Synthesis of a novel class of cdc25A inhibitors from Vitamin D3

Article abstract of DOI:10.1016/S0960-894X(00)00065-2

We have developed a novel class of cdc25A inhibitors by drastic modification of hydrophobic and hydrophillic substructures of dysidiolide. The unsaturated derivative 3b strongly inhibited cdc25A (IC₅₀=7.7 μM) and caused G1 arrest of HL60 cells. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00065-2

Bioorganic & Medicinal Chemistry Letters 10 (2000) 615±617
Synthesis of a Novel Class of cdc25A Inhibitors from
Vitamin D3
Kosuke Dodo, a Masato Takahashi, a Yuji Yamada, b Yoshikazu Sugimoto, b
Yuichi Hashimoto a and Ryuichi Shirai c,*
aInstitute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
bCancer Research Lab., 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 8 December 1999; accepted 20 January 2000
AbstractÐWe have developed a novel class of cdc25A inhibitors by drastic modi®cation of the hydrophobic and hydrophilic sub-
structures of dysidiolide. The unsaturated derivative 3b strongly inhibited cdc25A (IC50=7.7 mM) and caused G1 arrest of HL60
cells. # 2000 Elsevier Science Ltd. All rights reserved.
A novel group of dual-speci®city protein phosphatases,
consisting of three homologues termed cdc25A, -B and
-C, has been shown to play crucial roles in human cell
proliferation.1,2 Cdc25A is expressed in the early G1
phase of the cell cycle and is responsible for G1±S tran-
sition.3 Accumulating evidence suggests that inap-
propriate ampli®cation or activation of cdc25A is
characteristic of a number of human cancers.4,5 Thus,
increased cdc25A activity may contribute to the growth
of certain types of cancer. Consequently, inhibitors of
cdc25A are candidates for new therapeutic agents to
treat human cancers. Dysidiolide is the ®rst natural
inhibitor of cdc25A to be discovered (IC50=9.4 mM); it
was also shown to inhibit the growth of lung carcinoma
and murine leukemia cell lines.6 Total synthesis of dys-
idiolide was accomplished by us and other groups,7±10
but the structure±activity relationship remains unclear.
It has been suggested that the g-hydroxybutenolide
moiety (hydrophilic substructure) serves as a surrogate
phosphate and the long side chain (hydrophobic sub-
structure) occupies a hydrophobic binding pocket when
the molecule is bound to cdc25A (Fig. 1). Modi®cation of
these substructures may provide novel cdc25A inhibitors.
These analogues have a carboxylic acid moiety as the
hydrophilic substructure and a perhydroindan frame-
work, derived from Vitamin D3, as the hydrophobic
substructure. The complete ozonolysis of Vitamin D3
furnished 1, while partial ozonolysis provided a mixture
of 1 and 2. According to standard procedures as illu-
strated in Scheme 1, 3a±4b11 were prepared from 1.
Compounds 5 and 6 were prepared from 2 in the same
manner (Fig. 2). Compounds 4a, 4b and 6 were
obtained as inseparable mixtures of epimers.
Compounds 3a±6 were tested for cdc25A-inhibitory
activity12 and in vitro antitumor activity.13 As shown in
Table 1, compounds 3b and 6 showed strong cdc25A-
inhibitory activity, while 4b and 5 had weak activity.
The methyl ester analogues 3a and 4a did not inhibit
cdc25A at all. Compound 3b has antitumor activity as
well as cdc25A-inhibitory activity. It was suggested that
3b may exert antitumor activity by blocking cell cycle
progression.
Next, to establish the e€ect of these compounds on cell
cycle progression, ¯ow cytometric analysis was per-
formed.14 HL60 (human leukemia cell line) cells were
treated for 20 h with these compounds at 50 mM
concentration. The results are presented in Figure 3.
Simultaneous increase of G1 phase population and
decrease of S phase population indicates G1 arrest, an
inhibition of G1±S progression. As expected, compound
3b, which had strong cdc25A-inhibitory activity, caused
Here, we report the design, synthesis and biological
activities of novel inhibitors of cdc25A phosphatase.
*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)00065-2

616
K. Dodo et al. / Bioorg. Med. Chem. Lett. 10 (2000) 615±617
Figure 1.
Scheme 1.
Table 1. Inhibitory activity of cdc25A and antitumor activity12,13
Compound
cdc25A inhibition
IC50 (mM)
Antitumor activity
IC50 (mM)/SBC-5
3a
3b
4a
4b
5
>300
7.7
>300
32.0
94.0
9.0
71.0
47.0
70.0
>100
42.0
>100
Figure 2.
6
G1 arrest. Interestingly, the methyl ester 3a also caused
G1 arrest, though it showed no cdc25A-inhibitory
activity. Membrane-permeable 3a might be metabolized
by endogenous esterases to a€ord the active carboxylic
acid 3b.
an easily available intermediate from Vitamin D3, is
quite e€ective to construct such molecules. While this
project was undergoing, Zalkow and co-workers repor-
ted the synthesis of 7, a potent inhibitor of cdc25A
having characteristic nitrile side chain (Fig. 4).15 We
believe our strategy should open the easy access to
the future cdc25A inhibitors with minimal structure.
Exploration of the structure±activity relationship is
continuing.
The hydrophobic and hydrophilic substructure model
turned out to be useful to design cdc25A inhibitors. It
is also demonstrated that perhydroindan framework,

K. Dodo et al. / Bioorg. Med. Chem. Lett. 10 (2000) 615±617
617
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11. 1H NMR (500 MHz, CDCl3) data are as follows: 3a (clear
oil): d 5.47 (1H, s), 3.82±3.87 (1H, m), 3.69 (3H, s), 2.10 (1H,
t, J=8 Hz), 2.02 (1H, br d, J=6 Hz), 0.93 (3H, d, J=6 Hz),
0.87 (3H, d, J=7 Hz), 0.86 (3H, d, J=7 Hz), 0.57 (3H, s). 3b
(colorless crystals, mp. 117±118 ꢀC): d 5.50 (1H, s), 3.81±3.87
(1H, m), 2.13 (1H, t, J=7 Hz), 2.03 (1H, br d, J=6 Hz), 0.93
(3H, d, J=6 Hz), 0.87 (3H, d, J=7 Hz), 0.86 (3H, d, J=7 Hz),
0.59 (3H, s).
Figure 3. Flow cytometric analysis of HL60 cells. G1 Arrest on cell
cycle e€ected by the drugs.
12. 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 containing
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.
Figure 4.
Acknowledgements
13. In vitro antitumor activity evaluation: SBC-5 (human non-
small lung carcinoma cell line) cells were plated at the density
of lÂ103/well in ¯at-bottomed 96-well microplates and cul-
tured overnight. The cells were incubated with the test com-
pound at various concentrations for 72 h. The IC50 value was
de®ned as the drug concentration needed to cause 50% inhi-
bition of cell growth with respect to the control.
14. Flow cytometric analysis: HL60 cells (5Â105 cells/mL)
were treated with compounds for 20 h at 10 mM (5) or 50 mM
(others) concentration. Cell cycle distributions were deter-
mined by using a Becton Dickinson ¯uorescence-activated cell
analyzer. Data were interpreted using the ModFit LT software
provided by the manufacturer. Compound 5 induced apotosis
at 50 mM concentration.
We are grateful to Professor T. Tsuruo and Dr. A.
Tomida (Institute of Molecular and Cellular Bios-
ciences, The University of Tokyo) for helpful discus-
sions about the ¯ow cytometric analysis. 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.
References and Notes
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Med. Chem. 1998, 41, 4677.