Syntheses and biological activities of a novel group of steroidal derived inhibitors for human Cdc25A protein phosphatase

Article abstract of DOI:10.1021/jm0004401

Silica gel supported pyrolysis of an azido-homo-oxa steroid led to rearrangement, presumably by a mechanism similar to that of solution phase Schmidt fragmentation, to produce a group of novel inhibitors for the oncogenic cell cycle regulator Cdc25A phosp

Full text of DOI:10.1021/jm0004401

834
J . Med. Chem. 2001, 44, 834-848
Syn th eses a n d Biologica l Activities of a Novel Gr ou p of Ster oid a l Der ived
In h ibitor s for Hu m a n CDC25A P r otein P h osp h a ta se1
Hairuo Peng,‡,† Wenge Xie,‡ Diane M. Otterness,§ J ohn P. Cogswell,| Randy T. McConnell,| H. Luke Carter,|
Garth Powis, Robert T. Abraham,§ and Leon H. Zalkow*,‡
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, Department of Pharmacology
and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, Department of Functional Genetics,
GlaxoWellcome Inc., Research Triangle Park, North Carolina 27709, and Arizona Cancer Center, University of Arizona,
1515 North Campbell Avenue, Tucson, Arizona 85724-5024
Received October 10, 2000
Silica gel supported pyrolysis of an azido-homo-oxa steroid led to rearrangement, presumably
by a mechanism similar to that of solution phase Schmidt fragmentation, to produce a group
of novel inhibitors for the oncogenic cell cycle regulator Cdc25A phosphatase. Cyano-containing
acid 17, one of the best inhibitors in this group, inhibited the activity of Cdc25A protein
phosphatase reversibly and noncompetitively with an IC50 value of 2.2 µM. Structure-activity
relationships revealed that a phosphate surrogate such as a carboxyl or a xanthate group is
required for inhibitory activity, and a hydrophobic alkyl chain, such as the cholesteryl side
chain, contributes greatly to the potency. Without the cyano group, acid 26 and xanthate 27
were found to be more selective over Cdc25A (IC50 ) 5.1 µM and 1.1 µM, respectively) than
toward CD45 (IC50 > 100 µM, in each case), a receptor protein tyrosine phosphatase. Several
of these inhibitors showed antiproliferative activities in the NCI 60-human tumor cell line
screen. These steroidal derived Cdc25 inhibitors provide unique leads for the development of
dual-specificity protein phosphatase inhibitors.
In tr od u ction
and employ a similar catalytic mechanism.2 Cdc25 cell
cycle regulators are examples of DSPases which de-
phosphorylate contiguous pTyr and pThr on the cyclin
dependent kinases (cdks) and have been shown to play
crucial roles in cell proliferation.
Reversible phosphorylations of proteins are frequently
used in eukaryotic cells as convenient molecular switches
for the control of intracellular signaling events. The
attachment or removal of phosphate groups from intra-
cellular signaling proteins coordinates the relay of
signals to the next level by modulating the formation
of protein-protein interactions or by directly regulating
the enzyme activities via blocking or unmasking the
catalytic site.2a Two types of enzymes regulate protein
phosphorylation states: protein kinases, which catalyze
the covalent attachment of a phosphate group to the
amino acid side chain of a protein, and protein phos-
phatases, which reverse the procedure.
Eukaryotic protein phosphatases are represented by
two distinct families: protein serine/threonine phos-
phatases (PSTPase), which dephosphorylate phospho-
serine (pSer) and phosphothreonine (pThr) in a single-
step reaction using a metal-activated water molecule,
and protein tyrosine phosphatases (PTPase) which
dephosphorylate phosphotyrosine (pTyr) via a two-step
reaction involving a cysteinyl-phosphate enzyme inter-
mediate. Dual-specificity protein phosphatases (DSPase)
which dephosphorylate both pTyr and pThr residues are
considered as a subfamily of PTPase, because they
possess the conserved PTPase signature motif HCxxxxxR
In human cells, Cdc25 consists of three phase-specific
isoforms termed Cdc25A, Cdc25B, and Cdc25C. Both
Cdc25C and Cdc25B are considered as regulators of
G2/M transition,3 and Cdc25C was recently found to be
a downstream effector of the radiation induced G2 check
point.4 In contrast, Cdc25A is expressed early in the G1
phase and is responsible for the G1/S transition of cell
cycle.5 This DSPase dephosphorylates the cdk at Tyr-
15 and Thr-14, near the ATP-binding side, allowing
binding of ATP to the cyclin/cdk complex and rendering
the complex catalytically active to phosphorylate down-
stream components that initiate DNA synthesis. The
dephosphorylation of cyclin/cdk complexes by Cdc25
phosphatases is tightly regulated in normal cells. How-
ever, accumulating evidence suggests that inappropriate
amplification or activation of Cdc25A or Cdc25B is
characteristic of a number of human cancers, including
breast cancers.6 Enforced expression of Cdc25A or B
causes cellular transformation, and the transcription
and catalytic activities of both proteins are directly
regulated by two other protooncogenes products, c-Myc
and c-Raf, respectively.7 Thus, deregulation of Cdc25A
and Cdc25B activities, either through overexpression or
abnormal activation, may contribute to the growth of
certain types of cancer. Therefore, small molecule
inhibitors of these two enzymes may possess novel
antitumor activities.8
* To whom correspondence should be directed: Prof. Leon H.
Zalkow, School of Chemistry and Biochemistry, Georgia Institute of
Technology, Atlanta, GA 30332-0400. Phone: (404) 894 4045. Fax:
(404) 894 7452. E-mail:leon.zalkow@chemistry.gatech.edu.
‡ Georgia Institute of Technology.
§ Duke University Medical Center.
| GlaxoWellcome Inc.
University of Arizona.
In contrast to PSPTase, for which a number of very
potent inhibitors, such as Okadaic acid (IC50 ) 49 nM,
† Current address: Yale University, Department of Chemistry, P.O.
Box 208107, New Haven, CT 06520-8107.
10.1021/jm0004401 CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/25/2001

Steroidal Derived Inhibitors for Human CDC25A Phosphatase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 835
Sch em e 1. Schmidt and Beckmann (a) Rearrangement
and (b) Fragmentation
F igu r e 1. Chemical structures of dysidiolide, sulfircin, me-
nadione, and HP-19.
PP1),9 have been reported, the development of inhibitors
for PTPase, particularly for its DSPase subfamily, have
just recently attracted intense effort. Not long ago, a
broad-spectrum protein phosphatase inhibitor, sodium
othrovanadate,10 was the only readily available Cdc25
phosphatase inhibitor widely used. Later, a few small
molecule Cdc25A phosphatase inhibitors were reported,
including natural products dysidiolide (IC50 ) 9.4 µM,11a
pNPP; IC50 > 50 µM, mFP11b), sulfricin (IC50 ) 7.8 µM,
pNPP),12 and menadione (vitamin K3) and its naphtho-
quinone analogues,13 which inactivate Cdc25A irrevers-
ibly by forming Michael adducts (Figure 1). A combi-
natorial library of inhibitors based on the pharmacophore
of Ser/Thr phosphatase resulted in competitive Cdc25A
inhibitors showed antiproliferative activities in the NCI
60-human tumor cell line screen. On the basis of an
analysis of structure-activity-relationships, a group of
more potent Cdc25A inhibitors of simpler structures
were synthesized; the best one (34) showed IC50 value
of 0.7 µM (Table 9).
Resu lts a n d Discu ssion
Ch em istr y. The Beckmann and Schmidt rearrange-
ments are classical examples of molecular rearrange-
ments in which an alkyl or aryl group migrates with
their bonding electrons to adjacent electron-deficient
nitrogen atoms to form amides or lactams. (Scheme 1a).
However, if cleavage of the adjacent C-C bond leads to
the formation of a carbonium ion with considerable
stability, fragmentation reactions may occur resulting
in a nitrile and a positively charged fragment.16 These
types of fragmentation reactions have been referred to
as “abnormal”, “second order” Beckmann and Schmidt
rearrangements, or Beckmann and Schmidt fragmenta-
tions (Scheme 1b).17 Examples of these two types of
fragmentation reactions have been reported to yield
nitriles, and carbocations that eventually formed double
bonds, under Beckmann19 or Schmidt18 rearrangement
conditions. We decided to take advantage of this type
of fragmentation reaction to cleave the A and B rings
of the cholesteryl template to obtain favorable pharma-
cophores for Cdc25A inhibition: an R,â-unsaturated
cyano group which is an excellent substrate for Michael
addition by the cystein group in the active site, and a
carboxylic acid group that may coordinate with the
active site arginine.
For unknown reasons, the Schmidt fragmentation
(Figure 1b) of 5,6-seco-3-cholesten-6-oic acid (7) in
solution was unsuccessful. Therefore, compound 3 was
synthesized as a promising precursor. As illustrated in
Scheme 2, pyrolysis precursor 3 was prepared by
ozonolysis of cholesteryl acetate,20 followed by conver-
sion of the resulting keto acid 1 to 3â-acetoxy-B-homo-
6-oxa-4-cholesten-7-one (2) with thionyl chloride at room
temperature. Exposure of 2 to sodium azide (10%
aqueous) at room temperature gave 3 quantitatively.
inhibitors such as SC-RRδ9 (IC50 ) 15 µM, FDP; IC50
)
4 µM, pNPP).14 Libraries of dipeptidyl phosphonates and
malonates resulted in several noncompetitive Cdc25A
inhibitors.15 We have also recently reported steroidal
derived acids as Cdc25A inhibitors.1,22
We initiated a program of designing novel Cdc25A
inhibitors based on templates of readily available
natural products. The rigidity and well-defined stereo-
chemistry of the cholesteryl template makes it a good
scaffold for distal functionalization. It has been sug-
gested that, in dysidiolide, the γ-hydroxybutenolide
moiety likely serves as a surrogate phosphate, while the
long side chain occupies a hydrophobic binding pocket
near the active site. We visualized that the C and D
rings and the attached C8 side chain of cholesteryl
acetate could mimic the hydrophobic rings, with at-
tendant side chains, of dysidiolide, and the surrogate
phosphate could be constructed from the A and B rings
of the cholesteryl template via some type of fragmenta-
tion 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.
Herein we report the discovery of a group of novel
Cdc25A inhibitors synthesized by pyrolysis of a readily
available natural product derivative, 3R-azido-B-homo-
6-oxa-4-cholesten-7-one (3), on silica gel (Scheme 2). HP-
19 (17), one of the best inhibitors in this series, inhibited
the dephosphorylation of fluorescein diphosphate by
Cdc25A with an IC50 of 2.2 µM (Table 4). These

836 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Peng et al.
Sch em e 2a
arise from a common fragmentation intermediate. As
illustrated in Schemes 4 and 5, compounds 8-13 can
be visualized as arising from the common carbocation
intermediate C. Heating of the pyrolysis precursor 3 on
silica gel would lead to A by a 3,3-sigmatropic rear-
rangement and protonation. Fragmentation of A would
be initiated by opening of the B ring to give B that
already contains a favorable carboxyl group necessary
for Cdc25A inhibition. Intermediate B can be seen to
possess an ideal structure to undergo the Schmidt
fragmentation reaction: cleavage of the C-5, C-10 bond
leads to a stable tertiary carbocation. As a result, loss
of nitrogen gives an R,â-unsaturated cyano group and
a stable carbocation at C-10 in the common intermediate
C for the formation of compounds 8-13.
As shown in Scheme 4, trapping of the carbocation
by lone pair electrons on the carboxyl oxygen would give
rise to epimeric lactones 8 and 9, while elimination of
hydrogen atoms from adjacent carbons would result in
unsaturated isomers, 10-12. As illustrated in Scheme
5, recombination of the carbocation and the cyano group
in C followed by hydrolysis through a Ritta reaction21
would give iminol D, which upon reaction with the B
ring carboxyl group and loss of water, would result in
the formation of imide 13.
a
(a) O3, -60 °C, hexane, 45 min; piperidine, 0 °C, 3 h; 2 M HCl;
(b) SOCl2, CH2Cl2, 2 h; (c) 2 equiv of NaN3/H2O(10%), acetone, 2
h.
However, attempts to rearrange this compound in
different solvents at temperatures up to 160 °C were
unsuccessful, as well. Fragmentation of the A and B
rings of the cholesteryl template was eventually ac-
complished by pyrolyzing 3R-azido-B-homo-6-oxa-4-cho-
lesten-7-one (3) on silica gel. Silica gel (0.040-0.063 mm;
EM science) was evenly coated with 3 to give 20 wt %
solid, after evaporation of the solvent. Pyrolysis of this
solid at 180 °C for 1 h, followed by extraction, silica gel
column chromatography, and reverse phase HPLC, gave
products 4-13 (Scheme 3) with diverse structural
features.
Another compound (4) of unexpected structure was
formed in the same pyrolysis reaction. Compound 4
(Figure 2) contains a dihydro-quinone moiety, and
surprisingly, the stereochemistry at C-10 has been
inverted as compared to the cholesteryl acetate starting
material. It was reasoned that this compound might be
formed from one of the major products of the pyrolysis
reaction, compound 7, by rotation of the C10-C9 bond,
oxidation of the enone, and finally cyclization and loss
of water as seen in Scheme 6.
Formation of compounds 5-7 is seen to readily arise
through elimination of the azido group, and further
opening of the lactone B ring leads to 6 and 7. Com-
pounds 8-12, the fragmentation products, and 13 may
Sch em e 3a
a
(a) Compound 3 was coated on silica gel to give a 20% weight solid and pyrolyzed at 180 °C for 1 h.

Steroidal Derived Inhibitors for Human CDC25A Phosphatase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 837
Sch em e 6. Proposed Mechanism for the Formation of
Compound 4
F igu r e 2. ORTEP drawing of the X-ray structure for com-
pound 4.
Sch em e 4. Proposed Mechanism for the Formation of
Compounds 8-12
was removed to eliminate the possibility of Michael
addition, as in compound 16, the cyano group was
hydrolyzed into a carboxyl group to give compound 26.
Compounds 28-37 were synthesized as illustrated in
Schemes 8-10, in order to introduce other functional
groups that might serve as phosphate surrogates. As
seen in Scheme 8, cholestanyl xanthates 28-32 were
synthesized by treatment of potassium O-cholestanyl
xanthate (27) with 1 equiv of the appropriate alkyl
halide in DMSO. Compound 27 was obtained by treat-
ment of the potassium salt of cholestanol with carbon
disulfide.22 As shown in Scheme 9, compound 33 was
synthesized by reductive amination using cholestane-
3-one and δ-amino butyric acid. Cholestanyl-O-acetic
acid 34, cholestanyl thiocarbamates 35 and 36, and
cholestanyl carbamate 37 were synthesized by treat-
ment of cholestanol with sodium hydride followed by
reaction with bromo acetic acid, aryl isothiocynates, and
phenyl isocyanate, respectively (Scheme 10).
Sch em e 5. Proposed Mechanism for the Formation of
Compound 13
Sp ectr oscop ic Ch a r a cter iza tion . The products of
the pyrolysis reaction exhibited varied functionalities
in place of the previous A and B rings of the cholesterol
skeleton. Structures of compounds 4, 8, and 13 were
firmly established through X-ray crystallographic analy-
sis (Figures 2-4). Structures of other compounds were
identified by IR, MS, and a series of NMR experiments
including 1H and 13C NMR, DEPT, COSY, HMQC,
HMBC, and NOESY. The 1H and 13C NMR spectral
assignments of the characteristic resonances for repre-
sentative new compounds are listed in Tables 1-3. As
seen in Tables 1 and 2, the chemical shifts of the Me-
19 protons are the defining features distinguishing the
molecules, and the 13C signals of Me-19, with an R
configuration, as in compounds 4 and 8, appear at much
higher (δ 26 ppm) resonances than the ones with a â
configuration (Table 3).
Compounds 8-12 have common structural features
with the cholesteryl side chain and C, D rings intact,
while the A and B rings have been opened to give chains
containing a conjugated cyano group exhibiting absorp-
tions around 2220-2230 cm-1 in their IR spectra. Two
highly conjugated olefinic protons resonate as a down-
field doublet triplet (H-3) and a upperfield doublet (H-
4) in the 1H NMR spectra of these compounds, the
coupling constants (11.0 Hz) indicating cis-double bonds
Compound 5 was hydrogenated to remove the ∆3
double bond to give 14. Compounds 7 and 9-13 were
hydrogenated to remove the conjugated double bonds
using 10% Pd/C to give compounds 15-20, respectively.
Compound 10 (Scheme 3) was hydrogenated using 5%
Pt/C to saturate both double bonds to yield C-10 epimers
21 and 22.
As shown in Scheme 7, base-catalyzed hydrolysis of
lactone 9 in methanol gave tetrahydrofuran 23, pre-
sumably formed by Michael addition of the C-10 oxygen
to the conjugated cyano group. Michael addition of the
solvent, methanol, at the same position gave compound
24. Similarly, base-catalyzed solvolysis of compound 10
in ethanol gave 25. When the conjugated double bond

838 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Peng et al.
Sch em e 7. Base-Catalyzed Hydrolysis and Michael Addition of Compounds 9, 10, and 16a
a
(a) 0.6% NaOH/MeOH, reflux 10 h; 2 M HCl; (b) 6% NaOH/EtOH, reflux 10 h; 2 M HCl; (c) 6% NaOH/MeOH, reflux 10 h; 2 M HCl.
Sch em e 8a
Sch em e 10a
a
Reagents: (a) NaH, THF, BrCH2CO2H, 60%; (b) NaH, THF,
ArNCS or ArNCO, 75-80%.
a
Reagents: (a) RX (X ) Cl or Br), DMSO, 80-85%.
and the NOE observed between Me-19 protons and H-8
(δ 2.07, 1H, m), as well as that between H-2 (δ 2.55,
2H, m) and H-9 (δ 1.49, 1H, m), in the NOESY spectrum
of 9. Compounds 10-12 were assigned as carboxylic
acids for the characteristic absorptions around 3350-
2500 (br) and 1700 cm-1 in the IR spectra. The chemical
shifts for the H-1 and Me-19 protons (Table 1) indicated
different double bond configurations for compounds 10-
12. Compound 11 was assigned as 1,10Z based on the
NOE observed between H-1 and Me-19 protons while
12 was assigned as 1,10E by the NOE between H-2 and
Me-19 protons.
Sch em e 9a
a
Reagents: (a) δ-amino butyric acid, NaBH3CN, CH3OH/THF,
85%.
The configuration at C-3 in compound 23 (Scheme 7),
the product of intramolecular Michael addition of 9, was
determined as S by the NOE observed between the
signals for Me-19 protons and H-3 (Table 2). Compounds
21 and 22, the C-10 epimers derived from the hydro-
genation of 10 (Scheme 3), exhibit differentiated Me-19
signals at δ 0.89 (d, 3H, J ) 6.6 Hz) and 0.78 (d, 3H, J
) 6.6 Hz), respectively. However, without crystal struc-
tures for these compounds, it is not possible to assign
(Table 1). The C-10 quaternary carbons at δ 86.3 and
86.6 ppm (Table 3) in compounds 8 and 9, respectively,
are indicative of a newly formed stereocenter attached
to an oxygen atom. Compounds 8 and 9 were identified
as lactone epimers by IR absorptions of a six-membered
δ-lactone at 1720 and 1733 cm-1, respectively. The
relative stereochemistry of these two isomers were
confirmed by the single-crystal X-ray diffraction of 8,

Steroidal Derived Inhibitors for Human CDC25A Phosphatase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 839
Ta ble 1. 1H NMR Data for Compounds 8-12
chemical shift, ppm (CDCl3)
no.
19
8
9
10
11
12
1.43 (s, 3H)
1.34 (s, 3H)
4.93 (s, 1H), 4.84 (s,
1H)
1.67 (s, 3H)
1.60 (s, 3H)
1
2
1.85 (m, 2H)
2.63 (m, 2H)
1.75 (m, 2H)
2.55 (m, 2H)
2.17 (t, 7.2 Hz, 2H)
2.59 (m, 2H)
5.18 (t, 7.7 Hz, 2H)
3.10 (dd, 7.7, 5.5 Hz
2H)
5.17 (t, 7.2 Hz, 2H)
3.08 (t, 7.2 Hz, 2H)
3
6.54 (dt, 11.0, 7.8 Hz,
1H)
6.52 (dt, 10.8, 7.8 Hz,
1H)
6.50 (dt, 11.0, 7.2 Hz,
1H)
6.37 (dt, 10.9, 7.7 Hz,
1H)
6.38 (dt, 11.1, 7.5 Hz
1H)
4
7
5.35 (d, 11.1 Hz, 1H)
2.61 (dd, 18.0, 5.1 Hz,
1H)
5.32 (d, 10.5 Hz, 1H)
2.60 (dd, 17.7, 4.8 Hz,
1H)
5.33 (d, 11.0 Hz, 1H)
2.35 (dd, 15.0, 3.3 Hz,
1H)
5.27 (d, 10.9 Hz, 1H)
2.22 (dd, 15.4, 3.3 Hz,
1H)
5.27 (d, 10.4 Hz, 1H)
2.22 (dd, 15.4, 4.4 Hz,
1H)
2.02 (dd, 18.0, 11.5 Hz,
1H)
2.00 (dd, 17.7, 11.5 Hz,
1H)
2.10 (dd, 15.0, 6.6 Hz,
1H)
2.04 (dd, 15.4, 7.6 Hz,
1H)
2.07 (dd, 15.4, 6.6 Hz,
1H)
Ta ble 2. 1H NMR Data for Compounds 4, 6, 13, and 23
chemical shift, ppm (CDCl3)
1.30 (s, 3H)
position
4
6
13
23
19
1
1.43 (s, 3H)
1.05 (s, 3H)
1.25 (s, 3H)
NAa
3.20 (d, 21.1 Hz, 1H)
2.85 (d, 21.1 Hz, 1H)
5.68 (m, 1H)
2.41-2.22 (m, 2H)
2
3
4
6.69 (dd, 10.2, 1.5 Hz, 1H)b
6.50 (d, 10.2 Hz, 1H)b
NAa
NAa
5.68 (m, 1H)
6.74 (ddd, 10.4, 7.7, 2.7 Hz, 1H)
6.21 (dd, 10.4, 2.7 Hz, 1H)
4.20 (m, 1H)
2.46 (dd, 17.4, 5.5 Hz, 1H)
2.00 (buried in, 1H)
2.70-2.44 (m, 2H) NAa
5
7
3.39 (s, 1H)
NAa
2.27 (dd, 16.6, 2.7 Hz, 1H)
2.09 (dd, 16.6, 7.5 Hz, 1H)
2.43 (dd, 17.6, 4.4 Hz, 1H)
2.00 (dd, 17.6, 12.5 Hz, 1H)
2.70-2.44 (m, 2H) NAa
a
b
Not assigned. Assignment may be interchanged.
Ta ble 3. 13C Data for Compounds 4, 6, 8-10, 13, and 23
chemical shift, ppm (CDCl3)
carbon
4
6
8
9
10
13
23
19
10
1
2
3
4
5
6
26.1
55.3
16.8
50.9
26.0
86.3
18.6 111.2
86.6 150.2
18.7
62.0
21.2
89.7
NAa
NAa
74.4
201.0b
140.1
139.1
196.4b
71.6
40.9 NAa
NAa
32.5 NAa
124.4 NAa
126.0 154.4 154.5 154.7 149.6
39.1 100.2 99.8
NAa
29.9 NAa
99.8 128.8 NAa
217.1 115.8 115.9 116.0 169.3 116.8
206.0b 178.9 170.6 170.8 179.1 175.7 174.1
F igu r e 3. ORTEP drawing of the X-ray structure for com-
pound 8.
a
b
Not assigned. Assignment may be interchangeable.
inhibitors were tested for their inhibitory activities
against Cdc25A and CD45 receptor protein tyrosine
phosphatase23 using p-nitro phenyl phosphate (pNPP)
as substrate. The results are listed in Tables 4-9.
The orientations of the cyano-containing side chains
vary in the double bond isomers 10-12 as seen in the
energy minimized conformations (Hyperchem). These
differing conformations may account for the differenti-
ated inhibitory activities observed for compounds 10-
12 (9.7-36.3 µM, Table 4). Increasing the flexibility of
the cyano-containing side chain by saturating the double
bonds, as in compounds 17-19 and 21-22, significantly
increased the potencies of these compounds (2.5-1.9
µM, Table 4). Removal of the ∆3 double bond by Michael
addition, as in compound 25, also resulted in better
inhibitory activity (2.9 µM) than the parent compound.
However, formation of the tetrahydrofuran ring as in
23 resulted in reduced inhibitory activity (15.8 µM),
perhaps, because of the increased rigidity of the com-
pound. Compounds 4 and 13, although containing the
R,â-unsaturated ketone as Michael acceptor, did not
show Cdc25A inhibitory activity.
F igu r e 4. ORTEP drawing of the X-ray structure for com-
pound 13.
unequivocal absolute configurations at C-10 in these
isomers.
Biologica l Eva lu a tion . The inhibition effects of
compounds synthesized were evaluated as inhibitors of
human Cdc25A phosphatase at 25 °C using fluorescein
diphosphate (FDP) as substrate. Some of the best
The ready formation of compounds 23 and 25 through
base-catalyzed solvolysis illustrated the ease of forma-

840 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Peng et al.
F igu r e 6. Michaelis-Menten plot for the Cdc25A inhibition
by compound 17.
Ta ble 5. Vmax and Apparent Km for Cdc25A Inhibition by
Compound 17
inhibitor concn (µΜ)
Vmax
SE (Vmax
)
Km (µΜ) SE (Km)
F igu r e 5. Lineweaver-Burk plot for the CD45 inhibition by
compound 17.
control
0.625
1.25
2.5
4724
4607
4261
3074
632
70
84
84
99
141
37.4
37.4
32.3
33.6
23.1
1.6
1.96
1.91
3.24
Ta ble 4. Protein Phosphatase Inhibition Activities of
Compounds 10-12, 17-19, 22-23, and 25
5
17.3
CD45, a receptor tyrosine phosphatase. The kinetic
characteristics of CD45 inhibition with compound 17
were studied using pNPP as substrate. As shown in
Figure 5, the Lineweaver-Burk plot was most consis-
tent with a mixed inhibition model. However, the kinetic
characteristics of compound 17 for Cdc25A, using 3-OMe
FMP as substrate, exhibited a noncompetitive pattern.
Figure 6 shows the Michaelis-Menten plot, and the
Vmax and apparent Km derived from the plot are listed
in Table 5. Compound 17 inhibited the enzyme by
decreasing the Vmax, but the apparent Km was un-
changed within the experimental error. These observa-
tions suggest that compounds, like 17, which contain a
cyano group might not bind to Cdc25A or CD45 protein
phosphatase specifically at the active site. It is worth
noting that simple cholesteryl acid, 3-oxo-4-cholesten-
2â-acetic acid, has been shown to inhibit Cdc25A
competitively, suggesting that this type of simple ste-
roidal acid may interact with the arginine group at the
substrate binding site.22
Cdc25Aa Cdc25Ab CD45b
config.
C10
IC50
IC50
IC50
3
1,10
10,19
compd
R1
-H
-H
-H
-H
-H
-H
-H
∆
∆
∆
(µM)
(µM)
(µM)
10
11
12
17
18
19
21
22
23
25
cis
cis
cis
present
9.7
36.3
24.2
2.2
2.4
2.5
Z
E
present
8.3
9.3
9.1
12.2
13.2
8.6
Z
E
2.2
1.9
-H
-O-C10
-OEt
10R
15.8
2.9
present
a
Assay using fluorescein diphosphate (FDP) as substrate.
Assay using p-nitro phenyl phosphate (NPP) as substrate.
b
tion of Michael adducts at the R,â-unsaturated cyano
group. However, the observation that elimination of the
Michael addition potential, as in compounds 17-19, 21,
22, and 25, increased inhibitory activity suggests that
irreversible binding of these inhibitors to the cystein
group at the active site of Cdc25A is responsible for the
observed activity. The reversible inhibition of compound
10 was confirmed by a revised Cdc25A inhibition assay,
using Cdc25A bound GST beads. After preincubation
with the inhibitor, the enzyme bound beads were
washed with assay buffer before starting the phos-
phatase assay. Results indicated that removal of inhibi-
tor 10 by washing reversed inhibition of the enzyme.
It was unexpected that assays using pNPP exhibited
higher IC50 values than those obtained with FDP as
substrate (Table 4). In the literature,14 the IC50 reported
for SC-RRδ9 was higher with FDP (IC50 ) 15 µM, FDP;
IC50 ) 4 µM, pNPP). As shown in Table 4, compounds
17, 19, and 22 also showed moderate inhibition against
As shown in Table 6, repeated testing of lactone 9
gave an average IC50 value of 29 µM, which is larger
than previously reported.1 This suggested that the
carboxyl group in compound 10 and the related com-
pounds was important for inhibitory activity. Lactone
8, the 10S isomer of 9, is essentially inactive, presum-
ably due to the unfavorable orientation of the cyano-
containing side chain. However, when the conjugated
double bonds were removed as in derivatives 16 and 24,
the potency increased presumably because of the greater
flexibility of the side chains in these inhibitors. Com-
pound 26 (Scheme 7), containing both a carboxyl group
and a lactone ring, exhibited a much better IC50 (5.1
µM) compared to compound 9, suggesting that 26 may
orient differently in the enzyme, taking advantage of
the free carboxyl group. The fact that compound 26 was

Steroidal Derived Inhibitors for Human CDC25A Phosphatase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 841
Ta ble 6. Protein Phosphatase Inhibition Activities of
Compounds 8, 9, 16, 24, and 26
Ta ble 8. Protein Phosphatase Inhibitory Activities of
Compound 27-32
Cdc25Aa
IC50 (µM) IC50 (µM) IC50 (µM)
Cdc25Ab
CD45a
Cdc25Aa Cdc25Ab CD45b
compd
R
config.
C10
IC50
IC50
IC50
3
27
28
29
30
31
32
-H
1.1
0.9
>100
>100
>100
>100
11.7
>100
compd
R1
-H
-H
-H
-OMe -CN
-H -COOH
R2
-CN
-CN
-CN
∆
(µM)
(µM)
(µM)
-CH2CO2H
-CH2CO2Et
-CH(CH3)CO2Et
-CH2COCH2CO2Et
-CH2COPh
8
9
16
24
26
present
present
S
R
R
R
R
>50
29
24
>100
>100
10.5
5.1
11.6 >100
a
Assay using fluorescein diphosphate (FDP) as substrate.
Assay using p-nitro phenyl phosphate (NPP) as substrate.
a
Assay using fluorescein diphosphate (FDP) as substrate.
Assay using p-nitro phenyl phosphate (pNPP) as substrate.
b
b
Ta ble 9. Cdc25A Inhibitory Activities of Compounds 33 and 34
Ta ble 7. Cdc25A Inhibitory Activities of Compounds 1, 6, 7,
and 14
compd
R
config. C3
IC50 (µM)
33
34
-NH(CH2)3CO2H
-OCH2CO2H
R, S
S
16%a
0.7
Cdc25Aa
IC50 (µM)
compd
R1
double bond
a
Percentage inhibition at 11 µΜ.
1
6
7
-OAc
-H
-H
9.3
5.8
9.5
5.9
2
∆
Ta ble 10. Cdc25A Inhibitory Activities of Compounds 35-37
3
∆
15
-H
a
Assay using fluorescein diphosphate (FDP) as substrate.
inactive for CD45 (IC50 > 100 µΜ) makes it a good
selective inhibitor for Cdc25A.
As shown in Table 7, compounds 1, 6, 7, and 14, which
simply contain the carboxyl group and cholesteryl side
chain, also showed fairly good inhibitory activities
against Cdc25A, demonstrating that the cyano groups
in compounds 8-12, 16-19, and 21-25 were not
essential for inhibition. We have reported, however, a
hydrophobic side chain is crucial for inhibitory activity.22
In light of these considerations, a series of cholestanol
derivatives (27-37) were synthesized to evaluate con-
tributions of alternative phosphate surrogates. As shown
in Table 8, compound 27,22 with the dithiocaboxylic acid
group and the cholesteryl side chain, exhibited potent
selective inhibitory activity over Cdc25A (IC50 ) 1.1 µM,
Cdc25A; IC50 > 100µM, CD45). Addition of an acetic acid
group to the xanthate, as in 28, slightly increased
Cdc25A inhibitory activity (IC50 ) 0.9 µM). Similarly,
addition of an acetic acid group directly to cholestanol
resulted in excellent inhibitory activity for compound
34 (IC50 ) 0.7 µM, Table 9). However, introduction of
an γ-amino butyric acid group to the cholestane skel-
eton, as in compound 33, led to acute loss of activity as
compared to compounds 27, 28, and 34. When the
dithiocarboxlic acid group or the carboxylic acid group
was masked as in compounds 29-32, activity was
completely lost. Unexpectedly, without a free carboxyl
group, the N-phenyl thiocarbamate derivative of chole-
compd
X
Ar
IC50 (µM)
35
36
37
S
S
O
Ph
2.1
Naphth
Ph
19%a
18%a
a
Percentage inhibition at 10 µΜ.
phenyl carbamate 37 were essentially inactive (Table
10). The mechanism of inhibition for 35 is currently
unknown.
The growth inhibition effects of representative Cdc25A
inhibitors were tested in the NCI 60 human tumor cell
lines representing eight types of solid tumors and
leukemia. The cell growth and viability were measured
using a protein stain sulforhodamine (SRB), which binds
to basic amino acids of proteins in the living cells that
remain attached to the culture plate after washing.24-25
Table 11 shows the GI50 values (micromolar concen-
trations required to produce 50% growth inhibition) of
different compounds for sensitive subpanels in the NCI
panel. As shown in Table 11, the cyano-containing acids
(17, 19, 21, 22) that inhibit Cdc25A exhibited similar
growth inhibition patterns with mean GI50s (MG-MID)
at about 8 µM. Compound 23, which contains a tetrahy-
drofuran moiety, in addition to the cyano and carboxyl
groups, showed essentially the same growth inhibition
stanol (35) was a very active Cdc25A inhibitor (IC50
)
2.1 µM), while N-naphthyl thiocarbamate 36 and N-

842 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Peng et al.
Ta ble 11. Inhibition of in Vitro Cancer Cell Lines by Compounds 9, 13, 14, 16, 17, 19, 21-23, and 26
GI50 (µM)a
disease type and cell lines
9
13
14
16
17
19
21
22
23
26
non-small cell lung cancer
A549/ATCC
NCI-H23
NCI-H522
colon cancer
COLO205
HCC2998
HT29
15.0
14.8
0.89
4.27
4.85
6.10
10.8
19.0
>25
5.51
5.63
>25
5.21
5.12
7.33
7.40
17.3
5.09
5.27
>25
4.35
5.85
>25
8.25
5.75
0.94
8.30
4.92
>25
NDc
8.49
4.85
3.19
4.76
2.63
>25
18.8
15.7
4.75
4.71
4.05
5.10
6.33
7.77
5.92
4.31
6.49
5.29
5.36
4.69
5.55
4.77
5.18
5.26
5.10
5.52
6.13
4.43
7.76
CNS cancer
SF-268
SF-539
>25
>25
>25
14.0
>25
NDc
4.50
6.49
16.6
10.5
5.53
>25
10.8
>25
2.86
19.5
6.01
>25
14.6
6.28
>25
4.46
3.46
5.17
5.04
5.01
4.35
U251
9.49
melanoma
LOX IMVI
SK-MEL-5
ovarian cancer
OVCAR-3
OVCAR-8
renal cancer
786-0
SN12C
UO-31
prostate cancer
PC-3
5.15
4.57
0.84
4.37
16.7
>25
5.35
5.10
4.92
5.98
5.91
8.83
4.86
7.04
4.69
7.71
6.23
10.8
NDc
9.69
13.3
8.72
4.00
1.97
>25
13.5
4.73
5.19
6.64
4.78
6.77
5.53
5.25
4.88
5.19
8.02
5.66
4.76
5.78
5.13
7.54
5.08
0.39
1.44
3.46
2.95
18.5
13.4
>25
4.30
4.38
0.47
11.5
6.90
9.41
8.69
7.73
9.55
8.45
6.05
9.97
7.18
6.46
8.74
9.23
5.40
15.3
8.44
8.30
11.0
9.73
4.22
16.0
5.51
7.36
6.34
7.15
6.88
7.77
7.21
breast cancer
NCI/ADR-RES
BT-549
>25
>25
8.35
11.5
6.05
5.07
4.09
3.55
14.6
13.0
16.9
19.6
5.53
5.89
4.92
5.51
5.66
5.62
8.39
7.98
6.44
5.75
8.22
8.62
5.34
5.16
6.69
7.41
8.20
6.20
9.27
8.13
5.44
5.05
8.41
8.25
5.55
4.19
7.30
8.91
T-47D
MG-MIDb
a
b
GI50 represents the compound concentration (µM) required to achieve 50% inhibition of tumor cell growth. MG MID represents the
calculated mean GI50 for all panels. c Not determined.
pattern, except that the NCI-H522 nonsmall cell lung
cancer cell line is more sensitive to this compound (GI50
) 0.94 µM). The less potent Cdc25A inhibitors, lactone
9 and its double-bond-saturated derivative 16, also
showed comparable growth inhibition effects among the
eight different cancer types with MG-MID at 11.5 µM
and 5.51 µM, respectively. This may be due to hydrolysis
of the lactone into a hydroxyl carboxylic acid by cellular
esterases. Compounds 9 and 16 differed from each other
in that 9 potently inhibited the NCI-H522 nonsmall cell
lung cancer cell line (GI50 ) 0.89 µM) and the SF-268
central nerve system cancer cell line (GI50 ) 3.19 µM),
which were insensitive to compound 16. This may be
due to inhibition of some cellular enzymes, specifically
expressed in these cell lines, by compound 9 with a
mechanism related to the double bond conjugated to the
cyano group. Compounds 16 and 9 also showed distinct
pounds 9 and 10 reduced the HT-29 colon cancer cell
colony number to 50% of the control. In addition,
compound 9 reduced the colony number of A549 human
lung adenocarcinoma cells by 50% at 7.2 µM.
Con clu sion
Unusual chemical transformations of readily available
natural products such as cholesteryl acetate were used
to synthesize small molecule inhibitors for protein
phosphatases, especially for Cdc25A dual-specificity
protein phosphatase, an oncogenic protein that is over-
expressed in several human tumor cell lines. Silica gel
supported pyrolysis of an azido-homo-oxa steroid led to
rearrangement, presumably by a mechanism similar to
that of solution phase Schmidt fragmentation, to pro-
duce a group of novel and potent inhibitors of human
Cdc25A phosphatase.
Cyano-containing acid 17, one of the best inhibitors
in this group, inhibited the activity of Cdc25A protein
phosphatase, reversibly and noncompetitively, with an
IC50 value of 2.2 µM. These results suggest that com-
pounds such as 17 may interact with Cdc25A, presum-
ably with an arginine group at a site distinct from the
substrate binding site. Compound 17 inhibited human
Cdc25C phosphatase with an IC50 of 6.5 µM. The
inhibition of receptor protein tyrosine phosphatase
CD45 by compound 17 (IC50 ) 8.3 µM) was most
consistent with a mixed inhibition model.
selectivity in the UO-31 renal cancer cell lines (GI50
0.39 and 0.47 µM, respectively).
)
Compound 13 showed very good growth inhibitory
activity (GI50 ) 3.55 µM), although it is not active in
the Cdc25A enzyme inhibition assay. It was also ob-
served that the selectivity over different tumor cell lines
of 13 was different from other compounds tested. It was
more selective for the LOX IMVI melanoma cell line
(GI50 ) 0.84 µM), the OVCAR-8 ovarian cancer cell line
(GI50 ) 1.97 µM), the 786-0 (GI50 ) 1.44 µM) and UO-
31 (GI50 ) 2.95 µM) renal cancer cell lines, and the HT29
colon cancer cell line (GI50 ) 2.63 µM). The antiprolif-
erative effects of compound 13 might arise from inhibi-
tion of other cellular enzymes.
Other simpler steroidal derived acids were synthe-
sized, using conventional methods, to evaluate structure-
activity relationships. It was observed that a phosphate
surrogate such as a carboxyl or a xanthate group is
required for inhibitory activity. A hydrophobic alkyl
chain, such as the cholesteryl side chain, contributes
Compounds 10 and 9 were shown to block anchorage
independent growth of tumor cells in soft agar clono-
genic assays. At 10.9 and 12.0 µM, respectively, com-

Steroidal Derived Inhibitors for Human CDC25A Phosphatase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 843
(CH3), 11.6 (CH3); CIMS m/z (relative intensity) 477 (M+ + 1,
4), 417 (M+ - AcOH + 1, 88), 399 (100), 357 (40), 331 (45).
greatly to the potency.22 Acid 26 and xanthate 27
showed better selectivity over Cdc25A with an IC50 of
5.1 µM and 1.1 µM, respectively, and their IC50s toward
receptor tyrosine phosphatase, CD45, were greater than
100 µM.
3-â-Acetoxy-B-h om o-6-oxa-4-ch olesten -7-on e (2).26 SOCl2
(0.8 mL, 10 mmol) in 2 mL of CH2Cl2 was added dropwise to
a stirred and cooled (0 °C) solution of 3-â-acetoxy-5,6-seco-5-
oxo-cholestan-6-oic acid (1) (2.3 g, 5 mmol) in 12 mL of CH2-
Cl2. After the mixture was stirred at 0 °C for 30 min, 0.1 mL
of pyridine was added, and the resulting mixture was stirred
at room temperature for 2 h. Then the solvent was evaporated
in vacuo to yield a residue which was chromatographed on
silica gel to yield 3-â-acetoxy-B-homo-6-oxa-4-cholesten-7-one
(2) (1.92 g, 78%) as a colorless oil: UV λmax (CHCl3) 241.6 nm
(ꢀ ) 358); FTIR (neat film) 2939, 2873, 1766, 1740, 1667, 1648,
The steroidal derived Cdc25A inhibitors described
herein provide unique leads for the development of
PTPase inhibitors, which previously employ the common
approach of incorporation of nonhydrolyzable phosphate
groups in synthetic peptide15 or peptidomimetic sub-
strates.31 It is expected that compounds with structures
significantly different from natural steroids might be
better candidates for drug development since they are
less likely to interfere with or be eliminated by hormonal
pathways. It was observed that simple steroidal acids,
such as compounds 14 and 27,22 although exhibiting
very potent Cdc25A inhibition, did not inhibit in vitro
growth of tumor cells as well as other Cdc25A inhibitors
with more complex structures. This may result from
poor penetration through the plasma membrane or
increased ease of elimination by the metabolic path-
ways.
1
1375, 1242, 1123 cm-1; H NMR (CDCl3, 300 MHz) δ 5.50 (d,
J ) 4.2 Hz, 1 H), 5.33 (t, J ) 3.3 Hz, 1 H), 2.37 (m, 2 H), 2.04
(s, 3 H), 1.03 (s, 3 H), 0.88 (d, J ) 6.6 Hz, 3 H), 0.85 (d, J )
6.6 Hz, 6 H) 0.68 (s, 3 H); EIMS m/z (relative intensity) 458
(22), 498 (35), 370 (100), 110(62); EI HRMS calcd for C29H46O4
m/z 458.3396, found 458.3408.
3-R-Azid o-B-h om o-6-oxa -4, 5-ch olesten -7-on e (3). To a
stirred solution of 2.40 g (5 mmol) of crude 3-â-acetoxy-B-homo-
6-oxa-4-cholesten-7-one (2) in 35 mL of acetone was added
dropwise a solution of NaN3 (650 mg, 10 mmol) in 5 mL of
water. The reaction mixture was stirred at room temperature
for 2 h, poured into 50 mL water, and extracted with ether.
The ethereal extracts were dried over MgSO4 and concentrated
in vacuo to yield a residue which was subjected to silica gel
column chromatography (230-400 mesh, hexane/EtOAC ) 10:
Exp er im en ta l Section
1) to afford compound 3, 1.17 g (55.3%) as a yellowish oil: [R]20
D
Starting materials were purchased from Aldrich. Thin-layer
chromatography analysis (TLC) was performed on aluminum
sheets precoated with 0.2 mm of silica gel containing 60F254
indicator. Spots were detected with shortwave UV light or
Ceric sulfate spray. Flash chromatography was run using 230-
400 mesh silica gel. Reverse phase high performance liquid
chromatography (HPLC) was run on a Phenomenex LUNA 5
µm C18 semipreparative column. The homogeneity of all the
compounds was routinely checked by TLC on silica gel plates
and also by HPLC. Melting points were measured on a Kofler
hot stage apparatus attached to a digital thermometer and
were uncorrected. Optical rotations were taken on a J ASCO
DIP-360 digital polarimeter. Fourier transformed infrared
spectra were obtained on a Nicolet 520 FTIR spectrometer.
1H (300 or 400 MHz) and 13C (75 or 100 MHz) NMR and DEPT
spectra were recorded on either a Varian Gemini-300 or on a
Varian XL-400 spectrometer. Chemical shifts are reported
relative to CDCl3 (δ 7.24). High resolution mass spectra (EI
or FAB) were recorded on a VG Analytical 70-SE mass
spectrometer equipped with a 11-250J data system. Elemental
analyses were performed by Atlantic Microlab, Norcross, GA.
+98.3° (c 0.6, CHCl3); UV λmax (CHCl3) 245 nm (ꢀ ) 899); FTIR
(neat film): 2949, 2873, 2094, 1763, 1670, 1247 cm-1; 1H NMR
(CDCl3, 300 MHz) δ 5.43 (d, J ) 2.5 Hz, 1 H), 4.07 (m, 1 H),
2.44 (d, J ) 15.0, 11.0 Hz, 1 H), 2.38 (dd, J ) 15.0, 2.0 Hz, 1
H), 0.99 (s, 3 H), 0.89 (d, J ) 6.5 Hz, 3 H,), 0.84 (d, J ) 6.6
Hz, 6 H), 0.65 (s, 3 H); 13C NMR and DEPT (CDCl3, 100 Hz)
δ 172.1 (C), 160.0 (C), 113.5 (CH), 57.4 (CH), 56.3 (CH), 55.4
(CH), 51.7 (CH), 42.9 (C), 39.5 (CH2), 39.4 (CH2), 39.0 (C), 37.0
(CH2), 35.9 (CH2), 35.6 (CH), 34.9 (CH), 33.9 (CH2), 28.0 (CH),
27.6 (CH2), 25.0 (CH2), 23.8 (CH2), 23.7 (CH2), 22.8 (CH3), 22.6
(CH3), 22.5 (CH2), 19.2 (CH3), 18.5 (CH3), 11.8 (CH3); CIMS
m/z (relative intensity) 442 (M+ + 1, 17), 414 (M+ - N2 + 1,
100), 399 (M+ - N3, 26). Anal. (C27H44N3O2‚0.25 hexane) C,
H, N.
Gen er a l Meth od of Syn th eses a n d P u r ifica tion of
Com p ou n d s 4-13. A solution of 3 (4.4 g, 10 mmol) in 15 mL
of EtOAc was poured onto 16 g of silica gel (0.040-0.063 mm,
EM Science Co.). The solvent was allowed to evaporate in the
hood. Then, the 3-R-azido-B-homo-6-oxa-4,5-cholesten-7-one (3)
coated silica gel was pyrolyzed at 180 °C for 1 h and then
extracted with EtOAc (3 × 200 mL) and methanol (2 × 200
mL). The resulting solutions were combined and concentrated,
in vacuo, to yield a residue that was subjected to column
chromatography (silica gel 230-400 mesh, hexane/EtOAc )
20:1∼3:1) to afford (10S)-1,4,6-trioxo-2-cholesten (4) (12 mg,
0.29%), 6-oxa-2,4-cholest-dien-7-one (5) (60 mg, 1.5%), and
briefly separated eluents of compounds 6-13. Further flash
chromatography of these eluents on silica gel (hexane/EtOAc
) 2:1) afford 5,6-seco-5-oxo-2-cholesten-6-oic acid (6) (78 mg,
1.9%), 5,6-seco-5-oxo-3-cholesten-6-oic acid (7) (1.70 g, 41.9%),
cyano-containing lactones 8 (53 mg, 1.3%) and 9 (392 mg,
9.5%), A-Homo-6-aza-3-cholesten-5,6a-dione (13) (24 mg, 0.58%),
and mixtures of cyano-containing acid isomers 10-12 (841 mg,
20.4%). Even though the TLC Rf values of compounds 6-13
were about the same using hexane/EtOAc ) 2:1 on silica gel
plates, these compounds were eluted from a silica gel column
in the order of 6, 10-12, 7, 8, 9, and 13. Pure isomers were
obtained by reverse phase HPLC (Phenomenex LUNA 5 µm
C18) using 100% acetonitrile. From peak areas in the HPLC
chromatogram and known total weight, the following yields
were calculated: 10 (400 mg, 9.7%), 11 (215 mg, 5.2%), 12 (226
mg, 5.5%).
The syntheses of compounds 1,20 7,20 14,20 27,27 34,28
35,29and 3730 were performed as previously reported except
as indicated.
3â-Acetoxy-5,6-seco-5-oxo-ch olestan -6-oic acid (1). Ozone
was passed into a stirred and cooled (chloroform/dry ice bath)
solution of cholesteryl acetate (12 g, 28 mmol) in 300 mL of
petroleum ether for 30 min until the solution turned light blue.
Piperidine (5 mL, 50 mmol) was then added dropwise over 3
min. The volume of solution was reduced to 100 mL after being
stirred and warmed in room temperature for 3 h. The white
precipitates were separated and washed with 2 M HCl (3 ×
25 mL) and with water (1 × 30 mL) to obtain the free acid.
Flash chromatography of the crude acid over silica gel, using
hexanes-EtOAc (3:1), afforded compound 1 (5.5 g, 41%) as
colorless crystals: mp 115∼117 °C (lit.20 115-116 °C); FTIR
(neat film) 3400-2500 (br), 2960, 2868, 1736, 1716, 1249 cm-1
;
1H NMR (CDCl3, 300 MHz) δ 9.89 (broad), 5.29 (brs, 1 H), 3.11
(dd, J ) 14.4, 4.3 Hz, 1 H), 2.33 (d, J ) 14.5 Hz, 1 H), 2.08 (s,
3 H), 0.99 (s, 3 H), 0.88 (d, J ) 6.4 Hz, 3 H), 0.83 (d, J ) 6.6
Hz, 6 H), 0.65 (s, 3 H); 13C NMR and DEPT (CDCl3, 100 Hz)
δ 216.4 (C), 178.6 (C), 170.3 (C), 73.5 (CH), 55.9 (CH), 54.4
(CH), 52.3 (C), 43.1 (CH2), 42.5 (C), 41.5 (CH), 39.7 (CH2), 39.4
(CH2), 35.9 (CH2), 35.7 (CH), 35.5 (CH), 34.4 (CH2), 34.1 (CH2),
27.9 (CH), 27.9 (CH2), 25.1 (CH2), 24.3 (CH2), 23.7 (CH2), 23.0
(CH2), 22.8 (CH3), 22.5 (CH3), 21.5 (CH3), 18.5 (CH3), 17.6
(10S)-2-ch olesten -1,4,6-tr ion e (4) was obtained as light
yellow crystals: mp 162-164 °C; FTIR (neat film) 2924, 2862,
1712, 1687, 1457, 1377, 1265, 1072, 848 cm-1; 1H NMR (CDCl3,

844 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Peng et al.
300 Hz) δ 6.69 (dd, J ) 10.2, 1.5 Hz, 1 H), 6.50 (d, J ) 10.2
Hz, 1 H), 3.39 (s, 1 H), 2.42 (m, 2 H), 2.11 (dt, J ) 13.0, 3.0
Hz, 1 H), 1.97-1.81 (m, 3 H), 1.70 (m, 1 H), 1.43 (s, 3 H), 0.93
Hz, 1 H), 2.55 (m, 2 H), 2.07 (dt, J ) 12.0, 2.7 Hz, 1 H), 2.00
(dd, J ) 17.7, 11.5 Hz, 1 H), 1.99∼1.77 (m, 5 H), 1.34 (s, 3 H),
0.90 (d, J ) 6.0 Hz, 3 H), 0.86 (d, J ) 6.6 Hz, 6 H), 0.70 (s, 3
H); 13C NMR and DEPT (CDCl3, 100 Hz) δ 170.8 (C), 154.5
(CH), 115.9 (C), 99.8 (CH), 86.6 (C), 55.7 (CH), 55.6 (CH), 44.5
(CH), 42.5 (C), 39.4 (CH2), 38.9 (CH2), 38.2 (CH2), 36.0 (CH2),
35.7 (CH2), 35.6 (CH), 30.8 (CH), 28.0 (CH2), 28.0 (CH), 25.8
(CH2), 23.7 (CH2), 23.5 (CH2), 23.0 (CH3), 22.8 (CH3), 22.8
(CH2), 22.5 (CH3), 18.6 (CH3), 11.9 (CH3); EIMS m/z (relative
intensity) 413 (M+, 5), 398 (2), 385 (2), 353 (5), 333 (46), 290
(21), 248 (28), 135 (100); CIMS m/z (relative intensity) 414 (M+
+ 1, 100), 396 (37), 333 (18), 135 (22); EI HRMS calcd for
(d, J ) 6.5 Hz, 3H), 0.87 (d, J ) 6.6 Hz, 6 H), 0.82 (s, 3 H); 13
C
NMR (CDCl3, 100 MHz) δ 206.0 (C), 201.0 (C), 196.4 (C), 140.1
(CH), 139.1 (CH), 71.6 (CH), 57.0 (CH), 55.8 (CH), 55.3 (C),
51.0 (CH), 47.2 (CH2), 42.9 (C), 39.7 (CH2), 39.4 (CH2), 37.7
(CH), 36.1 (CH2), 35.7 (CH), 28.0 (CH2), 28.0 (CH), 26.1 (CH3),
24.0 (CH2), 23.8 (CH2), 22.8 (CH3), 22.5 (CH3), 21.5 (CH2), 18.7
(CH3), 11.7 (CH3); EI MS m/z (relative intensity) 412 (M+, 78),
397 (M+ - CH3, 20), 288 (25), 124 (100). Anal. (C27H40O3) C,
H.
C
27H43NO2 m/z 413.3294, found 413.3253. Anal. (C27H43NO2)
C, H, N.
Com p ou n d 10 was obtained as a colorless oil: [R]20D +31.0°
6-Oxa -2,3-4,5-ch olest-d ien -7-on e (5) was obtained as
colorless oil: FTIR (neat film) 2947, 2847, 1768, 1657 cm-1
;
1H NMR (CDCl3, 300 MHz) δ 5.91 (ddd, J ) 5.7, 3.6, 3.5 Hz,
1 H), 5.69 (d, J ) 5.5 Hz, 1 H), 5.67 (ddd, J ) 5.7, 5.4, 2.5 Hz,
1 H), 2.50 (d, J ) 12.3 Hz, 1H), 2.37 (d, J ) 12.4 Hz, 1 H),
2.47 (d, J ) 18.3 Hz, 1 H), 2.27 (d, J ) 17.5, 2.8 Hz, 1 H), 1.02
(s, 3 H), 0.89 (d, J ) 6.5 Hz, 3 H), 0.86 (d, J ) 6.5 Hz, 6 H),
0.68 (s, 3 H); 13C NMR and DEPT (CDCl3, 100 Hz) δ173.4 (C),
157.6 (C), 123.5 (CH), 123.2 (CH), 111.6 (CH), 56.4 (CH), 55.4
(CH), 45.9 (CH), 43.1 (C), 39.4 (CH2), 39.4 (CH2), 37.8 (C), 37.2
(CH2), 36.3 (CH2), 35.1 (CH), 35.1 (CH), 28.0 (CH), 27.7 (CH2),
25.0 (CH2), 23.8 (CH2), 22.8 (CH2), 22.5 (CH3), 18.6 (CH3), 18.0
(CH2), 11.9 (CH3); EIMS m/z (relative intensity) 398 (M+, 20),
370 (10), 329 (22), 247 (23), 149 (40), 109 (100); EI HRMS calcd
for C27H42O2 m/z 398.3185, found 398.3187.
(c 0.35, CHCl3); UV λmax (CHCl3) 255.6 nm (ꢀ ) 814); HPLC
(acetonitrile, 4 mL/min, 205 nm) 11.04 min; FTIR (neat film)
3380-2500 (br), 3728, 2953, 2874, 2229, 1703, 1459, 1288
cm-1; 1H NMR (CDCl3, 300 MHz) δ 6.50 (dt, J ) 11.0, 7.2 Hz,
1 H), 5.33 (d, J ) 11.0 Hz, 1 H), 4.93 (s, 1 H), 4.84(s, 1 H),
2.59 (m, 2 H), 2.35 (dd, J ) 15.0, 3.3 Hz, 1 H), 2.10 (dd, J )
15.0, 6.6 Hz, 1 H), 2.17 (t, J ) 7.2 Hz, 2 H), 1.97 (m, 3 H), 0.90
(d, J ) 6.6 Hz, 3H), 0.86 (d, J ) 6.6 Hz, 6 H), 0.68 (s, 3 H); 13
C
NMR and DEPT (CDCl3, 100 Hz) δ 179.1 (C), 154.7 (CH), 150.2
(C), 116.0 (C), 111.2 (CH2), 99.8 (CH), 56.0 (CH), 55.3 (CH),
51.4 (CH), 43.0 (C), 39.6 (CH2), 39.5 (CH2), 37.2 (CH2), 36.1
(CH2), 35.8 (CH), 35.7 (CH), 32.5 (CH2), 29.9 (CH2), 29.0 (CH2),
28.0 (CH), 28.0 (CH2), 24.4 (CH2), 23.8 (CH2), 22.8 (CH3), 22.5
(CH3), 18.7 (CH3), 11.9 (CH3); EIMS m/z (relative intensity)
413 (M+, 34), 395 (100), 380 (25), 354 (67), 333 (23), 282 (44),
240 (40), 43 (98); EI HRMS calcd for C27H43NO2 m/z 413.3294,
found 413.3279. Anal. (C27H43NO2) C, H, N.
5,6-Seco-5-oxo-2,3-ch olesten -6-oic a cid (6) was obtained
was obtained as colorless oil: FTIR (neat film) 3400-2500 (br),
2951, 2867, 1738, 1706, 1466, 1380, 1240, 1045, 926, 676 cm-1
;
1
H NMR (CDCl3, 300 Hz) δ 5.68 (m, 2 H), 3.21 (d, J ) 21.1
Hz, 1 H), 2.85 (d, J ) 22.5 Hz, 1 H), 2.46 (dd, J ) 17.4, 5.5 Hz,
1 H), 2.27 (dd, J ) 16.6, 2.7 Hz, 1 H), 2.09 (dd, J ) 16.6, 7.5
Hz, 1 H), 2.00 (m, 3 H), 1.99 (m, 1 H), 1.98 (m, 1 H), 1.88 (m,
3 H), 1.83-1.20 (m, 10 H), 1.05 (s, 3 H), 0.86 (d, J ) 6.6 Hz,
3 H), 0.82 (d, J ) 6.6 Hz, 6 H), 0.65 (s, 3 H); 13 C NMR (CDCl3,
100 MHz) δ 217.1 (C), 178.9 (C), 126.0 (CH), 124.4 (CH), 55.8
(CH), 55.6 (CH), 50.9 (C), 42.6 (C), 40.9 (CH), 40.4 (CH2), 39.8
(CH2), 39.4 (CH2), 39.1 (CH2), 36.0 (CH2), 35.7 (CH), 35.6 (CH),
35.4 (CH2), 28.0 (CH2), 28.0 (CH), 24.2 (CH2), 24.0 (CH2), 23.8
(CH2), 22.8 (CH3), 22.5 (CH3), 18.6 (CH3), 16.8 (CH3), 11.7
(CH3); FAB MS m/z (relative intensity) 417 (M+ + 1, 48), 399
(M+ + 1 - H2O, 100), 381 (30), 247 (50); FAB HRMS calcd for
Com p ou n d 11 was obtained as a colorless oil: [R]20D +12.7°
(c 0.15, CHCl3); UV λmax (CHCl3) 255.0 nm (ꢀ ) 1067); HPLC
(acetonitrile, 4 mL/min, 205 nm) 10.56 min; FTIR (neat film)
3400-2500 (br), 2955, 2874, 2224, 1711, 1649, 1470, 1386,
1
1292 cm-1; H NMR (CDCl3, 300 MHz) δ 6.37 (dt, J ) 10.9,
7.7 Hz, 1 H), 5.27 (d, J ) 10.9, 1 H,), 5.18 (t, J ) 7.7 Hz, 1 H)
3.10 (dt, J ) 7.7, 5.5 Hz, 2 H), 2,22 (dd, J ) 15.4, 3.3 Hz, 1H),
2.04 (dd, J ) 15.4, 7.6 Hz, 1H), 1.98 (m, 3H), 1.67 (s, 3 H),
0.90 (d, J ) 6.6 Hz, 3 H), 0.86 (d, J ) 6.6 Hz, 6 H), 0.68 (s, 3
H); EIMS m/z (relative intensity) 413 (M+, 15), 395 (28), 354
(25), 333 (12), 240 (17), 43 (100); EI HRMS calcd for C27H43
-
C
27H43NO2 m/z 417.3369 (M+ + 1), found 417.3337.
NO2 m/z 413.3294, found 413.3312. Anal. (C27H43NO2) C, H,
N.
5,6-Seco-5-oxo-3,4-ch olesten -6-oic a cid (7) was obtained
as colorless oil: FTIR (neat film) 3400-2500 (br), 2916, 1747,
1712, 1677, 1466, 1389, 1228 cm-1; 1H NMR (CDCl3, 300 MHz)
δ 6.73 (m, 1 H), 5.88 (dd, J ) 10.5, 1.5 Hz, 1 H), 2.40 (dd, J )
15.5, 5.0 Hz, 1 H), 1.10 (s, 3 H), 0.90 (d, J ) 6.5 Hz, 3 H), 0.86
(d, J ) 6.6 Hz, 6 H), 0.69 (s, 3 H); EIMS m/z (relative intensity)
416 (M+, 17), 398 (23), 354 (32), 333 (55), 247 (63), 110 (100);
EI HRMS calcd for C27H44O3 m/z 416.3290, found 416.3272.
Com p ou n d 12 was obtained as a colorless oil: [R]20D +26.6°
(c 0.30, CHCl3); UV λmax (CHCl3) 255.4 nm (ꢀ ) 1212.4); HPLC
(acetonitrile, 4 mL/min, 205 nm) 12.11 min; FTIR (neat film)
3400-2500 (br), 2946, 2871, 2221, 1704, 1704, 1647, 1470,
1
1388, 1294 cm-1; H NMR (CDCl3, 300 MHz) δ 6.38 (dt, J )
11.1, 7.5 Hz, 1 H), 5.27 (d, J ) 10.4 Hz, 1 H), 5.17 (t, J ) 7.2
Hz, 1 H), 3.08 (t, J ) 7.2 Hz, 2 H), 2.22 (dd, J ) 15.4, 4.4 Hz,
1H), 2.07 (dd, J ) 15.4, 6.6 Hz, 1H), 1.94 (m, 3H), 1.60 (s, 3
H), 0.90 (d, J ) 6.6 Hz, 3 H), 0.86 (d, J ) 6.6 Hz, 6 H), 0.68 (s,
3 H); EIMS m/z (relative intensity) 413 (M+, 20), 395 (57), 354
(50), 333 (12), 282 (32), 240 (32), 43 (100); EI HRMS calcd for
Com p ou n d 8 was obtained as colorless prisms: mp 132-
134 °C; [R]20 +39.3° (c 0.52, CHCl3); UV λmax (CHCl3) 242.6
D
nm (ꢀ ) 152); FTIR (neat film) 2954, 2870, 2224, 1733, 1473,
1
1389, 1256 cm-1; H NMR (CDCl3, 300 MHz) δ 6.54 (dt, J )
C
27H43NO2 m/z 413.3294, found 413.3271. Anal. (C27H43NO2)
C, H, N.
A-Hom o-6-a za -3-ch olesten -5,6a -d ion e (13) was obtained
11.0, 7.8 Hz, 1 H), 5.35 (d, J ) 11.1 Hz, 1 H,), 2.63 (m, 2 H),
2.61 (dd, J ) 18.0, 5.1 Hz, 1 H), 2.08 (dt, J ) 12.1, 3.0 Hz, 1
H), 2.02 (dd, J ) 18.0, 11.5 Hz, 1 H), 2.01∼1.68 (m, 5 H), 1.43
(s, 3 H), 0.91 (d, J ) 6.6 Hz, 3 H), 0.86 (d, J ) 6.3 Hz, 6 H),
0.70 (s, 3 H); 13C NMR and DEPT (CDCl3, 100 Hz) δ 170.6
(C), 154.4 (CH), 115.8 (C), 100.2 (CH), 86.3 (C), 55.9 (CH), 55.8
(CH), 49.3 (CH), 42.6 (C), 39.4 (CH2), 39.1 (CH2), 36.0 (CH2),
35.7 (CH2), 35.6 (CH), 34.8 (CH2), 30.9 (CH), 28.1 (CH2), 28.0
(CH), 26.1 (CH2), 26.0 (CH3), 23.8 (CH2), 23.4 (CH2), 22.9 (CH2),
22.8 (CH3), 22.5 (CH3), 18.7 (CH3), 12.0 (CH3); EIMS m/z
(relative intensity) 413 (M+, 45), 398 (24), 385 (27), 333 (31),
as white crystals: mp 187-188 °C; FTIR (neat film) 2949,
2864, 1721, 1645, 1391, 1221, 1179 cm-1; 1H NMR (CDCl3, 300
Hz) δ 6.74 (ddd, J ) 10.4, 7.7, 2.7 Hz, 1 H), 6.21 (dd, J ) 10.4,
2.7 Hz, 1 H), 2.43 (dd, J ) 17.6, 4.4 Hz, 1 H), 2.41-2.22 (m, 2
H), 2.16 (dd, J ) 14.8, 6.6 Hz, 1 H), 2.07 (dt, J ) 13.2, 3.3 Hz,
1 H), 1.92 (dd, J ) 17.6, 12.5 Hz, 1 H), 1.30 (s, 3 H), 0.90 (d,
J ) 6.6 Hz, 3 H), 0.86 (d, J ) 6.6 Hz, 6 H), 0.71 (s, 3 H); 13C
NMR (CDCl3, 100 MHz) δ 175.7, 169.3, 149.6, 128.8, 62.0, 56.2,
55.9, 42.8, 42.3, 39.4, 39.1, 39.1, 37.5, 36.0, 35.6, 30.2, 28.1,
28.0, 25.2, 23.7, 23.7, 23.1, 22.8, 22.5, 22.3, 18.7, 11.9; EI MS
m/z (relative intensity) 413 (M+, 30), 385 (20), 332 (100), 124
(60). Anal. (C27H43NO2) C, H, N.
248 (33), 135 (100); EI HRMS calcd for
C27H43NO2 m/z
413.3294, found 413.3308. Anal. (C27H43NO2) C, H, N.
Com pou n d 9 was obtained as colorless prisms: mp 109∼111
°C; [R]20 - 42.1° (c 0.73, CHCl3); UV λmax (CHCl3) 240.8 nm
D
(ꢀ ) 89.7); FTIR (neat film) 3071, 2956, 2871, 2219, 1720, 1390,
Hyd r ogen a tion of Com p ou n d s 5, 7, a n d 9-13. General
procedure of the hydrogenation: To a solution of a compound
in ethyl acetate was added 10% Pd/C. The resulting mixture
1
1259 cm-1; H NMR (CDCl3, 300 MHz) δ 6.52 (dt, J ) 10.8,
7.8 Hz, 1 H), 5.32 (d, J ) 10.5 Hz, 1 H), 2.60 (dd, J ) 17.7, 4.8

Steroidal Derived Inhibitors for Human CDC25A Phosphatase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 845
was stirred and hydrogenated at atmospheric pressure until
1 equiv of hydrogen was absorbed, and then the reaction
solution was filtered through Celite. The filtrate was concen-
trated in vacuo to yield the product.
Hz, 2 H), 2.45 (dd, J ) 17.4, 5.1 Hz, 1 H), 2.04 (m, 2 H), 1.94
(dd, J ) 17.4, 11.1, 1 H), 1.29 (s, 3 H), 0.89 (d, J ) 6.6 Hz, 3
H), 0.84 (d, J ) 6.6 Hz, 6 H), 0.68 (s, 3 H); FABMS m/z (relative
intensity): 416 (M+
+ 1, 100), 400 (30), 386 (5), 372 (10), 332
(10); FAB HRMS calcd for C27H46NO2 m/z 416.3529 (M+ + 1),
found 416.3515.
B-H om o-5-oxa -4-ch olest en -7-on e (14) was obtained
through hydrogenation of 5 as a colorless oil: FTIR (neat film)
2933, 2874, 1756, 1457, 1130 cm-1; 1H NMR (CDCl3, 300 MHz)
δ 5.38 (t, J ) 3.2 Hz, 1 H), 2.40 (m, 2 H), 1.89 (m, 2 H), 0.97
(s, 3 H), 0.87 (d, J ) 6.6 Hz, 3 H), 0.84 (d, J ) 6.6 Hz, 6 H),
0.67 (s, 3 H); 13C NMR (CDCl3, 100 MHz) 173.8 (C), 155.6 (C),
114.3 (CH), 56.4 (CH), 55.6 (CH), 51.3 (CH), 43.0 (C), 39.7
(CH2), 39.4 (CH2), 38.5 (C), 37.2 (CH2) 35.9 (CH2), 35.7 (CH),
34.9 (CH2), 34.8 (CH), 28.0 (CH), 27.7 (CH2), 25.1 (CH2), 24.2
(CH2), 23.8 (CH2), 22.8 (CH3), 22.5 (CH3), 22.1 (CH2), 19.1
(CH3), 18.6 (CH3), 17.1 (CH2), 11.9 (CH3); EI MS m/z (relative
intensity) 400 (M+, 15), 372 (100), 330 (73), 111 (50); EI HRMS
calcd for C27H44O2 m/z 400.3341, found 400.3361.
5,6-Seco-5-oxo-ch olesta n -6-oic a cid (15)20 was obtained
through hydrogenation of compound 7 as colorless oil: 1H NMR
(CDCl3, 300 MHz) δ 3.18 (m, 1 H), 2.26-1.99 (m, 6 H), 0.98 (s,
3 H), 0.88 (d, J ) 0.66 Hz, 3 H), 0.84 (d, J ) 0.66 Hz, 6 H),
0.66 (s, 3 H).
Com p ou n d s 21 a n d 22. To a solution of compound 10 (25
mg, 0.061 mmol) in 2 mL of ethyl acetate was added 5 mg of
5% Pt/C. The resulting mixture was stirred and hydrogenated
for 72 h and then filtered through Celite. The filtrate was
concentrated in vacuo and separated by reverse phase HPLC
using 100% acetonitrile to afford 10 mg of compound 21 as a
colorless oil: HPLC (acetonitrile, 4 mL/min, 205 nm) tR ) 14.21
1
min; H NMR (CDCl3, 300 MHz) δ 2.32 (t, J ) 6.9 Hz, 2 H),
2.24 (d, J ) 4.8 Hz, 2 H), 1.94 (m, 1 H), 0.89 (d, J ) 6.6 Hz, 3
H), 0.88 (d, J ) 6.6 Hz, 3 H), 0.84 (d, J ) 6.6 Hz, 6 H), 0.67 (s,
3 H); 13C NMR (CDCl3, 100 MHz) δ 178.6, 120.1, 56.2, 55.3,
48.9, 43.0, 39.6, 39.5, 37.1, 36.1, 35.8, 35.5, 29.5, 28.0, 27.9,
27.4, 25.7, 24.8, 23.8, 22.8, 22.5, 21.0, 19.0, 18.7, 17.1, 11.9;
FABMS m/z (relative intensity): 418 (M+ + 1, 32), 400 (100),
382 (55), 356 (35); FAB HRMS calcd for C27H48NO2 m/z
418.3771 (M+ + 1), found 418.3683. Compound 22 (8 mg) as
colorless oil: HPLC (acetonitrile, 4 mL/min, 205 nm) tR ) 12.96
Com p ou n d 16 was obtained through hydrogenation of
1
compound 9 as colorless oil: [R]20 -9.2° (c 0.5, CHCl3); FTIR
min; H NMR (CDCl3, 300 MHz) δ 2.31 (m, 4 H), 1.94 (m, 1
D
(neat film) 2952, 2873, 2250, 1726, 1468, 1388, 1256, 977 cm-1
;
H), 0.88 (d, J ) 6.6 Hz, 3 H), 0.84 (d, J ) 6.6 Hz, 6 H), 0.78 (d,
J ) 7.2 Hz, 3 H), 0.66 (s, 3 H); 13C NMR (CDCl3, 100 MHz) δ
179.1, 119.8, 56.1, 54.8, 44.8, 43.0, 39.5, 39.5, 39.4, 36.1, 35.7,
35.5, 34.9, 31.9, 28.0, 27.9, 26.8, 25.5, 24.8, 23.8, 22.8, 22.5,
20.2, 18.7, 17.1, 13.6, 11.7; FABMS m/z (relative intensity):
418 (M+ + 1, 25), 400 (100), 382 (45), 356 (20); FAB HRMS
calcd for C27H48NO2 m/z 418.3771 (M+ + 1), found 418.3679.
1H NMR (CDCl3, 300 MHz) δ 2.60 (dd, J ) 17.7, 4.8 Hz, 1 H),
2.36 (t, J ) 6.6 Hz, 2 H), 2.06 (m, 1 H), 1.97 (dd, J ) 17.7,
11.4 Hz 1 H), 0.90 (d, J ) 6.6 Hz, 3 H), 0.86 (d, J ) 6.6 Hz, 6
H), 0.70 (s, 3 H); EIMS m/z (relative intensity): 415(M+, 12),
400(22), 355 (15), 333 (38), 248 (30), 135 (100); CI HRMS m/z
calcd for C27H46NO2 m/z 416.3529 (M+ + 1), found 416.3520.
Ba se-Ca ta lyzed Hyd r olysis of Com p ou n d s 9, 10, a n d
16.. Com p ou n d s 23 a n d 24. A solution of 25 mg (0.06 mmol)
of compound 9 in 10 mL of 0.6% sodium hydroxide in methanol
was heated under reflux for overnight. The solution was
carefully acidified with 2 M HCl, and then 6 mL of ethyl ether
was added to precipitate the NaCl formed. The solution was
filtered, dried over anhydrous sodium sulfate, and evaporated
at reduced pressure. The residue was subjected to silica gel
(200-240 mesh) column chromatography and eluted with
hexane:EtOAc (3:1) to afford compound 23 (3S, 9 mg, 34.7%)
as a colorless crystal: mp 144-145 °C; FTIR (neat film) 3400-
Com p ou n d 17, 18, and 19. To a solution of a mixture of
compounds 10, 11, and 12 (2:1:1, 100 mg, 0.24 mmol) in 15
mL of ethyl acetate was added 10 mg of 10% Pd/C. The
resulting mixture was stirred and hydrogenated at atmo-
spheric pressure for 30 min until 1 equiv of hydrogen was
absorbed, and then the reaction solution was filtered through
Celite. The filtrate was concentrated in vacuo and purified by
reverse phase HPLC to afford 17 as a colorless oil: [R]20
D
+38.9° (c 0.15, CHCl3); HPLC (acetonitrile, 4 mL/min, 205 nm)
tR )11.09 min; 1H NMR (CDCl3, 300 MHz) δ 4.85 (s, 1 H), 4.78
(s, 1 H), 2.35 (m, 4 H), 0.95 (d, J ) 6.6 Hz, 3 H), 0.85 (d, J )
6.6 Hz, 6 H), 0.75 (s, 3 H); 13C NMR (CDCl3, 100 MHz) δ 179.1,
151.3, 119.7, 110.5, 56.0, 55.4, 51.4, 43.1, 39.7, 39.5, 37.3, 36.1,
35.9, 35.7, 29.3, 28.0, 28.0, 27.9, 26.9, 25.2, 24.4, 23.8, 22.8,
22.5, 18.7, 17.1, 12.0; EIMS m/z (relative intensity): 415 (M+,
15), 397 (53), 369 (28), 355 (50), 305 (100), 242 (80), 135 (93);
EI HRMS calcd for C27H45NO2 m/z 415.3450, found 415.3451.
Compound 18 was obtained as colorless oil: HPLC (aceto-
2500 (br), 2956, 2937, 2869, 2289, 1700, 1464, 1414, 1383, 1289
1
cm-1
;
H NMR (CDCl3, 300 Hz) δ 4.20 (m, 1 H), 2.70-2.58
(m, 3 H), 2.44 (dd, J ) 14.3, 4.4 Hz, 1 H,), 2.16-2.03 (m, 1 H),
1.96-1.78 (m, 6 H), 1.25 (s, 3 H), 0.87 (d, J ) 6.6 Hz, 3 H),
0.85 (d, J ) 6.6 Hz, 6 H), 0.68 (s, 3 H); 13C NMR (CDCl3, 100
MHz) δ 174.1, 116.8, 89.7, 74.4, 55.9, 53.2, 50.6, 42.8, 39.6,
39.5, 39.4, 36.9, 35.9, 35.7, 34.9, 30.2, 28.0, 27.8, 25.6, 24.6,
24.2, 23.7, 22.8, 22.5, 21.2, 18.6, 11.9; FAB MS m/z (relative
intensity) 432 (40, M+ + 1), 414 (M+ + 1 - H2O, 100), 396
(M+ + 1 - 2H2O, 20); FAB HRMS calcd for C27H46NO3 m/z
432.3478 (M+ + 1), found 432.3429. Compound 24 was
obtained as a mixture of two epimers (13 mg, 37%, 3R:3S )
1:1) as colorless oil: FTIR (neat film) 2951, 2869, 2278, 1725,
1466, 1380, 1281, 1111 cm-1; 1H NMR (CDCl3, 300 Hz) δ 3.43
(m, 2 H), 3.38 (s, 3 H), 3.37 (s, 3 H), 2.58 (dd, J ) 18.6, 5.0 Hz,
2 H), 2.53 (m, 4 H), 1.95 (dd, J ) 17.4, 12.0 Hz, 2 H), 1.32 (s,
3 H), 1.30 (s, 3 H), 0.88 (d, J ) 6.6 Hz, 6 H), 0.83 (dd, J ) 6.6
Hz, 12 H), 0.68 (s, 6 H); CI MS m/z (relative intensity) 446
(M+ + 1, 100), 414 (M+ + 1 - MeOH, 67), 333 (15), 135 (50),
1
nitrile, 4 mL/min, 205 nm) tR )10.65 min; H NMR (CDCl3,
300 MHz) δ 5.11 (t, J ) 6.9 Hz, 1 H), 2.30 (t, J ) 7.2 Hz, 2 H),
2.21∼1.93 (m, 6 H), 1.65 (s, 3 H), 0.88 (d, J ) 6.6 Hz, 3H),
0.84 (d, J ) 6.6 Hz, 6 H), 0.72 (s, 3 H); 13C NMR (CDCl3, 100
MHz) δ 178.2, 139.8, 125.1, 119.7, 56.0, 55.2, 44.4, 43.1, 39.5,
39.5, 37.0, 36.1, 35.7, 34.9, 28.0, 27.9, 26.8, 26.2, 25.6, 24.4,
23.8, 22.8, 22.5, 19.2, 18.7, 16.6, 11.9; FAB MS m/z (relative
intensity) 416 (M+ + 1, 100), 398 (M+ + 1 - H2O, 60), 369
(28), 356 (45), 305 (18), 242 (25); FAB HRMS calcd for C27H46
-
NO2 m/z 416.3529 (M+ + 1), found 416.3546.
Compound 19 was obtained as colorless oil: HPLC (aceto-
1
nitrile, 4 mL/min, 205 nm) tR )12.01 min; H NMR (CDCl3,
114 (53); CI HRMS calcd for C28H48NO3 m/z 446.3634 (M+
1), found 446.3641.
+
300 MHz) δ5.13 (t, J ) 6.9 Hz, 1 H), 2.30 (t, J ) 7.2 Hz, 2 H),
2.26-2.02 (m, 4 H), 1.94 (m, 2 H), 1.55 (s, 3 H), 0.88 (d, J )
6.6 Hz, 3 H), 0.84 (d, J ) 6.6 Hz, 6 H), 0.72 (s, 3 H); 13C NMR
(CDCl3, 100 MHz) δ 178.5, 140.0, 124.2, 119.9, 56.1, 55.0, 54.0,
43.1, 39.5, 39.5, 37.3, 36.0, 35.7, 35.2, 28.0, 28.8, 27.8, 26.5,
25.3, 24.6, 23.8, 22.8, 22.5, 18.7, 16.5, 12.9, 11.9; FAB MS m/z
(relative intensity) 416 (M+ + 1, 90), 398 (M+ + 1 - H2O, 100),
56 (60), 305 (40), 242 (45); FAB HRMS calcd for C27H46NO2
m/z 416.3529 (M+ + 1), found 416.3537.
Com p ou n d 25. A solution of 25 mg (0.06 mmol) of com-
pound 10 in 10 mL of 6% sodium hydroxide in aqueous ethanol
was heated under reflux for overnight. The solution was
carefully acidified with 2 M HCl until there are white
precipitates formed. The solution was extracted with ethyl
ether, dried over anhydrous sodium sulfate, and evaporated
under pressure. The residue was purified using preparative
TLC to afford compound 25 as a mixture of two epimers (17
mg) (3R:3S ) 1:1) (61.8%) as colorless oil: FTIR (neat film)
Com p ou n d 20 was obtained through hydrogenation of 13
in methanol as a white solid: FTIR (neat film) 2947, 2868,
1742, 1673, 1459, 1400, 1176, 1163, 1150, 735 cm-1; 1H NMR
(CDCl3, 300 MHz) δ 2.71 (m, 2 H), 2.53 (ddd, J ) 8.1, 5.2 4.1
1
3400-2500 (br), 2926, 2860, 2250, 1700, 1375, 1103, 891; H
NMR (CDCl3, 300 Hz) δ 4.87 (s, 2 H), 4.79 (s, 2 H), 3.65-3.46

846 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Peng et al.
(m, 6 H), 2.52 (d, J ) 6.1 Hz, 3 H?), 2.33 (dd, J ) 15.9 Hz, 2
H), 2.12-1.92 (m, 8 H), 1.20 (t, J ) 6.6 Hz, 6 H), 0.88 (d, J )
6.6 Hz, 6 H), 0.83 (d, J ) 6.6 Hz, 12 H), 0.73 (s, 6 H); EI MS
m/z (relative intensity) 459 (M+, 10), 441 (M+ - H2O), 413 (M+
- CH3CH2OH), 305 (100); EI HRMS calcd for C29H49NO3 m/z
459.3712, found 459.3700.
4-aminobutyric acid amino acid. The pH was adjusted to 6 by
adding glacial acetic acid and then 20 mL of THF, and 1.1
equiv of NaBH3CN was added. After the mixture was stirred
under reflux overnight, 2 M NaOH was added in to a pH of 7.
The residue obtained after evaporation was extracted with
ethyl acetate, and the organic layer was washed with brine
and then dried over anhydrous sodium sulfate. Evaporation
gave a residue which was washed with petroleum ether to give
compound 33 in 85% yield as an amorphous solid: 1H NMR
(CDCl3) δ 3.32 (m, 1 H), 3.05 (m, 2 H), 0.86 (d, J ) 6.0 Hz, 3
H), 0.83 (d, J ) 7.2 Hz, 6 H), 0.80 (s, 3 H), 0.62 (s, 3 H); FABMS
m/z (relative intensity) 474 (M+ + 1, 100), 430 (M+ + 1 - CO2,
10), 386 [M+ - (CH2)3CO2H, 10]; FAB HRMS m/z calcd for
C31H56NO2 474.4311 (M+ + 1), found 474.4315.
Com p ou n d 34. To a solution of 100 mg (0.27 mmol) of 3â-
cholestanol and cat. n-Bu4NI in THF was added 1.1 equiv of
NaH at room temperature under N2. After the mixture was
stirred for 1/2 h, 1.0 equiv of bromo acetic acid was added
dropwise to the solution. After further stirring for 2 h, 2 M
HCl was added to give a pH of 6. The residue obtained after
evaporation was extracted with ethyl acetate, and the organic
layer was washed with brine and then dried over anhydrous
sodium sulfate. After evaporation, the residue was washed
with petroleum ether to gave compound 34 in 60% yield as
colorless solid: mp 169-171 °C (lit.28 168 °C); 1H NMR (CDCl3)
δ 4.11 (s, 1 H), 3.35 (m, 1 H), 0.85 (d, J ) 6.0 Hz, 3 H), 0.83 (d,
J ) 7.2 Hz, 6 H), 0.77 (s, 3 H), 0.62 (s, 3 H); EIMS m/z (relative
intensity) 446 (M+ + 1, 95), 431 (10), 389 (10), 388 (100); FAB
HRMS m/z calcd for C29H51O3 446.3760 (M+ + 1), found
446.3779. Anal. (C29H50O3‚1.2H2O), C, H.
Ar yl Th ioca r ba m a te a n d Ar yl Ca r ba m a te Der iva tives
of Ch olesta n ol (35-37). General procedure: To a solution
of 100 mg (0.27 mmol) of 3â-cholestanol and cat. n-Bu4NI in
THF was added 1.1 equiv of NaH at room temperature under
N2. After the mixture was stirred for 1/2 h, 1.0 equiv of
isothiocyanate or isocyanate was added dropwise. After the
mixture was stirred for 2 h, the residue obtained by evapora-
tion was extracted with ethyl acetate, and the organic layer
was washed with brine and dried over anhydrous sodium
sulfate. Evaporation gave a residue that was washed with
petroleum ether to yield compounds 35-37 in 75-80% yield.
Using phenyl isothiocyanate, compound 35 was obtained as
yellowish crystals: mp 176-178 °C (lit.29 180-181 °C); 1H
NMR (CDCl3) δ 7.32 (m, 4 H), 7.22 (m, 1 H), 5.30 (m, 1 H),
0.85 (d, J ) 6.0 Hz, 3 H), 0.83 (d, J ) 7.2 Hz, 6 H), 0.77 (s, 3
H), 0.62 (s, 3 H); FABMS m/z (relative intensity) 524 (M+ + 1,
5), 388 [M+ + 1 - C(S)NHPh, 4], 371 [(M+ + 1 - OC(S)NHPh,
100]; FAB HRMS m/z calcd for C34H54NOS (M+ + 1) 524.3926,
found 524.3884. Anal. (C34H53NOS‚2H2O), C, N; H: calcd,
10.26; found 9.68.
Using 1-naphthyl isothiocyanate, compound 36 was obtained
as yellowish crystals: mp 137-139 °C; 1H NMR (CDCl3) δ 7.87
(m, 4 H), 7.52 (m, 3 H), 5.31 (m, 1 H), 0.87 (d, J ) 6.0 Hz, 3
H), 0.85 (d, J ) 6.6 Hz, 6 H), 0.84 (s, 3 H), 0.62 (s, 3 H); FABMS
m/z (relative intensity) 574 (M+ + 1, 60), 388 [M+ + 1 - C(S)-
NHNaphth, 10], 371 [(M+ + 1 - OC(S)NHNaphth, 100];
HRMS (FAB) m/z calcd for C38H56NOS 574.4083 (M+ + 1),
found 524.4111. Anal. (C38H55NOS‚1.6H2O), C, N; H: calcd
9.73; found 9.30. Using phenyl isocyanate, compound 37 was
obtained as yellowish crystals: 146-148 °C (lit.30 151 °C); 1H
NMR (CDCl3) δ 7.32 (m, 4 H), 7.04 (m, 1 H), 4.66 (m, 1 H),
0.87 (d, J ) 6.0 Hz, 3 H), 0.83 (d, J ) 6.6 Hz, 6 H), 0.80 (s, 3
H), 0.62 (s, 3 H); FABMS m/z (relative intensity) 508 (M+ + 1,
30), 428 (100), 371 (M+ + 1 - O2CNHPh, 95); FAB HRMS m/z
calcd for C34H54NO2 508.4155 (M+ + 1), found 508.4149. Anal.
(C34H53NO2‚0.25H2O), C, H. N.
Com p ou n d 26. A solution of 25 mg (0.06 mmol) of com-
pound 16 in 10 mL of 6% sodium hydroxide in aqueous
methanol was heated under reflux for overnight. The solution
was carefully acidified with 2 M HCl until there are white
precipitates formed. The solution was extracted with ethyl
ether, dried over anhydrous sodium sulfate, and evaporated
under pressure. The residue was purified using preparative
TLC to afford 19 mg of compound 26 (73.0%) as colorless oil:
FTIR (neat film) 3400-2500 (br), 2950, 2870, 1733, 1716, 1459,
1
1382, 1259 cm-1; H NMR (CDCl3, 300 MHz) δ 2.58 (dd, J )
17.7, 5.1 Hz, 1 H), 2.35 (t, J ) 7.2 Hz, 2 H), 2.04 (brd, J ) 12
Hz, 2 H), 1.95 (dd, J ) 17.7, 12.0 Hz, 1 H), 1.30 (s, 3 H), 0.90
(d, J ) 6.6 Hz, 3 H), 0.85 (d, J ) 6.6 Hz, 6 H), 0.69 (s, 3 H);
FAB MS m/z (relative intensity) 435 (M+ + 1, 100), 417 (18),
399 (15), 375 (12), 323 (10); EI HRMS calcd for C27H46O4 m/z
434.3396, found 434.3384.
Alk yl O-Ch olesta n yl Xa n th a tes (28-32). General pro-
cedures for the syntheses of compound 28-32: potassium
O-cholestanyl xanthate (27)22 (100 mg, 0.20 mmol) was dis-
solved in 4 mL of warm DMSO, and 1.0 equivalent of alkyl
halide was added. After being stirred for 2 h at room temper-
ature, the reaction solution was poured into water and
extracted with ethyl acetate. The organic layer was washed
with brine and then dried over anhydrous sodium sulfate. After
evaporation, the residue was washed with a 5:1 solution of
ethyl acetate and petroleum ether to give the corresponding
alkyl O-cholestanyl xanthate in 80-85% yield. All the xan-
thates were isolated as amorphous solids. Using bromo acetic
acid, compound 28 was obtained as colorless solid: mp 136-
138 °C; 1H NMR (CDCl3) δ 5.42 (m, 1 H), 3.88 (s, 2 H), 0.85 (d,
J ) 5.4 Hz, 3 H), 0.83 (d, J ) 6.6 Hz, 6 H), 0.82 (s, 3 H), 0.62
(s, 3 H); FABMS m/z (relative intensity) 523 (M+ + 1, 5), 506
(M+ + 1 - OH, 3), 464 (M+ + 1 - CH2CO2H, 35), 448 (M+
+
1 - CH2CO2H - CH4, 40), 371 (M+ + 1 - OC(S)SCH2CO2H,
100). Anal. (C30H50O3S2) C, H. Using ethyl bromoacetate,
compound 29 was obtained as a gum: 1H NMR (CDCl3) δ 5.43
(m, 1 H), 4.18 (q, J ) 7.2 Hz, 2 H), 3.84 (s, 2 H), 1.26 (t, J )
7.2 Hz, 3 H), 0.86 (d, J ) 6.6 Hz, 3 H), 0.83 (d, J ) 6.6 Hz, 6
H), 0.82 (s, 3 H), 0.62 (s, 3 H); FABMS m/z (relative intensity)
551(M+ + 1, 2), 550 (M+, 5), 534 (M+ - CH4, 3), 521 (M+
-
CH2CH3, 3), 477 (M+ - CH3CH2OCO, 5), 463 (M+ - CH3CH2-
OCOCH2, 5), 371 (M+ + 1 - OC(S)SCH2CO2Et, 100). Anal.
(C32H54O3S2) C, H. Using ethyl 2-bromopropionate, compound
30 was obtained as a gum: 1H NMR (CDCl3) δ 5.43 (m, 1 H),
4.28 (q, J ) 7.2 Hz, 1 H), 4.15 (q, J ) 7.2 Hz, 2 H), 1.22 (t, J
) 7.2 Hz, 3 H), 0.85 (d, J ) 6.6 Hz, 3 H), 0.83 (t, J ) 7.2 Hz,
3 H), 0.82 (d, J ) 6.6 Hz, 6 H), 0.81 (s, 3 H), 0.61 (s, 3 H);
FABMS m/z (relative intensity) 565 (M+ + 1, 2), 549 (M+ + 1
- CH4, 5), 520 (M+ + 1 - OC2H5, 15), 464 (M+ + 1 - CH-
(CH3)CO2Et, 20), 388 (M+ + 1 - C(S)SCH(CH3)CO2Et, 35), 371
(M+ + 1 - OC(S)SCH(CH3)CO2Et, 100). Anal.(C33H54O3S2) C,
H. Using ethyl chloroacetoacetate, compound 31 was obtained
as a gum: 1H NMR (CDCl3) δ 5.40 (m, 1 H), 4.17 (q, J ) 7.2
Hz, 2 H), 4.02 (s, 2 H), 3.60 (s, 2 H), 1.25 (t, J ) 7.2 Hz, 3 H),
0.86 (d, J ) 6.6 Hz, 3 H), 0.83 (d, J ) 6.6 Hz, 6 H), 0.81 (s, 3
H), 0.61 (s, 3 H); FABMS m/z (relative intensity) 592 (M+, 2),
477 (M+ - COCH2CO2Et, 5), 463 (M+ - CH2COCH2CO2Et, 3),
371 (M+ +1 -OC(S)SCH2COCH2CO2Et,100).Anal.(C34H56O4S2‚
0.3 hexane) C, H. Using bromo acetophenone, compound 32
1
was obtained as a yellowish solid: mp 108-110 °C; H NMR
(CDCl3) δ 8.00 (m, 2 H), 7.60 (m, 1 H), 7.48 (m, 2 H), 5.42 (m,
1 H), 4.61 (s, 2 H), 0.85 (d, J ) 6.6 Hz, 3 H), 0.83 (d, J ) 6.6
Hz, 6 H), 0.78 (s, 3 H), 0.62 (s, 3 H); FABMS m/z (relative
intensity) 582 (M+, 5), 477 (M+ - COC6H5, 5), 463 (M+ - CH2-
COC6H5, 3), 371 (M+ + 1 - OC(S)SCH2COC6H5, 100). Anal.
(C34H56O2S2‚0.5H2O), C, H.
X-r a y An a lysis. Detailed crystal data and summaries of
data collection and structure refinement for compounds 4, 8,
and 13 are listed in Table 12 in the Supporting Information.
Crystals of compound 4 were obtained by slow evaporation
from a mixture of hexanes-ethyl acetate (10:1). Compound 4
was crystallized in space group P21 with a ) 11.0952(10) Å, R
) 90°, b ) 6.4084(6) Å, â ) 103.5220°, c ) 18.121(2) Å, γ )
90°, and it was refined to a conventional factor R ) 0.0547
Com p ou n d 33. To a solution of 250 mg (0.64 mmol) of
cholestan-3-one in 20 mL of methanol was added 6.0 equiv of

Steroidal Derived Inhibitors for Human CDC25A Phosphatase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 847
and goodness of fit on F∧2 ) 1.024. Data collection was at
173 K, with Z ) 2. The structure of compound 4 is shown in
Figure 2.
Crystals of compound 8 were obtained by slow evaporation
from a chloroform solution. Compound 8 was crystallized
orthorhombic in space group P212121 with a ) 6.4670(2) Å, b
) 34.1026(12) Å, c ) 35.4789(13) Å and refined to a conven-
tional factor R ) 0.0690 and goodness of fit on F∧2 ) 1.086.
Data collection was at 248 K, with Z ) 12; The structure of
compound 8 is shown in Figure 3.
Crystals of compound 13 were obtained by slow evaporation
from hexanes-ethyl acetate (1:1). Compound 13 was crystal-
lized orthorhombic in space group P212121 with a ) 6.2731(6)
Å, R ) 90°, b ) 9.8150(9) Å, â ) 90°, c ) 40.038(4) Å, γ ) 90°,
and it was refined to a conventional factor R ) 0.0758 and
goodness of fit on F∧2 ) 0.993. Data collection was at 233 K,
with Z ) 4. The structure of compound 13 is shown in Figure
4.
CD45 In h ibition Assa y a n d Stea d y-Sta te Kin etics.
CD45 assays in triplicate were performed with p-nitrophenyl
phosphate as substrate in 96-well plates at 25 °C in a final
volume of 100 µL using 10 ng of enzyme. Assay buffer was
composed of 50 mM HEPES, 100 mM NaCl, and 2 mM
dithiothreitol, pH 7.4. Compound 17 was added to the wells
dissolved in 5 µL of DMSO at a final concentration of 5, 10, or
15 µM, followed by 20 µL of enzyme and 75 µL of pNPP
substrate. Substrate concentration was varied between 5 and
30 µM (Km )15 µM). Absorbance at 405 nm was monitored
for 30 min, and reaction rates under linear conditions were
determined using Molecular Devices SOFTmax Pro. Under
these conditions, inhibition of CD45 by compound 17 appeared
to be mostly noncompetitive but having a mixed component
as indicated by the intersection above the x axis.
Cytotoxicity Assa ys. The in vitro cytotoxicity assays were
carried out at the National Cancer Institute. Details of the
assay procedures have been reported previously.25
Exp r ession a n d P u r ifica tion of th e Cytop la sm ic Re-
gion of CD45. The cytoplasmic region of CD45, fused at the
C-terminal to six histidines, was expressed in the pET23D
vector (Novagen). The 705 amino acids of CD45 being ex-
pressed begins MAGNLDEQQELV and ends ALNQGSHHH-
HHH and has a molecular weight of 83,664. Escherichia coli
BL21 (DE3) transformed with the expression plasmid was
grown in a 10 L fermentor at 28 °C to an optical density at
600 nm of 0.6 and induced by the addition of 0.6 g of IPTG
(isopropyl-â-thiogalactopyranoside). After 5 h the cells were
harvested by centrifugation and stored at -80 °C. The CD45
was purified by metal affinity and gel filtration chromatogra-
phy. Briefly, cells were lysed by two passes through a French
pressure cell at 16 000 psi, and the lysate clarified by cen-
trifugation at 47000g for 60 min at 4 °C. The supernatant was
applied to a 50 mL Chelating Sepharose Fast Flow (Pharma-
cia) column charged with NiCl2 and equilibrated in 25 mM
Hepes, 250 mM NaCl and 25 mM imidazole at pH 7.5. The
column was eluted with a linear gradient from 25 to 250 mM
imidazole. Fractions containing CD45 phosphatase activity
(50-100 mM imidazole fractions) were pooled and concen-
trated with a Centriprep 30 (Amicon). The concentrate was
applied to a 2 L Superdex 75 (Pharmacia) column equilibrated
in 20 mM Tris and 100 mM NaCl at pH 8.0. Fractions
containing CD45 phosphatase activity were pooled (approxi-
mately 80% pure), aliquoted, and stored at -80 °C.
Cd c25A In h ibition Assa y. The procedures for the prepa-
ration of GST-Cdc25A fusion protein and Cdc25A inhibition
assay have been described in detail previously.22 Briefly, each
batch of GST-Cdc25A fusion protein was calibrated such that
the quantity added to each well dephosphorylated FDP in a
linear fashion over a 20 min reaction time course in the
preliminary study. The phosphatase assay was performed in
96-well plates. The standard reaction time and conditions were
15 min at 25 °C in a darkened chamber. The reactions were
terminated with 15 µL of sodium orthovanadate (285 mM)
dissolved in distilled water. The fluorescence emission of the
reaction product (fluorescein monophosphate) was measured
with a Millipore Cytofluor 2350 fluorimeter (excitation wave-
length, 485 nm; emission wavelength, 530 nm). The IC50 values
were determined from at least two independent determina-
tions, each run in triplicate, where the variation from the mean
did not exceed (20%.
Ack n ow led gm en t. We gratefully acknowledge sup-
port of this research by the NCI /NIH (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.
Su p p or tin g In for m a tion Ava ila ble: Atomic coordinate
information for compounds 4, 9, and 13. This material is
available free of charge via the Internet at http://pubs.acs.org.
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final assay volume. Excitation wavelength was 485 nm,
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Excel.

848 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
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