Synthesis of new xanthone analogues and their biological activity test-Cytotoxicity, topoisomerase II inhibition, and DNA cross-linking study

Article abstract of DOI:10.1016/j.bmcl.2006.12.030

In this report, we prepared some 3-(2′,3′-epoxypropoxy)xanthones and their epoxide ring opened halohydrin analogues, and evaluated their cytotoxicity and topoisomerase II inhibition activity using doxorubicin and etoposide as references, respectively. Another xanthone compound 9, 1,3-di(2′,3′-epoxypropoxy)xanthone, was also synthesized and its DNA cross-linking property including other two biological activities investigated. The biological test results showed compound 9 possessed excellent cytotoxic and topoisomerase II inhibitory activity than other compounds tested. It also exhibited significant DNA cross-linking activities.

Full text of DOI:10.1016/j.bmcl.2006.12.030

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1163–1166
Synthesis of new xanthone analogues and their biological
activity test—Cytotoxicity, topoisomerase II inhibition,
and DNA cross-linking study
a
b
c
b,
a,
*
*
Sangwook Woo, Ji Jung, Chongsoon Lee, Youngjoo Kwon and 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
Department of Biochemistry, College of Natural Sciences, Yeungnam University, Gyeongsan 712-749, Republic of Korea
c
Received 19 October 2006; revised 7 December 2006; accepted 11 December 2006
Available online 13 December 2006
0
0
Abstract—In this report, we prepared some 3-(2 ,3 -epoxypropoxy)xanthones and their epoxide ring opened halohydrin analogues,
and evaluated their cytotoxicity and topoisomerase II inhibition activity using doxorubicin and etoposide as references, respectively.
0
0
Another xanthone compound 9, 1,3-di(2 ,3 -epoxypropoxy)xanthone, was also synthesized and its DNA cross-linking property
including other two biological activities investigated. The biological test results showed compound 9 possessed excellent cytotoxic
and topoisomerase II inhibitory activity than other compounds tested. It also exhibited significant DNA cross-linking activities.
ꢀ
2006 Elsevier Ltd. All rights reserved.
Xanthone (1) compounds, secondary metabolites from
higher plants and microorganisms, have very diverse bio-
logical profiles including anti-hypertensive, anti-oxida-
tive, anti-thrombotic, and anti-cancer activity, based on
Psorospermin (4), an ingredient of African plant
Psorospermum febrifugum, is another natural compound
showing good anti-cancer activity against human and
3
murine cancer cell lines. From the structural viewpoint,
1
their diverse structures. The interesting structural scaf-
4 also possessed xanthone and 2,3-epoxypropoxy group
moieties. According to the literatures, psorospermin has
been known to show biological activities via intercala-
tion of xanthone group into DNA base pair and alkyl-
ation of epoxide by N7-guanine in the presence of
fold and biological efficacy of xanthones enforced many
scientists to isolate or synthesize these compounds for
the development of prospective new drug candidates.
Among these, poly-oxygenated xanthones (2) either syn-
thesized or isolated from natural resources showed effec-
4
topoisomerase II.
2
tive inhibitory activity against several cancer cell lines.
Especially, 2,3-epoxypropoxy substituted xanthoneshave
efficiently prohibited growth of cancer cells and xanthone
In this report, first, we tried to synthesize and study anti-
cancer activities of some epoxypropoxy xanthones 7–9
and their epoxy ring opened halohydrin compounds
10–13. In the literature, epoxide ring opened halohydrin
compounds enhanced cytotoxic activities than their
(
position showed most active anti-cancer activity in the
3) possessing two 2,3-epoxypropoxy groups at 3 and 5
2
a,b
series prepared.
of these compounds has not been reported yet.
However, the exact action mechanism
5
parent compounds. We, therefore, expected that
O
O
O
O
O
OH
O
O
OCH3
8
1
9
2
7
6
3
O
O
O
R'
5
10
4
R
O
OH
H
O
1
2
3
4
O
O
Keywords: Xanthones; Topoisomerase II inhibition; DNA cross-linking; Anti-cancer agents.
*
Corresponding authors. Tel.: +82 2 3277 4653; fax: +82 2 3277 2851 (Y. K.); tel.: +82 53 850 3616; fax: +82 53 850 3602 (Y. N.); e-mail addresses:
ykwon@ewha.ac.kr; yna7315@cu.ac.kr
0
960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.030

1164
S. Woo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1163–1166
8
employment of good leaving group (Br or Cl) instead of
unstable epoxide group could modulate the biological
capacity. Second, topoisomerase II inhibition study
was also conducted to check possible action mechanism
of the xanthone compounds. Third, we performed DNA
cross-linking experiment to see that introduction of two
2,3-epoxypropoxy groups at 1 and 3 position in xan-
thone core, compound 9, might generate double alkyl-
ated DNA adduct with linearized pBR322 DNA.
MTT assay procedure according to the literature. The
result is indicated in Table 1. Compound 9 showed most
effective cytotoxic activity among the series compounds
comparable to the reference. Compound 13 also showed
moderate activity but lower than those of compound 9
and adriamycin. In this study, we found two epoxyprop-
oxy group substitutions at 1 and 3 carbon on xanthone
core generating better cytotoxic activity than mono-
substituted one. This finding is parallel to the previous
report of compound 3 though the locations of two
2
a,b
The synthetic method employed for the compounds is
depicted in Scheme 1. Xanthone core was prepared
using phloroglucinol and salicylic acid by the literature
epoxypropoxy groups are different.
Based on the test
result, we asked what pathway might contribute for the
activity of xanthone compounds. In order to understand
the possible action mechanism of the compounds 7–13,
we conducted topoisomerase II inhibition and DNA
cross-linking experiment.
6
method . Introduction of epoxypropoxy group was
conducted in the Cs CO basic acetone condition, subse-
2
3
quently. In this step we could get mono- and bis-epoxy-
propoxy group substituted xanthone compounds, 8 and
9
, at the same time. Compound 9 was obtained as dia-
Topoisomerase relaxation assay was conducted using
human topoisomerase II (Topogen) with etoposide as
a positive control. The data were analyzed and calculat-
ed with LabWork 4.5 Software for the inhibition ratio.
Compound 13 effectively inhibited the topoisomerase
II action with 57% inhibition ratio at 100 lM (Fig. 1).
But this value is lower than that of etoposide. Further-
more, the inhibitory activity of compound 13 is almost
stereomeric mixture. Finally, epoxide ring open reaction
was conducted in aqueous 1 M-HCl or -HBr in EtOAc
to produce halohydrin compounds 10–13.
7
Compounds 7–13 were tested for the cytotoxicity
against several human cancer cell lines using adriamycin
as a reference. The method applied for the test is typical
OH
O
O
OH
CO H
Epichlorohydrin
2
ZnCl2
+
Cs CO , Acetone
2
3
POCl3
OH
R = H, OCH3
OH
Reflux
HO
OH
9
3-95%
2
7-32%
R
R
Phloroglucinol
5 R = OCH
3
6
R = H
O
O
OH
O
OH
1
M-HCl or HBr
EtOAc
O
O
O
O
8
0-99%
R
R
R
OH
O
O
7
8
R = OCH3
R = H
+
X
1
0 R = OCH , X = Cl
3
11 R = OCH3, X = Br
1
2 R = H,
X =Cl
O
O
O
13 R = H,
X = Br
O
9
R = H
Scheme 1. Synthetic method for target compounds.
Table 1. Cytotoxicities of compounds 7–13 against various human cancer cells
a
Cells (origin)/compound
IC50 (lM)
7
8
10
11
12
13
9
Adriamycin
LnCap (prostate)
MCF-7 (breast)
HCT 116 (colon)
MDA-MB231 (breast)
Hela (cervix)
>100
>100
>100
>100
>100
93.1 ± 16.9
68.4 ± 4.8
80.8 ± 3.1
>100
>100
>100
>100
97.8 ± 0.2
>100
>100
>100
>100
>100
>100
64.4 ± 3.1
53.5 ± 5.4
16.9 ± 0.5
76.3 ± 5.0
98.1 ± 6.3
9.0 ± 0.2
3.2 ± 0.8
4.6 ± 0.6
4.5 ± 0.3
7.7 ± 0.2
17.6 ± 0.3
3.3 ± 0.4
31.4 ± 3.0
81.4 ± 3.5
60.3 ± 3.3
10.2 ± 0.7
12.8 ± 0.9
23.3 ± 1.7
99.8 ± 11.1
>100
68.7 ± 8.7
a
Each value is the average of four experiments.

S. Woo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1163–1166
10 11 12 13 14 15 16 17 18
1165
1
2
3
4
5
6
7
8
9
Figure 1. Effects of compounds on human topoisomerase II-mediated DNA relaxation. Supercoiled pBR322 plasmid DNA (0.3 lg) was incubated
with 0.2 unit of topoisomerase II in the presence of compound (20 and 100 lM) at 37 ꢁC for 30 min. Lane 1: DNA only; lane 2: DNA + topo II; lanes
3 and 4: DNA + topo II + etoposide, 20 and 100 lM; lanes 5–18: DNA + topo II + compounds, 7, 8, 10, 11, 12, 13, 9, 20 lM and 10 lM,
respectively.
1
2
3
4
5
6
References and notes
1
. (a) Pinto, M. M. M.; Sousa, M. E.; Nascimento, M. S. J.
Curr. Med. Chem. 2005, 12, 2517; (b) Peres, V.; Nagem, T.
J.; de Oliveira, F. F. Phytochemistry 2000, 55, 683; (c)
Wang, L.-W.; Kang, J.-J.; Chen, I.-J.; Teng, C.-M.; Lin, C.-
N. Bioorg. Med. Chem. 2002, 10, 567; (d) Lin, C.-N.; Liou,
S.-S.; Ko, F.-N.; Teng, C.-M. J. Pharm. Sci. 1992, 81, 1109;
9
4
.4kb
.4kb
Figure 2. DNA cross-linking activity of compound 9 was determined
by 1.2 % alkaline agarose gel electrophoresis. 0.5 lg of the linearized
pBR322 was incubated with various concentrations of compound 9
(
J. Pharm. Sci. 1993, 82, 11.
e) Lin, C.-N.; Liou, S.-S.; Ko, F.-N.; Teng, C.-M.
2
. (a) Liou, S.-S.; Shieh, W.-L.; Cheng, T.-H.; Won, S.-J.; Lin,
C.-N. J. Pharm. Pharmacol. 1993, 45, 791; (b) Lin, C.-N.;
Liou, S.-S.; Lee, T.-H.; Chuang, Y.-C.; Won, S.-J.
J. Pharm. Pharmacol. 1996, 48, 539; (c) Pedro, M.;
Cerqueira, F.; Sousa, M. E.; Nascimento, M. S. J.; Pinto,
M. Bioorg. Med. Chem. 2002, 10, 3725; (d) Saraiva, L.;
Fresco, P.; Pinto, E.; Sousa, E.; Pinto, M.; Goncalves, J.
Bioorg. Med. Chem. 2003, 11, 1215; (e) Zhou, Y.-S.; Hou,
A.-J.; Zhu, G.-F.; Chen, Y.-F.; Sun, H.-D.; Zhao, Q.-S.
Bioorg. Med. Chem. 2004, 12, 1947.
. (a) Kupchan, S. M.; Streelman, D. R.; Sneden, A. T. J. Nat.
Prod. 1980, 43, 296; (b) Cassady, J. M. J. Nat. Prod 1990, 53, 23.
. (a) Hansen, M.; Lee, S.-J.; Cassady, J. M.; Hurley, L. H.
J. Am. Chem. Soc. 1996, 118, 5553; (b) Kwok, Y.; Hurley,
L. H. J. Biol. Chem. 1998, 273, 33020.
(lanes 2–6: 0, 10, 50, 100, 200 lM) for 1 h at room temperature. Lane
1: k HindIII DNA Marker (Promega).
half of that of the etoposide at 20 lM. Although com-
pound 9 exhibited strong topoisomearse II inhibition
at 100 lM (almost 100% inhibition), we could not make
clear decision regarding the inhibition activity because
of the smeared band. Other compounds have not
showed significant inhibition pattern. This topoisomer-
ase relaxation assay data suggested that the epoxide ring
opening of compounds might enhance inhibition activity
than epoxide ring possessing ones (compounds 12 and
3
4
13 vs 7 and 8). It also showed that bromohydrin intro-
duced compounds generate higher activity than chloro-
hydrin ones (compounds 11 and 13 vs 10 and 12).
5. Kim, M.-Y.; Na, Y.; Vankayalapati, H.; Gleason-Guzman,
M.; Hurley, L. H. J. Med. Chem. 2003, 46, 2958.
6
7
. Grover, P. K.; Shah, G. D.; Shah, R. C. J. Chem. Soc. 1955,
982.
. Spectral data for compounds. Compound 8: H NMR
3
Due to its bis-epoxypropoxy groups in the structure,
only compound 9 was tested for DNA cross-linking
property using linearized pBR322 DNA. In this test,
compound 9 showed concentration dependent DNA
cross-linking activity (Fig. 2). The effective cross-linking
activity below 50 lM implicates the possible application
of this compound as a new DNA cross-linking agent.
1
(
(
3
250 MHz, CDCl ) d 2.77 (dd, J = 2.6, 4.6 Hz, 1H), 2.93
dd, J = 4.3, 4.6 Hz, 1H), 3.36–3.39 (m, 1H), 3.99 (dd,
9
J = 5.9, 11.1 Hz, 1H), 4.33 (dd, J = 2.8, 11.1 Hz, 1H), 6.34
d, J = 2.2 Hz, 1H), 6.44 (d, J = 2.2 Hz, 1H), 7.36 (dd,
(
J = 7.8, 8.3 Hz, 1H), 7.40 (d, J = 8.7 Hz, 1H), 7.70 (ddd,
J = 1.5, 8.3, 8.7 Hz, 1H), 8.22 (dd, J = 1.5, 7.8 Hz, 1H),
1
3
1
69.2, 93.4, 97.4, 104.2, 117.6, 120.6, 124.1, 125.9, 135.1,
2.85 (s, 1H); C NMR (62.5 MHz, CDCl ) 44.6, 44.7,
3
In conclusion, compound 9 showed best biological activ-
ity than other compounds in the test. The test results
suggest that compound 9 might exert its cytotoxic activ-
ity via topoisomerase II inhibition or DNA cross-linking
process. But other mechanisms cannot be excluded.
Because of small numbers of compounds, we could
not determine structure–activity relationship of com-
pounds tested. We are currently pursuing synthesis
and biological study of expanded series of these com-
pounds and results will be reported in the course of time.
1
2
56.0, 157.7, 163.6, 165.4, 180.9 ppm; LC–MS (ESI) m/e
85.2 [M+1] .
+
1
Compound 9: H NMR (250 MHz, CDCl ) d 2.75–2.78 (m,
3
1
1
H), 2.91–2.96 (m, 2H), 3.12–3.16 (m, 1H), 3.35–3.38 (m,
H), 4.43–4.48 (m, 1H), 3.95 (d, J = 6.0, 11.1 Hz, 1H), 4.13
(
dd, J = 4.4, 11.1 Hz, 1H), 4.32–4.37 (m, 2H), 6.38 (d,
J = 1.9 Hz, 1H), 6.47 (d, J = 1.9 Hz, 1H), 7.24–7.33 (m,
H), 7.58 (ddd, J = 1.2, 7.0, 8.2 Hz, 1H), 8.23 (dd, J = 1.2,
2
7.7 Hz, 1H); (400 MHz, CDCl ) d 2.80 (dd, J = 2.8, 4.8 Hz,
3
1H), 2.96 (dd, J = 4.4, 4.8 Hz, 1H), 2.98 (dd, J = 4.0,
4
3
.8 Hz, 1H), 3.16–3.18 (m, 1H), 3.39–3.41 (m, 1H), 3.48–
.50 (m, 1H), 4.01 (ddd, J = 1.6, 6.0, 11.2 Hz, 1H), 4.19 (dd,
Acknowledgment
J = 4.4, 11.2 Hz, 1H), 4.25–4.41 (m, 2H), 6.44 (d,
J = 2.4 Hz, 1H), 6.53 (d, J = 2.4 Hz, 1H), 7.33 (ddd,
J = 1.2, 7.0, 8.0 Hz, 1H), 7.36 (d, J = 7.6 Hz, 1H), 7.63
(ddd, J = 1.6, 7.0, 7.6 Hz, 1H), 8.27 (dd, J = 1.6, 8.0 Hz,
This research was supported by research grant from
Catholic University of Daegu, Korea in 2004.

1
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S. Woo et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1163–1166
H); C NMR (62.5 MHz, CDCl ) 44.5, 45.0, 49.7, 50.0, Korea) under 5% CO
13
1
6
1
3
2
in a humidified atmosphere at 37 ꢁC.
8.8, 69.2, 94.1, 96.7, 107.7, 117.0, 122.9, 123.8, 126.6,
33.8, 154.9, 159.5, 160.6, 163.4, 175.2 ppm; (100 MHz,
On day 1, culture medium in each well was exchanged with
0.1 mL aliquots of medium containing graded concentra-
tions of compounds 7–13. On day 4, each well was added
with the MTT (Sigma) solution (final concentration 0.5 mg/
mL in media) and then incubated for additional 4 h under
the same condition. 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) with a
570 nm wavelength. For determination of the IC50 values,
the absorbance readings at 570 nm were fitted to the four-
parameter logistic equation.
CDCl
9
1
3
) 44.8, 45.2, 49.9, 50.3, 69.2(69.1), 69.5, 94.5(94.4),
7.1, 108.1, 117.2, 123.2, 124.1, 126.9, 134.0, 155.2, 159.8,
61.0, 163.7, 175.4 ppm; The values in the parentheses
correspond to the peaks of diastereomer. LC–MS (ESI) m/e
3
+
41.2 [M+1] .
1
Compound 12: H NMR (250 MHz, CDCl
3
) d 3.70–3.80
m, 2H), 4.13–4.19 (m, 2H), 4.25 (d, J = 4.5 Hz, 1H), 6.34
d, J = 1.9 Hz, 1H), 6.44 (d, J = 1.9 Hz, 1H), 7.34 (dd,
(
(
J = 8.2, 7.4 Hz, 1H), 7.41 (d, J = 8.2 Hz, 1H) 7.71 (dd,
J = 7.4, 8.1 Hz, 1H), 8.23 (d, J = 8.1 Hz, 1H), 12.85 (s, 1H);
C NMR(62.5 MHz, CDCl ) 45.0, 69.1, 69.6, 93.4, 97.6,
1
3
9. Alkaline agarose gel (1.2%) was prepared with the solution
(pH 8.0) containing 50 mM NaCl and 2 mM EDTA. 0.5 lg
of the linearized pBR322 was incubated with variable
concentrations of compounds 9 for 1 h at room tempera-
ture under the buffered condition of 10 mM Tris–HCl (pH
7.5). The mixture of DNA and compound was loaded with
agarose loading dye into the gel which was soaked in an
alkaline running buffer (40 mM NaOH and 1 mM EDTA).
The gel was run in fresh alkaline running buffer, then
neutralized for 45 min in the solution (pH 7.0) containing
100 mM Tris and 150 mM NaCl refreshing every 15 min.
The gel was subsequently stained in an ethidium bromide
solution (2.5 lL of a 10 mg/mL ethidium bromide in 50 mL
of the solution (pH 7.5) containing 100 mM Tris and
150 mM NaCl). The gel was visualized by UV and
3
1
1
04.4, 117.7, 120.7, 124.2, 126.0, 135.2, 156.1, 157.8, 163.7,
65.2, 180.9 ppm; LC–MS (ESI) m/e 321.2 [M+1] .
+
1
Compound 13: H NMR (250 MHz, CDCl
3
) d 3.56–3.69
m, 2H), 4.15–4.21 (m, 3H), 6.34 (d, J = 1.9 Hz, 1H), 6.43
d, J = 1.9 Hz, 1H), 7.37 (dd, J = 7.4, 8.1 Hz, 1H), 7.41 (d,
(
(
J = 8.1 Hz, 1H), 7.71 (dd, J = 7.4, 8.1 Hz, 1H), 8.23 (d,
J = 8.1 Hz, 1H), 12.85 (s, 1H); C NMR (62.5 MHz,
CDCl ) 35.0, 69.2, 69.8, 93.4, 97.6, 104.2, 117.7, 120.7,
3
124.2, 126.0, 135.3, 156.1, 157.8, 163.7, 165.1, 180.9 ppm;
LC–MS (ESI) m/e 365.1 [M+1] .
. Cytotoxicity was determined by MTT assay. Cancer cells
were purchased from the American Tissue Culture Collec-
tion (Rockville, MD) and cultured according to the
1
3
+
8
4
supplier’s instructions. 2–4 · 10 cells per well in 96-well
TM
microplates were attached for overnight in 0.1 mL of media
supplemented with 10% Fetal Bovine Serum (Welgene,
photographed using ChemiImager Ready (Alpha Innotech
Corp.).