Synthesis of 1-/2-substituted-[1,2,3]triazolo[4,5-g]phthalazine-4,9-diones and evaluation of their cytotoxicity and topoisomerase II inhibition

Article abstract of DOI:10.1016/j.bmc.2008.02.049

Studies on antitumor heterocyclic quinones containing nitrogens revealed that the number and position of nitrogens on the heterocyclic ring have significance on cytotoxicity of quinones. In our continuous effort to find more cytotoxic quinone compounds, w

Full text of DOI:10.1016/j.bmc.2008.02.049

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4545–4550
Synthesis of 1-/2-substituted-[1,2,3]triazolo[4,5-g]phthalazine-
4,9-diones and evaluation of their cytotoxicity
and topoisomerase II inhibition
Jin Sung Kim,a Hee-Kyung Rhee,a Hyen Joo Park,a Sang Kook Lee,a
Chong-Ock Leeb and Hea-Young Park Chooa,*
aSchool of Pharmacy, Ewha Womans University, Seoul 120-750, South Korea
bPharmaceutical Screening Division, Korea Research Institute of Chemical Technology, Taejon 305-606, South Korea
Received 3 December 2007; revised 13 February 2008; accepted 13 February 2008
Available online 19 February 2008
Abstract—Studies on antitumor heterocyclic quinones containing nitrogens revealed that the number and position of nitrogens on
the heterocyclic ring have significance on cytotoxicity of quinones. In our continuous effort to find more cytotoxic quinone com-
pounds, we designed triazolophthalazine analogues in order to introduce more nitrogens on the heterocyclic quinones. 1-/2-Substi-
tuted-[1,2,3]triazolo[4,5-g]phthalazine-4,9-diones were synthesized by 1,3-dipolar addition of phthalazine-5,8-dione and 4-
methoxybenzyl azide by modification of previously reported method. The cytotoxicity of the synthesized compounds was evaluated
by a SRB (sulforhodamine B) assay against nine types of human cancer cell lines and inhibition against topoisomerase II (Topo II)
of them was assessed by a decatenation assay. Most of the synthesized compounds showed considerably higher cytotoxicity than
that of doxorubicin. Also, topoisomerase II inhibitory activity of the tested compounds was higher than that of etoposide and
IC50 values of the compounds were 19.4–64.5 lM.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
The precise mechanism of ellipticine, a natural antitu-
mor alkaloid has not yet been fully understood but
was suggested including DNA intercalation and inhibi-
tion of topo II activity. Also, the antitumor activity of
anthracyclines, such as doxorubicin, mainly resulted
from an inhibition of mammalian topo II.5,6 The topoi-
somerase is involved in the control of the shape of
DNA. The DNA intercalators have cytotoxocity by
inhibiting topoisomerase. The representative DNA
intercalators are acridines, alkaloids, anthracyclines,
anthracenediones, arylaminoalcohols, coumarins, in-
doles, phenanthridines, quinolines and quinoxalines.7
In our previous reports, the heterocyclic quinones, tricy-
cles such as imidazoquinoxalines 1, pyrazinoquinoxa-
lines 2,8 imidazoquinolines 39 and imidazophthalazines
410 were synthesized and their cytotoxicity was evalu-
ated (Fig. 1).
Quinones have been widely known to have various phys-
iological activities as antimicrobial and anticancer
agents. Especially, heterocyclic quinones have occupied
large classes of antitumor agents. Although the toxicity
mechanism of these compounds is still to be elucidated,
their mechanisms have been proposed as redox cycling
and alkylation.1 The study on quinone reductases has
focused on DT-diaphrase or NAD(P)H:quinone oxido-
reductase 1 (NQO1). Streptonigrin, a quinone antitu-
mor, is metabolized by NQO1,2 and the compound is
regarded to lead to Topo II-mediated DNA cleavage.3
DNA topoisomerases are nuclear enzymes regulating
the topological state of DNA by breaking and rejoining
of DNA strands. Topo II breaks both strands of the du-
plex. A DNA intercalator has cytotoxic activity by sta-
bilizing the ternary DNA intercalator–Topo complex.4
Shaikh et al. emphasized that the number or position of
nitrogen atoms in the heterocyclic quinone compound
plays an important role in an anticancer activity and
that DNA intercalation activity increases in the order
of naphthalene, quinoline and diazanaphthalene.11 Qui-
none structure constituted with 3 or 4 planar fused
Keywords: Triazolophthalazine; Cytotoxicity; Topoisomerase II.
*
Corresponding author. Fax: +82 2 3277 2851; e-mail: hypark@
ewha.ac.kr
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.049

4546
J. S. Kim et al. / Bioorg. Med. Chem. 16 (2008) 4545–4550
O
O
O
O
N
N
N
N
N
N
R1
R2
N
N
N
N
N
N
N
N
N
R
R
R
O
O
O
O
1
2
3
4
Figure 1. Structures of the heterocyclic quinones, tricycles developed in our laboratory.
cycles including two or more nitrogen atoms and conju-
gated carbonyl groups in para is required as DNA inter-
calator. The planarity of heterocycles is susceptible to
intercalate into DNA helix, which leads to the extension
of p electron stacking effect and the stability of the
DNA-intercalator complex. The nitrogen atoms and
carbonyl groups presented in heterocyclic compounds
also increase the stability of the complex through form-
ing hydrogen bonds with DNA. Based on such reasons
the number or position of nitrogen atoms in the hetero-
cyclic compounds plays an important role in exhibiting
an anticancer activity.12,13
2.2. Cytotoxicity by SRB assay
The in vitro cytotoxic activities were evaluated by SRB
assay method. The various human tumor cell lines (lung;
A549, ovarian; SK-OV-3, melanoma; SK-MEL-2, CNS;
XF498, colon; HCT15, stomach; SNU-638, fibro sar-
coma; HT1080 and myeloid leukemic; HL-60) were
used. The cell growth inhibitory potential was deter-
mined as described.18 The inhibitory activities were pre-
sented as micromolar concentrations of the compounds
that cause 50% inhibition per unit of enzyme (IC50) un-
der the assay conditions and compared with that of
doxorubicin and etoposide, the clinically used agents
for the treatment of solid tumors (Table 1). In general,
the cytotoxicity of most 2-substituted-triazolophthal-
azine-4,9-diones (9a–9f) was superior to that of doxoru-
bicin and showed higher activity than the corresponding
1-substituted-triazolophthalazine-diones (8a–8f) against
all kinds of cancer cell lines. Especially, 2-methyl-substi-
tuted compound 9a showed significantly high cytotoxic-
ity against almost all cell lines, and its cytotoxicity
against human ovarian cell line (SK-OV-3) was 100
times of doxorubicin. 1-iso-Butyl-triazolophthalazine-
dione (8f) showed low cytotoxicity among the synthe-
sized compounds. This might be caused by the reduced
hydrogen-bonding of carbonyl oxygen in the cells,
which is caused by the bulky isobutyl group. The
iso-propyl derivatives (8e and 9e) represented similar
or predominant cytotoxicity to the other derivatives.
However, cytotoxicity of compound 6 having bulky
methoxybenzyl group as 1-substituent was weaker than
that of corresponding alkyl derivatives. Also, non-
substituted triazolo-phthalazine-dione 7 did not show
any activity against all tumor cell lines.
In this paper, we report the synthesis, cytotoxicity,
and inhibitory activity against Topo II of 1-/2-substi-
tuted-[1,2,3]triazolo[4,5-g]phthalazine-4,9-diones 8, 9
(Scheme 1). We suggest that the coplanar tricyclic het-
eroquinones possessing five nitrogen atoms can be novel
DNA-intercalating heterocyclics.
2. Results and discussion
2.1. Chemistry
Previously reported10 synthetic method of 1H-
[1,2,3]triazolo[4,5-g]phthalazine-4,9-dione
derivatives
included monoamination, acylation, amination and
diazotization of 6,7-dichlorophthalazine-5,8-dione,
which had been prepared from phthalazine. However,
this scheme comprises too many complicated steps and
inevitably utilizes ammonia gas for direct amination of
halide. Therefore, we employed 1,3-dipolar addition
reaction of naphthoquinone for the synthesis of triazol-
ophthalazinedione. Synthesis of 1-/2-substituted-
[1,2,3]triazolo[4,5-g]phthalazine-4,9-diones 8, 9 has been
achieved by the modified reaction of reported method14
starting from phthalazine-5,8-dione 515 and 4-methoxy-
benzyl azide.16 The 1-(4-methoxybenzyl)-1H-[1,2,3]triaz-
olo[4,5-g]phthalazine-4,9-dione 6 was produced by 1,3-
dipolar addition of the 4-methoxybenzyl azide to the
phthalazine-5,8-dione.17 The 4-methoxybenzyl group
was removed by trifluoroacetic acid. The N-alkylation
of the triazolo-phthalazine-dione by alkylhalides gave
both isomers 8 and 9. Generally, the alkylation activity
decreased with increase in the alkyl chain length.
Also the alkylating agents with strong electron-with-
drawing group, such as trifluoroethyliodide, gave very
poor yield. Two geometric isomers were obtained, and
generally 2-substituted isomers were obtained in higher
yield than 1-substituted isomer. The two isomers were
separated by column chromatography (hexane/
EtOAc = 1:2) (Scheme 1).
2.3. Inhibitory activity for topoisomerase II
To investigate the cytotoxic mechanism of tested com-
pounds, inhibitory activity for topo II, essential enzymes
for DNA metabolism was evaluated. The decatenation
assay19,20 is specific in measuring topo II activity be-
cause it is based on the conversion of catenated DNA
to its decatenated form, which requires DNA double
strand breakage followed by strand rotation and liga-
tion activities uniquely done by topo II. The removal
of these KDNA by the enzyme can be seen in agarose
gels. Based on this information, effects of the synthesized
compounds for the catalytic activity of topo II were
evaluated. The decatenation of KDNA was induced by
topo II. The results of topo II inhibition of the tested
compounds and etoposide21 are shown in Table 2. Also,
the electrophotogram on the inhibitory activity of com-
pounds is presented in Figure 2. When these compounds

J. S. Kim et al. / Bioorg. Med. Chem. 16 (2008) 4545–4550
4547
OMe
O
O
N
N
N
N
a
N
N
N
N
N
O
6
Phthalazine
O
5
b
O
O
O
O
R
H
N
N
N
N
c
N
N
N
N
N
N
N
N
+
N R
N
N
O
7
O
8
9
a. 4-MeOPhCH2N3/EtOAc, b. TFA, c. RI, K2CO3/ DMF
No
R
No
8d
R
CH2CH2CH2CH3
8a CH3
CH3
CH2CH2CH2CH3
CH(CH3)2
9a
8b
9b
8c
9c
9d
8e
9e
8f
CH2CH3
CH2CH3
CH(CH3)2
CH2CH2CH3
CH2CH2CH3
CH2CH(CH3)2
CH2CH(CH3)2
9f
Scheme 1. Synthesis of 1-/2-substituted-[1,2,3]triazolo[4,5-g]phthalazine-4,9-diones.
Table 1. Cytotoxicity of 1-/2-substituted-[1,2,3]triazolo[4,5-g]phthalazine-4,9-diones against human tumor cell lines
Compound
IC50 (lM)
XF498
A549
SK-OV-3
SK-MEL-2
HCT15
SNU-638
HT1080
HL-60
Doxorubicin
Etoposide
0.022
1.16
0.041
4.28
0.019
5.72
0.044
3.48
0.251
2.80
0.052
0.23
0.022
0.25
0.044
0.37
0.130
6
0.054
9.02
0.012
7.64
0.041
>10.00
0.008
0.002
0.008
0.004
0.020
0.009
0.023
0.015
0.016
0.007
0.066
0.015
0.118
>10.00
0.035
0.004
0.034
0.017
0.085
0.018
0.181
0.019
0.049
0.014
0.14
0.059
>10.00
0.103
0.020
0.039
0.018
0.176
0.018
0.177
0.018
0.033
0.014
0.11
0.257
>5
0.330
>5
7
>5
8a
9a
8b
9b
8c
9c
8d
9d
8e
9e
8f
9f
0.004
0.002
0.006
0.018
0.026
0.019
0.081
0.018
0.032
0.009
0.11
0.003
0.0004
0.003
0.003
0.014
0.009
0.021
0.017
0.049
0.031
0.19
0.241
0.054
0.126
0.046
0.212
0.043
0.155
0.050
0.057
0.011
0.069
0.014
0.073
0.0078
0.068
0.204
0.075
0.033
0.099
0.030
0.114
0.055
0.077
0.085
0.082
0.124
0.056
0.090
0.083
0.157
0.089
0.151
0.050
0.018
0.075
0.038
0.019
0.043
0.019
0.029
A549, human lung tumor cell line; SK-OV-3, human ovarian tumor cell line; SK-MEL-2, human melanoma tumor cell line; XF498, human CNS
tumor cell line; HCT15, human colon tumor cell line; SNU-638, human stomach tumor cell line; HT1080, human fibro sarcoma tumor cell line; HL-
60, human myeloid leukemic tumor cell line.
were tested at a concentration of 50 lM, most of the
compounds showed topo II inhibition activity. IC50 val-
ues of the compounds were 19.4–64.5 lM. As a result of
these tests, all tested 1/2-substituted-1H-[1,2,3]triazolo
[4,5-g]phthalazine-4,9-diones were considered as potent
topo II inhibitors higher than etoposide. Although the

4548
J. S. Kim et al. / Bioorg. Med. Chem. 16 (2008) 4545–4550
1
apparatus (Buchi). H NMR spectra were recorded on
¨
Table 2. Inhibitory activity of 1-/2-substituted-[1,2,3]triazolo[4,5-
g]phthalazine-4,9-diones against on topoisomerase II
a 400 MHz Varian FT-NMR spectrometer using tetra-
methylsilane as an internal standard. Samples were dis-
solved in CDCl3 or DMSO-d6. Mass spectra were
obtained on the Mass spectrometer JMS-700 (Jeol, Ja-
pan) at the Korea Basic Science Institute (Seoul). Most
of the reagents were purchased from Sigma–Aldrich
Chemical Company.
Compound Decatation activity Compound Decatation activity
(for Topo II)
(for Topo II)
IC50 (lM)
IC (lM)
50
6
28.6
52.8
39.0
55.6
55.6
61.5
49.0
44.4
8d
9d
8e
9e
8f
43.2
64.8
33.3
54.5
29.8
19.4
7
8a
9a
8b
9b
8c
9c
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 were from
Invitrogen (Grand Island, NY). Topo II and assay kits
were purchased from Topogen, Inc. (Columbus, OH,
USA). Stock solutions of all test agents were dissolved
in DMSO for topo II decatenation assay.
9f
Etoposide 92.9
Decatenation assay for topo II catalytic activity was done as described
in Section 4.
correlation of the cytotoxicity and the inhibitory activity
of DNA decatenation by topo II is not clear, DNA
topoisomerase related to DNA intercalation is consid-
ered as an important target for inhibition of human can-
cer cells.22
4.2. Methods
3. Conclusion
4.2.1. Synthesis
4.2.1.1. 1-(4-Methoxybenzyl)-1H-[1,2,3]triazolo[4,5-g]-
phthalazine-4,9-dione (6). A solution of phthalazine-5,8-
dione (1.00 g, 6.20 mmol) and 4-methoxybenzyl azide
(338 mg, 2.07 mmol) in EtOAc (60.0 mL) was refluxed over-
night. The solvent was evaporated off, and the residue was
purified by column chromatography under ethyl acetate to
give 6 (415 mg, 62%; based on azide) as a brown solid:
1-/2-Substituted-[1,2,3]triazolo[4,5-g]phthalazine-4,9-
diones, the coplanar tricyclic heteroquinones with five
nitrogen atoms, were synthesized and evaluation for
their cytotoxic activity using SRB assay was performed
in vitro. In general, most triazolophthalazines showed
great cytotoxic effects against human cancer cell lines
and 2-substituted-triazolophthalazine-4,9-diones exhib-
ited higher cytotoxicity against all kinds of cancer cell
lines than the corresponding 1-substituted-triazoloph-
thalazine-diones. The cytotoxic effects of most 2-substi-
tuted-triazolophthalazine-4,9-diones were superior to
that of doxorubicin. At a concentration of 50 lM,
most compounds showed good topo II inhibition
activity and IC50 values of the compounds were 19.4–
64.5 lM. As a result of these tests, the 1/2-substituted-
1
mp: >300 ꢁC; H NMR (CDCl3) d 10.04 (d, J = 1.2 Hz,
1H), 9.94 (d, J = 1.2 Hz, 1H), 7.47 (d, J = 8.8 Hz, 2H),
6.87 (d, J = 8.8 Hz, 2H), 5.96 (s, 2H), 3.78 (s, 3H); 13C
NMR (CDCl3) d 175.81, 174.79, 160.66, 147.28 (2C),
146.32 (2C), 130.53 (2C), 125.34, 124.96, 124.83, 114.75
(2C), 55.55, 54.26; HR-FABMS Calcd for C16H12N5O3
(M+H)+: 322.0940. Found: 322.0943.
4.2.1.2. 1H-[1,2,3]Triazolo[4,5,-g]phthalazine-4,9-dione
solution of the 1-(4-methoxybenzyl)-1H-
1H-[1,2,3]triazolo[4,5-g]phthalazine-4,9-diones
were
(7).
A
considered as a topo II inhibitor with potential cytotoxic
effect.
[1,2,3]triazolo[4,5-g]phthalazine-4,9-dione (415 mg, 1.29
mmol) in CF3CO2H (30.0 mL) was refluxed for 2 days
and evaporated to dryness under reduced pressure. One
molar aqueous NaOH (100 mL) was added to the resi-
due, and the aqueous layer was washed with EtOAc.
The aqueous phase was acidified to pH 1 with HCl,
and the product was extracted into EtOAc. Evaporation
of the dried (MgSO4) extract gave 7 (150 mg, 58%) as a
4. Experimental
4.1. Materials
1
4.1.1. Synthesis. All melting points were taken in Pyrex
capillaries using electrothermal digital melting point
pale brown solid: mp: >300 ꢁC; H NMR (DMSO-d6) d
9.89 (s, 2H); 13C NMR (DMSO-d6) d 177.37 (2C),
Figure 2. Effects of 1-/2-substituted-[1,2,3]triazolo[4,5-g]phthalazine-4,9-diones on the decatenation of KDNA by topoisomerase II. Lane A; KDNA
without enzyme (catenated form); lane B; KDNA with 1 unit of topoisomerase II (decatenated form); lane C; decatenated KDNA marker; lane D;
linear KDNA marker; lane E; etoposide, 200 lM; lane F; etoposide, 100 lM; lane 8a–9f; KDNA with 1 U of topoisomerase II in the presence of the
prepared compounds at a concentration of 50 lM, respectively.

J. S. Kim et al. / Bioorg. Med. Chem. 16 (2008) 4545–4550
4549
146.76 (4C), 126.91 (2C); HR-FABMS Calcd for
C8H4N5O2 (M+H)+: 202.0365. Found: 202.0362.
(2C), 59.39, 23.24, 11.13; HR-FABMS Calcd for
C11H10O2N5 (M+H)+: 244.0834. Found: 244.0835.
4.2.1.3. General procedure for the preparation of 1-
substituted-1H-[1,2,3]triazolo[4,5-g]phthalazine-4,9-diones
(8a–8f) and 2-substituted-2H-[1,2,3]triazolo[4,5-g]phthal-
azine-4,9-diones (9a–9f). Corresponding alkyl halide (3
equiv) was added to a solution of 1H-[1,2,3]triazolo[4,5-
g]phthalazine-4,9-dione (1 equiv) in DMF (0.23 M), and
the reaction mixture was stirred for 5 h at room temper-
ature. The reaction mixture was passed through a short
pad of Celite (EtOAc) and the filtrate was concentrated
to dryness. Chromatography of the residue with hex-
ane/EtOAc (1:2) gave 1/2-substituted-1/2H-[1,2,3]triazol-
o[4,5-g]phthalazine-4,9-diones.
4.2.1.7. 1-n-Butyl-1H-[1,2,3]triazolo[4,5-g]phthalazine-
4,9-dione (8d) and 2-n-butyl-2H-[1,2,3] triazolo[4,5-
g]phthalazine-4,9-dione (9d). Compounds 8d and 9d (8d;
6 mg and 9d; 20 mg, 36%) were obtained from 7 (57.0 mg,
0.28 mmol) as a orange solid 8d and a yellow solid 9d: 8d;
1
mp: >300 ꢁC; H NMR (CDCl3) 10.05 (d, J = 1.2 Hz,
1H), 9.95 (d, J = 1.2 Hz, 1H), 4.88 (t, J = 7.6 Hz, 2H),
1.97–2.06 (m, 2H) 1.37–1.47 (m, 2H), 1.00 (t, J = 7.2 Hz,
3H); 13C NMR (CDCl3) d 175.87, 174.91, 147.33 (2C),
146.35 (2C), 125.01, 124.85, 51.18, 32.11, 19.80, 13.58;
HR-FABMS Calcd for C12H12O2N5 (M+H)+: 258.0991.
1
Found: 258.0989. 9d; mp: >300 ꢁC; H NMR (CDCl3) d
10.03 (s, 2H), 4.75 (t, J = 7.2 Hz, 2H), 2.08–2.17 (m, 2H),
1.37–1.44 (m, 2H), 0.99 (t, J = 7.2 Hz, 3H); 13C NMR
(CDCl3) d 176.33 (2C), 147.07 (4C), 125.52 (2C), 57.62,
31.61, 19.79, 13.54; HR-FABMS Calcd for C12H12O2N5
(M+H)+: 258.0991. Found: 258.0988.
4.2.1.4. 1-Methyl-1H-[1,2,3]triazolo[4,5-g]phthalazine-
4,9-dione (8a) and 2-methyl-2H-[1,2,3]triazolo[4,5-
g]phthalazine-4,9-dione (9a). Compounds 8a and 9a
(8a; 27 mg and 9a; 15 mg, 56%) were obtained from 7
(70.0 mg, 0.35 mmol) as a apricot solid 8a and a pale
1
brown solid 9a: 8a; mp: >300 ꢁC; H NMR (CDCl3) d
4.2.1.8. 1-iso-Propyl-1H-[1,2,3]triazolo[4,5-g]phthal-
azine-4,9-dione (8e) and 2-iso-propyl-2H-[1,2,3]triazol-
o[4,5-g]phthalazine-4,9-dione (9e). Compounds 8e and 9e
(8e; 6 mg and 9e; 11 mg, 20%) were obtained from 7
(70.0 mg, 0.35 mmol) as a brown solid 8e and 9e: 8e;
10.07 (d, J = 1.2 Hz, 1H), 9.96 (d, J = 1.2 Hz, 1H),
4.55 (s, 3H); 13C NMR (CDCl3) d 175.72, 175.08,
147.37 (2C), 146.24 (2C), 124.91 (2C), 37.74; HR-FAB-
MS Calcd for C9H6O2N5 (M+H)+: 216.0521. Found:
1
1
216.0522. 9a; mp: >300 ꢁC; H NMR (CDCl3) d 10.03
mp: >300 ꢁC; H NMR (CDCl3) d 10.07 (s, 1H), 9.97
(s, 2H), 4.54 (s, 3H); 13C NMR (CDCl3) d 176.18
(2C), 147.06 (4C), 125.46 (2C), 44.19; HR-FABMS
Calcd for C9H6O2N5 (M+H)+: 216.0521. Found:
216.0517.
(s, 1H), 5.52–5.63 (m, 1H), 1.78 (d, J = 6.4 Hz, 6H);
13C NMR (CDCl3) d 176.08, 174.88, 147.27 (2C),
146.47 (2C), 125.15, 124.69, 55.44, 22.47 (2C); HR-
FABMS Calcd for C11H10O2N5 (M+H)+: 244.0834.
1
Found: 244.0831. 9e; mp: >300 ꢁC; H NMR (CDCl3)
4.2.1.5. 1-Ethyl-1H-[1,2,3]triazolo[4,5-g]phthalazine-
4,9-dione (8b) and 2-ethyl-2H-[1,2,3]triazolo[4,5-g]phthal-
azine-4,9-dione (9b). Compounds 8b and 9b (8b; 24 mg
and 9b; 33 mg, 52%) were obtained from 7 (100 mg,
0.50 mmol) as a pale brown solid 8b and a yellow solid
9b: 8b; mp: >300 ꢁC; 1H NMR (CDCl3) d 9.98 (d,
J = 1.2 Hz, 1H), 9.89 (d, J = 1.2 Hz, 1H), 4.87 (q,
J = 7.2 Hz, 2H), 1.62 (t, J = 7.2 Hz, 3H); 13C NMR
(CDCl3) d 175.86, 174.88, 147.32 (2C), 146.32 (2C),
124.98, 124.87, 46.87, 15.41; HR-FABMS Calcd for
C10H8O2N5 (M+H)+: 230.0678. Found: 230.0683. 9b;
d 10.04 (s, 2H), 5.13–5.25 (m, 1H), 1.76 (d, J = 6.8 Hz,
6H); 13C NMR (CDCl3) d 176.44 (2C), 147.08 (4C),
125.55 (2C), 61.25, 22.52 (2C); HR-FABMS Calcd for
C11H10O2N5 (M+H)+: 244.0834. Found: 244.0834.
4.2.1.9.
1-iso-Butyl-1H-[1,2,3]triazolo[4,5-g]phthal-
azine-4,9-dione (8f) and 2-iso-butyl-2H-[1,2,3] triazol-
o[4,5-g]phthalazine-4,9-dione (9f). Compounds 8f and 9f
(8f; 9 mg and 9f; 13 mg, 24%) were obtained from 7
(70.0 mg, 0.35 mmol) as a brown solid 8f and 9f: 8f;
1
mp: >300 ꢁC; H NMR (CDCl3) d 10.08 (s, 1H), 9.96
1
mp: >300 ꢁC; H NMR (CDCl3) d 9.96 (s, 2H), 4.73
(s, 1H), 4.72 (d, J = 7.6 Hz, 2H), 2.34–2.46 (m, 1H),
1.03 (d, J = 6.8 Hz, 6H); 13C NMR (CDCl3) d 175.90,
174.95, 147.33 (2C), 146.37 (2C), 125.04, 124.82, 58.00,
30.07, 19.91 (2C); HR-FABMS Calcd for C12H12O2N5
(M+H)+: 258.0991. Found: 258.0988. 9f; mp: >300 ꢁC;
1H NMR (CDCl3) d 10.04 (s, 2H), 4.56 (t, J = 7.6 Hz,
2H), 2.48–2.62 (m, 1H), 1.02 (d, J = 6.8 Hz, 6H); 13C
NMR (CDCl3) d 176.36 (2C), 147.09 (4C), 125.54
(2C), 64.72, 29.87, 19.95 (2C); HR-FABMS Calcd for
C12H12O2N5 (M+H)+: 258.0991. Found: 258.0991.
(q, J = 7.2 Hz, 2H), 1.69 (t, J = 7.2 Hz, 3H); 13C NMR
(CDCl3) d 176.32 (2C), 147.07 (4C), 125.51 (2C),
53.14, 14.83; HR-FABMS Calcd for C10H8O2N5
(M+H)+: 230.0678. Found: 230.0676.
4.2.1.6.
1-n-Propyl-1H-[1,2,3]triazolo[4,5-g]phthal-
azine-4,9-dione (8c) and 2-n-propyl-2H-[1,2,3] triazol-
o[4,5-g]phthalazine-4,9-dione (9c). Compounds 8c and 9c
(8c; 15 mg and 9c; 23 mg, 45%) were obtained from 7
(70.0 mg, 0.35 mmol) as a brown solid 8c and 9c: 8c;
1
mp: >300 ꢁC; H NMR (CDCl3) d 10.07 (s, 1H), 9.96
4.2.2. In vitro antitumor activity evaluation by SRB
assay15. The in vitro cytotoxic activities were evaluated
by SRB method. Human tumor cell lines; lung; A549,
ovarian; SK-OV-3, melanoma; SK-MEL-2, CNS;
XF498, colon; HCT15, stomach; SNU-638, fibro sar-
coma; HT1080 and myeloid leukemic; HL-60
(5 · 104 cells/mL) was treated with different concentra-
tions of the test agents for 3 days. After treatment, cells
were fixed with TCA and cell viability was determined
(s, 1H), 4.86 (br s, 2H), 2.04–2.12 (m, 2H), 1.03 (t,
J = 7.6 Hz, 3H); 13C NMR (CDCl3) d 176.19, 175.22,
147.64 (2C), 146.66 (2C), 125.33, 125.17, 53.20, 24.00,
11.41; HR-FABMS Calcd for C11H10O2N5 (M+H)+:
244.0834. Found: 244.0831. 9c; mp: >300 ꢁC; 1H
NMR (CDCl3) d 10.02 (s, 2H), 4.71 (t, J = 7.2 Hz,
2H), 2.14–2.21 (m, 2H) 1.01 (t, J = 7.6 Hz, 3H); 13C
NMR (CDCl3) d 176.33 (2C), 147.06 (4C), 125.52

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4.2.3. Decatenation assay for topoisomerase II activity19.
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assay buffer [50 mM Tris–HCl, pH 8, 120 mM KCl,
10 mM MgCl2, 0.5 mM ATP, 0.5 mM dithiothreitol,
30 lg/mL bovine serum albumin (BSA)] containing
200 ng of KDNA (TopoGEN), and a solution of the test
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and then the reaction mixture was analyzed on a 1% aga-
rose gel by running at 40 V for 3.5 h in TBE buffer (89 mM
Tris, 89 mM borate, 2 mM Na–EDTA, pH 8.3). Gels
were stained with SYBR Green I (Molecular Probes, Eu-
gene, OR), and observed under UV illumination. Gels
were photographed, and remaining KDNA from photo-
graphic negatives was scanned using AlphaImager 2200
(AlphaEase version 5.5). The IC50 values were calculated
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Acknowledgment
19. Park, H. J.; Lee, H.-J.; Lee, E.-J.; Hwang, H. J.;
Shin, S.-H.; Suh, M.-E.; Kim, C.; Kim, H. J.; Seo,
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This work was supported by the Korea Research Foun-
dation (KRF-2005-005-J01501).
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