Inhibitory activities against topoisomerase I and II by isoaurostatin derivatives and their structure-activity relationships

Article abstract of DOI:10.1016/j.bmcl.2005.02.052

Isoaurostatin A (IAS-A) isolated from Thermomonospora alba showed weak inhibition against topoisomerase (topo) I (IC₅₀ = 307 μM). To get more strong inhibition, derivatives of IAS-A were prepared and their structure-activity relationships against topo I and II were investigated. The addition of hydroxyl group on aromatic rings increased the activities, 3-(3,4,5-trihydroxybenzylidene)-5-hydroxy-3H-benzofuran-2-one (IAS-9) showed strong inhibition (IC₅₀ = 3 μM) against topo I. And also, the increasing of hydroxyl group increased growth inhibition against a variety of cancer cells, and IAS-9 showed most potent inhibition. Unlike camptothecin and etoposide, IAS-9 neither stabilized DNA-topo cleavable complex nor intercalated into DNA, and it inhibited topo I and II noncompetitively. The inhibitory activities also increased by opening of lactone ring in the molecule of IAS-9.

Full text of DOI:10.1016/j.bmcl.2005.02.052

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2065–2068
Inhibitory activities against topoisomerase I and II by
isoaurostatin derivatives and their structure–activity relationships
Keitarou Suzuki,a,* Tadashi Okawara,b Tatuya Higashijima,a Kazumi Yokomizo,a
Tohru Mizushimaa and Masami Otsukab
aDepartment of Pharmaceutical Microbiology, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University,
Kumamoto 862-0973, Japan
bDepartment of Bioorganic Medicinal Chemistry, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University,
Kumamoto 862-0973, Japan
Received 27 December 2004; revised 17 February 2005; accepted 17 February 2005
Available online 18 March 2005
Abstract—Isoaurostatin A (IAS-A) isolated from Thermomonospora alba showed weak inhibition against topoisomerase (topo) I
(IC50 = 307 lM). To get more strong inhibition, derivatives of IAS-A were prepared and their structure–activity relationships
against topo I and II were investigated. The addition of hydroxyl group on aromatic rings increased the activities, 3-(3,4,5-trihydr-
oxybenzylidene)-5-hydroxy-3H-benzofuran-2-one (IAS-9) showed strong inhibition (IC50 = 3 lM) against topo I. And also, the
increasing of hydroxyl group increased growth inhibition against a variety of cancer cells, and IAS-9 showed most potent inhibition.
Unlike camptothecin and etoposide, IAS-9 neither stabilized DNA–topo cleavable complex nor intercalated into DNA, and it
inhibited topo I and II noncompetitively. The inhibitory activities also increased by opening of lactone ring in the molecule of
IAS-9.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
2. Chemistry
DNA topoisomerases (topo) are a family of enzymes
that are essential for a variety of DNA associated pro-
cesses such as replication, recombination and transcrip-
tion.1 Topo are principal intercellular targets for a
number of clinically important anticancer drugs.2–7
Topo inhibitors like camptothecin,8 etoposide9 and
doxorubicin10 bind to the cleavable complex formed be-
tween topo and DNA, and keep it from going back to
the original DNA. This action is associated with severe
side effects as well as other anticancer drugs targeted at
DNA.11,12 Now drugs directly inhibiting topo are ur-
gently being requested. We have previously reported iso-
aurostatin A (IAS-A) isolated from Thermomonospora
alba directly inhibited topo I.13 In this short communi-
cation, we tried to prepare the modified compounds in
order to enhance topo inhibition, and discussed their
structure–activity relationships.
Synthetic methods for the preparation of IAS-A deriva-
tives (IAS-1–15) are summarized in Scheme 1. IAS-1–5
and 10–15 are prepared by reaction of 2-hydroxyphenyl-
acetic acid with various benzaldehyde in the presence
of p-toluene sulfonic acid. IAS-6–9 are prepared by
Keywords: Isoaurostatin; Topoisomerase; Anticancer; Structure–activ-
ity relationship.
*
Corresponding author. Tel./fax: +81 96 371 4327; e-mail: keitarou@
gpo.kumamoto-u.ac.jp
Scheme 1.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.052

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K. Suzuki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2065–2068
Table 2. Growth inhibition of IAS-4, -5, -8 and -9 against various
cancer cells
reaction of 5-hydroxybenzofurane-2(3H)-one with vari-
ous benzaldehyde in existence of p-toluene sulfonic acid.
Cancer (cell)
Growth inhibition (GI50, lM)
IAS-4
IAS-5
IAS-8
IAS-9
3. Inhibitory activity
Breast
HBC4
HBC-5
>100
>100
70
45
23
15
26
16
24
21
30
29
34
75
19
16
20
18
Inhibition against relaxation activity of topo was mea-
sured by detecting the conversion of supercoiled
pBR322 DNA to its relaxed form.14,15 As shown in Ta-
ble 1, IAS-9, which have four hydroxyl groups showed
most potent topo I inhibitory activity with IC50 3 lM,
one hundred times stronger than IAS-A. Trihydroxyl
derivatives (IAS-5, -8) indicated IC50 4 and 6 lM,
respectively. Dihydroxyl and monohydroxyl derivatives
indicated weak inhibition. The benzoyl derivatives
without hydroxyl group showed no activities. From
these results, it is suggested that hydroxyl group is
important for inhibitory activity. Additionally, the
hydroxyl group on benzene ring is more related to inhib-
itory activity than hydroxyl group on benzofluoro ring.
BSY-1
MCF-7
MDA-MB-231
>100
>100
Brain
U251
>100
68
32
66
20
22
44
35
40
28
27
22
14
21
31
30
20
20
23
21
SF-268
SF-295
SF-539
SNB-75
SNB-78
>100
48
74
>100
Colon
HCC2998
KM-121>100
HT-29
82
33
19
23
492
17
>100
>100
>100
28
61
22
95
25
21
29
67
21
HCT-15
HCT-116
4. Growth inhibition of various human cancer cells
Lung
Growth inhibition of IAS-A derivatives were investi-
gated on a human cancer cell line panel (HCC panel)
of Cancer Chemotherapy Center, Japanese Foundation
for Cancer Research, consisting of 39 cell lines as shown
in Table 2.16,17 As well as topo inhibition, growth inhi-
bition increased with the increase of hydroxyl group.
Especially, IAS-9 showed potent growth inhibition
against lung cancer cell. It is also similar to topo inhibi-
tion that hydroxyl group on benzene ring is more deeply
related to the cell growth inhibition than hydroxyl group
on benzofluoro ring.
NCI-H23
NCI-H226
NCI-H522
NCI-H460
A549
>100
>100
21
41
9
70
46
22
56
63
22
18
14
9
11
20
51
19
14
6
>100
>100
68
33
99
19
16
DMS273
DMS114
24
Melanoma
LOX-IMVI
>100
21
66
21
Ovary
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
80
23
17
20
29
56
12
15
17
>100
>100
>100
>100
>100
622
>100
19
31
3
5. Inhibitory property
38
74
Inhibitory properties of IAS-9 were examined as the fol-
lowing four methods.
Renal
RXF-631L
ACHN
>100
59
55
>100
66
5285
Stomach
St-4
Table 1. Inhibition of topoisomerase I and II by IAS derivatives
>100
99
19
17
17
40
23
20
>100
20
38
17
25
27
29
20
19
MKN1
MKN7
MKN28
MKN45
MKN74
IAS
R1
R2
R3
R4
R5
Inhibition
(IC50, lM)
>100
>100
49
55
19
Topo I Topo II
>100
34
1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
>1000 >400
2
OH
H
H
H
H
262
250
160
4
>400
327
272
Prostate
DU-145
PC-3
3
OH
OH
OH
H
H
H
>100
78
35
19
>100
29
29
23
4
OH
OH
H
H
H
5
OH
H
H
63
6
OH
OH
OH
960
555
6
>400
>400
240
7
H
OH
OH
OH
OCH3
OCH3
H
5.1. Inhibitory manner of IAS-9 against topo I and II
8
OH
OH
H
H
9
OH OH
3
>1000 >400
120
The type of inhibition was determined by Lineweaver–
Burk plots18 of substrate concentrations against the rate
of relaxation of supercoiled pBR322 DNA by topo I and
II in the presence or absence of IAS-9. IAS-9 inhibited
the relaxation activities of topo I and II noncompeti-
tively with respect to pBR322 DNA exhibiting Ki values
of 3.6 lM and 34.3 lM, respectively. The Michaelis con-
10
11
12
13
14
15
IAS-A
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
800
630
>400
351
>400
OCH3 OH
H
H
H
H
NO2
F
120
392>400
CH2CO2Et
OH
>1000 >400
>400
OH 307

K. Suzuki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2065–2068
2067
stants (Km values) of topo I and II were 3.6 nM and
8.3 nM, respectively. In view of inhibitory potency (Ki/
Km) against DNA relaxation by topo I and II, IAS-9
was 8-fold potent against topo I than topo II. From
these results, IAS-9 was considered to bind with a differ-
ent site from the binding site of the substrate DNA in
the enzyme molecule.
5.2. Stabilization of topo-cleavable complex by IAS-9
Topo inhibitors of the cleavable complex-forming type
such as camptothecin and etoposide stabilize the cleav-
able complex (topo–DNA reaction intermediate) and in-
hibit the DNA rejoining reaction of topo, which is the
inhibitory mechanism of the inhibitors, therefore the
inhibitors induce nicked DNA in the cleavage assay.8,19
To determine whether IAS-9 is an inhibitor of the cleav-
able complex-forming type or not, cleavage assays were
carried out. Camptothecin used as the controls of cleav-
able complex-forming inhibitors against topo I induced
nicked DNA with increasing concentrations. Unlike
camptothecin, IAS-9 could not induce the nicked
DNA even at 100 lM. This results suggests that IAS-9
is an inhibitor of the cleavable-nonforming type. IAS-9
may directly acts on topo I molecule in earlier step than
the formation of the topo–DNA complex and inhibits
the DNA breaking and rejoining reactions by the
enzyme.
Figure 1. Effects of IAN-9, camptothecin and etoposide on cell cycle of
HeLa cells.
from late S. The results suggest that the cytotoxicity of
IAS-9 is different from that of camptothecin and
etoposide.
6. Effect of pH on the structure and inhibitory activity
of IAS-9
5.3. DNA interaction by IAS-9
It has been reported that camptothecin is hydrolyzed in
the blood, and converted to the carboxylate form (open
ring). The carboxylate form is inactive with respect to
topo inhibition.24,25 IAS-9 also has a lactone ring.
Therefore, it was investigated whether carboxylate form
of IAS-9 inhibits topo I or not. The lactone and carbox-
ylate forms of IAS-9 were detected by HPLC. As shown
in Figure 2, the lactone form decreases the rise of pH
and the carboxylate form increases above pH 7. The
inhibitory activity of IAS-9 went up based on increasing
of carboxylate form. The result suggest that IAS-9 is
activated by open ring contrary to camptothecin.
Some topo inhibitors such as doxorubicin and amsa-
crine are DNA intercalators. To determine whether
IAS-9 has the ability to intercalate into DNA strands,
CD (circular dichroism) spectral change of DNA by
addition of IAS-9 was measured, because the spectrum
is sensitive to the conformation changes of DNA by
intercalators.20,21 The spectrum of DNA changed
greatly with increasing concentrations of doxorubicin
used as control of intercalator. On the other hand, the
spectral changes by IAS-9 did not occur, therefore, it
is clear that IAS-9 has no ability to intercalate into
DNA. Thus, IAS-9 is different from inhibitors causing
DNA damage such as cleavable complex-forming inhib-
itors and DNA intercalators.
100
Lactone form
5.4. Effect of IAS-9 on the growth and cell cycle of HeLa
cells
The cell growth inhibition of IAS-9 was determined in
HeLa cells by Alamar Blue assay.22 The values of cell
growth inhibition (GI50) of IAS-9, camptothecin and
etoposide were 24, 0.6 and 40 lM, respectively. Campto-
thecin and etoposide arrest the cell cycle progression.
The cell cycle progression was analyzed with a flow
cytometer (Becton Dickinson FACS Calibur) using the
ModFit LT, which is a software to determine the per-
centage of cells in G0/G1, S and G2/M phases as shown
in Figure 1.23 HeLa cells were arrested at S phase and
G2/M phase when cultured with 0.1 lM camptothecin
and 2 lM etoposide, respectively. On the other hand,
IAS-9 showed weak arrest against the cell cycle. When
100 lM of IAS-9 was added, it arrested G2/M phase
75
50
25
Inhibition
Carboxylate
form
0
6
7
8
9
pH
Figure 2. Effects of pH on the structure and topo I inhibition of IAS-9.

2068
K. Suzuki et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2065–2068
5. Deady, L.; Desneves, J.; Kaye, A.; Thompson, M.; Finlay,
7. Materials and general experimental procedures
G.; Baguley, B.; Denny, W. Bioorg. Med. Chem. 1999, 7,
2801.
6. Matsui, S.; Endo, W.; Wrzosek, C.; Haridas, K.; Seetha-
ramulu, P.; Hausheer, F.; Rustum, Y. Eur. J. Cancer 1999,
35, 984.
7. Dingemans, A.; Pinedo, H.; Giaccone, G. Biochem.
Biophys. Acta 1998, 275, 1400.
8. Hsiang, Y. H.; Hertzberg, R.; Hecht, S.; Liu, L. F. J. Biol.
Chem. 1985, 260, 14873.
2-Hydroxyphenylacetic acid, 5-hydroxybenzofurane-
2(3H)-one, and 3,4,5-tri-hydroxybenzaldehyde were ob-
tained from Aldrich. Other aldehydes were purchased
from Tokyo Kasei and Wako.
7.1. Preparation of IAS-1–5 and 10–15
A solution of 2-hydroxyphenylacetic acid (0.76 g,
5 mmol) and benzaldehyde (5 mmol) in toluene
(15 mL) was refluxed for 3–4 h in the presence of a cat-
alytic amount of p-toluenesulfonic acid. In the case of
IAS-1–4, 10 and 12–15 toluene was removed under re-
duced pressure and the residue was recrystallized from
toluene to give yellow powder in yields of 6.2–82.5%.
The residue of IAS-5 and -11 was purified by column
chromatography on silica gel with an eluent of
CH2Cl2/MeOH (19:1) to give the product as a yellow
powder in yields of 4.0% and 23.0%, respectively.
9. Chow, K. C.; MacDonald, T. L.; Ross, W. E. Mol.
Pharmacol. 1988, 34, 467.
10. Tewey, K. M.; Rowe, T. C.; Yang, L.; Halligan, B. D.;
Liu, L. F. Science 1984, 226, 466.
11. Chino, M.; Nishikawa, K.; Yamada, A.; Ohsono, M.;
Sawa, T.; Hanaoka, F.; Ishizuka, M.; Takeuchi, T. J.
Antibiot. 1998, 51, 480.
12. Ogiso, Y.; Tomida, A.; Lei, S.; Omura, S.; Tsuruo, T.
Cancer Res. 2000, 60, 2429.
13. Suzuki, K.; Yahara, S.; Maehata, K.; Uyeda, M. J. Nat.
Prod. 2001, 64, 204.
14. Muller, M. T.; Spitzner, J. R.; DiDanato, J. A.; Metha,
V. B.; Tsutsui, K.; Tsutsui, K. Biochemistry 1988, 27,
8369.
15. Spitzner, J. R.; Muller, M. T. Nucleic Acids Res. 1988, 16,
5533.
16. Katayama, K.; Kiryu, Y.; Kaneko, K.; Ohshima, R.
Chem. Pharm. Bull. 2000, 48, 1628.
7.2. Preparation of IAS-6–9
A solution of 5-hydroxybenzofurane-2(3H)-one (0.30 g,
2mmol) and benzaldehyde (2mmol) in xylene (10 mL)
was heated at 140 °C for 3–4 h in the presence of a cat-
alytic amount of p-toluenesulfonic acid. After removal
of xylene under reduced pressure, the residue was trea-
ted on silica gel column chromatography with an eluent
of CH2Cl2/MeOH (19:1) to afford the product as a yel-
low powder in yields of 34.0–53.0%.
17. Yamori, T.; Matsunaga, A.; Sato, S.; Yamazaki, A.;
Komi, A.; Ishizu, K.; Mita, I.; Edatsugi, H.; Matsuba, Y.;
Takezawa, K.; Nakanishi, O.; Kohno, H.; Nakajima, Y.;
Komatsu, H.; Andoh, T.; Tsuruo, T. Cancer Res. 1999, 59,
4042.
18. Lineweaver, H.; Burk, D. J. Am. Chem. Soc. 1934, 56,
658.
19. Yamashita, Y.; Kawada, S.; Fujii, N.; Nakano, H. Cancer
Res. 1990, 50, 5841.
20. Suzuki, K.; Uyeda, M. Biosci. Biotechnol. Biochem. 2002,
66, 1706.
Structures of all compounds were confirmed on the basis
of spectral data and elemental analyses.
References and notes
21. Uno, T.; Hamasaki, K.; Tanigawa, M.; Shimabayashi, S.
Inorg. Chem. 1997, 36, 1676.
22. OÕBrien, J.; Wilson, I.; Orton, T.; Prognan, F. Eur. J.
Biochem. 2000, 267, 5421.
23. Suzuki, K.; Doi, S.; Yahara, S.; Uyeda, M. J. Enzyme
Inhib. Med. Chem. 2003, 18, 497.
24. Blaney, S. M.; Balis, F. M.; Cole, D. E.; Craig, C.; Reid, J.
M.; Ames, M. M.; Krailo, M.; Reaman, G.; Hammond,
D.; Poplack, D. G. Cancer Res. 1993, 53, 1032.
25. Jaxel, C.; Kohn, K. W.; Wani, M. C.; Wall, M. E.;
Pommier, Y. Cancer Res. 1989, 49, 1465.
1. Wang, J. C. Annu. Rev. Biochem. 1996, 65, 635.
2. Alper, S.; Arpaci, T.; Aki, E.; Yalcin, I. Il Farmaco 2003,
58, 497.
3. Ishida, K.; Asao, T. Biochem. Biophys. Acta 2002, 155,
1587.
4. Akao, A.; Hiraga, S.; Iida, T.; Kamatani, A.; Kawasaki,
M.; Mase, T.; Nemoto, T.; Satake, N.; Weissman, S.;
Tschaen, D.; Rossen, K.; Petrillo, D.; Reamer, R.;
Volante, R. Tetrahedron 2001, 57, 8917.