Synthesis and biological evaluation of dialkylsubstituted maleic anhydrides as novel inhibitors of Cdc25 dual specificity phosphatases

Article abstract of DOI:10.1016/j.ejmech.2006.09.014

An efficient synthesis of dialkylsubstituted maleic anhydrides 1a-j is described. The inhibitory potential of these original anhydride derivatives was tested toward the three human isoforms A, B and C of dual specific phosphatases Cdc25. A micromolar rang

Full text of DOI:10.1016/j.ejmech.2006.09.014

European Journal of Medicinal Chemistry 42 (2007) 243e247
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and biological evaluation of dialkylsubstituted
maleic anhydrides as novel inhibitors of Cdc25
dual specificity phosphatases
1
Laurent Brault , Mickael Denance , Estelle Banaszak, Souhayla El Maadidi,
1
´
Eric Battaglia, Denyse Bagrel, Mohammad Samadi
¨
,2
*
LIMBP, Universite´ Paul Verlaine e Metz, 1, bd Arago, Metz Technopoˆle, 57078 Metz, France
Received 5 July 2006; received in revised form 18 September 2006; accepted 21 September 2006
Available online 2 November 2006
Abstract
An efficient synthesis of dialkylsubstituted maleic anhydrides 1aej is described. The inhibitory potential of these original anhydride de-
rivatives was tested toward the three human isoforms A, B and C of dual specific phosphatases Cdc25. A micromolar range inhibition of
Cdc25s was observed with the maleic anhydrides bearing simple alkyl side chains longer than C9, to reach the optimal activity with
a C17 chain length.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: Barton decarboxylation; Maleic anhydride; Cdc25; Inhibition
1. Introduction
radioresistance [6]. Hence, Cdc25 inhibitors constitute attrac-
tive agents with potential anticancer activity and therefore an
increasing number of natural and synthetic small molecules
has been reported as inhibitors of Cdc25s [7e10]. Among
them, we have recently synthesized coscinosulfate which
exhibits a significant inhibition of Cdc25A with an IC50 of
3 mM [11]. Continuing our program devoted to the search
of Cdc25 phosphatase inhibitors, we investigated the effect
of dialkylsubstituted maleic anhydride toward Cdc25s. It
has been suggested that in dysidiolide, the g-hydroxybuteno-
lide residue likely serves as a phosphate surrogate, while the
long side chain occupies a hydrophobic binding pocket
[12,13]. Based on this consideration, and given that our
derivatives and dysidiolide analogues possess the same
constrained furan unit (maleic anhydride and g-hydroxybute-
nolide), we postulated that the anhydride moiety might also
act as phosphate mimic toward Cdc25s. Therefore, we
synthesized a series of maleic anhydrides, bearing various
simple alkyl side chains at C-4 position to examine their in-
hibitory activity toward Cdc25s. Herein we report an efficient
Dual specificity phosphatases Cdc25 play a crucial role in
the cell cycle progression [1]. They allow activation of cy-
clin/CDK complexes by dephosphorylating the phospho-
Thr14 and phospho-Tyr15 residues of the CDK subunit. The
Cdc25 family consists of three members: Cdc25A, Cdc25B
and Cdc25C, each of them acting on distinct phases of the
cell cycle. Whereas the G2-M transition is regulated through
CDK1 dephosphorylation by Cdc25B and Cdc25C, Cdc25A
mainly activates CDK2, allowing in particular progression
from G1 to S phase [2e4]. Interestingly, Cdc25A has been de-
scribed as an oncogene since this protein is over expressed in
a wide variety of human tumors [5] and is also implicated in
* Corresponding author. Tel.: þ33 3 87 54 77 93; fax: þ33 3 87 31 58 01.
E-mail address: samadi@sciences.univ-metz.fr (M. Samadi).
1
These authors contributed equally to the work.
Present address: Laboratoire de Chimie et Applications, Universite Paul
2
´
Verlaine e Metz, 1 bd Arago, Metz Technopole, 57078, Metz Cedex 3, France.
ˆ
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.09.014

244
L. Brault et al. / European Journal of Medicinal Chemistry 42 (2007) 243e247
synthesis of anhydrides 1aej using Barton radical decarbox-
ylation, which led to the discovery of a novel class of Cdc25
inhibitors.
derivatives indicates that none of these compounds inhibited
alkaline phosphatase, whereas an inhibitory potential was
found toward Cdc25s and was dependent upon the chain
length. The inhibitory activity was detected for compounds
1dej bearing an alkyl side chain of C10 and higher. In con-
trast, the anhydrides 1aec possessing aliphatic groups
shorter than C10 were inactive toward Cdc25s. The highest
activity was found for compounds 1hej that displayed
IC50 < 10 mM toward Cdc25s. Compounds 1g, 1h and 1j ex-
hibited a higher inhibitory activity toward Cdc25A than
Cdc25B (ratio ꢀ 1.5) and Cdc25C (ratio ꢀ 1.8). Interestingly,
the presence of the double bond in anhydride 1j bearing
a C17 chain length similar to 1i, seemed to reduce its activ-
ity toward Cdc25B and Cdc25C. This indicates that not
only the chain length but also its conformation may be in-
volved in activity toward Cdc25s. Our results highlight, for
these derivatives, the importance of the alkyl side chain,
which could interact with hydrophobic residues of the en-
zymes as already mentioned in the literature for another
Cdc25 inhibitor presenting an alkyl chain [8] or an inhibitor
bearing a benzene ring [17].
We further examined the kinetics of inhibition of human
Cdc25A by the potent derivative 1i (Fig. 1). The inhibition
profiles best fit with a mixed inhibition kinetic model
(R2 ¼ 0.945 ꢁ 0.987). This model implies that the inhibitor
combines with the free enzyme (dissociation constant KI)
and with the enzymeesubstrate complex (dissociation
constant KI0). Values of 0.58 ꢂ 0.07 mM for KI and
5.50 ꢂ 0.66 mM for KI0 were deduced from four independent
experiments. This inhibition model was already proposed for
structurally different Cdc25 inhibitors [9,18].
2. Chemistry
The dialkylsubstituted maleic anhydrides 1aej were synthe-
sized based on a two-step radical addition to phenyl maleimide
by means of Barton decarboxylation [14e16]. In the first step,
the readily available succinic acid monomethyl ester 2 was
transformed into its thiohydroxamic ester 3, in the presence of
2-mercaptopyridine-N-oxide using the DCC coupling method.
Irradiation of 3 in situ with tungsten light, in the presence of
N-phenyl maleimide, gave the addition product 4 as a mixture
of isomers. Treatment of 4 with mCPBA, followed by elimina-
tion of the resulting sulfoxide produced the monosubstituted an-
hydride 5 in 90% yield. Compound 5 was subjected as the olefin
trap to a second step radical decarboxylation. Thus acid 6 was
transformed into its thiohydroxamic ester 7 as was described
for 2, and irradiation in situ with tungsten light in the presence
of olefin 5, produced the intermediate addition product 8 as
a mixture of isomers, which was further treated with KOH in
MeOHeTHF to give the desired dialkylsubstituted maleic an-
hydride 1 as the sole isomer (Scheme 1).
3. Biology
The dialkylsubstituted maleic anhydrides 1aej were as-
sessed for in vitro inhibition of Cdc25s and horse liver al-
kaline phosphatase using 4-nitrophenyl phosphate ( p-NPP)
as substrate. As shown in Table 1, the screening of these
Ph
HO
COOH
N
N
O
O
O
O
C
Spy
S
i
MeOOC
MeOOC
NPh
O N
S
h
MeOOC
O
2
3
4
ii
O
iii
Spy: S
N
NPh
N
S
O
O
C
MeOOC
5
R
Spy
OH
O
O N
RCOOH
R
NPh
iv
h
MeOOC
S
6a-j
O
7
8
v
O
f : R = CH3(CH2)12
g: R = CH3(CH2)13
h: R = CH3(CH2)14
i : R = CH3(CH2)16
a: R = CH3(CH2)5
b: R = CH3(CH2)7
c: R = CH3(CH2)8
d: R = CH3(CH2)9
R
O
HOOC
O
1a-j
j : R = (Z)CH3(CH2)7CH=CH(CH2)7
e: R = CH3(CH2)10
Scheme 1. (i) DCC, CH2Cl2, rt, 2 h, then phenyl maleimide (5 equiv), hn, 15 ꢃC, 30 min (82%); (ii) mCPBA, CH2Cl2, 0 ꢃC, 1 h; (iii) toluene, 110 ꢃC, 1 h (90%
from 4); (iv) DCC, CH2Cl2, rt, 2 h, then 5 equiv of 5, hn, 15 ꢃC, 30 min; and (v) KOH, THFeMeOH, reflux, 3 h, 1a (44%); 1b (42%); 1c (43%); 1d (46%); 1e
(48%); 1f (51%); 1g (49%); 1h (50%); 1i (53%); 1j (45%), two steps from 6.

L. Brault et al. / European Journal of Medicinal Chemistry 42 (2007) 243e247
245
Table 1
Phosphatases inhibitory profile of dialkylsubstituted maleic anhydrides
5. Experimental section
Compound
IC50 (mM)a
5.1. Chemistry
Cdc25A
Cdc25B
Cdc25C
AP
All reactions were carried out under an argon atmosphere.
All reagents were obtained from commercial suppliers and
used without further purification. THF and toluene were
freshly distilled from sodium/benzophenone. Methylene chlo-
ride was distilled from CaH2. Flash chromatography was car-
ried out using silica gel 60 (Merck) with mixtures of ethyl
acetate and cyclohexane as eluent unless specified otherwise.
TLC analyses were performed on thin-layer analytical plates
60F254 (Merck).
1a
1b
1c
1d
1e
1f
NAb
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
53.9 ꢂ 1.7*
NA
44.4 ꢂ 1.4
44.3 ꢂ 2.4
8.1 ꢂ 0.3
6.3 ꢂ 0.2
4.7 ꢂ 0.1
5.1 ꢂ 0.3
4.5 ꢂ 1.7
55.2 ꢂ 1.4*
47.7 ꢂ 3.0
8.7 ꢂ 0.8
11.6 ꢂ 0.4*
7.6 ꢂ 0.6*
3.6 ꢂ 0.4*
6.7 ꢂ 1.9
5.7 ꢂ 0.2*
12.6 ꢂ 0.3*
8.6 ꢂ 1.5*
4.3 ꢂ 1.1
8.8 ꢂ 1.3
1g
1h
1i
1j
AP: horse liver alkaline phosphatase. *: Significantly different from Cdc25A
inhibitory activity at p < 0.05.
Note: The N-hydroxy-2-thiopyridone derivatives are some-
what sensitive to daylight. It is advisable to cover the reaction
flask with an aluminum foil.
a
IC50 values are means ꢂ SD calculated using a multiple comparison test
with the Bonferroni’s correction for at least two independent experiments.
Each of them was performed with four determinations for all inhibitor concen-
trations (0e100 mM).
5.1.1. Methyl 3-(2,5-dioxo-1-phenyl-4-(pyridin-2-ylthio)
pyrrolidin-3-yl)propanoate (4)
b
NA: not active, IC50 > 100 mM.
To a solution of acid 2 (1.320 g, 10 mmol) and 2-mercapto-
pyridine N-oxide (1.466 g, 12 mmol) in dry CH2Cl2 (50 mL)
was added DCC (2.475 g, 12 mmol) under argon and the mix-
ture was stirred at room temperature for 2 h. Phenyl maleimide
(8.658 g, 50 mmol) was added, the aluminum foil was re-
moved, and the mixture was irradiated with a tungsten lamp
(500 W) at 10e15 ꢃC for 30 min. CH2Cl2 was evaporated un-
der reduced pressure and the residue was dissolved in ether.
The solution was filtered to remove the urea and the filtrate
was washed with saturated NaHCO3, water, brine, dried over
MgSO4, and concentrated in vacuo. The residue was purified
over silica gel using cyclohexaneeEtOAc (80:20) as an eluent
to yield compound 4 (3.047 g, 82%) as a whꢁit1e solid. Mp
;
1H NMR
4. Conclusion
In summary, a series of novel maleic anhydride derivatives
were synthesized and evaluated as inhibitors of Cdc25 dual
specificity phosphatases. The inhibitory activity of these com-
pounds may result from the presence of two distinct structural
features. Their anhydride moiety could serve as a phosphate
surrogate as proposed for the g-hydroxybutenolide residue
of dysidiolide analogs [13]. The results obtained herein indi-
cate that the hydrophobic alkyl chain length contributes to
the inhibitory potency of these molecules, since a chain length
of at least 13 carbons is necessary to reach a significant activ-
ity. This is in good agreement with the enhanced Cdc25B in-
hibitory activity when tetronic acid derivatives are substituted
with an increasing alkyl chain length [19]. Considering the
compounds synthesized and tested, the results described
herein indicate that maleic anhydride derivatives could be re-
garded as attractive Cdc25 inhibitors.
128e129 ꢃC; IR (KBr) 1733, 1714, 1176 cm
(CDCl3), d 8.30 (d, J ¼ 5 Hz, 1H), 7.53e7.00 (m, 8H), 3.96
(d, J ¼ 6 Hz, 1H), 3.68 (s, 3H), 3.30 (q, J ¼ 6 Hz, 1H), 2.68
(t, 2H), 2.37 (m, 1H), 2.25 (m, 1H); 13C NMR (CDCl3),
d 176.5, 173.7, 173.0, 155.8, 149.0, 136.7, 132.4, 129.1,
128.5, 126.4, 122.1, 120.3, 51.8, 47.0, 46.4, 31.2, 25.6.
5.1.2. Methyl 3-(2,5-dioxo-1-phenyl-2,5-dihydro-1H-pyrrol-
3-yl)propanoate (5)
1e+6
To a solution of 4 (3.047 g, 8.198 mmol) in CH2Cl2 (40 ml)
was added mCPBA (2.230 g, 9.018 mmol) at 0 ꢃC. The reac-
tion mixture was stirred at 0 ꢃC for 1 h, diluted with
CH2Cl2, washed with saturated NaHCO3, water, brine, dried
over MgSO4, and concentrated in vacuo. The crude sulfoxide
was dissolved in dry toluene (40 mL) and the solution was re-
fluxed for 1 h. The solvent was evaporated under reduced pres-
sure and the residue was purified over silica gel using
cyclohexaneeEtOAc (60:40) as an eluent to yield compound
5 (1.890 g, 90%) as a white solid. Mp 71e72 ꢃC; IR (KBr)
1.5 µM
8e+5
1.2 µM
6e+5
4e+5
2e+5
1 µM
0.5 µM
0.2 µM
0 µM
1737, 1708, 1698, 1409, 873 cmꢁ1 1H NMR (CDCl3),
;
d 7.49e7.32 (m, 5H), 6.48 (t, J ¼ 1.5 Hz, 1H), 3.73 (s, 3H),
2.87 (t, J ¼ 7 Hz, 2H), 2.72 (t, J ¼ 7 Hz, 2H); 13C NMR
(CDCl3), d 172.1, 170.0, 169.3, 148.0, 131.5, 129.1, 127.8,
127.2, 125.9, 52.0, 31.2, 21.0. Anal. calcd for C14H13NO4:
C, 64.86; H, 5.05. Found: C, 64.61; H, 5.36.
0.05
0.1
0.15
0.2
1/[p-NPP] (mM-1)
-2e+5
Fig. 1. Kinetic analysis of Cdc25A inhibition by 1i. LineweavereBurk plot il-
lustrating a mixed inhibition.

246
L. Brault et al. / European Journal of Medicinal Chemistry 42 (2007) 243e247
5.1.3. General procedure for synthesis of dialkylsubstituted
maleic anhydrides 1aej
146.0, 141.4, 31.7, 30.9, 29.5, 29.4, 29.3, 29.2, 29.1, 27.9,
24.6, 22.4, 19.6, 13.9.
To a solution of acid 6 (1 mmol) and 2-mercaptopyri-
dine-N-oxide (147 mg, 1.2 mmol) in dry CH2Cl2 (10 mL)
was added DCC (248 mg, 1.2 mmol) under argon. The mix-
ture was stirred at room temperature for 2 h. Olefin 5
(1.296 g, 5 mmol) was then added, the aluminum foil was
removed, and the mixture irradiated with a tungsten lamp
(500 W) at 10e15 ꢃC for 30 min. CH2Cl2 was evaporated
under reduced pressure and the residue was dissolved in
ether. The solution was filtered to remove the urea and
the filtrate was washed with saturated NaHCO3, water,
brine, dried over MgSO4, and concentrated in vacuo. The
residue was purified over silica gel using cyclohexanee
EtOAc (80:20) as an eluent to yield compound 8 which
was used in the next step without further characterization.
To a solution of imide 8 in a THFeMeOH (6 mL, 1:2)
was added a solution of KOH (0.6 g) in water (2 mL),
and the reaction was refluxed for 3 h with stirring. The sol-
vent mixture was removed under reduced pressure, and the
residue was dissolved in water and extracted with ether. The
aqueous phase layer was acidified with diluted HCl and ex-
tracted with ether. The organic layer was washed with wa-
ter, brine, dried over MgSO4 and concentrated. The residue
was purified by column chromatography on silica gel using
cyclohexaneeEtOAc as an eluent to yield compound 1.
5.1.3.5. 3-(4-Undecyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propa-
noic acid (1e). Mp 54e55 ꢃC; IR (KBr) 2917, 2850, 1768,
1
1712, 1257, 910 cmꢁ1; H NMR (CDCl3), d 2.76 (s, 4H),
2.49 (t, J ¼ 7 Hz, 2H), 1.58 (m, 2H), 1.28 (m, 16H), 0.88 (t,
J ¼ 6.5 Hz, 3H); 13C NMR (CDCl3), d 177.1, 165.6, 165.4,
146.3, 141.4, 31.9, 30.9, 29.6, 29.5, 29.4, 29.32, 29.28, 29.2,
27.9, 24.6, 22.7, 19.6, 14.1; HRMS m/z: calcd for C18H29O5
[MH] 325.2015, found 325.2022.
5.1.3.6. 3-(4-Tridecyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propa-
noic acid (1f). Mp 63e64 ꢃC; IR (KBr) 2915, 2848, 1768,
1
1712, 1259, 910 cmꢁ1; H NMR (CDCl3), d 2.77 (s, 4H),
2.50 (t, J ¼ 7 Hz, 2H), 1.57 (m, 2H), 1.28 (m, 20H), 0.88 (t,
J ¼ 6.5 Hz, 3H); 13C NMR (CDCl3), d 176.4, 165.6, 165.4,
146.3, 141.4, 31.9, 30.9, 29.7, 29.66, 29.6, 29.58, 29.4, 29.3,
29.2, 27.9, 24.6, 22.7, 19.6, 14.1; HRMS m/z: calcd for
C20H33O5 [MH] 353.2328, found 353.2315.
5.1.3.7. 3-(4-Tetradecyl-2,5-dioxo-2,5-dihydrofuran-3-yl)pro-
panoic acid (1g). Mp 61e62 ꢃC; IR (KBr) 2917, 2850,
1
1768, 1712, 1259, 910 cmꢁ1; H NMR (CDCl3), d 2.77 (s,
4H), 2.50 (t, J ¼ 7 Hz, 2H), 1.57 (m, 2H), 1.27 (m, 22H),
0.88 (t, J ¼ 6.5 Hz, 3H); 13C NMR (CDCl3), d 176.1, 165.6,
165.4, 146.2, 141.5, 31.9, 30.9, 29.7, 29.63, 29.6, 29.5, 29.4,
29.3, 29.2, 27.9, 24.6, 22.7, 19.6, 14.1; HRMS m/z: calcd for
C21H35O5 [MH] 367.2484, found 367.2519.
5.1.3.1. 3-(4-Hexyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propanoic
acid (1a) [20,21]. Oil; IR (neat) 2915, 2848, 1768, 1712,
1
1257, 910 cmꢁ1; H NMR (CDCl3), d 2.77 (s, 4H), 2.49 (t,
J ¼ 7 Hz, 2H), 1.57 (m, 2H), 1.28 (m, 6H), 0.88 (t,
J ¼ 6.5 Hz, 3H); 13C NMR (CDCl3), d 176.1, 165.6, 165.4,
146.3, 141.4, 31.3, 30.8, 29.2, 27.9, 24.6, 22.4, 19.6, 13.9.
5.1.3.8. 3-(4-Pentadecyl-2,5-dioxo-2,5-dihydrofuran-3-yl)pro-
panoic acid (1h). Mp 68e69 ꢃC; IR (KBr) 2917, 2848,
1
1768, 1712, 1257, 910 cmꢁ1; H NMR (CDCl3), d 2.77 (s,
4H), 2.49 (t, J ¼ 7 Hz, 2H), 1.57 (m, 2H), 1.26 (m, 24H),
0.88 (t, J ¼ 6.5 Hz, 3H); 13C NMR (CDCl3), d 176.2, 165.6,
165.4, 146.2, 141.5, 31.9, 30.9, 29.7, 29.66, 29.63, 29.6,
29.44, 29.4, 29.2, 27.9, 24.6, 22.7, 19.6, 14.1; HRMS m/z:
calcd for C22H37O5 [MH] 381.2641, found 381.2625.
5.1.3.2. 3-(4-Octyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propanoic
acid (1b) [20,21]. Oil; IR (neat) 2915, 2848, 1770, 1714,
1
1259, 910 cmꢁ1; H NMR (CDCl3), d 2.77 (s, 4H), 2.50 (t,
J ¼ 7 Hz, 2H), 1.57 (m, 2H), 1.28 (m, 10H), 0.88 (t,
J ¼ 6.5 Hz, 3H); 13C NMR (CDCl3), d 176.8, 165.6, 165.4,
146.3, 141.4, 31.7, 30.9, 29.6, 29.1, 29.0, 27.9, 24.6, 22.6,
19.6, 14.0.
5.1.3.9. 3-(4-Heptadecyl-2,5-dioxo-2,5-dihydrofuran-3-yl)pro-
panoic acid (1i). Mp 76e77 ꢃC; IR (KBr) 2915, 2850, 1770,
1
1712, 1259, 906 cmꢁ1; H NMR (CDCl3), d 2.76 (s, 4H),
5.1.3.3. 3-(4-Nonyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propanoic
2.49 (t, J ¼ 7 Hz, 2H), 1.57 (m, 2H), 1.26 (m, 28H), 0.88 (t,
J ¼ 6.5 Hz, 3H); 13C NMR (CDCl3), d 176.1, 165.1, 164.8,
146.6, 141.1, 31.9, 30.9, 29.7, 29.63, 29.6, 29.57, 29.4, 29.3,
29.2, 27.9, 24.6, 22.7, 19.6, 14.1; HRMS m/z: calcd for
C24H41O5 [MH] 409.2954, found 409.3000.
acid (1c). Oil; IR (neat) 2917, 2850, 1768, 1712, 1257,
910 cmꢁ1 1H NMR (CDCl3), d 2.76 (s, 4H), 2.49 (t,
;
J ¼ 7 Hz, 2H), 1.58 (m, 2H), 1.28 (m, 12H), 0.88 (t,
J ¼ 6.5 Hz, 3H); 13C NMR (CDCl3), d 176.2, 165.4, 165.2,
146.1, 141.2, 31.6, 30.8, 29.5, 29.4, 29.2, 29.0, 27.7, 24.4,
22.4, 19.4, 13.9. Anal. calcd for C16H24O5: C, 64.84; H,
8.16. Found: C, 64.65; H, 8.30.
5.1.3.10. (Z )-3-(4-(Heptadec-8-enyl)-2,5-dioxo-2,5-dihydro-
furan-3-yl)propanoic acid (1j). Oil; IR (neat) 2927, 2856,
1
5.1.3.4. 3-(4-Decyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propanoic
1768, 1714, 1274, 912 cmꢁ1; H NMR (CDCl3), d 5.35 (m,
acid (1d) [22]. Oil; IR (neat) 2917, 2850, 1768, 1712, 1257,
2H), 2.77 (s, 4H), 2.50 (t, J ¼ 7 Hz, 2H), 2.01 (m, 4H), 1.57
(m, 2H), 1.26 (m, 20H), 0.88 (t, J ¼ 6.5 Hz, 3H); 13C NMR
(CDCl3), d (ppm) 176.4, 165.6, 165.4, 146.3, 141.4, 130.1,
129.6, 31.9, 30.9, 30.3, 29.8, 29.7, 29.6, 29.5, 29.3, 29.1,
910 cmꢁ1 1H NMR (CDCl3), d 2.77 (s, 4H), 2.49 (t,
;
J ¼ 7 Hz, 2H), 1.57 (m, 2H), 1.28 (m, 14H), 0.88 (t,
J ¼ 6.5 Hz, 3H); 13C NMR (CDCl3), d 176.1, 165.4, 165.2,

L. Brault et al. / European Journal of Medicinal Chemistry 42 (2007) 243e247
247
29.0, 27.9, 27.2, 27.1, 24.6, 22.7, 19.6, 14.1; HRMS m/z: calcd
for C24H39O5 [MH] 407.2797, found 407.2851.
Cdc25B2, Cdc25C. This study was supported by grants from
´
the Ligue contre le Cancer (Comites de Moselle, de Meurthe
´
and Moselle et de Meuse) and the Conseil Regional de Lor-
raine. L.B. was recipient of a fellowship provided by the Ligue
5.2. Biological evaluations
´
contre le Cancer (Comite de Moselle).
5.2.1. Expression and purification of Cdc25s
The Escherichia coli BL21(DE3) strain containing a pGex-
2T plasmid encoding the genes fusion construct of glutathione
S-transferase (GST) and human Cdc25s whose expression was
under the control of IPTG was used. These constructs corre-
spond to the entire Cdc25A and Cdc25C, and to the catalytic
domain of Cdc25B. E. coli was first grown in 50 mg of ampicil-
lin/mL of LB medium. When the optical density at 600 nm
reached a value between 0.4 and 0.6, IPTG (50 mg/mL) was
added and the culture incubated for 5 h. Cells were then har-
vested by a 3000g centrifugation for l5 min at 4 ꢃC. Pellets
were kept frozen at ꢁ80 ꢃC until extraction. The bacterial
pellet was disrupted by sonication in lysis buffer (50 mM
TriseHCl (pH 7.5) 250 mM NaCl, 1 mM EDTA, 0.1% Triton
X-100, 0.1 mM PMSF, 3% protease inhibitor cocktail (Sigma))
at 4 ꢃC. The homogenate was centrifuged for 45 min at 4 ꢃC at
100,000g. The supernatant was then frozen at ꢁ80 ꢃC until pu-
rification. Elution was performed using a Biologic low-pres-
sure chromatography (BioRad). Supernatant was incubated
with glutathioneeagarose gel previously packed in a column
and equilibrated with lysis buffer for 3 h. The glutathioneeaga-
rose gel was washed with five volumes of lysis buffer, followed
by a wash with five volumes of Tris buffer A (50 mM TriseHCl
(pH: 8.0), 50 mM NaCl, 1 mM EDTA, and 10 mM DTT). Elu-
tion of the fusion protein was performed by 20 mM glutathione
in Tris buffer A at a flow rate of 0.5 mL/min.
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Acknowledgements
The authors thank Laurent Meijer and Ingrid Hoffmann for
providing the expression plasmids for GSTeCdc25A and