Novel naphthoquinone and quinolinedione inhibitors of CDC25 phosphatase activity with antiproliferative properties

Article abstract of DOI:10.1016/j.bmc.2008.08.009

CDC25 phosphatases are considered as attractive targets for anti-cancer therapy. To date, quinone derivatives are among the most potent inhibitors of CDC25 phosphatase activity. We present in this paper the synthesis and the biological evaluation of new q

Full text of DOI:10.1016/j.bmc.2008.08.009

Bioorganic & Medicinal Chemistry 16 (2008) 9040–9049
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel naphthoquinone and quinolinedione inhibitors of CDC25
phosphatase activity with antiproliferative properties
Emmanuelle Braud a,b, Mary-Lorène Goddard a,b, Stéphanie Kolb a,b, Marie-Priscille Brun a,b
,
Odile Mondésert c, Muriel Quaranta c, Nohad Gresh a,b, Bernard Ducommun c, Christiane Garbay a,b,
*
a Université Paris Descartes, UFR biomédicale, Laboratoire de Pharmacochimie Moléculaire et Cellulaire, 45 rue des Saints-Pères, Paris F-75006, France
b INSERM U648, Paris F-75006, France
c Université Paul Sabatier, Laboratoire de Biologie Cellulaire et Moléculaire du Contrôle de la Prolifération, UMR 5088-IFR109, Route de Narbonne, Toulouse, F-31062, France
a r t i c l e i n f o
a b s t r a c t
Article history:
CDC25 phosphatases are considered as attractive targets for anti-cancer therapy. To date, quinone deriv-
atives are among the most potent inhibitors of CDC25 phosphatase activity. We present in this paper the
synthesis and the biological evaluation of new quinolinedione and naphthoquinone derivatives, contain-
ing carboxylic or malonic acids groups introduced to mimic the role of the phosphate moieties of Cyclin-
Dependent Kinase complexes. The most efficient compounds show inhibitory activity against CDC25B
Received 5 May 2008
Revised 29 July 2008
Accepted 4 August 2008
Available online 7 August 2008
with IC50 values in the 10
l
M range, and are cytotoxic against HeLa cells.
Ó 2008 Published by Elsevier Ltd.
Keywords:
Cancer
Phosphatase CDC25
Cell cycle regulation
Quinone derivatives
1. Introduction
gastric cancers, hepatocarcinomas and non-hodgkinian lympho-
mas.9,17 Such an abnormal expression often correlates with poor
prognosis and since isoforms A and B possess oncogenic proper-
ties,18 CDC25 phosphatases are now considered as particularly
attractive targets toward novel approach in cancer treatment.9
Several classes of inhibitors have been described so far (for re-
cent reviews, see Boutros et al.9 and Contour-Galcera et al.19).
The most potent ones share the para-quinone moiety such as the
thio-ether derivative Cpd 5 that inhibits CDC25 activity in Hep3B
cells20,21 or NSC 95397 that blocks cell cycle at the G2/M transi-
tion.22 Among the quinolinedione derivatives, NSC 663284 inhibits
CDC25 activity in an irreversible manner by binding covalently to a
serine residue.23,24 Adociaquinone B is a natural product isolated
from a marine sponge that inhibits in vitro CDC25B activity with
an IC50 value of 70 nM.25 More recently, BN82685 was demon-
strated to inhibit in vivo the growth of human pancreatic MiaPaCa2
tumors xenografted on athymic nude mice.26,27 IRC-083864 con-
taining two quinone nuclei is the most potent CDC25 inhibitor that
also exhibits antiproliferative properties against MiaPaCa2 and
CDC25 dual-specificity phosphatases are major actors of
eukaryotic cell cycle regulation and are involved in essential
cellular events such as growth, division, or replication. They thus
activate Cyclin-Dependent Kinase (CDK) complexes by dephospho-
rylating two adjacent threonine and tyrosine residues allowing
progression of the cell cycle.1,2
The human genome encodes three CDC25 homologues, namely
CDC25A, B, and C.3–5 Though their entire role has not been eluci-
dated yet, these three isoforms have been shown to play multiple
roles and to cooperate at various stages of the cell cycle. CDC25A
has been demonstrated to activate CDK2/cyclin E and CDK2/cyclin
A complexes allowing progression into S phase6–8 and is also in-
volved at mitosis in the control of CDK1/cyclin B.9 Isoform B cata-
lyzes the dephosphorylation of CDK1/cyclin A to drive the cell
through the G2/M transition10 and has also been shown to play a
role in centrosome duplication and in S-phase.11,12 CDC25B expres-
sion is also required for checkpoint recovery after DNA repair.13,14
CDC25 C is involved in cell progression in mitosis due to its ability
to activate CDK1/cyclin B complex15 and might also be involved in
the control of S-phase.16
28
LNCaP cell lines and xenografted tumors.(Chart 1)
We have previously reported the synthesis and the inhibitory
activity of several naphthoquinone derivatives containing carboxyl-
ate groups designed to interact with CDC25B through arginine resi-
dues R482 and R544 involved in the recognition of the CDKs (Chart
2).29 These inhibitors displayed anti-CDC25 activity with IC50 values
in the micromolar range as well as anti-proliferative properties to-
ward HeLa cells. They also caused hyperphosphorylation of CDK1
Overexpression of CDC25A and B was observed in a wide variety
of tumors and related cancer cell lines including prostate, breast, or
* Corresponding author. Tel.: +33 142 864 080; fax: +33 142 864 082.
E-mail address: christiane.garbay@univ-paris5.fr (C. Garbay).
0968-0896/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.08.009

E. Braud et al. / Bioorg. Med. Chem. 16 (2008) 9040–9049
9041
Chart 1. Structures of CDC25 phosphatase inhibitors.
ing from 80% to 89%. The naphthalene derivatives 1a,b–2a,b were
then oxidized by a mixture of periodic acid and chromic anhydride
into 1,4-naphthoquinone derivatives 1c,d–2c,d with low to moder-
ate yields (15–39%).31 The diterbutyl malonates 1d and 2d were
hydrolyzed using trifluoroacetic acid in dichloromethane to pro-
vide the corresponding malonic acids 1e and 2e in 70% and 82%
yields, respectively.
Compound 3 was obtained in 13% yield following the protocol
described by Salmon-Chemin and co-workers by reacting menadi-
one and glutaric acid in the presence of silver nitrate as a catalyst
and ammonium salt (Scheme 2).32
Chart 2. CDC25 inhibitors designed to interact with the phosphate binding pocket.
Compound 4 was prepared using a 1,4-Michael type addition of
mercaptopropanoic acid to 2-methyl-[1,4]-naphthoquinone in the
presence of DBU in ether in 20% yield (Scheme 2).29
The 6,7-dibromo-5,8-quinolinedione 5 was obtained by oxidiz-
ing the commercially available 5-amino-8-hydroxyquinoline with
NaBrO3/HCl in 43% yield.33 Subsequently, the 6,7-disubstituted
quinoline-5,8-diones 6 and 7 were synthesized in 33% and 63%
yields, respectively, using a 1,4-Michael-type addition of 2.5 equiv-
alents of mercaptoacetic acid or mercaptopropanoic acid to 5 in the
presence of triethylamine in THF at room temperature (Scheme 2).34
showing that they were actually targeting CDC25 in HeLa cells. In or-
der to improve the potency of these compounds, we have developed
new series of naphthoquinone and quinolinedione derivatives by
introducinga malonicacid group atthe extremityof an aliphatic side
chain to optimize interactions between the carboxylate groups and
the arginine residues.
2. Chemistry
Naphthoquinones 1e and 2e were prepared following a three-
step procedure using the commercially available 2-bromomethyl-
naphthalene 1 or 2-(2-naphthyl)ethyl bromide 2 which was ob-
tained by bromination of naphthaleneethanol (Scheme 1) as
starting material.30 Bromide derivatives 1 and 2 were converted
into the corresponding dibenzyl or diterbutyl malonate analogues
1a,b–2a,b using the malonic synthesis conditions with yields rang-
3. Results and discussion
3.1. Enzymatic activity
In a previous work, we demonstrated that naphthoquinone
derivatives bearing carboxylic groups introduced to mimic the role
Scheme 1. Reagents and conditions: (i) NaH, CH2(CO2R)2, DMF, rt; (ii) HIO4, CrO3, CH3CN, 5 °C then rt; (iii) TFA, CH2Cl2, rt.

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E. Braud et al. / Bioorg. Med. Chem. 16 (2008) 9040–9049
Scheme 2. Reagents and conditions: (i) HO2C–(CH2)3–CO2H (3 equiv), AgNO3, (NH4)2S2O8 (1.3 equiv), CH3CN/H2O (30/70), 60 °C; (ii) HS–(CH2)2–CO2H (1 equiv), DBU, ether,
rt; (iii) NaBrO3, concd HCl, 50 °C; (iv) HS–(CH2)n–CO2H (2.5 equiv), Et3N, THF, rt.
of the phosphate moieties of CDKs showed CDC25 phosphatase
inhibitory activity with IC50 values in the micromolar range.29
Molecular dynamics calculations were performed on these carbox-
ylic acid derivatives using the program Discover (Accelrys) and the
available 3D crystal structure of CDC25B catalytic domain (PDB
1QB0) to propose a binding mode for these inhibitors. Strong ionic
interactions between the carboxylate groups and arginine residues
R479, R482, or R544 were observed while the naphthoquinone
core was located out of the floor of the active site. In order to opti-
mize these interactions and enhance activity, we replaced carbox-
ylic groups by malonic acids and similar docking calculations were
performed on malonic acid 1e to examine possible interactions.
These studies suggested that 1e displayed a similar binding mode
with both carboxylate groups interacting with the three arginine
residues R479, R482, and R544 while the naphthoquinone core
was still placed outside the active site (Fig. 1). We thus expected
that this series of new malonic compounds would be most or at
least as efficient as their previously reported carboxylic analogs.29
All our compounds were evaluated for their CDC25 inhibitory
activity on an enzymatic assay using the human recombinant fused
MBP-CDC25B3 protein with fluorescein 3,6-diphosphate (FDP) as
the substrate (Table 1). Cpd 5 and NSC 95397 were evaluated as
references in the same conditions. We previously discussed the
discrepancies observed between the IC50 value of NSC 95397 that
we measured and the one reported in the literature.29
group and the arginine residues. Thus, in the malonic acid series,
only one of the two carboxylates may be required for inhibitory
activity. By contrast, introduction of a thio-alkyl side chain con-
taining only one carboxylate group retained activity, as compound
4 was the most active derivative with an IC50 value of 4.55 lM. The
presence of a sulfur atom that links the aliphatic side chain to the
naphthoquinone core was therefore essential for activity.
Lazo et al. previously demonstrated that substitution with ami-
no groups at positions 6 or 7 on the quinolinedione core was re-
quired for efficient CDC25 inhibition.23 Since the presence of the
thio-ether side chain is also significant for CDC25 inhibitory activ-
ity, quinolinediones 6 and 7 were accordingly synthesized. Unfor-
tunately, neither of them led to the expected increase of potency as
both compounds displayed comparable activities with IC50 values
of 11.2 and 10 lM, respectively. The length of the alkyl side chains
had no effect on the inhibitory properties.
To further investigate the effects of the thio-ether side chains,
the 6,7-dibromoquinolinedione 5 was also evaluated on the enzy-
matic assay and exhibited an IC50 value of 5.3 lM. Thus, for this
limited family of quinolinedione derivatives, substitution at the
6- and 7-positions with thiol groups led to a slight loss of activity.
In fact, the 6,7-dibrominated quinolinedione 5 was as active as Cpd
5 and NSC 95397 in the present test. On the other hand, the ana-
logue of compound 6 in the naphthoquinone series displayed an
IC50 value of 1.75
We next determined the kinetic properties of CDC25 inhibition
by compounds 4 and 6. The Km value with FDP was 2.89 0.30 M.
l
M, lower than that of 5.29
In the malonic acid series, an increase of the aliphatic side-chain
length was correlated with a stronger potency since compound 2c
was about fourfold more active than 1c, and 2e displayed a twofold
stronger inhibitory activity against CDC25B than 1e. Unexpectedly,
the malonic acid derivatives 1e and 2e were less active than the
corresponding dibenzyl esters 1c and 2c, 2e inhibiting CDC25
l
Inhibition of CDC25B by the potent naphthoquinone 4 and quinol-
inedione 6 appeared to be most consistent with a mixed-competi-
tive inhibition kinetic model (Fig. 2). This inhibition model was
previously reported for NSC 95397,22 NSC 66328423, and also for
other types of CDC25 inhibitors such as steroidal or maleic anhy-
drides derived inhibitors.35,36
activity with an IC50 value of 24.7 lM versus 10.7 lM for 2c. These
results suggest that benzyl derivatives 1c and 2c should adopt a
different binding mode than the malonic acids 1e and 2e.
Since quinone derivatives are often responsible for ROS genera-
tion, we investigated the effect of increased dithiothreitol (DTT)
concentrations on the inhibitory activity of compounds 4 and 6.
Enzymatic assays were conducted as described in Section 5 replac-
ing the normally used 1 mM DTT concentration by 10 and 20 mM.
Replacement of the malonic group by a carboxylic moiety sup-
pressed enzymatic activity, as compound 3 was shown to display
no activity against CDC25B. Such dramatic decrease could be
linked to a loss of interactions between the terminal carboxylate

E. Braud et al. / Bioorg. Med. Chem. 16 (2008) 9040–9049
9043
Figure 1. Molecular dynamics calculations. Docked pose of compound 1e in CDC25B active site obtained using Discover (Accelrys).
Table 1
Results indicated that an increase of DTT concentration was corre-
lated with an augmentation of the IC50 values for both compounds
consistent with ROS production in vitro (Table 2). Similar observa-
tions were already reported by Cossy et al. for some quinolinedione
derivatives.37
In vitro activity against recombinant MBP-CDC25B3 and cytotoxicity measured on
HeLa cell line.
Compound
Enzymatic assaya IC50 SD (
lM)
Cytotoxicityb IC50 SD (
lM)
GpdS
NSC 95397
3.65 0.2
3.77 0.9
37.9 4.6
10.7 0.5
52.7 6.0
24.7 3.0
>100
4.55 0.16
5.3 0.3
11.2 1.2
10.0 5.5
8.8 3.8
10.8 2.9
21.5 2.3
12.2 3.7
210.7 13.6
37.7 2.2
54.2 2.9
51.3 4.1
21.5 0.3
>100
lc
2c
le
2e
3
4
5
6
7
3.2. Cellular activity
The naphthoquinone and quinolinedione derivatives were eval-
uated for their cytotoxic effects on HeLa cells treated with increas-
ing concentrations of inhibitors using the WST-1 cell viability
colorimetric assay. The results are reported in Table 1 and show
these compounds to be moderately cytotoxic against HeLa cells.
The most active compound was the dibenzyl malonate 2c which
exhibited an IC50 value similar to those of Cpd 5 and NSC 95397.
In the malonic acid series, compounds with the longer aliphatic
chain appeared to be the most cytotoxic ones. Benzyl malonate 1c
>100
a
Enzyme inhibition was assayed using fluorescein 3,6-diphosphate as the sub-
strate. The IC50 values and the SD were calculated from at least two independent
experiments with three determinations per tested concentration.
b
Cytotoxicity was evaluated on HeLa cells using the colorimetric assay WST-1.
The IC50 values and the SD were calculated from three independent experiments
with eight determinations per tested concentration.
displayed an IC50 value of 21.5
active with an IC50 value of 12.2
l
l
M whereas 2c was slightly more
M. Concerning the malonic acids,

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E. Braud et al. / Bioorg. Med. Chem. 16 (2008) 9040–9049
Figure 2. Kinetic analyses of CDC25B inhibition by compounds 4 and 6. Concentrations of compound 4: (h) 0
l
M; (
D
) 0.3
l
M; (
r
) 1 lM; (e) 3 lM; (o) 6 lM; (ꢀ) 10 lM.
Concentrations of compound 6: (h) 0 M; ( ) 0.3 M; ( ) 1 M; (e) 3 M; (o) 6 M. (A) Michaelis-Menten plot of CDC25B inhibition by 4. (B) Lineweaver–Burk plot of
l
D
l
r
l
l
l
CDC25B inhibition by 4. (C) Michaelis–Menten plot of CDC25B inhibition by 6. (D) Lineweaver–Burk plot of CDC25B inhibition by 6.
Table 2
The 6,7-dibromoquinolinedione 5 showed moderate cytotoxic
activity with an IC50 value of 21.5 M. Quinolinedione derivative
6 with anti-phosphatase activity did not display potent cytotoxic
Effect of DTT on in vitro CDC25B inhibition by compounds 4 and 6.
l
Compound
% Inhibition at 100
l
Ma
DTT (20 mM)
activity on HeLa cells with an IC50 value above 100 lM as observed
DTT (10 mM)
for its naphthoquinone analog.29 Compound 7 with increased side
chain lengths did not either show cytotoxicity. For these two com-
pounds, the presence of the two carboxylic groups may impede cell
penetration.
4
6
42.45 0.05
51.63 0.31
65.79 2.70
73.45 1.72
a
The % of inhibition were calculated from two independent experiments with
three determinations per tested concentration.
The antiproliferative effects of malonic acid 2e were also exam-
ined on HeLa cells using a colony formation assay. HeLa cells were
treated with increasing concentrations of 2e and plated for ten
additional days. After crystal violet staining and colony counting,
the dose-response curve was drawn and the IC50 value determined.
Compound 2e was able to inhibit colony formation with an IC50 va-
compound 1e did not show significant cytotoxicity with an IC50 va-
lue of 210 lM versus 37.7 lM for compound 2e. Thus, in this ser-
ies, a correlation between the in vitro enzymatic activity of the
compounds and their cytotoxic effects on HeLa cell line was ob-
served. Furthermore and as expected, the di-benzyl malonates 1c
and 2c synthesized in order to improve cell penetration were more
efficient than their acid analogs 1e and 2e. Surprisingly, compound
3 containing one carboxylic group was less active than its malonic
lue of 3.3 lM and was more potent than Cpd 5 and NSC 95397,
which displayed IC50 values of 4.1 and 8.5
lM, respectively in the
same conditions.29
3.3. Effects on cell cycle progression
acid analog 2e with IC50 values of 54.2 and 37.7 lM, respectively.
The effects of vitamin K3 analogues and especially of Cpd 5 on
cell cycle progression were previously examined on different tu-
mor cell lines.21,38,39 Studies carried out on asynchronous human
hepatoma Hep3B cells showed that Cpd 5 was able to arrest cell cy-
cle progression at the G2/M and G1 steps.38 Similar results were
Concerning the monoacid derivatives, the presence of a sulfur or
a carbon atom had no effect on the cytotoxic properties since com-
pounds 3 and 4 exhibited comparable efficiencies with IC50 values
of 51.3 and 54.2
lM, respectively. Thus, compound 3 triggers tar-
gets other than CDC25 phosphatase in HeLa cells.

E. Braud et al. / Bioorg. Med. Chem. 16 (2008) 9040–9049
9045
Figure 3. Cpd 5, NSC 95397, and naphthoquinone 2e hardly modify cell cycle distribution. Asynchronous HeLa cells were treated with Cpd 5 and NSC95397 at a concentration
of 10 M and with compound 2e at a concentration of 50 M for 24 h and then stained for flow cytometry analysis. The cell distribution reported in each figure was measured
l
l
as the percentage of cells containing DNA of G0/G1, S, and G2/M phases.
obtained on synchronous murine tsFT210 cells.21 More recently,
Han et al. demonstrated that Cpd 5 slightly modified cell cycle dis-
tribution of HCT-116 cell line at a concentration of 7.5 lM,
whereas it appeared more efficient on HCT-116R30A as it arrested
cell cycle at the G2/M transition at the same concentration. In this
study, the authors also demonstrated that NSC 95397 was equally
efficient on both cell lines and blocked cell cycle progression at the
G2/M phase.39
erate effects on cell cycle distribution in accordance with CDC25
inhibitory effects. Further chemical investigation carried out on
these derivatives could lead to new promising potent CDC25
inhibitors.
5. Experimental
5.1. Abbreviations
The effects of Cpd 5, NSC 95397, and compound 2e were exam-
ined in this study on cell cycle distribution using flow cytometry
analysis on asynchronous HeLa cells (Fig. 3).
DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DMF, dimethylform-
amide; DMSO, dimethylsulfoxide; THF, tetrahydrofuran; TMS,
tetramethylsilane.
After 24 h treatment at a concentration of 10 lM of Cpd 5 and
NSC 95397, cell cycle distribution hardly changed. In the presence
of the reference compounds, cells were arrested at various stages
of cell cycle progression suggesting that the activity of the three
CDC25 isoforms was inhibited. Concerning compound 2e, only a
slight accumulation in the G2/M and S phases was observed at a
5.2. Chemicals
The intermediate 2-(2-naphthyl)ethyl bromide 230 as well as
Cpd 5,21 NSC 95397,34 6,7-dibromoquinolinedione 533, and 4-[3-
methyl-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)]butanoic acid 332
were synthesized as previously reported. All other chemicals were
purchased from Acros Organics and Aldrich and used without fur-
ther purification unless otherwise specified.
concentration of 50
of CDC25 activity.
lM that may also be correlated with a decrease
4. Conclusion
We have reported the synthesis and the CDC25 phosphatase
inhibitory activity of novel naphthoquinone and quinolinedione
derivatives. These compounds were designed to increase the inter-
action with the arginine residues involved in the recognition of the
CDKs upon introducing malonic or acid groups at the extremity of
an alkyl or a thio-alkyl side chain. Although these compounds did
not show the expected increase of activity with IC50 value for the
most potent one in the 10 lM range, they displayed moderate
cytotoxicities against HeLa cell line. Interestingly, compound 2e
exhibited antiproliferative properties against HeLa cells and mod-
5.3. General
All melting points were determined on a Kofler apparatus and
are uncorrected. Kieselgel 60F254 plates (Merck) were used for ana-
lytical thin layer chromatography and were visualized with ultra-
violet light (254 nm). Flash chromatography was performed on
silica gel 60 (0.04–0.063 mm) purchased from Carlo Erba—SDS.
1H 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

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E. Braud et al. / Bioorg. Med. Chem. 16 (2008) 9040–9049
were recorded on a LCQ Advantage spectrometer (ThermoElectron,
France) at the Laboratoire de Chimie et Biochimie Pharmacologi-
ques et Toxicologiques, Université Paris Descartes, Paris, France.
Elemental analyses (C, H, N), performed at the Service de Microan-
alyse, Pierre et Marie Curie Université, Paris, France, were within
0.4% of the theoretical values.
3H, Har). 13C NMR (CDCl3): d 28.32 (6C), 29.75, 30.54, 54.59,
80.46 (2C), 124.56, 124.96, 126.02, 126.09, 126.32, 126.45,
126.68, 131.54, 132.68, 135.06, 167.65 (2C).
5.9. General procedure for the preparation of compounds (1c,
1d) and (2c, 2d)
5.4. General procedure for the synthesis of compounds (1a, 1b)
and (2a, 2b)
Periodic acid (4.2 mmol) was dissolved in acetonitrile (12 mL)
under vigorous stirring and CrO3 (0.1 mmol) was added. The solu-
tion was cooled to 5 °C and a solution of the previously synthesized
naphthalene derivatives 1a,b–2a,b (1 mmol) dissolved in acetoni-
trile (5 mL) was added. The resulting mixture was stirred at room
temperature for 30 min and the precipitate filtered. The filtrate
was evaporated under reduced pressure and the residue dissolved
in a mixture of CH2Cl2/H2O. The aqueous layer was extracted with
dichloromethane. The organic layers were then washed with a
solution of saturated aqueous NaCl, dried (Na2SO4), and the solvent
removed under reduced pressure. The residue was finally purified
on silica gel column chromatography (EtOAc/cyclohexane, 1:9) to
give the expected naphthoquinones 1c,d–2c,d.
A solution of diterbutyl or dibenzyl malonate (1 mmol) in dry
DMF (10 mL) was added dropwise to a chilled suspension of NaH
(1.1 mmol) in DMF (5 mL). The reaction mixture was stirred at
0 °C for an hour before a solution of 2-bromomethylnaphthalene
or 2-(2-naphthyl)ethyl bromide 2 (1.1 mmol) dissolved in DMF
(10 mL) was added dropwise. The resulting mixture was stirred
at room temperature for 12 h and then diluted into water. The
aqueous phase was extracted with ethyl acetate (3ꢀ). The organic
layers were then washed with water and saturated aqueous NaCl,
dried (Na2SO4), and evaporated to dryness. The residue was puri-
fied on silica gel column chromatography (EtOAc/cyclohexane,
1:9) to give the expected malonate derivatives 1a,b–2a,b.
5.10. Dibenzyl [(1,4-dioxo-1,4-dihydronaphthalen-2-yl)methyl]
malonate (1c)
5.5. Dibenzyl (2-naphthylmethyl)malonate (1a)
Compound 1c was obtained from compound 1a (1 g,
2.36 mmol) as a yellow powder (0.15 g, 28%); mp 72 °C; 1H NMR
(CDCl3): d 3.19 (d, 2H, J = 7.8 Hz, CH2), 3.97 (t, 1H, CH), 5.16 (s,
4H, 2ꢀ CH2 benzyl), 6.77 (s, 1H, H3), 7.25–7.45 (m, 10H, Har),
7.74–7.77 (m, 2H, H6 and H7), 8.03–8.10 (m, 2H, H5 and H6); 13C
NMR (DMSO-d6): d 28.44, 49.96, 67.11 (2C), 125.99, 126.58,
128.24 (5C), 128.49 (2C), 128.71 (5C), 131.81, 134.53, 135.68
(2C), 136.39, 147.33, 168.29 (2C), 184.63 (2C); MS (ESI) m/z 477
(M+Na)+. Anal. Calcd for C28H22O6: C, 74.00; H, 4.88. Found: C,
73.83; H, 5.03.
Compound 1a was obtained from 2-bromomethylnaphthalene
(3 g, 13.57 mmol) and dibenzyl malonate (3.73 mL, 14.9 mmol) as
a colorless oil (5.12 g, 89%); 1H NMR (CDCl3): d 3.4 (t, 2H,
J = 7.8 Hz, CH2-CH), 3.88 (t, 1H, CH2-CH), 5.09 (s, 4H, 2ꢀ CH2 ben-
zyl), 7.15–7.30 (m, 10H, Har), 7.42–7.46 (m, 2H, Har), 7.59 (s, 1H,
Har), 7.65–7.88 (m, 4H, Har). 13C NMR (CDCl3): d 35.35, 54.24,
67.66 (2C), 126.10, 126.51, 127.47, 127.96, 128.06, 128.14,
128.55 (5C), 128.73 (2C), 128.93 (5C), 132.86, 133.94, 135.51,
135.66, 168.98 (2C).
5.6. Diterbutyl (2-naphthylmethyl)malonate (1b)
5.11. Diterbutyl [(1,4-dioxo-1,4-dihydronaphthalen-2-yl)methyl]
malonate (1d)
Compound 1b was obtained from 2-bromomethylnaphthalene
(3 g, 13.57 mmol) and diterbutyl malonate (3.33 mL, 14.9 mmol)
as a colorless oil (4.10 g, 85%); 1H NMR (CDCl3): d 1.38 (s, 18H,
Compound 1d was obtained from compound 1b (0.5 g,
1.4 mmol) as a yellow powder (0.18 g, 37%); mp 120 °C; 1H NMR
t
t
2ꢀ Bu), 3.28 (d, 2H, J = 7.8 Hz, CH2), 3.54 (t, 1H, CH), 7.31–7.34
(CDCl3): d 1.41 (s, 18H, 2ꢀ Bu), 3.05 (d, 2H, J = 7.8 Hz, CH2), 3.58
(m, 1H, Har), 7.39–7.43 (m, 2H, Har), 7.64 (m, 1H, Har), 7.72–
7.77 (m, 3H, Har). 13C NMR (CDCl3): d 26.46 (6C), 33.35, 54.20,
80.21 (2C), 124.03, 124.56, 125.95, 125.99, 126.14, 126.21,
126.59, 130.90, 132.09, 134.53, 166.90 (2C).
(t, 1H, CH), 6.81 (s, 1H, Har), 7.70–7.74 (m, 2H, H6 and H7), 8.02–
8.09 (m, 2H, H5 and H6). 13C NMR (CDCl3): d 28.28 (6C), 29.65,
52.58, 82.52 (2C), 126.52, 127.05, 132.40, 132.54, 134.18 (2C),
136.78, 148.39, 167.99 (2C), 185.06 (2C).
5.7. Dibenzyl [2-(2-naphthyl)ethyl]malonate (2a)
5.12. Dibenzyl [2-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)ethyl]
malonate (2c)
Compound 2a was obtained from 2-(2-naphthyl)ethyl bromide
2 (0.6 g, 2.55 mmol) and dibenzyl malonate (0.7 mL, 2.8 mmol) as a
white powder (0.89 g, 80%); mp 75 °C; 1H NMR (CDCl3): d 2.29–2.36
(m, 2H, CH2–CH2–CH), 2.78 (t, 2H, J = 7.6 Hz, CH2–CH2–CH), 3.47 (t,
1H, CH), 5.13 (s, 4H, 2ꢀ CH2 benzyl), 7.30 (s, 10H, Har), 7.40–7.51
(m, 3H, Har), 7.68–7.79 (m, 4H, Har). 13C NMR (CDCl3): d 30.62,
33.77, 51.64, 67.56 (2C), 125.77, 126.40, 127.23, 127.51, 127.89,
128.01, 128.65 (5C), 128.79 (2C), 128.99 (5C), 132.56, 133.95,
135.78, 138.31, 169.42 (2C).
Compound 2c was obtained from compound 2a (0.3 g,
0.68 mmol) as a yellow oil (50 mg, 15%); 1H NMR (CDCl3): 2.21–
2.30 (m, 2H, CH2–CH2–CH), 2.64 (t, 2H, J = 7.5 Hz, CH2–CH2–CH),
3.56 (t, 1H, J = 7.4 Hz, CH), 5.18 (s, 4H, 2ꢀ CH2 benzyl), 6.78 (s,
1H, H3), 7.35 (m, 10H, Har),7.74–7.78 (m, 2H, H6 and H7), 8.07–
8.13 (m, 2H, H5 and H6); 13C NMR (DMSO-d6): d 26.99, 27.02,
50.82, 66.90 (2C), 125.88, 126.50, 128.27 (5C), 128.52 (2C),
128.79 (5C), 132.00, 132.27, 134.36, 135.46, 135.86, 150.02,
168.93 (2C), 184.9 (2C); MS (ESI) m/z 491 (M+Na)+. Anal. Calcd
for C29H24O6: C, 74.35; H, 5.16. Found: C, 74.23; H, 5.12.
5.8. Diterbutyl [2-(2-(naphthyl)ethyl]malonate (2b)
Compound 2b was obtained from 2-(2-naphthyl)ethyl bromide
2 (1.5 g, 6.38 mmol) and diterbutyl malonate (1.57 mL, 7 mmol) as
a white powder (2 g, 85%); mp 81 °C; 1H NMR (CDCl3): d 1.46 (s,
18H, 2ꢀ tBu), 2.18–2.21 (m, 2H, CH2–CH2–CH), 2.80 (t, 2H,
J = 7.4 Hz, CH2–CH2–CH), 3.15–3.17 (m, 1H, CH), 7.31–7.34 (m,
1H, H3), 7.42–7.43 (m, 2H, Har), 7.62 (m, 1H, Har), 7.74–7.80 (m,
5.13. Diterbutyl [2-(1,4-dioxo-1,4-dihydronaphthalen-2-yl)ethyl]
malonate (2d)
Compound 2d was obtained from compound 2b (1.3 g,
3.51 mmol) as a yellow oil (150 mg, 39%); 1H NMR (CDCl3): d
1.45 (s, 18H, 2ꢀ tBu), 2.06–2.12 (m, 2H, CH2–CH2–CH), 2.61 (t,

E. Braud et al. / Bioorg. Med. Chem. 16 (2008) 9040–9049
9047
2H, J = 7.4 Hz, CH2CH2CH), 3.19 (t, 1H, J = 7.8 Hz, CH), 6.81 (s, 1H,
H3), 7.69–7.73 (m, 2H, H6 and H7), 8.03–8.08 (m, 2H, H5 and H6).
13C NMR (CDCl3): d 27.30, 27.69, 28.29 (6C), 53.71, 82.14 (2C),
126.44, 127.01, 132.47, 132.63, 134.06 (2C), 135.63, 150.85,
168.66 (2C), 185.20, 185.33.
5.19. 2,20-[(5,8-Dioxo-5,8-dihydroquinoline-6,7-diyl)disulfanediyl]
diacetic acid (6)
Compound 6 was obtained from 6,7-dibromo-5,8-quinolinedi-
one 5 (400 mg, 1.26 mmol) and mercaptoacetic acid (0.22 mL,
3.8 mmol) as a yellow powder (141 mg, 33%); mp 195 °C; 1H
NMR (CD3OD): d 3.78 (s, 2H, CH2), 3.90 (s, 2H, CH2), 7.57 (dd, 1H,
J = 4.4 Hz, J = 8.4 Hz, H6), 8.62 (d, 1H, H7), 8.87 (d, 1H, H5); 13C
NMR (DMSO-d6): d 35.66, 38.20, 116.04, 119.46, 120.00, 122.17,
132.27, 139.08, 147.52, 147.72, 150.13, 171.27, 172.44; MS (ESI)
m/z 342 (M+2H)+. Anal. Calcd for C13H9NO6S.0.5H2O: C, 44.78; H,
2.87; N, 4.02. Found: C, 44.58; H, 2.93; N, 3.97.
5.14. General procedure for the preparation of malonic acids 1e
and 2e
Trifluoroacetic acid (0.3 mL) was added to
a solution of
diterbutyl malonate 1d or 2d (0.3 mmol) dissolved in dichloro-
methane (10 mL). The reaction mixture was then stirred at
room temperature for 12 h. The solvent was removed under
reduced pressure and the residue was taken up in ether. Final-
ly, the precipitate was filtered to give the expected acids 1e
and 2e.
5.20. 3,30-[(5,8-Dioxo-5,8-dihydroquinoline-6,7-diyl)disulfanediyl]
dipropanoic acid (7)
Compound 7 was obtained from 6,7-dibromo-5,8-quinolinedi-
5.15. [(1,4-Dioxo-1,4-dihydronaphthalen-2-yl)methyl]malonic
acid (1e)
one
5
(400 mg, 1.26 mmol) and mercaptopropanoic acid
(0.28 mL, 3.8 mmol) as a red powder (291 mg, 63%); mp 205 °C;
1H NMR (CD3OD): d 2.70 (s, 4H, 2ꢀ CH2), 3.50 (s, 4H, 2ꢀ CH2),
7.80 (dd, 1H, J = 4.4 Hz, J = 8.4 Hz, H6), 8.48 (d, 1H, H7), 8.90 (d,
1H, H5); 13C NMR (DMSO-d6): d 30.29, 35.97 (2 C), 45.81, 128.59,
130.76, 135.35, 149.40, 154.67, 173.72 (4 C), 177.92, 179.19; MS
(ESI) m/z 366 (MꢁH)ꢁ. Anal. Calcd for C15H13NO6S2: C, 47.87; H,
3.72; N, 3.72. Found: C, 48.05; H, 3.78; N, 4.18.
Compound 1e was obtained from compound 1d (140 mg,
0.36 mmol) as a yellow powder (45 mg, 70%); mp 212 °C; 1H
NMR (CDCl3): d 3.02-3.05 (d, 2H, J = 7.4 Hz, CH2), 3.71 (t, 1H,
CH), 6.96 (s, 1H, H3), 7.91–7.95 (m, 2H, H6 and H7), 8.03–8.11
(m, 2H, H5 and H6); 13C NMR (DMSO-d6):
d 28.69, 50.52,
126.00, 126.61, 131.81, 132.06, 134.57, 135.85, 135.88, 148.43,
170.31 (2C), 184.63, 184.81; MS (ESI) m/z 273 (M+H)+. Anal.
Calcd for C14H10O6 0.5H2O: C, 59.36; H, 3.88. Found: C, 59.28;
5.21. Enzyme, cell culture, chemicals, and antibodies
.
H, 3.94.
The recombinant MBP-CDC25B3 protein was produced in bacte-
ria as previously described.40 Human cancer cell line HeLa was ob-
tained from Aptanomics (Lyon, France). Cells were cultured at
37 °C in Dulbecco’s minimum essential medium complemented
with 10% fetal bovine serum and 100 U/ml penicillin/streptomycin
in a humidified atmosphere of 5% CO2. The tetrazolium salt WST-1
was purchased from Roche Diagnostics (Mannheim, Germany) and
propidium iodide from Sigma–Aldrich (St Louis, MO, USA). The fol-
lowing antibodies were used for flow cytometry analysis: anti-H3P
antibody from Upstate Biotechnology (Lake Placid, NY, USA) and
anti-rabbit Alexa 488 antibody from Sigma-Aldrich (St Louis, MO,
USA).
5.16. [2-(1,4-Dioxo-1,4-dihydronaphthalen-2-yl)ethyl]malonic
acid (2e)
Compound 2e was obtained from compound 2d (0.13 g,
0.33 mmol) as a gray powder (78 mg, 82%); mp 238 °C; 1H NMR
(CDCl3): d 2.04–2.10 (m, 2H, CH2–CH2–CH), 2.56 (t, 2H, J = 7.4 Hz,
CH2–CH2–CH), 3.30 (m, 1H, CH), 6.84 (s, 1H, H3), 7.90–7.96 (m,
2H, H6 and H7), 8.02–8.10 (m, 2H, H5 and H6); 13C NMR (DMSO-
d6): d 26.82, 27.33, 51.41, 125.89, 126.50, 132.01, 132.30, 134.35,
135.17, 135.21, 150.44, 170.98 (2C), 184.84, 184.89; MS (ESI) m/z
287 (M+H)+. Anal. Calcd for C15H12O6 0.5H2O: C, 60.60; H, 4.20.
.
Found: C, 60.18; H, 4.21.
5.22. In vitro enzymatic assay and Steady-State Kinetics
5.17. 3-[(3-Methyl-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)sulfanyl]propanoic acid (4)
The activity of the MBP-CDC25B3 recombinant enzyme was
monitored using fluorescein diphosphate. The assay was per-
formed in 96-well plates in a final volume of 200 lL. MBP-
Compound 4 was obtained according to reported procedure21
from menadione (1 g, 8.71 mmol) and mercaptopropanoic acid
(0.5 ml, 8.71 mmol) as an orange powder (470 mg, 20%); mp
163 °C; 1H NMR (DMSO-d6): d 2.26 (s, 3H, CH3), 2.61 (t, 2H,
J = 6.7 Hz, S–CH2), 3.30 (t, 2H, CH2–CO2H), 7.83–7.86 (m, 2H, H6
and H7), 8.00–8.04 (m, 2H, H5 and H6); 13C NMR (DMSO-d6): d
15.99, 29.86, 36.14, 126.88, 127.27, 132.43, 133.37, 134.55,
134.75, 147.56, 173.64, 181.42, 182.55; MS (ESI) m/z 275 (M-H)ꢁ.
Anal. Calcd for C14H12O4S: C, 60.87; H, 4.34. Found: C, 61.60; H,
4.40.
CDC25B3 was diluted in assay buffer [30 mM Tris–HCl (pH 8.2),
75 mM NaCl, 0.67 mM EDTA, 0.033% BSA, 1 mM DTT] so that the fi-
nal concentration was 90 ng/well. The reaction was initiated by
addition of 30 lM of fluorescein diphosphate followed by immedi-
ate measurement of fluorescein monophosphate emission with a
Fluoroskan Ascent (Lab Systems; excitation filter: 485 nm, emis-
sion filter: 530 nm). For each compound, the drug concentration
required for 50% inhibition (IC50) was determined from a sigmoidal
dose-response curve using GraphPad Prism 4 (GraphPad Software,
San Diego, CA). Compounds 4 and 6 were added to the wells at a
final concentration of 0.3, 1, 3, 6 and 10
lM. Substrate concentra-
5.18. General procedure for the preparation of quinolinediones
(6) and (7)
tion was varied between 1 and 50 M (Km = 2.89 l
are expressed as the mean of two independent experiments with
l
M). The results
three determinations per tested concentration and per experiment.
To a solution of 6,7-dibromo-5,8-quinolinedione 5 (1 mmol)
dissolved in THF (6 mL) were added mercaptoacetic acid or merca-
ptopropanoic acid (2.5 mmol) and triethylamine (3 mmol). The
reaction mixture was stirred at room temperature for 2 h and then
poured into water. The precipitate was filtered to give the expected
quinolinediones 6 and 7.
5.23. Cell proliferation assay
The inhibition of cell proliferation was determined using a col-
orimetric assay based on the cleavage of the WST-1 tetrazolium
salt by mitochondrial dehydrogenases in viable cells, leading to

9048
E. Braud et al. / Bioorg. Med. Chem. 16 (2008) 9040–9049
formazan formation. On day 1, HeLa cells were plated at 3000 cells/
well in 96-well culture plates with 95 L of medium/well. On day
2, cells were treated for 96 h with 5 L of increasing concentrations
of drug. On day 6, after addition of 10 L of WST-1 per well, cells
were incubated for 2 h at 37 °C in a humidified atmosphere of 5%
CO2. Absorbance was measured at 430 nm. The results are ex-
pressed as the mean of three independent experiments with eight
determinations per tested concentration and per experiment. For
each compound, the IC50 value was determined from a sigmoidal
dose-response using GraphPad Prism (GraphPad Software, San Die-
go, CA).
(Accelrys) and the cff91force field. Energy minimizations and
molecular dynamics were realized without considering water mol-
ecules and by fixing the value of the dielectric constant to 4. The
protein backbone was held frozen, while the lateral chains and
the inhibitor were relaxed. After 5000-fs initialization at 300 K,
l
l
l
100 steps of molecular dynamics calculations were performed.
41
Each was done at 300 K during 5000 steps of 1 fs. As in Ref.
,
the resulting conformations were submitted to a conjugate-
gradient energy minimization and stored.
Acknowledgments
5.24. Clonogenic assay
We are grateful to Dr. Wang-Qing Liu for mass spectra
recording. This research is supported by La Ligue Nationale contre
le Cancer, Equipe Labellisée 2006 (U648) and Equipe Labellisée
2008 (UMR 5088-IFR109).
For cloning inhibition assays, HeLa cells were plated at 100
cells/well in 6-well culture plates with 2.5 mL of medium/well.
After 24 h growing at 37 °C in a humidified atmosphere of 5%
CO2, the culture medium was replaced by control medium or med-
ium containing inhibitors at increasing concentrations (up to
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