Discovery and biological evaluation of a new family of potent inhibitors of the dual specificity protein phosphatase Cdc25

Article abstract of DOI:10.1021/jm0102046

The Cdc25 dual specificity phosphatases have central roles in coordinating cellular signaling processes and cell proliferation, but potent and selective inhibitors are lacking. We experimentally examined the 1990 compound National Cancer Institute Diversity Set and then computationally selected from their 140 000 compound repository 30 quinolinediones of which 8 had in vitro mean inhibitory concentrations < 1 μM. The most potent was 6-chloro-7-(2-morpholin-4-ylethylamino)quinoline-5,8-dione (NSC 663284), which was 20- and 450-fold more selective against Cdc25B₂ as compared with VHR or PTP1B phosphatases, respectively. NSC 663284 exhibited mixed competitive kinetics against Cdc25A, Cdc25B₂, and Cdc25C with K_i values of 29, 95, and 89 nM, respectively. As compared with NSC 663284, the regioisomer 7-chloro-6-(2-morpholin-4-ylethylamino)quinoline-5,8-dione was 3-fold less active against Cdc25B₂ in vitro and less potent as a growth inhibitor of human breast cancer cells. Computational electrostatic potential mapping suggested the need for an electron-deficient 7-position for maximal inhibitor activity. Using a chemical complementation assay, we found that NSC 663284 blocked cellular Erk dephosphorylation caused by ectopic Cdc25A expression.

Full text of DOI:10.1021/jm0102046

4042
J . Med. Chem. 2001, 44, 4042-4049
Articles
Discover y a n d Biologica l Eva lu a tion of a New F a m ily of P oten t In h ibitor s of
th e Du a l Sp ecificity P r otein P h osp h a ta se Cd c25
J ohn S. Lazo,*,† Diana C. Aslan,‡ Eileen C. Southwick,† Kathleen A. Cooley,† Alexander P. Ducruet,†
Beomjun J oo,‡ Andreas Vogt,† and Peter Wipf‡
Departments of Pharmacology and Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
Received May 8, 2001
The Cdc25 dual specificity phosphatases have central roles in coordinating cellular signaling
processes and cell proliferation, but potent and selective inhibitors are lacking. We experi-
mentally examined the 1990 compound National Cancer Institute Diversity Set and then
computationally selected from their 140 000 compound repository 30 quinolinediones of which
8 had in vitro mean inhibitory concentrations <1 µM. The most potent was 6-chloro-7-(2-
morpholin-4-ylethylamino)quinoline-5,8-dione (NSC 663284), which was 20- and 450-fold more
selective against Cdc25B2 as compared with VHR or PTP1B phosphatases, respectively. NSC
663284 exhibited mixed competitive kinetics against Cdc25A, Cdc25B2, and Cdc25C with Ki
values of 29, 95, and 89 nM, respectively. As compared with NSC 663284, the regioisomer
7-chloro-6-(2-morpholin-4-ylethylamino)quinoline-5,8-dione was 3-fold less active against
Cdc25B2 in vitro and less potent as a growth inhibitor of human breast cancer cells.
Computational electrostatic potential mapping suggested the need for an electron-deficient
7-position for maximal inhibitor activity. Using a chemical complementation assay, we found
that NSC 663284 blocked cellular Erk dephosphorylation caused by ectopic Cdc25A expression.
In tr od u ction
HCX5R motif, they are unique in their ability to
hydrolyze both phosphoserine/threonine and phospho-
tyrosine residues on the same protein substrate.1 Im-
portant members of the DSPase family are the Cdc25
phosphatases, which control cell cycle progression by
activating cyclin-dependent kinases (Cdk)2 and partici-
pate in Raf-1-mediated cell signaling.3
Reversible protein phosphorylation is a ubiquitous
intracellular process required for mammalian cell com-
munication and growth. Determined by the dynamic
balance between protein kinases and phosphatases,
serine, threonine, or tyrosine phosphorylation can affect
catalytic activity or promote protein-protein interac-
tions that influence subcellular location and functional-
ity. Small molecule inhibitors have provided valuable
tools to decode the role of kinases and phosphatases
participating in specific cellular signaling pathways,
because they are generally reversible and readily enter
cells. Natural product inhibitors of serine/threonine
protein phosphatases, such as okadaic acid and calycu-
lin A, have been extremely valuable reagents to probe
serine/threonine phosphatase function; there is however
an absence of potent and selective inhibitors for the
other major mammalian phosphatase class, namely, the
protein tyrosine phosphatases (PTPase), which includes
the dual specificity protein phosphatase (DSPase) sub-
family. The PTPases are defined by the active site
signature sequence motif HCX5R, where H is a highly
conserved histidine residue, C is the catalytic cysteine,
the five X residues form a loop in which all of the amide
nitrogens hydrogen-bond to the phosphate of the sub-
strate, and R is a highly conserved arginine that
hydrogen bonds to the phosphorylated amino acid of the
substrate.1 While the DSPases retain the conserved
Three Cdc25 homologues exist in humans: Cdc25A,
Cdc25B, and Cdc25C.2,4-6 Cdc25A and B have oncogenic
properties,7 are transcriptional targets of the c-myc
oncogene,8 and are overexpressed in many human
tumors.9,10 Both Cdc25B and Cdc25C are thought to be
regulators of G2/M transition through their ability to
dephosphorylate and activate the Cdk1/cyclin B mitotic
kinase complex, which is required for cell entry into
mitosis.2,11 Cdc25A is likely to be important for G1/S
phase transition and in preserving genomic integrity,12-14
although Cdc25A may also have some role in the
initiation of mitosis.14 Cdc25A is rapidly degraded in
response to DNA damage, which impairs the G1/S
transition.15 Cdc25A may also have other cellular roles
as it has been shown to regulate the tyrosine phospho-
rylation status and kinase activity of Raf-1, which has
a central role in the mitogen-activated kinase signal
transduction pathway.3
Although two crystal structures have been published
for the Cdc25 catalytic domain,16,17 none expose the
nature of interactions with small molecule inhibitors.
Moreover, the protein substrate may initiate key con-
formational changes and provide an important catalytic
acid.18,19 Thus, rational design parameters for potential
inhibitors are lacking. The current work was initiated
* Corresponding author telephone: (412)648-9319; fax: (412)648-
2229; e-mail: lazo@pitt.edu.
† Department of Pharmacology.
‡ Department of Chemistry.
10.1021/jm0102046 CCC: $20.00 © 2001 American Chemical Society
Published on Web 10/18/2001

Inhibitors of Protein Phosphatase Cdc25
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4043
Ta ble 1. Chemical Structures of Quinolinedione Cdc25 Inhibitors and In Vitro Activity against Recombinant Human Cdc25B2, VHR,
or PTP1Bb
a
b
Single determination. IC50 values were from 3 or more determinations with SEM indicated.
based on the belief that selective Cdc25 inhibitors could
be obtained by using a general and unbiased approach
to evaluate a chemically diverse compound library.
Thus, we probed a small subset of the National Cancer
Institute’s (NCI) 140 000 compound library for a poten-
tial inhibitory pharmacophore and used this information
to identify a pharmacophore yielding the most potent
and selective inhibitor of Cdc25 reported to date. This
compound had marked antiproliferative activity against
human tumor cells and blocked the biochemical actions
of ectopic Cdc25A expression.
at 10 µM. Upon reevaluation, 5 of the 24 compounds
had IC50 values <1 µM; the most potent Cdc25B2
inhibitor was the quinolinedione NSC 668394, which
had an IC50 of 640 ( 130 nM against Cdc25B2 and had
g8-fold preference for Cdc25B2 as compared to the
DSPase VHR or the PTPase PTP1B (Table 1). Moreover,
the mean IC50 for growth inhibition by NSC 668394 was
2.1 ( 2.0 µM when assayed in the NCI 60 Tumor Cell
Panel (see http://www.dtp.nci.nih.gov/). NSC 668394
was the sole quinolinedione in the Diversity Set. The
importance of the 5,8-dione pharmacophore for Cdc25
inhibition was emphasized by the lack of significant
anti-phosphatase activity at 10 µM with any of the other
118 quinoline structures evaluated in the Diversity Set.
To test the hypothesis that the quinolinedione pharma-
cophore was important for Cdc25 inhibitory activity, J ill
J ohnson (NCI, Developmental Therapeutics Program,
Rockville, MD) computationally identified 30 additional
quinolinediones in the NCI Compound Repository, and
we examined them experimentally for anti-phosphatase
activity. When tested, all of these compounds had in
vitro IC50 values <40 µM for Cdc25B2, and seven had
IC50 values <1 µM. The mean IC50 value was 5.4 ( 1.5
µM. Six of the seven most active compounds were
Resu lts
Qu in olin ed ion es fr om th e NCI Com p ou n d Li-
br a r y In h ibited Hu m a n Cd c25. Initially, we exam-
ined the 1990 compounds comprising the NCI Diversity
Set, which was designed to be representative of the
140 000 NCI compound repository. Members of the
Diversity Set have been evaluated by the NCI for growth
inhibition against the NCI 60 Tumor Cell Panel, and
these data are publicly available at http//www.dtp.
nci.nih.gov/. We examined the Diversity Set for in vitro
inhibition of recombinant full-length human Cdc25B2
and found 24 compounds that caused >80% inhibition

4044 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Lazo et al.
Ta ble 2. Structures and Activity of Synthesized
Heteroarenedione Analoguesb
F igu r e 1. Lineweaver-Burk plots for NSC 663284 inhibition
of Cdc25 isoforms. (A) Cdc25A. Concentrations of NSC 663284
are 1, 15; [, 25; b, 50; 0, 100; 4, 150; 3, 200; ], 300 nM. (B)
Cdc25B2, 1, 25; [, 50; b, 100; 0, 150; 4, 200; 3, 300; ], 500
nM. (C) Cdc25C. 1, 25; [, 50; b, 100; 0, 150; 4, 200; 3, 300;
], 500 nM.
7-substituted quinolinediones, with the most potent
(NSC 663284) having an IC50 value of 206 ( 75 nM
(Table 1). All of the submicromolar inhibitors displayed
a strong preference for Cdc25B2 as compared with VHR
or PTP1B. For example, with NSC 663284, the relative
IC50 values for Cdc25B2 were 20- and 450-fold lower
than for VHR or PTP1B, respectively.
We examined further the kinetics and relative sen-
sitivity of each of the full-length human Cdc25 isoforms
to NSC 663284. For all three Cdc25 isoforms, NSC
663284 inhibition kinetics fit best to a partial mixed
competitive model, which may reflect the potential for
interactions at the two anionic binding sites as observed
in the crystal structure of Cdc25B17 (Figure 1). The
calculated Ki values for Cdc25A, Cdc25B, and Cdc25C
were 29 ( 7, 95 ( 14, and 89 ( 18 nM (n ) 4-6, (
SEM), respectively, suggesting that NSC 663284 had
3-fold selectivity toward Cdc25A as compared with
either Cdc25B or Cdc25C.
An a logu e An a lyses Revea led th e Im p or ta n ce of
th e C-7 Su bstitu tion for P oten t Cd c25 In h ibition .
To interrogate further the quinolinedione pharmacoph-
ore, we resynthesized NSC 663284, DA3003-1, and
synthesized the regioisomer of NSC 663284, namely,
a
b
Single determination. IC50 values were from 3 or more
determinations with SEM indicated. ND ) not determined.
DA3003-2, as well as several other analogues. A com-
parative study with the minimal quinolinedione, iso-
quinolinedione, phthalazinedione, and quinazolinedione
(namely, DA276, DA3002, DA295, DA3044, and DA3045)
revealed that a heterocyclic quinone pharmacophore
alone was insufficient for potent Cdc25 inhibition (Table
2). NSC 663284 had identical inhibitory properties as
compared to DA3003-1 against Cdc25A, Cdc25B, and
Cdc25C as well as HPLC and spectral profiles. Thus,
we used NSC 663284 for most of the subsequent
biological studies because of the much greater quantities

Inhibitors of Protein Phosphatase Cdc25
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4045
F igu r e 2. Growth inhibition of NSC 663284 and DA3003-2.
Cells were treated for 3 h with NSC 663284 (hatched bars) or
the regioisomeric DA3003-2 (black bars), and cell growth was
determined 48 h later as described in the Experimental
Section.
F igu r e 3. Reduction of phospho-Erk levels by Cdc25A over-
expression and inhibition of Cdc25A activity by NSC 663284.
(Upper Panel) Western blot of phospho-Erk levels after mock
transfection (lanes 1 and 2) or wild-type human Cdc25A
transfection (lanes 3 and 4). Cells were treated for 1 h with
vehicle (lanes 1 and 3) or 10 µM NSC 663284 (lanes 2 and 4).
(Middle Panel) â-tubulin was used as a loading control. (Lower
Panel) Quantification of the phospho-Erk levels in cells after
treatment with NSC 663284. n ) 4; bars ) SEM. Only Cdc25A
control was less than mock control (p < 0.05, Student’s t-test).
available to us. Addition of the 2-morpholin-4-ylethyl-
amino moiety at the 7-position clearly enhanced inhibi-
tory activity against Cdc25B as indicated by comparing
the IC50 values of DA3049 with DA3044, of DA296 with
DA295, or of NSC 663284 with either DA3002 or DA276.
Other 7-substituted analogues, such as DA3018 and
DA3020, were poor inhibitors of Cdc25B, although
DA3100 exhibited significant inhibitory activity indicat-
ing that the 2-morpholin-4-ylethylamino moiety was one
of at least two unique enhancers. For this limited series,
the quinolinedione pharmacophore provided a superior
scaffold as compared to the other heterocycles. Thus,
the quinoline analogue NSC 663284 was a more potent
inhibitor than either the isoquinoline analogue DA296
or the phthalazine analogue DA3049. The 7-substituted
quinolinedione NSC 663284, which contained the 2-mor-
pholin-4-ylethylamino moiety at the 7-position, was also
approximately 3-fold better as an inhibitor of Cdc25B2
than its regioisomer DA3003-2 and was the most potent
inhibitor of all compounds tested.
HeLa cells resulted in a >50% decrease in Erk phos-
phorylation (Figure 3). Treatment of mock (no Cdc25)
transfected cells with 10 µM NSC 663284 caused no
significant increase in Erk phosphorylation while ex-
posure of Cdc25A transfected cells for 1 h to 10 µM NSC
663284 reversed the depression of Erk phosphorylation
caused by Cdc25A transfection and returned phospho-
Erk to constitutive levels (Figure 3). These results
support the hypothesis that NSC 663284 blocked the
biological effects of Cdc25A within cells.
The substitution with amino groups at 6- and 7-posi-
tions, respectively, of the quinone cores of NSC 663284
and DA3003-2 was significant for increasing inhibitory
activity. This effect could be due to a polarization of the
electron distribution in the quinolinedione, since the
decrease in activity of the chlorinated derivative is
considerable and the difference in activity between
DA3100 and NSC 663284 was much less pronounced.
The latter two compounds would be expected to exhibit
a closely related electronic polarization of the quinone
core. IC50 values of NSC 663284, NSC 668394, DA3100,
DA296, DA3049, and DA3003-2 vary by a factor of 3,
while IC50 values for the corresponding dichloro ana-
logues DA3002, DA295, and DA3044 are about an order
of magnitude higher. Electron density surfaces encoded
with the electrostatic potential obtained by ab initio
computation of the core pharmacophore structure dem-
onstrate that, despite significant differences in the
position and the number of nitrogen atoms, NSC
663284, DA296, DA3049, and DA3003-2 have closely
related, polarized electron distributions (Figure 4). In
comparison, the analogous graphs for dichloro analogues
DA3002, DA295, and DA3044 show a very different,
much less polarized electrostatic potential surface (Fig-
ure 5). On the basis of this analysis, it becomes clear
that replacing a chloride substituent with an amino
group leads to a considerable perturbation of the
electron distribution in heteronaphthoquinones and that
NSC 663284 Block ed Cell P r olifer a tion a n d th e
Action s of Cellu la r Cd c25A. NSC 663284 had a mean
IC50 value in the NCI 60 Cell Human Tumor Panel of
1.5 ( 0.6 µM when cells were treated for 48 h. Most
sensitive were human breast cancer MDA-MB-435 and
MDA-N cells, which had IC50 values of 0.2 µM. We
observed an IC50 for growth inhibition of 1.7 µM with
continuous 48-h NSC 663284 treatment of human
breast MCF-7 cells in culture (data not shown). Even
after only a 3-h exposure to NSC 663284, we observed
an IC50 for growth inhibition of ∼35 µM with MCF-7
cells (Figure 2). Consistent with in vitro Cdc25 inhibi-
tion, the IC50 for growth inhibition after a 3-h exposure
to NSC 663284 was 3-fold lower than that seen with
the 6-regioisomer DA3003-2 (Figure 2). To probe for
inhibition of cellular Cdc25 activity, we used a recently
described chemical complementation assay,20 which
revealed the ability of a small molecule to complement
or reverse a biochemical effect caused by ectopic Cdc25A
expression. The advantage of this assay for studying
small molecules that also may affect cell cycle progres-
sion is the requirement for only a brief exposure of an
asynchronous cell population to an agent. Cdc25A has
been implicated in controlling the phosphorylation
status of Raf-1, which is a proximal effector of the
mitogen-activated protein kinase (Erk1/2) pathway.3
Thus, transient expression of full-length Cdc25A in

4046 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Lazo et al.
several small molecule Cdc25 phosphatase inhibitors
have been reported such as dysidiolide (IC50 ) 9.4 µM),21
sulfircin (IC50 ) 7.8 µM),22 SC-RRδ9 (IC50 ) 15 µM),23
FY21-RR09 (IC50 ) 7 µM),24 a cyano-containing choles-
teryl derivative (IC50 ) 2.2 µM),25 and a vitamin K
analogue called 5 (IC50 ) 3.8 µM).26 Of interest to our
study was the previous identification of tetrahydroiso-
quinolines with some inhibitory activity against Cdc25B
(IC50 ) 15-35 mM).27 Most of the above-mentioned
agents have not been evaluated for specificity and, with
the exception of 5 and SC-RRδ9, none have been shown
to inhibit Cdc25 within cells. Moreover, all of these
compounds lack the desired potency for exceptional
inhibitors.
Our studies illustrate how a subset of compounds
selected from a chemical library based on chemical
diversity can be used to screen against a predicted end
point and yield potent bioactive compounds. The quino-
line-5,8-diones have been the focus of previous studies
because of their wide spectrum of biological activity
including antitumor, antifungal, and antimalarial ef-
fects.28 Despite considerable investigation, their mech-
anism of action is largely not understood. For quino-
linediones, such as streptonigrin and lavendamycin,
depletion of NADPH/NADH, uncoupling of oxidative
phosphorylation, and/or DNA cleavage has been sug-
gested.29 Although we cannot formally exclude such
mechanisms for the cytotoxic effects of NSC 663284, one
advantage of our chemical complementation assay is
that it provides one with the ability to examine cellular
events after only a relatively brief exposure to an agent.
This is especially important for agents that block cell
proliferation. Thus, the chemical complementation assay
provided experimental evidence that NSC 663284 can
block the intracellular actions on the expressed target,
namely, Cdc25A. Moreover, the regioisomer that was a
superior inhibitor of Cdc25 in vitro was more cytotoxic
after a brief cell exposure. Interestingly, we found no
significant difference in the cytotoxicity of the two
regioisomers when cells were treated continuously with
these two regioisomers, suggesting that prolonged ex-
posure may cause other toxic actions. Consistent with
this notion is the observation that after prolonged
exposure isoquinolinediones are more toxic to cells than
their respective quinolinediones30 even though in our
studies the quinolinediones were more potent as in vitro
inhibitors of Cdc25 than the corresponding isoquino-
linediones (Table 2). Preliminary molecular modeling
studies with the available crystal structures for Cdc25B
are consistent with the quinolinedione pharmacophore
binding in at least one of the Cdc25 anionic binding sites
and provide a hypothetical explanation for the regioi-
someric preference that can be tested with newer
analogues (manuscript in preparation). As shown in the
electrostatic potential maps, the nitrogen-substituted
carbon of the ene-5,8-dione moiety in these inhibitors
is the most electron-deficient center of the quinone
substructure. While these computational electrostatic
potential mapping studies suggest the need for an
electron-deficient 7-position for maximal inhibitor activ-
ity, it is evident that the potency of inhibitors was not
simply correlated with increasing electrophilicity at this
position, which follows the series NSC 663284 <
DA3003-2 < DA296 < DA3049. This would be consis-
F igu r e 4. Electrostatic potential-encoded electron density
surfaces of the core structures of NSC 663284 (A), DA296 (B),
DA3003-2 (C), and DA3049 (D). The surfaces were generated
with Spartan 5.1.1 (Wavefunction, Inc.) after ab initio mini-
mization with a 3-21G* basis set. The coloring represents
electrostatic potential with red indicating the strongest at-
traction to a positive point charge and blue indicating the
strongest repulsion. The electrostatic potential is the energy
of interaction of the positive point charge with the nuclei and
electrons of a molecule. It provides a representative measure
of overall molecular charge distribution.
F igu r e 5. Electrostatic potential-encoded electron density
surfaces of less active core structures: DA3002 (A), DA295 (B),
and DA3044 (C). The surfaces were generated with Spartan
5.1.1 (Wavefunction, Inc.) after ab initio minimization with a
3-21G* basis set.
this electrostatic effect is likely to provide a better match
for the complementary surface in the enzyme binding
pocket. The core structure of NSC 663284 could there-
fore represent a general motif for the electronic config-
uration that is necessary to achieve selective and potent
Cdc25 inhibition.
Discu ssion
Although there is consensus that DSPases have
important roles in controlling cell proliferation and
signaling pathways, the absence of potent and selective
inhibitors has limited our ability to probe their biological
function. For example, until recently the only readily
available Cdc25 inhibitor was the broad spectrum
PTPase inhibitor sodium orthovanadate. More recently,

Inhibitors of Protein Phosphatase Cdc25
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4047
141.7, 135.7, 123.4, 118.0. MS (EI) m/z (rel intensity): 227 (M+,
100), 199 (20), 192 (80), 164 (80). HRMS (EI) m/z: calcd for
C9H3NO2Cl2, 226.9541; found, 226.9544.
tent with the notion that the pendant moieties on the
7-position also are important.
We noted some modest selectivity for inhibition of
Cdc25A as compared with Cdc25B and Cdc25C. Al-
though there is no available crystal structure of a ligand
with any Cdc25 isoform, structural differences in the
catalytic region of Cdc25A and Cdc25B have been
noted.16,17 Moreover, protein substrate preferences have
been reported with Cdc25A and Cdc25C, which reflect
not only residues within the catalytic domain but others
as yet undefined outside of the catalytic domain.17,18 Our
results suggest that it may be feasible to identify small
molecules with significant specificity for individual
Cdc25 isoforms. These would be valuable reagents as
there continues to be controversy about the precise
cellular actions of some of the isoforms. We suggest that
quinolinediones have a rich potential as a pharmacoph-
ore for inhibitors of the DSPase Cdc25 and that they
could represent useful biochemical probes as well as
possible lead compounds for the design of agents for the
treatment of cancer or other diseases.2,18
DA296 (6-Ch lor o-7-(2-m or p h olin -4-yleth yla m in o)iso-
qu in olin e-5,8-d ion e). A solution of DA295 (114 mg, 0.500
mmol) and 2-morpholin-4-ylethylamine (65 mg, 0.50 mmol) in
5 mL of THF was treated with triethylamine (0.07 mL, 0.5
mmol) at room temperature. The reaction mixture was stirred
for 20 h at room temperature, concentrated under reduced
pressure, diluted with ethyl acetate, and washed with water.
The organic layer was dried (Na2SO4) and concentrated in
vacuo. The crude residue was purified by chromatography on
SiO2 (CH2Cl2/MeOH, 15:1) to give a 4:1 mixture of DA296 and
its regioisomer (110 mg, 69%). The major regioisomer was
assigned based on 1H NMR analysis.32 Separation of isomers
by chromatography on SiO2 (CH2Cl2/MeOH, 50:1) or recrys-
tallization from Et2O/hexanes provided DA296 in ca. 90%
purity as a dark red solid: mp 125 °C (dec). IR (neat): 3271,
2958, 2851, 1683, 1640, 1600, 1564, 1113 cm-1 1H NMR: δ
.
9.24 (s, 1 H), 9.00 (d, 1 H, J ) 5.0 Hz), 7.94 (d, 1 H, J ) 5.0
Hz), 7.13 (br, 1 H), 4.02-3.96 (m, 2 H), 3.78-3.75 (m, 4 H),
2.69 (t, 2 H, J ) 5.6 Hz), 2.54 (m, 4 H). 13C NMR: δ 179.9,
175.0, 156.3, 154.3, 148.5, 138.2, 123.7, 119.1, 118.2, 67.0 (2C),
56.6, 53.0 (2C), 40.9. MS (EI) m/z (rel intensity): 323 ([M +
2]+, 5), 221 (35), 101 (100). HRMS (EI) m/z: calcd for
C
15H18N3O3Cl (M + 2H), 323.1037; found: 323.1037.
DA3002 (6,7-Dich lor oqu in olin e-5,8-d ion e). Prepared in
Exp er im en ta l Section
Libr a r y Ch em ica ls. The Diversity Set and selected quino-
linediones were the kind gift of J ill J ohnson (NCI, National
Institutes of Health, Developmental Therapetuics Program,
Rockville, MD). The Diversity Set was generated by the NCI
from the 140 000 compounds in the NCI Compound Repository
based first on the availability of g1 g in their Repository, which
reduced the total eligible compounds to 71 756. Chem-X
(Oxford Molecular Group) was then used to define hydrogen
bond acceptor, hydrogen bond donor, positive charge, aromatic,
hydrophobic, acid, base, and distance intervals to create a
particular finite set of pharmacophores. The default setting
of 3-point pharmacophores resulted in almost 106 possible
pharmacophores. The Chem-X diverse subset generating func-
tion was used to determine the acceptable conformations of
individual structures and the acceptable pharmacophore for
the conformation. The Diversity Set was populated in a
comparative iterative manner with a requirement of e5
rotatable bonds. Because the selection procedure was order
dependent, the order in which the structures were considered
was randomized. This procedure resulted in the selection of
1990 compounds; more information about the NCI Diversity
Set can be found at http://dtp.nci.nih.gov/.
Syn th esis of Com p ou n d s. All moisture-sensitive reactions
were performed under an atmosphere of dry nitrogen, and all
glassware were dried in an oven prior to use. THF and ether
were dried by distillation over sodium benzophenone and CH2-
Cl2 was dried by distillation over CaH2. Unless otherwise
stated, all commercially available materials were used without
purification. IR spectra were recorded neat using NaCl cells.
NMR spectra were obtained at 300 MHz/75 MHz (1H/13C NMR)
in CDCl3 unless noted otherwise. High- and low-resolution
masses were determined by introduction with a direct insertion
probe into a VG-70-70 HF spectrometer operating in the
electron ionization mode. The purity of synthetic DA3002,
DA296, DA3003-1, DA3003-2, and DA3049 was ascertained
by HRMS and HPLC analyses in two different mobile phases
to be at least 94%, in most cases >99% (see Supporting
Information for details of the analyses).
30-40% yield from quinoline-8-ol according to literature
procedures:20 1H NMR (DMSO-d6, δ): 9.01 (dd, 1 H, J ) 3.8,
1.2 Hz), 8.41 (dd, 1 H, J ) 7.3, 1.2 Hz), 7.86 (dd, 1 H, J ) 7.3,
3.8 Hz). 13C NMR (DMSO-d6, δ): 176.0, 174.3, 154.5, 147.1,
143.1, 141.6, 134.9, 128.5, 128.3. MS (EI) m/z (relative
intensity): 227 (M+, 100), 199 (80), 192 (25), 136 (100). HRMS
(EI) m/z: calcd for C9H3NO2Cl2, 226.9541; found: 226.9545.
DA3003-1 (NSC 663284, 6-Ch lor o-7-(2-m or p h olin -4-yl-
eth ylam in o)qu in olin e-5,8-dion e) an d DA3003-2 (7-Ch lor o-
6-(2-m or p h olin -4-yleth yla m in o)qu in olin e-5,8-d ion e). A
solution of DA3002 (228 mg, 1.00 mmol) and 2-morpholin-4-
ylethylamine (130 mg, 1.00 mmol) in 5 mL of THF was treated
at room temperature with triethylamine (0.14 mL, 1.0 mmol).
The reaction mixture was stirred for 20 h at room temperature,
concentrated under reduced pressure, diluted with ethyl
acetate, and washed with water. The organic layer was dried
(Na2SO4) and concentrated in vacuo. The crude residue was
purified by chromatography on SiO2 (CH2Cl2/MeOH, 15:1) to
give a 2:1 mixture (1H NMR) of DA3003-1 and DA3003-2 (260
mg, 80%). Further separation by chromatography on SiO2
(CH2Cl2/MeOH, 50:1) gave pure DA3003-1 and DA3003-2 as
dark red, amorphous solids. The 5,8-quinolinedione regioiso-
mers were assigned based on the known preference for
nucleophilic addition at the 7-position in aprotic solvents and
the observation that ∆δ (H2-H4) of the 6-isomer is higher
than that of the 7-isomer in 1H NMR.32 5,8-Isoquinolinedione
regioisomers were assigned based on the known preference for
nucleophilic additions at the 7-position33 and the apparent
trend that ∆δ (H1-H4) of the 6-isomer is higher than that of
the 7-isomer in 1H NMR.
DA3003-1. IR (neat): 3275, 2958, 2855, 1695, 1636, 1600,
1
1568, 1109 cm-1. H NMR: δ 8.92 (dd, 1 H, J ) 4.7, 1.6 Hz),
8.48 (dd, 1 H, J ) 7.8, 1.6 Hz), 7.66 (dd, 1 H, J ) 7.8, 4.7 Hz),
7.09 (br, 1 H), 4.05-3.95 (m, 2 H), 3.80-3.70 (m, 4 H), 2.70 (t,
2 H, J ) 5.7 Hz), 2.65-2.45 (m, 4 H). 13C NMR: δ 179.0, 175.4,
153.4, 146.2, 145.3, 134.6, 129.8, 128.4, 67.0 (2C), 56.7, 53.0
(2C), 40.9. MS (EI) m/z (rel intensity): 323 ([M + 2]+, 7), 210
(25), 100 (100). HRMS (EI) m/z: calcd for C15H18N3O3Cl (M +
2H), 323.1037; found: 323.1034. A direct comparison between
DA3003-1 and NSC 663284 showed no detectable difference
in HPLC profiles, chemical properties, enzyme inhibitory
effects, or growth inhibition. Thus, most of the reported cellular
and kinetic studies were performed with NSC 663284 because
of the limited amount of DA3003-1.
DA276 (Qu in olin e-5,8-d ion e). Prepared in 56% yield from
quinoline-8-ol according to literature procedures:31 1H NMR:
δ 9.06 (dd, 1 H, J ) 4.6, 1.6 Hz), 8.43 (dd, 1 H, J ) 7.8, 1.6
Hz), 7.72 (dd, 1 H, J ) 7.8, 4.6 Hz), 7.16 (d, 1 H, J ) 10.0 Hz),
7.07(d, 1 H, J ) 10.0 Hz). HRMS (EI) m/z: calcd for C9H5-
NO2, 159.0320; found: 159.0321.
DA295 (6,7-Dich lor oisoqu in olin e-5,8-d ion e). Prepared
according to literature procedures:30 1H NMR (MeOH-d4, δ):
9.33 (s, 1 H), 9.08 (d, 1 H, J ) 5.1 Hz), 8.08 (d, 1 H, J ) 5.1
Hz). 13C NMR (MeOH-d4, δ): 173.8 (2C), 153.5, 146.7, 141.8,
DA3003-2. IR (neat): 3275, 2958, 2851, 1687, 1647, 1600,
1
1564, 1106 cm-1. H NMR: δ 9.02 (dd, 1 H, J ) 4.6, 1.6 Hz),
8.36 (dd, 1 H, J ) 8.0, 1.6 Hz), 7.59 (dd, 1 H, J ) 8.0, 4.6 Hz),

4048 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24
Lazo et al.
6.98 (br, 1 H), 4.0-3.9 (m, 2 H), 3.8-3.7 (m, 4 H), 2.70 (t, 2 H,
J ) 5.4 Hz), 2.6-2.45 (m, 4 H). 13C NMR: δ 180.3, 175.2, 155.4,
148.6, 144.4, 134.8, 127.0, 126.7, 67.1 (2C), 56.8, 53.1 (2C), 40.8.
MS (EI) m/z (rel intensity): 323 ([M + 2]+, 40), 285 (8) 267
(10), 100 (100). HRMS (EI) m/z: calcd for C15H18N3O3Cl (M +
2H), 323.1037; found: 323.1027.
DA3044 (6,7-Dich lor op h th a la zin e-5,8-d ion e). Prepared
according to literature procedures:33 1H NMR: δ 9.93 (s, 2 H).
MS (EI) m/z (rel intensity): 228 (M+, 100), 200 (30). HRMS
(EI) m/z: calcd for C8H2N2O2Cl2, 227.9493; found: 227.9492.
DA3045 (6,7-Dich lor oqu in a zolin e-5,8-d ion e). Prepared
according to literature procedures:33 1H NMR: δ 9.75 (s, 1 H),
9.64 (s 1 H). MS (EI) m/z (rel intensity): 228 (M+, 100), 200
(30), 165 (30), 110 (30), 87 (40). HRMS (EI) m/z: calcd for
C8H2N2O2Cl2, 227.9493; found: 227.9497.
and the total cell number was determined spectrophotometri-
cally at 540 nm as previously described.34
For the chemical complementation assay, HeLa cells were
obtained from ATCC and were maintained in Dulbecco’s
minimum essential medium (DMEM) containing 10% fetal
bovine serum (FBS, HyClone, Logan, UT) and 1% penicillin-
streptomycin (Life Technologies, Inc., Rockville, MD) in a
humidified atmosphere of 5% CO2 at 37 °C. We used a
mammalian expression plasmid encoding full-length wild-type
Cdc25A in a pcDNA3 vector generously provided by Thomas
Roberts (Dana Farber Cancer Institute, Boston, MA)3 as
previously described.20
Ack n ow led gm en t. This work was supported by the
U.S. Department of Defense, the Fiske Drug Discovery
Fund, and the National Institutes of Health. We thank
Daniel Zaharevitz and J ill J ohnson of the NCI for
providing compounds and advice.
DA3049 (6-Ch lor o-7-(2-m or p h olin -4-yleth yla m in o)p h -
th a la zin e-5,8-d ion e). A solution of DA3044 (67 mg, 0.29
mmol) and 2-morpholin-4-ylethylamine (38 mg, 0.29 mmol) in
5 mL of THF was treated at room temperature with triethy-
lamine (0.04 mL, 0.3 mmol). The reaction mixture was stirred
for 2 h at room temperature, concentrated under reduced
pressure, diluted with ethyl acetate, and washed with water.
The organic layer was dried (Na2SO4) and concentrated in
vacuo. The crude residue was purified by chromatography on
SiO2 (CH2Cl2/MeOH, 15:1) to give pure DA3049 (42 mg, 45%)
as a dark red, amorphous sticky solid. IR (neat): 3271, 2958,
Su p p or tin g In for m a tion Ava ila ble: HPLC analysis
results for DA compounds (with HRMS data). This material
is available free of charge via the Internet http://pubs.acs.org.
Refer en ces
(1) Denu, J . M.; Stuckey, J . A.; Saper, M. A.; Dixon, J . E. Form and
function in protein dephosphorylation. Cell 1996, 87, 361-364.
(2) Nilsson, I.; Hoffmann, I. Cell cycle regulation by the Cdc25
phosphatase family. Prog. Cell Cycle Res. 2000, 4, 107-114.
(3) Xia, K.; Lee, R. S.; Narsimhan, R. P.; Mukhopadhyay, N. K.;
Neel, B. G.; Roberts, T. M. Tyrosine phosphorylation of the proto-
oncoprotein Raf-1 is regulated by Raf-1 itself and the phos-
phatase Cdc25A. Mol. Cell. Biol. 1999, 19, 4819-4824.
(4) Galaktionov, K.; Beach, D. CDC25 phosphatases as potential
human oncogenes. Cell 1991, 67, 1181-1194.
(5) Sadhu, K.; Reed, S. I.; Richardson, H.; Russell, P. Human
homolog of fission yeast Cdc25 mitotic inducer is predominantly
expressed in G2. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 5139-
5143.
(6) Nagata, A.; Igarashi, M.; J inno, S.; Suto, K.; Okayama, H. An
additional homolog of the fission yeast cdc25+ gene occurs in
humans and is highly expressed in some cancer cells. New Biol.
1991, 3, 959-968.
(7) Galaktionov, K.; Lee, A. K.; Eckstein, J .; Draetta, G.; Meckler,
J .; Loda, M.; Beach, D. CDC25 phosphatases as potential human
oncogenes. Science 1995, 269, 1575-1577.
2923, 2855, 2808, 1695, 1636, 1556, 1311, 1295, 1113 cm-1
.
1H NMR: δ 9.83 (s, 1 H), 9.65 (s, 1 H), 7.18 (br, 1 H), 4.00-
3.90 (m, 2 H), 3.75-3.70 (m, 4 H), 2.69 (t, 2 H, J ) 6.0 Hz),
2.55-2.50 (m, 4 H). 13C NMR: δ 180.5, 174.2, 171.4, 147.2,
145.3, 144.5, 124.8, 123.3, 67.1 (2C), 56.3, 53.0 (2C), 41.0. MS
(EI) m/z (rel intensity): 324 ([M + 2]+, 10), 286 (12), 256 (8),
235 (12) 100 (100). HRMS (EI) m/z: calcd for C14H17N4O3Cl
(M + 2H), 324.0989; found: 324.0989.
DA3100 (6-Ch lor o-7-(in d a n -2-yla m in o)qu in olin e-5,8-d i-
on e). According to the procedure described for DA3003-1,
DA3100 (192 mg, 59%) was obtained from DA3002 (228 mg,
1.00 mmol) and 1-aminoindane (133 mg, 1.00 mmol) as a dark
red, amorphous sticky solid. IR (neat): 3323, 3065, 2939, 2844,
1
1695, 1640, 1592, 1560, 1315, 721 cm-1. H NMR: δ 8.92 (dd,
1 H, J ) 4.6, 1.4 Hz), 8.49 (dd, 1 H, J ) 7.8, 1.4 Hz), 7.67 (dd,
1 H, J ) 7.8, 4.6 Hz), 7.29-7.22 (m, 4 H), 6.40 (br, 1 H), 6.18-
6.10 (m, 1 H), 3.13-2.92 (m, 2 H), 2.78-2.72 (m, 1 H), 2.13-
2.06 (m, 1 H). 13C NMR: δ 178.8, 175.8, 153.5, 146.0, 144.2,
143.5, 142.4, 134.8, 130.0, 128.8, 128.5, 127.3, 125.2, 124.5,
59.8, 36.3, 20.2. MS (EI) m/z (rel intensity): 324 (M+, 30), 287
(15), 220 (15), 205(35), 117 (100). HRMS (EI) m/z: calcd for
(8) Galaktionov, K.; Chen, X.; Beach, D. Cdc25 cell-cycle phos-
phatase as a target of c-myc. Nature 1996, 382, 511-517.
(9) Wu, W.; Fan, Y.-H.; Kemp, B. L.; Walsh, G.; Mao, L. Overex-
pression of cdc25A and cdc25B is frequent in primary non-small
cell lung cancer but is not associated with overexpression of
c-myc. Cancer Res. 1998, 58, 4082-4085.
C
18H13N2O2Cl, 324.0666; found: 324.0656.
(10) Giulia Cangi, M.; Cukor, B.; Soung, P.; Signoretti, S.; Moreira,
J ., G.; Ranashinge, M.; Cady, B.; Pagano, M.; Loda, M. Role of
the Cdc25A phosphatase in human breast cancer. J . Clin. Invest.
2000, 106, 753-761.
(11) Millar, J . B.; Blevitt, J .; Gerace, L.; Sadhu, K.; Featherstone,
C.; Russell, P. p55CDC25 is a nuclear protein required for the
initiation of mitosis in human cells. Proc. Natl. Acad. Sci. U.S.A.
1991, 88, 10500-10504.
(12) Hoffman, I.; Draetta, G.; Karsenti, E. Activation of the phos-
phatase activity of human cdc25A by a cdk2-cyclin E dependent
phosphorylation at the G1/S transition. EMBO J . 1994, 13,
4302-4310.
(13) J inno, S.; Suto, J .; Nagata, A.; Igarashi, M.; Kanaoka, Y.; Nojima,
H.; Okayama, H. Cdc25A is a novel phosphatase functioning
early in the cell cycle. EMBO J . 1994, 13, 1549-1556.
(14) Molinari, M.; Mercurilo, C.; Dominguez, J .; Goubin, F.; Draetta,
G. F. Human Cdc25 A inactivation is response to S phase
inhibition and its role in preventing premature mitosis. EMBO
Rep. 2000, 1, 71-79.
(15) Mailand, N.; Falck, J .; Lukas, C.; Syljuasen, R. G.; Welcker, M.;
Lukas, J . Rapid destruction of human Cdc25A in response to
DNA damage. Science 2000, 288, 1425-1429.
(16) Fauman, E. B.; Cogswell, J . P.; Lovejoy, B.; Rocque, W. J .;
Holmes, W.; Montana, V. G.; Piwnica-Worms, H.; Rink, M. J .;
Saper, M. A. Crystal structure of the catalytic domain of the
human cell cycle control phosphatase, Cdc25A. Cell 1998, 93,
617-625.
(17) Reynolds, R. A.; Yem, A. W.; Wolfe, C. L.; Deibel, J . M. R.;
Chidester, C. G.; Watenpaugh, K. D. Crystal structure of the
catalytic subunit of Cdc25B required for G2.M phase transition
of the cell cycle. J . Mol. Biol. 1999, 293, 559-568.
In Vitr o En zym e Assa ys. The activities of the GST-fusion
Cdc25A, Cdc25B2, Cdc25C, and VHR as well as human
recombinant PTP1B were measured using o-methyl fluorescein
phosphate (Sigma, St. Louis, MO) as substrate and a minia-
turized, 96-well microtiter plate assay based on previously
described methods.23 The final incubation mixtures (25 µL)
were prepared using a Biomek 2000 laboratory automation
workstation (Beckman Coulter, Inc., Fullerton, CA). Fluores-
cence emission from the product was measured after a 60-min
incubation period at ambient temperature with a multiwell
plate reader (PerSeptive Biosystems Cytofluor II; Framing-
ham, MA; excitation filter, 485/20; emission filter, 530/30). Best
curve fit for Lineweaver-Burk plots and Ki values were
determined by using the curve-fitting programs Prism 3.0
(GraphPad Software, Inc., San Diego, CA) and SigmaPlot 2000
Enzyme Kinetics (SSPS Science, Richmond, CA).
An tip r olifer a tive a n d Ch em ica l Com p lem en ta tion As-
sa ys. The proliferation of human MCF-7 breast cancer cells
was measured by a previously described colorimetric assay.34
Briefly, we seeded 2000-4000 cells per well in microtiter plates
and allowed them to attach overnight. Cells were then treated
with vehicle or compound continuously or for 3 h. After a 48-h
incubation in 5% CO2 atmosphere and 100% humidity, the
medium was replaced with serum-free medium containing
0.1% 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide. Plates were incubated for an additional 3 h in the dark,

Inhibitors of Protein Phosphatase Cdc25
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 24 4049
(18) Rudolph, J .; Epstein, D. M.; Parker, L.; Eckstein, J . Specificity
of natural and artificial substrates for human Cdc25A. Anal.
Biochem. 2001, 289, 43-51.
(19) Chen, W.; Wilborn, M.; Rudolph, J . Dual-specific Cdc25B phos-
phatase in search of the catalytic acid. Biochemistry 2000, 39,
10781-10789.
a synthetic thioalkyl vitamin K analogue. Cancer Res. 2000, 60,
1317-1325.
(27) Fritzen, E. L.; Brightwell, A. S.; Erickson, L. A.; Romero, D. L.
The solid phase synthesis of tetrahydroisoquinolines having
Cdc25B inhibitory activity. Bioorg. Med. Chem. Lett. 2000, 649-
652.
(20) Vogt, A.; Adachi, T.; Ducruet, A. P.; Chesebrough, J .; Nemoto,
K.; Carr, B. I.; Lazo, J . S. Spatial analysis of key signaling
proteins by high-content solid-phase cytometry in Hep3B cells
treated with an inhibitor of Cdc25 dual specificity phosphatases.
J . Biol. Chem. 2001, 276, 20544-20550.
(21) Gunasekera, S. P.; McCarthy, P. J .; Kelly-Borges, M. Dysid-
iolide: A novel protein phosphatase inhibitor from the Caribbean
sponse dysidea etheria de laubenfels. J . Am. Chem. Soc. 1996,
118, 8759-8760.
(22) Cebula, R. E.; Blanchard, J . L.; Boisclair, M. D.; Pal, K.;
Bockovich, N. J . Synthesis and phosphatase inhibitory activity
of analogs of sulfircin. Bioorg. Med. Chem. Lett. 1997, 7, 2015-
2020.
(23) Rice, R. L.; Rusnak, J . M.; Yokokawa, F.; Yokokawa, S.; Messner,
D. J .; Boynton, A. L.; Wipf, P.; Lazo, J . S. A targeted library of
small molecule, tyrosine and dual specificity phosphatase inhibi-
tors derived from a rational core design and random side chain
variation. Biochemistry 1997, 36, 15965-15974.
(28) Behforouz, M.; Haddad, J .; Cai, W.; Gu, Z. Chemistry of
quinoline-5,8-diones. J . Org. Chem. 1998, 63, 343-346.
(29) Boger, D. L.; Yasuda, M.; Mitscher, L. A.; Drake, S. D.; Kitos,
P. A.; Thompson, S. C. Streptonigrin and lavendamycin partial
structures. Probes for the minimal, potent pharmacophore of
streptonigrin, lavendamycin, and synthetic quinoline-5,8-diones.
J . Med. Chem. 1987, 30, 1918-1928.
(30) Ryu, C.-K.; Lee, I.-K.; J ung, S.-H.; Lee, C.-O. Synthesis and
cytotoxicity of 6-chloro-7-arylamino-5,8-isoquinolinediones. Bioorg.
Med. Chem. Lett. 1999, 9, 1075-1080.
(31) Cameron, D. W.; Deutscher, K. R.; Feutrill, G. I. Nucleophilic
alkenes. IX. Addition of 1,1-dimethoxyethene to azanaphtho-
quinones: Synthesis of bostrycoidin and 8-O-methylbostrycoidin.
Aust. J . Chem. 1982, 35, 1439-1450.
(32) Yoon, E. Y.; Choi, H. Y.; Shin, K. J .; Yoo, K. H.; Chi, D. Y.; Kim,
D. J . The regioselectivity in the reaction of 6,7-dihaloquinoline-
5,8-diones with amine nucleophiles in various solvents. Tetra-
hedron Lett. 2000, 41, 7475-7480.
(24) Ducruet, A. P.; Rice, R. L.; Tamura, K.; Yokokawa, F.; Yokokawa,
S.; Wipf, P.; Lazo, J . S. Identification of new Cdc25 dual
specificity phosphatase inhibitors in a targeted small molecule
array. Bioorg. Med. Chem. 2000, 8, 1451-1466.
(33) Shaikh, I. A.; J ohnson, F.; Grollman, A. P. Streptonigrin. 1.
Structure-activity relationships among simple bicyclic ana-
logues. Rate dependence of DNA degradation on quinone reduc-
tion potential. J . Med. Chem. 1986, 29, 1329-1340.
(34) Vogt, A.; Rice, R. L.; Settineri, C. E.; Yokokawa, F.; Yokokawa,
S.; Wipf, P.; Lazo, J . S. Disruption of insulin-like growth factor-1
signaling and down-regulation of Cdc2 by SC-RRδ9, a novel small
(25) Peng, H.; Xie, W.; Otterness, D. M.; Cogswell, J . P.; McConnell,
R. T.; Carter, H. L.; Powis, G.; Abraham, R. T.; Zalkow, L. H.
Synthesis and biological activities of a novel group of steroidal
derived inhibitors for human Cdc25A protein phosphatase. J .
Med. Chem. 2001, 44, 834-848.
molecule antisignaling agent identified in
a targeted array
library. J . Pharmacol. Exp. Therap. 1998, 287, 806-813.
(26) Tamura, K.; Southwick, E. C.; Kerns, J .; Rosi, K.; Carr, B. I.;
Wilcox, C.; Lazo, J . S. Cdc25 inhibition and cell cycle arrest by
J M0102046