PIGA (N,N-Di-n-butyl-5-chloro-2-(4-chlorophenyl)indol-3-ylglyoxylamide), a New Mitochondrial Benzodiazepine-Receptor Ligand, Induces Apoptosis in C6 glioma Cells

Supporting Information © Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2005

© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2005

Supporting Information for

PIGA (N,N-di-n-butyl-5-chloro-2-(4-chlorophenyl)indol-3-ylglyoxylamide), a New Mitochondrial Benzodiazepine Receptor Ligand, Induces Apoptosis in C6 Glioma Cells. B. Chelli, L. Rossi, E. Da Pozzo, B. Costa, F. Spinetti, M. Rechichi, A. Salvetti, A. Lena, F. Simorini, R. Vanacore, F. Scatena, F. Da Settimo, V. Gremigni, and C. Martini*

Chemistry. Melting points were determined using a Reichert Köfler ho t-stage apparatus and are uncorrected. Infrared spectra were recorded with a Nicolet-Avatar 360 FTIR spectrophotometer in Nujol mulls. Routine nuclear magnetic resonance spectra were recorded in [D 6]DMSO solution on a Varian Gemini 200 spectrometer operating at 200 MHz. Evaporation was performed in vacuo (rotary evaporator). Analytical TLC was carried out on Merck 0.2 mm precoated silica gel aluminum sheets (60 F-254). Elemental analyses were performed by our Analytical Laboratory and agreed with theoretical values to within ± 0.4%. 2-Phenylindole was from Sigma-Aldrich. Synthesis of 5-Chloro-2-(4-chlorophenyl)indole 2.[1] A stirred solution of 4-chloro-N-(4chloro-2-methylphenyl)benzamide 1[1] (1.4 g, 5.0 mmol) in dry THF (35 mL), maintained under an N2 atmosphere and at an internal temperature of –25 to 0°C, was treated dropwise with 9.3 mL (15 mmol) of nBuLi (1.6 M solution in hexane). The stirred mixture was kept at room temperature for 48 h (TLC analysis), cooled in an ice bath and acidified to pH 5-6 with diluted HCl. The organic layer was separated and the aqueous phase was extracted with CH2Cl2 . After drying (MgSO4), the combined organic phases were evaporated to dryness, furnishing the crude indole 2, which was purified by using Biotage Flash 40i (cyclohexane/ethyl acetate 85:15) to give 1.11 g, yield 85%, m.p. 190-192°C.

Synthesis of (5-Chloro-2-(4-chlorophenyl)indol-3-yl)glyoxylyl Chloride 3. Oxalyl chloride (0.51 mL, 5.9 mmol) was added dropwise, at 0°C, to a well-stirred mixture of indole 2 (1.11 g, 4.23 mmol) in anhydrous ethyl ether (20 mL). The reaction was maintained at room temperature for 2 h (TLC analysis). The precipitate formed was collected and washed with portions of petroleum ether 30-50°C to give the acid chloride 3 (0.97 g, yield 65%), which was directly used for the next reaction. M.p. 125-128°C. Synthesis

of

N,N-Di-n-butyl-5-chloro-2-(4-chlorophenyl)indol-3-ylglyoxylamide

(PIGA). A solution of di-n-butylamine (0.41 mL, 2.5 mmol) in dry toluene (5.0 mL) was added dropwise to a stirred suspension, cooled to 0°C, of indolylglyoxylyl chloride 3 (0.88 g, 2.5 mmol) in the same solvent (50 mL). A solution of triethylamine (0.35 mL, 2.5 mmol) in dry toluene (5.0 mL) was successively added. The reaction mixture was left to warm to room temperature, stirred for 24 h (TLC analysis) and then filtered to eliminate the triethylamine hydrochloride. The toluene solution was washed with water, dried with MgSO4 and evaporated to dryness to yield the crude compound PIGA, which was purified by recrystallization from cyclohexane (1.0 g, yield 90%). M.p. 178-181°C. IR (Nujol): ν = 3230, 1610, 1580, 1420, 1100, 730 cm- 1 . 1 H NMR : δ = 0.74 (t, J=7.2 Hz, 3H; CH3 ); 0.87 (t, J=6.3 Hz, 3H; CH3 ); 1.05-1.50 (m, 8H; 2 CH2 (CH 2 ) 2CH3 ); 2.90-3.08 (m, 4H; 2 CH 2 (CH2 )2 CH3 ); 7.33 (dd, J=8.7,2.1 Hz, 1H; 6-H); 7.51 (d, J=8.6 Hz, 1H; 7-H); 7.59-7.62 (m, 4H; Ar-H); 8.05 (d, J=2.2 Hz, 1H; 4-H); 12.72 (br s, 1H; NH, exch. with D2 O). Elemental analysis calcd (%) for C24 H26Cl2 N2 O2 (444.14): C 64.72, H 5.88, N 6.29; found C 64.87, H 5.84, N 6.42. Synthesis of (2-Phenylindol-3-yl)glyoxylyl Chloride.[2] Oxalyl chloride (0.12 mL, 1.4 mmol) was added dropwise, at 0°C, to a well-stirred mixture of 2-phenylindole (1.93 g, 10.0 mmol) in anhydrous ethyl ether (50 mL). The reaction was maintained at room temperature for 1-2 h (TLC analysis). The precipitate formed was collected and washed with portions of anhydrous ethyl ether to give the acid chloride (1.93 g, yield 68%), which was directly used for the next reaction. M.p. 132-134°C. Synthesis of N-n-Propyl-2-phenylindol-3-ylglyoxylamide. A solution of n-propylamine (0.23 mL, 2.75 mmol) in dry toluene (50 mL) was added dropwise to a stirred suspension, at 0°C, of (2-phenylindol-3-yl)glyoxylyl chloride (0.78 g, 2.5 mmol) and triethylamine (0.42 mL, 3.0 mmol) in the same solvent (50 mL). The reaction mixture was left to warm to room temperature, stirred for 24 h (TLC analysis) and the obtained suspension was filtered. The precipitate was triturated with a saturated NaHCO3 aqueous solution and washed with water to give a first portion of crude product. The toluene solution was washed with diluted

HCl, and then with a saturated NaHCO3 aqueous solution and water, dried (MgSO4) and evaporated to dryness to yield the crude compound, which was purified by recrystallization from benzene (0.42 g, yield 55%). M.p. 218-220°C. IR (Nujol): ν = 3350, 3100, 1645, 1590, 1420, 740.

1

H NMR: δ = 0.76 (t, 3H, J=7.4 Hz, CH3 ); 1.19-1.26 (m, 2H,

CH2CH2CH3); 2.66-2.74 (m, 2H, CH2CH2CH3); 7.23-7.27 (m, 2H, 5-H, 6-H); 7.46-7.55 (m, 6H, Ar-H); 8.05-8.10 (m, 1H, 4-H); 8.49 (t, 1H, J=5.6 Hz, CONH, exch. with D2O). Elemental analysis calcd (%) for C 19 H18 N2 O 2 (306.36): C 74.49, H 5.92, N 9.14; found C 74.73, H 6.03, N 9.07. Synthesis of 1-(2-Phenyl-1H-indol-3-yl)-2-pyrrolidin-1-ylethane-1,2-dione. A solution of pyrrolidine (0.23 mL, 2.75 mmol) in dry toluene (50 mL) was added dropwise to a stirred suspension, at 0°C, of (2-phenylindol-3-yl)glyoxylyl chloride (0.78 g, 2.5 mmol) and triethylamine (0.42 mL, 3.0 mmol) in the same solvent (50 mL). The reaction mixture was left to warm to room temperature, stirred for 24 h (TLC analysis) and the obtained suspension was filtered to eliminate the triethylamine hydrochloride. The toluene solution was washed with diluted HCl, and then with a saturated NaHCO3 aqueous solution and water, dried (MgSO4 ) and evaporated to dryness to yield the crude compound, which was purified by recrystallization from toluene (0.56 g, yield 71%). M.p. 217-221°C. IR (Nujol): ν = 3200, 1630, 1590, 1410, 1180, 750. 1 H NMR: δ = 1.54-1.72 (m, 4H, CH2 (CH2)2CH2); 2.79 (t, 2H, J=6.4 Hz, CH2 N); 3.21 (t, 2H, J=6.8 Hz, CH2 N); 7.22-7.36 (m, 2H, 5-H, 6-H); 7.46-7.51 (m, 6H, Ar-H); 8.14-8.18 (m, 1H, 4-H); 12.45 (bs, 1H, NH, exch. with D2O). Elemental analysis calcd (%) for C 20 H18 N2 O 2 (318.37): C 75.45, H 5.70, N 8.80; found C 75.71, H 5.89, N 8.58.

References [1] K. Dinnel, J.M. Elliott, G.J. Hollingworth, M.P. Ridgill, Shaw, D.E., U.S. Pat. Appl. Publ. 20010039286; Chem. Abstr. 2001, 135, 357843. [2] M. Julia, R. Melamed, R. Gombert, Research in the Indole Series. XVI. 2Aryltryptamines and homologous Amines. Ann. Inst. Pasteur 1965, 109(3), 343-362. Beilstein Registry Number 410984.