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J. Ungvarsky et al. / Bioorganic Chemistry 57 (2014) 13–29
0
2.2.12. Synthesis of 3,6-bis[3-(2-dimethylaminoethyl)thioureido]-9-
[40-(N,N-dimethylamino)phenylamino]acridine (15)
2ꢀCH3 ), 2.47 (t, 4H, 2ꢀCH2AN(CH3)2, J = 6.60), 2.21 (s, 12H,
4ꢀCH3). 13C NMR (150 MHz, CD3OD): 156.00 (2ꢀC@NH), 153.38
(C9), 150.47 (C4a, C10a), 149.64 (C3, C6), 148.79 (C40), 138.57
(C10), 127.22 (C1, C8), 122.95 (C20, C60), 122.50 (C2, C7), 115.97
(C30, C50), 115.89 (C4, C5), 114.69 (C8a, C9a), 59.67 (2ꢀCH2-
The diisothiocyanate 14 (0.1 g, 0.23 mmol) was dissolved in
20 mL of chloroform and cooled to ꢁ20 °C. Triethylamine (0.4 mL,
2.86 mmol)
and
N,N-dimetylethylenediamine
(0.07 mL,
0
0.64 mmol) were added to the mixture which was then placed in
a freezer to react overnight. The end of the reaction was monitored
with TLC (eluent MeOH/NH4OH 50:4). Chloroform was evaporated
in vacuo and a crude bisthiourea 15 was purified by column chro-
matography on silica gel eluted by a mixture of methanol–25%
water solution of ammonia (50:4). After evaporation of the eluent
in vacuo, the bisthiourea 15 was diluted with a small quantity of
methanol and subsequently poured into a saturated solution of
sodium hydrogencarbonate. The precipitate 15 was then filtered
off.
AN(CH3)2), 45.50 (4ꢀCH3), 41.88 (2ꢀCH3 ), 40.13 (2ꢀCH2ANH).
2.2.15. Synthesis of 3,6-bis[3-(2-dimethylaminoethyl)-2-
methylguanidino]-9-[40-(N,N-dimethylamino)phenylamino]acridine
(18)
Triethylamine (0.5 mL, 3.58 mmol), anhydrous sodium sulfate
(1 g, 7.04 mmol), calcium chloride (0.05 g, 0.45 mmol), mercury(II)
oxide (0.12 g, 0.58 mmol), and methylamine hydrochloride (0.2 g,
2.09 mmol) were added to the bisthiourea 15 (0.05 g, 0.08 mmol)
in 10 mL of methanol with dosing intervals of 5 min for each reac-
tant. The reaction mixture was refluxed for 1 h. An insoluble resi-
due was filtered off and a crude product 18 was purified by
column chromatography on silica gel eluted by a methanol–25%
water solution of ammonia (50:4).
Yield: 65%, brown solid, mp: 155–157 °C. Anal. Calcd for
C31H41N9S2 (603.85): 61.66% C, 6.84% H, 20.88% N. Found: 61.55%
C, 6.69% H, 20.80% N. 1H NMR (600 MHz, CD3OD): 7.97 (s, 2H, H-
4,5), 7.78 (d, 2H, H-1,8, J = 7.20), 7.12 (s, 2H, 2H, H-2,7), 6.91 (d,
2H, H-20,60, J = 9.00), 6.73 (d, 2H, H-30,50, J = 8.40), 3.74 (s, 4H,
Yield: 25%, light red solid, mp: 133–135 °C. Anal. Calcd for
0
2ꢀCH2ANH), 2.90 (s, 6H, 2ꢀCH3 ), 2.61 (t, 4H, 2ꢀCH2AN(CH3)2,
C33H47N11 (597.80): 66.30% C, 7.92% H, 25.77% N. Found: 66.22%
J = 6.00), 2.30 (s, 12H, 4ꢀCH3). 13C NMR (150 MHz, CD3OD):
182.14 (2ꢀC@S), 151.14 (C9), 149.58 (C40), 147.39 (C4a, C10a),
143.74 (C3, C6), 136.26 (C10), 127.00 (C1, C8), 124.26 (C20, C60),
119.66 (C2, C7), 115.17 (C30, C50), 114.40 (C4, C5), 113.58 (C8a,
C9a), 58.54 (2ꢀCH2AN(CH3)2), 45.61 (4ꢀCH3), 43.10 (2ꢀCH2ANH),
C, 7.83% H, 25.70% N. 1H NMR (600 MHz, CD3OD): 8.25 (d, 2H, H-
1,8, J = 9.00), 8.15 (d, 2H, H-4,5, J = 8.40), 7.03 (m, 6H, H-2,7, H-
0
20,60, H-30,50), 3.64 (s, 6H, 2ꢀCH3 ), 3.35 (t, 4H, 2ꢀCH2ANH,
J = 6.60), 2.83 (s, 6H, 2ꢀCH3AN@C), 2.54 (t, 4H, 2ꢀCH2AN(CH3)2,
J = 6.60), 2.29 (s, 12H, 4ꢀCH3). 13C NMR (150 MHz, CD3OD):
157.86 (2ꢀCNACH3), 157.05 (C9), 156.73 (C40), 149.75 (C4a,
C10a), 144.55 (C3, C6), 141.53 (C10), 127.19 (C1, C8), 125.91 (C4,
C5), 122.73 (C2, C7), 122.03 (C30, C50), 109.35 (C20, C60), 108.76
(C8a, C9a), 60.09 (2ꢀCH2ANA(CH3)2), 45.52 (4ꢀCH3), 40.81
0
41.45 (2ꢀCH3 ).
2.2.13. Synthesis of 3,6-bis[3-(2-dimethylaminoethyl)ureido]-9-[40-
(N,N-dimethylamino)phenylamino]acridine (16)
0
Mesitylnitrile oxide (0.05 g, 0.33 mmol) was added to a suspen-
sion of the bisthiourea 15 (0.05 g, 0.08 mmol) in 7 mL of methanol
and stirred well overnight at room temperature. A resultant bisu-
rea 16 was filtered off and purified by column chromatography
on silica gel eluted by a mixture of methanol–25% water solution
of ammonia (50:4).
(2ꢀCH2ANH), 36.38 (2ꢀCH3 ), 28.97 (2ꢀCH3AN@C).
2.2.16. Synthesis of 4-(acetylamino)-2-{[3-
(acetylamino)phenyl]amino}-N-benzyl benzamide (22)
A mixture of the acid 7 (1 g, 3.05 mmol) and carbodiimidazole
(1.5 g, 9.15 mmol) in dimethylformamide (10 mL) was stirred at
room temperature. After the reaction was completed (approxi-
mately 2 h, checked by TLC using a mobile phase methanol–ethyl
acetate (6:1 v/v)), benzylamine (1 mL, 9.51 mmol) was added.
The resultant mixture was stirred at room temperature for 24 h
and subsequently poured into distilled water. A precipitated crude
product 19 was filtered off and dried on air overnight. The product
19 was purified by column chromatography using a mobile phase
methanol–ethyl acetate (6:1 v/v).
Yield: 30%, dark yellow solid, mp: 105–106 °C. Anal. Calcd for
C
31H41N9O2 (571.72): 65.13% C, 7.23% H, 22.05% N. Found:
65.25% C, 7.15% H, 21.99% N. 1H NMR (600 MHz, CD3OD): 7.64 (d,
2H, H-4,5, J = 1.20), 7.56 (d, 2H, H-1,8, J = 8.40), 7.09 (d, 2H, H-
20,60, J = 9.00), 6.90 (d, 2H, H-2,7, J = 9.00), 6.76 (d, 2H, H-30,50,
J = 8.40), 3.55 (t, 4H, 2ꢀCH2ANH, J = 6.60), 3.00 (t, 4H, 2ꢀ
0
CH2AN(CH3)2, J = 6.00), 2.98 (s, 6H, 2ꢀCH3 ), 2.67 (s, 12H,
4ꢀCH3). 13C NMR (150 MHz, CD3OD): 157.07 (2ꢀC@O), 153.90
(C9), 151.30 (C40), 147.04 (C4a, C10a), 142.66 (C3, C6), 130.54
(C10), 127.24 (C20, C60), 127.12 (C1, C8), 117.13 (C2, C7), 114.10
(C30, C50), 108.86 (C8a, C9a), 103.53 (C4, C5), 59.14 (2ꢀCH2AN
Yield: 62%, gray solid, mp: 214–216 °C. Anal. Calcd for
C
24H24N4O3 (416.47): 69.21% C, 5.81% H, 13.45% N. Found:
69.33% C, 5.60% H, 13.23% N. 1H NMR (600 MHz, DMSO–D6):
10.01 (s, 1H, NH0), 10.00 (s, 1H, PhANHAPh), 9.90 (s, 1H, NH00),
9.00 (t, 1H, NH, J = 6.00), 7.69 (d, 1H, H-6, J = 8.40), 7.54 (s, 1H,
H-3), 7.41 (s, 1H, H-200), 7.33–7.32 (m, 4H, H-2000,6000, H-3000,5000),
7.25–7.21 (m, 3H, H-4000, H-400, H-500), 7.13 (d, 1H, H-5, J = 8.40),
6.88–6.86 (m, 1H, H-600), 4.45 (d, 2H, CH2, J = 6.00), 2.03 (s, 3H,
0
(CH3)2), 44.59 (4ꢀCH3), 40.73 (2ꢀCH3 ), 37.07 (2ꢀCH2ANH).
2.2.14. Synthesis of 3,6-bis[3-(2-dimethylaminoethyl)guanidino]-9-
[40-(N,N-dimethylamino)phenylamino]acridine (17)
Triethylamine (0.5 mL, 3.58 mmol), anhydrous sodium sulfate
(1 g, 7.04 mmol), calcium chloride (0.05 g, 0.45 mmol), mercury(II)
oxide (0.12 g, 0.58 mmol), and ammonium hydrogencarbonate
(0.25 g, 3.16 mmol) were added to the bisthiourea 15 (0.05 g,
0.08 mmol) in 10 mL of methanol with dosing intervals of 5 min
for each reactant. The reaction mixture was refluxed for 1 h. An
insoluble residue was filtered off and a crude product 17 was puri-
fied by column chromatography on silica gel eluted by a metha-
nol–25% water solution of ammonia (50:4).
CH3 ), 2.01 (s, 3H, CH300). 13C NMR (150 MHz, DMSO–D6): 168.69
0
(C@O0), 168.54 (C@O), 168.31 (C@O00), 145.66 (C2), 142.69 (C4),
141.60 (C100), 140.45 (C300), 139.62 (C1000), 129.52 (C6, C500), 128.31
(C3000, C5000), 127.24 (C2000, C6000), 126.75 (C4000), 114.37 (C600), 112.81
(C400), 112.38 (C1), 110.70 (C200), 108.67 (C5), 104.21 (C3), 42.39
(CH2), 24.18 (CH300), 24.11 (CH3 ).
0
2.3. UV–vis absorption and fluorescence measurement
Yield: 35%, light red solid, mp: 148–149 °C. Anal. Calcd for
C
31H43N11 (569.75): 65.35% C, 7.61% H, 27.04% N. Found: 65.50%
UV–vis absorption spectra were measured on a Varian Cary 100
UV–vis spectrophotometer equipped with a thermostat cell com-
partment using quartz cuvettes with 1 cm path lengths in a
0.01 M Tris buffer (pH 7.4). The concentration of ctDNA (Sigma
Chemical Co.) ranged from 0 to 2.1 ꢀ 10ꢁ5 M bp. The BRACO19
C, 7.69% H, 26.99% N. 1H NMR (600 MHz, CD3OD): 7.82 (d, 2H, H-
1,8, J = 9.60), 7.16 (d, 2H, H-4,5, J = 1.80), 6.83 (d, 2H, H-20,60,
J = 9.00), 6.75 (dd, 2H, H-2,7, J1 = 2.40, J2 = 7.20), 6.71 (d, 2H,
H-30,50, J = 9.00), 3.29 (t, 4H, 2ꢀCH2ANH, J = 6.00), 2.80 (s, 6H,