Steroidal derived acids as inhibitors of human Cdc25A protein phosphatase

Article abstract of DOI:10.1016/S0968-0896(99)00284-9

A group of steroidal derived acids were synthesized and found to be human Cdc25A inhibitors. Their potency ranged from 1.1 to > 100 μM; the best ones compare very favorably with that of the novel cyano-containing 5,6-seco- cholesteryl acid 1 (IC₅₀

Full text of DOI:10.1016/S0968-0896(99)00284-9

Bioorganic & Medicinal Chemistry 8 (2000) 299±306
Steroidal Derived Acids as Inhibitors of Human Cdc25A
Protein Phosphatase
Hairuo Peng, a Wenge Xie, a Deog-Il Kim, a Leon H. Zalkow, a,* Garth Powis, b
Diane M. Otterness c and Robert T. Abraham c
aSchool of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA
bArizona Cancer Center, University of Arizona, 1515 North Campbell Avenue, Tucson, AZ 85724-5024, USA
cDepartment of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA
Received 17 May 1999; accepted 14 September 1999
AbstractÐA group of steroidal derived acids were synthesized and found to be human Cdc25A inhibitors. Their potency ranged
from 1.1 to >100 mM; the best ones compare very favorably with that of the novel cyano-containing 5,6-seco-cholesteryl acid 1
(IC50=2.2 mM) reported by us recently (Peng, H.; Zalkow, L. H.; Abraham, R. T.; Powis, G. J. Med. Chem. 1998, 41, 4677).
Structure±activity relationships of these compounds revealed that a hydrophobic cholesteryl side chain and a free carboxyl group
are crucial for activity. The distance between these two pharmacophores is also important for the potency of these compounds.
Several of the compounds showed selective growth inhibition e€ects in the NCI in vitro cancer cell line panel. # 2000 Elsevier
Science Ltd. All rights reserved.
Introduction
products dysidiolide,11 menadione (vitamin K3) and its
naphthoquione analogues,12,13 SC-aad9 and its ana-
logues,14 and our recently published novel cyano-con-
taining 5,6-seco-cholesteryl derivatives,15 represented by
1, the most potent Cdc25A inhibitor reported (IC50=
2.2 mM) (Fig. 1). Menadione and its naphthoquinone
analogues were considered to inactivate Cdc25A irre-
versibly by modifying its active site. Vanadate and SC-
aad9 were reported to be competitive to the Cdc25A
substrate binding,10,14 presumably by mimicking the
phosphate group on the substrate with the carboxyl or
vanadate moiety. Compound 1, which contains a free
carboxyl group, may inhibit Cdc25A with a similar
mechanism as SC-aad9.
Accumulating evidence suggests that cell cycle-related
oncogenes contribute to the neoplastic transformation
of normal cells. Cdc25A is now considered as such an
oncogene1,2 that was found overexpressed in a number
of human cancers, including head and neck cancer,
colon cancer, non-small cell lung cancer and breast
cancer.3±5 It is also found signi®cantly associated with
lymphomas of more aggressive phenotypes.6 Cdc25A is
a protein phosphatase expressed early in the G1 phase
of the cell cycle, and it acts to remove inhibitory phos-
phate groups from the cyclin/CDK complexes which, in
turn, trigger cell cycle progression from G1 to S
phase.7,8 Cdc25A protein phosphatase is considered as a
promising target for the development of potential
chemotherapeutic anti-cancer agents because of its high
speci®city toward substrate and its unique phase speci®-
city in the cell cycle.9 However, the mechanisms that lead
to Cdc25A dysregulation, and the resulting neoplastic
transformations in human tumors are not clear yet.
To identify unique chemical templates for the design of
inhibitors active against oncogenic components of cell
cycle and intracellular signaling pathways, unusual
chemical transformations of readily available natural
product sca€olds were investigated. As a result, a group of
novel cyano-containing 5,6-seco-cholesteryl derivatives
represented by 1 were discovered as potent reversible
Cdc25A phosphatase inhibitors, possessing antitumor
activities. In search of new Cdc25A inhibitors with sim-
pler structures and ready availability, a series of ster-
oidal derived acids were synthesized and evaluated in
the Cdc25A enzyme inhibition assay. The potencies of
the best inhibitors in this group compared very favorably
A number of Cdc25A phosphatase inhibitors (Fig. 1)
have been reported, including vanadate,10 the natural
Keywords: antitumor compds; antiproliferatives agents; enzyme inhi-
bitors; steroids and sterols.
*Corresponding author.
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00284-9

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H. Peng et al. / Bioorg. Med. Chem. 8 (2000) 299±306
spectrum of compound 14. The b-isomer 14 was easily
epimerized to the a-isomer when treated with KOH.
Compounds 11 and 12 were obtained from treatment of
a mixture of 13 and 14 (5:1) with saturated KOH solu-
tion in water and THF (v/v, 5:2).
The syntheses of compounds 1519,20±19 are illustrated in
Scheme 3. Compound 15 was obtained by treatment of
the potassium salt of cholestanol with carbon disul®de.
Alkyl O-cholestanyl xanthates 16±19 were synthesized
by treatment of compound 15 with 1 equivalent of alkyl
halide in DMSO.
Results and Discussion
Compounds 2±16 were evaluated as inhibitors of the
recombinant human Cdc25A phosphatase at 25 ꢀC,
using ¯uorescein diphosphate (FDP) as the substrate.
The inhibitory activities of these compounds are shown
in Chart 1. The B ring opened acid (2), 3b-acetoxy-5,6-
seco-5-oxo-cholestan-6-oic acid, resembles the pre-
viously reported compound 1, but the A ring of the
cholestane skeleton remains intact. It showed fairly
good inhibitory activity against Cdc25A (IC50=
9.3 mM). However, when the free carboxyl group was
methylated, the resulting compound 3 was found to be
inactive, suggesting that the free carboxylic acid func-
tionality of compound 2 is necessary for activity. The
carboxyl group may serve as a surrogate phosphate
group which interacts with the active site arginine
(Arg-436), thus, competing with substrate binding.
Compound 4 inhibited Cdc25A at the same level
(IC50=9.5 mM) as compound 2 suggesting that the 3-
acetoxy group was not critical for activity.
Figure 1. Chemical structures for dysidiolide, SC-aad, menadione,
and compound 1.
with that of compound 1. Some of these also showed
signi®cant growth inhibition activities in human tumor
cell cultures.
Chemistry
The synthesis of 7 is illustrated in Scheme 1. Ozonolysis
of pregnenolone acetate in ethyl acetate:petroleum ether
at 60 ꢀC a€orded 3b-acetoxy-5-oxo-5,6-seco-pregnane-
6-oic acid 5 in 60% yield. Reductive amination of 5
using 4 equiv of N,N-dimethylethylenediamine and 1.5
equiv of sodium cyanoboro hydride gave 7 in a ratio of
20R:20S=3:1, the major isomer was assigned the 20R
con®guration based on literature precedent.16,17
In contrast, when the 5,6-seco-6-acid moiety was present
in the pregnenolone nucleus, 5 and 6, essentially all
activity was lost, con®rming the hypothesis that a
hydrophobic pocket exists close to the active site of this
enzyme. Replacement of carbon atoms in the side chain
by more polar atoms, as in compound 7, likewise led to
The syntheses of compounds 1118±14 are illustrated in
Scheme 2. Compounds 1318 and 14 were synthesized
from 4-cholesten-3-one in a ratio of 5:1 (13:14), and the
relative con®gurations were determined by the NOE
observed between Me-19 and H-28 in the NOESY
Scheme 1. Reagents: (a) O3, 60 ꢀC, petroleum ether:ethyl acetate 1:1; piperidine; 2 M HCl, 60%; (b) N,N-dimethylethylenediamine, CH3OH:THF,
NaB(CN)H3, 25%.
Scheme 2. Reagents: (a) LDA, THF; ethyl bromoacetate; 93%; (b) KOH, H2O:THF; 64%.

H. Peng et al. / Bioorg. Med. Chem. 8 (2000) 299±306
301
Scheme 3. Reagents: (a) K, (CH3OCH2CH2)2O, CS2, 85%; (b) RX, DMSO (X=Cl, Br, or I), 80±85%.
Chart 1. IC50 values were determined from at least two independent determinations, each run in triplicate, where the variation from the mean is
‹20% or less. NA stands for IC50 value larger than 100 mM. ND stands for not determined.
loss of activity. Lacking the steroidal skeleton, the nat-
ural product podocarpic acid 8 and its methyl ester 9
were found to be inactive.
to be slightly more potent (IC50=1.1 mM). This may be
due to the better interaction between the larger nega-
tively charged sulfur atoms and the active site arginine
group, or to the improved binding anity associated
with the longer distance between the hydrophobic side
chain and the surrogate phosphate group. Again, when
the carboxyl group of 11 was protected as in compound
13, or when the xanthate group of 15 was alkylated as
in compounds 16±19, inhibition was lost. The fact that
more lipophilic compounds, such as 13, 16±19 were
inactive as compared to their acid counterparts 11 and
15 demonstrated that the inhibitory activities of these
compounds are not due to the lipophilicity of the ster-
oidal structure. Lipophilic compounds sometime disrupt
enzyme activity by aggregating or stacking e€ects.
To determine if the structural features introduced by
opening of the B ring are critical for activity, an A ring
opened cholesteryl acid, 4,5-seco-5-oxo-cholestan-4-oic
acid (10), was synthesized and found to be more potent
(IC50=1.9 mM) than the
B ring cleaved acid 2
(IC50=9.2 mM) in the Cdc25A inhibition assay. In
addition, compound 11, which was envisioned to hold
the carboxyl group at the same position as in compound
10, but has an intact cholesteryl ring system, was found
to have the same potency level (IC50=1.4 mM) as 10,
suggesting that one of the most important features for
Cdc25A inhibition is the relative positioning of the
cholesteryl side chain and the free carboxylic acid, while
opening of either the A or B ring is not necessary. The
kinetic characteristic of Cdc25A inhibition with com-
pound 11 was studied using FDP as substrate. The
inhibition pattern is most consistent with a competitive
inhibition model (Ki=0.57 mM), suggesting that the
carboxyl group mimics a phosphate of the substrate.
Compound 12, the 2b isomer of 11, exhibited similar
inhibition potency at IC50=2.1 mM.
In summary, three crucial features emerged from ana-
lysis of the structure±activity relationship of the com-
pounds described above. (1) A free alkyl carboxyl or
xanthate functionality is required for inhibitory activity.
(2) A completely hydrophobic alkyl chain, such as the
cholesteryl side chain contributes greatly to the potency.
(3) The inhibitory activity is sensitive to the distance
between the carboxyl or xanthate functionality and the
alkyl side chain.
In light of this consideration, the dithiocarboxylic acid
derivative (15) of cholestanol was synthesized and found
Only the crystal structure of the catalytic domain of
Cdc25A, which lies at the edge of the crystal structure,

302
H. Peng et al. / Bioorg. Med. Chem. 8 (2000) 299±306
has been reported;21 therefore, it is dicult, at this time,
to verify the SAR by molecular modeling. In attempted
modeling (data not shown), when the carboxyl group
forms a reasonable interaction with the active site argi-
nine (Arg 436), then the cholesteryl side chain hangs
outside the reported structure, making it impossible to
obtain any useful information. We therefore, for com-
parison purposes, simply determined the distances
between the carboxyl group and the end of the side
chain in the lowest energy conformations of compounds
2, 10, 11 and 15, to see if this information could be
related to the activities of the various compounds. As
illustrated in Figure 2, the distances between the carbon
atom (labeled as A) that holds the two oxygen atoms of
the carboxylate, or the carbon atom (labeled as A) that
holds the two sulfur atoms of the xanthate function-
ality, and the atom (labeled as B) that holds the two
methyl groups on the cholesteryl side chain were mea-
sured. Interestingly, the distances appeared to be well
correlated with their inhibitory activities. In compound
15, the most potent Cdc25A inhibitor (IC50=1.1 mM) in
this series, a distance of 17.7 A was observed between A
and B (Fig. 2). This series of compounds are either A or
B-ring cleaved, or with an intact steroidal skeleton. The
correlation is however not applicable to compounds like
1, in which both the A and B rings are opened, and the
cyano-containing side chain contributes additionally to
its inhibitory activity.15 Of course, it is recognized that
the gas phase modeling of distances may not relate to
conditions in solution.
(micromolar concentrations required to produce 50%
growth inhibition) are shown in Table 1 for sensitive
subpanels.
Compound 4 showed a mean GI50 value (MG MID) at
3.95 mM for 54 tumors. It was more selective against the
HOP-92 non-small cell lung cancer (GI50=0.66 mM),
the OVCAR-8 ovarian cancer (GI50=1.62 mM), two
breast cancer cell lines NCI/ADR-RES (GI50=1.37 mM)
and BT549 (GI50=1.12 mM), and the leukemia HL-60
(GI50=56 nM).
Compound 11, which is a more potent Cdc25A inhib-
itor than 4, showed an average GI50 of 7.07 mM. It
exhibited selectivity over HOP-92 non-small cell lung
cancer (GI50=3.56 mM), HCC2998 colon cancer
(GI50=3.96 mM), OVCAR8 ovarian cancer (GI50=
4.63 mM), and two breast cancer cell lines, NCI/ADR-
RES (GI50=4.20 mM) and BT-549 (GI50=4.08 mM).
Compound 12, the 2b epimer of 11 showed a similar
sensitivity pattern to that of 11, but was less active in
each panel. In contrast to their potent Cdc25A inhibi-
tion activities, the MG MIDs of compounds 10 and 15
were at 19.5 and 18.2 mM, respectively. Compound 10
showed good selectivity for leukemia SR (1.89 mM) and
compound 15 exhibited good selectivity for HT-29
colon cancer (1.22 mM). The reasons that the more
potent Cdc25A inhibitors 10, 11, and 15 were less active
in the in vitro cytotoxicity screening are unknown at this
time. Poor penetration of plasma membrane is possible,
and we can not rule out the possibility that the observed
cytotoxicity is not related to Cdc25A inhibition. The
®nding that compounds 4, 11, 12 and 15 were more
selective for the subpanels of non-small cell lung cancer,
colon cancer, and breast cancer as compared to other
tumor cell lines, is consistent with reports that Cdc25A
phosphatase is overexpressed in these cancer cell lines.3±5
Compound 2 was tested for its growth inhibition e€ects
on HT-29 colon cancer in cell culture22 and showed an
IC50 of 16.2 mM.23 The in vitro cytotoxicity of compounds
4, 10±12 and 15 was evaluated at the National Cancer
Institute against a panel of 60 human tumor cell lines
representing nine di€erent cancer types. The GI50 values
Figure 2. Calculated lowest-energy conformations for compounds 2, 10, 11 and 15.

H. Peng et al. / Bioorg. Med. Chem. 8 (2000) 299±306
303
Table 1. Inhibition of in vitro cancer cell lines by compounds 4, 10±
12 and 15
was run using 230±400 mesh silica gel. Reverse phase
high performance liquid chromatography (HPLC) was
run on a Phenomenex1 LUNA 5 m C18 semi-preparative
column (Phenomenex, Torrance, CA). The homogeneity
of all the compounds was routinely checked by TLC on
silica gel plates, and also by HPLC. Fourier trans-
formed infrared spectra were obtained on a Nicolet 520
Cell lines
GI50 (mM)a
4
10
11
12
15
Leukemia
HL60TB
SR
0.056
5.55
6.27
1.89
8.28
6.16
6.11
5.73
>25
>25
1
FTIR spectrometer. H (300 or 400 MHz), 13C (75 or
100 MHz) NMR and DEPT spectra were recorded on
either a Varian Gemini-300 or on a Varian XL-400
spectrometer. High-resolution mass spectra (EI or
FAB) were recorded on a VG Analytical 70-SE mass
spectrometer equipped with a 11-250J data system. Ele-
mental analyses were performed by Atlantic Microlab,
Norcross, GA.
Non-small cell
lung cancer
EKVX
HOP-92
NCI-H23
4.21
0.66
4.51
>25
>25
>25
4.86
3.56
>25
6.91
6.52
12.3
6.67
>25
>25
Colon cancer
COLO205
HCC2998
HT29
NDc
4.36
4.44
>25
NDc
5.19
3.96
4.87
5.36
5.88
6.94
11.7
3.92
1.22
19.4
>25
9.32
Synthesis and spectral data for steroidal inhibitors 2±19
Ovarian cancer
OVCAR8
1.62
3.65
4.63
7.75
8.90
>25
5.75
>25
>25
>25
18.2
The syntheses of compounds 2,25 3,26 4,27 5,28 7,16,17
10,24 11, 13,18 16 and 1719,20 were performed as pre-
viously reported except as indicated.
Prostate cancer
PC-3
10.7
Breast cancer
NCI/ADR-RES
BT-549
T-47D
MG-MIDb
1.37
1.12
3.90
3.95
>25
>25
9.18
19.5
4.20
4.08
7.35
7.07
16.5
10.7
11.2
12.0
3ꢀ-Acetoxy-5,6-seco-5-oxo-cholest-6-oic acid (2). Com-
pound 2 was obtained as colorless crystals: mp 114±
116 ꢀC (Lit.24 mp 115±117 ꢀC); FTIR (neat ®lm) 3400±
1
2500 (br), 2960, 2868, 1736, 1716, 1249 cm 1; H NMR
aGI50 represents the compound concentration (mM) required to
achieve 50% inhibition of tumor cell growth.
bMG MID represents the calculated mean GI50 for all panels.
cNot determined.
(CDCl3, 300 MHz) d 9.89 (broad), 5.29 (brs, 1H), 3.11
(dd, J=14.4, 4.3 Hz, 1H), 2.33 (d, J=14.5 Hz, 1H), 2.08
(s, 3H), 0.99 (s, 3H), 0.88 (d, J=6.4 Hz, 3H), 0.83 (d,
J=6.6 Hz, 6H), 0.65 (s, 3H); 13C NMR and DEPT
(CDCl3, 100 MHz) d 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 (CH3), 11.6 (CH3); EIMS m/z (relative
intensity) 416.4 (M+-AcOH, 43), 398.4 (M+-H2O, 21),
306.3 (34), 247.3 (97), 110.1 (100); CIMS m/z (relative
intensity) 477.4 (M++1, 4), 417.4 (M+-AcOH+1, 88),
399.3 (100), 357.3 (40), 331.3 (45).
Conclusion
In summary, a group of steroidal derived acids were
found to be potent Cdc25A inhibitors (IC50=1.1±
9.5 mM). Structure±activity relationships of these com-
pounds revealed that potency depended on the presence
of a free carboxyl group and an alkyl side chain. Potas-
sium xanthate 15, with a distance of about 17.7 A
between the surrogate phosphate group (xanthate) and
the hydrophobic side chain, was found to be the most
potent Cdc25A inhibitor to date. The lead compounds
identi®ed in this study provide readily available Cdc25A
inhibitors (10±12, 15) and control compounds (3, 5 and
6) for Cdc25A and cell cycle studies. Although the
selectivities of these inhibitors over the other two iso-
forms (B, C) of Cdc25 phosphatase or other protein
tyrosine phosphatases have not been evaluated at this
time, it is important to have selective inhibitors since
Cdc25C, a normal gene product, is a critical component
for DNA damage induced cell cycle check points.24 It is
noteworthy that compounds 4, 10±12 showed selective
antitumor activities for non-small cell lung, colon,
breast and ovarian cancers in in vitro tumor cell
screening.
3ꢀ-Acetoxy-5,6-seco-5-oxo-pregnan-6-oic acid (5).
Ozone was passed into a stirred and cooled (chloro-
form/dry ice bath) solution of 5 g pregnenolone acetate
in a 300 mL mixture of ethyl acetate:petroleum ether
(1:1) for 40 min, until the solution turned light blue. To
the cold solution was added 5 mL piperidine. The mix-
ture was stirred for 2 h at 60 ꢀC. The resulting white
precipitate was isolated by ®ltration, then dissolved in
80 mL chloroform which was washed with 2 M HCl
(3Â10 mL), then with water, dried over anhydrous
Na2SO4, and the solution was evaporated. The residue
was subjected to ¯ash chromatography (petroleum
ether:ethyl acetate 3:1) to yield 3b-acetoxy-5-oxo-5,6-
seco-pregnan-6-oic acid (5), 3.1 g (60%), as an oil: IR
(neat ®lm) 3400±2500 (br), 2951, 1735, 1710, 1703,
1697 cm 1; 1H NMR (CDCl3, 300 MHz) d 5.35 (br, 1H),
3.16 (dd, J=14.3, 4.5 Hz, 1H), 2.09 (s, 3H), 1.97 (s, 3H),
1.06 (s, 3H), 0.61 (s, 3H); 13C NMR (CDCl3, 100 MHz)
d 212.1, 209.2, 176.5, 170.3, 73.3, 63.4, 53.9, 52.2, 43.9,
43.2, 41.1, 38.7, 35.4, 34.4, 33.2, 31.4, 25.2, 24.6, 23.0,
22.6, 21.2, 17.7, 13.0; EIMS m/e (relative intensity) 346
Experimental
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. Flash chromatography

304
H. Peng et al. / Bioorg. Med. Chem. 8 (2000) 299±306
(M+-CH3COOH, 30), 328 (M+-CH3COOH±H2O, 10),
110 (100).
0.83 (d, J=5.4 Hz, 6H), 0.66 (s, 3H); HRMS (FAB) m/z
calcd for C31H51O3 471.3838, found 471.3873.
3ꢀ-Acetoxy-5,6-seco-5-oxo-3-pregnen-6-oic acid (6).
Compound 6 was prepared from pregnenolone acetate
by ozonolysis in ethyl acetate, as described for 5, and
isolated as a colorless oil (65%): IR (neat ®lm) 3400±
3-Oxo-4-cholesten-2ꢁ-acetic acid (11). A mixture of 13
and 14 (5:1, 120 mg) was dissolved in THF (2 mL) and
H2O (5 mL). A saturated KOH solution (5 drops) was
added and the solution was stirred for 12 h at room
temperature. The solution was adjusted to pH 2 with
10% HCl and diluted with H2O. Aqueous layer was
washed with ethyl acetate several times and the com-
bined organic layer was dried (MgSO4), ®ltered and
concentrated. The concentrate was chromatographed
and eluted with MeOH:EtOAc:hexane:CH2Cl2 1:3:7:10
to a€ord 72 mg of 3-oxo-4-cholesten-2a-acetic acid 1118
(64%) as colorless crystals: mp 174±176 ꢀC; [a] +41.0ꢀ
(c 0.5, CHCl3); HPLC (acetonitrile, 4 mL/min, 205 nm)
17.27 min; FTIR (neat ®lm) 3500±2500 (br), 2948, 2874,
1715, 1681 cm 1; 1H NMR (CDCl3, 300 MHz) d 5.73 (s,
1H), 2.90 (m, 2H), 2.34 (dt, J=4.5 Hz, 13.2 Hz, 1H),
2.27 (m, 2H), 2.01 (dt, J=3.6 Hz, 9.9 Hz, 2H), 1.81
(m, 2H), 1.65±0.96 (m, 20H), 1.22 (s, 3H), 0.88 (d,
J=6.6 Hz, 3H), 0.83 (d, J=6.6 Hz, 6H), 0.67 (s, 3H);
13C NMR (CDCl3, 125 MHz,) d 200.0, 178.1, 172.3,
123.1 56.5, 56.2, 54.5, 42.8, 42.7, 39.9 (3C, 39.92,
39.90, 39.85), 39.6, 36.5, 36.1, 35.8, 35.1, 33.1, 32.3,
28.6, 28.4, 24.6, 24.2, 23.2, 23.0, 21.3, 19.0, 17.8, 12.4;
HRMS (EI) calcd for C29H46O3 m/z 442.3447, found
442.3446.
1
2500 (br), 2972, 2939, 1753, 1706, 1673 cm 1; H NMR
(CDCl3, 300 MHz) d 6.76 (m, 1H), 5.89 (m, 1H), 2.50
(m, 2H), 2.08 (s, 3H), 1.20 (s, 3H), 0.62 (s, 3H); 13C
NMR (CDCl3, 100 MHz) d 209.4, 208.2, 178.0, 147.1,
28.5, 63.5, 54.7, 47.7, 43.8, 41.6, 38.7, 35.1, 34.8, 34.6,
31.4, 21.7, 24.7, 23.2, 22.5, 18.1, 12.9; CIMS m/z (rela-
tive intensity) 347 (M++1, 80), 329 (M+-H2O, 50), 110
(100); HRMS (CI) calcd for C21H31O4 (M+1)+ m/z
347.2222, found 347.2234.
3ꢀ-Hydroxy-5-oxo-5,6-seco-22, 25-diaza-cholestan-6-oic
acids (7). A solution 1 g of acid 5 and 4 equiv N,N-
dimethylethylenediamine in 20 mL methanol was adjus-
ted to pH 6 by the addition of glacial acetic acid. The
solution was diluted with 20 mL THF and treated with
1.5 equiv NaB(CN)H3. The resulting mixture was
heated at re¯ux for 18 h. After cooling, saturated
Na2CO3 solution was added, the solution stirred at
room temperature for 2 h, adjusted to pH 7±8 by adding
saturated NH4Cl solution. After evaporation of the
solution, the residue was extracted with chloroform,
which was washed with aq FeSO4 and dried over anhyd
Na2SO4. The residue of the evaporated solution was
washed with ethyl acetate:petroleum ether (1:1) to give a
epimeric mixture of 3b-hydroxy-5-oxo-5,6-seco-22, 25-
3-Oxo-4-cholesten-2ꢀ-acetic acid (12). Compound 14
(25 mg) was dissolved in THF (1 mL) and H2O (2.5 mL).
A saturated KOH solution (5 drops) were added to the
solution and stirred for 4 h at room temperature. The
mixture was adjusted to pH 2 with 10% HCl and dilu-
ted with H2O. The aq layer was washed with ethyl ace-
tate by several times and the combined organic layer
was dried (MgSO4), ®ltered and concentrated. The con-
centrate was chromatographed on silica gel and eluted
with MeOH:EtOAc:hexane:CH2Cl2 1:3:7:10 to a€ord
8.1 mg (34%) of 11 and 6.0 mg (26%) of 12, 3-oxo-4-
cholesten-2b-acetic acid: HPLC (acetonitrile, 4 mL/min,
1
diaza-cholestan-6-oic acids 20R and 20S (ca. 3:1 by H
1
NMR analysis) as a brown solid: H NMR (CDCl3,
300 MHz) d 3.46 (m, 6H), 2.28, 2.26 [1/3, (2s, 6H,
-N(CH3)2], 0.97 (s, 3H), 0.72 (s, 3H); CIMS m/z (relative
intensity) 419 (M++1-H2O, 55), 402 (M+-CH3, 100),
.
362 (80). Anal. (C25H44N2O4 2H2O) C, H, N.
3-Oxo-4-cholesten-2-acetic acid ethyl esters (13, 14). To a
solution of 4-cholesten-3-one (1.8 g) in THF (15 mL)
was added 3.5 mL of LDA (2 M) at 10 ꢀC under argon
and the solution was stirred for 3 h. HMPA (2.4 mL)
was added to the solution, and then ethyl bromoacetate
(780 mL) was added at 10 ꢀC. After stirring for 2 h, the
mixture was quenched with H2O and the aqueous layer
was washed with ethyl acetate three times. The com-
bined organic layer was dried (MgSO4), ®ltered and
concentrated. The concentrate was chromatographed
on silica gel, and elute with EtOAc:hexane (1:10) to
a€ord ethyl 3-oxo-4-cholesten-2-acetic acids 13 (1.71 g,
79%) and 14 (0.34 g, 14%) with a ratio of 5:1. Com-
pound 13:18 1H NMR (CDCl3, 300 MHz) d 5.69 (s, 1H),
4.14 (m, 2H), 2.86 (m, 2H), 2.39±2.12 (m, 3H), 2.00 (m,
2H), 1.80 (m, 2H), 1.68±0.89 (m, 20H), 1.24 (t,
J=6.9 Hz, 3H), 1.21 (s, 3H), 0.87 (d, J=6.6 Hz, 3H),
0.83 (d, J=6.6 Hz, 6H), 0.67 (s, 3H); HRMS (EI) m/z
calcd for C31H50O3 470.3760, found 470.3725; Com-
pound 14: HPLC (acetonitrile, 4 mL/min, 205 nm)
1
205 nm) 17.09 min; H NMR (CDCl3, 300 MHz) d 5.77
(s, 1H), 2.85 (m, 2H), 2.48 (dt, J=4.5 Hz, 12.6 Hz, 1H),
2.20 (m, 3H), 1.97 (m, 2H), 1.81 (m, 1H), 1.66±0.92 (m,
20H), 1.12 (s, 3H), 0.88 (d, J=6.6 Hz, 3H), 0.84 (d,
J=6.6 Hz, 6H), 0.67 (s, 3H); HRMS (EI) calcd for
C29H46O3 m/z 442.3447, found 442.3456.
Potassium O-cholestanyl xanthate (15).19 3b-Cholestanol
(5 g, 12.9 mmol) was dissolved in 120 mL of 2-methoxy-
ethyl ether, then 500 mg (1.0 equiv) of potassium metal
was added. The reaction mixture was stirred under
re¯ux, for 1 h. The reaction mixture was cooled to 50 ꢀC,
then 0.9 mL (1.1 equiv) of carbon disul®de was added
and the solution was stirred for an additional hour. The
reaction mixture was concentrated to give the crude
product as a brown powder. The residue was washed
with petroleum ether and driedꢀto give 5.7 g (85%) of 15
as yellow powder: mp 279±281 C; 1H NMR (DMSO) d
0.61 (s, 3H), 0.77 (s, 3H), 0.82 (d, J=6.6 Hz, 6H), 0.85
(d, J=5.1 Hz, 3H), 5.19 (m, 1H); CIMS m/z (relative
intensity) 502 (M+, 5), 388 (M++1 (S)CSK, 45), 387
(M+ (S)CSK, 45), 371 (M+ O(S)CSK, 100).
1
22.13 min; H NMR (CDCl3, 300 MHz) d 5.71 (s, 1H),
4.13 (m, 2H), 2.86 (m, 2H), 2.46 (dt, J=4.8 Hz, 13.2 Hz,
1H), 2.17±1.74 (m, 6H), 1.65±0.94 (m, 20H), 1.24 (t,
J=6.9 Hz, 3H), 1.10 (s, 3H), 0.87 (d, J=6.6 Hz, 3H),

H. Peng et al. / Bioorg. Med. Chem. 8 (2000) 299±306
305
Alkyl O-cholestanyl xanthates (16±19). General pro-
cedures for the syntheses of compounds 16±19: Com-
pound 15 (100 mg, 0.20 mmol) was dissolved in 4 mL
warm DMSO, and 1.0 equiv of alkyl halide was added.
After stirring for 2 h at room temperature, 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. Evaporation
gave the corresponding alkyl O-cholestanyl xanthate in
80±85% yield. Using methyl iodide, O-cholestanyl
methyl xanthate (16) was obtained: mp 86±87 ꢀC (Lit.19
well as those in the B, C and D ring system. Ninety-two
low-energy conformations were obtained. For com-
pound 11, the torsion angles varied were 21-20-17-16,
29-28-2-1, as well as those in the A, B, C, and D
ring system. Fifteen low-energy conformations were
obtained. For compound 15, the torsion varied were 21-
20-17-16, 29-28-3-2, as well as those in the A, B, C, and
D ring system. Five low energy conformations were
obtained. The lowest-energy conformations obtained
for compounds 2, 10, 11 and 15 are shown in Figure 2,
and the distances between the atom labeled as A and the
atom labeled as B were measured and given in Figure 2.
87.5±88 ꢀC); H NMR (CDCl3) d 0.62 (s, 3H), 0.82 (s,
1
3H), 0.83 (d, J=6.6 Hz, 6H), 0.87 (d, J=5.4Hz, 3H),
5.47 (m, 1H); FABMS m/z (relative intensity) 479
(M++1, 5), 371 (M+ O(S)CSMe, 100); HRMS (FAB):
calcd for (M++1) m/z 479.3381, found 479.3381. Using
benzyl bromide, benzyl O-cholestanyl xanthate (19) was
obtained: mp 99±101 ꢀC (Lit.19 102±102.5 ꢀC); 1H NMR
(CDCl3) d 0.60 (s, 3H), 0.80 (s, 3H), 0.81 (d, J=7.5 Hz,
6H), 0.85 (d, J=5.4 Hz, 3H), 4.26 (s, 2H), 5.47 (m, 1H),
7.26 (m, 5H); FABMS m/z (relative intensity) 555
(M++1, 6), 478 (M++1-C6H5, 5), 371 (M+ O(S)CSBn,
100); HRMS (FAB) calcd for (M++1) m/z 555.3694,
found 555.3701. Using allyl bromide, allyl O-cholest-
anyl xanthate (17) was obtained: mp 90±91 ꢀC; 1H NMR
(CDCl3) d 0.62 (s, 3H), 0.82 (s, 3H), 0.83 (d, J=6.6 Hz,
12H), 0.87 (d, J=5.4 Hz, 3H), 3.72 (d, J=6.6 Hz, 2H),
5.13 (d, J=9.6 Hz, 1H), 5.25 (d, J=16.8 Hz, 1H), 5.47
(m, 1H), 5.85 (m, 1H); FABMS m/z (relative inten-
sity): 505 (M++1, 4), 464 (M++1 C3H5, 3), 371
(M+ O(S)CSC3H5, 100); HRMS (FAB) calcd for
(M++1) m/z 505.3540, found 505.3526. Using isoamyl
bromide, O-cholestanyl isoamyl xanthate (18) was
Cdc25A inhibition assay
The full-length human Cdc25A cDNA (a kind gift of
Dr. Laurent Meijer, CNRS, Rosco€, France) was
cloned into the pGEX-KG expression vector. The
resulting pGEX-cdc25A plasmid was transformed into
E. coli strain BL21(DE3). The bacteria were grown in a
medium containing 100 mg per mL ampicillin at 30 ꢀC
until the cell suspension reached an optical density
(OD600 nm) of 0.7. The incubation temperature was then
decreased to 25 ꢀC, and after 1 h, production of the
fusion protein was induced by addition of 0.1 mM iso-
propyl-b-d-thiogalactopyranoside (IPTG). After 4 h,
the bacteria were harvested by centrifugation. The
bacterial pellet was resuspended in phosphate-bu€ered
saline (PBS), pH 7.2, containing 1% (wt/vol) Triton
X-100, 1 mM EDTA, 5 mM dithiothreitol, 10 mg per mL
leupeptin, 10 mg per mL aprotinin, and 10 mg per mL
soybean trypsin inhibitor. The bacteria were disrupted
by sonication on ice, and the cell lysates were cleared by
centrifugation for 30 min at 100,000 g. The glutathione
S-transferase (GST)-Cdc25A fusion protein was pur-
i®ed from the cleared supernatant by chromatography
over glutathione-agarose. The eluted protein was dia-
lyzed into assay bu€er (20 mM Tris±HCl, 150 mM
NaCl, 1 mM EDTA, and 5 mM dithiothreitol), and ali-
quots of puri®ed enzyme were brought to 40% (wt/vol)
glycerol for storage at 70 ꢀC.
obtained: mp 105±107 ꢀC; H NMR (CDCl3) d 0.62 (s,
1
3H), 0.82 (s, 3H), 0.83 (d, J=6.6 Hz, 12H), 0.87 (d,
J=5.4 Hz, 3H), 3.06 (m, 2H), 5.47 (m, 1H); FABMS m/z
(relative intensity) 535 (M++1, 4), 507 (M++1-C2H4,
3), 371 (M+-O(S)CSC5H11, 100); HRMS (FAB) calcd
for (M++1) m/z 535.4009, found 535.3970.
Conformational search
Compounds 2, 10, 11 and 15 were computer-built using
ISIS/Draw (version 2.1; MDL Information System,
Inc., San Leandro, CA) and input into Hyperchem
Molecular Modeling Package (version 5.1; Hypercube
Inc., Gainesville, FL) with Chemplus. Conformation
searches were performed by varying torsion angles
de®ned by atoms in the side chains as well as those in
the ring system using usage directed searching method.
The acyclic torsion angles were varied between ‹60 and
180ꢀ, the torsion angles in the ring system were varied
between ‹30 and 120ꢀ. The conformations obtained
were geometry optimized to reach a rms gradients of
<0.01 kcal/(A mol) with the MM+ force ®eld, and with
the Polak-Ribiere optimizer. A total of 500 optimiza-
tions were performed for each compound.
The Cdc25A phosphatase assay was performed in 96-
well plates. The substrate, ¯uorescein diphosphate
(FDP; Molecular Probes, Inc.), was dissolved in water
to yield a ®nal concentration of 46.2 mM. The FDP
stock solution was aliquoted and stored at 70 ꢀC. Each
well received 100 mL assay bu€er modi®ed to contain
20 mM dithiothreitol. The indicated compounds were
dissolved in dimethylsulfoxide and 1±2 mL of the con-
centrated stock solutions were added per well. Control
incubations contained an equivalent volume of dime-
thylsulfoxide only. Puri®ed GST-Cdc25A was diluted in
assay bu€er, and 20 mL of the diluted enzyme was added
per well. In preliminary studies, each batch of enzyme
was calibrated such that the quantity added to each well
dephosphorylated FDP in a linear fashion over a 20-min
reaction time course. After 5 min at room temperature,
the reactions were initiated by addition of 65 mL of FDP
solution. The plates were incubated for 15 min at 25 ꢀC
in the dark. The reactions were terminated with 35 mL of
sodium orthovanadate (285 mM) dissolved in distilled
water. The ¯uorescence emission of the reaction product
For compound 2, the torsion angles varied were 21-20-
17-16, 9-8-7-6, 1-10-9-8, 29-28-3-4, as well as those in
the A, C and D rings. Seven low-energy conformations
were obtained. For compound 10, the torsion angles
varied were 21-20-17-16, 2-1-10-5, 3-2-1-10, 4-3-2-1, as

306
H. Peng et al. / Bioorg. Med. Chem. 8 (2000) 299±306
(¯uorescein monophosphate)14,29 was measured with a
Millipore Cyto¯uor 2350 ¯uorimeter (excitation wave-
length, 485 nm; emission wavelength, 530 nm).
12. Ham, S. W.; Park, H. J.; Lim, D. H. Bioorg. Chem. 1997,
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Cytotoxicity assays
The in vitro cytotoxicity assays were carried out at the
National Cancer Institute. Details of the assay pro-
cedures have been reported previously.30
16. Kirk, D. N.; Hartshorn, M. P. Steroid Reaction Mechan-
isms. Elsevier: Amsterdam, 1968, pp 131.
17. Schmitt, J.; Panhouse, J. J. Fr. Patent 1 459 719 (Chem.
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Acknowledgements
18. Lingham, R. B.; Silverman, K. C.; Layasuriya, H.; Kim, B.
M.; Amo, S. E.; Wilson, F. R.; Rew, D. J.; Schaber, M. D.;
Bergstrom, J. D.; Koblan, K. S.; Graham, S. L.; Kohl, N. E.;
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M. J.; Saper, M. A. Cell 1998, 93, 617.
We gratefully acknowledge support of this research by
the NCI/NIH (Grant 5U19 CA52995). We are grateful
to Dr. Leslie T. Gelbaum, Mr. David E. Bostwick and
Ms. Sarah J. Shealy for their assistance in obtaining
high resolution NMR, mass spectra.
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