Novel CDC25A phosphatase inhibitors from pyrolysis of 3-alpha-azido-B-homo-6-oxa-4-cholesten-7-one on silica gel.

Full text of DOI:10.1021/jm980500r

© Copyright 1998 by the American Chemical Society
Volume 41, Number 24
November 19, 1998
Communications to the Editor
normal cells. However, accumulating evidence suggests
Novel CDC25A P h osp h a ta se In h ibitor s
fr om P yr olysis of
3-r-Azid o-B-h om o-6-oxa -4-ch olesten -7-on e
on Silica Gel
that inappropriate amplification or activation of CDC25A
is characteristic of a number of human cancers, includ-
ing breast cancers.3,4 Thus, increased CDC25A activity
may contribute to the dysregulated growth of certain
types of cancer cells. Small molecule inhibitors of
CDC25A may possess novel antitumor activities.5
Hairuo Peng and Leon H. Zalkow*
School of Chemistry and Biochemistry, Georgia Institute of
Technology, Atlanta, Georgia 30332
Except for the widely used broad-spectrum protein
phosphatase inhibitor vanadate,6 few dual specificity
protein phosphatase inhibitors have been reported.
Dysidiolide (Figure 1) was the first natural CDC25A
phosphatase inhibitor (IC50 ) 9.4 µM) with antitumor
activity discovered.7 The promising biological activities
of this compound soon resulted in total syntheses by
three groups.8 A combinatorial library of small molecule
phosphatase inhibitors based on the pharmacophore of
natural PSTPase inhibitors has been reported. SC-
RRδ9, the best CDC25A inhibitor in the library, showed
an IC50 of 15 µM.9
Thus, a need remains for the discovery and develop-
ment of more potent CDC25A inhibitors. We have
sought novel structures, active as signal transduction
and cell cycle inhibitors, with antitumor activities. To
shorten the time from initial discovery to clinic, we have
investigated unusual chemical transformations of readily
available complex natural product scaffolds. It has been
suggested7 that in dysidiolide, the γ-hydroxybutenolide
moiety likely serves as a surrogate phosphate, while the
long side chain occupies a hydrophobic binding pocket
associated with the normal substrate. We visualized
that a cholesteryl moiety might provide the scaffold for
constructing a CDC25A inhibitor, with the C and D
rings and attached C8 side chain mimicking the hydro-
phobic rings, with attendant side chains, of dysidiolide,
and the surrogate phosphate could be constructed from
the A and B rings via some type of fragmentation
reaction of these rings. The C-3 acetoxy group and C-5
double bond of cholesteryl acetate would obviously serve
as entries in this approach.
Robert T. Abraham
Department of Immunology and Pharmacology,
Mayo Clinic, Rochester, Minnesota 55905
Garth Powis
Arizona Cancer Center, University of Arizona,
1515 North Campbell Avenue, Tucson, Arizona 85724-5024
Received August 27, 1998
In tr od u ction . Posttranslational modifications of
intracellular signaling proteins by addition and removal
of phosphate groups provide eukaryotic cells with
convenient molecular switches for the control of the
normal functions of intracellular signaling pathways.
Protein serine/threonine phosphatases (PSTPase) and
protein tyrosine phosphatases (PTPase) catalyze the
removal of phosphate groups from phosphoserine/phos-
phothreonine and phosphotyrosine residues, respec-
tively. Recently, a novel group of dual specificity protein
phosphatases, CDC25 phosphatases, which dephospho-
rylate contiguous phosphothreonine and phosphoty-
rosine residues on the cyclin-dependent kinases (cdk),
has been shown to play crucial roles in cell prolifera-
tion.1,2
In human cells, CDC25 consists of three phase-specific
isoforms termed CDC25A, CDC25B, and CDC25C.
CDC25A is expressed early in the G1 phase of the cell
cycle and is responsible for the activation of at least two
cyclin-dependent kinases required for G1-to-S phase
progression.1,2 CDC25A dephosphorylates the catalytic
cdk subunit at Tyr-15 and Thr-14 near the ATP-binding
site, allowing binding of ATP to the cyclin-cdk complex
and rendering the complex catalytically active. The
dephosphorylation of both cyclin E-cdk2 and cyclin
A-cdk2 complexes by CDC25A is tightly regulated in
Herein, we report the discovery of a group of novel
CDC25A inhibitors, synthesized by pyrolysis of a readily
available natural product derivative, 3-R-azido-B-homo-
10.1021/jm980500r CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/31/1998

4678 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24
Communications to the Editor
Sch em e 2. Proposed Mechanism for Formation of
Compounds 2-6
Ta ble 1. Chemical Shifts for H-19 of Compounds 2-6
2
3
4
5
6
δH-19
4.93, s
4.84, s
1.34, s
1.43, s
1.67, brsa
1.60, brsa
a
brs stands for broad singlet.
F igu r e 1. Chemical structures of dysidiolide, SC-RRδ9, and
compound 7.
compound 1 was identified by its IR spectrum (azide,
2094 cm-1; enol lactone, 1763 cm-1) and confirmed by
1H NMR, 13C NMR, DEPT spectra, and high-resolution
CIMS spectroscopy. Silica gel (0.040-0.063 mm; EM
Science) was evenly coated with 1 (ethyl acetate solu-
tion) to give 20 wt % solid, after evaporation of the
solvent. Heating of this solid at 180 °C for 1 h, followed
by extraction (ethyl acetate), silica gel column chroma-
tography, and reverse-phase HPLC, gave products 2-6.
These products can be visualized as arising from the
common carbocation intermediate C as illustrated in
Scheme 2. Thus, heating of compound 1 on silica gel
would lead to A by 3,3-sigmatropic rearrangement and
protonation. Fragmentation of A would be initiated by
opening of ring A to give B, which after loss of molecular
nitrogen would give C, the common intermediate in the
formation of products 2-6. These compounds share the
structural features of a cholesteryl side chain and intact
C and D rings, while rings A and B have been opened
to give one chain containing a conjugated cyano group
exhibiting absorptions around 2220-2230 cm-1 in the
IR spectra. Elemental analysis and high-resolution
Sch em e 1a
EIMS confirmed the same molecular formula, C27H43
-
NO2, for compounds 2-6. Compound 2 was hydroge-
nated to remove the conjugated double bond using Pd
on carbon to give compound 7.
The structures of compounds 2-7 were determined
by IR, MS, and a series of NMR experiments, including
1H NMR, 13C NMR, DEPT, COSY, HMBC, and HMQC.
As shown in Table 1, the chemical shifts of H-19 are
indicative of the structures. The configurations of the
conjugated double bonds in these compounds were
assigned as cis based on the coupling constants. The
configurations of the double bonds between C-1 and
C-10 in acids 5 and 6 were assigned from the NOSEY
spectrum of 5. The relative stereochemistry of the
lactone epimers 3 and 4 was determined by the single-
crystal X-ray diffraction11 of 3 and the NOESY spectrum
of 4.
a
(a) O3, -60 °C, petroleum ether, 2 M HCl, 72%; (b) SOCl2,
CH2Cl2, 2 h, 84%; (c) 2 equiv of NaN3 (10%), acetone, 1 h, 98%; (d)
compound 1 was coated on silica gel, heated at 180 °C, 1 h.
6-oxa-4-cholesten-7-one (1). Compound 7, the best
inhibitor in this series, inhibited the dephosphorylation
of fluorescein diphosphate by CDC25A with an IC50 of
2.2 µM and is thus far the most potent CDC25A
inhibitor to be reported.
Resu lts a n d Discu ssion . As illustrated in Scheme
1, pyrolysis precursor 1 was prepared by ozonolysis of
cholesteryl acetate,10 followed by conversion of the
resulting keto acid to 3â-actoxy-B-homo-6-oxa-4-choles-
ten-7-one and exposure of the latter to sodium azide
(10% aqueous) at room temperature. The structure of
Compounds 2-7 were tested for their CDC25A in-
hibitory activity by measuring the inhibition of dephos-

Communications to the Editor
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 24 4679
observed in the X-ray structure7 of dysidiolide, wherein
the diaxial orientations of the two side chains lead to
their almost vertical projections from the plain formed
by the two rings. This suggests that a flattened
structure may fit better into the binding site of CDC25A.
The recent publication of the crystal structure of
CDC25A13 and the deposition of the atomic coordinates
in the Brookhaven Protein Data Bank have permitted
us to initiate the modeling studies on the compounds
described in this communication, and these studies will
be reported in a future publication.
Ta ble 2. CDC25A Inhibition by Compounds 2-7
dysidi-
olide SC-RRδ9
2
3
4
5
6
7
IC50
(µM)
9.7a 13.3a >50a 36.3a 24.2a 2.2a
9.4b 15c, 4d
a
IC50 value obtained by assays using fluorescein diphosphate
b
as substrate. IC50 value reported in ref 7 by assays using
p-nitrophenylphosphate as substrate. c IC50 value reported in ref
d
9 by assays using fluorescein diphosphate as substrate. IC50
value reported in ref 9 by assays using p-nitrophenylphosphate
as substrate.
At this time, only compounds 2 and 4 have been tested
for their growth inhibition activities against tumor cell
cultures, as described previously.14 Acid 2 and lactone
4 were found to suppress growth of HT-29 colon cancer
cells with IC50 values of 10.9 and 12.0 µM, respectively.
Lactone 4 also inhibited the growth of A-549 lung
adenocarcinoma cells with an IC50 of 7.0 µM.
In conclusion, we have employed silica gel-supported
pyrolysis of an azido-homo-oxa steroid to synthesize a
group of novel, small molecule inhibitors of human
CDC25A phosphatase, using the readily available natu-
ral product cholesteryl acetate as starting material. Acid
7, the best inhibitor in this group, inhibited the activity
of CDC25A protein phosphatase reversibly and more
potently (IC50 ) 2.2 µM) than the well-known marine
natural product dysidiolide. Compounds 2 and 4 in-
hibited proliferation of some human tumor cell lines in
a dose-dependent manner. These compounds provide
unique leads for the design of more potent CDC25A
inhibitors as potential anticancer agents, and the
synthesis and screening of structural variants based on
these motifs are currently underway.
F igu r e 2. ORTEP drawing of the X-ray structure for com-
pound 3.
F igu r e 3. Computed low-energy conformation of compound
7.
Ack n ow led gm en t. We gratefully acknowledge sup-
port of this research by the NCI/NIH (Grant 5U19-
CA52995). We are grateful to Dr. Donald G. VanDerveer
and Dr. Leslie T. Gelbaum for their valuable assistance
in obtaining single-crystal X-ray and high-resolution
NMR data. We thank Mr. David E. Bostwick and Ms.
Sarah J . Shealy for mass spectroscopic services.
phorylation of fluorescein diphosphate by this enzyme
as described previously.9 Results of the in vitro CDC25A
inhibition assay of compounds 2-7 presented in Table
2 revealed the importance of the orientations of the
cyano-containing side chains in these compounds. With
the 10S configuration (Figure 2), lactone 3 showed very
little inhibitory activity (IC50 > 50 µM), while its 10R
isomer, compound 4, is approximately 4-fold more
potent (IC50 ) 13.3 µM). A similar tendency can be
observed within the acid series of compounds 2 and 5-7,
wherein the different configurations of double bonds
vary the positioning of the cyano group and result in
differentiated inhibitory activities (IC50 ) 9.7, 36.3, and
24.2 µM for 2, 5, and 6, respectively). When the
conjugated double bond was hydrogenated to give
compound 7, a significant enhancement of CDC25A
inhibitory activity was obtained (IC50 ) 2.2 µM).
Some common structure features can be observed
among compounds 2-7, dysidiolide, and SC-RRδ9. They
all contain long alkyl side chains that may fit into a
hydrophobic pocket in the substrate binding site, and
they either contain a carboxyl group (compounds 2, 5-7,
and SC-RRδ9) or a lactone moiety (compounds 3, 4, and
dysidiolide), which may interact with the active site
arginine in place of a phosphate. Figure 3 illustrates
one of the lowest-energy conformations of compound 7
found by a conformational search using the MM+ force
field.12 This conformation shows a flattened structure
which is very different from the 3D conformation
Su p p or tin g In for m a tion Ava ila ble: Full experimental
details and spectroscopic data for compounds listed in Table
2 and atomic coordinate information for compound 3 (19
pages). Ordering information is given on any current mast-
head page.
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Communications to the Editor
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(11) Compound 3 crystallized in space group P212121 with a ) 6.467-
(2) Å, b ) 34.103(1) Å, c ) 35.479(1) Å and refined to a
conventional factor R ) 0.0069 for 867 parameters and 10 471
reflections with Fo > 4 σ (Fo). Data collection was at 248 K, with
Z ) 12. There was some disorder in the side chains of all three
unique molecules.
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(12) Compound 7 was computer-built using ISIS/Draw (version 2.1;
J M980500R