Design, synthesis, and biological evaluation of novel naphthoquinone derivatives with CDC25 phosphatase inhibitory activity

Article abstract of DOI:10.1016/j.bmc.2005.05.005

CDC25 dual-specificity phosphatases are essential key regulators of eukaryotic cell cycle progression and the CDC25A and B isoforms are over-expressed in different tumors and related cancer cell lines. CDC25s are now considered to be interesting targets in the search for novel anticancer agents. We describe new compounds derived from vitamin K₃ that inhibit CDC25B activity with IC₅₀ values in the low micromolar range. These naphthoquinone derivatives also display antiproliferative activity on HeLa cells as expected for CDC25 inhibitors and inhibit cell growth in a clonogenic assay at submicromolar concentrations. They increase inhibitory tyrosine 15 phosphorylation of CDK and induce the cleavage of PARP, a hallmark of apoptosis.

Full text of DOI:10.1016/j.bmc.2005.05.005

Bioorganic & Medicinal Chemistry 13 (2005) 4871–4879
Design, synthesis, and biological evaluation
of novel naphthoquinone derivatives with CDC25
phosphatase inhibitory activity
a
a
a
b
´
Marie-Priscille Brun, Emmanuelle Braud, Delphine Angotti, Odile Mondesert,
Muriel Quaranta,b Matthieu Montes,a Maria Miteva,a Nohad Gresh,a
Bernard Ducommunb and Christiane Garbaya,*
a
´
´
Laboratoire de Pharmacochimie Moleculaire et Cellulaire INSERM U648—CNRS FRE 2718, UFR Biomedicale,
`
45 rue des Saints Peres, 75270 Paris Cedex 06, France
´
´
Universite Paul Sabatier, Route de Narbonne, 31062 Toulouse, France
b
ˆ
´
Laboratoire de Biologie Cellulaire et Moleculaire du Controle de la Proliferation UMR 5088—IFR 109
Received 21 March 2005; revised 27 April 2005; accepted 3 May 2005
Available online 24 May 2005
Abstract—CDC25 dual-specificity phosphatases are essential key regulators of eukaryotic cell cycle progression and the CDC25A
and B isoforms are over-expressed in different tumors and related cancer cell lines. CDC25s are now considered to be interesting
targets in the search for novel anticancer agents. We describe new compounds derived from vitamin K3 that inhibit CDC25B activity
with IC50 values in the low micromolar range. These naphthoquinone derivatives also display antiproliferative activity on HeLa cells
as expected for CDC25 inhibitors and inhibit cell growth in a clonogenic assay at submicromolar concentrations. They increase
inhibitory tyrosine 15 phosphorylation of CDK and induce the cleavage of PARP, a hallmark of apoptosis.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
injection of CDC25A antibodies into cells induces cell
arrest by blocking G1/S transition.8,9 It has also been
suggested that CDC25A is involved in mitosis.10
CDC25B and CDC25C are activators of Cdc2/cyclin
B, Cdc2/cyclin A, and CDK2/cyclin A complexes and
play a role in the G2/M transition and in the control
of entry into mitosis.11,12
The accurate transduction of intracellular signals is
achieved through processes of phosphorylation–dephos-
phorylation of cyclin-dependent kinase (CDK) com-
plexes, which are key regulators of essential cellular
events such as cell cycle regulation, growth, or differenti-
ation. CDKs are activated by the CDC25 dual-specificity
phosphatases, which catalyze the dephosphorylation of
two adjacent phosphothreonine and phosphotyrosine
residues on the CDK subunit.1,2
CDC25A and B have been reported to present onco-
genic properties.13 Furthermore, they are found to be
over-expressed in many tumors with poor prognosis
including head and neck cancers, colon cancers, non-
small cell lung, and breast cancers.14–16 Thus, the discov-
ery of potent CDC25s inhibitors appears as an attractive
approach in the search for new targeted anticancer
agents.
Three genes expressing CDC25 have been identified in
human cells, namely CDC25A, CDC25B, and
CDC25C,3–5 and several splice variants have been char-
acterized.6,7 CDC25A, expressed in the G1 phase, allows
the progression of the cell into the S phase by dephos-
phorylation of the CDK2/cyclin A complex.8–10 Micro-
Several classes of CDC25s inhibitors have been reported
so far showing cytotoxicity in the micromolar concen-
tration range (Chart 1). They include natural substances
Keywords: Cancer; Signal transduction; CDC25 phosphatases;
Naphthoquinones.
and their synthetic derivatives such as dysidiolide17 or
sulfiricins,18 analogs of vitamin K3
and D3,21,22 as
19,20
*
Corresponding author. Tel.: +33 142 864 080; fax: +33 142 864
082; e-mail: christiane.garbay@univ-paris5.fr
well as naphthoquinones,23,24 quinolinediones,25 or
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.05.005

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M.-P. Brun et al. / Bioorg. Med. Chem. 13 (2005) 4871–4879
Chart 1. Inhibitors of CDC25 phosphatases.
to 2-methyl-[1,4]-naphthoquinone or 2,3-dichloro-[1,4]-
naphthoquinone, followed by subsequent air-oxidation
of naphthohydroquinones to the naphthoquinone core.
The carboxylic acid 1 was obtained by nucleophilic at-
tack of mercaptoacetic acid at position 3 of menadione
in the presence of DBU following the method described
by Lazo et al., applied to synthesize Cpd 5 (Scheme 1).31
To prepare the di-acid compound 2, which is an analog
of NSC 95397, 2,3-dichloronaphthoquinone was reacted
with 2 equivalents of mercaptoacetic acid, DBU being
replaced by pyridine.33 The temperature was maintained
at 65 ꢁC and 2 precipitated in high yield (89%) within
15 min (Scheme 1). We applied the same conditions to
synthesize the benzyl ester 4 but in this case we did
not observe the formation of a precipitate. However,
monitoring the reaction by TLC revealed the presence
of two major products and the heating was maintained
at 65 ꢁC for 1 h. The mono-ester 3 and the di-ester 4
were isolated after purification in, respectively, 42%
and 36% yields, with a greater solubility of the ester ad-
ducts and a thermodynamic equilibration between 3 and
4 and the starting material explaining the orientation of
the reaction in favor of compound 3 (Scheme 1).
Chart 2. Chemical structures of novel vitamin K3 derivatives.
naphthofurane-diones.26 Recently, Brezak et al. de-
scribed a new CDC25 inhibitor BN82002 (IC50
=
6.3 lM on CDC25B3) and its analogs.27,28 In our search
for new CDC25 inhibitors, we were interested in vitamin
K3 and its thio-analogs Cpd 5 and NSC 95397 (Chart 1).
Indeed, Cpd 5 has been reported to inhibit CDC25A
with an IC50 value of 5.6 lM in Hep3B hepatoma
cells and to arrest cell cycle progression at G1 and
G2/M,29,30 whereas NSC 95397 was claimed to be one
of the most potent CDC25 inhibitors with an IC50 value
in the nanomolar range and to block the G2/M
transition.31
The recently solved X-ray crystallographic structure of
the catalytic domain of CDC25B32 has revealed the
presence of four arginine residues (Arg479, 482, 544,
and 548) located in the active site of the enzyme. As
the substrate is recognized by its phosphate group, we
decided to introduce carboxylate groups, bioisosteres
of the phosphate, to target these residues. Thus, we de-
scribe in the present paper the design and synthesis of
novel derivatives of vitamin K3 (Chart 2) as well as
the biological results obtained in terms of inhibition of
CDC25B3, antiproliferative activity on HeLa cells, and
the effect on cell cycle progression. Since our design of
carboxylic derivatives did not result in increased activ-
ity, we performed molecular modeling studies to under-
stand the potential roles of these negatively charged
groups with regard to binding affinity. Moreover, we re-
placed the sulfur atom by a nitrogen atom to examine
the effects of a restricted conformation of the substituted
alkyl chain.
In the N-substituted series, the 3-methyl compounds 5,
6, and 7 were obtained by addition of the corresponding
primary amines to menadione whereas the chloro deriv-
atives 8 and 9 were synthesized from 2,3-dichloro-[1,4]-
naphthoquinone in the presence of the corresponding
amines and triethylamine in, respectively, 46% and
59% yields (Scheme 2). Finally, the hydroxyl derivative
8 was converted into the bromo derivative 10 using mild
conditions (CBr4/PPh3) in 72% yield (Scheme 2).34 This
compound was originally synthesized to introduce other
bioisosteric groups of the phosphate such as malonate
or phosphonate groups. Unfortunately, these com-
pounds could not be obtained by this method.
3. Results and discussion
3.1. Enzymatic activity
2. Chemistry
All our compounds were evaluated for in vitro enzymatic
inhibition using recombinant fused MBP-CDC25B3
(one of the identified splicing variants of CDC25B) with
The sulfur and nitrogen series were synthesized using
Michael-type 1,4-addition of primary amines and thiols

M.-P. Brun et al. / Bioorg. Med. Chem. 13 (2005) 4871–4879
4873
Scheme 1. Reagents and conditions: (a) HS–CH2–CO2H (1 equiv), DBU, ether, rt; (b) HS–CH2–CO2H (2 equiv), pyridine, toluene, 65 ꢁC; (c) HS–
CH2–CO2Bn (2 equiv), pyridine, toluene, 65 ꢁC.
The four derivatives of the S-series 1–4 are the most po-
tent in vitro CDC25B3 inhibitors with IC50 values rang-
ing from 1.75 to 4.73 lM, similar or inferior to those of
Cpd 5 and NSC 95397. The di-acid 2 and the di-ester 4
with IC50 values of 1.75 and 3.40 lM are slightly more
potent CDC25 inhibitors than their mono-substituted
analogs 1 and 3 with IC50 values of 4.13 and 3.95 lM,
respectively. Thus, the replacement of the hydroxyl
groups of Cpd 5 and NSC 95397 by carboxylic groups
maintains the inhibitory potency but does not lead to
the expected increase of activity.
To analyze the possible interactions of vitamin K3 and
compounds 1 and 2 with the catalytic domain of
CDC25, molecular modeling was performed using the
docking programs Surflex36 and Ligandfit.37 The docked
Scheme 2. Reagents and conditions: (a) R–NH2, Et2O, rt; (b) R–NH2,
Et3N, Et2O, rt; (c) CBr4, PPh3, anhydrous CH2Cl2, rt.
poses with the most energetically favorable binding are
fluorescein diphosphate (FDP) as the substrate (Table 1).
shown in Figure 1. Our results obtained by both docking
We synthesized Cpd 5 and NSC 95397, which were tested
methods propose that vitamin K3 binds in the inhibitor
as references and showed, respectively, IC50 values of
binding pocket defined by Kristjansdottir et al.35 (Fig.
3.65 and 3.77 lM under our assay conditions. Surpris-
1A). In such a binding mode, vitamin K3 is involved
ingly, NSC 95397 gave an in vitro IC50 value in the
in strong vdW interactions with the pocket residues. In
micromolar range, although it was previously reported
addition, a salt bridge between one oxygen of the naph-
to inhibit CDC25B with a nanomolar concentration.31
thoquinone core and Arg482 was predicted by both Sur-
However, such discrepancies have already been reported
flex and Ligandfit methods. Our docking experiments
for several CDC25 inhibitors of the naphthoquinone
carried out with both programs suggest that compounds
series.35
1 (Fig. 1B) and 2 (Fig. 1C) bind into the active site and
that the carboxylate group might occupy the phosphate
binding pocket attracted by Arg479. Compound 2, con-
taining a second carboxylic group, could interact with
Table 1. In vitro inhibition of purified recombinant CDC25B3
IC50 SD (lM)
Cpd 5
NSC 95397
3.65 0.2
3.77 0.9
4.13 0.8
1.75 0.1
3.95 0.5
3.40 0.3
8.85 0.2
31.50 9.9
ꢀ100
Arg482 (proposed by Ligandfit) or Arg544 (proposed
by Surflex) and form a salt bridge. We hypothesized that
such a salt bridge should stabilize the binding of the li-
gand in the active site, thus higher activity of compound
2 in comparison with compound 1 should be expected,
to a higher extent than experimentally observed.
1
2
3
4
5
6
The results of our docking experiments propose different
binding modes for compounds 1 and 2 compared to
vitamin K3. The strong interaction of the carboxylate
group of compounds 1 or 2 with Arg479 of the active
site most likely brings the naphthoquinone core out of
the floor of the inhibitor binding pocket. Presence of hy-
droxyl groups should not lead to such effects. Thus, a
similar binding mode for vitamin K3 and the hydroxyl
7
8
15.40 2.7
10.55 0.4
10.50 0.1
9
10
Enzyme inhibition was assayed on recombinant MBP-CDC25B3
protein using fluorescein 3,6-diphosphate as the substrate. The IC50
and the SD were calculated from at least three independent experi-
ments with three determinations per tested concentration.

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M.-P. Brun et al. / Bioorg. Med. Chem. 13 (2005) 4871–4879
than the sulfur atom, unfortunately led this second ser-
ies of molecules to a low, but general, decrease of the
inhibitory potency. Thus, the hydroxyl derivative 5
exhibited an IC50 value of 8.85 lM versus 3.65 lM for
Cpd 5. Contrary to the sulfur derivatives, the replace-
ment of the methyl group by a chlorine on the quinone
moiety (compound 8) significantly diminished the inhib-
itory activity (IC50 = 15.40 lM vs 8.85 lM for com-
pound 5). Increasing the length of the alkyl chain led
to naphthoquinone 6, which also showed a severe de-
crease of its inhibitory activity with an IC50 value of
31.50 lM. To evaluate the potential role of a hydrogen
bond, the replacement of the terminal hydroxyl group
by a methyl led to compound 7, which showed no activ-
ity at doses up to 100 lM. The morpholino analog 9 was
less potent than its parent compound NSC 663284
(IC50 = 0.2 lM on CDC25B2) with an IC50 value of
10.55 lM, confirming the importance of a quinolinedi-
one nucleus.25 Finally, replacement of the hydroxyl
group of compound 6 by a bromine atom led to 10,
which interestingly showed an increase of activity with
an IC50 value of 10.50 lM.
3.2. Cellular activity
The cytotoxicity of all compounds was evaluated on
HeLa cells treated with increasing concentrations of
inhibitors, using a WST-1 colorimetric test (Table 2).
Cpd 5 and NSC 95397 were also tested under our assay
conditions showing IC50 values of 8.8 and 10.8 lM,
respectively. Most of our compounds displayed cyto-
toxic activity (IC50 values ranging from 3.5 to 30 lM),
as expected for CDC25 inhibitors, the most active being
the bromo derivative 10. In the S-series, the benzyl ester
derivatives 3 and 4, prepared as prodrugs, were more
cytotoxic than their free-carboxylic homologs 1 and 2
as expected. Thus, the di-acid 2 and its benzyl
ester analog 4 showed IC50 values of 85.3 and
11.2 lM, respectively. We could also observe that the
Figure 1. Molecular modeling. Inhibitor docked poses obtained with
Surflex (in green) and Ligandfit (gray). CDC25B is shown as ribbon
(white/yellow). The inhibitor binding pocket and the active site region
are shown in purple. (A) Vitamin K3; (B) compound 1; and (C)
compound 2.
Table 2. In vitro growth inhibition of HeLa cell line
derivatives (Cpd 5 and NSC 95397) could be suggested.
Hence, the hydroxyl and the carboxylic derivatives
could interact differently with CDC25, and thus the sub-
stitution of hydroxyl (in Cpd 5 and NSC 95397) by car-
boxylic groups (in compounds 1 and 2) does not lead to
an increase in inhibitory activity.
Antiproliferative assay
IC50 SD (lM)
Clonogenic assay
IC50 SD (lM)
Cpd 5
NSC 95397
8.8 3.8
10.8 2.9
17.8 1.3
85.3 6.0
9.6 1.7
4.1 0.1
8.5 0.7
n.d.a
n.d.a
1
2
3
3.9 0.6
4.77 1.3
n.d.a
4
11.2 1.0
25.7 2.9
69.5 20.1
>100 lM
22.7 1.5
16.3 3.2
3.5 0.2
The benzyl esters 3 and 4, initially synthesized to im-
prove cell penetration, were revealed to be as potent as
their carboxylic free analogs 1 and 2, supporting a differ-
ent mode of binding for these compounds. In fact, if the
carboxylic group of compounds 1 and 2 interacts just
like the phosphate of CDK, as suggested by our model-
ing results, compounds 3 and 4 should not interact in the
same way with CDC25. Finally, the introduction of a
chlorine atom at position 2 (compound 3), instead of a
methyl group, had no significant effect on the inhibitory
activity.
5
6
n.d.a
7
n.d.a
8
n.d.a
9
10
n.d.a
0.85 0.1
Growth inhibition was assayed on HeLa cells using either the WST-1
cell proliferation assay or the crystal violet clonogenic assay. For the
antiproliferative assay, the IC50 and the SD reported were calculated
from at least three independent experiments with eight determinations
per tested concentration. For the clonogenic assay, the IC50 and the
SD reported were calculated from at least two independent experi-
ments with two determinations per tested concentrations.
a Not determined.
In the N-substituted series, replacement of the sulfur by
a nitrogen atom, more conjugated to the quinone moiety

M.-P. Brun et al. / Bioorg. Med. Chem. 13 (2005) 4871–4879
4875
mono-substituted naphthoquinones Cpd 5, 1, and 3 were
more efficient than the corresponding di-substituted ana-
logs NSC 95397, 2, and 4. In the N-series, increasing the
alkyl chain length induced a severe decrease of toxicity
when compared to compound 5 (IC50 = 25.7 lM) and
compound 6 (IC50 = 69.5 lM). Moreover, suppressing
the terminal hydroxyl group led to 7, which showed no
cytotoxicity (IC50 > 100 lM), confirming the importance
of a H-bond donor group to anchor the inhibitor in the
catalytic domain of the enzyme. Finally, the replacement
of the methyl group at position 3 on the quinone ring of
compound 5 by a chlorine increased slightly the activity,
as 8 has an IC50 of 22.7 lM. In conclusion, we could ob-
serve a correlation between in vitro inhibitory potency
and antiproliferative activity of the synthesized com-
pounds. Compound 10 displayed an antiproliferative
activity higher than its in vitro phosphatase inhibitory
activity, suggesting that it might trigger targets other
than CDC25s. Indeed, the presence of the reactive bro-
momethyl group can lead to substitution reactions with
nucleophilic species within the cells.
Figure 2. Effect of compound 1 on CDK phosphorylation status. After
treatment with compound 1 at the indicated concentrations for 8 h,
HeLa cells were lysed and processed for Western blot analysis using
antibodies against tyrosine 15-phosphorylated CDKs and actin.
Phospho-cdc2 (Tyr15) detects endogenous levels of Cdc2, CDK1,
and CDK5 only when phosphorylated at tyrosine 15.
The effects on cell proliferation were further investigated
in a clonogenic assay for the more cytotoxic com-
pounds, namely compounds 3, 4, and 10 (Table 2).
HeLa cells were treated at day 1 with increasing concen-
trations of drugs and then plated for 10 additional days.
After crystal violet staining and colony counting, dose–
response curves were drawn and the IC50 was deter-
mined. Each of the tested compounds was effective at
inhibiting clonal proliferation of the HeLa cells in a con-
centration-dependent manner with IC50 ranging from
0.85 to 8.5 lM, the most potent being the bromo deriv-
ative 10 with a submicromolar IC50 value. As in the pro-
liferation assay, the mono-substituted derivatives Cpd 5
and compound 3 exhibited IC50 values of 4.1 and
3.9 lM inferior to those of the di-substituted NSC
95397 and compound 4, which showed, respectively,
IC50 values of 8.5 and 4.77 lM. Moreover, the three
tested compounds 3, 4, and 10 were more potent than
NSC 95397.
Figure 3. Effect of compound 1 on apoptosis. After treatment with
compound 1 at the indicated concentrations for 8 or 16 h, HeLa cells
were lysed and processed for Western blot analysis using antibodies
against full and cleaved PARP and actin.
inhibits CDC25 phosphatase activity in these cells.
The effect of this compound on cell cycle progression
was therefore examined.
HeLa cells were used to investigate the effect of com-
pounds 1 and 10 on cell cycle progression by flow
cytometry determination of their DNA content after
propidium iodide staining. After 24 h of treatment with
10 lM of inhibitor, a concentration that inhibited prolif-
eration (Table 2), the cell cycle distribution was only
modestly affected with a slight increase in cells contain-
ing a G2 DNA content, suggesting that the cells treated
with either compound 1 or 10 were arrested at various
stages of the cell cycle (data not shown).
3.3. Molecular characterization of the cellular effects
Inhibition of CDC25 phosphatases should induce the
accumulation of the hyperphosphorylated form of their
natural substrate, namely the CDK kinases. As CDK1
(Cdc2) tyrosine 15 phosphorylation is experimentally
accessible, we investigated the molecular basis of the
inhibitory activity of our compounds by monitoring,
on HeLa cells, the effect of compound 1 on this phos-
phorylation status. HeLa cells were treated with
increasing concentrations of compound 1 for 8 h, lysed,
and processed for Western blot analysis using an anti-
body directed against the 15-phosphorylated tyrosine
form of the CDK kinases. Actin was used as loading
control. As shown in Figure 2, compound 1 at
10 lM induced a moderate accumulation of the 15-
phosphorylated tyrosine form of CDK in a concentra-
tion-dependent manner. The inhibition of CDK
dephosphorylation suggests that compound 1 down-
regulates the phosphatase activity of CDC25 toward
these kinases, indicating that treatment with this drug
As we noticed that compound 1 induced a high level of
mortality on HeLa cells at high concentration, we inves-
tigated whether cell death occurred through an apopto-
sis process by monitoring the cleavage of poly-(ADP-
ribose)-polymerase (PARP). This death substrate is a fi-
nal common pathway of drug-induced apoptosis and is
specifically cleaved during apoptosis to produce a
85 kDa fragment.38 As shown in Figure 3, both concen-
trations of compound 1 induced cleavage of the 116 kDa
polypeptide to the 85 kDa fragment after 8 h of
treatment.
4. Conclusion
In conclusion, we have developed two new series of vita-
min K3 derivatives that exhibited in vitro inhibition of
CDC25 activity in the low micromolar range. The
introduction of a carboxylic group, bioisostere of the

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M.-P. Brun et al. / Bioorg. Med. Chem. 13 (2005) 4871–4879
phosphate, did not significantly increase the CDC25
inhibitory activity (compounds 1 and 2). The corre-
sponding esters 3 and 4 proved to be interesting, as they
were as potent as their carboxylic analogs, suggesting a
different way of interacting with the enzyme. The substi-
tution of the alkyl chain also seemed to be essential, as
removing a H-bond donor group led to a decrease of
activity that may be linked to a loss of a hydrogen bond
(compounds 6/7). As compound 10 exhibited the best
antiproliferative activity but might inhibit targets other
than CDC25s, it will be further investigated. Finally,
we could observe that compound 1 caused increased
phosphorylation of CDK, indicating that the CDC25
phosphatase family is, at least, one of the targets of
our compounds and that it induced the cleavage of
PARP, indicating that apoptosis was triggered by this
molecule.
(30 mL). The solution turned red and the stirring was
maintained for 12 h at room temperature. HCl 3.6 M
(4 mL) was then added and the aqueous phase was ex-
tracted with ether (2 · 20 mL). The organic layers were
combined, dried (Na2SO4), and the solvent was evapo-
rated under reduced pressure. The residue was purified
by flash chromatography (EtOAc/cyclohexane, 3:7) to
give 1 as an orange powder (638 mg, 42%); mp:
156 ꢁC; 1H NMR (CDCl3): d 8.09–8.04 (2H, m, H5
and H8), 7.73–7.65 (2H, m, H6 and H7), 3.96 (2H, s,
CH2), 2.36 (3H, s, CH3). Anal. Calcd for C14H10O6S2:
C, 59.54; H, 3.81. Found: C, 59.44; H, 4.16.
5.5. (3-Carboxymethylsulfanyl-1,4-dioxo-1,4-dihydro-
naphthalen-2-ylsulfanyl) acetic acid (2)
Compound 2 was prepared following a reported proce-
dure with minor modifications.33 Pyridine (0.8 mL)
was added to a solution of 2,3-dichloro-[1,4]-naphtho-
quinone (1 g, 4.4 mmol) and mercaptoacetic acid
(0.70 mL, 10.12 mmol) in toluene (16 mL) heated at
55 ꢁC. After pyridine addition, the temperature in-
creased and was maintained at 65 ꢁC. Within 15 min, a
red precipitate formed. The reaction mixture was then
cooled to room temperature, filtered, and the precipitate
rinsed with water before being purified by flash chro-
matography (CH2Cl2/MeOH/AcOH, 95:5:2) to give 2
5. Experimental
5.1. Abbreviations
DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene;DMF, dimethyl-
formamide; DMSO, dimethylsulfoxide; TEA, triethyl-
amine; and TFA, trifluoroacetic acid.
1
5.2. Chemicals
as a red powder (1.31 g, 89%); mp: 194 ꢁC; H NMR
(DMSO-d6): d 8.02–7.99 (2H, m, H5 and H8), 7.89–
7.86 (2H, m, H6 and H7), 4.13 (4H, s, 2· S–CH2); 13C
NMR (DMSO-d6): d 178.63 (2C), 170.23 (2C), 145.67
(2C), 134.28 (2C), 132.78 (2C), 126.83 (2C), 35.98
(2C). Anal. Calcd for C14H10O6S2 Æ 0.5H2O: C, 48.41;
H, 3.17. Found: C, 48.11; H, 3.12.MS (ESI) m/z 339
(M+1)+.
The intermediates 2-(naphthyl)ethyl bromide39 and ben-
zyl (2-methylthio)acetate,40 as well as the final com-
pounds Cpd 530 and NSC 95397,33 were synthetized
according to reported procedures. All other chemicals
were purchased from Acros and Aldrich, and used with-
out further purification unless otherwise specified.
5.3. General
5.6. (3-Chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl-
sulfanyl) acetic acid benzyl ester (3) and (3-benzyloxy-
carbonyl-methylsulfanyl-1,4-dioxo-1,4-dihydronaphtha-
len-2-ylsulfanyl) acetic acid benzyl ester (4)
All melting points were determined with the Kofler
apparatus and are uncorrected. Analytical thin-layer
chromatography was performed on Kieselgel 60F254
plates (Merck). Spots were visualized under UV
(254 nm). Flash chromatography was performed on sil-
ica gel 60 (0.04–0.063 mm) purchased from Carlo Erba–
SDS. 1H NMR and 13C NMR spectra were recorded on
a Bruker WMFT-250 MHz spectrometer. Chemical
shifts were expressed in parts per million (ppm) using
TMS as internal standard. Mass spectra were recorded
on a Bruker Esquire 3000 Ion Trap spectrometer using
an electrospray source at the Laboratoire de Chimie
Structurale Organique et Biologique, Universite Pierre
et Marie Curie, Paris, France. Elemental analyses (C,
H, and N), performed at the Service de Microanalyse,
´
Pierre et Marie Curie Universite, Paris, France, were
within 0.4% of the theoretical values.
Pyridine (0.66 mL) was added to a solution of 2,3-di-
chloro-[1,4]-naphthoquinone (820 mg, 3.6 mmol) and
benzyl mercaptoacetate (1.5 g, 7.2 mmol) heated at
55 ꢁC in toluene (20 mL). The solution was heated at
55 ꢁC for 1 h, cooled to room temperature, and filtered.
The filtrate was evaporated under reduced pressure and
purified by flash chromatography (EtOAc/cyclohexane,
1:9) to provide 3 as a yellow powder (564 mg, 42%);
mp: 108 ꢁC. 1H NMR (CDCl3): d 8.14–8.11 (1H, m,
H5), 8.01–7.98 (1H, m, H8), 7.79–7.70 (2H, m, H6 and
H7), 7.29–7.27 (5H, m, Hphenyl), 5.14 (2H, s, CH2),
4.21 (2H, s, S–CH2); 13C NMR (DMSO-d6): d 179.54,
174.82, 168.72, 147.47, 139.06, 135.85, 134.76, 134.59,
132.61, 131.00, 128.67 (2C), 128.49, 128.36 (2C),
127.31, 126.97, 67.00, 35.10. Anal. Calcd for
C19H13ClO4S: C, 61.21; H, 3.50. Found: C, 61.01; H,
3.37. MS (ESI) m/z 373.2 (M+1)+. Compound 4 was iso-
´
5.4. (3-Methyl-1,4-dioxo-1,4-dihydronaphthalen-2-yl-
sulfanyl) acetic acid (1)
1
lated as a red powder (681 mg, 36%); mp: 90 ꢁC; H
Compound 1 was obtained according to published pro-
cedure.30 DBU (0.02 mL, 0.14 mol) was added to a solu-
tion of 2-methyl-[1,4]-naphthoquinone (1 g, 5.8 mmol)
and mercaptoacetic acid (0.4 mL, 5.8 mmol) in ether
NMR (CDCl3): d 7.98–7.94 (2H, m, H5 and H8), 7.70–
7.66 (2H, m, H6 and H7), 7.28 (10H, m, Hphenyl),
5.14 (4H, s, 2· CH2), 4.07 (4H, s, 2· S–CH2); 13C
NMR (DMSO-d6): d 178.51 (2C), 168.82 (2C), 145.74

M.-P. Brun et al. / Bioorg. Med. Chem. 13 (2005) 4871–4879
4877
(2C), 135.88 (2C), 134.31 (2C), 132.62 (2C), 128.66 (3C),
5.11. 2-Chloro-3-[2-(morpholin-4-yl)ethylamino]-[1,4]-
naphthoquinone (9)
128.41 (2C), 128.35 (3C), 126.87 (2C), 66.90 (2C), 35.52
(2C). Anal. Calcd for C28H22O6S2: C, 64.86; H, 4.25.
Found: C, 64.72; H, 4.12. MS (ESI) m/z 519 (M+1)+.
To a suspension of 2,3-dichloro-[1,4]-naphthoquinone
(1 g, 4.4 mmol) in ether (40 mL) were added 2-amino-
ethylmorpholine (0.6 mL, 4.4 mmol) and TEA
(0.61 mL, 1 equiv). The reaction mixture was stirred
at room temperature for 12 h, concentrated under re-
duced pressure, diluted with ethyl acetate, and washed
with water. The organic layer was dried (Na2SO4) and
the solvent was evaporated in vacuo. The residue was
purified by flash chromatography (EtOAc/cyclohexane,
1:1) to give 9 as an orange powder (830 mg, 59%);
mp: 112 ꢁC; 1H NMR (CDCl3): d 8.17 (1H, d,
J = 8.6 Hz, H5), 8.05 (1H, d, J = 9.6 Hz, H8), 7.77–
7.64 (m, 2H, H6 and H7), 6.90 (1H, br s, NH), 3.99
(2H, m, O–CH2), 3.78 (4H, t, J = 4.55 Hz, 2· O–
CH2morpholine), 2.70 (2H, t, J = 6 Hz, N–CH2), 2.55
(4H, t, J = 4.55 Hz, 2· N–CH2morpholine); 13C NMR
(DMSO-d6): d 180.37, 175.56, 146.11, 135.20, 132.98,
132.31, 130.31, 126.82, 126.13, 66.58 (2C), 57.80, 53.20
(2C), 41.08. Anal. Calcd for C16H17ClN2O3: C, 59.90;
H, 5.30; N, 8.73. Found: C, 60.09; H, 5.33; N, 8.61.
MS (ESI) m/z 321 (M+1)+.
5.7. 2-(2-Hydroxyethyl)amino-3-methyl-[1,4]-
naphthoquinone (5)
Compound 5 was obtained according to published pro-
cedure41 from 3-methyl-[1,4]-naphthoquinone (0.5 g,
2.9 mmol) and ethanolamine (0.18 mL, 2.9 mmol) as
an orange powder (160 mg, 24%); mp: 131 ꢁC; 1H
NMR (DMSO-d6): d 7.89 (2H, m, H5 and H8), 7.78–
7.63 (2H, m, H6 and H7), 6.47 (1H, br s, NH), 4.90
(1H, br s, OH), 3.59 (4H, m, 2· CH2), 2.09 (3H, s,
CH3); 13C NMR (DMSO-d6): d 182.43, 182.05, 147.19,
134.69, 133.09, 132.37, 130.55, 125.90, 125.70, 111.19,
60.80, 47.12, 11.05.
5.8. 2-(3-Hydroxypropyl)amino-3-methyl-[1,4]-
naphthoquinone (6)
Compound 6 was obtained according to published pro-
cedure41 from 3-methyl-[1,4]-naphthoquinone (1.5 g,
8.72 mmol) and propanolamine (0.67 mL, 8.71 mmol)
as a dark red powder (410 mg, 20%); mp: 74 ꢁC; H
NMR (CDCl3): d 8.11 (1H, d, J = 7.6 Hz, H5), 8.01
(1H, d, J = 7.5 Hz, H8), 7.73–7.56 (2H, m, H6 and H7),
5.75 (1H, br s, NH), 3.62–3.54 (2H, m, N–CH2), 2.27
(3H, s, CH3), 1.71–1.63 (2H, m, CH2), 1.53–1.41 (2H,
m, CH2), 0.99 (3H, t, J = 7.2 Hz, CH3); 13C NMR
(DMSO-d6): d 182.46, 181.96, 147.00, 134.68, 133.13,
132.31, 130.52, 125.87, 125.68, 110.84, 58.97, 42.75,
33.64, 10.98.
1
5.12. 2-(2-Bromoethylamino)-3-chloro-[1,4]-
naphthoquinone (10)
Compound 10 was obtained according to the procedure
described by Nishizawa et al.34 To a mixture of 8 (1 g,
4 mmol) and CBr4 (2 g, 6 mmol) dissolved in anhydrous
CH2Cl2 (70 mL), PPh3 (1.26 g, 4.8 mmol) was added
portionwise. The reaction mixture was stirred at room
temperature for 2 h. The organic phase was washed with
water (3 · 50 mL), dried (Na2SO4), and the solvent was
evaporated under reduced pressure. The crude residue
was purified by flash chromatography (EtOAc/cyclohex-
ane, 1:5) to give 10 as an orange powder (909 mg, 72%);
mp: 180 ꢁC; 1H NMR (CDCl3): d 8.18 (1H, d,
J = 7.5 Hz, H5), 8.08 (1H, d, J = 7.7 Hz, H8), 7.80–7.64
(2H, m, H6 and H7), 6.33 (1H, br s, NH), 4.27 (2H, m,
CH2Br), 3.64 (2H, t, J = 6 Hz, N–CH2); 13C NMR
(DMSO-d6): d 180.37, 175.83, 145.50, 135.18, 133.16,
132.01, 130.39, 126.88, 126.13, 45.56, 32.69. Anal. Calcd
for C12H9BrClNO2: C, 45.78; H, 2.86; N, 4.45. Found
C, 45.67; H, 2.93; N, 4.43. MS (ESI) m/z 316 (M+1)+.
5.9. 2-Butylamino-3-methyl-[1,4]-naphthoquinone (7)
Compound 7 was prepared according to published pro-
cedure41 from 3-methyl-[1,4]-naphthoquinone (1.5 g,
8.72 mmol) and butylamine (0.86 mL, 8.71 mmol) as a
dark red powder (576 mg, 27%); mp: 79 ꢁC; H NMR
(CDCl3): d 8.10 (1H, d, J = 7.5 Hz, H5), 8.01 (1H, d,
J = 7.5 Hz, H8), 7.72–7.56 (2H, m, H6 and H7), 5.98
(1H, br s, NH), 3.87–3.72 (4H, m, O–CH2 and N–
CH2), 2.26 (3H, s, CH3), 1.98–1.88 (2H, m, CH2); 13C
NMR (DMSO-d6): d 182.46, 181.87, 146.82, 134.63,
133.09, 132.27, 130.53, 125.85, 125.65, 110.95, 44.49,
33.07, 19.71, 14.00, 10.92.
1
5.13. Enzymes, cell culture, chemicals, and antibodies
5.10. 2-Chloro-3-(2-hydroxyethyl-amino)-[1,4]-
naphthoquinone (8)
The maltose binding protein-CDC25B3 recombinant
protein was produced in bacteria, as previously de-
scribed.27 The human cancer cell line HeLa was obtained
from Aptanomics (Lyon, France). Cells were cultured at
37 ꢁC in Dulbeccoꢀs minimum essential medium comple-
mented with 10% fetal bovine serum and 100 U/mL pen-
icillin/streptomycin in a humidified atmosphere of 5%
CO2. The CDC25 inhibitors were solubilized in dimeth-
ylsulfoxide so that the DMSO final concentration was
<0.1%. The tetrazolium salt WST-1 was purchased from
Roche Diagnostics (Mannheim, Germany). The follow-
ing antibodies were used in this study: phospho-
cdc2(Tyr15) antibody was purchased from Cell Signaling
Technology (Beverly, MA), human full and cleaved
Compound 8 was obtained according to published pro-
cedure42,43 from 2,3-dichloro-[1,4]-naphthoquinone
(0.5 g, 2.2 mmol) and ethanolamine (0.18 mL, 2.2 mmol)
in the presence of TEA (0.3 mL, 2.2 mmol) as an orange
1
powder (257 mg, 46%); mp: 147 ꢁC; H NMR (CDCl3):
d 8.17 (d, 1H, J = 7.8 Hz, H5), 8.05 (1H, d, J = 7.5 Hz,
H8), 7.78–7.62 (m, 2H, H6 and H7), 6.43 (1H, br s,
NH), 4.11–4.05 (2H, m, O–CH2), 3.93–3.91 (2H, m,
N–CH2); 13C NMR (DMSO-d6): d 180.44, 175.74,
145.97, 135.21, 132.97, 132.33, 131.69, 130.23, 126.80,
126.14, 60.71, 46.66.

4878
M.-P. Brun et al. / Bioorg. Med. Chem. 13 (2005) 4871–4879
a-PARP antibody from BD Pharmingen (San Jose, CA),
actin antibody from Sigma (St. Louis, MO), and the sec-
ondary antibodies, namely anti-rabbit and anti-mouse
IgG-horseradish peroxidase antibodies, from Amersham
Biosciences (Buckinghamshire, England).
violet (2 g in 100 mL ethanol, then 2 mL in 100 mL
water) for 30 min, and finally rinsed with water. The
plating efficiency was determined by counting the colo-
nies. The results are expressed as means of two indepen-
dent experiments for Cpd 5 and NSC 95397 and as
means of three independent experiments for 3, 4, and
10. For each compound, the IC50 was determined from
a sigmoidal dose–response using GraphPad Prism
(GraphPad Software, San Diego, CA).
5.14. In vitro enzymatic assay
The activity of the MBP-CDC25B3 recombinant en-
zyme was monitored using fluorescein diphosphate.
The assay was performed in 96-well plates in a final vol-
ume of 200 lL. The maltose binding protein-CDC25B3
was kept in elution buffer [50 mM phosphate, pH 7.5,
300 mM NaCl, 500 mM imidazole, and 10% glycerol].
It was diluted in assay buffer [30 mM Tris–HCl (pH
8.2), 75 mM NaCl, 0.67 mM EDTA, 0.033% BSA, and
1 mM DTT]. The final concentration of CDC25B3 was
75 ng/well. Products were studied in concentration re-
sponse up to 100 lM. The reaction was initiated by
addition of 30 lM fluorescein diphosphate, followed
by an immediate measurement of fluorescein monophos-
phate emission with a Fluoroskan Ascent (Lab Systems;
excitation filter: 485 nm, emission filter: 530 nm). The re-
sults are expressed as means of at least three indepen-
dent experiments with three determinations per tested
concentration and per experiment. For each compound,
the drug concentration required for 50% inhibition
(IC50) was determined from a sigmoidal dose–response
curve using GraphPad Prism (GraphPad Software,
San Diego, CA).
5.17. Immunoblotting
HeLa cells were plated at 2 · 106 cells on a 10 cm plate
and incubated for 24 h. They were treated with 1, 5,
10, 15, and 20 lM compound 1 for 8 h for experiments
concerning the phosphorylation status of Cdc2. As far
as apoptosis is concerned, cells were treated with 10
and 30 lM compound 1 for 8 and 16 h. Cells were
washed four times with ice-cold phosphate-buffered sal-
ine and lysed in 250 lL lysis buffer [50 mM HEPES, pH
7.5, 1% Triton X-100, 150 mM NaCl, 10% glycerol,
1 mM MgCl2, 1 mM EGTA, phosphatase inhibitor
(5 mM Na3VO4), kinase inhibitor (10 mM NaF), and
protease inhibitor (1 mM phenylmethylsulfonyl fluoride
and CompleteTM protease inhibitors mixture, Roche
Molecular Biochemicals)]. After a 15-min incubation
at 4 ꢁC, the lysates were collected and centrifuged at
13,200g for 10 min at 4 ꢁC to remove insoluble material.
Protein concentration of the lysates was determined
using the Bradford assay (Sigma). Lysate proteins
(25 lg/lane for Cdc2 and 50 lg/lane for PARP) were
separated on a 12% (Cdc2) or 10% (PARP) gel by
SDS–polyacrylamide gel electrophoresis and electro-
blotted onto a nitrocellulose membrane. Membranes
were saturated overnight at 4 ꢁC with Superblock buffer
(Pierce) and blotted for 1 h at room temperature with
the appropriate primary antibody: rabbit polyclonal
anti-phospho-Cdk1 diluted at 1:2000, mouse monoclo-
nal full and cleaved anti-PARP diluted at 1:1000, and
mouse monoclonal anti-actin diluted at 1:10,000. After
four washes with TBS–0.1% Tween, membranes were
incubated with the secondary antibody (horseradish per-
oxidase-conjugated anti-rabbit or anti-mouse IgG di-
luted at 1:10,000) for 30 min at room temperature.
Immunoreactive bands were detected using the ECL
immunodetection system (Amersham Biosciences). The
results are expressed as means of at least three indepen-
dent experiments.
5.15. Cell proliferation assay
The inhibition of cell proliferation was determined using
a WST-1 colorimetric assay based on the cleavage of the
tetrazolium salt WST-1 by mitochondrial dehydroge-
nases in viable cells, leading to formazan formation.
At day 0, HeLa cells were plated at 3000 cells/well in
96-well tissue culture plates with 95 lL medium/well.
At day 1, cells were treated for 96 h with 5 lL of increas-
ing concentrations of drug (up to 100 lM). At day 6,
10 lL of WST-1 was added per well, and cells were incu-
bated for 2 h at 37 ꢁC in a humidified atmosphere of 5%
CO2. Absorbance was measured at 430 nm. The results
are expressed as means of at least three independent
experiments with eight determinations per tested con-
centration and per experiment. For each compound,
the IC50 was determined from a sigmoidal dose–re-
sponse using GraphPad Prism (GraphPad Software,
San Diego, CA).
5.18. Molecular modeling studies
5.16. Clonogenic assay
The crystal structure of the catalytic subunit of CDC25B
(PDB code 1CWT) was used to dock vitamin K3 and the
compounds 1 and 2 in CDC25. All docking calculations
were carried out with Surflex36 and Ligandfit.37 The pro-
gram Surflex defines a binding site called a protomol
tuned to produce a docking target consistent with the
binding pocket properties. Flexible docking of the li-
gand was performed with the incremental construction
algorithm as described. The best 10 poses for each li-
gand were ranked by using the Surflex scoring function
involving hydrophobic, polar, entropic, and solvation
terms. The best energy ranked pose was selected. The
For cloning inhibition assays, HeLa cells were plated at
100 cells/well in six-well tissue culture plates with 2.5 mL
medium/well. After 24 h growth at 37 ꢁC in a humidified
atmosphere of 5% CO2, the culture medium was re-
placed by control medium or medium containing
inhibitors at increasing concentrations (up to 100 lM)
and incubated for 10 days. Cells were then washed with
phosphate-buffered saline and fixed in 10% formalde-
hyde for 30 min at room temperature. They were care-
fully rinsed with water, stained with 1 mL crystal

M.-P. Brun et al. / Bioorg. Med. Chem. 13 (2005) 4871–4879
4879
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Yamada, Y.; Hashimoto, Y.; Shirai, R. Bioorg. Med.
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Workstation. The Ligandfit approach combines a shape
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CDC25 was determined with the implemented cavity
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Acknowledgment
We thank la Ligue Nationale contre le Cancer for its
´
financial support (Equipes ‘‘labellisees’’).
´
27. Brezak, M. C.; Quaranta, M.; Mondesert, O.; Galcera,
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