3. Materials and Methods
Solvents were distilled prior to use as follows: dichloromethane was distilled from phosphorous pentoxide; ethyl acetate was distilled from potassium carbonate; ethanol and methanol were distilled from magnesium in the presence of iodine; toluene was distilled from sodium and benzophenone; hexane was distilled prior to use; tetrahydrofuran was freshly distilled from sodium and benzophenone. Diethyl ether was obtained pure from Riedel-de Haën. Organic phases were dried using anhydrous magnesium sulphate. All commercial reagents were used without further purification unless otherwise stated. All samples were confirmed as >95% pure by use of high resolution LCMS analysis.
Infrared spectra were recorded as a thin film on sodium chloride plates for liquids or potassium bromide (KBr) disc for solids on a Perkin Elmer Spectrum 100 FT-IR spectrometer.
1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded on a Bruker Avance 300 NMR spectrometer. 1H (600 MHz) and 13C (150.9 MHz) NMR spectra were recorded on a Bruker Avance III 600 MHz NMR spectrometer equipped with a dual CH cryoprobe. All spectra were recorded at room temperature (~20 °C) in deuterated dimethylsulfoxide (DMSO-d6) were assigned using the DMSO-d6 peak as the reference peak. Chemical shifts (δH and δC) are expressed in parts per million (ppm) relative to the reference peak. Coupling constants (J) are expressed in Hertz (Hz). Splitting patterns in 1H NMR spectra are designated as s (singlet), br s (broad singlet), d (doublet), br d (broad doublet), t (triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets), ddd (doublet of doublet of doublets), ddt (doublet of doublet of triplets) and m (multiplet).
Low resolution mass spectra were recorded on a Waters Quattro Micro triple quadrupole spectrometer (QAA1202) in electrospray ionisation (ESI) mode using 50% acetonitrile:water containing 0.1% formic acid as eluent. High resolution mass spectra (HRMS) were recorded on a Waters LCT Premier Time of Flight spectrometer (KD-160) in electrospray ionisation (ESI) mode using 50% acetonitrile:ater containing 0.1% formic acid as eluent.
Melting points were measured in a uni-melt Thomas Hoover capillary melting point apparatus and are uncorrected. Thin layer chromatography (TLC) was carried out on precoated silica gel plates (Merck 60 PF254) or aluminium oxide TLC paltes (Sigma). Visualisation was achieved by UV light detection (254 nm).
5-Methyl-6H-pyrido[4,3-b]carbazole-11-carboxylic acid 9. 5-Methyl-6H-pyrido[4,3-b]carbazole-11-carbaldehyde 8 (92 mg, 0.353 mmol) in acetonitrile (9 mL) was treated with dimethylsulfoxide (0.03 mL, 0.423 mmol) and conc. sulfuric acid (0.3 mL, 0.622 mmol). A solution of sodium chlorite (48 mg, 0.530 mmol) in water (3 mL) was added dropwise and the reaction mixture was stirred at room temperature overnight. The reaction was quenched with sodium sulfite (22 mg, 0.177 mmol) in water (1 mL). The acetonitrile was evaporated to leave an aqueous solution, which was carefully adjusted to pH 5 with saturated aqueous sodium bicarbonate to precipitate the product. The mixture was cooled to 0 °C, filtered and washed with water (3 mL), to give 5-methyl-6H-pyrido[4,3-b]carbazole-11-carboxylic acid 9. The orange solid was dried at 0.02 mbar for 24 h (71 mg, 73.2%). m.p. >300 °C; vmax/cm−1 (KBr): 3161 (NH), 3010 (OH broad), 1689 (C=O), 1648 (C=C arom.), 1597 (C=C arom.), 1488, 1463, 1417, 1241 (C-O stretch), 1109; δH (300 MHz, DMSO-d6): 2.88 [3H, s, C(5)CH3], 7.26 [1H, t, J = 7.9, C(9)H], 7.55–7.62 [2H, m, C(7)H, C(8)H], 8.03 [1H, d, J = 5.9, C(3)H], 8.21 [1H, d, J = 7.8, C(10)H], 8.47 [1H, d, J = 5.9, C(4)H], 9.43 [1H, s, C(1)H], 11.64 [1H, s, N(6)H]; δC (75.5 MHz, DMSO-d6): 12.3 [3H, s, C(5)CH3], 111.5 [CH, C(7)H], 113.3 (C, aromatic C), 117.7 [CH, C(3)H], 118.5 (C, aromatic C), 120.0 [CH, C(9)H], 120.3 (C, aromatic C), 122.3 (C, aromatic C), 123.1 [CH, C(10)H], 124.9 (C, aromatic C), 129.1 [CH, C(8)H], 132.2 (C, aromatic C), 136.1 [CH, C(4)H], 141.9 (C, aromatic C), 143.1 (C, aromatic C), 147.9 [CH, C(1)H], 168.8 [C, C(11)COOH]; m/z (ESI+): 277 [(M+H)+ 40%], 115 (100%); HRMS (ESI+): Exact mass calculated for [C17H13N2O2]+ 277.0977. Found 277.0977.
5-Methyl-6H-pyrido[4,3-b]carbazole-11-carbonylchloride 10. 5-Methyl-6H-pyrido[4,3-b]carbazole-11-carboxylic acid 9 (99 mg, 0.358 mmol) in dichloromethane (50 mL), under N2, was treated with oxalyl chloride (0.04 mL, 0.459 mmol). Slight fizzing was observed on addition and mixture was stirred at room temperature for 20 h. The bright orange suspension was evaporated under reduced pressure. IR analysis showed the carbonyl peak at 1785 cm−1 indicating acid chloride formation.
N-Benzyl-5-methyl-6H-pyrido[4,3-b]carbazole-11-carboxamide 11. 5-Methyl-6H-pyrido[4,3-b]carbazole-11-carboxylic acid 9 (99 mg, 0.358 mmol) in dichloromethane (50 mL), under N2, was treated with oxalyl chloride (0.04 mL, 0.459 mmol) and stirred at room temperature for 20 h. The mixture was evaporated under reduced pressure, cooled to 0 °C under N2, and treated drop-wise with benzylamine (2 mL, 18.3 mmol). After stirring for 1 h, diethyl ether (40 mL) was added, the mixture was cooled and filtered to give a cream solid (220 mg) containing the desired product and residual benzylamine. This was stirred with diethyl ether and decanted (3 × 40 mL). Purification by column chromatography, eluting with dichloromethane:methanol (100:0–95:5), gave product 11 as a yellow solid (39 mg, 30%). m.p. 294–296 °C; vmax/cm−1 (KBr): 3159 (NH), 3051 (CH) 1731 (C=O), 1621 (C=C arom.), 1600 (C=C arom.), 1542, 1466, 1410, 1245; δH (600 MHz, DMSO-d6): 2.79 [3H, s, C(5)CH3], 4.65–4.70 [2H, m, CONHCH2], 6.98 [1H, t, J = 7.4, C(9)H], 7.28 [1H, t, J = 7.2, N-benzyl-C(4)H], 7.36 [2H, t, J = 7.3, N-benzyl-C(2)H, C(6)H], 7.43–7.50 [4H, m, C(7)H, C(8)H, N-benzyl-C(3)H, C(5)H], 7.72 [1H, d, J = 7.8, C(10)H], 7.93 [1H, d, J = 5.9, C(3)H], 8.37 [1H, d, J = 5.1, C(4)H], 9.19 [1H, s, C(1)H], 9.41 [1H, t, J = 5.9, C(11)CONH], 11.46 [1H, s, N(6)H]; δC (150.9 MHz, DMSO-d6): 12.2 [CH3, C(5)CH3], 43.1 [CH2, C(11)CONHCH2], 110.9 (CH, aromatic CH), 111.3 (C, aromatic C), 116.0 [CH, C(3)H], 119.1 [CH, C(9)H], 119.8 (C, aromatic C), 120.9 (C, aromatic C), 121.1 (C, aromatic C), 122.8 [CH, C(10)H], 127.1 [CH, N-benzyl-C(4)H], 127.2 (C, aromatic C), 127.9 (CH, aromatic CH), 128.0 (CH, aromatic CH), 128.2 (CH, aromatic CH), 128.4 (CH, aromatic CH), 128.5 (CH, aromatic CH), 131.7 (C, aromatic C), 138.9 (C, aromatic C), 140.4 (C, aromatic C), 140.7 [CH, C(4)H], 142.7 (C, aromatic C), 150.1 [CH, C(1)H], 167.1 [C, C(11)CONH]; m/z (ESI+): 366 [(M+H+), 100%]; HRMS (ESI+): Exact mass calculated for [C24H20N3O]+ 366.1606. Found 366.1615.
Methyl 5-methyl-6H-pyrido[4,3-b]carbazole-11-carboxylate 12. 5-Methyl-6H-pyrido[4,3-b]carbazole-11-carboxylic acid 9 (99 mg, 0.358 mmol) in dimethylformamide (8 mL) under N2, was heated gently until the acid dissolved. Carbonyldiimidazole (70 mg, 0.430 mmol) was added and the solution was heated at 120 °C for 4 h. The mixture was cooled to 0 °C and diethyl ether (20 mL) was added. The resulting precipitate was filtered and washed (diethyl ether 10 mL). The solid was cooled to 0 °C and treated dropwise with methanol (10 mL). The mixture was stirred at r.t. for 1 h and heated to reflux for 5 h. On cooling, a precipitate formed which was filtered to give the methyl ester, methyl 5-methyl-6H-pyrido[4,3-b]carbazole-11-carboxylate 12 (14 mg, 14%). m.p. 247–250 °C; vmax/cm−1 (KBr): 3147 (NH), 3008 (CH), 1742 (C=O), 1634 (C=C arom.), 1589 (C=C arom.), 1457, 1422, 1246 (C-O stretch), 1095; δH (300 MHz, DMSO-d6): 2.89 [3H, s, C(5)CH3], 4.22 [3H, s, C(11)COOCH3], 7.27 [1H, overlapping ddd, J = 8.0, 5.8, 2.3, C(9)H], 7.57–7.63 [2H, m, C(7)H, C(8)H], 7.99 [1H, d, J = 8.0, C(10)H], 8.04 [1H, d, J = 6.1, C(3)H], 8.50 [1H, br s, C(4)H], 9.38 [1H, br s, C(1)H], 11.73 [1H, s, N(6)H]; δC (150.9 MHz, DMSO-d6): 12.4 [CH3, C(5)CH3], 53.1 [CH3, C(11)COOCH3], 111.3 [CH, C(7)H or C(8)H], 113.7 (C, aromatic C), 116.2 [CH, C(3)H], 119.6 [CH, C(9)H], 120.2 (C, aromatic C), 121.3 (C, aromatic C), 121.8 (C, aromatic C), 122.5 [CH, C(10)H], 128.7 [CH, C(7)H or C(8)H], 131.6 (C, aromatic C), 140.2 (C, aromatic C), 140.9 [CH, C(4)H], 143.1 (C, aromatic C), 149.8 [CH, C(1)H], 168.4 [C, C(11)C=O]; m/z (ESI+): 291 [(M+H)+, 100%]. HRMS (ESI+): Exact mass calculated for [C18H15N2O2]+ 291.1134. Found 291.1121.
(E)-4-(5-methyl-6H-pyrido[4,3-b]carbazol-11-yl)but-3-en-2-one 13. A stirred solution of 5-methyl-6H-pyrido[4,3-b]carbazole-11-carbaldehyde 8 (300 mg, 1.15 mmol) in acetone (70 mL) was treated drop-wise with potassium permanganate (364 mg, 2.30 mmol) in water (70 mL). The mixture was stirred at r.t. for 1 h and heated to reflux for 22 h. Sodium bicarbonate (244 mg, 2.30 mmol) was added and stirred for 20 min. The mixture was filtered through celite and washed with water (100 mL) and acetone (200 mL). The filtrate was concentrated under reduced pressure to remove the acetone and then acidified to pH 5 with 20% aqueous HCl. The aqueous layer was extracted with chloroform:methanol (90:10, 3 × 100 mL). Organic extracts were combined and washed with water (1 × 100 mL) and brine (1 × 100 mL), dried over magnesium sulphate and evaporated under reduced pressure to give an orange solid (300 mg). Analysis showed that the desired carboxylic acid 9 had not formed but instead the condensation product (E)-4-(5-methyl-6H-pyrido[4,3-b]carbazol-11-yl)but-3-en-2-one 13 (87.0%). m.p. 263–265 °C; vmax/cm−1 (KBr): 3149(NH), 3087 (CH), 2982 (asymm. CH3 stretch), 2883 (symm. CH3 stretch), 1664 (C=O), 1620 (C=C), 1592 (C=C arom.), 1464, 1404, 1383, 1243; δH (300 MHz, DMSO-d6): 2.61 [3H, s, COCH3], 2.84 [3H, s, C(5)CH3], 6.75 [1H, d, J = 16.6, C(11)CH=CH], 7.25 [1H, overlapping ddd, J = 8.0, 6.6, 1.5, C(9)H], 7.53–7.61 [2H, m, C(7)H, C(8)H], 7.98 [1H, dd, J = 6.1, 0.7, C(3)H], 8.14 [1H, d, J = 8.0, C(10)H], 8.47 [1H, d, J = 6.1, C(4)H], 8.59 [1H, d, J = 16.6, C(11)CH=CH], 9.58 [1H, s, C(1)H], 11.57 [1H, s, N(6)H]; δC (75.5 MHz, DMSO-d6): 12.2 [CH3, C(5)CH3], 27.8 (CH3, COCH3), 111.1 [CH, C(7)H], 111.7 (C, aromatic C), 115.9 [CH, C(3)H], 119.4 [CH, C(9)H], 120.5 (C, aromatic C), 121.9 (C, aromatic C), 122.4 (C, aromatic C), 123.6 [CH, C(10)H], 126.0 (C, aromatic C), 127.9 [CH, C(8)H], 132.3 (C, aromatic C), 135.9 [CH, C(11)CH=CH], 138.4 [CH, C(11)CH=CH], 140.2 (C, aromatic C), 140.9 [CH, C(4)H], 143.0 (C, aromatic C), 150.1 [CH, C(1)H], 197.8 (C, C=O); m/z (ESI+): 301[(M+H)+ 100%]; HRMS (ESI+): Exact mass calculated for [C20H17N2O]+ 301.1341. Found 301.1348.
(E)-N-(4-(5-methyl-6H-pyrido[4,3-b]carbazol-11-yl)but-3-en-2-yl)formamide 14. (E)-4-(5-Methyl-6H-pyrido[4,3-b]carbazol-11-yl)but-3-en-2-one 13 (91 mg, 0.303 mmol), formamide (0.2 mL, 5.03 mmol) and formic acid (0.1 mL, 2.65 mmol) were heated to 150 °C for 1.5 h, at which point TLC analysis indicated consumption of the starting material. On cooling, saturated aqueous sodium bicarbonate (8 mL) was added and extraction was attempted with dichloromethane:methanol (90:10), however the organic extracts contained no material. The aqueous layer was treated with 20% aqueous HCl to pH 1, stirred for 20 min and then adjusted to pH 10 with 20% aqueous NaOH. The aqueous layer was extracted with dichloromethane–methanol (90:10 3 × 30 mL and 80:20 4 × 30 mL), dried and evaporated under reduced pressure to give an orange solid (64 mg). Purification by column chromatography on alumina, eluting with dichloromethane:methanol (97:3–90:10) gave three fractions. The largest of these (34 mg) was repurified by chromatography on silica to give an orange solid (19 mg). This was found to be the formamide, (E)-N-(4-(5-methyl-6H-pyrido[4,3-b]carbazol-11-yl)but-3-en-2-yl)formamide 14. δH (300 MHz, DMSO-d6): 1.47 [3H, d, J = 6.9, C(11)CH=CH–CH(CH3)], 2.83 [3H, s, C(5)CH3], 4.88–4.95 [1H, m, C(11)CH=CH–CH(CH3)], 6.14 [1H, dd, J = 16.2, 5.6, C(11)CH=CH], 7.21 [1H, overlapping ddd, J = 8.0, 6.7, 1.4, C(9)H], 7.36 [1H, d, J = 16.5, C(11)CH=CH], 7.52 [1H, td, J = 7.4, 1.1, C(8)H], 7.56–7.59 [1H, m, C(7)H], 7.99 [1H, br s, NHCHO], 8.23 [1H, br s, C(3)H], 8.33 [1H, d, J = 7.7, C(10)H], 8.43 [1H, br s, NHCHO], 8.54 [1H, d, J = 7.5, C(4)H], 9.58 [1H, br s, C(1)H], 11.51 [1H, s, N(6)H]; m/z (ESI+): 330 [(M+H)+ 60%], 169 (100%); HRMS (ESI): Exact mass calculated for [C21H20N3O]+ 330.1606. Found 330.1619.
9-Formyl-5-methyl-6H-pyrido[4,3-b]carbazole-11-carboxylic acid 15. 5-Methyl-6H-pyrido[4,3-b]carbazole-11-carboxylic acid 9 (497 mg, 1.80 mmol) in trifluoroacetic acid (60 mL), was treated with hexamethylenetetramine (2.522 g, 18.0 mmol) portionwise over 5 min and heated to reflux for 25 min. On cooling, the reaction mixture was concentrated to approx. one quarter volume, water (30 mL) was added and the solution was transferred to a large conical flask (500 mL). The solution was cooled to 0 °C and neutralized with solid sodium bicarbonate while stirring vigorously. The mixture was stirred at 0 °C for 1 h, readjusted to pH 7 and filtered to give a brown solid which was dried at 0.02 mbar for 2 days (449 mg, 81.9%). m.p. 315–317 °C; vmax/cm−1 (KBr): 3069 (OH broad), 1677 (C=O × 2, broad), 1583 (C=C arom.), 1472, 1404, 1349, 1242 (C-O stretch), 1128, 808; δH (600 MHz, DMSO-d6): 2.88 [3H, s, C(5)CH3], 7.72 [1H, d, J = 8.3, C(7)H], 8.04 [1H, d, J = 6.0, C(3)H], 8.10 [1H, d, J = 8.4, C(8)H], 8.50 [1H, d, J = 6.0, C(4)H], 8.80 [1H, s, C(10)H], 9.51 [1H, s, C(1)H], 10.04 [1H, s, C(9)CHO], 12.14 [1H, s, N(6)H]; δC (150.9 MHz, DMSO-d6): 12.3 [3H, s, C(5)CH3], 111.4 [CH, C(7)H], 113.0 (C, aromatic C), 116.2 [CH, C(3)H], 119.7 (C, aromatic C), 120.4 (C, aromatic C), 121.3 (C, aromatic C), 125.7 [CH, C(10)H], 128.5 (C, aromatic C), 129.8 [CH, C(8)H], 129.9 (C, aromatic C), 132.4 (C, aromatic C), 140.8 [CH, C(4)H], 141.1 (C, aromatic C), 146.9 (C, aromatic C), 150.7 [CH, C(1)H], 169.2 [C, C(11)COOH], 191.6 [CH, C(9)CHO]; m/z (ESI+): 305 [(M+H)+ 70%], 155 (40%), 64 (100%); HRMS (ESI+): Exact mass calculated for [C18H13N2O3]+ 305.0926. Found 305.0940.
Benzylammonium 9-((benzylimino)methyl)-5-methyl-6H-pyrido[4,3-b]carbazole-11-carboxylate 16. A suspension of 9-formyl-5-methyl-6H-pyrido[4,3-b]carbazole-11-carboxylic acid 15 (194 mg, 0.638 mmol) in dichloromethane (65 mL), under N2, was treated with oxalyl chloride (0.08 mL, 0.917 mmol) and stirred at room temperature overnight. Additional oxalyl chloride (0.08 mL, 0.917 mmol), was added and the reaction was stirred for 4 h. The mixture was evaporated under reduced pressure, cooled to 0 °C under N2, and treated drop-wise with benzylamine (2 mL, 18.3 mmol). After stirring for 1 h, diethyl ether (40 mL) was added, the mixture was cooled and filtered to give a yellow solid. Crude analysis showed this to contain residual benzylamine. Recrystallisation from dichloromethane followed by a second recrystallisation from methanol gave product still containing benzylamine in 1:1 ratio with product. To investigate whether an amide or imine had formed, the product (27 mg) was dissolved in dichloromethane:methanol (90:10, 10 mL) and washed with 1M HCl (10 mL). The organic layer was dried and evaporated under reduced pressure. NMR and MS analysis showed that the compound had hydrolysed to the starting material 15, indicating imine rather than amide formation. Full analysis (along with subsequent reduction of the imine) confirmed the product as the imine salt, benzylammonium 9-((benzylimino)methyl)-5-methyl-6H-pyrido[4,3-b]carbazole-11-carboxylate 16 (208 mg, 82.9%). m.p. 241–243 °C; vmax/cm−1 (KBr): 3029 (NH), 2864 (OH, broad), 1598 (C=O), 1572 (C=C arom.), 1494, 1450, 1402, 1347, 1278, 1244 (C-O stretch); δH (300 MHz, DMSO-d6): 2.81 [3H, s, C(5)CH3], 4.06 [2H, s, benzylammmonium-CH2], 4.74 [2H, s, C(9)CH=NCH2], 7.23–7.29 [1H, m, iminobenzyl-C(4)H], 7.33–7.42 [7H, m, iminobenzyl-C(2)H, C(3)H, C(5)H, C(6)H, benzylammmonium-C(3)H, C(4)H, C(5)H], 7.48 [2H, dd, J = 7.7, 1.7, benzylammmonium-C(2)H, C(6)H], 7.56 [1H, d, J = 8.3, C(7)H], 7.91 [1H, dd, J = 6.1, 0.7, C(3)H], 7.97 [1H, dd, J = 8.4, 1.6, C(8)H], 8.38 [1H, d, J = 6.1, C(4)H], 8.52 [1H, s, C(9)CH=N], 8.73 [1H, d, J = 1.1, C(10)H], 9.50 [1H, s, C(1)H], 11.64 [1H, s, N(6)H]; δC (75.5 MHz, DMSO-d6): 12.0 [CH3, C(5)CH3], 42.3 [CH2, benzylammonium CH2], 64.1 [CH2, C(9)CH=N–CH2], 110.6 (CH, aromatic CH), 115.7 (CH, aromatic CH), 119.2 (C, aromatic C), 119.4 (C, aromatic C), 122.2 (C, aromatic C), 124.3 (CH, aromatic CH), 125.1 (C, aromatic C), 126.7 (CH, aromatic CH), 127.3 (C, aromatic C), 127.9 (CH, 2 × aromatic CH), 128.2 (CH, aromatic CH), 128.3 (CH, 2 × aromatic CH), 128.5 (CH, 2 × aromatic CH), 128.6 (CH, aromatic CH), 128.7 (CH, 2 × aromatic CH), 131.3 (C, aromatic C), 132.1 (C, aromatic C), 134.6 (C, aromatic C), 139.9 (CH, aromatic CH), 140.5 (CH, aromatic CH), 141.1 (C, aromatic C), 144.3 (C, aromatic C), 144.5 (C, aromatic C), 152.0 (CH, aromatic CH), 162.2 (C, C=O); m/z (ESI+): 394 [(M+H)+ 20%], 305 (20%), 108 (100%); HRMS (ESI+): Exact mass calculated for [C25H20N3O2]+ 394.1556. Found 394.1560.
9-((Benzylamino)methyl)-5-methyl-6H-pyrido[4,3-b]carbazole-11-carboxylic acid 17. A solution of benzylammonium 9-((benzylimino) methyl)-5-methyl-6H-pyrido[4,3-b]carbazole-11-carboxylate 16 (88 mg, 0.224 mmol) in absolute ethanol (12 mL) was treated with sodium cyanoborohydride (21 mg, 0.338 mmol) and heated to reflux for 4.5 h. The solution was evaporated under reduced pressure and water (10 mL) was added. The mixture was stirred for 15 min, cooled and filtered. The orange solid was washed with water (5 mL) and diethyl ether (10 mL) and dried at 0.1 mbar for 24 h (56 mg, 62.9%). m.p. >300 °C (without melting); vmax/cm−1 (KBr): 3370 (NH), 3188 (OH, broad), 3056 (CH), 1596 (C=O), 1565 (C=C arom.), 1482, 1403, 1348, 1247 (C-O stretch); δH (600 MHz, DMSO-d6): 2.73 [3H, s, C(5)CH3], 3.89 [2H, s, one of CH2], 4.04 [2H, s, one of CH2], 7.21–7.26 [3H, m, N-benzyl-C(3)H, C(4)H, C(5)H], 7.39–7.40 [2H, m, N-benzyl-C(2)H, C(6)H], 7.46 [1H, d, J = 7.3, C(7)H], 7.53 [1H, d, J = 7.5, C(8)H], 7.82 [1H, d, J = 5.6, C(3)H], 8.29 [1H, d, J = 5.6, C(4)H], 8.42 [1H, s, C(10)H], 9.44 [1H, s, C(1)H], 11.39 [1H, s, N(6)H]; δC (150.9 MHz, DMSO-d6): 12.0 [CH3, C(5)CH3], 50.3 (CH2), 51.4 (CH2), 108.3 (C, aromatic C), 110.4 [CH, C(7)H], 115.6 [CH, C(3)H], 119.1 (C, aromatic C), 119.3 (C, aromatic C), 122.4 (C, aromatic C), 124.5 [CH, C(10)H], 127.7 [CH, N-benzyl-C(4)H], 128.1 (CH, C(8)H], 128.3 [CH, N-benzyl-C(3)H, N-benzyl-C(5)H], 129.0 [CH, N-benzyl-C(2)H, N-benzyl-C(6)H], 129.1 (C, aromatic C), 132.0 (C, aromatic C), 133.7 (C, aromatic C), 136.0 (C, aromatic C), 140.2 [CH, C(4)H], 141.3 (C, aromatic C), 142.2 (C, aromatic C), 152.2 [CH, C(1)H], 170.8 (C, C=O); m/z (ESI+): 396 [(M+H)+ 80%], 306 (100%); HRMS (ESI+): Exact mass calculated for [C25H22N3O2]+ 396.1712. Found 396.1729.
Topoisomerase II decatenation assay. The decatenation assay kit was obtained from Inspiralis, Norwich Bioincubator, Norwich Research Park, Colney, Norwich, UK. The kit comprised of the following: assay buffer (supplied as 10× stock) containing 50 mM Tris.HCl (pH 7.5), 125 mM NaCl, 10 mM MgCl2, 5 mM DTT and 100 µg/mL albumin; dilution buffer containing 50 mM Tris. HCl (pH 7.5), 100 mM NaCl, 1 mM DTT, 0.5 mM EDTA, 50% (v/v) glycerol, 50 µg/mL albumin; ATP 30 mM; kDNA (100 ng/µl); 10 U/µL human topoisomerase II in dilution buffer; 5× stop buffer containing 2.5% SDS, 15% Ficoll-400, 0.05% bromophenol blue, 0.05% xylene cyanol and 25 mM EDTA. Tris-acetate-EDTA buffer (supplied as 10X buffer) and agarose were obtained from Sigma Life Sciences (Dublin, Ireland) and Safe View Stain was supplied by NBS Biologicals, Cambridgeshire, England.
The topo II decatenation assay protocol involved initial incubation of each inhibitor candidate (100 µM) along with a stock solution containing water, ATP, assay buffer, kDNA obtained from the mitochondrial DNA of Crithidia fasciculate, and topo II, at 37 °C for 1 h. Following addition of stop buffer, agarose DNA gel electrophoresis was run at 50 V for 2 h using a Consort EV243 power pack, to determine the relative amounts of decatenated DNA bands obtained in each compound lane. Positive (water), as well as negative controls (ellipticine) were incorporated in order to validate the results of each run. The resulting gels were viewed under UV light using a DNR Bio-Imaging System and photographed using GelCapture software.
NCI-60 Anti-cancer screening. Tested compounds were initially solubilised in DMSO, diluted into RPMI 1640 and 5% fetal bovine serum/L-glutamine, and added to 96-well plates containing cell lines previously cultured for 24 h. After 48-h incubation, the media were removed, and the cells were fixed and stained with sulforhodamine B to determine overall percent growth/total protein content. Unbound dye was removed with five washes of 1% acetic acid, and the plates were allowed to air dry. The dye was then resolubilised in Tris buffer, and the colorimetric absorbance was measured (515 nm). Growth inhibition was measured relative to the response generated from proliferating cells cultured under identical conditions for 48 h. In the five dose study, serial 5 × 10-fold dilution from an initial DMSO stock solution was performed, prior to incubation at each individual concentration (10 nM, 100 nM, 1 µM, 10 µM and 100 µM).
Using seven absorbance measurements (time zero (Tz), control growth (C), and test growth in the presence of drug at the five concentration levels (Ti)), the percentage growth was calculated at each of the drug concentrations levels. Percentage growth inhibition was calculated as:
Three dose response parameters were calculated for each experimental agent. Growth inhibition of 50% (GI50) was calculated from [(Ti−Tz)/(C−Tz)] × 100 = 50, which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by Sulforhodamine B staining) in control cells during the drug incubation. The drug concentration resulting in total growth inhibition (TGI) was calculated from Ti = Tz. The LC50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment was calculated from [(Ti−Tz)/Tz] × 100 = −50. Values were calculated for each of these three parameters if the level of activity was reached; however, if the effect was not reached or was exceeded, the value for that parameter was expressed as greater or less than the maximum or minimum concentration tested [
36,
37]. Data from one dose experiments pertains to the percentage growth at 10 µM.
COMPARE analysis. COMPARE analysis was conducted using the private access system provided by the National Cancer Institute (
https://dtp.cancer.gov/databases_tools/compare.htm). Seed compounds were analysed using a number of target sets: synthetic compounds, mechanistic set, standard agents, marketed drugs and diversity set. While the minimum correlation was set to 0.4, correlations of less than 0.5 were discounted. All other criteria were unchanged. Experiments that were carried out at different concentrations to the seed compound were ignored unless the concentration deviated by ±0.1, as was usual for older testing methods. COMPARE analysis was conducted solely on five dose data for compounds
11 and
13 [
38].