EXPERIMENTAL
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Materials and methods
Melting points were determined using a Reichert Köfler hot-stage apparatus and are uncorrected. Infrared spectra were recorded with a Nicolet/Avatar FT-IR spectrometer in Nujol mulls. Routine nuclear magnetic resonance spectra were recorded in DMSO-d6 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). Silica gel 60 Merck (230-400 mesh ASTM) was used for column chromatography. Anhydrous reactions were performed in flame-dried glassware under N2. All compounds showed ≥ 95% purity.
All reagents used were obtained from commercial sources. All solvents were of an analytical grade.
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Chemistry
5-Nitro-2-phenylindole 2
A solution of NaNO3 (1.40 g; 16.5 mmol) in 50 ml of H2SO4 conc., previously prepared and cooled at 5oC, was added dropwise to a stirred solution of 2-phenylindole (3.00 g; 15.5 mmol) in 100 ml of H2SO4 conc. cooled at 5oC. The reaction mixture was stirred for 5 minutes (TLC analysis: hexane/AcOEt 5:5). Then the solution was poured into ice and the yellow precipitate formed was filtered at reduced pressure and dried over P2O5.
5-nitro-2-phenylindole 2: yield: 95%. m.p. = 194-196 oC. lit. ref n° 49.
5-amino-2-phenylindole 3
A mixture of 5-nitro-2-phenylindole 2 (0.500 g; 3.7 mmol) and 10% Pd/C (approximately 0.050 mg), in 150 ml of absolute EtOH, has been placed to react to catalytic hydrogenator for 3-4 h (TLC analysis: hexane/AcOEt 5:5). Once hydrogen absorbition was ceased (3-4 h), the catalyst was filtered off and the solvent was evaporated to afford a violet colored solid. The residue was purified by crystallization from toluene.
5-amino-2-phenylindole 3: yield: 80%. m.p.= 223-225 oC. IR (nujol, cm-1): 3347, 3413 1631, 1537, 1071, 757. 1H-NMR (DMSO-d6, ppm): 4.87 (bs exch. D2O, 2H, NH2); 6.52 (d, 1H, J = 8.2 Hz, Ar-H); 6.63 (s, 1H, Ar-H); 6.70 (s, 1H, H); 7.10 (d, 1H, J = 8.4 Hz, H); 7.22-7.29 (m, 1H, Ar-H); 7.38-7.45 (m, 2H, Ar-Ar-H); 7.78 (d, 2H, J = 7.4 Hz, Ar-Ar-H); 11.06 (bs exch. D2O, 1H, NH).
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t-Butyl-(2-phenylindol-5-yl)-carbamate 4
Di-tert-butyl-dicarbonate (0.209 g; 0.96 mmol) was added to a stirred solution of 5-amino-2-phenylindole 3 (0.200 g; 0.96 mmol) in a mixture of H2O (3ml) / Acetone (6 ml). The reaction mixture, was stirred at room temperature for 24 h (TLC analysis: hexane/AcOEt 7:3). The solvent was reduced to half volume under reduced pressure and the precipitate formed was collected and purified by crystallization from toluene.
t-Butyl-(2-phenylindole-5-yl)-carbamate 4: yield: 80%. m.p. = 165-167oC. IR (nujol, cm-1): 3437, 3326, 3289, 1686, 1531, 1050, 754. 1H-NMR (DMSO-d6, ppm): 1.48 (s, 9H, C(CH3)3); 6.81 (s, 1H, Ar-H); 7.10-7.15 (m, 1H, Ar-H); 7.23-7.32 (m, 2H, Ar-H); 7.40-7.47 (m, 2H, Ar-H); 7.65 (s, 1H, Ar-H); 7.80-7.85 (m, 2H, Ar-H); 9.05 (bs exch. D2O, 1H, CONH); 11.36 (bs exch. D2O, 1H, NH).
t-Butyl-[(2-phenylindol-3-ylglyoxyl)-5-yl]-carbamate 5
Oxalyl chloride (0.084 ml; 0.97 mmol), in 2 ml of anh. THF, was added dropwise to a stirred solution of t-Butyl-(2-phenylindole-5-yl)-carbamate 4 (0.200 g; 0.65 mmol) in anhydrous THF, cooled at 0oC. The mixture was maintained at room temperature for 2 h (TLC analysis: hexane/AcOEt 7:3). The organic solvent was removed under reduced pressure and the residue was washed with three portions of dry distilled THF. The glyoxylyl chloride 5 obtained was particularly instable, so it was utilized in the following reaction without any further purification.
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N-(7-Nitro-2,1,3-benzoxadiazol-4-yl)-N'-[5-(N-Boc-amino)-2-phenylindol-3-ylglyoxyl]-1,6-diaminohexane
12
A solution of 8 (0.084 g; 0.3 mmol) in anhydrous DMF (2-3 ml), was added dropwise to a stirred solution, previously prepared and cooled at 0oC, of glyoxylyl chloride 5 (0.199 g; 0.5 mmol) in 3 ml of the same solvent, followed by addition of triethylamine solution (0.16 ml; 1.2 mmol). The reaction mixture was stirred at room temperature for 12 h (TLC analysis: hexane/AcOEt 3:7), and then filtered to eliminate the hydrochloride triethylamine precipitate. The organic solvent was evaporated under reduced pressure and the residue was washed with water. The suspension was filtered, and the brown colored solid obtained was washed with cold diethyl ether. N-(7-Nitro-2,1,3-benzoxadiazol-4-yl)-N'-[5-(N-Boc-amino)-2-phenylindol-3-ylglyoxylyl]-1,6-diaminohexane 12: yield: 79%. m.p. = 153-155oC. IR (nujol, cm-1): 3324, 1696, 1580, 1290, 1153, 897, 726. 1H-NMR (DMSO-d6, ppm): 1.06-1.80 (m, 19H, CH2(CH2)4CH2, C(CH3)3, CONHCH2); 2.68-2.74 (m, 2H, CH2NH); 6.42 (d, 1H, J = 9.2 Hz, 5'-H); 7.34-7.53 (m, 7H, Ar-H); 8.28 (s, 1H, Ar-H); 8.42 (bs exch. D2O, 1H, COCONH); 8.51 (d, 1H, J = 9.0 Hz, 6'-H); 9.28 (s, 1H, ArNHCO); 9,57 (bs exch. D2O, 1H, CH2NH); 12.21 (bs exch. D2O, 1H, NH).
N-(7-Nitro-2,1,3-benzoxadiazol-4-yl)-N'-[5-amino-2-phenylindol-3-ylglyoxylyl]-1,6-diaminohexane 13
Trifluoroacetic acid (1.9 ml; 20 mmol) was added dropwise to a stirred solution of 12 (0.240 g; 0.3 mmol) in anhydrous dichloromethane cooled at 0oC. The reaction mixture was stirred at room temperature for 24 h (TLC analysis: AcOEt/hexane 7:3). Then the reaction mixture was concentrated
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under r.p.. The resulting solid residue was taken up with water and cooled at 0oC. Upon addition of a 5% solution of NaHCO3, a precipitate formed was filtered. N-(7-Nitro-2,1,3-benzoxadiazol-4-yl)-N'-[5-amino-2-phenylindol-3-ylglyoxylyl]-1,6-diaminohexane 13: yield: 91 %. m.p. = 195-197 oC. IR (nujol, cm-1): 3338, 3242, 1621, 1587, 1135, 801, 723. 1H-NMR (DMSO-d6, ppm): 1.04-1.36 (m, 8H, CH2(CH2)4CH2); 1.60-1.69 (m, 2H, CONHCH2); 2.68-2.74 (m, 2H, CH2NH); 6.42 (d, 1H, J = 9.6 Hz, 5'-H); 6.76-6.80 (m, 1H, Ar-H); 7.25-7.55 (m, 9H, Ar-H, NH2); 8.36 (bs exch. D2O, 1H, COCONH); 8.52 (d, 1H, J = 9.8 Hz, 6'-H); 9.58 (bs exch. D2O, 1H, CH2NH); 12.11 (bs exch. D2O, 1H, NH).
N-(7-Nitro-2,1,3-benzoxadiazol-4-yl)-N'-[5-isothiocyanate-2-phenylindol-3-ylglyoxylyl]-1,6-diaminohexane 14
To a stirred solution of compound 13 (0.119 g; 0.3 mmol) in 20 ml of 6% NaHCO3, was added 20 ml of dichloromethane. After 20 minutes of vigorous stirring at 0oC, thiofosgene (0.056 ml; 0.1 mmol) was added dropwise. The reaction mixture was allowed to stir for 4 h (TLC analysis: AcOEt/hexane 7:3). A precipitate formed was collected by vacuum filtration and washed with cold ether.
N-(7-Nitro-2,1,3-benzoxadiazol-4-yl)-N'-[5-isothiocyanate-2-phenylindol-3-ylglyoxylyl]-1,6-diaminohexane 14: yield: 67%. m.p. = 152-154oC. IR (nujol, cm-1): 3345, 2123, 2041, 1648, 1614, 1586, 1177, 1105, 811, 719. 1 H-NMR (DMSO-d6, ppm): 1.15-1.36 (m, 8H, CH2(CH2)4CH3); 1.60-1.69 (m, 2H, CONHCH2); 2.72-2.80 (m, 2H, CH2NH); 6,41 (d, 1H, J = 7.8 Hz, 5'-H); 7.31-7.56 (m, 8H, Ar-H, CONH); 8.01 (s, 1H, Ar-H); 8.50 (d, 1H,
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J = 7.2 Hz, 6'-H); 9.57 (bs exch. D2O, 1H, CH2NH); 12.68 (bs exch. D2O, 1H, NH).
General procedure for the synthesis of
2-oxo-2-(2-phenyl-1H-indol-3-glyoxylyl)–N-Boc-1,6-diaminohexane 17
or
2-oxo-2-(2-phenyl-1H-indol-3-glyoxylyl)–N-Boc-1,4-diaminobutane 18
Oxalyl chloride (0.084 ml; 0.97 mmol) in 1 ml of anhydrous Et2O was added to a stirred solution of 2-phenylindole (0.200 g; 0.65 mmol) in 10 ml of anhydrous Et2O, cooled at 0oC. This reaction was maintained at room temperature for 2 h (TLC analysis: hexane/AcOEt 7:3). The generated precipitate was collected and washed with portion of anhydrous Et2O, to give the acylchloride which was dried over P2O5 in vacuo, and then suspended in 50 ml of dry toluene. A solution of N-Boc-1,6-diaminohexane or N-Boc-1,4-diaminobutane (0.120 g; 0.5 mmol) in 3 ml of the same solvent was then added dropwise at 0oC, followed by the addition of triethylamine (0.10 ml; 1.2 mmol). The reaction mixture was allowed to warm to room temperature, stirred for 12 h (TLC analysis: hexane/AcOEt 3:7) and then filtered. The collected precipitate was washed with a 5% solution of NaHCO3 to yield the indolylglyoxylamide 17 or 18 and filtered at r.p..
2-oxo-2-(2-phenyl-1H-indol-3-glyoxylyl)–N-Boc-1,6-diaminohexane 17:
yield: 79%. 1H-NMR (DMSO-d6, ppm): 1.10-1.48 (m, 15H, CH2(CH2)4CH2, C(CH3)3); 2.65-3.08 (m, 6H, CH2(CH2)4CH2); 6.80 (bs exch. D2O, 1H, NHCOO); 7.10-7.30 (m, 3H, H); 7.40-7.70 (m, 5H, Ar-H); 8.12 (d, 1H, J = 8.6 Hz, Ar-Ar-H); 8.23 (bs exch. D2O, 1H, COCONH); 12.40 (bs exch. D2O, 1H, NH).
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2-oxo-2-(2-phenyl-1H-indol-3-glyoxylyl)–N-Boc-1,4-diaminobutane 18:
yield: 79%. 1H-NMR (DMSO-d6, ppm): 1.10-1.65 (m, 13H, CH2(CH2)2CH2, C(CH3)3); 2.65-2.98 (m, 4H, CH2(CH2)2CH2); 6.80 (bs exch. D2O, 1H, NHCOO); 7.10-7.40 (m, 3H, H); 7.50-7.70 (m, 5H, Ar-H); 8.11 (d, 1H, J = 7.4 Hz, Ar-Ar-H); 8.50 (bs exch. D2O, 1H, COCONH); 12.40 (bs exch. D2O, 1H, NH).
General procedure for the synthesis of
N-(6-aminohexyl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide 19
or
N-(4-aminobutyl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide 20
HCl 3M (4.5 ml; 6.0 mmol) was added to a stirred solution of 17 or 18 (0.513 g; 1.3 mmol) in AcOEt (10 ml). The reaction mixture was stirred for about 2 h at 60oC (TLC analysis: hexane/AcOEt 5:5). Then the solvent was evaporated under reduced pressure, and the resulting product was washed with dichloromethane. The aqueous phase was alkalized with NaOH 50% until pH = 10. Then the solution was extracted with dichloromethane. The organic phase was dried over MgSO4, filtered and then evaporated to dryness.
N-(6-aminohexyl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide 19 : yield:
70%. 1H-NMR (DMSO-d6, ppm): 1.10-1.50 (m, 8H, CH2(CH2)4CH2); 2.78 (s, 2H, CH2(CH2)4 CH2); 3.30-3.50 (m, 2H, NH2); 7.30-7.40 (m, 2H, Ar-H); 7.45-7.65 (m, 6H, Ar-H); 8.12 (d, 1H, J = 6.6 Hz, Ar-H); 8.45 (bs exch. D2O, 1H, NHCO).
N-(4-aminobutyl)-2-oxo-2-(2-phenyl-1H-indol-3-yl)acetamide 20 : yield:
70%. 1H-NMR (DMSO-d6, ppm): 1.18-1.50 (m, 4H, CH2(CH2)2CH2); 2.60-2.85 (m, 4H, CH2(CH2)2CH2); 3.25-3.60 (m, 2H, NH2); 7.22-7.38 (m, 2H,
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Ar-H); 7.50-7.70 (m, 6H, Ar-H); 8.15 (d, 1H, J = 2.4 Hz, Ar-H); 8.58 (bs exch. D2O, 1H, NHCO).
General procedure for the synthesis of
N-(1,4,7-triazonane-1,4,7-triyl)triacetic
acid)-[2-phenyl-1H-indol-3-ylglyoxylyl]-1,6-diaminohexane 21
or N-(1,4,7-triazonane-1,4,7-triyl)triacetic
acid)-[2-phenyl-1H-indol-3-ylglyoxylyl]-1,4-diaminobutane 22
CDI (0.027 g; 0.16 mmol) was added portion-wise, under a nitrogen atmosphere, to a solution of NOTA (0.050 g; 0.16 mmol) dissolved in 5 ml of anhydrous DMF. Once CO2 evolution had ceased, a solution of compound 19 or 20 (0.054 g; 0.16 mmol) in 1 ml of dry DMF was added dropwise. The reaction mixture was maintained under stirring at room temperature, until the disappearance of the starting material (TLC analysis: CHCl3/MeOH 9:1). The solvent was eliminated under vacuum and the residue was triturated with crushed ice and extracted with AcOEt.
N-(1,4,7-triazonane-1,4,7-triyl) triacetic acid)-[2-phenyl-1H-indol-3-ylglyoxylyl]-1,6-diaminohexane 21: Yield: 60%. 1H-NMR (DMSO-d6, ppm): 0.95-1.60 (m, 12H, CH2(CH2)4CH2); 2.60-3.60 (m, 18H, CH2(CH2)4CH2, CH2CH2); 6.95-7.75 (m, 9H, Ar-H); 8.05 (bs exch. D2O, 1H, NHCO); 8.45 (bs exch. D2O, 1H, COCONH); 11.79 (bs exch. D2O, 1H, NHCO).
N-(1,4,7-triazonane-1,4,7-triyl) triacetic acid)-[2-phenyl-1H-indol-3-ylglyoxylyl]-1,4-diaminobutane 22
