Synthesis of the novel analogues of dysidiolide and their structure-activity relationship

Article abstract of DOI:10.1016/S0960-894X(00)00527-8

The novel analogues of natural cdc25A inhibitor dysidiolide were synthesized. To investigate the structure-activity relationship, the inhibitory activity to enzyme and cell cycle was examined. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00527-8

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2571±2574
Synthesis of the Novel Analogues of Dysidiolide and their
Structure±Activity Relationship
Masato Takahashi,a Kosuke Dodo,a Yoshikazu Sugimoto,b Yoshimi Aoyagi,b
Yuji Yamada,b Yuichi Hashimotoa and Ryuichi Shiraic,*
aInstitute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
bCancer Research Laboratory, Hanno Research Center, Taiho Pharmaceutical Co., Ltd, 1-27, Misugidai, Hanno-shi,
Saitama 357-8527, Japan
cResearch and Education Center for Materials Science, Nara Institute of Science and Technology, 8916-5, Takayama-cho,
Ikoma-shi, Nara 630-0101, Japan
Received 12 July 2000; accepted 9 September 2000
AbstractÐThe novel analogues of natural cdc25A inhibitor dysidiolide were synthesized. To investigate the structure±activity
relationship, the inhibitory activity to enzyme and cell cycle was examined. # 2000 Elsevier Science Ltd. All rights reserved.
Dysidiolide (1) is the ®rst natural inhibitor of dual-spe-
ci®city phosphatase cdc25A,1 which is expressed in the
early G1 phase of the cell cycle and promotes G1/S
transition by dephosphorylation of the cyclin/CDK
complex.2 Cdc25A also proved to be oncogenic and
overexpressed in a number of tumor cell lines.3 There-
fore, the cdc25A-speci®c inhibitor is expected as the
hopeful candidate for the chemotherapy of human can-
cers. As dysidiolide has attracted much attention
because of its bioactivity and complex structure, four
groups have already reported the total syntheses of
natural,4 unnatural5 and racemic6 dysidiolide. However,
its structure±activity relationship remains unclear.
Recently, we have completed the asymmetric total
synthesis of dysidiolide.7 Herein, we report the synthesis
of potent dysidiolide analogues and their structure±
activity relationship.
Our strategy for structural modi®cation is shown in
Figure 1. It is assumed that the g-hydroxybutenolide
Figure 1. Modi®cation of dysidiolide.
*Corresponding author. Tel.: +81-743-72-6171; fax: +81-743-72-
6179; e-mail: rshirai@ms.aist-nara.ac.jp
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00527-8

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moiety serves as a surrogate phosphate and the long
side chain occupies a hydrophobic binding pocket when
the molecule is bound to cdc25A. Therefore, we con-
sidered that the stereochemistry of the C-6 quaternary
carbon center bearing a g-hydroxybutenolide side chain,
which is in the center of the molecule, is signi®cant for
the expression of cdc25A inhibition. Furthermore, the
interaction between the C-4 hydroxy group and the cat-
alytic domain of cdc25A may be concerned with cdc25A
inhibitory activity. In order to prove this hypothesis, a
series of dysidiolide analogues such as 4-epi-dysidiolide
(2), 3, 4 and 5 were synthesized based on our ecient
synthesis of natural dysidiolide (Fig. 2). Diastereomers
of dysidiolide 2, 3 and 4 were synthesized according to
our developed total synthesis of dysidiolide (Scheme 1).7
The key steps of this synthesis are the intermolecular
Diels±Alder reaction of the triene 6 with crotonaldehyde
and construction of a quaternary center at C-6 by
methylation of the exocyclic enolate generated from
the aldehyde. Synthesis of both 4-epi-3 and 4-epi-4 was
not successful because corresponding furylcarbinol
intermediates were too labile to isolate. Monocyclic
Scheme 1. Reagents and conditions: (a) i. EtAlCl2 THF (1:1), CH2Cl2, 30 ꢀC to 0 ꢀC; ii. AlH3, THF, 0 ꢀC and separation; (b) i. TPAP, NMO, MS
4 A, rt; ii. CH3I, t-BuOK, THF, rt; iii. NaBH4, MeOH, 0 ꢀC and separation; (c) i. TsCl, pyridine, 0 ꢀC; ii. NaCN, HMPA, 90 ꢀC; iii. DIBAL-H,
CH2Cl2, 78 ꢀC; iv. 3-bromofuran, n-BuLi, THF, 78 ꢀC; (d) O2, hn, Rose Bengal, i-Pr2EtN, CH2Cl2, 78 ꢀC.
.

M. Takahashi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2571±2574
2573
Figure 2. Structure of dysidiolide and its derivatives.
analogue (5) was prepared in racemic form from 1-
methyl-1-cyclohexanecarboxylic acid (Scheme 2). The
structures of these compounds were characterized by
NMR and MS.8 The C-4 carbinol absolute con®gura-
tions of 10 and 11 were determined by modi®ed
Mosher's method using a-methoxy-a-tri¯uoromethyl-
phenylacetic acid (MTPA) esters.9
be quite concerned with cdc25A inhibitory activity and
enzyme speci®city. Unexpectedly, both 3 and 4 had
stronger activity against cdc25A and B than 1, and 3
also exhibited much stronger antitumor activity. Inter-
estingly, analogue 4, a C-6-desmethyl analogue of 3,
showed moderate speci®city to cdc25A than 2 and 3.
These results suggested that the stereochemistry of the
C-6 quaternary carbon center is not critical and the
original orientation of the two side chains is not neces-
sary for the expression of cdc25A inhibition.
These compounds were tested for the inhibitory activity
of cdc25A, its isoform cdc25B10 and in vitro anti-
proliferative activity.11 As shown in Table 1, 1 showed
cdc25A speci®c inhibition with moderate activity.
Monocyclic analogue 5 did not inhibit both cdc25A and
B at all. The bicyclic structure and/or the alkenyl side
chain of dysidiolide may be necessary to inhibit cdc25A.
Although C-4 epimer 2 showed higher inhibitory
activity toward both cdc25A and cdc25B, its inhibition
of proliferation of tumor cells was relatively weak. The
absolute con®guration of the C-4-hydroxy group may
To con®rm the e€ect of these compounds on cell cycle
progression, cell cycle analysis was performed.12 After
20 h treatment with compounds, cell cycle distribution
of HL60 (human leukemia cell line) cells was analyzed
by ¯ow cytometry. The percentage of cells in each phase
of the cell cycle is shown in Figure 3. Simultaneous
increase of G1 phase population and decrease of S
phase population indicates G1 arrest, an inhibition of
G1/S progression. Dysidiolide (1) and its derivatives 2
and 3 induced G1 arrest in accordance with the cell
growth inhibition. Especially, compound 3, which
showed stronger activity than 1 in both enzyme inhibi-
tion and cell growth inhibition, induced G1 arrest at
very low concentration (0.5 mM).
Table 1. Inhibition of cdc25A, B and proliferation of tumor cells by
dysidiolide analogues
Compound
IC50 (mM)
cdc25B
SBC-5a
cdc25A
HL60b
Thus, we could synthesize the potent dysidiolide analo-
gues with unnatural con®gurations having higher activ-
ity of cdc25A inhibition than natural dysidiolide. These
®ndings on the structure±activity relationship should
provide crucial information for the future design of
cdc25A inhibitor. Synthesis of advanced analogues and
further studies are in progress.
Dysidiolide (1)
4-epi-Dysidiolide (2)
3
4
5
35
15
13
15
87
19
18
27
5.4
16
1.3
4.4
7.1
13.1
1.0
±
>300
>300
12
±
aHuman lung cancer cell line.
bHuman leukemia cell line.
Scheme 2. Reagents and conditions: (a) LiAlH4, THF, 0 ꢀC; (b) i. TsCl, pyridine, 0 ꢀC; ii. NaCN, HMPA, 160 ꢀC; iii. DIBAL-H, CH2Cl2, 78 ꢀC;
iv. 3-bromofuran, n-BuLi, THF, 78 ꢀC; (c) O2, hn, Rose Bengal, i-Pr2EtN, CH2Cl2, 78 ꢀC.

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M. Takahashi et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2571±2574
5. Boukouvalas, J.; Cheng, Y. X.; Robichaud, J. J. Org.
Chem. 1998, 63, 228.
6. (a) Magnuson, S. R.; Sepp-Lorenzino, L.; Rosen, N.;
Danishefsky, S. J. J. Am. Chem. Soc. 1998, 120, 1615. (b)
Miyaoka, H.; Kajiwara, Y.; Yamada, Y. Tetrahedron Lett.
2000, 41, 911.
7. Takahashi, M.; Dodo, K.; Hashimoto, Y.; Shirai, R. Tet-
rahedron Lett. 2000, 41, 2111.
8. 1H NMR (500 MHz, CDCl3) data are as follows: 2 (clear
oil, d6-DMSO): d 0.77 (3H, d, J=7.0 Hz), 0.93 (3H, s), 1.08
(3H, s), 1.69 (3H, s), 1.80 (1H, m), 1.90±2.10 (3H, m), 2.70
(1H, m), 4.50 (1H, brs), 4.60 (2H, s), 5.24 (1H, brs), 5.92 (1H,
brs), 6.10 (1H, brs), 7.86 (1H, s). 3 (clear oil, CDCl3): d 0.81
(3H, d, J=6.5 Hz), 0.96 (3H, s), 0.98 (3H, s), 1.08±1.34 (4H,
m), 1.69 (3H, s), 1.94±2.00 (3H, m), 2.10 (2H, m), 4.67 (2H, s),
4.80 (1H, m), 5.29 (1H, s), 6.06 (1H, s), 6.22 (1H, s). 4 (clear
oil, CDCl3): d 0.93 (3H, d, J=6.5 Hz), 0.97 (3H, s), 1.69 (3H,
s), 1.85 (1H, m), 1.96 (2H, m), 2.09±2.17 (2H, m), 4.67 (2H, s),
4.70 (1H, m), 5.30 (1H, s), 6.05 (1H, s), 6.19 (1H, s). 5 (clear
oil, CDCl3): d 1.00 (3H, s), 1.56±1.67 (2H, m), 4.75 (1H, d,
J=9.0 Hz), 6.00 (1H, brs), 6.14 (1H, brs).
Figure 3. E€ect on the cell cycle of HL60 cells.
9. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am.
Chem. Soc. 1991, 113, 4092.
Acknowledgements
10. Cdc25A phosphatase assay: Catalytic domain protein of
human cdc25A (283-523 aa) was produced from Escherichia
coli strain DH5a using pGEX-2T glutathione-S-transferase
(GST)-fusion protein expression vector (Pharmacia) according
to the instructions provided by the manufacturer. Phosphatase
activity of cdc25A was assayed in 100 mL of bu€er containg
10 mM HEPES (pH 8.0), 50 mM NaCl, and 1 mM dithio-
threitol (DTT), with 10 mM p-nitrophenol phosphate (Sigma)
as a substrate, using 96-well microtiter plates. Cdc25B phos-
phatase assay was performed in a similar manner as above.
11. In vitro antitumor activity evalution: The cells (SBC-5 and
HL60) were incubated with the test compound at various
concentrations for 72 h. The IC50 value was de®ned as the
drug concentration needed to cause 50% inhibition of cell
growth with respect to the control.
This work was supported in part by a Grant-in-Aid for
Scienti®c Research from the Ministry of Education,
Science, Sports and Culture, Japan.
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12. Flow cytometric analysis: HL60 cells were treated with
compounds for 20 h at various concentrations. Cell cycle dis-
tributions were determined using a Becton Dickinson ¯uores-
cence-activated cell analyzer. Data were interpreted using the
ModiFit LT software provided by the manufacturer.