J. S. Kim et al. / Bioorg. Med. Chem. 15 (2007) 451–457
455
synthesized and their cytotoxicity was evaluated. In gen-
eral, the cytotoxicity of the pyrido[3,4-b]phenazinedi-
ones was higher than that of the corresponding
chloroisoquinolinediones.
J = 6.8 Hz), 1.27 (6H, d, J = 6.8 Hz); IR (CH2Cl2)
;
3208, 1681, 1585 cmꢀ1
HR-FABMS Calcd for
C18H16ClO2N2 (M++1): 327.0900. Found: 327.0896.
4.2.1.2. 4-(6-Chloro-5,8-dihydro-5,8-dioxoisoquinolin-
7-ylamino)benzamide (5f). Yield: 42%; red powder; mp
297–299 ꢁC; H NMR (DMSO) d 9.64 (2H, br s), 9.18
(1H, d, J = 0.8 Hz), 9.08 (1H, d, J = 5.2 Hz), 7.90 (1H,
dd, J = 5.2 and 0.8 Hz), 7.82 (2H, d, J = 8.4 Hz), 7.29
(1H, br s), 7.18 (2H, d, J = 8.4 Hz); IR (KBr) 3417,
3263, 1679, 1658, 1588, 1556 cmꢀ1; HR-FABMS Calcd
for C16H11ClO3N3 (M++1): 328.0489. Found: 328.0491.
Highly permeable compounds in a series showed greater
cytotoxic effects against five different cancer cell lines.
However, the cytotoxicity of these compounds was not
clearly correlated with DNA topoisomerase inhibitory
activity. Whether these compounds exert the cytotoxici-
ty through binding by DNA intercalation or other
mechanisms will be clarified by further study.
1
4.2.1.3. 6-Chloro-7-[N-(3-fluorophenyl)amino]isoquin-
oline-5,8-dione (5g). Yield: 67%; brick brown powder;
mp 253–254 ꢁC; 1H NMR (CDCl3) d 9.36 (1H, d,
J = 0.8 Hz), 9.09 (1H, d, J = 4.8 Hz), 8.00 (1H, dd,
J = 4.8 and 0.8 Hz), 7.71 (1H, br s), 7.34 (1H, m), 6.97
(1H, m), 6.89 (1H, m), 6.82 (1H, m); IR (CH2Cl2)
4. Experimental
4.1. Materials and methods
4.1.1. Synthesis. Melting points were taken in Pyrex cap-
illaries using electrothermal digital melting point appa-
ratus (Buchi) and are not corrected. The IR spectra
¨
3220, 1678, 1557 cmꢀ1
;
HR-FABMS Calcd for
C15H9ClFO2N2 (M++1): 303.0337. Found: 303.0333.
were recorded on a FT-Infrared spectrometer (Bio-
Rad Co., USA) using KBr pellet. 1H NMR spectra were
recorded on a 400 MHz Varian FT-NMR spectrometer
facility by using tetramethylsilane as an internal stan-
dard. Mass spectra were obtained on a Tandem Mass
spectrometer JMS-HX110/110A (Jeol). Most of the re-
agents were purchased from Aldrich Chemical Compa-
ny and Merck Company.
4.2.1.4. 6-Chloro-7-[N-(3-hydroxyphenyl)amino]iso-
quinoline-5,8-dione (5i). Yield: 56%; dark red powder;
mp 286–287 ꢁC; 1H NMR (acetone) d 9.22 (1H, d,
J = 0.8 Hz), 9.08 (1H, d, J = 4.8 Hz), 8.65 (1H, br s),
8.48 (1H, br s), 7.94 (1H, dd, J = 4.8 and 0.8 Hz), 7.17
(1H, m), 6.69–6.74 (3H, m); IR (KBr) 3268, 1686,
1554 cmꢀ1
; HR-FABMS Calcd for C15H10ClO3N2
(M++1): 301.0380. Found: 301.0381.
4.1.2. Cytotoxicity and topoisomerase inhibitory assay.
Trichloroacetic acid (TCA) and sulforhodamine B
(SRB) were purchased from Sigma Chemical Co. (St.
Louis, MO). Minimal essential medium with Eagles’ salt
(MEME), fetal bovine serum (FBS), non-essential ami-
no acid solution (10 mM, 100·), trypsin–EDTA solution
(1·), and antibiotic–antimycotic solution (PSF) were
from Gibco-BRL (Grand Island, NY). Topo I, II and
assay kits were purchased from Topogen, Inc. (Colum-
bus, OH, USA). Stock solutions of all test agents were
dissolved in dimethylsulfoxide (DMSO) for topo I relax-
ation assay and 10% DMSO in acetone for topo II
decatenation assay.
4.2.2. General procedure for the preparation of pyri-
do[3,4-b]phenazinediones (6a–e).14 To the solution of
chloroisoquinoline-5,8-dione
5
(1 equiv) in DMF
(0.02 M), sodium azide (1.5 equiv), which was suspend-
ed in little amount of distilled water, was added. The
mixture was heated at 90 ꢁC on a steam bath overnight.
The reaction mixture was cooled, the filtered precipitate
was extracted with ethyl acetate. The organic layer was
washed with water, dried with anhydrous MgSO4, con-
centrated, and then the residue was purified by recrystal-
lization or column chromatography.
4.2.2.1. 8-Methoxypyrido[3,4-b]phenazine-5,12-dione
(6a). The general procedure was followed with 6-chlo-
ro-7-[N-(4-methoxyphenyl)-amino]isoquinoline-5,8-
dione 5a (24.0 mg, 0.076 mmol), and the concentrated
residue was yellow solid (15.0 mg, 68%). Mp >300 ꢁC;
1H NMR (DMSO) d 9.45 (1H, s), 9.19 (1H d,
J = 5.2 Hz), 8.32 (1H, d, J = 9.2 Hz), 8.14 (1H, d,
J = 5.2 Hz), 7.76–7.82 (2H, m), 4.07 (3H, s); IR
(CH2Cl2) 1683, 1613 cmꢀ1; HR-FABMS Calcd for
C16H10O3N3 (M++1): 292.0722. Found: 292.0720.
4.2. Synthesis
4.2.1. General procedure for the preparation of 6-chloro-
7-arylaminoisoquinoline-5,8-diones (5a–i). To a solution
of 6,7-dichloro-5,8-isoquinolinedione, prepared accord-
ing to the literature16,17 (10 mmol) in 95% ethanol
(100 mL), appropriate arylamine (11 mmol) was added
and heated under reflux. The reaction mixture was
cooled and then filtered. The filtered precipitate was
washed with cold 95% ethanol. The physical data of
the prepared compounds, 5a, 5c, 5d, 5e, and 5h, were
previously reported.16,17
4.2.2.2. 8-Isopropylpyrido[3,4-b]phenazine-5,12-dione
(6b). The general procedure was followed with 6-chlo-
ro-7-[N-(4-isopropylphenyl)-amino]isoquinoline-5,8-
dione 5b (40.0 mg, 0.12 mmol), and the concentrated
residue was yellow solid (25.0 mg, 68%). Mp >300 ꢁC;
1H NMR (acetone) d 9.58 (1H, d, J = 0.8 Hz), 9.22
(1H, d, J = 4.8 Hz), 8.33 (1H, d, J = 8.8 Hz), 8.20–8.22
(2H, m), 8.13 (1H, dd, J = 8.8 and 2.2 Hz), 3.33 (1H,
quin, J = 6.8 Hz), 1.45 (6H, d, J = 6.8 Hz); IR (CH2Cl2)
4.2.1.1. 6-Chloro-7-[N-(4-isopropylphenyl)amino]iso-
quinoline-5,8-dione (5b). Yield: 84%; shining dark violet
powder; mp 179–180 ꢁC; 1H NMR (CDCl3) d 9.33
(1H, d, J = 0.8 Hz), 9.06 (1H, d, J = 4.8 Hz), 8.00 (1H,
dd, J = 4.8 and 0.8 Hz) 7.77 (1H, br s), 7.22 (2H, d,
J = 8.4 Hz), 7.03 (2H, d, J = 8.4 Hz), 2.92 (1H, quin,