EXPERIMENTAL
SECTION
55
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 (230-400 mesh) was used
for column chromatography. Elemental analyses were performed by
our analytical laboratory and agreed with theoretical values to within
±0.4%.
2-hydrazinyl-1H-benzo[d]imidazole
(
62),
(Z)-2-(2-(1H-benzo[d]imidazol-2-yl)hydrazono)-2-phenylacetic acid
(
63)
and3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-4(10H)-one
(
64)
were prepared in accordance with a reported procedure. 100,1012-hydrazinyl-1H-benzo[d]imidazole
62
.
Yield: 60%; mp= 148-151 °C (lit. ref. n. 101 mp=146-147 °C).(Z)-2-(2-(1H-benzo[d]imidazol-2-yl)hydrazono)-2-phenylacetic acid
63
.
Yield: 83%; mp = 240-243 °C (lit. ref. n. 100 mp = 247-250 °C)3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-4(10H)-one
64
.
Yield: 91%; mp = >300°C (lit. ref. n. 100 mp = > 300 °C)Yield, melting point and spectral data of compounds 62, 63 and 64 were reported in literature. 100,101
56
General procedure for:
N10
-[(ethoxycarbonyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] (65) and
N1
-[(ethoxycarbonyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one (66).
Sodium hydride (3 mmol) was added in small portions to a solution of 3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-4(10H)-one (64) (3 mmol) in 10 mL of anhydrous DMF, cooled at 0 °C. After the development of H2 (about 1h),
Ethyl bromo acetate (3 mmol), dissolved in 2 mL of the same solvent, was added dropwise. The reaction mixture was maintained under stirring at room temperature for 24h (TLC analysis). The solution was dripped into ice and once the ice dissolved, the formed precipitate was collected by filtration (TLC analysis) and purified by flash chromatography (Toluene/Acetonitrile= 9/1 as eluent). We obtained two regioisomers. Yield: 65: 70%; 66: 30%. N10 -[(ethoxycarbonyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] 65. Yield: 70%; 1H-NMR (DMSO-d6, ppm): 1.23 (t, 3H, CH3,
J=7,1 Hz); 4.17-4.27 (m, 2H, CH2); 5.37 (s, 2H, CH2); 7.42-7.59 (m, 5H, ArH);
7.75 (d, 1H, ArH, J=7,8 Hz); 8.03-8.06 (m, 2H, ArH); 8.38 (d, 1H, ArH, J=7,8 Hz).
N1 -[(ethoxycarbonyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one 66. Yield: 30%; 1H-NMR (DMSO-d6, ppm): 1.22 (t, 3H, CH3,
J=6,9 Hz); 4.18-4.25 (m, 2H, CH2); 5.32 (s, 2H, CH2); 7.48-7.53 (m, 4H, ArH);
57
General Procedure for:
N10 -[BOC-(amminobutylcarbamoyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] (67), N10 -[BOC-(amminohexylcarbamoyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] (68), N1 -[BOC-(amminobutylcarbamoyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] (73) and N1 -[BOC-(amminohexylcarbamoyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] (74).
Compound 65 or 66 (0,6 mmol) was heated at 80 °C in a Pyrex capped tube with N-Boc-1,4-butanediamine or N-Boc-1,6-hexanediamine (1,2 mmol), for 3h (TLC analysis). The precipitate was dissolved with CH2Cl2 and washed with
HCl 10%. After drying with MgSO4, the dichloromethane solution was
evaporated under reduced pressure, yielding the crude product that was purified by flash-chromatography (AcOEt/Hexane=5/5).
N10
-[BOC-(amminobutylcarbamoyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] 67. Yield: 70%; 1H-NMR (DMSO-d6, ppm):
1.23-1.51 (m, 13H, 3CH3, 2CH2); 2.85-3.09 (m, 4H, 2CH2); 5.09 (s, 2H, CH2);
6.77 (m, 1H, NH, exch D2O); 7.41-7.58 (m, 5H, ArH); 7.74 (d, 1H, J=8 Hz,
ArH); 8.02-8.05 (m, 2H, ArH); 8.16-8.18 (m, 1H, NH, exch D2O); 8.39 (d, 1H,
J=8Hz, ArH).
N10
-[BOC-(amminohexylcarbamoyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] 68. Yield: 74%; 1H-NMR (DMSO-d6, ppm):
1.24-1.37 (m, 17H, 4CH2, 3CH3); 2.56-2.59 (m, 4H, 2CH2); 5.10 (s, 2H, CH2);
6.75-6.76 (m, 1H, NH, exch D2O); 7.37-7.44 (m, 1H, ArH); 7.46-7.58 (m, 4H,
ArH); 7.74 (d, 1H, ArH, J=8 Hz); 8.03-8.05 (m, 2H, ArH); 8.15 (t, NH, exch D2O, J=5.6 Hz); 8.40 (d, 1H, ArH, J=6 Hz).
N1
-[BOC-(amminobutylcarbamoyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] 73. Yield: 79%; 1H-NMR (DMSO-d6, ppm):
1.22-1.34 (m, 13H, 2CH2, 3CH3); 2.89-3.07 (m, 4H, 2CH2); 5.07 (s, 2H, CH2);
6.76-6.79 (m, 1H, NH, exch D2O); 7.46-7.82 (m, 5H, ArH); 8.14-8.20 (m, 2H,
58 N1
-[BOC-(amminohexylcarbamoyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] 74. Yield: 81%; 1H-NMR (DMSO-d6, ppm):
1.18-1.30 (m, 17H, 4 CH2 , 3CH3); 2.80-3.06 (m, 4H, 2CH2); 5.03 (s, 2H, CH2);
6.70-6.74 (m, 1H, NH, exch D20); 7.45-7.73 (m, 5H, ArH); 8.15-8.19 (m, 2H, ArH);
8.24-8.27 (m, 1H, NH, exch D2O); 8.50 (d, 2H, ArH, J=7,8 Hz).
General Procedure for:
N10 -[(amminobutylcarbomoyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] (69), N10 -[(amminohexylcarbomoyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] (70), N1 -[(amminobutylcarbomoyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] (75) and N1 -[(amminohexylcarbomoyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] (76).
Trifluoroacetic acid (2,1 mmol) was added to a stirred solution of derivatives
67
,
68,
73or
74 (0,21 mmol) dissolved in 10 mL of CH2Cl2. The reactionmixture was stirred at room temperature until the disappearance of the starting material (8h, TLC analysis). The dichloromethane was evaporated under reduced pressure and then the product obtained was diluted with ice. Once the ice dissolved, the solution was treated with saturated NaOH aqueous solution to pH=10 and extracted with CH2Cl2. After drying with
MgSO4,the organic solution was evaporated under reduced pressure, yielding
the crude product.
N10
-[(amminobutylcarbomoyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] 69. Yield: 87%; 1H-NMR (DMSO-d6, ppm):
1.21-1.50 (m, 6H, 5CH2); 3.07 (q, 2H, CH2, J=8,8 Hz); 5.07 (s, 2H, CH2);
7.39-7.57 (m, 5H, ArH); 7.73 (d, 1H, ArH, J=8 Hz); 8.01 (d, 2H, ArH, J=4.8 Hz); 8.18 (m, 1H, NH, exch D2O); 8.37(d, 1H, ArH, J=8,2 Hz).
N10
-[(amminohexylcarbomoyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] 70. Yield: 83%; 1H-NMR (DMSO-d6, ppm):
7.40-59 7.55 (m, 4H, ArH); 7.72 (d, 1H, ArH, J=8,4 Hz); 7.99-8.01 (m, 2H, ArH); 8.14-8.18 (m, 1H, NH, exch D2O); 8.37-8.40 (m, 2H, ArH).
N1
-[(amminobutylcarbomoyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] 75. Yield: 88%; 1H-NMR (DMSO-d6, ppm):
1.32-1.46 (m, 6H, 3CH2); 3.10 (q, 2H, CH2, J=9,7Hz); 5.10 (s, 2H, CH2);
7.47-7.54 (m, 4H, ArH); 7.68 (m, 1H, ArH); 7.73 (d, 1H, ArH, J=8Hz); 8.19-8.21 (m, 2H, ArH); 8.33 (t, 1H, J=6 Hz, NH, exch D2O); 8.53 (d, 1H, ArH, J=8Hz).
N1
-[(amminohexylcarbomoyl)methyl]-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] 76. Yield: 84%; 1H-NMR (DMSO-d6, ppm):
1.24-1.52 (m, 10H, 5CH2); 3.08 (q, 2H, CH2, J=9,8 Hz); 5.08 (s, 2H, CH2);
7.46-7.53 (m, 4H, ArH); 7.63-7.73 (m, 2H, ArH); 8.19 (d, 2H, ArH, J=8 Hz); 8.28 (t, 1H, NH, J=6,8 Hz, exch D2O); 8.52 (d, 1H, ArH, J=8 Hz).
General Procedure for:
N10 -((4-(4-nitrobenzo[c][1,2,5]oxadiazol-7- ylamino)hexylcarbamoyl)methyl)-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] (71), N10 -((6-(4-nitrobenzo[c][1,2,5]oxadiazol-7- ylamino)butylcarbamoyl)methyl)-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] (72), N1 -((4-(4-nitrobenzo[c][1,2,5]oxadiazol-7- ylamino)butylcarbamoyl)methyl)-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] (77) and N1 -((6-(4-nitrobenzo[c][1,2,5]oxadiazol-7- ylamino)hexylcarbamoyl)methyl)-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] (78).
4-Chloro-7-nitrobenzofurazan (0,28 mmol) dissolved in 1 mL of anhydrous DMF was added dropwise to a stirred solution of derivatives 69, 70, 75 or
76 (0,28 mmol), in 10 mL of the same solvent, cooled at 0 °C, followed by addition of a solution of Triethylamine (0,30 mmol). The reaction mixture was allowed under stirring for 48h at room temperature (TLC analysis). The organic solvent was eliminated under vacuum, the product obtained was diluted with ice and the solution was extracted with CH2Cl2. After drying
60 pressure, yielding the crude product that was purified by flash-chromatography (AcOEt=10).
N10 -((4-(4-nitrobenzo[c][1,2,5]oxadiazol-7-ylamino)hexylcarbamoyl)methyl)-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] 71. Yield: 51%;
1H-NMR (DMSO-d6, ppm): 1.54-1.58 (m, 2H, CH2); 1.68-1.72 (m, 2H, CH2);
3.20-3.21 (m, 2H, CH2); 3.42-3.45 (m, 2H. CH2); 5.11 (s, 2H, CH2); 6.32-6.34 (m,
1H, ArH); 7.32-7.36 (m, 1H, ArH); 7.45-7.49 (m, 4H, ArH); 7.65-7.66 (m, 1H, ArH); 8.03-8.05 (m, 2H, ArH); 8.26 (t, 1H, NH, J=5,4 Hz, exch D2O);
8.32-8.36 (m, 2H, ArH); 9.46-9.48 (m, 1H, NH, exch D2O).
N10 -((6-(4-nitrobenzo[c][1,2,5]oxadiazol-7-ylamino)butylcarbamoyl)methyl)-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] 72. Yield: 48%;
1H-NMR (DMSO-d6, ppm): 1.30-1.42 (m, 6H, 3CH2); 1.57-1.60 (m, 2H, CH2); 3.12
(q, 2H, CH2, J=9Hz); 3.36-3.39 (m, 2H, CH2); 5.08 (s, 2H, CH2); 6.26-6.29 (m,
1H, ArH); 7.38 (t, 1H, ArH, J=7,9Hz); 7.48-7.54 (m, 4H, ArH); 7.72 (d, 1H, ArH, J=7,8Hz); 8.03-8.05 (m, 2H, ArH); 8.18 (t, 1H, NH, J=6Hz, exch D2O);
8.36 (d, 1H, ArH, J=8,3Hz); 8.45 (d, 1H, ArH, J=8Hz); 9.46-9.55 (m, 1H, NH, exch D2O). N1 -((4-(4-nitrobenzo[c][1,2,5]oxadiazol-7-ylamino)butylcarbamoyl)methyl)-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] 77. Yield: 54%; 1H-NMR (DMSO-d6, ppm): 1.49-1.54 (m, 4H, 2CH2); 1.55-1.69 (m, 2H, CH2); 3.47-3.48 (m, 2H, CH2); 5.10 (s, 2H, CH2); 6.38-6.40 (m, 1H, ArH); 7.45-7.50
(m, 3H, ArH); 7.62-7.66 (m, 1H, ArH); 7.74 (d, 1H, ArH, J=8,4 Hz); 7.72-7.75 (m, 1H, ArH); 7.96 (s, 1H, ArH); 8.15-8.16 (m, 1H, ArH); 8.35 (t, 1H, NH, exch D20, J=5,4 Hz); 8.43 (d, 1H, ArH, J=9,2 Hz); 8.52 (d, 1H, ArH, J=8 Hz); 9.51-9.55 (m, 1H, NH, exch D2O). N1 -((6-(4-nitrobenzo[c][1,2,5]oxadiazol-7-ylamino)hexylcarbamoyl)methyl)-3-phenyl[1,2,4]Triazino[4,3-a]benzimidazol-[4(10H)-one] 78. Yield: 49%; 1H-NMR (DMSO-d6, ppm): 1.19 (s, 2H, CH2); 1.31-1.37 (m, 2H, CH2); 1.41-1.53 (m, 2H, CH2); 1.62-1.67 (m, 2H, CH2); 3.10-3.15 (m, 2H, CH2); 3.42-3.44 (m,2H, CH2); 5.10 (s, 2H, CH2); 6.34 (d, 1H, ArH, J=8,4Hz); 7.43-7.49 (m, 4H, ArH);
7.64-7.67 (m, 1H, ArH); 7.73 (d, 1H, ArH, J=8Hz); 8.16 (d, 2H, ArH, J=6,8Hz); 8.30 (t, 1H, NH, exch D2O; J=7,8Hz); 8.44 (d, 1H, ArH, J=8,8Hz);
