New benzoxanthone derivatives as topoisomerase inhibitors and DNA cross-linkers

Article abstract of DOI:10.1016/j.bmc.2009.12.069

We synthesized 12 benzoxanthone derivatives classified as three different groups based on the tetracyclic ring shapes and evaluated their pharmacological activities to find potential anticancer agents. In the cytotoxicity test, most compounds showed effective cancer cell growth inhibition against the HT29 and DU145 cell lines. Among the compounds tested, compound 19 was the most effective in the cancer cell lines tested. Compound 9 showed dual inhibitory activities against DNA relaxation by topoisomerases I and II. The% inhibition of compound 9 on topoisomerase I was comparable to that of camptothecin. Compound 9 efficiently blocked topoisomerase II function by almost threefold than etoposide at 20 μM. Compound 19 had selective topoisomerase II inhibitory activity at 100 μM. The DNA cross-linking test revealed that only compounds 8 and 19, which possess epoxy groups, cross-linked DNA duplex, while 14 did not. From the combined pharmacological results, we proposed that the target through which compound 19 inhibits cancer cell growth may be the DNA duplex itself and/or DNA-topoisomerase II complex.

Full text of DOI:10.1016/j.bmc.2009.12.069

Bioorganic & Medicinal Chemistry 18 (2010) 1010–1017
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New benzoxanthone derivatives as topoisomerase inhibitors and DNA
cross-linkers
Hee-Ju Cho a, , Mi-Ja Jung b, , Sangwook Woo a, Jungsook Kim c, Eung-Seok Lee d, Youngjoo Kwon b,
,
*
Younghwa Na a,
*
a College of Pharmacy, Catholic University of Daegu, Gyeongsan, Gyeongbuk 712-702, Republic of Korea
b College of Pharmacy, Ewha Womans University, Seoul 120-750, Republic of Korea
c Department of Chemistry, College of Science and Technology, Dongguk University, Gyeongju Campus 707, Sekjang-Dong, Gyeongju, Gyeongbuk 780-714, Republic of Korea
d College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We synthesized 12 benzoxanthone derivatives classified as three different groups based on the tetracyclic
ring shapes and evaluated their pharmacological activities to find potential anticancer agents. In the cyto-
toxicity test, most compounds showed effective cancer cell growth inhibition against the HT29 and DU145
cell lines. Among the compounds tested, compound 19 was the most effective in the cancer cell lines tested.
Compound 9 showed dual inhibitory activities against DNA relaxation by topoisomerases I and II. The% inhi-
bition of compound 9 on topoisomerase I was comparable to that of camptothecin. Compound 9 efficiently
Received 10 December 2009
Revised 24 December 2009
Accepted 30 December 2009
Available online 4 January 2010
Keywords:
Anticancer activities
Topoisomerase inhibition
DNA cross-linking
Oxiranylmethoxybenzoxanthones
Thiiranylmethoxybenzoxanthones
blocked topoisomerase II function by almost threefold than etoposide at 20
lM. Compound 19 had selective
topoisomerase II inhibitory activity at 100 M. The DNA cross-linking test revealed that only compounds 8
l
and 19, which possess epoxy groups, cross-linked DNA duplex, while 14 did not. From the combined phar-
macological results, we proposed that the target through which compound 19 inhibits cancer cell growth
may be the DNA duplex itself and/or DNA–topoisomerase II complex.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Topoisomerases, generally classified as types I and II, are critical
cellular enzymes necessary for cell proliferation that remove topo-
Xanthones1 (1), which are mainly found as secondary metabo-
lites from higher plants and microorganisms, have diverse pharma-
cological activities such as anti-hypertensive,2 anti-thrombotic,3
and anticancer activity,4,5 based on their diverse structures. Simple
and interesting structural scaffolds and diverse biological spectra
of xanthones have led many scientists to isolate or synthesize xan-
thone analogues for the development of prospective drug candi-
dates. Previously, we reported the synthesis and biological
activities of some epoxy-tethered xanthone analogues.5 The pre-
pared compounds showed significant topoisomerase II inhibitory
activities and the bis-oxiranylmethoxy-substituted compound (2)
was the most efficient among the compounds tested.
A quinobenzoxazine, A-62176 (3), a fluoroquinolone analogue,
is a catalytic inhibitor of topoisomerase II and a poison under cer-
tain conditions.6,7 Hurley and co-workers reported that ring expan-
sion of 3 increased the poison activity and reduced the catalytic
function. FQA-CS (4) was the most effective topoisomerase II
poison.7
logical hurdles in the process of DNA replication.8 Topoisomerase I
mediates the breaking and rejoining of a single strand of the DNA
duplex to relax supercoiled chromosomes. On the other hand,
topoisomerase II relaxes DNA double helices by working on two
strands. Due to the critical role of these enzymes in the cell prolif-
erative process, topoisomerases have been one of the major targets
in anticancer drug development.
Interstrand DNA cross-linking of two strands of DNA inhibits
cascade of the cell proliferation, such as replication and transcrip-
tion, and finally causes cell death.9,10 Some antitumor compounds
possessing two electrophilic functions, such as mitomycin C and
nitrogen mustard, can act as DNA cross-linking agents, which is
one of the important classes of antitumor drugs targeting DNA.11,12
In the continued effort to develop anticancer drug candidates
that target topoisomerases and DNA, we designed a series of new
benzoxanthone analogues possessing epoxy or thioepoxy groups.
For this purpose we synthesized three different groups of benzox-
anthones according to the position of the fourth phenyl ring to the
xanthone core. We expected that the shapes of resulting benzox-
anthones would affect their mode of interaction such as intercala-
tion and/or binding to DNA and enzymes. Here, we report the
synthesis of 12 benzoxanthone analogues and their pharmacolog-
ical activities including cytotoxicity, topoisomerase inhibition,
and DNA cross-linking.
* Corresponding authors. Tel.: +82 2 3277 4653; fax: +82 2 3277 3051 (Y.K.); tel.:
+82 53 850 3616; fax: +82 53 850 3602 (Y.N.).
E-mail addresses: ykwon@ewha.ac.kr (Y. Kwon), yna7315@cu.ac.kr (Y. Na).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.069

H.-J. Cho et al. / Bioorg. Med. Chem. 18 (2010) 1010–1017
1011
O
O
O
N
O
O
O
O
O
F
OH
N
O
O
O
H2N
1
2
3
O
N
O
F
O
OR1
R1 = H, Oxiranylmethoxy, Thiiranylmethoxy
R2 = Oxiranylmethoxy, Thiiranylmethoxy
OH
C
N
B
O
O
OR2
A
FQA-CS
H2N
Target structures
4
Generally, most compounds effectively inhibited cancer cell prolif-
eration. Among these, compounds 13 and 19 showed stronger sup-
pression of the HT29 cancer cell line than did adriamycin,
etoposide, and camptothecin, which were used as references. Com-
pound 19, especially, exhibited significant cytotoxicity in the
tested cancer cell lines when compared to other compounds.
2. Results and discussion
2.1. Chemistry
Synthetic methods used for the preparation of benzoxanthone
derivatives are shown in Scheme 1. Starting dihydroxybenzoxant-
hones 5,13 10,14 and 1514 were synthesized by modifying or
employing the reported methods. Coupling reactions of benzoxant-
hones with epichlorohydrin or epithiochlorohydrin were accom-
plished as reported previously by our research group.5,15 First,
coupling of the 8,10-dihydroxy-7H-benzo[c]xanthen-7-one (5)
with chlorohydrins gave the monoepoxy- and bisepoxy-benzox-
anthone compounds depending on the amount of K2CO3 and chlo-
rohydrins used. One equivalent K2CO3 and three equivalents of
epichlorohydrin or epithichlorohydrin provided monoepoxy-
substituted 6 or monothioepoxy-substituted 7, respectively. On
the other hand, increment of K2CO3 and chlorohydrins by two
and fivefold each furnished bisepoxy 8 (23.5% yield) or bisthioep-
oxy-benzoxanthone 9 (13.2% yield), respectively. Second, coupling
reaction of 9,11-dihydroxy-12H-benzo[a]xanthen-12-one (10)
with 5–6-fold of chlorohydrins in acetone in the presence of
3 equiv of Cs2CO3 afforded mono- and bisepoxy-benzoxanthone
compounds 11, 13, 12, and 14 as a mixture. Finally, 1,3-dihy-
droxy-12H-benzo[b]xanthen-12-one (15) with epithiochlorohy-
drin or epichlorohydrin in the presence of K2CO3 gave
monothioepoxy or monoepoxy derivatives 16 and 17. But replace-
ment of K2CO3 with Cs2CO3 base afforded bisthioepoxy or bisepoxy
derivatives 18 and 19, respectively. In the 1H NMR spectra, most
mono-substituted benzoxanthones showed singlet peaks at d
ꢀ13.00 corresponding to the hydroxyl protons adjacent to the car-
bonyl group in the benzoxanthone ring. These singlet peaks disap-
peared in the bisepoxy derivatives, which indicates introduction of
two chlorohydrins groups at two hydroxyl groups. All other spec-
troscopic data were consistent with the proposed structures.
2.2.2. Topoisomerases I and II inhibition test
Topoisomerase relaxation inhibitory activities were evaluated
using human topoisomerases I and II (Topogen) with camptothecin
and etoposide as positive controls.17 Data were analyzed and eval-
uated with LabWork 4.5 Software to calculate the inhibition ratio.
Test results are indicated in Figure 1 and Table 2. Compound 9
showed comparable topoisomerase I inhibitory activity to campto-
thecin at the test concentrations. Interestingly, it was a better
topoisomerase II inhibitor than etoposide, which was used as posi-
tive control, exhibiting about 2.5-fold higher efficiency at 20 lM.
On the other hand, compound 19 exerted selective inhibitory activ-
ity on topo II function. It has almost the same capacity for topo II
inhibition as the reference at 100
lM, but its inhibitory capacity
was lower at 20 M. All other compounds were inactive in the
l
topoisomerase relaxation pathway. Although there were no clear
correlations between topoisomerase inhibitory activities and
structures of the benzoxanthone derivatives, the observed results
suggest that the shape of the tetracyclic system and the incorpo-
rated functional groups is an important factor for interaction be-
tween the DNA–protein complex and benzoxanthone analogues
during DNA unwinding process.
2.2.3. DNA cross-linking test
Previously we reported that 1,3-bisepoxyxanthone (2) effi-
ciently cross-linked DNA duplex due to the electrophilic nature
of bisepoxy groups. DNA cross-linking property was measured
using linearized pBR 322 DNA by alkaline agarose gel electropho-
resis.18 Only selected bis-substituted compounds were tested.
The DNA cross-linking profiles for the benzoxanthone compounds
are shown in Figure 2. Compounds 8 and 19 possessing epoxy
groups showed strong DNA cross-linking activities, but others that
had thiiranyl groups were inactive to nucleophiles. This observa-
tion was consistent with previous results.5 Interestingly, com-
pound 14 did not generate any DNA cross-linked adducts,
although it had bisepoxy groups. These results implied that the
intercalation pattern of the benzoxanthone was attributed to the
DNA cross-linking process, which means that the location of the
compound in the DNA–compound complex is important to provide
2.2. Pharmacological evaluation
2.2.1. Cytotoxicity test
For the evaluation of cytotoxicity, five different cancer cell lines
were tested: human breast adenocarcinoma cell line (MDA-
MB231), human cervix tumor cell line (HeLa), human prostate tu-
mor cell line (DU145), human colorectal adenocarcinoma cell line
(HT29), and human myelogenous leukemia cell line (HL60). The
typical MTT assay procedure was applied for this assessment.16
The inhibitory activities of the compounds are shown in Table 1.

1012
H.-J. Cho et al. / Bioorg. Med. Chem. 18 (2010) 1010–1017
O
OH
1)
OH
CO2H
O
O
O
O
OH
Epi(thio)chlorohydrin
K2CO3
X
6. X = O
P2O5
+
7. X = S
DMF
OH
+
CH3SO3H
OH
O
O
O
5
X
HO
OH
OH
O
X
8. X = O
9. X = S
O
OH
2)
O
O
O
OH
Epi(thio)chlorohydrin
K2CO3 or Cs2CO3
X
11. X = S
ZnCl2
+
12. X = O
POCl3
acetone
O
OH
+
OH
O
O
O
10
CO2H
OH
X
HO
O
X
13. X = S
14. X = O
O
OH
3)
CO2H
OH
O
O
O
O
OH
Epi(thio)chlorohydrin
K2CO3 or Cs2CO3
X
16. X = S
ZnCl2
+
17. X = O
POCl3
acetone or DMF
OH
+
OH
O
O
O
15
X
HO
OH
O
X
18. X = S
19. X = O
Scheme 1. Synthetic methods of benzoxanthone compounds.
proper distance necessary for nucleophilic attack of the DNA base
to epoxy groups. Expansion of the phenyl ring toward the upper
side of xanthone core might disfavor intercalation of benzoxanth-
one into DNA base pairs because of disrupted p–p stacking interac-
tion between DNA base pairs and compound 14.
effective cancer cell growth inhibition against the HT29 and
DU145 cell lines. Among the compounds, compound 19 was the
most effective in the cancer cell lines tested. Generally, the pre-
pared compounds were not active against topoisomerase func-
tions. Compound 9, however, showed dual inhibitory activities
on DNA relaxation by topoisomerases I and II. The% inhibitory val-
ues of compound 9 on topoisomerase I were comparable to those
of camptothecin at test concentrations. Compound 9 efficiently
blocked topoisomerase II function by almost threefold than etopo-
3. Conclusions
We synthesized 12 benzoxanthone derivatives classified as
three different groups based on the tetracyclic ring shapes and
evaluated their pharmacological activities to find potential anti-
cancer agents. In the cytotoxicity test, most compounds showed
side at 20
itory activity at 100
only compounds 8 and 19 possessing epoxy groups cross-linked
DNA duplex but 14 did not. These observations suggested that
l
M. Compound 19 has selective topoisomerase II inhib-
M. The DNA cross-linking test revealed that
l

H.-J. Cho et al. / Bioorg. Med. Chem. 18 (2010) 1010–1017
1013
Table 1
Cytotoxicities of compounds 6–9, 11–14, and 16–19 against various cancer cells
a
Compd/cells
IC50
DU145
(lM)
HeLa
HT29
MDA-MB231
HL60
Adriamycin
Etoposide
Camptothecin
6
7
8
9
11
12
13
14
16
17
18
19
0.71 0.01
0.47 0.07
0.86 0.03
>50
0.95 0.00
1.42 0.06
0.85 0.03
18.93 1.82
>50
1.19 0.16
1.63 0.08
3.57 1.03
2.56 0.37
10.84 0.60
1.29 0.06
5.66 0.76
8.76 2.21
1.40 0.39
1.32 0.16
3.44 1.42
1.55 0.03
36.20 0.31
19.37 4.26
3.01 0.22
3.15 0.23
2.87 0.29
0.92 0.01
0.73 0.04
>50
7.43 0.20
>50
29.13 7.66
>50
18.86 0.78
>50
14.84 1.77
15.08 0.21
5.16 0.82
1.64 0.06
0.94 0.07
0.97 0.07
0.065 0.0011
3.67 0.71
22.58 0.14
5.40 0.12
37.04 7.05
3.48 0.80
2.31 0.07
1.85 0.00
6.47 0.15
2.48 0.15
9.40 0.46
5.13 0.72
5.13 0.07
>50
24.17 0.46
25.60 0.49
>100
8.20 1.38
>50
3.58 1.75
0.96 0.05
0.75 0.27
7.38 2.98
5.40 2.91
2.34 0.07
1.66 0.06
0.60 0.31
>100
24.95 1.74
10.15 1.19
8.52 0.44
>100
8.01 0.76
2.19 0.70
a
Each data point represents mean SD from three different experiments performed in triplicate.
Figure 2. Cross-linking assay of selected compounds. Lane M: DNA HindIII marker,
Lane D: linearized pBR322 plasmid DNA only, Lanes 8, 9, 13, 14, 18, and 19;
linearized pBR322 + each compound at a final concentration of 100 lM.
shape of the tetracyclic ring system of benzoxanthone derivatives.
From the combined pharmacological results, we proposed that the
target of compound 19 action by which it inhibited cancer growth
was the DNA duplex itself and/or DNA–topoisomerase II complex.
In summary, oxiranylmethoxy- or thiiranylmethoxy-substi-
tuted benzoxanthone derivatives show potential for development
as new anticancer agents following elaborate optimization of sub-
stituent functionalities.
Figure 1. Topo I (A) and II (B) inhibitory activities of compounds. Compounds were
examined at a final concentration of 20 and 100 lM as designated. (A) Lane D:
pBR322 only, Lane T: pBR322 + Topo I, Lane C: pBR322 + Topo I + camptothecin,
Lanes 6–19: pBR322 + Topo I + compounds at designated concentrations. (B) Lane
D: pBR322 only, Lane T: pBR322 + Topo II, Lane E: pBR322 + Topo II + etoposide,
Lanes 6–19: pBR322 + Topo II + compounds at designated concentrations.
4. Experimental
4.1. General
The solvents and reactants were of the best commercial grade
available and were used without further purification unless noted.
TLC plates were Kieselgel 60 F254 (art A715, Merck). Silica gel 60
(0.040–0.063 mm ASTM, Merck) was used for column chromatog-
raphy. 1H and 13C NMR spectra were recorded on Varian NMR AS
400 MHz instrument. Chemical shifts (d) are in parts per million
(ppm) relative to tetramethylsilane as an internal standard, and
coupling constants (J values) are in hertz. Mass spectral investiga-
tions were performed on a LCQ advantage-trap mass spectrometer
equipped with an electrospray ionization (ESI) source or a GC-2010
(Shimadzu, USA) mass spectrometer equipped with an electron
ionization (EI) source. Melting points were measured on a Barn-
stead International MEL-TEMP 1202D instrument without
correction.
Table 2
Topo I and II inhibitory activities of compounds 6–9, 11–14, and 16–19
Compounds
Topo I (% inhibition)
Topo II (% inhibition)
Concentration
100
lM
20
l
M
100
lM
20
—
lM
Camptothecin
Etoposide
6
7
8
9
11
12
13
14
16
17
18
19
78.3
—
75.5
—
—
54.4
9.0
13.9
5.7
71.9
5.3
3.7
4.4
1.0
0.0
27.9
5.5
0.0
0.1
72.7
0.0
0.0
8.4
0.0
1.7
10.6
2.5
0.0
56.5
2.4
9.7
0.0
0.0
0.0
2.6
0.0
8.8
15.6
11.9
79.0
0.0
0.0
0.0
18.0
0.5
13.7
4.3
7.2
0.0
4.1.1. 8,10-Dihydroxy-7H-benzo[c]xanthen-7-one (5)
0.0
The mixture of phosphorous pentoxide (2.84 g, 0.02 mol) and
methanesulfonic acid (40 mL) was stirred at 90 °C for 1 h until
the mixture changed to a clear solution. To this solution were
added 1-hydroxy-2-naphthoic acid (3.76 g, 0.02 mol) and phloro-
glucinol (3.02 g, 0.02 mmol). After stirring of the mixture at 90 °C
for 30 min, the reaction mixture was poured into ice-water. The so-
lid formed was left for 1 d and then filtered, washed with water
1.9
60.1
13.5
8.9
12.3
DNA cross-linking occurs after intercalation of the tetracyclic ring
into DNA base pairs and this process might be dependent on the

1014
H.-J. Cho et al. / Bioorg. Med. Chem. 18 (2010) 1010–1017
and dried under vacuum to give a red solid. Purification by silica
gel column chromatography (eluant: ethyl acetate/n-hexane = 1:3)
afforded compound 5 as a yellow solid (535 mg, 9.61%). Rf: 0.43
(ethyl acetate/n-hexane = 1:3); 1H NMR (400 MHz, DMSO-d6) d
6.16 (s, 1H), 6.35 (s, 1H), 7.52 (dd, J = 7.2, 6.8 Hz, 1H), 7.65 (dd,
J = 7.2, 6.8 Hz, 1H), 7.99 (s, 2H), 8.17 (d, J = 8.0 Hz, 1H), 8.76 (s,
1H), 12.84 (s, 1H).
1H), 3.50–3.53 (m, 1H), 4.05 (ddd, J = 1.2, 6.0, 11.2 Hz, 1H), 4.20
(dd, J = 4.0, 11.2 Hz, 1H), 4.38–4.43 (m, 2H), 6.48 (d, J = 2.2 Hz,
1H), 6.72 (d, J = 2.2 Hz, 1H), 7.65–7.70 (m, 2H), 7.70 (d, J = 8.8 Hz,
1H), 7.91 (dd, J = 2.2, 7.0 Hz, 1H), 8.24 (d, J = 8.8 Hz, 1H), 8.55 (dd,
J = 2.0, 7.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) 44.8, 45.2, 50.0,
50.3, 69.3 (69.2), 69.5, 94.6, 97.6, 108.7, 118.9, 121.9, 122.7,
123.8, 124.1, 126.9, 128.2, 129.3, 136.4, 159.4, 160.8, 163.4,
152.3, 175.4 ppm; LC-ESI: m/e 391.2 [M+1]+.
4.1.2. 8-Hydroxy-10-(oxiran-2-ylmethoxy)-7H-
benzo[c]xanthen-7-one (6)
4.1.5. 8,10-Bis(thiiran-2-ylmethoxy)-7H-benzo[c]xanthen-7-
one (9)
To a mixture of compound 5 (150 mg, 0.54 mmol) and K2CO3
(74.5 mg, 0.54 mmol) was added DMF (20 mL). After addition of
epichlorohydrin (0.13 mL, 1.62 mmol), the reaction mixture was
stirred for 15 h at 70 °C and then cooled to room temperature.
The mixture was diluted with water (20 mL) and extracted with
ethyl acetate. The combined organic layer was washed with brine
and dried over Na2SO4. After evaporation of the solvent under re-
duced pressure, the residue was applied to silica gel column chro-
matography (eluant: ethyl acetate/n-hexane = 1:1) to afford
compound 6 as a pale yellow solid (37 mg, 20.6%). Mp 266 °C; Rf
0.84 (eluant: Ethyl acetate/n-hexane = 1:1); 1H NMR (400 MHz,
CDCl3) d 2.80 (dd, J = 2.4, 4.8 Hz, 1H), 2.96 (dd, J = 4.6, 4.8 Hz),
3.40–3.42 (m, 1H), 4.05 (dd, J = 2.0, 10.9 Hz, 1H), 4.38 (dd, J = 2.6,
10.9 Hz, 1H), 6.42 (d, J = 2.0 Hz, 1H), 6.66 (d, J = 2.0 Hz, 1H), 7.69–
7.75 (m, 2H), 7.74 (d, J = 8.6 Hz, 1H), 7.93 (d, J = 7.6 Hz, 1H), 8.16
(d, J = 8.6 Hz, 1H), 8.61 (d, J = 8.0 Hz, 1H), 12.93 (s, 1H); 13C NMR
(100 MHz, CDCl3) 44.8, 50.0, 69.5, 93.7, 98.2, 105.0, 116.5, 120.6,
123.0, 123.9, 124.4, 127.3, 128.4, 130.0, 136.9, 154.0, 157.5,
163.5, 165.2, 180.9 ppm; LC-ESI: m/e 335.2 [M+1]+.
To a mixture of compound 5 (200 mg, 0.72 mmol) and K2CO3
(198.67 mg, 1.44 mmol) was added DMF (20 mL). After addition
of epithiochlorohydrin (0.39 g, 3.59 mmol) the reaction mixture
was kept stirring for 24 h at 70 °C and then cooled to room temper-
ature. The mixture was diluted with water and extracted with
ethyl acetate. The combined organic layer was washed with brine
and dried over Na2SO4. After evaporation of the solvent under re-
duced pressure, the residue was applied to silica gel column chro-
matography (eluant: ethyl acetate/n-hexane = 2:1) to afford
compound 9 as a white solid (40 mg, 13.2%). Mp 202 °C; Rf 0.59
(ethyl acetate/n-hexane = 1:1); 1H NMR (400 MHz, CDCl3) d 2.39
(dd, J = 1.4, 5.0 Hz, 1H), 2.55 (d, J = 5.2 Hz, 1H), 2.68 (d, J = 6.4 Hz,
1H), 2.73 (d, J = 6.0 Hz, 1H), 3.30–3.35 (m, 1H), 3.47–3.53 (m,
1H), 3.97 (dd, J = 7.6, 10.1 Hz, 1H), 4.09 (dd, J = 6.6, 10.3 Hz, 1H),
4.31 (dd, J = 5.6, 10.3 Hz, 1H), 4.47 (dd, J = 4.6, 10.1 Hz, 1H), 6.42
(d, J = 2.4 Hz, 1H), 6.71 (d, J = 2.4 Hz, 1H), 7.66–7.69 (m, 2H), 7.72
(d, J = 8.6 Hz, 1H), 7.92 (d, J = 2.0, 8.8 Hz, 1H), 8.25 (d, J = 8.6 Hz,
1H), 8.57 (dd, J = 1.6, 8.8 Hz, 1H); 13C NMR (100 MHz, CDCl3)
23.9, 24.7, 30.9, 31.1 73.2, 74.0, 94.5, 97.8, 108.7, 118.9, 121.9,
122.7, 123.8, 124.1, 126.9, 128.2, 129.3, 136.3, 152.3, 159.5,
160.8, 163.2, 175.4 ppm; LC-ESI: m/e 423.0 [M+1]+ 445.1 [M+Na]+.
4.1.3. 8-Hydroxy-10-(thiiran-2-ylmethoxy)-7H-
benzo[c]xanthen-7-one (7)
To a mixture of compound 5 (100 mg, 0.36 mmol) and K2CO3
(49.7 mg, 0.36 mmol) was added DMF (20 mL). After addition of
epithiochlorohydrin (117 mg, 1.08 mmol), the reaction mixture
was stirred for 15 h at 70 °C and then cooled to room temperature.
The mixture was diluted with water (20 mL) and extracted with
ethyl acetate. The combined organic layer was washed with brine
and dried over Na2SO4. After evaporation of the solvent under re-
duced pressure, the residue was applied to silica gel column chro-
matography (eluant: CH2Cl2) to afford compound 7 as a pale yellow
solid (54 mg, 42.8%). Mp 214 °C; Rf 0.76 (eluant: CH2Cl2); 1H NMR
(400 MHz, CDCl3) d 2.32 (dd, J = 2.4, 5.5 Hz, 1H), 2.60 (d, J = 5.5 Hz,
1H), 3.20–3.29 (m, 1H), 3.99 (dd, J = 6.8, 10.0 Hz, 1H), 4.25 (dd,
J = 5.6, 10.0 Hz, 1H), 6.36 (d, J = 2.4 Hz, 1H), 6.59 (d, J = 2.4 Hz,
1H), 7.63–7.69 (m, 2H), 7.69 (d, J = 8.8 Hz, 1H), 7.88 (dd, J = 1.2,
8.8 Hz, 1H), 8.11 (d, J = 8.8 Hz, 1H), 8.55 (dd, J = 1.4, 8.2 Hz, 1H),
12.87 (s, 1H); 13C NMR (100 MHz, CDCl3) 31.0, 35.8, 73.2, 93.6,
98.2, 105.0, 116.5, 120.6, 123.0, 123.9, 124.4, 127.3, 128.4, 130.0,
136.9, 154.0, 157.6, 163.5, 165.1, 180.9 ppm; LC-ESI: m/e 351.2
[M+1]+.
4.1.6. 9,11-Dihydroxy-12H-benzo[a]xanthen-12-one (10)
The reaction mixture of 2,4,6-trihydroxybenzoic acid (1.88 g,
0.01 mol), 2-naphthol (1.44 g, 0.01 mol), ZnCl2 (5.0 g, 0.036 mol),
and POCl3 (40 mL) was refluxed for 5 h at 80 °C, cooled to room
temperature and then poured into iced water very slowly. The so-
lid formed was kept for 1 d at room temperature, collected and
washed with water to give a red solid. After drying under vacuum,
the solid was applied to silica gel column chromatography (eluant:
ethyl acetate/n-hexane = 1:3) to give a yellow solid (0.20 g, 7.2%).
Rf: 0.28 (ethyl acetate/n-hexane = 1:3); 1H NMR (400 MHz, CDCl3)
d 6.26 (s, 1H), 6.38 (s, 1H), 7.42 (d, J = 8.8 Hz, 1H), 7.52 (dd,
J = 7.6, 6.8 Hz, 1H), 7.69 (dd, J = 7.2, 7.2 Hz, 1H), 7.83 (d,
J = 8.0 Hz, 1H), 8.04 (d, J = 8.8 Hz, 1H), 9.87 (d, J = 8.4 Hz, 1H).
4.1.7. 11-Hydroxy-9-(thiiran-2-ylmethoxy)-12H-
benzo[a]xanthen-12-one (11) and 9,11-bis(thiiran-2-
ylmethoxy)-12H-benzo[a]xanthen-12-one (13)
To a mixture of compound 10 (0.1 g, 0.36 mmol) and Cs2CO3
(0.36 g, 1.15 mmol), in anhydrous acetone (13 mL) was added epit-
hiochlorohydrin (0.2 g, 1.84 mmol) in acetone (2 mL). The reaction
mixture was stirred over night at 55–60 °C under nitrogen and
then cooled to room temperature. The solid in the mixture was re-
moved by filtration and the filtrate was concentrated under re-
duced pressure. The residue was purified by silica gel column
chromatography (eluant: ethyl acetate/n-hexane = 1:3) to give
compounds 11 (27.3 mg, 21.7%) and 13 (31.2 mg, 20.5%) as yellow
solids. Compound 11: Mp 167–168 °C; Rf: 0.66 (ethyl acetate/n-
hexane = 1:3); 1H NMR (400 MHz, CDCl3) d 2.37 (d, J = 5.2 Hz,
1H), 2.66 (d, J = 5.2 Hz, 1H), 3.28–3.44 (m, 1H), 4.03 (dd, J = 10.0,
6.8 Hz, 1H), 4.29 (dd, J = 10.0, 5.6 Hz, 1H), 6.40 (d, J = 2.0 Hz, 1H),
6.49 (d, J = 2.0 Hz, 1H), 7.51 (d, J = 9.2 Hz, 1H), 7.61 (dd, J = 8.0,
7.2 Hz, 1H), 7.78 (dd, J = 8.8, 7.2 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H),
8.14 (d, J = 8.0 Hz, 1H), 9.96 (d, J = 8.8 Hz, 1H), 13.48 (s, 1H); 13C
4.1.4. 8,10-Bis(oxiran-2-ylmethoxy)-7H-benzo[c]xanthen-7-
one (8)
To a mixture of compound 5 (100 mg, 0.36 mmol) and K2CO3
(99.4 mg, 0.72 mmol) was added DMF (15 mL). After addition of
epichlorohydrin (0.14 mL, 1.8 mmol), the reaction mixture was
stirred for 24 h at 70 °C and then cooled to room temperature.
The mixture was diluted with water and extracted with ethyl ace-
tate. The combined organic layer was washed with brine and dried
over Na2SO4. After evaporation of the solvent under reduced pres-
sure, the residue was applied to silica gel column chromatography
(eluant: ethyl acetate/n-hexane = 2:1) to afford compound 8 as an
orange solid (33 mg, 23.5%). Mp 154 °C; Rf 0.30 (eluant: ethyl ace-
tate/n-hexane = 1:1); 1H NMR (400 MHz, CDCl3) d 2.83 (dd, J = 2.4,
8.8 Hz, 1H), 2.97–3.01 (m, 2H), 3.19–3.22 (m, 1H), 3.41–3.45 (m,

H.-J. Cho et al. / Bioorg. Med. Chem. 18 (2010) 1010–1017
1015
NMR (100 MHz, CDCl3) 24.1, 31.0, 73.1, 92.7, 98.2, 105.7, 109.8,
4.1.10. 1-Hydroxy-3-(thiiran-2-ylmethoxy)-12H-
benzo[b]xanthen-12-one (16)
117.8, 126.5, 127.0, 128.8, 129.8, 130.4, 131.0, 137.3, 156.7,
157.9, 163.8, 164.7, 183.2 ppm; GC–MS (EI): m/e 350 [M]+. Com-
pound 13: Mp 225–226 °C; Rf: 0.35 (ethyl acetate/n-hexane = 1:3);
1H NMR (400 MHz, CDCl3) d 2.38 (dd, J = 5.0, 1.6 Hz, 1H), 2.55 (d,
J = 5.0 Hz, 1H), 2.66 (dd, J = 6.0 Hz, 1H), 2.74 (d, J = 6.0 Hz, 1H),
3.28–3.34 (m, 1H), 3.51–3.57 (m, 1H), 4.02 (dd, J = 10.4, 7.2 Hz,
1H), 4.05 (dd, J = 10.4, 7.2 Hz, 1H), 4.27 (dd, J = 10.4, 5.2 Hz, 1H),
4.50 (dd, J = 10.4, 4.8 Hz, 1H), 6.45 (d, J = 2.0 Hz, 1H), 6.57 (d,
J = 2.0 Hz, 1H), 7.46 (d, J = 8.8 Hz, 1H), 7.58 (dd, J = 7.6, 7.2 Hz,
1H), 7.75 (dd, J = 8.8, 8.0 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 8.06 (d,
J = 8.8 Hz, 1H), 10.06 (d, J = 8.4 Hz, 1H); 13C NMR (100 MHz, CDCl3)
24.0, 24.8, 31.0, 31.3, 73.1, 74.4, 94.0, 98.4, 110.0, 115.9, 117.5,
126.1, 127.3, 128.5, 129.4, 130.6, 131.3, 135.9, 156.3, 158.4,
160.7, 162.8, 177.5 ppm; GC–MS (EI): m/e 422 [M]+.
To a mixture of compound 15 (0.05 g, 0.18 mmol) and K2CO3
(0.07 g, 0.54 mmol) in anhydrous acetone (18 mL) was added epit-
hiochlorohydrin (0.07 g, 0.65 mmol) in acetone (2 mL). The reac-
tion mixture was stirred overnight at 55–60 °C under nitrogen
and then cooled to room temperature. The solid in the mixture
was removed by filtration and the filtrate was concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (eluant: ethyl acetate/n-hexane = 1:3) to give
compound 16 (13.1 mg, 20.8%) as a yellow solid. Mp 189–190 °C;
Rf: 0.61 (ethyl acetate/n-hexane = 1:3); 1H NMR (400 MHz, CDCl3)
d 2.38 (d, J = 5.2 Hz, 1H), 2.67 (d, J = 6.0 Hz, 1H), 3.31–3.34 (m,
1H), 4.05 (dd, J = 10.4, 7.2 Hz, 1H), 4.30 (dd, J = 10.4, 6.0 Hz, 1H),
6.35 (d, J = 1.6 Hz, 1H), 6.47 (d, J = 1.6 Hz, 1H), 7.53 (dd, J = 7.6,
7.2 Hz, 1H), 7.64 (dd, J = 8.0, 7.2 Hz, 1H), 7.84 (s, 1H), 7.92 (d,
J = 8.4 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 8.86 (s, 1H), 12.94 (s, 1H);
13C NMR (100 MHz, CDCl3) 24.0, 30.9, 73.2, 93.8, 96.3, 97.3,
113.5, 120.4, 126.0, 127.3, 127.9, 129.5, 129.8, 130.0, 137.0,
152.6, 158.4, 164.3, 166.0, 181.6 ppm; GC–MS (EI): m/e 350 [M]+.
4.1.8. 11-Hydroxy-9-(oxiran-2-ylmethoxy)-12H-
benzo[a]xanthen-12-one (12) and 9,11-bis(oxiran-2-
ylmethoxy)-12H-benzo[a]xanthen-12-one (14)
To a mixture of compound 10 (0.1 g, 0.36 mmol) and Cs2CO3
(0.36 g, 1.15 mmol), in anhydrous acetone (20 mL) was added epi-
chlorohydrin (0.2 g, 2.16 mmol) with a syringe. The reaction mix-
ture was stirred for 2 d at 55–60 °C under nitrogen and then
cooled to room temperature. The solid in the mixture was removed
by filtration and the filtrate was concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatogra-
phy (eluant: ethyl acetate/n-hexane = 1:3) to give compound 12
(24.3 mg, 20.3%) and compound 14 (32 mg, 22.1%) as yellow solids.
Compound 12: Mp 191–193 °C; Rf: 0.63 (ethyl acetate/n-hex-
ane = 1:1); 1H NMR (400 MHz, CDCl3) d 2.81 (dd, J = 4.8, 2.4 Hz,
1H), 2.97 (dd, J = 4.8, 4.4 Hz, 1H), 3.40–3.43 (m, 1H), 4.04 (dd,
J = 11.2, 6.0 Hz, 1H), 4.35 (dd, J = 11.2, 3.2 Hz, 1H), 6.42 (d,
J = 2.0 Hz, 1H), 6.52 (d, J = 2.0 Hz, 1H), 7.51 (d, J = 8.8 Hz, 1H),
7.61 (dd, J = 8.0, 3.2 Hz, 1H), 7.78 (ddd, J = 8.4, 8.4, 1.6 Hz, 1H),
7.91 (d, J = 8.0 Hz, 1H), 8.13 (d, J = 8.8 Hz, 1H), 9.96 (d, J = 8.4 Hz,
1H), 13.48 (s, 1H); 13C NMR (100 MHz, CDCl3) 44.9, 50.0, 69.4,
92.9, 98.2, 105.8, 113.5, 117.8, 126.5, 127.0, 128.8, 129.8, 130.4,
131.0, 137.3, 156.7, 157.9, 163.8, 164.8, 183.3 ppm; GC–MS (EI):
m/e 334 [M]+. Compound 14: Mp 198–200 °C; Rf: 0.30 (ethyl ace-
4.1.11. 1,3-Bis(thiiran-2-ylmethoxy)-12H-benzo[b]xanthen-12-
one (18)
To a mixture of compound 15 (0.05 g, 0.18 mmol) and Cs2CO3
(0.23 g, 0.74 mmol) in anhydrous acetone (18 mL) was added epit-
hiochlorohydrin (0.12 g, 1.11 mmol) in acetone (2 mL). The reac-
tion mixture was stirred overnight at 55–60 °C under nitrogen
and then cooled to room temperature. The solid in the mixture
was removed by filtration and the filtrate was concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (eluant: ethyl acetate/n-hexane = 1:3) to give
compound 18 (13.1 mg, 17.2%) as an off-white solid. Mp 196–
197 °C; Rf: 0.39 (ethyl acetate/n-hexane = 1:3); 1H NMR
(400 MHz, CDCl3) d 2.37 (d, J = 5.2 Hz, 1H), 2.53 (d, J = 4.8 Hz, 1H),
2.65 (d, J = 6.0 Hz, 1H), 2.71 (d, J = 6.0 Hz, 1H), 3.28–3.31 (m, 1H),
3.46–3.50 (m, 1H), 3.98 (dd, J = 10.0, 7.2 Hz, 1H), 4.06 (dd,
J = 10.0, 7.2 Hz, 1H), 4.25 (dd, J = 10.0, 5.6 Hz, 1H), 4.44 (dd,
J = 10.0, 4.4 Hz, 1H), 6.34 (d, J = 2.4 Hz, 1H), 6.51 (d, J = 2.4 Hz,
1H), 7.46 (dd, J = 8.4, 7.2 Hz, 1H), 7.58 (dd, J = 8.0, 7.2 Hz, 1H),
7.75 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 8.84
(s, 1H); 13C NMR (100 MHz, CDCl3) 23.9, 24.6, 31.0, 31.1, 73.1,
73.8, 94.4, 96.8, 107.3, 112.8, 123.6, 125.6, 127.1, 128.4, 128.9,
130.0 (Â2), 136.4, 151.7, 160.4, 161.3, 164.1, 176.2 ppm; GC–MS
(EI): m/e 422 [M]+.
tate/n-hexane = 1:1); 1H NMR (400 MHz, CDCl3)
d 2.80 (dd,
J = 4.8, 2.4 Hz, 1H), 2.96 (dd, J = 4.4, 4.4 Hz, 1H), 3.00 (dd, J = 4.8,
4.0 Hz, 1H), 3.15–3.17 (m, 1H), 3.38–3.42 (m, 1H), 3.52–3.55 (m,
1H), 4.02 (dd, J =10.8, 6.2 Hz, 1H), 4.20 (dd, J = 11.2, 4.4 Hz, 1H),
4.36 (dt, J = 11.2, 2.4 Hz, 1H), 4.47 (dt, J = 11.2, 4.2 Hz, 1H), 6.52
(d, J = 2.4 Hz, 1H), 6.57 (d, J = 2.4 Hz, 1H), 7.45 (d, J = 8.8 Hz, 1H),
7.56 (ddd, J = 7.2, 7.2, 1.2 Hz, 1H), 7.72 (ddd, J = 7.2, 7.2, 1.2 Hz,
1H), 7.87 (d, J = 7.2 Hz, 1H), 8.05 (d, J = 8.8 Hz, 1H), 10.05 (d,
J = 8.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) 44.8, 45.3, 50.0, 50.5,
69.5, 69.7, 94.0, 97.9, 109.8, 115.9, 117.5, 126.1, 127.3, 128.5,
129.4, 130.6, 131.3, 135.9, 156.2, 158.3, 160.8, 162.9, 177.5 ppm;
GC–MS (EI): m/e 390 [M]+.
4.1.12. 1-Hydroxy-3-(oxiran-2-ylmethoxy)-12H-
benzo[b]xanthen-12-one (17)
To a mixture of compound 15 (0.1 g, 0.36 mmol) and K2CO3
(0.2 g, 1.44 mmol) in anhydrous DMF (5 mL) was added epichloro-
hydrin (0.19 g, 2.05 mmol) with a syringe. The reaction mixture
was stirred overnight at 75 °C under nitrogen and then cooled to
room temperature. The mixture was diluted with water and ex-
tracted with ethyl acetate (two times). The combined organic layer
was washed with brine and dried over anhydrous Na2SO4. The sol-
vent was removed under reduced pressure and the residue was
purified by silica gel column chromatography (eluant: ethyl ace-
tate/n-hexane = 1:3) to give compound 17 (13.0 mg, 10.8%) as a
yellow solid. Mp 201–202 °C; Rf: 0.58 (ethyl acetate/n-hex-
ane = 1:1); 1H NMR (400 MHz, CDCl3) d 2.82 (dd, J = 4.4, 2.8 Hz,
1H), 2.98 (dd, J = 4.4, 4.4 Hz, 1H), 3.41–3.44 (m, 1H), 4.04 (dd,
J = 10.8, 6.0 Hz, 1H), 4.39 (dd, J = 11.2, 3.2 Hz, 1H), 6.36 (d,
J = 2.0 Hz, 1H), 6.50 (d, J = 2.0 Hz, 1H), 7.52 (dd, J = 7.2, 6.8 Hz,
1H), 7.64 (dd, J = 7.2, 6.8 Hz, 1H), 7.84 (s, 1H), 7.92 (d, J = 8.4 Hz,
1H), 8.07 (d, J = 8.4 Hz, 1H), 8.85 (s, 1H); 13C NMR (100 MHz,
CDCl3+CD3OD) 44.8, 50.0, 69.4, 94.0, 97.2, 113.4, 113.5, 120.3,
4.1.9. 1,3-Dihydroxy-12H-benzo[b]xanthen-12-one (15)
The reaction mixture of 3-hydroxy-2-naphthoic acid (2.0 g,
10.63 mmol), phloroglucinol (1.34 g, 10.63 mmol), ZnCl2 (3.30 g,
24.24 mmol), and POCl3 (40 mL) was refluxed for 5 h at 80 °C,
cooled to room temperature and then poured into iced water very
slowly. The solid formed was kept for 1 d at room temperature, col-
lected and washed with water to give a brown solid. After drying
under vacuum, the solid was applied to silica gel column chroma-
tography (eluant: ethyl acetate/n-hexane = 1:3) to give a yellow
solid (0.29 g, 9.8%). Rf: 0.47 (ethyl acetate/n-hexane = 1:3); 1H
NMR (400 MHz, DMSO-d6) d 6.16 (s, 1H), 6.35 (s, 1H), 7.52 (dd,
J = 7.2, 6.8 Hz, 1H), 7.65 (dd, J = 7.2, 6.8 Hz, 1H), 7.99 (s, 2H), 8.17
(d, J = 8.0 Hz, 1H), 8.76 (s, 1H), 12.84 (s, 1H).

1016
H.-J. Cho et al. / Bioorg. Med. Chem. 18 (2010) 1010–1017
126.0, 127.3, 127.8, 129.6, 129.8, 129.9, 137.0, 152.1, 158.4, 163.9,
human cervix tumor cell line (HeLa), human prostate tumor cell
line (DU145), human colorectal carcinoma cell line (HCT116),
and human myelogenous leukemia cell line (HL60). Cancer cells
were cultured according to supplier’s instructions. Cells were
seeded in 96-well plates at a density of 2–4 Â 104 cells per well
and incubated overnight in 0.1 mL of media supplied with 10% fetal
bovine serum (Hyclone, USA) in 5% CO2 incubator at 37 °C. On day
2, culture medium in each well was exchanged with 0.1 mL ali-
quots of medium containing graded concentrations of compounds.
166.1, 181.7 ppm; GC–MS (EI): m/e 334 [M]+.
4.1.13. 1,3-Bis(oxiran-2-ylmethoxy)-12H-benzo[b]xanthen-12-
one (19)
To a mixture of compound 15 (0.15 g, 0.54 mmol) and Cs2CO3
(0.53 g, 1.63 mmol) in anhydrous acetone (30 mL) was added epi-
chlorohydrin (0.25 g, 2.7 mmol) with syringe. The reaction mixture
was stirred overnight at 55–60 °C under nitrogen and then cooled to
room temperature. The solid in the mixture was removed by filtra-
tion and the filtrate was concentrated under reduced pressure. The
residue was purified by silica gel column chromatography (eluant:
ethyl acetate/n-hexane = 1:3) to give compound 19 (15.3 mg, 7.3%)
as a yellow solid. Mp 181–182 °C; Rf: 0.26 (ethyl acetate/n-hex-
ane = 1:1); 1H NMR (400 MHz, CDCl3) d 2.80 (dd, J = 4.8, 2.4 Hz,
1H), 2.96 (dd, J = 4.0, 2.4 Hz, 1H), 2.99 (dd, J = 5.2, 4.4 Hz, 1H),
3.21–3.25 (m, 1H), 3.35–3.41 (m, 1H), 3.47–3.52 (m, 1H), 4.00
(ddd, J = 10.4, 5.2, 2.0 Hz, 1H), 4.18 (dd, J = 11.2, 4.4 Hz, 1H), 4.36
(dt, J = 9.6, 2.4 Hz, 1H), 4.40 (dt, J = 11.2, 2.4 Hz, 1H), 6.37 (d,
J = 2.4 Hz, 1H), 6.49 (d, J = 2.4 Hz, 1H), 7.45 (ddd, J = 8.0, 6.8, 1.2 Hz,
1H), 7.56 (ddd, J = 8.0, 6.8, 1.2 Hz, 1H), 7.72 (s, 1H), 7.85 (d,
J = 8.0 Hz, 1H), 8.01 (d, J = 8.4 Hz, 1H), 8.82 (s, 1H); 13C NMR
(100 MHz, CDCl3) 44.8, 45.2, 50.0, 50.3, 69.0, 69.5, 94.5, 96.6,
107.3, 112.8, 122.7, 125.5, 127.1, 128.3, 128.9, 130.0 (Â2), 136.3,
151.7, 160.3, 161.2, 164.1, 175.9 ppm; GC–MS (EI): m/e 390 [M]+.
On day 4, 5 lL of the cell counting kit-8 solution (Dojindo, Japan)
were added to each well and then incubated for 4 h under the same
conditions. The absorbance of each well was determined by an
Automatic Elisa Reader System (Bio-Rad 3550) at a wavelength
of 450 nm. For determination of IC50 values, the absorbance read-
ings at 450 nm were fitted to the four-parameter logistic equation.
Adriamycin, etoposide, and camptothecin were purchased from
Sigma and used as positive controls.
4.5. DNA cross-linking assay
DNA cross-linking property of each compound was tested using
linearized pBR322 plasmid DNA and denaturing 1.2% alkaline aga-
rose gel electrophoresis. The linear plasmid DNA was obtained by
treating circular pBR322 plasmid DNA with EcoRI (Invitrogen,
USA) and purified by ethanol precipitation. Alkaline agarose gel
(1.2%) was prepared with the solution (pH 8.0) containing 50 mM
4.2. Human topoisomerase I relaxation assay
NaCl and 1 mM EDTA. 0.5 lg of the linearized pBR322 was incu-
bated with designated concentrations of compound for 2 h at room
temperature in buffered 10 mM Tris–HCl and 1 mM EDTA adjusted
to pH 8.0. After the mixture of DNA and compound was loaded
with agarose loading dye, the gel was soaked in an alkaline running
buffer containing 50 mM NaOH and 1 mM EDTA. The gel was run in
fresh alkaline running buffer then neutralized for 1 h in neutraliz-
ing buffer solution containing 100 mM Tris and 150 mM NaCl ad-
justed to pH 7.6 with refreshing every 20 min. The gel was
All test compounds were dissolved in DMSO at a concentration
of 20 mM as stock solutions and kept at À20 °C until used. DNA
topo I inhibition assay was performed as described previously with
minor modifications. The activity of DNA topo I was determined by
assessing the relaxation of supercoiled pBR322 DNA. The mixture
of 100 ng of plasmid pBR322 DNA and 0.4 units of recombinant hu-
man DNA topoisomerase I (TopoGEN Inc., USA) was incubated
without and with the prepared compounds at 37 °C for 30 min in
relaxation buffer [10 mM Tris–HCl (pH 7.9), 150 mM NaCl, 0.1% bo-
vine serum albumin, 1 mM spermidine, 5% glycerol]. The reaction
subsequently stained with ethidium bromide solution (2.5 lL of
10 mg/mL ethidium bromide in 50 mL of neutralizing buffer solu-
tion). The gel was visualized by UV transillumination and photo-
graphed using ChemiImager™ Ready (Alpha Innotech Corp.).
(final volume = 10 lL) was terminated by adding 2.5 lL of stop
solution containing 5% sarcosyl, 0.0025% bromophenol blue, and
25% glycerol. DNA samples were then electrophoresed on 1% aga-
rose gels at 15 V for 7 h with TAE running buffer. Gels were stained
Acknowledgements
for 30 min in an aqueous solution of ethidium bromide (0.5 lg/
mL). DNA bands were visualized by transillumination with UV light
and were quantified using AlphaImager™ (Alpha Innotech Corp.).
This work was supported by the Korea Research Foundation
Grant funded by the Korean Government (MOEHRD) (KRF-2006-
521-E00159). M.-J.J. was partially supported by the Brain Korea
21 project.
4.3. Human topoisomerase IIa relaxation assay
References and notes
DNA topo II inhibitory activity of compounds was measured as
follows. A mixture of 200 ng of supercoiled pBR322 plasmid DNA
and 1 unit of human DNA topoisomerase IIa (Usb Corp., USA) was
incubatedwithoutand with the prepared compoundsin assay buffer
[10 mM Tris–HCl (pH 7.9) containing 50 mM NaCl, 50 mM KCl,
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5 mM MgCl2, 1 mM EDTA, 1 mM ATP, and 15
albumin] for 30 min at 30 °C. The reaction (final volume = 20
was terminated by the addition of 3 L of 7 mM EDTA. Reaction
lg/mL bovine serum
l
L)
l
products were electrophoresed on 1% agarose gels at 25 V for 4 h
with TAE running buffer. Gels were stained for 30 min in an aqueous
solution of ethidium bromide (0.5 lg/mL). DNA bands were visual-
ized by transillumination with UV light and supercoiled DNA was
quantified using AlphaImager™ (Alpha Innotech Corp.).
4.4. Cytotoxicity test
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