Synthesis of small molecule CDC25 phosphatases inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.09.041

A targeted library of 2-(4-nitrophenyl)ethylbenzylamines has been prepared to optimize the biological activity of BN82002, our initial lead compound, recently described as an original inhibitor of CDC25 phosphatases. The most potent of these compounds inhibit CDC25 in the micromolar range. A targeted library of small molecules has been prepared to optimize the biological activity of BN82002, our initial lead compound, recently described as an original inhibitor of CDC25 phosphatases. Some of these compounds inhibit CDC25 in the micromolar range and therefore reinforce the interest of CDC25 as an anticancer target.

Full text of DOI:10.1016/j.bmcl.2004.09.041

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5809–5812
Synthesis of small molecule CDC25 phosphatases inhibitors
a
a,
Marie-Odile Contour-Galcera, Olivier Lavergne, Marie-Christine Brezak,
a
*
´
Bernard Ducommun and Gregoire Prevost
b
a
´
´
a
´
Ipsen Research Laboratories, Institut Henri Beaufour, 5, Avenue du Canada, F-91966 Les Ulis Cedex, France
´ ´
LBCMCP-CNRS UMR5088-IFR109, ‘Institut d’Exploration Fonctionnelle des genomes’, Universite PaulSabatier,
b
118 route de Narbonne, 31062 Toulouse, France
Received 29 June 2004; revised 26 August 2004; accepted 17 September 2004
Abstract—A targeted library of small molecules has been prepared to optimize the biological activity of BN82002, our initial lead
compound, recently described as an original inhibitor of CDC25 phosphatases. Some of these compounds inhibit CDC25 in the
micromolar range and therefore reinforce the interest of CDC25 as an anticancer target.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
N
O
O
4
5
6
3
Protein phosphorylation/dephosphorylation is one of
the most important mechanisms used by cells to regulate
crucial biological events such as metabolism, gene
expression, cell division, differentiation and develop-
ment. CDC25 phosphatases, dual specificity enzymes,
which can dephosphorylate both phospho-Ser/Thr and
phospho-Tyr residues,1 are essential regulators by de-
phosphorylating and activating CDK/cyclin complexes
at key transition steps of the cell cycle. It has been
shown that overexpression of CDC25 is characteristic
of a number of human cancers2 and consequently, inhi-
bition of these phosphatases could represent a new and
valuable approach in cancer therapy.3 Although several
inhibitors have been described in the literature,4,5 only a
few molecules have shown in vivo efficacy in xenograft
models.
9
11
N
12
13
2
MeO
10
1
8
7
OH
11'
NO2
12'
1
2
Figure 1. Structure of menadione (1) and BN82002 (2).
BN82002 (2, Fig. 1), was identified as a hit with inhibi-
tory properties at micromolar concentrations, and a
family of related compounds has been prepared to
explore the structure–activity relationships in the series.
2. Chemistry
Synthesis of BN82002 was performed by reductive
amination of 5-(dimethylamino)-2-hydroxy-3-methoxy-
benzaldehyde (3)8 with N-methyl-2-(4-nitrophenyl)-
ethylamine (4) (Scheme 1).9,10
To identify new inhibitors, we have established a high-
throughput screening of our molecular library, using
recombinant human CDC25C6 and one of the first
CDC25 inhibitors described in the literature, Menadi-
one (1, Fig. 1), as a positive control.7
These conditions are well suited for parallel synthesis
strategies and in particular, for the use of polymer sup-
ported reagents and scavengers. This methodology,
starting from a carboxaldehyde (5), and an amine (6),
allows the rapid preparation of secondary or tertiary
amines (7) containing the required benzylamine moiety
(Scheme 2). Aldehydes 5 in the presence of amines 6 lead
to an imine intermediate, which is reduced11 in situ by
treatment with borohydride supported on resin.12
Keywords: CDC25 inhibitor; Dual specificity phosphatases inhibitor;
Antiproliferative agent.
*
Corresponding author. Tel.: +33 160922000; fax: +33 169073802;
e-mail: m-odile.galcera.contour@ipsen.com
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.041

´
M.-O. Contour-Galcera et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5809–5812
5810
N
N
a
HN
+
H
N
HCl
O
O
NO2
OH
O
OH
NO2
3
4
2
Scheme 1. Synthesis of 2. Reagents and conditions: (a) (i) 4 (1.1equiv), Et3N (1.5equiv), MeOH, 25°C, 18h; (ii) NaBH4 (1.1equiv), 25°C, 4h (34%
for steps (i) and (ii)).
R1
R1
R4
R5
HN
R2
R3
a
R2
R3
R5
N
+
H
HCl
NO2
R4
O
NO2
5
6
7
Scheme 2. Synthesis of 7. Reagents and conditions: (a) (i) 6 (1.2equiv), Et3N (1.3equiv), MeOH, 25°C, 18h; (ii) polymer supported borohydride
(2equiv), 25°C, 2h.
Compounds 7 are obtained directly by this Ôone potÕ
process with acceptable purity using electrophilic scav-
enger resins: 4-benzyloxybenzaldehyde polystyrene
resin,13 selective for primary amines in the presence of
secondary ones and methylisothiocyanate polystyrene
resin14 for secondary amines.15 Purities were determined
by LC/MS.16 UV purity of the compounds presented is
greater than 80%.
the dimethylamino and hydroxy substituents are para
or ortho to each other on the phenyl ring.
Furthermore, the comparison between 7m and 7j and
between 7n and BN82002 (2) shows that the secondary
amines (7m,n) are somewhat better CDC25C inhibitors
than the corresponding tertiary amines.
BN82002 has been further studied, to confirm the mech-
anism of action.17 BN82002 has been shown to inhibit
the phosphatase activity of recombinant CDC25A,
CDC25B2, CDC25B3, as well as CDC25C through irre-
versible linkage to its catalytic domain. From a mecha-
nistic point of view, it has been checked by western-blot
analysis that BN82002 inhibits tyrosine dephosphoryl-
ation on CDK1, the natural CDC25C substrate, and
thereby inactivates the CDK1/cyclin B1 complex in cul-
tured Mia PaCa-2 cells. In addition, it has been shown,
in synchronized HeLa cells, that BN82002 delays cell
cycle progression at G1/S, in S-phase and at the G2/M
transition. This observation is in accordance with
BN82002 being active against several members of the
CDC25 phosphatase family. Thus, cell cycle inhibition
is observed at the various stages of the cell cycle where
CDC25 phosphatases activities are thought to be
required.
3. Results and discussion
Enzyme inhibition was assayed using recombinant fused
MBP–CDC25 with 3-O-methylfluorescein phosphate
(OMFP) as the substrate.6 In this test, BN82002 (2)
showed in vitro inhibition of CDC25C phosphatase
in a dose dependent manner with an IC50 value of
5.4lM; menadione, under the same conditions, showed
an IC50 of 18.8lM.
Using the parallel synthesis methodology described, a
series of 2-(4-nitrophenyl)ethylbenzylamines (7a–n)
incorporating the Rgroups present in BN82002 has
been prepared. The starting benzaldehydes were chosen
with one, two or three substituents present on the benzyl
moiety of the BN82002: dimethylamino, methoxy and
hydroxy groups. No activity was observed with the
unsubstituted phenyl ring or with monosubstituted com-
pounds (7a–e, Table 1). Among the disubstituted com-
pounds, hydroxy and methoxy groups at R3 and R4
positions led to a loss of biological activity (7f) as did
1,2- and 1,4-dimethoxy and 1-methoxy-3-dimethyl-
amino substitution (7g,h,i). On the other hand, the
1-hydroxy-4-dimethylaminophenyl ring (7j) gave appre-
ciable activity. The replacement of the N-dimethylamino
by a nitro group (7k) induced a loss of enzyme inhibi-
tion. The trisubstituted phenyl ring: 2-methoxy-3-hydr-
oxy-4-dimethylaminophenyl (7l) was moderately active.
In summary, positive results were obtained only when
On the basis of the targeted mechanism of action, inhibi-
tors of CDC25 such as BN82002 and the other inhibi-
tors of the series should prevent cell proliferation of a
broad spectrum of cell types. Preliminary results have
confirmed that compounds with significant CDC25
inhibitory activity are antiproliferative in cell culture
assays.18 Studies on the profile of the antiproliferative
activity are on-going as a prelude to the choice of appro-
priate xenograft models.
In conclusion, through the synthesis of a series of ana-
logs of our CDC25 inhibitor, BN82002, we have identi-

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M.-O. Contour-Galcera et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5809–5812
5811
Table 1. CDC25C inhibition by a series of general structure (7) (IC50: 50% inhibitory concentration)
R1
R2
R5
N
R3
R4
NO2
7
Compound
R1
R2
R3
R4
R5
IC50 (lM)a
BN82002 (2)
7a
NMe2
H
H
OMe
H
OH
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
5.4 1.2
NAb
NA
H
7b
7c
NMe2
H
H
H
H
NMe2
H
H
H
NA
NA
7d
7e
H
OMe
H
H
H
H
OH
OH
OMe
OMe
OMe
OH
OH
H
NA
NA
7f
7g
H
H
OMe
OMe
H
H
H
NA
NA
7h
7i
OMe
H
H
NMe2
H
H
NA
5.6 1.6
NA
7j
7k
NMe2
NO2
NMe2
NMe2
NMe2
H
H
H
7l
7m
OH
H
OMe
H
OMe
20.7 0.6
3.9 0.4
4.0 0.6
18.8 1.1
OH
OH
7n
Menadione (1)
H
H
a The IC50 values reported are calculated from at least two independent experiments. Data represent the mean SEM.
b NA: not active, IC50 > 80lM.
7. Ham, S. W.; Park, H. J.; Lim, D. H. Bioorg. Chem. 1997,
25, 33–36.
8. Ando, M.; Emoto, S. Bull. Chem. Soc. Jpn. 1978, 51,
2433–2434.
9. Auvin, S.; Chabrier de Lassauniere, P.-E.; Harnett, J.;
Pons, D.; Ulibarri, G. WO 0017190, 2000.
fied the key moiety of the molecule, and have prepared,
using parallel synthesis strategy, further inhibitors of
phosphatase CDC25C. Studies are in progress to evalu-
ate their in vitro and in vivo behaviour. These results
reinforce the interest of considering inhibiting cell cycle
driving enzymes as a therapeutic strategy in oncology.
10. Compound 2: 3 (1g, 5.12mmol) was added to a mixture of
4 (1.22g, 5.63mmol) and triethylamine (1.1mL, 7.7mmol)
in anhydrous methanol (30mL) at room temperature
under argon, and the resulting mixture was stirred for 18h.
Sodium borohydride (0.213g, 5.63mmol) was then added
and stirring was maintained for a further 4h. The reaction
mixture was then partitioned between cold water (10mL)
and dichloromethane (50mL), and the aqueous phase was
further extracted with dichloromethane. The combined
organic layers were dried over magnesium sulfate, filtered
and concentrated to give 1.10g of a brown viscous oil.
Purification by chromatography on silica gel using 3%
methanol in dichloromethane gave 0.626g (34%) of 2 as a
Acknowledgements
We thank Jose Camara, Denis Giraud and Gilles Mario
for analytical support and Jerry Harnett and Christine
Dolo for the initial preparation of BN82002.
References and notes
1
1. (a) Kumagai, A.; Dunphy, W. G. Cell 1991, 64, 903–914;
(b) Strausfeld, U.; Labbe, J.-C.; Fesquet, D.; Cavadore,
J.-C.; Picard, A.; Sadhu, K.; Russell, P.; Doree, M. Nature
1991, 351, 242–245; (c) Gautier, J.; Solomon, M. J.;
Booher, R. N.; Bazan, J. F.; Kirschner, M. W. Cell 1991,
67, 197–211.
brown solid, mp 92–93°C; H NMR(DMSO): d 8.90 (s,
1H, OH), 8.15–8.12 (d, 2H, J = 8.7Hz, 12, 120-H), 7.52–
7.50 (d, 2H, J = 8.7Hz, 11, 110-H), 6.28 (s, 1H, 3-H), 6.04
(s, 1H, 5-H), 3.70 (s, 3H, OCH3), 3.57 (s, 2H, 7-CH2), 2.94
(t, 2H, J = 7.1Hz, 8-CH2), 2.74–2.70 (m, 8H, 9-CH2,
N(CH3)2), 2.21 (s, 3H, NCH3).
2. (a) Gasparotto, D.; Maestro, R.; Piccinin, S.; Vukosavl-
jevic, T.; Barzan, L.; Sulfaro, S.; Boiocchi, M. Cancer Res.
1997, 57, 2366–2368; (b) Guo, J.; Kleeff, J.; Li, J.; Ding, J.;
11. Kaldor, S. W.; Siegel, M. G.; Fritz, J. E.; Dressman, B. A.;
Hahn, P. J. Tetrahedron Lett. 1996, 37, 7193–7196.
12. Borohydride, polymer supported (on AmberliteÒ IRA-
400), supplier: Aldrich Chemical Company, Inc.
13. 4-Benzyloxybenzaldehyde polystyrene HL (200–400
mesh), 2% DVB, supplier Novabiochem.
Hammer, J.; Zhao, Y.; Giese, T.; Korc, M.; Buchler, M.
¨
W.; Friess, H. Oncogene 2004, 23, 71–81.
3. Eckstein, J. W. Invest. New Drugs 2000, 18, 149–156.
4. Pestell, K. E.; Ducruet, A. P.; Wipf, P.; Lazo, J. S.
Oncogene 2000, 19, 6607–6612.
14. Methylisothiocyanate polystyrene HL (200–400mesh), 2%
DVB, supplier Novabiochem.
5. Prevost, G. P.; Brezak, M.-C.; Goubin, F.; Mondesert, O.;
Galcera, M.-O.; Quaranta, M.; Alby, F.; Lavergne, O.;
Ducommun, B. Prog. Cell Cycle Res. 2003, 5225–5234.
6. Mondesert, A.; Lemaire, M.; Brezak, M.-C.; Galcera-
Contour, M.-O.; Prevost, G.; Ducommun, B.; Bugler, B.
Curr. Genet. 2004, 45, 283–288.
15. Compounds 7a–n: 50lmol of aldehyde 5 was added to a
mixture of amine 6 (60lmol, 1.2equiv) and triethylamine
(66lmol, 1.3equiv), in anhydrous methanol (1mL) and the
resulting mixture was stirred at room temperature for 18h.
Borohydride (100lmol, 2equiv) supported on resin were
then added and stirring was maintained for 2 additional

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M.-O. Contour-Galcera et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5809–5812
5812
hours. Compounds 7a–l: 60 lmol (1.2equiv) of 4-benzyl-
model) and HPLC retention times were acquired on an
HPLC system (HP 1100) equipped with a photodiode
array UV detector.
oxybenzaldehyde polystyrene resin was added and the
mixture stirred for 18h at room temperature. Compounds
7m and 7n: 60lmol (1.2equiv) of methylisothiocyanate
polystyrene resin was added and the mixture stirred for
18h at room temperature. In both cases, the resins were
filtered and washed with MeOH. The solvent was evapo-
rated and compounds 7a–n were obtained as yellow oils.
´
17. Brezak, M.-C.; Quaranta, M.; Mondesert, O.; Galcera,
M.-O.; Lavergne, O.; Alby, F.; Cazales, M.; Baldin, V.;
Thurieau, C.; Harnett, J.; Lanco, C.; Kasprzyk, P. G.;
Prevost, G. P.; Ducommun, B. Cancer Res. 2004, 64,
3320–3325.
16. Mass spectra were acquired on a single quadrupole
electrospray mass spectrometer (Micromass, Platform
18. Prasad, K. N.; Edwards-Prasad, J.; Sakamoto, A. Life Sci.
1981, 29, 1387–1392.