Synthesis and biological evaluation of novel coumarin-based inhibitors of Cdc25 phosphatases

Article abstract of DOI:10.1016/j.bmcl.2010.07.130

The cell division cycle 25 (Cdc25) family of proteins are dual specificity phosphatases that activate cyclin-dependent kinase (CDK) complexes, which in turn regulate progression through the cell division cycle. Overexpression of Cdc25 proteins has been reported in a wide variety of cancers; their inhibition may thus represent a novel approach for the development of anticancer therapeutics. Herein we report new coumarin-based scaffolds endowed with a selective inhibition against Cdc25A and Cdc25C, being 6a and 6d the most efficient inhibitors and worthy of further investigation as anticancer agents.

Full text of DOI:10.1016/j.bmcl.2010.07.130

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5827–5830
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel coumarin-based inhibitors
of Cdc25 phosphatases
*
Sergio Valente, Emilie Bana, Elodie Viry, Denyse Bagrel, Gilbert Kirsch
Laboratoire d’Ingénierie Moléculaire et Biochimie Pharmacologique, Institut Jean Barriol, Université Paul Verlaine, FR CNRS 2843, 1 Boulevard Arago, 57070 Metz, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The cell division cycle 25 (Cdc25) family of proteins are dual specificity phosphatases that activate cyclin-
dependent kinase (CDK) complexes, which in turn regulate progression through the cell division cycle.
Overexpression of Cdc25 proteins has been reported in a wide variety of cancers; their inhibition may
thus represent a novel approach for the development of anticancer therapeutics. Herein we report new
coumarin-based scaffolds endowed with a selective inhibition against Cdc25A and Cdc25C, being 6a
and 6d the most efficient inhibitors and worthy of further investigation as anticancer agents.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 11 June 2010
Revised 28 July 2010
Available online 3 August 2010
Keywords:
Cdc25 phosphatases
Coumarins
Chalcones
Heck coupling
Cell division cycle 25 (Cdc25) phosphatases are key actors in
eukaryotic cell cycle control. The three human Cdc25 (Cdc25A,
Cdc25B, and Cdc25C) are dual specificity phosphatases responsible
for dephosphorylating Cdk/cyclins on pThr14 and/or pTyr15
residues, so activating Cdk/cyclin during normal cell cycle progres-
sion.1 So far, Cdk–cyclin complexes are the only known substrates
for Cdc25 phosphatases.2
Cdc25A seems to be implicated in the control of G1/S and G2/M
transitions, whereas Cdc25B and Cdc25C seems to be more impli-
cated in regulation of G2/M transition.3 The transitions between
each cell cycle phase must be strictly regulated in order to
maintain genomic stability. Over activity of these phosphatases is
associated with checkpoint bypass and upset this genomic equilib-
rium.1 Thus, experimental data show that overexpression of
Cdc25B (but not Cdc25C) rapidly pushes S or G2 phase cells into
mitosis with incompletely replicated DNA4 and overexpression of
Cdc25A can induces mitotic events.3,5,6 Cdc25 phosphatases are
also central targets and regulators of the G2/M checkpoint mecha-
nisms activated in response to DNA injury including ATM/ATR
pathways.7
in many high-grade tumors and is correlated with poor prognosis
in human cancers.8 Cell division cycle 25 proteins are reported to
be overexpressed in primary tumor samples from patients with
breast, prostate, ovarian, endometrial, colorectal, oesophageal, thy-
roid, gastric and hepatocellular cancers, glioma, neuroblastoma or
non-Hodgkin lymphoma.1 Until now, a lot of inhibitors have been
tested for Cdc25 inhibition.9–11 Three non-quinone and quinone
based compounds (BN 82002, BN82685 and IRC083864) are
reported as active on, respectively HeLa, miaPaCa2 and LNCap cell
lines with an in vivo activity in xenografted mice12–14 (Fig. 1).
Nevertheless, in 2009 a new class of thiazolopyrimidines
( Fig. 1), like Cdc25B inhibitors were disclosed by parallel click
Cdc25 phosphatases can thus serve as central regulators of the
cell cycle with the role of driving each state of cell division. The
expression and activity of these enzymes are finely regulated by
multiple mechanisms including post-translational modifications,
interactions with regulatory partners, control of their intracellular
localization (all Cdc25s shuttle between the nucleus and the cyto-
plasm during the cell cycle6), and cell cycle-regulated degradation.
Accordingly, increased expression of Cdc25A and Cdc25B is found
* Corresponding author. Tel.: +33 (3) 87315295; fax: +33 (3) 87325801.
E-mail address: kirsch@univ-metz.fr (G. Kirsch).
Figure 1. Cdc25 phosphatases inhibitors active in cultured cells.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.130

5828
S. Valente et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5827–5830
Scheme 2. Reagents and conditions: (a) dry DMF, 0 °C, 1 h, 85–90%.
(Scheme 2) The reverse chalcone–coumarin (cinnamoyl coumarin)
10a–c were prepared with a different synthetic approach: indeed,
Figure 2. Novel coumarin-based inhibitors of Cdc25 phosphatases.
through a-carbon regioselective Heck coupling onto the 4-tosylate
intermediate 724 as reported,25 followed by acidic hydrolysis,
4-acetylcoumarin 826 was provided. The final compounds 10a–c
were furnished by aldolic condensation of 8 with the opportune
aldehydes 9a–c (Scheme 3).
At last we investigated the introduction of a sulfur atom be-
tween coumarin and benzoyl moieties, as shown by derivatives
14a–c, which were synthesized reacting 4-chloromethylcoumarin
11, prepared according to the method previously by us reported,27
with thiourea and, after hydrolysis, yielded 4-mercaptomethyl-
coumarin 12. Finally 12 was converted to 14a–c through alkylation
chemistry and in situ screening and shown to display cytotoxic
properties against HeLa cells15 and against prostatic LNCaP cells.16
Furthermore, recently several flavonolignan compounds isolated
from silymarin showed a strong cell cycle arrest in PC3 cells and
differential effect of these compounds on the levels of some cell cy-
cle regulators-cyclins among these Cdc25A, Cdc25B and Cdc25C.17
Finally it has been reported in Caenorhabditis elegans CDC-25.1 was
inhibited by flavones.18 So, prompted by these findings and by the
structural basis of the flavone nucleus, and with the attempt to
identify new scaffolds able to inhibit Cdc25 phosphatases, we
developed the synthesis of novel coumarin-based small molecules
(Fig. 2) and we tested such derivatives against all of three Cdc25
isoenzymes (Fig. 2).
Our approach started with the introduction of 4-methoxy(hy-
droxy)phenyl and 4-methoxy(hydroxy)styryl groups at C3 position
of the 4-methylcoumarin (compounds 2a–b, 3a–b). Compound 119
underwent a Suzuki coupling reacting with 4-methoxyphenylbo-
ronic acid, palladium acetate, potassium hydrogen phosphate to af-
ford compound 2a,20 whereas, through Heck coupling reacting
with 4-methoxy styrene and palladium chloride, gave compound
3a. Finally, carrying out onto the methoxy derivatives 2a and 3a
a cleavage with boron tribromide 2b20 and 3b, respectively, were
obtained (Scheme 1).
with the opportune
x-bromoacetophenones 13a–c (Scheme 4).
Human glutathione-S-transferase (GST)–Cdc25 recombinant
enzymes were used to evaluate the inhibitory potential of com-
pounds. Each isoenzyme was prepared as previously described.10,28
Briefly, the GST-tagged Cdc25s were expressed in a bacterial
expression system via an IPTG induction. After lysis of bacteria,
purification on GSH-Agarose column allowed to obtain pure
GST–Cdc25 recombinant proteins. Recombinant Cdc25A and
Cdc25 C are full length enzymes whereas Cdc25 B is truncated
one (active site only). The enzymatic activity was measured by a
dephosphorylation assay with 3-O-methyl fluorescein phosphate
as described.29 The results are expressed as percentage of inhibi-
tion of Cdc25 phosphatase activity in presence of the tested
compounds. All compounds were tested at 100 lM final concentra-
tion, except for BN82002 (Sigma–Aldrich), used as reference drug,
The synthesis of the chalcone–coumarin (benzoylvinyl couma-
rin) derivatives 6a–f was performed through Wittig condensation
between aldehydes 4a21 or 4b22 and phosphoranes 5a–e, previ-
ously prepared according to methods described in literature.23
Scheme 1. Reagents and conditions: (a) p-MeO-Ph-boronic acid, K2HPO4, Pd(OAc)2,
MeOH, 60 °C, 2 h, 85%; (b) p-MeO-styrene, PdCl2, Et3N, reflux, 12 h, 78%; (c) BBr3,
CH2Cl2, À20 °C, overnight, 70%.
Scheme 3. Reagents and conditions: (a) (1) n-butyl vinyl ether, DIPEA, DPPF,
Pd2(dba)3, dioxane, 85 °C, 22 h, 75% (2) 2 N HCl, EtOH, rt, 3 h, 80%; (b) piperidine,
glacial acetic acid, EtOH, 70 °C, 3 h, 80–85%.

S. Valente et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5827–5830
5829
Table 2
Inhibitory activity (IC50 values) against Cdc25A and
against Cdc25C phosphatases of compounds 6a–fa
Compds
Phosphatases (IC50, lM)
Cdc25A
Cdc25C
6a
6b
6c
6d
6e
6f
27
70
78
28
96
62
49
47
67
26
>150
72
a
The IC50 values were determined by testing seven
different concentrations of compounds (from to
150 M). For each compound, each concentration was
0
l
separately tested in three independent microplates, at
the rate of three wells per microplate. The statistical
evaluation of IC50 was made with a software specially
designed for calculating the median inhibitory con-
centration for toxicity tests.31
Scheme 4. Reagents and conditions: (a) (1) NH2CSNH2, EtOH/Et2O, reflux, over-
night, (2) 2 N NaOH, rt, 10 min, (3) 2 N HCl, rt,75%; (b) KOH, EtOH, rt, 15 min,
85–90%.
which was tested at 10 lM (Table 1). Inhibition values percentage
of all tested derivatives are reported in Table 1.
or hydroxy groups onto the benzoyl moiety decreased the inhibi-
tory activity for Cdc25A and Cdc25 C.
With the exception of compounds 6a, 6b, 6d and 14a displaying
a weak inhibition of Cdc25B (35.0%, 21.3%, 23.2% and 16.1%, respec-
tively), these derivatives did not show affinity for recombinant
Cdc25 B. This result can be explained by the fact that this recombi-
nant protein is truncated and limited to its active site. Considering
first series of compounds (2a–b, 3a–b), 3-phenyl derivatives (2a)
inhibited feebly Ccd25 A and Cdc25 C or showed selectivity of
inhibition regarding Cdc25A (2b); when we introduced a vinyl
group between phenyl ring and coumarin ring the capability of
inhibition increased twofold against Cdc25A (from 20.5% (2a) to
45.3% (3a)) and almost threefold against Cdc25 C (from 17.9%
(2a) to 48.4% (3a)).
When we shifted the carbonyl group from phenyl ring to the
coumarin moiety, obtaining reverse chalcone–coumarin com-
pounds (10a–c), the inhibitory activity reduced up to half (10a)
or it was almost completely lost (10b–c) against all enzymes. The
insertion of a sulfur atom between coumarin moiety and benzoyl
group, as described for compounds 14a–c, did not ameliorate the
activity, but, interestingly, the trend was upset: indeed the addi-
tion of one or two methoxy groups onto the phenyl ring increased
the inhibition and, in particular, against Cdc25A until almost 40%
(14c).
These results were confirmed by establishing IC50 values (con-
centrations inhibiting 50% of enzyme activity) for the most potent
compounds (6a–f). Statistical calculations were performed using
generalized Poisson regression model, as described by Maul.31
(Table 2)
Furthermore we examined the reversible fixation of these small
coumarin-based molecules. Cdc25A phosphatase was pre-incu-
bated for several times with compound 6a at active concentration
In series of compounds 6, the introduction of benzoylvinyl moi-
ety at C4 position of coumarin nucleus (chalcone–coumarin 6a–f)
gave us an interesting increase of inhibition against both Cdc25A
and Cdc25 C up to 94.2% or 79.3% (6a) and 94.3% or 94.2% (6d),
respectively, being compounds 6a and 6d the most potent inhibi-
tors in this assay. The substitution of the methoxy group with hy-
droxy group onto the coumarin moiety didn’t improve the potency,
nevertheless it provided an higher inhibition for Cdc25 C (from
79.3% (6a) to 94.2% (6d)). The addition of just one or two methoxy
of 100
lM. Then, the reaction mixture was diluted 100-fold to ob-
tain a final non inhibitory concentration of 1
lM (data not shown).
It is assumed that the mechanism of action should be considered as
irreversible if the inhibitory effects persist after dilution13 (Fig. 3).
Table 1
Inhibitory activity (expressed as percentage of inhibition) of compounds 2a–b, 3a–b,
6a–f, 10a–c and 14a–c tested at 100 lM against Cdc25A, Cdc25B and Cdc25C
phosphatasesa
Compds30
Cdc25A
Phosphatases
Cdc25B
Cdc25C
2a
2b
3a
3b
6a
6b
6c
6d
20.5 ( 11.0)
22.0 ( 10.4)
45.3 ( 7.6)
27.5 ( 1.6)
94.2 ( 7.3)
66.4 ( 3.8)
61.5 ( 3.0)
94.3 ( 7.9)
55.0 ( 10.1)
68.7 ( 4.9)
45.0 ( 8.0)
ND
7.4 ( 9.9)
ND
26.7 ( 3.8)
39.8 ( 9.7)
87.9 ( 4.6)
ND
ND
ND
8.5 ( 3.1)
35.0 ( 5.5)
21.3 ( 4.2)
6.5 ( 4.5)
23.2 ( 10.5)
9.2 ( 7.0)
ND
ND
ND
9.1 ( 2.5)
16.1 ( 3.3)
ND
17.9 ( 0.8)
ND
48.4 ( 1.3)
15.6 ( 6.9)
79.3 ( 2.8)
64.8 ( 4.1)
69 ( 2)
94.2 ( 7.6)
23.9 ( 10.3)
43.7 ( 10.5)
31.3 ( 3.5)
4.8 ( 2.3)
ND
6e
6f
10a
10b
10c
14a
14b
14c
BN82002
Figure 3. Reversibility assay of Cdc25A inhibition by 6a. DMSO was used as control
and BN82002 as reference for irreversible inhibition. Cdc25A was pre-incubated
21.2 ( 7.2)
27.9 ( 4.5)
26.2 ( 6.8)
92.9 ( 9.6)
with inhibitory concentrations of 6a (100
time intervals (0–90 min). After incubation, the reaction mixture was diluted 100-
fold to reach inactive concentrations of 6a (1 M) and BN82002 (0.1 M). The
phosphatase activity was determined by adding the substrate OMFP in each well
containing 900 ng of Cdc25A after dilution. Data are representative of three
independent experiments (M SD).
lM), and BN82002 (10 lM) for various
ND
86.3 ( 2.1)
l
l
ND: not detectable
a
Values are means of three independent experiments, standard deviation is
given in round brackets.

5830
S. Valente et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5827–5830
12. Brezak, M. C.; Quaranta, M.; Mondesert, O.; Galcera, M. O.; Lavergne, O.; Alby,
Compound 6a displayed
a time-dependent inhibition of
F.; Cazales, M.; Baldin, V.; Thurieau, C.; Harnett, J.; Lanco, C.; Kasprzyk, P. G.;
Prevost, G. P.; Ducommun, B. Cancer Res. 2004, 64, 3320.
13. Brezak, M. C.; Quaranta, M.; Contour-Galcera, M. O.; Lavergne, O.; Mondesert,
O.; Auvray, P.; Kasprzyk, P. G.; Prevost, G. P.; Ducommun, B. Mol. Cancer Ther.
2005, 4, 1378.
14. Brezak, M. C.; Valette, A.; Quaranta, M.; Contour-Galcera, M. O.; Jullien, D.;
Lavergne, O.; Frongia, C.; Bigg, D.; Kasprzyk, P. G.; Prevost, G. P.; Ducommun, B.
Int. J. Cancer 2009, 124, 1449.
15. Duval, R.; Kolb, S.; Braud, E.; Genest, D.; Garbay, C. J. Comb. Chem. 2009, 11, 947.
16. Kolb, S.; Mondesert, O.; Goddard, M. L.; Jullien, D.; Villoutreix, B. O.;
Ducommun, B.; Garbay, C.; Braud, E. Chem. Med. Chem. 2009, 4, 633.
17. Deep, G.; Oberlies, N. H.; Kroll, D. J.; Agarwal, R. Int. J. Cancer 2008, 123, 41.
18. Kim, K. S.; Kawasaki, I.; Chong, Y.; Shim, Y. H. Mol. Cell 2009, 27, 345.
19. Kelkar, R. M.; Joshi, U. K.; Paradkar, M. V. Synthesis 1986, 3, 214.
20. Cook, C. E.; Corley, R. C.; Wall, M. E. J. Org. Chem. 1965, 30, 4120.
21. Ito, K.; Sawanobori, J. Synth. Commun. 1982, 12, 665.
Cdc25A, and the maximal inhibitory effect is observed for a
90 min pre-incubation (35% of residual activity) as indicated in
Figure 3. As the inhibitory activity persisted after the dilution pro-
cedure, compound 6a could be considered as acting as irreversible
inhibitor of the Cdc25 phosphatases in vitro.
In summary, we have identified new coumarin-based derivatives
as Cdc25 phosphatases inhibitors, among these the chalcone–cou-
marin series 6a–f showed the most interesting results, being 6a
and 6d endowed with lowest IC50 as reported in Table 2: 27 and
28 lM against Cdc25A, respectively. Compounds 6a and 6b can be
considered two new lead compounds for further work leading to
optimization, but also for in cell evaluation that will be reported in
due course.
22. Binda, C.; Wang, J.; Pisani, L.; Caccia, C.; Carotti, A.; Salvati, P.; Edmondson, D.
E.; Mattevi, A. J. Med. Chem. 2007, 50, 5848.
23. Marqués-López, E.; Herrera, R. P.; Marks, T.; Jacobs, W. C.; Könning, D.; de
Figueiredo, R. M.; Christmann, M. Org. Lett. 2009, 11, 4116.
Acknowledgments
24. Kuroda, J.; Inamoto, K.; Hiroya, K.; Doi, T. Eur. J. Org. Chem. 2009, 14, 2251.
25. Lindhardt, H. A.; Troels, S. J. Org. Chem. 2005, 70, 5997.
26. Ito, K.; Nakajima, K. J. Heterocycl. Chem. 1988, 25, 511.
27. Rodriguez-Dominguez, J. C.; Kirsch, G. Synthesis 2006, 11, 1895.
28. Brault, L.; Denance, M.; Banaszak, E.; El Maadidi, S.; Battaglia, E.; Bagrel, D.;
Samadi, M. Eur. J. Med. Chem. 2007, 42, 243.
29. The enzymatic activity of the GST–Cdc25 recombinant enzyme was performed
in 96-well plates in [50 mM Tris–HCl, 50 mM NaCl, 1 mM EDTA and 0.1% SAB,
The research leading to these results has received funding from
the [European Community’s] Seventh Framework Programme
([FP7/2007-2013] under Grant Agreement No. 215009.
Region de Lorraine, Conseil Général de la Moselle, and Ligue
contre le Cancer (54, 55, 57 Departmental comitees) are acknowl-
edged for financial support.
pH 8,1] buffer containing 3-O-methylfluorescein phosphate 500
substrate. The Gst–Cdc25 proteins, diluted in assay buffer, were used at a
final concentration of g/well. After 2 h at 30 °C, 3-O-methylfluorescein
fluorescent emission was measured with CytoFluor system Perspective
lM as
E.B. is recipient of an AFR grant from the ‘‘Fonds national de la
Recherche (Luxembourg)”.
1
l
a
Applied Biosystems; excitation filter: 475 nm and emission filter: 510 nm.
30. (a) All compounds were characterized by HRMS (ESI) and NMR analysis. (b)
Analytical data for compounds 6a and 6d: for 6a 1H NMR (250 MHz, DMSO-d6)
d ppm 3.90 (s, 3H, OCH3), 6.94–7.05 (m, 3H, coumarin protons), 7.55–7.70 (m,
3H, benzene protons), 7.88–8.20 (m, 5H, coumarin proton, benzene protons, –
CH@CHCOPh and –CH@CHCOPh); 13C NMR (250 MHz, CDCl3) d ppm 55.85,
98.20, 101.35, 109.85, 111.55, 112.70, 125.66, 128.70 (2C), 129.04 (2C), 129.62,
133.77, 136.45, 136.96, 148.78, 155.69, 160.96, 163.18, 179.61, 188.82; HRMS
(ESI) m/z 329.09 [M+Na]+; for 6d 1H NMR (250 MHz, DMSO-d6) d ppm 6.76–
6.85 (m, 3H, coumarin protons), 7.55–7.79 (m, 4H, benzene protons and
coumarin proton), 7.90–7.96 (d, 1H, –CH@CHCOPh), 8.07–8.20 (m, 3H, benzene
protons and –CH@CHCOPh), 10.70 (bs, 1H, OH); 13C NMR (250 MHz, DMSO-d6)
d ppm 102.67, 108.03, 110.05, 113.26, 126.30, 128.85 (2C), 128.88 (2C), 130.31,
133.75, 135.28, 136.65, 147.93, 155.22, 160.36, 161.52, 188.78; HRMS (ESI) m/z
315.06 [M+Na]+.
References and notes
1. Boutros, R.; Lobjois, V.; Ducommun, B. Nat. Rev. Cancer 2007, 7, 495.
2. Kristjansdottir, K.; Rudolph, J. Chem. Biol. 2004, 11, 1043.
3. Rudolph, J. Biochemistry 2007, 46, 3595.
4. Karlsson, C.; Katich, S.; Hagting, A.; Hoffmann, I.; Pines, J. J. Cell Biol. 1999, 146,
573.
5. Molinari, M.; Mercurio, C.; Dominguez, J.; Goubin, F.; Draetta, G. F. EMBO Rep.
2000, 1, 71.
6. Boutros, R.; Dozier, C.; Ducommun, B. Curr. Opin. Cell Biol. 2006, 18, 185.
7. Niida, H.; Nakanishi, M. Mutagenesis 2006, 21, 3.
8. Aressy, B.; Ducommun, B. Anticancer Agents Med. Chem. 2008, 8, 818.
9. Lavecchia, A.; Di Giovanni, C.; Novellino, E. Curr. Med. Chem. 2009, 16, 1831.
10. Brault, L.; Bagrel, D. Life Sci. 2008, 82, 315.
11. Lazo, J. S.; Aslan, D. C.; Southwick, E. C.; Cooley, K. A.; Ducruet, A. P.; Joo, B.;
Vogt, A.; Wipf, P. J. Med. Chem. 2001, 44, 4042.
31. Maul, A. Environ. Monit. Assess. 1992, 23, 153.