5.36 (s, 2H), 6.12 (dd, J = 3.85 and 2.6 Hz, 1H), 6.27 (s, 2H), 6.85 (t, J = 2.0 Hz, 1H), 7.04 (dd, J = 3.9 and 1.7 Hz, 1H), 10.69 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1703, 2995 cm −1 . Anal.(C 15 H 17 NO 5 (291.30)) C, H, N. 2-(2-Hydroxyethoxy)ethyl 3,4,5-Trimethyl-1-(3,4,5-trimethoxybenzyl)-1 H -pyrrole-2-carboxylate (55) A mixture of 48 (0.05 g, 0.0014 mol) and potassium hydroxide (0.043 g, 0.00077 mol) in diethylene glycol (1 mL) was heated at 140 °C for 3 h. After cooling, water and ethyl acetate were added and the layers separated. The organic layer was washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by silica gel column chromatography (ethyl acetate as eluent) to give 55 as a yellow oil (0.02 g, 34%). 1 H NMR (CDCl 3 ): δ 1.89 (s, 3H), 2.02 (s, 3H), 2.23 (s, 3H), 3.50 (m, 2H), 3.62 (m, 2H), 3.66 (m, 3H), 3.68 (s, 6H), 3.73 (s, 3H), 4.28 (m, 2H), 5.41 (s, 2H), 6.06 ppm (s, 2H). IR: ν 1705, 2836 cm −1 . Anal. (C 22 H 31 NO 7 (421.28)) C, H, N. 1-(3,4,5-Trimethoxybenzyl)-1 H -imidazole-4-carboxylic Acid Hydrochloride (56) A mixture of 50 (0.08 g, 0.00025 mol) and 37% hydrochloric acid (1.5 mL) was refluxed for 1.5 h. After cooling, the reaction mixture was washed with ethyl acetate. The aqueous layer was evaporated in vacuo. The residue was dissolved in 2-propanol (1.5 mL) and cooled to −78 °C. Upon addition of n -hexane, the suspension was filtered to give 56 as a white solid (0.05 g, 68%), mp 163–166 °C (from methanol). 1 H NMR (CD 3 OD): δ 3.77 (s, 3H), 3.85 (s, 6H), 5.73 (s, 2H), 6.82 (s, 2H), 8.24 (s, 1H), 9.22 (s, 1H), 12.10 (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1705, 3100 cm −1 . Anal. (C 14 H 16 N 2 O 5 · HCl (328.75)) C, H, Cl, N. Ethyl 2-(2-(4-Bromophenyl)hydrazono)propanoate (57) A mixture of 4-bromophenylhydrazine hydrochloride (5.7 g, 0.0255 mol), ethyl pyruvate (2 g, 1.91 mL, 0.017 mol), and sodium acetate (2.79 g, 0.034 mol) in ethanol (40 mL) was placed into the MW cavity (open vessel mode). MW irradiation at 250 W was used, the temperature being ramped from 25 to 100 °C. Once 100 °C was reached, taking about 2 min, the reaction mixture was held at this temperature for 5 min while stirring. The reaction mixture was cooled at 0 °C, with stirring, and filtered to give 57 , yield 90%, mp 142–144 °C (from ethanol). 1 H NMR (CDCl 3 ): δ 1.39 (t, J = 7.2 Hz, 3H), 2.12 (s, 3H), 4.33 (q, J = 7.1 Hz, 2H), 7.10 (d, J = 6.7 Hz, 2H), 7.40 (d, J = 6.9 Hz, 2H), 7.64 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1576, 1675, 3290 cm −1 . Anal. (C 11 H 13 BrN 2 O 2 (285.14)). Ethyl 5-Bromo-1 H -indole-2-carboxylate (58) Compound 57 (35.15 g, 0.123 mol) was added in portions to PPA (350 g) preheated at 110 °C. The mixture was stirred at the same temperature for 30 min and then quenched with ice–water. The solid was filtered, washed with water, dried, and recrystallized from ethanol to give 58 as a brown solid (32.95 g, 60%), mp 160–161 °C. 1 H NMR (CDCl 3 ): δ 1.43 (t, J = 7.1 Hz, 3H), 4.43 (q, J = 7.1 Hz, 2H), 7.15–7.16 (m, 1H), 7.31 (d, J = 8.1 Hz, 1H), 7.41 (dd, J = 7.0 Hz, 1H), 7.84 (m, 1H), 9.98 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1690, 3311 cm −1 . Anal. (C 11 H 10 BrNO 2 (268.11)). 5-Bromo-1 H -indole-2-carboxylic Acid (59) A mixture of 58 (0.25 g, 0.001 mol) in 3 N sodium hydroxide (2 mL) was placed into the MW cavity (closed vessel mode, P max = 250 PSI). MW irradiation at 150 W was used, the temperature being ramped from 25 to 110 °C. Once 110 °C was reached, taking about 1 min, the reaction mixture was held at this temperature for 2 min while stirring. After cooling, the reaction mixture was made acidic with 3 N hydrochloric acid (pH = 2) and extracted with ethyl acetate; the organic layer was washed with brine, dried, filtered, and evaporated to give 59 as a brown solid (0.23 g, 95%), mp 188 °C (from ethanol). 1 H NMR (DMSO-d 6 ): δ 7.05 (s, 1H), 7.32 (dd, J = 8.9 Hz, 1H), 7.39 (d, J = 8.8 Hz, 1H), 7.85 (d, J = 2.1 Hz, 1H), 11.94 (broad s, disappeared on treatment with D 2 O, 1H), 12.61 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1652, 2850, 3422 cm −1 . Anal. (C 9 H 6 BrNO 2 (240.05)). Methyl 5-Bromo-1 H -indole-2-carboxylate (60) To a cold suspension of 59 (3.4 g, 0.014 mol) in anhydrous methanol (13.4 mL) was added thionyl chloride (1.6 mL) dropwise. The reaction mixture was stirred at the same temperature for 20 min and then heated at 65 °C for 2 h under an Ar stream, then cooled and diluted with water and ethyl acetate. The layers were separated, and the organic phase was washed with a saturated solution of sodium hydrogen carbonate and brine, dried, filtered, and evaporated to give 60 as a white solid (3.43 g, 96%), mp 217–220 °C (from ethanol/cyclohexane), lit. 25 211.8–213.6. 2-(3,4,5-Trimethoxyphenyl)acetyl Chloride This compound was synthesized by treating a cold solution of 3,4,5-trimethoxyphenylacetic acid (0.25 g, 0.0011 mol) in anhydrous THF (5 mL) with oxalyl chloride (0.28 g, 0.19 mL, 0.0022 mol). Immediately afterward, a catalytic amount (2 drops) of anhydrous DMF was added, and vigorous bubbling ensued. After 10 min, the reaction mixture was carefully concentrated in vacuo to provide a viscous oil. Fresh anhydrous THF was added, and the solution was concentrated in vacuo again to furnish 2-(3,4,5-trimethoxyphenyl)acetyl chloride (0.27 g, yellow oil), which was used without further purification.

Molecular Modeling

All molecular modeling studies were performed on a MacPro dual 2.66 GHz Xeon running Ubuntu 8. The tubulin structure was downloaded from the PDB ( http://www.rcsb.org/ : PDB code 1SA0). 3 Hydrogen atoms were added to the protein, using Molecular Operating Environment (MOE) 2007.09, 27 and minimized, keeping all the heavy atoms fixed until a rmsd gradient of 0.05 kcal mol −1 Å −1 was reached. Ligand structures were built with MOE and minimized using the MM-FF94x forcefield until a rmsd gradient of 0.05 kcal mol −1 Å −1 was reached. The docking simulations were performed using FlexX 28 with the MOE interface. The rmsd of the trimethoxyphenyl moiety for each of the compounds evaluated was calculated in comparison with ring A of DAMA-colchicine, and the results from the docking were scored using this value. 29 The images presented here were created with Zodiac. 30 Biology.

Tubulin Assembly

The reaction mixtures contained 0.8 M monosodium glutamate (pH 6.6 with hydrochloric acid in 2 M stock solution), 10 μM tubulin, and varying concentrations of drug. Following a 15 min preincubation at 30 °C, samples were chilled on ice, GTP to 0.4 mM was added, and turbidity development was followed at 350 nm in a temperature controlled recording spectrophotometer for 20 min at 30 °C. Extent of reaction was measured. Full experimental details were previously reported. 31 [ 3 H]Colchicine Binding Assay The reaction mixtures contained 1.0 μM tubulin, 5.0 μM [ 3 H]colchicine, and 5.0 μM inhibitor and were incubated 10 min at 37 °C. Complete details were described previously. 32 Cell Lines Methodology for the evaluation of the growth of human MCF-7 breast carcinoma cells was previously described. 32 Human HeLa cells (from carcinoma of the uterine cervix) and HCT116/chr3 cells (from colon carcinoma) were grown at 37 °C in a humidified atmosphere containing 5% carbon dioxide in D-MEM (Gibco BRL, UK) supplemented with 10% fetal calf serum, 4 mM glutamine, 2 mM sodium pyruvate, 100 U/mL penicillin, and 0.1 mg/mL streptomycin (all reagents were from Celbio, Italy). For the HCT116/chr3 cells, the medium also included 500 μg/mL of the selection compound G418 (Gibco). Cells were trypsinized when subconfluent and seeded in T75 flasks at a concentration of 10 5 /mL. U937 (human monocytic leukemia cell line) and J774.1 cells (murine macrophage cell line) were cultured in RPMI 1640 medium containing 10% fetal calf serum, 50 μg/mL each of penicillin and streptomycin, and 2 mM glutamine. Human melanoma M14 cells and human embryonic kidney (HEK) 293 cells were grow at 37 °C in Dulbecco's modified Eagle's medium containing 10 mM glucose (DMEM-HG) supplemented with 10% fetal calf serum and 50 μg/mL each of penicillin and streptomycin. The P. tridactylis PtK2 cells, the A10 rat embryonic aortic smooth muscle cells, the human umbilical vein endothelial cells, and the human aortic smaooth muscle cells were obtained from the American Type Tissue Collection and grown as recommended by the supplier, except that a 5% CO 2 atmosphere was used with all cells.

Morphological Analysis

Cells grown in T75 flasks and treated with 100 μM 24 for 24 h were observed with an Olympus IX71 microscope and photographed with a Cool SNAPES ES Photo-metric digital camera.

Cell Viability Assay

HeLa and HCT116/chr3 cells. Cells were seeded in 96-well plates at a density of 1 × 10 3 cells/100 μL well. Twenty-four hours later, cells were treated with the test compound (stock solution: 10 mM in DMSO, kept at room temperature in the dark) used at increasing concentrations (0.1–100 μM) for 24 h. Cells were harvested at the end of the treatment or washed with phosphate-buffered saline and incubated in drug-free medium for an additional 24 h. To rule out a possible effect of the solvent, parallel samples were incubated in complete medium containing 1% DMSO. At the end of the treatments, 20 μL of CellTiter96 Aqueous One Solution Reagent (Promega) was added to each well. The plates were then incubated for 4 h at 37 °C and analyzed with a microplate reader (Gio. De Vita, Roma, Italy) at 492 nm. Experiments were performed in quadruplicate and repeated three times. M14, HEK, U937, and J774.1 cells. Cell viability was determined using the 3-[4,5-demethylthiazol-2,5-diphenyl-2 H -tetrazolium bromide (MTT) colorimetric assay. The test is based on the ability of mitochondrial dehydrogenase to convert, in viable cells, the yellow MTT reagent into a soluble blue formazan dye. Cells were seeded into 96-well plates to a density of 10 5 cells/100 μL well. After 24 h of growth to allow attachment to the wells, compounds were added at various concentrations (10, 30, 50, 100, 150, and 200 nM). After 24 or 48 h of growth and after removal of the culture medium, 100 μL/well of medium containing 1 mg/mL of MTT was added. Cell cultures were further incubated at 37 °C for 2 h in the dark. The MTT solution was then removed, and 100 μL DMSO was added to dissolve formazan crystals. Absorbance at 550 nm was measured using a plate reader. Experiments were performed in triplicate. As a control, 0.5% DMSO was added to untreated cells.

Time-Lapse Microscopy

Effect of drug treatment on HEK and M14 cell morphology was determined by TLM using a Leica CTR6500 microscope. Images were captured every hour for a 48 h period. Plates (96-well) were incubated under standard culture conditions and kept at 37 °C in a 5% carbon dioxide atmosphere for the observation period (up to 48 h). Yeast S. cerevisiae yeast wild type strain BY4741 ( Mat a his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0 ) was used. Cells were grown at 28 °C in minimal medium (0.67% yeast nitrogen base without amino acids) containing 2% glucose (synthetic dextrose, SD) supplemented with 20 μg/μL of the appropriate nutritional supplements. A fresh exponential culture (5 × 10 6 cells/mL) of the yeast were incubated for 18 h at 28 °C with the compound of interest at 20 μM. The cells were then centrifuged for 5 min at 13000g at 4 °C, washed twice with distilled water, and resus-pended in SD (1 mL). Cell suspensions (15 μL) were transferred in 60 μL of fresh SD medium, serially diluted 10-fold, incubated overnight at 28 °C, and spotted onto YP plates (1% yeast extract, 2% peptone) supplemented with 2% glucose (YPD). The plates were incubated at 28 °C for three days before recording cell growth.

General Procedure for the Synthesis of Compounds 4, 7, 10, 13, 16, 19, 21, 25, 28, 31, 34, 39, and 43. Example: (1 H -Indol-3-yl)-(3,4,5-trimethoxyphenyl)methanone (4) A mixture of AlCl 3 (0.57 g; 0.0043 mol), the appropriate 1 H -indole (0.5 g, 0.0043 mol) and 3,4,5-trimethoxybenzoyl chloride (0.99 g, 0.0043 mol) in 1,2-dichloroethane (2 mL) was placed into the MW cavity (closed vessel mode, P max = 250 PSI). MW irradiation of 150 W was used, the temperature being ramped from 25 to 110 °C while stirring. Once 110 °C was reached, taking about 1 min, the reaction mixture was held at this temperature for 2 min, then cooled, diluted cautiously with water, and extracted with chloroform; the organic layer was washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by silica gel column chromatography (ethyl acetate: n -hexane = 1:1 as eluent) to furnish 4 as a white solid (0.71 g, 53%), mp 210–213 °C (from ethanol), differing from lit. 22 174–176 °C and lit. 23 132–133 °C. 1 H NMR (DMSO- d 6 ): δ 3.73 (s, 3H), 3.83 (s, 6H), 7.06 (s, 2H), 7.20–7.24 (m, 2H), 7.48 (d, J = 9.1 Hz, 1H), 8.06 (d, J = 3.0 Hz, 1H), 8.21 (d, J = 8.5 Hz, 1H), 11.99 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1571, 3179 cm −1 .

General Procedure for the Synthesis of Compounds 5, 14, 26, and 35. Example: 3-(3,4,5-trimethoxybenzyl)-1 H -indole (5) Sodium borohydride (0.80 g, 0.021 mol) was added to a solution of 4 (0.66 g, 0.0021 mol) in ethanol (85 mL). The reaction mixture was refluxed for 3 h, then cooled, cautiously diluted with water, and extracted with ethyl acetate; the organic layer was washed with brine, dried, and filtered. Evaporation of the solvent gave a residue that was purified by silica gel column chromatography (ethyl acetate: n -hexane = 1:1 as eluent) to furnish 5 as a white solid (0.26 g, 42%), mp 133–136 °C (from ethanol), lit. 22 125–126 °C and lit. 24 128–130 °C.

General Procedure for the Synthesis of Compounds 8, 11, 17, 20, 23, 29, 32, 41, and 45. Example: Methyl 3-(3,4,5-trimethoxybenzyl)-1 H -indole-2-carboxylate (8) To a cold solution of 7 (0.31 g, 0.00084 mol) in trifluoroacetic acid (0.96 g, 0.65 mL, 0.0084 mol) was added dropwise triethylsilane (0.22 g, 0.30 mL, 0.0019 mol). The reaction mixture was stirred at 25 °C for 24 h, then neutralized with a saturated solution of sodium hydrogen carbonate and extracted with ethyl acetate; the organic layer was washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by silica gel column chromatography (ethyl acetate: n -hexane = 3:2 as eluent) to furnish 8 as a yellow solid (0.15 g, 50%), mp 156–161 °C (from ethanol/ n -hexane). 1 H NMR (CDCl 3 ): δ 3.69 (s, 6H), 3.72 (s, 3H), 3.89 (s, 3H), 4.38 (s, 2H), 6.46 (s, 2H), 7.05 (t, J = 8.1 Hz, 1H), 7.25 (t, J = 8.3 Hz, 1H), 7.33 (d, J = 7.4 Hz, 1H), 7.57 (d, J = 8.2 Hz, 1H), 8.75 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1690, 3318 cm −1 . Anal. (C 20 H 21 NO 5 (355.38)) C, H, N.

General Procedure for the Synthesis of Compounds 40 and 44. Example: Methyl 3-(Hydroxy(3,4,5-trimethoxyphenyl)methyl)-5-methoxy-1 H -indole-2-carboxylate (40) A mixture of 39 (0.34 g, 0.00085 mol) and sodium borohydride (0.03 g, 0.00085 mol) in THF (2.1 mL) and water (0.12 mL) was refluxed for 2 h. After cooling, water and ethyl acetate were added. The organic layer was removed and washed with brine, dried, and filtered. Removal of the solvent gave a residue that was purified by silica gel column chromatography (ethyl acetate: n -hexane=1:1 as eluent) to furnish 40 as a brown solid (0.1 g, 29%), mp 100–103 °C (from ethanol). 1 H NMR (CDCl 3 ): δ 3.76 (s, 3H), 3.79 (s, 6H), 3.82 (s, 3H), 3.97 (s, 3H), 4.53 (d, J = 1.2 Hz, 1H), 6.50 (d, J = 7.4 Hz, 1H), 6.77 (s, 2H), 6.95 (d, J = 2.4 Hz, 1H), 7.02 (dd, J = 6.5 Hz, 1H), 7.28 (d, J = 8.4 Hz, 1H), 8.78 ppm (broad s, disappeared on treatment with D 2 O, 1H). IR: ν 1707, 2937, 3322 cm −1 . Anal. (C 21 H 23 NO 7 (401.41)) C, H, N.