Synthesis and pharmacological evaluation of new methyloxiranylmethoxyxanthone analogues

Article abstract of DOI:10.1016/j.ejmech.2010.06.017

In order to develop potential anti-cancer agents that act on topoisomerase II and DNA, we have synthesized 12 new xanthone derivatives. In the cytotoxicity test, compounds 17 and 31 exhibited 2- to 7-fold stronger inhibitory activity than adriamycin again

Full text of DOI:10.1016/j.ejmech.2010.06.017

European Journal of Medicinal Chemistry 45 (2010) 4221e4228
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and pharmacological evaluation of new
methyloxiranylmethoxyxanthone analogues
,
,
Sangwook Woo a 1, Da-hye Kang b 1, Jung Min Nam b, Chong Soon Lee c, Eun-Mi Ha a, Eung-Seok Lee d,
**
,
a,
*
Youngjoo Kwon b , Younghwa Na
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 Biochemistry, College of Natural Sciences, Yeungnam University, Gyeongsan 712-749, 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:
In order to develop potential anti-cancer agents that act on topoisomerase II and DNA, we have
synthesized 12 new xanthone derivatives. In the cytotoxicity test, compounds 17 and 31 exhibited 2- to
7-fold stronger inhibitory activity than adriamycin against most cancer cell lines tested. Halohydrin
group-tethered compounds 19, 21 and 27 showed comparable topoisomerase II inhibitory activity to
Received 17 April 2010
Received in revised form
6 June 2010
Accepted 9 June 2010
Available online 17 June 2010
etoposide at 100 mM concentration. In the DNA cross-linking test, compounds 20, 30 and 31 produced
DNA cross-linked adducts and compound 30 was the strongest DNA cross-linker. Based on the combined
pharmacological results, we suspected that the strong anti-cancer activity of compounds 16, 17, 20, 30
and 31 originated from the DNA mono-alkylation or cross-linking properties of the compounds.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Anticancer activities
Topoisomerase II inhibition
DNA cross-linking
Oxiranylmethoxyxanthones
Methyloxiranylmethoxyxanthones
1. Introduction
halohydrin (4) analogues showed significant cytotoxic and topo-
isomerase II relaxation inhibitory activities [3]. The 1,3-bis-oxir-
Xanthones (1) are mainly identified and isolated as secondary
metabolites from higher plants and microorganisms. They have
diverse biological activities including anti-hypertensive, anti-
oxidative, anti-thrombotic, and anti-cancer activities according to
their diverse structures [1]. Because of the simple and interesting
structural scaffolds and wide biological profiles of xanthones,
researchers have endeavored to isolate or synthesize numerous
derivatives of xanthone compounds as potential drug candidates.
Among the xanthones reported to date, oxygenated xanthones
synthesized or isolated from natural resources inhibit the prolif-
eration of several cancer cell lines [2]. Especially, 20,30-epoxy-
propoxy-substituted xanthones efficiently prohibit the growth of
cancer cells, and xanthone (2) possessing two 20,30-epoxypropoxy
groups at the 3 and 5 positions show the most active anti-cancer
activity in the series prepared [2a,b]. We previously reported that
the 20,30-epoxypropoxyxanthones (3) and their ring-opened
anylmethyloxy-substituted compound (5) was particularly efficient
among the compounds tested. Compound 5 also showed strong
DNA cross-linking activity. The findings suggested that the epoxide
ring coupling and its ring-opened compounds might modulate the
biological efficacy of xanthone compounds.
Topoisomerases are critical cellular enzymes necessary for cell
proliferation that remove topological hurdles in the process of DNA
replication [4]. Among them, topoisomerase II relaxes supercoiled
DNA double helices by mediating the cleavage of double-stranded
DNA and the process of religation. Because of the importance of
these enzymes in the cell proliferative process, topoisomerases have
been one of the major targets in anti-cancer drug development.
Interstrand DNA cross-linking of two strands of DNA inhibits the
cascade of cell proliferation, such as replication and transcription,
and finally causes cell death [5,6]. Some anti-tumor compounds
that possess two electrophilic functions, such as mitomycin C and
nitrogen mustard, can act as DNA cross-linking agents, which are an
important class of anti-tumor drugs targeting DNA [7,8].
* Corresponding authors. Tel.: þ82 53 850 3616; fax: þ82 53 850 3602.
** Corresponding authors. Tel.: þ82 2 3277 4653; fax: þ82 2 3277 3051.
E-mail addresses: ykwon@ewha.ac.kr (Y. Kwon), yna7315@cu.ac.kr (Y. Na).
Psorospermin (6) isolated from an African plant is a natural
compound that shows good anti-cancer activity against human
and murine cancer cell lines [9]. In its structure, 6 possesses
1
These authors equally contributed to this work.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.06.017

4222
S. Woo et al. / European Journal of Medicinal Chemistry 45 (2010) 4221e4228
O
O
O
O
OH
O
O
OH
O
O
OH
8
1
9
2
7
6
B
A
4
3
O
O
O
5
10
O
OH
Cl
O
O
4
3
2
1
O
O
O
O
OR2
O
O
OCH3
R1 = H, OCH3
R2 = H, CH3
R3 = H, CH3
O
O
O
O
O
O
R1
R3
O
OH
H
O
7
5
6
O
a 20,30-epoxypropoxy-xanthone skeleton. Its biological activities
are known to be expressed via intercalation of the xanthone group
into a DNA base pair and alkylation of the epoxide by N7-guanine in
the presence of topoisomerase II [10].
change the DNA alkylation pattern compared to the parent
compound. Here, we report the synthesis of new xanthone
analogues and their pharmacological activities, including cytotox-
icity, topoisomerase II inhibition and DNA cross-linking.
In our continued effort to develop anti-cancer drug candidates
that target topoisomerases and DNA, we designed a series of new
xanthone analogues possessing methyloxyranyl-methoxy groups.
For this purpose, we synthesized three different groups of
xanthones (7) based on the addition of 1) a methoxyl group at the
C5-position on the B ring, 2) a methoxyl group at the C1-position
instead of a hydroxyl group on the A ring, and 3) a [2-(methyl)
oxiranyl]methoxy group at the C3-position in the xanthone core.
We suspected that the introduction of methoxyl groups in the
xanthone core would affect their mode of interaction such as
intercalation and/or binding to DNA and enzymes. On the other
hand, the addition of a methyl group into the oxiranyl part would
2. Chemistry
2.1. Synthesis of 3-(oxiran-2-ylmethoxy)-9H-xanthen-9-one
analogues
First, 3-hydroxy-1-methoxyxanthone (14) and 3-hydroxy-1,5-
dimethoxyxanthone (15) were used as starting compounds for
preparation of 1-methoxy-3-(oxiran-2-ylmethoxy)-9H-xanthen-9-
one analogues and were obtained from the corresponding 1,3-
dihydroxyxanthone (8) [3] and 1,3-dihydroxy-5-methoxyxanthone
(9). The 3-hydroxyl groups of compounds 8 and 9 were protected
O
O
OH
O
O
OH
O
O
OCH3
O
O
OCH3
H2
BnBr
CH3I
Pd/C
K2CO3
Cs2CO3
DMF
OH
OBn
OH
OBn
acetone
R
R
R
R
8 R = H
9 R = OCH3
10 R = H
14 R = H
12 R = H
13 R = OCH3
11 R = OCH3
15 R = OCH3
Scheme 1. Synthesis of 3-Hydroxy-1-methoxyxanthone (14) and 3-Hydroxy-1,5-dimethoxyxanthone (15).
OR2
O
O
OR2
O
O
OR2
A
O
O
Cl
K2CO3
1M-HCl/EtOAc
RT, 30 min
O
+
Acetone
reflux
O
O
OH
R1
R1
OH
Cl
R1
O
18 R1 = H,
R2 = CH3
14 R1 = H,
R2 = CH3
16 R1 = H,
R2 = CH3
19 R1 = OCH3, R2 = CH3
15 R1 = OCH3, R2 = CH3
17 R1 = OCH3, R2 = CH3
OH
O
O
OH
O
O
O
B
O
O
Cl
O
NaH
Cl
+
O
+
DMF, 70 O
C
OH
O
O
O
OCH3
OCH3
OCH3
O
O
20
21
9
Scheme 2. Synthesis of 3-(oxiran-2-ylmethoxy)-9H-xanthen-9-one analogues.

S. Woo et al. / European Journal of Medicinal Chemistry 45 (2010) 4221e4228
4223
O
OH
A
O
O
OH
K2CO3
Cl
+
Acetone, Reflux
O
OH
O
R
R
8 R = H
22 R = H
9 R = OCH3
23 R = OCH3
m-CPBA
CH2Cl2, RT
O
O
OH
O
O
OH
1M-HCl/EtOAc
O
O
R
R
OH
Cl
O
26 R = H
24 R = H
27 R = OCH3
25 R = OCH3
B
O
O
OH
O
O
O
O
O
O
O
Cs2CO3
Cl
+
m-CPBA
Acetone
Reflux
OH
O
O
CH2Cl2, RT
R
R
R
O
8 R = H
9 R = OCH3
28 R = H
30 R = H
29 R = OCH3
31 R = OCH3
Scheme 3. Synthesis of 3-[(2-methyloxiran-2-yl)methoxy]-9H-xanthen-9-one analogues.
with benzyl bromide, and subsequent methylation with methyl
iodide under Cs2CO3 basic conditions gave compounds 12 and 13.
Final deprotection of the benzyl group was accomplished under
hydrogenolysis, and the compounds obtained were applied to the
next step without purification (Scheme 1).
and oxyranylmethoxy groups were observed in the gCOSY experi-
ment. In the 13C NMR spectrum, we observed a signal at 71.6 ppm
corresponding to C200, but peak separation between diastereomers
was not recorded. The molecular ion peak (m/e 407.1 [Mþ1]þ)
supported the formation of one halohydrin (Scheme 2B).
The coupling reaction of the prepared xanthones and epichloro-
hydrin (4e5 equiv.) was conducted in basic K2CO3 (2e2.5 equiv.) in
acetone. In the 1H NMR spectrum, compound 16 showed two doublet
2.2. Synthesis of 3-[(2-methyloxiran-2-yl)methoxy]-9H-xanthen-9-
one analogues
of doublet peaks at d 2.81 and 2.97 and one multiplet at d 3.36e3.39
corresponding to the two methylene protons and the methine proton
of the epoxide ring. We confirmed the correlation of the protonpeaks
by gCOSY experiment. In the 13C NMR spectrum, we observed three
signals at 69.4 (C-10), 50.0 (C-20), and 44.8 (C-30) ppm. Based on the
observation of the molecular ionpeak(m/e 299.1 [Mþ1]þ) in the mass
spectrum, we identified the structure of compound 16. Other
compounds also showed similar spectroscopic patterns (Scheme 2A).
Conversion of epoxide ring to halohydrin was accomplished in
1 M HCl aqueous ethyl acetate. For example, in the 1H NMR spec-
In order to prepare mono 3-[(2-methyloxiran-2-yl)methoxy]-
9H-xanthen-9-one analogues, we tried a two-step method. First,
compounds 22 and 23 were synthesized with 3-chloro-2-methyl-
propene (1.1 equiv.) and 8 or 9 under basic K2CO3 (3.5 equiv.)
acetone. In the 1H NMR spectrum, the appearance of a singlet peak
at d 12.88, corresponding to C1eOH, supported one methylpropene
Table 1
trum, the C-20 methine proton of compound 16 near
down-field shifted to around 4.20 due to the halohydrin formation
in compound 18. C-30 methylene also down-field shifted from
2.81
d 3.40 was
Cytotoxicity of prepared compounds.
a
d
Compounds
Cell lines/IC50
(
m
M)
d
MDA-MB231 MCF-7
HCT-116
DU-145
HeLa
and 2.97 to 3.76e3.8. In the 13C NMR spectrum, weobserveda down-
field shift of C-20 signal from 50.0 to 69.1 ppm by halohydrin
formation. The molecular ion peak (m/e 335.2 [Mþ1]þ) in the mass
spectrum confirmed the identity of compound 18. Other halohydrin
compounds showed similar spectroscopic patterns (Scheme 2A).
Bis-epoxy compound 20 was prepared with 11 equiv. of
epichlorohydrin in DMF under basic NaH (3 equiv.) conditions. In
the 1H and 13C NMR spectra, two sets of peaks derived from two
oxiranylmethoxy groups at the C1 and C3 positions were observed.
All spectral data (1H and 13C NMR, COSY, and Mass spectra) were
consistent with the proposed structures. Compound 21 was iso-
lated from the same reaction. In the 1H NMR spectrum, two
Adriamycin
Etoposide
Camptothecin
4.4 ꢀ 0.2
16.3 ꢀ 1.8
4.2 ꢀ 0.3
10.9 ꢀ 0.4
3.8 ꢀ 1.4
>100
56.0 ꢀ 2.1
5.4 ꢀ 0.7
18.9 ꢀ 2.4
41.7 ꢀ 2.0
>100
18.9 ꢀ 0.8
4.0 ꢀ 0.8
1.4 ꢀ 0.2 10.1 ꢀ 0.8
17.5 ꢀ 1.2 11.9 ꢀ 2.1 13.8 ꢀ 0.8 18.2 ꢀ 1.2
14.2 ꢀ 2.1
14.0 ꢀ 0.8
8.0 ꢀ 0.2
>100
2.3 ꢀ 0.2
6.6 ꢀ 2.3
1.5 ꢀ 0.1
3.2 ꢀ 0.2
1.5 ꢀ 0.3 10.5 ꢀ 0.3
0.8 ꢀ 0.1 6.2 ꢀ 0.3
1.1 ꢀ 0.6
16
17
18
19
20
21
24
25
26
27
30
31
21.6 ꢀ 3.4 40.1 ꢀ 2.3 >100
51.7 ꢀ 5.0 16.4 ꢀ 0.4 15.3 ꢀ 1.1 59.5 ꢀ 3.8
9.9 ꢀ 0.6
2.0 ꢀ 0.4
9.9 ꢀ 0.7
2.4 ꢀ 0.6
4.7 ꢀ 0.7
69.0 ꢀ 1.9
3.9 ꢀ 0.5 41.7 ꢀ 2.0
15.0 ꢀ 0.8 12.1 ꢀ 1.3 18.9 ꢀ 1.5 14.3 ꢀ 0.3
>100
>100
69.1 ꢀ 3.3 >100
47.1 ꢀ 4.3 >100
>100
>100
>100
15.4 ꢀ 1.2
68.2 ꢀ 5.4
5.1 ꢀ 0.1
88.4 ꢀ 5.4 15.8 ꢀ 1.0
7.2 ꢀ 0.6 68.2 ꢀ 5.4
15.2 ꢀ 1.7
2.5 ꢀ 0.4
2.6 ꢀ 0.9
0.8 ꢀ 0.0
3.9 ꢀ 0.4 15.8 ꢀ 2.3
methine protons of C-20 and C-200 appeared at
d 3.33e3.37 and
0.9 ꢀ 0.1
1.5 ꢀ 0.5
a
4.12e4.18; one of them was down-field shifted by hydroxyl group
Each data point represents mean ꢀ standard deviation, from three different
introduction at C200. Two correlation sets for protons of halohydrin
experiments performed in triplicate. Cell lines used are described in the text.

4224
S. Woo et al. / European Journal of Medicinal Chemistry 45 (2010) 4221e4228
Fig. 1. Topoisomerase II
a
inhibitory activities of compounds. Compounds were examined at a final concentration of 20 and 100
mM as indicated. Lane D: pBR322 only, Lane T:
pBR322 þ Topoisomerase II
a, Lane E: pBR322 þ Topoisomerase II
a
þ etoposide at indicated concentrations, Lanes with compounds, pBR322 þ Topoisomerase II
a
þ compounds at
indicated concentrations.
group substitution on C3eOH in compound 22. Subsequent
epoxidation with mCPBA afforded desired compounds 24 and 25
(Scheme 3A). In the 1H NMR spectrum of compound 24, two singlet
in the cell proliferation inhibition. Compounds 24 and 25, however,
which possessed one (2-methyloxiran-2-yl)methoxy group at the C3
position, showed lower activity than 16 and 17. Compound 25 lost
activity almost completely.
Inhibition of topoisomerase relaxation was evaluated using
human topoisomerase II (Topogen) with etoposide as a positive
control. Data were analyzed and evaluated with LabWork 4.5
Software to calculate the inhibition ratio. Test results are indicated
in Fig. 1 and Table 2. Compounds 19, 21 and 27 showed comparable
sp2 protons at
to
d 5.07 and 5.10 of compound 22 were up-field shifted
d
2.71 and 2.82 as two doublet peaks indicating complete
epoxidation. In the 13C NMR spectrum, two epoxide ring carbons
were detected at 52.1 and 55.4 ppm, respectively.
1,3-Bis[(2-methyloxiran-2-yl)methoxy]-9H-xanthen-9-one
analogues were also synthesized by the same method as the mono
analogues. But, Cs2CO3 (4 equiv.) was used as a base instead of
K2CO3, and 10 equiv. of 3-chloro-2-methylpropene were used.
Compounds 28 and 29 did not show C1eOH proton in the 1H NMR
spectrum, which supported introduction of two methylpropene
group on C1e and C3eOH. Subsequent epoxidation with mCPBA
afforded desired compounds 30 and 31 (Scheme 3B). All spectral
data were consistent with the desired structures.
inhibitory activity to etoposide at 100
mM concentration. Interest-
ingly, all of these compounds hold a halohydrin group at the C3
position and a methoxy group at the C5 position. In contrast,
compounds possessing one epoxide ring at the C3 position were
not good topoisomerase II inhibitors. However, the bis-epoxy
substituted compounds, 30 and 31, showed moderate inhibitory
activity at 100 mM concentration.
Because compound 5 previously showed excellent DNA cross-
linking activity, compounds possessing an epoxide ring were tested
for DNA cross-linking property. In the test, compounds 20, 30 and 31
produced DNA cross-linked adducts (Fig. 2). In further experiments
with various concentrations, the compounds generated DNA cross-
linked adducts in concentration-dependent way (Fig. 3). Compound
30, which lacks the C5-methoxygroup, was the strongest DNA cross-
3. Results and discussion
Synthetic procedures are quite straight-forward and we have
prepared 12 new xanthone derivatives. For the pharmacological
evaluationaspotentialanti-canceragents, wehavetested cytotoxicty,
topoisomerase II inhibitory activity and DNA cross-linking ability of
prepared compounds.
For the evaluation of cytotoxicity, five different cancer cell lines
were used: human breast adenocarcinoma cell line (MDA-MB231),
human cervix tumor cell line (HeLa), human prostate tumor cell line
(DU-145), human colorectal carcinoma cell line (HCT-116), and
human breast cancer cell line (MCF-7). The inhibitory activities of the
compounds are shown inTable 1. Among these, compounds 16,17, 20,
30, and 31 showed excellent suppression of the cancer cell line tested.
In particular, compounds 17 and 31 exhibited at least 2- to 7-fold
stronger inhibitory activity than Adriamycin (used as a reference),
except in the MDA-MB231 cell line. From a structural viewpoint, all
five compounds possessing one or two epoxide rings at C1 and C3
positiononthexanthoneshowedstrongcytotoxicityinthecancercell
lines tested, which indicates thatepoxide rings playan important role
Fig. 2. DNA cross-linking activities of prepared compounds were tested at a concen-
tration of 100 mM. M: l DNA/Hind III Marker (Promega) B; only DNA.
Table 2
The % inhibition of prepared compounds against topoisomerase IIa.
Compds/concentration
20
m
M
100 mM
Etoposide
16
17
40
5
0
71
51
4
18
19
20
21
24
25
26
27
1
2
9
27
11
3
20
41
24
15
33
83
71
65
25
23
52
85
41
48
Fig. 3. DNA cross-linking activities of compounds 20, 30 and 31 at various concen-
trations. M: Hind III DNA Marker (Promega). Lanes 2e6: 0, 10, 25, 50, 100
concentrations of compounds.
30
31
l
mM

S. Woo et al. / European Journal of Medicinal Chemistry 45 (2010) 4221e4228
4225
linker among the compounds tested. Based on our results, the
C5-methoxy group and the methyl group in the epoxide ring (20 vs.
31) reduced the DNA cross-linking ability of the xanthone
compounds. The findings suggest that the C5-methoxy group
hinders the intercalation of the planar xanthone ring into the DNA
base pair and finally positions the compound unfavorably for elec-
trophilic interactionwith DNA. The methyl group in the epoxide ring
might also sterically affect the position of the epoxide ring or the
hydrophobic interaction during compound-DNA interaction.
(200 mL) were added to a dry round-bottom flask. The reaction
mixture was refluxed overnight and cooled to room temperature
(RT). After addition of H2O (200 mL), the reaction mixture was
acidified with 3 M HCl. The mixture was extracted with ethyl
acetate (2 times) and the combined organic layer was washed with
H2O and brine and then dried over Na2SO4. Solvent was evaporated
under reduced pressure and the residue was purified by silica gel
column chromatography (eluant: CH2Cl2) to give compound 10
(1.05 g, 42.6%) as a pale yellow solid. 1H NMR (250 MHz, CDCl3)
d
5.14 (s, 2H), 6.42 (d, J ¼ 2.3 Hz, 1H), 6.49 (d, J ¼ 2.3 Hz, 1H),
4. Conclusions
7.24e7.44 (m, 7H), 7.70 (ddd, J ¼ 1.6, 7.2, 8.3 Hz, 1H), 8.23 (dd,
J ¼ 1.6, 7.9 Hz, 1H), 12.85 (s, 1H).
We have synthesized 12 new xanthone derivatives. In the
cytotoxicity test, compounds 16, 17, 20, 30, and 31 showed excellent
anti-cancer activities. In particular, compounds 17 and 31 exhibited
2- to 7-fold stronger inhibitory activity than Adriamycin against
most cancer cell lines tested. Compounds 19, 21 and 27, which
possess a halohydrin group at the C3 position and a methoxy group
at the C5 position, showed comparable topoisomerase II inhibitory
5.3. 3-Benzyloxy-1-hydroxy-5-methoxy-9H-xanthen-9-one (11)
Compound 9 (2.0 g, 7.75 mmol), benzyl bromide (1.38 mL,
11.63 mmol), K2CO3 (2.76 g, 0.02 mol), and anhydrous acetone
(200 mL) were added to a dry round-bottom flask. Reaction
mixture was refluxed overnight and cooled to RT. After the addition
of H2O (200 mL), the reaction mixture was acidified with 3 M HCl.
The mixture was extracted with ethyl acetate (2 times), and the
combined organic layer was washed with H2O and brine and then
dried over Na2SO4. Solvent was evaporated under reduced pressure
and the residue was purified by silica gel column chromatography
(eluant: CH2Cl2) to give compound 11 (1.29 g, 47.4%) as a pale
activities to etoposide at 100 mM concentration. The test results
revealed that epoxide ring-tethered compounds have strong cyto-
toxic activity and halohydrin compounds were efficient topo-
isomerase II inhibitors. In the DNA cross-linking test, compounds
20, 30 and 31 yielded DNA cross-linked adduct and compound 30
was the strongest DNA cross-linker. These observations suggest
that DNA cross-linking occurs after intercalation of the tricyclic
xanthone ring into the DNA base pairs, and the substituents on the
xanthone control the intercalation and DNA alkylation properties of
the xanthone analogues. Although the correlation between DNA
cross-linking and topoisomerase II inhibitory activities is not clear,
the topoisomerase II inhibition might occur through formation of
compoundeenzyme binary complex or compoundeenzymeeDNA
ternary complex by the VandereWaals interaction and/or hydrogen
bond, not by DNA alkylation. However, the DNA alkylation can
inhibit topoisomerase II function indirectly and the cross-linked
double helix DNA can be hardly relaxed by topoisomerase II. From
the combined pharmacological results, we suspect that the strong
anti-cancer activity of compounds 16, 17, 20, 30 and 31 originated
from the DNA mono-alkylation or cross-linking properties of the
compounds. In summary, with elaborate calibration, oxir-
anylmethoxy- or halohydrin-tethered xanthone derivatives show
potential for development as new anti-cancer agents.
yellow solid. 1H NMR (250 MHz, CDCl3)
d 4.05 (s, 3H), 5.18 (s, 2H),
6.46 (d, J ¼ 2.2 Hz, 1H), 6.66 (d, J ¼ 2.2 Hz, 1H), 7.23e7.48 (m, 7H),
7.84 (dd, J ¼ 1.8, 7.8 Hz, 1H), 12.86 (s, 1H).
5.4. 3-Benzyloxy-1-methoxy-9H-xanthen-9-one (12)
A DMF (10 mL) solution of compound 10 (0.40 g, 1.26 mmol) and
Cs2CO3 (0.82 g, 2.52 mmol) was added to methyl iodide (535.68 mg,
3.77 mmol). The mixture was stirred at 50 ꢁC (3 h), transferred to an
Erlenmeyer flask and then acidified with 2 M HCl in an ice bath. The
mixture was extracted with CH2Cl2 and H2O, and the combined
organic layer was washed with H2O and brine and then dried over
Na2SO4. Solvent was evaporated under reduced pressure and
sludge residue was triturated with ethanol. The solid formed was
filtered, collected and dried to give compound 12 (0.41 g, 97.3%) as
a white solid. 1H NMR (250 MHz, CDCl3)
d 3.95 (s, 3H), 5.15 (s, 2H),
6.42 (d, J ¼ 2.2 Hz, 1H), 6.56 (d, J ¼ 2.2 Hz, 1H), 7.24e7.47 (m, 7H),
7.61 (ddd, J ¼ 1.4, 6.9, 7.0 Hz, 1H), 8.27 (dd, J ¼ 1.4, 7.9 Hz, 1H).
5. Experimental
5.5. 3-Benzyloxy-1,5-dimethoxy-9H-xanthen-9-one (13)
5.1. General
To a DMF (10 mL) solution of compound 11 (0.5 g, 1.45 mmol)
and Cs2CO3 (0.94 g, 2.86 mmol) was added methyl iodide (0.62 g,
4.35 mmol). The mixture was stirred at 50 ꢁC (3 h), transferred to
an Erlenmeyer flask and then acidified with 2 M HCl in an ice bath.
The mixture was extracted with CH2Cl2 and H2O, and the
combined organic layer was washed with H2O and brine and then
dried over Na2SO4. The solvent was evaporated under reduced
pressure and the sludge residue was triturated with ethanol. The
solid formed was filtered, collected and dried to give compound 13
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) and silica gel for
column chromatography was Silica gel 60 (0.040e0.063 mm ASTM,
Merck).1H and 13C NMR spectra were taken on 250 MHz (Bruker) or
400 MHz (Varian NMR AS) instruments. 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 investigations were run on a LCQ advantage-trap mass
spectrometer equipped with an electrospray ionization (ESI) source
(Thermo Finnigan, San Jose, CA, USA). Melting points were
measured on a Barnstead International MEL-TEMPÒ 1202D instru-
ment without correction.
(0.29 g, 55.9%) as a white solid. 1H NMR (250 MHz, CDCl3)
d 4.00 (s,
3H), 3.95 (s, 3H), 5.13 (s, 2H), 6.42 (d, J ¼ 2.2 Hz, 1H), 6.70 (d,
J ¼ 2.2 Hz, 1H), 7.12e7.26 (m, 2H), 7.35e7.45 (m, 5H), 7.85 (dd,
J ¼ 1.6, 7.9 Hz, 1H).
5.6. 3-Hydroxy-1-methoxy-9H-xanthen-9-one (14)
5.2. 3-Benzyloxy-1-hydroxy-9H-xanthen-9-one (10)
A bottle containing compound 12 (120 mg, 0.36 mmol), Pd/C
(60 mg), and absolute ethanol (30 mL) was shaken under hydrogen
(50 psi) for 12 h. The reaction mixture was filtered with celite and
Compound 8 (1.77 g, 7.75 mmol), benzyl bromide (1.38 mL,
11.63 mmol), K2CO3 (2.76 g, 0.02 mol), and anhydrous acetone

4226
S. Woo et al. / European Journal of Medicinal Chemistry 45 (2010) 4221e4228
washed with DMF (20 mL). The combined filtrate was evaporated
under reduced pressure to remove the ethanol and the remaining
solution was directly used for the next reaction.
5.11. 3-(3-Chloro-2-hydroxypropoxy)-1,5-dimethoxy-9H-xanthen-
9-one (19)
A mixture of compound 17 (10 mg, 0.03 mmol) in 1 M HCl in
aqueous ethyl acetate (3 mL) was stirred at RT (30 min). Solvent was
removed under reduced pressure and residue was dried under
vacuum to give compound 19 (10 mg, 90%) as a brown jelly. 1H NMR
5.7. 3-Hydroxy-1,5-dimethoxy-9H-xanthen-9-one (15)
A bottle containing compound 13 (100 mg, 0.28 mmol), Pd/C
(50 mg), and absolute ethanol (30 mL) was shaken under hydrogen
(50 psi) for 12 h. The reaction mixture was filtered with celite and
washed with DMF (20 mL). The combined filtrate was evaporated
under reduced pressure to remove ethanol and the remaining
solution was directly used for the next reaction.
(400 MHz, CDCl3) d 3.75e3.82 (m, 2H), 3.99 (s, 3H), 4.01 (s, 3H), 4.20
(m, 2H), 4.28e4.31 (m,1H), 6.37 (d, J ¼ 2.0 Hz,1H), 6.53 (d, J ¼ 2.0 Hz,
1H), 7.17 (dd, J ¼ 1.2, 7.6 Hz,1H), 7.25 (dd, J ¼ 7.6, 8.0 Hz,1H), 7.86 (dd,
J ¼ 1.2, 8.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) 46.0, 69.3, 69.8, 56.6,
56.7, 93.8, 95.9, 107.9, 114.8, 118.0, 123.6, 124.2, 145.4, 148.2, 159.7,
162.2, 163.6, 175.5 ppm; LC-ESI: m/e 365.1 [Mþ1]þ.
5.8. 1-Methoxy-3-(oxiran-2-ylmethoxy)-9H-xanthen-9-one (16)
5.12. 5-Methoxy-1,3-bis(oxiran-2-ylmethoxy)-9H-xanthen-9-one
(20) and 1-(3-chloro-2-hydroxypropoxy)-5-methoxy-3-(oxiran-2-
ylmethoxy)-9H-xanthen-9-one (21)
A mixture of compound 14 and K2CO3 (99.8 mg, 0.72 mmol) in
DMF (20 mL) was added to epichlorohydrin (0.11 mL, 1.44 mmol).
The reaction mixture was stirred at 70 ꢁC (15 h) and H2O (20 mL)
was added. After extraction of the mixture with ethyl acetate (2
times), the organic layer was washed with brine and dried over
Na2SO4. Solvent was evaporated under reduced pressure and the
residue was purified by silica gel column chromatography (eluant:
ethyl acetate/n-hexane ¼ 3:1) to give compound 16 (28 mg, 26%) as
an orange solid. Mp 122 ꢁC; Rf 0.09 (ethyl acetate/n-hexane ¼ 1:1);
A mixture of compound 9 (100 mg, 0.39 mmol) and NaH
(27.9 mg, 1.16 mmol) in DMF (15 mL) was added to epichlorohydrin
(0.3 mL, 4.09 mmol) under nitrogen. The reaction mixture was
stirred at 70 ꢁC (15 h) and then quenched by slow addition of H2O.
The mixture was repeatedly extracted with CH2Cl2, and the
combined organic layer was washed with brine and dried over
Na2SO4. Solvent was evaporated and the residue was purified by
silica gel column chromatography (eluant: ethyl acetate/n-
hexane ¼ 2:1) to afford compound 20 (35 mg, 24.4%) as a brown
jelly and compound 21 (19 mg, 12.1%) as a white solid. Compound
20: Rf 0.19 (ethyl acetate/n-hexane ¼ 1:1); 1H NMR (400 MHz,
1H NMR (400 MHz, CDCl3)
d
2.81 (dd, J ¼ 2.6, 4.8 Hz, 1H), 2.97 (dd,
J ¼ 4.6, 4.8 Hz, 1H), 3.39e3.42 (m, 1H), 3.98 (s, 3H), 4.02 (dd, J ¼ 5.6,
11.4 Hz, 1H), 4.38 (dd, J ¼ 3.0, 11.4 Hz, 1H), 6.40 (d, J ¼ 2.2 Hz, 1H),
6.50 (d, J ¼ 2.2 Hz, 1H), 7.31e7.37 (m, 2H), 7.63 (ddd, J ¼ 1.6, 7.2,
8.4 Hz, 1H), 8.28 (dd, J ¼ 1.6, 7.6 Hz, 1H); 13C NMR (100 MHz, CDCl3)
44.8, 50.0, 56.6, 69.4, 93.7, 95.7, 107.8, 117.1, 123.3, 124.1, 127.0, 134.0,
155.1, 159.9, 162.3, 163.8, 175.7 ppm; LC-ESI: m/e 299.1 [Mþ1]þ.
CDCl3)
d
2.77 (dd, J ¼ 2.6, 4.6 Hz, 1H), 2.93e2.97 (m, 2H), 3.16e3.18
(m, 1H), 3.37e3.41 (m, 1H), 3.45e3.49 (m, 1H), 3.97e4.01 (m, 1H),
4.00 (s, 3H), 4.17 (dd, J ¼ 4.6, 11.0 Hz, 1H), 4.33e4.37 (m, 2H), 6.44
(d, J ¼ 2.0 Hz, 1H), 6.63 (d, J ¼ 2.0 Hz, 1H), 7.15 (dd, J ¼ 1.2, 8.4 Hz,
1H), 7.24 (dd, J ¼ 7.8, 8.4 Hz, 1H), 7.84 (dd, J ¼ 1.4, 7.8 Hz, 1H); 13C
NMR (100 MHz, CDCl3) 44.8, 45.3, 49.9, 50.3, 56.6, 69.2 (69.1), 69.5,
94.4, 97.51, 108.0, 114.8, 117.9, 123.6, 124.2, 145.4, 148.2, 159.6, 160.9,
163.6, 175.3 ppm; LC-ESI: m/e 371.1 [Mþ1]þ. Compound 21: Mp
180 ꢁC; Rf 0.09 (ethyl acetate/n-hexane ¼ 1:2); 1H NMR (400 MHz,
5.9. 1,5-Dimethoxy-3-(oxiran-2-ylmethoxy)-9H-xanthen-9-one (17)
A mixture of compound 15 and K2CO3 (76.29 mg, 0.55 mmol) in
DMF (20 mL) was added to epichlorohydrin (0.09 mL, 1.10 mmol).
The reaction mixture was stirred at 70 ꢁC (15 h) and H2O (20 mL)
was added. After extraction of the mixture with ethyl acetate (2
times), the organic layer was washed with brine and dried over
Na2SO4. Solvent was evaporated under reduced pressure and the
residue was purified by silica gel column chromatography (eluant:
ethyl acetate/n-hexane ¼ 3:1) to give compound 17 (29 mg, 32%) as
a white solid. Mp 186 ꢁC; Rf 0.09 (ethyl acetate/n-hexane ¼ 1:1); 1H
CDCl3)
d
2.71e2.74 (m, 1H), 2.89 (dd, J ¼ 4.4, 4.4 Hz, 1H), 3.33e3.37
(m, 1H), 3.72e3.74 (m, 1H), 3.92e3.97 (m, 1H), 3.95 (s, 3H),
4.12e4.18 (m, 1H), 4.20 (dd, J ¼ 3.2, 8.8 Hz, 1H), 4.29 (dd, J ¼ 3.2,
8.8 Hz, 1H), 4.31 (dd, J ¼ 2.8, 11.2 Hz, 1H), 6.41 (d, J ¼ 2.0 Hz, 1H),
6.62 (d, J ¼ 2.0 Hz, 1H), 7.12 (dd, J ¼ 1.4, 8.0 Hz, 1H), 7.20 (dd, J ¼ 8.0,
8.0 Hz, 1H), 7.76 (dd, J ¼ 1.2, 8.0 Hz, 1H); 13C NMR (100 MHz, CDCl3)
44.5, 44.7, 49.9, 56.6, 69.8, 69.6 (69.5), 71.6, 95.2 (95.1), 99.5 (99.4),
108.3, 115.1, 117.9, 123.8, 123.9, 145.6, 148.3, 159.4, 161.3, 164.0,
176.2 ppm; LC-ESI: m/e 407.1 [Mþ1]þ.
NMR (400 MHz, CDCl3)
d
2.79 (dd, J ¼ 2.6, 4.6 Hz, 1H), 2.96 (dd,
J ¼ 4.6, 4.6 Hz, 1H), 3.40e3.42 (m, 1H), 3.98 (s, 3H), 4.01 (s, 3H), 4.02
(dd, J ¼ 5.5, 11.2 Hz, 1H), 4.37 (dd, J ¼ 2.8, 11.2 Hz, 1H), 6.42 (d,
J ¼ 2.0 Hz, 1H), 6.61 (d, J ¼ 2.0 Hz, 1H), 7.16 (dd, J ¼ 0.4, 7.6 Hz, 1H),
7.25 (dd, J ¼ 7.6, 8.0 Hz, 1H), 7.86 (dd, J ¼ 0.4, 8.0 Hz, 1H); 13C NMR
(100 MHz, CDCl3) 44.8, 49.9, 56.6, 56.6, 69.4, 93.7, 96.0, 107.8, 114.8,
118.0, 123.6, 124.2, 145.4, 148.1, 159.7, 162.2, 163.8, 175.5 ppm; LC-
ESI: m/e 329.2 [Mþ1]þ.
5.13. 1-Hydroxy-3-(2-methylallyloxy)-9H-xanthen-9-one (22)
A mixture of compound 8 (0.75 g, 3.29 mmol) and K2CO3 (1.63 g,
11.8 mmol) in anhydrous acetone (40 mL) was added to 3-chloro-2-
methylpropene (0.36 mL, 3.72 mmol) in acetone (10 mL). The
reaction mixture was refluxed overnight and the solvent was
removed under reduced pressure. H2O (50 mL) was added and
acidified with 1 M HCl, and then the mixture was extracted with
CH2Cl2 (2 times). The combined organic layer was washed with
brine and dried over Na2SO4. Solvent was evaporated under
reduced pressure and the residue was purified by silica gel column
chromatography (eluant: ethyl acetate/n-hexane ¼ 1:1) to afford
compound 22 (0.62 g, 66.8%) as a yellow solid. 1H NMR (200 MHz,
5.10. 3-(3-Chloro-2-hydroxypropoxy)-1-methoxy-9H-xanthen-9-
one (18)
A mixture of compound 16 (25 mg, 0.08 mmol) in 1 M HCl in
aqueous ethyl acetate (3 mL) was stirred at RT (30 min). Solvent was
removed under reduced pressure and the residue was dried under
vacuum to give compound 18 (20 mg, 71.3%) as a brown solid. Mp
107 ꢁC; 1H NMR (400 MHz, CDCl3)
d 3.76e3.85 (m, 2H), 3.99 (s, 3H),
4.20e4.22 (m, 2H), 4.26e4.34 (m, 1H), 6.38 (d, J ¼ 2.4 Hz, 1H), 6.53
(d, J ¼ 2.4 Hz, 1H), 7.30e7.35 (m, 2H), 7.63 (ddd, J ¼ 1.6, 7.2, 8.3 Hz,
1H), 8.29 (dd, J ¼ 1.6, 8.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) 46.1,
56.6, 69.1, 69.8, 93.7, 95.5,108.0,117.2,123.3,124.1,127.0,134.0,155.2,
159.9, 162.4, 163.6, 175.6 ppm; LC-ESI: m/e 335.2 [Mþ1]þ.
CDCl3)
d 1.87 (s, 3H), 4.55 (s, 2H), 5.07 (s, 1H), 5.14 (s, 1H), 6.40 (d,
J ¼ 2.0 Hz, 1H), 6.48 (d, J ¼ 2.0 Hz, 1H), 7.36e7.47 (m, 2H), 7.74 (ddd,
J ¼ 1.4, 7.0, 8.4 Hz, 1H), 8.27 (dd, J ¼ 1.4, 8.0 Hz, 1H), 12.88 (s, 1H).

S. Woo et al. / European Journal of Medicinal Chemistry 45 (2010) 4221e4228
4227
5.14. 1-Hydroxy-5-methoxy-3-(2-methylallyloxy)-9H-xanthen-9-
one (23)
8.0 Hz, 1H), 7.44 (d, J ¼ 8.4 Hz, 1H), 7.73 (ddd, J ¼ 1.6, 7.1, 8.4 Hz,
1H), 8.26 (dd, J ¼ 1.6, 8.0 Hz, 1H), 12.86 (s, 1H); 13C NMR
(100 MHz, CDCl3) 22.5, 50.3, 71.9, 72.0, 93.5, 97.8, 104.6, 117.8,
120.8, 124.1, 126.1, 135.4, 156.2, 157.9, 163.8, 165.4, 181.1 ppm; LC-
ESI: m/e 335.2 [Mþ1]þ.
A mixture of compound 9 (848.8 mg, 3.29 mmol) and K2CO3
(1.63 g, 11.8 mmol) in anhydrous acetone (50 mL) was added 3-
chloro-2-methylpropene (0.36 mL, 3.72 mmol) in acetone (10 mL).
The reaction mixture was refluxed overnight and solvent was
removed under reduced pressure. H2O (50 mL) was added and
acidified with 1 M HCl and then the mixture was extracted with
CH2Cl2 (2 times). The combined organic layer was washed with
brine and dried over Na2SO4. Solvent was evaporated under
reduced pressure and the residue was purified by silica gel column
chromatography (eluant: ethyl acetate/n-hexane ¼ 1:1) to afford
compound 23 (0.61 g, 59.6%) as a pale yellow solid. 1H NMR
5.18. 3-(3-Chloro-2-hydroxy-2-methylpropoxy)-1-hydroxy-5-
methoxy-9H-xanthen-9-one (27)
A mixture of compound 25 (20 mg, 0.06 mmol) in 1 M HCl in
aqueous ethyl acetate (3 mL) was stirred at RT (30 min). Solvent
was removed under reduced pressure and the residue was dried
under vacuum to give compound 27 (19 mg, 85.5%) as a pale yellow
solid. Mp 142 ꢁC; 1H NMR (400 MHz, CDCl3)
d 1.44 (s, 3H), 2.48
(200 MHz, CDCl3)
d 1.86 (s, 3H), 4.05 (s, 3H), 4.54 (s, 2H), 5.05 (s,
(s, 1H), 3.70 (d, J ¼ 1.6 Hz, 2H), 4.00e4.07 (m, 2H), 4.03 (s, 3H), 6.38
(d, J ¼ 1.8 Hz,1H), 6.59 (d, J ¼ 1.8 Hz,1H), 7.25 (d, J ¼ 8.0 Hz,1H), 7.31
(dd, J ¼ 7.2, 8.0 Hz, 1H), 7.82 (d, J ¼ 7.2 Hz, 1H), 12.81 (s, 1H); 13C
NMR (100 MHz, CDCl3) 22.5, 50.3, 56.7, 71.9, 72.1, 93.4, 98.4, 104.6,
116.0, 117.0, 121.7, 123.9, 146.5, 148.5, 157.8, 163.6, 165.4, 181.1 ppm;
LC-ESI: m/e 365.2 [Mþ1]þ.
1H), 5.13 (s, 1H), 6.40 (d, J ¼ 2.4 Hz, 1H), 6.59 (d, J ¼ 2.4 Hz, 1H),
7.26e7.36 (m, 2H), 7.84 (dd, J ¼ 2.2, 7.6 Hz, 1H), 12.84 (s, 1H).
5.15. 1-Hydroxy-3-[(2-methyloxiran-2-yl)methoxy]-9H-xanthen-9-
one (24)
A CH2Cl2 (10 mL) solution of compound 22 (30 mg, 0.11 mmol)
was added to mCPBA (55 mg, 0.32 mmol). The mixture was stirred
at RT (6 h) and mCPBA (60 mg, 0.35 mmol) was added one more
time. After stirring for 3 h, solvent was removed under reduced
pressure and the residue was purified by silica gel column chro-
matography (eluant: ethyl acetate/n-hexane ¼ 1:1) to afford
compound 24 (12 mg, 38.0%) as a pale yellow solid. Mp 124 ꢁC; Rf
0.71 (ethyl acetate/n-hexane ¼ 1:1); 1H NMR (400 MHz, CDCl3)
5.19. 1,3-Bis(2-methylallyloxy)-9H-xanthen-9-one (28)
A mixture of compound 8 (532 mg, 2.33 mmol) and Cs2CO3
(3.04 g, 9.32 mmol) in anhydrous acetone (30 mL) was added to
3-chloro-2-methylpropene (2.11 g, 23.3 mmol). The solid was
removed through filtration and solvent was evaporated under
reduced pressure. The residue was purified by silica gel column
chromatography (eluant: CH2Cl2) to afford compound 28 (285 mg,
d
1.44 (s, 3H), 2.71 (d, J ¼ 4.6 Hz, 1H), 2.82 (d, J ¼ 4.6 Hz, 1H), 3.95 (d,
36.4%) as a pale yellow solid. 1H NMR (400 MHz, CDCl3)
d 1.86
J ¼ 10.8 Hz,1H), 4.09 (d, J ¼ 10.8 Hz,1H), 6.30 (d, J ¼ 2.0 Hz,1H), 6.41
(d, J ¼ 2.0 Hz, 1H), 7.32 (dd, J ¼ 7.5, 7.8 Hz, 1H), 7.37 (d, J ¼ 8.4 Hz,
1H), 7.65 (ddd, J ¼ 1.5, 7.8, 8.4 Hz, 1H), 8.19 (dd, J ¼ 1.5, 7.5 Hz, 1H),
12.79 (s, 1H); 13C NMR (100 MHz, CDCl3) 18.6, 52.1, 55.4, 72.1, 93.6,
97.8, 104.4, 117.8, 120.8, 124.3, 126.1, 135.3, 156.2, 157.9, 163.3, 165.8,
181.1 ppm; LC-ESI: m/e 299.2 [Mþ1]þ.
(s, 3H),1.93 (s, 3H), 4.52 (s, 2H), 4.56 (s, 2H), 5.06 (s,1H), 5.08 (s,1H),
5.14 (s, 1H), 5.43 (s, 1H), 6.37 (d, J ¼ 2.4 Hz, 1H), 6.49 (d, J ¼ 2.4 Hz,
1H), 7.26e7.36 (m, 2H), 7.60 (ddd, J ¼ 1.6, 6.8, 7.0 Hz, 1H), 8.29 (dd,
J ¼ 1.6, 8.0 Hz, 1H).
5.20. 5-Methoxy-1,3-bis(2-methylallyloxy)-9H-xanthen-9-one (29)
5.16. 1-Hydroxy-5-methoxy-3-[(2-methyloxiran-2-yl)methoxy]-
9H-xanthen-9-one (25)
A mixture of compound 9 (458 mg, 1.77 mmol) and Cs2CO3
(2.31 g, 7.09 mmol) in anhydrous acetone (25 mL) was added to
3-chloro-2-methylpropene (1.6 g, 17.74 mmol). The solid was
removed through filtration and solvent was evaporated under
reduced pressure. The residue was purified by silica gel column
chromatography (eluant: CH2Cl2) to afford compound 29 (176 mg,
A CH2Cl2 (15 mL) solution of compound 23 (70 mg, 0.22 mmol)
was added to mCPBA (120 mg, 0.70 mmol). The mixture was stirred
at RT (6 h) and mCPBA (60 mg, 0.35 mmol) was added one more
time. After stirring for 3 h, solvent was removed under reduced
pressure and the residue was purified by silica gel column chro-
matography (eluant: ethyl acetate/n-hexane ¼ 1:1) to afford
compound 25 (32 mg, 43.5%) as a yellow jelly. Rf 0.65 (ethyl acetate/
27.1%) as a yellow solid. 1H NMR (400 MHz, CDCl3)
d 1.86 (s, 3H),
1.93 (s, 3H), 4.01 (s, 3H), 4.51 (s, 2H), 4.56 (s, 2H), 5.04 (s, 1H), 5.08
(s, 1H), 5.13 (s, 1H), 5.42 (s, 1H), 6.39 (d, J ¼ 2.2 Hz, 1H), 6.61
(d, J ¼ 2.2 Hz, 1H), 7.16 (dd, J ¼ 1.4, 7.8 Hz, 1H), 7.60 (dd, J ¼ 7.8,
8.0 Hz, 1H), 8.29 (dd, J ¼ 1.6, 8.0 Hz, 1H).
n-hexane ¼ 1:1); 1H NMR (400 MHz, CDCl3)
d 1.53 (s, 3H), 2.79 (d,
J ¼ 4.6 Hz, 1H), 2.91 (d, J ¼ 4.6 Hz, 1H), 4.02 (d, J ¼ 10.8 Hz, 1H), 4.05
(s, 3H), 4.16 (d, J ¼ 10.8 Hz, 1H), 6.40 (d, J ¼ 2.1 Hz, 1H), 6.60 (d,
J ¼ 2.1 Hz, 1H), 7.16e7.26 (m, 2H), 7.84 (dd, J ¼ 7.6, 1.8 Hz, 1H), 12.84
(s, 1H); 13C NMR (50 MHz, CDCl3) 18.6, 52.1, 55.4, 56.7, 72.1, 93.6,
98.3, 104.4, 116.0, 117.0, 121.9, 123.9, 147.6, 148.5, 157.7, 163.3, 165.8,
181.1 ppm; LC-ESI: m/e 329.2 [Mþ1]þ.
5.21. 1,3-Bis[(2-methyloxiran-2-yl)methoxy]-9H-xanthen-9-one (30)
A CH2Cl2 (15 mL) solution of compound 28 (200 mg, 0.60 mmol)
was added to mCPBA (537 mg, 2.97 mmol). The mixture was stirred
at RT (6 h) and solvent was removed under reduced pressure. The
residue was purified by silica gel column chromatography (eluant:
ethyl acetate/n-hexane ¼ 2:1) to afford compound 30 (78 mg,
38.6%) as a yellow jelly. Rf 0.29 (ethyl acetate/n-hexane ¼ 1:1); 1H
5.17. 3-(3-Chloro-2-hydroxy-2-methylpropoxy)-1-hydroxy-9H-
xanthen-9-one (26)
A mixture of compound 24 (5 mg, 0.02 mmol) in 1 M HCl in
aqueous ethyl acetate (3 mL) was stirred at RT (30 min). Solvent
was removed under reduced pressure and the residue was dried
under vacuum to give compound 26 (5 mg, 89.1%) as a pale
NMR (400 MHz, CDCl3)
d
1.52 (s, 3H), 1.61 (s, 3H), 2.78 (d, J ¼ 4.6 Hz,
1H), 2.82 (d, J ¼ 4.6 Hz, 1H), 2.91 (d, J ¼ 4.6 Hz, 1H), 3.28
(d, J ¼ 4.6 Hz, 1H), 4.01 (dd, J ¼ 2.4, 10.4 Hz, 1H), 4.10e4.19 (m, 3H),
6.41 (d, J ¼ 2.2 Hz, 1H), 6.52 (d, J ¼ 2.2 Hz, 1H), 7.32 (ddd, J ¼ 1.0, 7.2,
8.0 Hz,1H), 7.34 (d, J ¼ 8.3 Hz, 1H), 7.63 (ddd, J ¼ 1.6, 7.2, 8.3 Hz, 1H),
8.28 (dd, J ¼ 1.6, 8.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) 18.6, 18.9,
52.1, 52.5, 55.4, 55.8, 72.0, 72.2(72.1), 94.4, 96.8, 108.3, 117.2, 123.3,
yellow solid. Mp 142 ꢁC; 1H NMR (400 MHz, CDCl3)
d 1.45 (s, 3H),
2.48 (s, 1H), 3.71 (d, J ¼ 2.8 Hz, 2H), 4.01e4.08 (m, 2H), 6.38 (d,
J ¼ 2.2 Hz, 1H), 6.47 (d, J ¼ 2.2 Hz, 1H), 7.39 (ddd, J ¼ 0.8, 7.1,

4228
S. Woo et al. / European Journal of Medicinal Chemistry 45 (2010) 4221e4228
124.1, 126.9, 134.0, 155.2, 159.8, 161.0, 163.8, 175.4 ppm; LC-ESI: m/e
369.2[Mþ1]þ.
5.25. DNA cross-linking assay [12]
The DNA cross-linking property of each compound was tested
using linearized pBR322 plasmid DNA and denaturing 1.2%
alkaline agarose gel electrophoresis. 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 solution (pH 8.0) containing
5.22. 5-Methoxy-1,3-bis[(2-methyloxiran-2-yl)methoxy]-9H-
xanthen-9-one (31)
A CH2Cl2 (15 mL) solution of compound 29 (120 mg, 0.33 mmol)
was added to mCPBA (283 mg,1.64 mmol). The mixture was stirred at
RT (6 h) and solvent was removed under reduced pressure. The
residue was purified by silica gel column chromatography (eluant:
ethyl acetate/n-hexane ¼ 2:1) to afford compound 31 (42 mg, 32.1%)
as a pale yellow jelly. Rf 0.22 (ethyl acetate/n-hexane ¼ 1:1); 1H NMR
50 mM NaCl and 1 mM EDTA. 0.5 mg of the linearized pBR322 was
incubated with designated concentrations of compounds for 2 h
at RT in buffer containing 10 mM TriseHCl 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 and then
neutralized for 1 h in neutralizing buffer solution containing
100 mM Tris and 150 mM NaCl adjusted to pH 7.6 with refreshing
every 20 min. The gel was subsequently stained with ethidium
(400 MHz, CDCl3)
d
1.51 (s, 3H), 1.61 (s, 3H), 2.78 (d, J ¼ 4.6 Hz, 1H),
2.82 (d, J ¼ 4.8 Hz,1H), 2.90 (d, J ¼ 4.6 Hz,1H), 3.29 (d, J ¼ 4.8 Hz,1H),
3.99e4.01 (m, 1H), 4.01 (s, 3H), 4.10e4.18 (m, 3H), 6.41 (d, J ¼ 2.4 Hz,
1H), 6.63 (d, J ¼ 2.4 Hz, 1H), 7.17 (dd, J ¼ 1.4, 7.8 Hz, 1H), 7.25 (dd,
J ¼ 7.8, 7.8 Hz, 1H), 7.86 (dd, J ¼ 1.4, 7.8 Hz, 1H); 13C NMR (100 MHz,
CDCl3) 18.6, 18.9, 52.0, 52.6, 55.4, 55.8, 56.6, 71.8(71.9), 72.1(72.0),
94.3, 97.1, 107.9, 114.7, 117.9, 123.6, 124.2, 145.4, 148.2, 159.6, 160.8,
163.8, 175.3 ppm; LC-ESI: m/e 399.1 [Mþ1]þ.
bromide solution (2.5 mL of 10 mg/mL ethidium bromide in 50 mL
of neutralizing buffer solution). The gel was visualized by UV
transillumination and photographed using ChemiImagerÔ Ready
(Alpha Innotech Corp.).
5.23. Cytotoxicity test [3]
For the evaluation of cytotoxicity, five different cancer cell lines
were used: human breast adenocarcinoma cell line (MDA-
MB231), human cervix tumor cell line (HeLa), human prostate
tumor cell line (DU-145), human colorectal carcinoma cell line
(HCT-116), and human breast cancer cell line (MCF-7). Cancer cells
were cultured according to the supplier’s instructions. 2e4 ꢂ104
cells per well in 96-well microplates were attached overnight in
0.1 mL of media supplied with 10% Fetal Bovine Serum (Welgene,
Korea) under 5% CO2 in a humidified atmosphere at 37 ꢁC. On day
1, the culture medium in each well was exchanged with 0.1-mL
aliquots of medium containing graded concentrations of
compounds. On day 4, each well was treated with MTT (Sigma)
solution (final concentration 0.5 mg/mL in media) then incubated
for an additional 4 h under the same conditions. The culture
medium in each well was discarded and replaced with 0.1 mL of
dissolving solution (DMSO). The absorbance of each well was
determined by an Automatic ELISA Reader System (Bio-Rad 3550)
at 570 nm wavelength. For determination of the IC50 values, the
absorbance readings at 570 nm were fitted to the four-parameter
logistic equation.
Acknowledgements
This work was supported by the Korea Research Foundation Grant
funded by the Korean Government (MOEHRD) (KRF-2006-521-
E00159). J.M.N. was partially supported by the Brain Korea 21 project.
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DNA topoisomerase II inhibition was measured by assessing
relaxation of supercoiled pBR322 plasmid DNA. The reaction
mixture contained 50 mM TriseHCl (pH 8.0), 120 mM KCl, 10 mM
MgCl2, 0.5 mM ATP, 0.5 mM dithiothreitol, 30
albumin, 0.3 g pBR322 plasmid DNA, 0.3 U human DNA topo-
isomerase II (TopoGEN) in a final volume of 20 L. The reactions
were incubated for 30 min at 37 ꢁC and terminated by the addition
of 3 L of 0.77% sodium dodecyl sulfate, 77 mM EDTA. Samples were
mixed with 2 L of 30% sucrose, 0.5% bromophenol blue and 0.5%
mg/ml bovine serum
m
a
m
m
m
xylene cyanol, and subjected to electrophoresis on a 1% agarose gel
at 1.5 V/cm for 10 h. Gels were stained for 30 min in an aqueous
solution of ethidium bromide (0.5 mg/mL). DNA bands were visual-
ized by transillumination with UV light and quantitated by an image
analyzer and LabWork 4.5 software (UVP).