Structure-activity relationship study of glaziovianin A against cell cycle progression and spindle formation of HeLa S3 cells

Article abstract of DOI:10.1016/j.bmcl.2010.07.111

Various derivatives of glaziovianin A, an antitumor isoflavone, were synthesized, and the cytotoxicity of each against HeLa S₃ cells was investigated. Compared to glaziovianin A, the O⁷-allyl derivative was found to be more cytotoxic

Full text of DOI:10.1016/j.bmcl.2010.07.111

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5402–5404
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Structure–activity relationship study of glaziovianin A against cell cycle
progression and spindle formation of HeLa S3 cells
Akiyuki Ikedo a, Ichiro Hayakawa a, Takeo Usui b, Sayaka Kazami c,d, Hiroyuki Osada d, Hideo Kigoshi a,
*
a Department of Chemistry, Graduate School of Pure and Applied Sciences, University of Tsukuba, Tennodai, Tsukuba 305-8571, Japan
b Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennodai, Tsukuba 305-8572, Japan
c Graduate School of Science and Engineering, Saitama University, 255, Shimo-okubo, Sakura-ku, Saitama 338-0825, Japan
d Chemical Biology Department, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Various derivatives of glaziovianin A, an antitumor isoflavone, were synthesized, and the cytotoxicity of
each against HeLa S3 cells was investigated. Compared to glaziovianin A, the O7-allyl derivative was found
to be more cytotoxic against HeLa S3 cells and a more potent M-phase inhibitor.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 22 June 2010
Revised 22 July 2010
Accepted 26 July 2010
Available online 1 August 2010
Keyword:
Structure–activity relationship study
Glaziovianin A
Isoflavone
Antitumor activity
In 2007, glaziovianin A (1) was isolated from the leaves of the
Brazilian tree Astelia glazioviana by Yokosuka et al. (Fig. 1).1 Glazi-
ovianin A (1) exhibited cytotoxicity against HL-60 cells with an IC50
zene-1,2-diol (6) was converted to compound 7. The bromination
of compound 7 gave a monobromo compound, which was con-
verted into arylboronate 8.4 2,3,4,5-Tetramethoxyphenylboronic
acid (10) was prepared by the lithiation of 1,2,3,4-tetramethoxy-
benzene (9) followed by treatment with trimethyl borate.5 The
Suzuki–Miyaura coupling6 of 3-iodo-6,7-dimethoxy-4H-chromen-
4-one (3)3 with boron compounds, such as arylboronate 8,
2,3,4,5-tetramethoxyphenylboronic acid (10), or commercially
available 3,4-(methylenedioxy)phenylboronic acid (11), afforded
glaziovianin A analogues 12–14, respectively.
Next, we prepared A-ring analogues. Selective protection of the
hydroxy group at the C7 position of 157 afforded compound 16
(Scheme 3). Condensation of 16 with N,N-dimethylformamide di-
methyl acetal gave an enamine, which was converted to iodochro-
mone 17.8 We tried a cross coupling reaction with arylboronate 53
and iodochromone compounds, such as 17 or 189, to provide com-
pounds 19 and 20, respectively. The THP group in 19 was removed
by using p-TsOHꢀH2O to give a 7-hydroxy derivative (21), which is
value of 0.29 lM. Also, glaziovianin A (1) was evaluated against a
panel of 39 human cancer cell lines (termed JFCR39) at the Japa-
nese Foundation for Cancer Research. The pattern of the differen-
tial cytotoxicities of glaziovianin A (1) has suggested that the
activity of glaziovianin A (1) involves the inhibition of tubulin
polymerization as a mechanism of action.2 Inhibitors of tubulin
polymerization have become clinically important drugs against
breast cancer. Because glaziovianin A showed antitumor activities
in a mouse xenograft model (unpublished data), we think that
modification of glaziovianin A (1) can lead to the discovery of novel
compounds that possess antitumor activity and that inhibit tubulin
polymerization. In this paper, we report the structure–activity
relationship study of glaziovianin A (1).
We previously reported the synthesis of glaziovianin A (1) by
using Suzuki–Miyaura coupling as a key step (Scheme 1).3 The
method of synthesizing glaziovianin A analogues was based on
our previous strategy. To develop analogues of glaziovianin A (1),
its structure can be divided into two structural moieties: an A-ring
and a B-ring (Fig. 2). Therefore, we synthesized 3-iodochromone
derivatives as an A-ring and borone compounds as a B-ring.
First, we tried to modify a methylene acetal part at the B-ring of
glaziovianin A (Scheme 2). The diol group in 3,6-dimethoxyben-
OMe
5'
4'
O
O
6'
O
B
5
A
8
4
3
MeO
MeO
6
10
9
3'
1'
2'
C
O
OMe
2
7
glaziovianin A (1)
Figure 1. Structure of glaziovianin A (1).
* Corresponding author.
E-mail address: kigoshi@chem.tsukuba.ac.jp (H. Kigoshi).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.111

A. Ikedo et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5402–5404
5403
OMe
OMe
OMe
OMe
O
O
O
O
OH
O
O
a
b,c
MeO
MeO
O
O
B
O
OH
OMe
OMe
4
OMe
OH
OMe
7
2
6
8
OMe
OMe
OMe
OMe
OMe
OMe
OMe
O
d
O
(HO)2B
MeO
MeO
I
OMe
9
OMe
10
O
O
B
O
O
OMe
3
5
R2
R2
R1
R1
R1
O
e
Suzuki–Miyaura
coupling
MeO
MeO
R3
R1
OMe
R2
R2
=
O
O
O
O
O
O
O
O
8 R1, R1
1
1
12
13
R , R
=
, R2 = OMe
MeO
MeO
O
R2 = OMe, R3 = Bpin
1
2
OMe
R = R = OMe
O
10 R1 = R2 = OMe
R3 = B(OH)2
O
14 R1, R1
=
, R2 = H
glaziovianin A (1)
O
1
1
11
R , R
=
Scheme 1. Total synthesis of glaziovianin A (1) by our group.
O
R2 = H, R3 = B(OH)2
B-ring part
Scheme 2. Synthesis of B-ring analogues of glaziovianin A. Reagents and condi-
tions: (a) 2-methoxypropene, PPTS, benzene, rt, 72%; (b) NBS, DMF, rt, 69%; (c)
bis(pinacolato)diboron, PdCl2(dppf), KOAc, DMF, 150 °C, 28%; (d) n-BuLi, B(OMe)3,
THF, rt; (e) 3, PdCl2(dppf), 1 M Na2CO3 aq, 1,4-dioxane, rt {64% for 12, 11% for 13
(from 9), 41% for 14}.
OMe
5'
A-ring part
4'
O
O
O
O
5
MeO
3'
2'
OMe
which indicated that the electron density of the B-ring might be
essential for cytotoxicity. On the other hand, compound 20, which
has an extra methoxy group at C5 of the A-ring, showed no
MeO
7
glaziovianin A (1)
cytotoxicity at 100 lM. This result showed that the steric hin-
O
R1
drance and electron density of the A-ring reduced cytotoxicity to
a large extent. While the 7-demethyl derivative 21 exhibited no
cytotoxicity, compounds 22–24, which each have an alkyl group
at O7 instead of the methyl group, showed cytotoxicity with IC50
R2
O
values of 0.75, 0.74, and 0.19
lM, respectively. Furthermore, com-
glaziovianin A analogues
pound 19, which has a THP group at O7, showed no cytotoxicity
O
even at 100 lM. These results indicated that the hydrophobicity
R1
I
of the O7-alkyl group in glaziovianin derivatives is important for
cytotoxicity. However, the THP group seems to be too large. It is
worth noting that allyl ether 2411 is more active than glaziovianin
A (1) itself.
R3O
R2
+
B
R3O
O
3-iodochlomone derivatives borone compounds
We previously reported that glaziovianin A (1) inhibited the cell
cycle progression in the M-phase with abnormal spindle struc-
tures.1 Therefore, we next investigated the effects of the most cyto-
toxic compound, 24, on both cell cycle progression12 and spindle
structures13 (Fig. 3). As with glaziovianin A (1), compound 24
inhibited cell cycle progression in M-phase, and 24-treated cells
showed abnormal spindle structures with unaligned chromosomes
Figure 2. Key structural moieties of glaziovianin A.
a suitable precursor for the synthesis of glaziovianin derivatives.
Conversion of the hydroxy group at C7 in 21 into various ethers
afforded benzyl ether 22, propargyl ether 23, and allyl ether 24.
Table 1 summarizes the cytotoxicity of glaziovianin A (1) and its
analogues against HeLa S3 cells.10 Compound 12, which has an ace-
tonide group instead of the methylene acetal group, showed no
at the concentration of 1
types were stronger than those of 1
l
M after 18 h treatment: these pheno-
M glaziovianin A (1) treat-
l
cytotoxicity even at 100 lM. Also, compound 13, which has four
ment, suggesting that compound 24 is a more potent M-phase
methoxy groups at the B-ring, was about 40-fold less cytotoxic
than glaziovianin A (1). These results indicated that steric hin-
drance of C30 and C40 at the B-ring part was shown to reduce cyto-
toxicity to a large extent. Compound 14, which lacks methoxy
groups at C20 and C50, was less cytotoxic than glaziovianin A (1),
inhibitor than the original compound glaziovianin A (1).
In conclusion, we have investigated the structure–cytotoxicity
relationships of glaziovianin A (1). From this work, we developed
the O7-allyl compound 24 as much more cytotoxic than glaziovia-
nin A (1) against HeLa S3 cells. Further studies on the synthesis of

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A. Ikedo et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5402–5404
O
O
O
MeO
MeO
I
b,c
7
RO
a
OH
THPO
15 R = H
17
16
R = THP
OMe
O
R4
O
O
R4
O
O
MeO
R5O
MeO
R5O
I
O
d
OMe
17 R4 = H, R5 = THP
19 R4 = H, R5 = THP
Figure 3. Effects of glaziovianin A (1) and compound 24 on cell cycle progression
and spindle structures in HeLa S3 cells. Effects of 1 and 24 on cell cycle progression
(A–C) and spindle structures (D–F) in HeLa S3 cells. HeLa S3 cells were treated with
4
4 = OMe, R5 = Me
R
= OMe, R5 = Me
18
20
R
DMSO (A and D), 1
lM of glaziovianin A (1) (B and E), or compound 24 (C and F) for
19
18 h. Microtubules (red) and chromosomes (blue) are shown in D–F. Microtubules
and chromosomes were stained with anti-
Hoechst 33258, respectively.
a-tubulin antibody (DM1A, Sigma) and
e
OMe
OMe
OMe
O
O
O
O
O
O
MeO
HO
MeO
f
Biologically Functional Molecules’ from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT), Japan.
OMe
OMe
7
O
21
BnO
g
O
22
References and notes
h
OMe
OMe
1. Yokosuka, A.; Haraguchi, M.; Usui, T.; Kazami, S.; Osada, H.; Yamori, T.; Mimaki,
Y. Bioorg. Med. Chem. Lett. 2007, 17, 3091.
O
O
O
O
2. Yamori, T.; Matsunaga, A.; Saito, S.; Yamazaki, K.; Komi, A.; Ishizu, K.; Mita, I.;
Edatsugi, H.; Matsuda, Y.; Takezawa, K.; Nakanishi, O.; Kohno, H.; Nakajima, Y.;
Komatsu, H.; Andoh, T.; Tsuruo, T. Cancer Res. 1999, 59, 4042.
3. Hayakawa, I.; Ikedo, A.; Kigoshi, H. Chem. Lett. 2007, 36, 1382.
4. Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem. 1995, 60, 7508.
5. Tremblay, S. M.; Sames, D. Org. Lett. 2005, 7, 2417.
6. Hoshino, Y.; Miyaura, N.; Suzuki, A. Bull. Chem. Soc. Jpn. 1988, 61, 3008.
7. Adityachaudhury, N.; Kirtaniya, C. L.; Mukherjee, B. Tetrahedron 1971, 27,
2111.
MeO
O
MeO
O
O
O
OMe
O
24
O
23
Scheme 3. Synthesis of A-ring analogues of glaziovianin A. Reagents and condi-
tions: (a) DHP, PPTS, CH2Cl2, rt, 80%; (b) Me2NCH(OMe)2, 90 °C, quant; (c) I2, pyr,
CHCl3, rt, 70%; (d) 5, PdCl2(dppf), 1 M Na2CO3 aq, 1,4-dioxane, rt (66% for 19, 16% for
20); (e) p-TsOHꢀH2O, MeOH, CHCl3, rt, 85%; (f) benzyl bromide, K2CO3, MeCN, rt,
80%; (g) allyl bromide, K2CO3, MeCN, rt, 78%; (h) propargyl bromide, K2CO3, MeCN,
rt, 70%.
8. Gammill, R. B. Synthesis 1979, 901.
9. Igarashi, Y.; Kumazawa, H.; Ohshima, T.; Satomi, H.; Terabayashi, S.; Takeda, S.;
Aburada, M.; Miyamoto, K. Chem. Pharm. Bull. 2005, 53, 1088.
10. Cell survival was determined by a WST-8 assay kit (Dojindo Laboratories,
Kumamoto, Japan). HeLa S3 cells (3 ꢁ 103 cells/well) in 96-well plates were
incubated overnight. Then, cells were treated with various concentrations of
each compounds. After 48 h incubation, 10 ll of WST-8 reagents were added to
the culture. After 2 h incubation, the absorbance at 450 nm was measured with
iMark microplate reader (BioRad Laboratories, Inc). Absorbance correlates with
the number of living cells. The number of living cells (% control) was calculated
with the following formula: (each absorbance–absorbance of blank well)/
Table 1
Cytotoxicity of glaziovianin A (1) and its analogues against HeLa S3 cells
Compound
Cytotoxicity
absorbance of 0
lM well ꢁ 100.
IC50
(
lM)
Relative value
11. Chemical data for compound 24: 1H NMR (400 MHz, CDCl3) d 7.89 (s, 1H), 7.62
(s, 1H), 6.89 (s, 1H), 6.52 (s, 1H), 6.11 (ddt, J = 17.6, 10.5, 5.4 Hz, 1H), 6.02 (s,
2H), 5.48 (ddt, J = 17.6, 1.4, 1.4 Hz, 1H), 5.38 (ddt, J = 10.5, 1.4, 1.4 Hz, 1H), 4.72
(dt, J = 5.4, 1.4 Hz, 2H), 3.98 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H); 13C NMR
(67.8 MHz, CDCl3) d 175.2, 154.4, 153.4, 152.0, 147.8, 139.0, 138.9, 137.0,
136.6, 135.8, 121.5, 118.0, 117.7, 116.5, 110.0, 105.2, 101.8, 100.9, 73.3, 62.1,
57.0, 56.4; IR (CHCl3) 3008, 2938, 1639, 1607, 1503, 1469, 1430, 1399, 1349,
1298, 1267, 1231, 1195, 1153, 1099, 1063, 1035, 995, 833, 697 cmꢂ1; ESIMS m/
z 435.1057, calcd for C22H20NaO8 [M+Na]+ 435.1056.
Glaziovianin A (1)
0.59
>100
22.0
56.2
>100
>100
>100
0.75
0.74
0.19
1
—
0.027
0.010
—
—
—
0.79
0.80
3.1
12
13
14
19
20
21
22
23
24
12. Flow cytometry was used to analyses the distribution of DNA content in the
cell populations. The cells were fixed with cold (ꢂ20 °C) 70% EtOH (v/v) and
stained with propidium iodide (Sigma). Total fluorescence intensities were
determined by quantitative flow cytometry with CyFlow PA (Partec GmbH,
Munster, Germany).
13. Immunofluorescence observation of tubulin was performed as described in
previous paper.14 The DNA and microtubules were photographed with Leica
AF6000 (Leica Microsystems GmbH, Wetzlar, Germany).
14. Kondoh, M.; Usui, T.; Nishikiori, T.; Mayumi, T.; Osada, H. Biochem. J. 1999, 340,
411.
O7-modified probe molecules of glaziovianin A (1) for searching
target biomolecules are currently in progress.
Acknowledgments
This work was supported in part by grants-in-aid for Scientific
Research (B), and Scientific Research on Priority Area ‘Creation of