Synthesis and biological evaluation of novel heterocyclic quinones as inhibitors of the dual specificity protein phosphatase CDC25C

Article abstract of DOI:10.1016/j.bmcl.2005.09.030

A focused set of heterocyclic quinones based on the benzothiazole, benzoxazole, benzimidazole, indazole and isoindole was prepared and screened with respect to the inhibition of the phosphatase activity of CDC25C. Benzoxazole- and benzothiazole-diones wer

Full text of DOI:10.1016/j.bmcl.2005.09.030

Bioorganic & Medicinal Chemistry Letters 16 (2006) 171–175
Synthesis and biological evaluation of novel heterocyclic quinones
as inhibitors of the dual specificity protein phosphatase CDC25C
a
a
a
Olivier Lavergne,a, Anne-Cecile Fernandes, Laetitia Brehu, Alban Sidhu,
*
´
´
a
a
b
´
´
´
Marie-Christine Brezak, Gregoire Prevost, Bernard Ducommun and
Marie-Odile Contour-Galceraa
aIpsen Research Laboratories, Institut Henri Beaufour, 5 avenue du Canada, 91960 Les Ulis, France
b
´
LBCMCP-CNRS UMR5088- IFR109, Institut dÕExploration Fonctionnelle des Genomes,
´
Universite Paul Sabatier, 118 route de Narbonne, 31062 Toulouse, France
Received 26 May 2005; revised 8 September 2005; accepted 12 September 2005
Available online 10 October 2005
Abstract—A focused set of heterocyclic quinones based on the benzothiazole, benzoxazole, benzimidazole, indazole and isoindole was
prepared and screened with respect to the inhibition of the phosphatase activity of CDC25C. Benzoxazole- and benzothiazole-diones
were at least 50 times more potent in inhibiting CDC25C than their benzimidazole-indazole- or isoindole-dione counterparts. These
in vitro activities were in good correlation with the anti-proliferative effects observed with Mia PaCa-2 and DU-145 human tumor cell
cultures. The IC50 values obtained by WST-1 colorimetric assay ranged from 0.10 to 0.50 lM for the benzoxazole- or benzothiazole-
diones and were above 10 lM for the other heterocyclic diones. This study further illustrates how the activity of the quinone pharma-
cophore can be selectively modulated by changing the type of five-membered heterocycle fused to the quinone ring.
Ó 2005 Elsevier Ltd. All rights reserved.
The CDC25 dual-specificity phosphatases play an
important role in regulating the cell cycle by dephospho-
rylating and activating CDK/cyclin complexes at key
check points, and therefore constitute interesting biolog-
ical targets for the development of novel anti-prolifera-
tive agents.1–4 For the moment, the subtype of CDC25
that should be targeted remains speculative and the
means to obtain selectivity among the various CDC25
isoforms have not been reported. Although we use re-
combinant CDC25C to screen and optimize our com-
pounds, a pan-CDC25 inhibitory approach is actually
pursued. We have identified BN82002 as a new inhibitor
of the CDC25 phosphatase activity and observed its
characteristic effects at the molecular and cellular levels,
as well as in animal models.5,6 Previously, Lazo and
colleagues had reported the potent CDC25 inhibition
induced by substituted quinolinediones such as NSC
663284 (1a) and other closely related analogues.7 The
bare aza-naphthoquinone moieties of these molecules
were insufficient for potent CDC25 inhibition and
required an enhancing substituent at position 7 of the
quinolinedione template such as 2-morpholin-4-ylethyl-
amino or 1-indan-1-ylamino. These compounds also fea-
tured a chloro substituent at the other available site of
the quinone ring, position 6 of quinolinedione. Recently,
we have identified and characterized BN82685 (2a) as
thiazole-quinone CDC25 inhibitor which is active in
vitro and in vivo.8 The present letter reports the evalua-
tion as CDC25C phosphatase inhibitors of a focused set
of isoesters of 2a, formed by five-membered heterocycles
fused to a benzoquinone (3–6, Fig. 1). The fused hetero-
cyclic rings consist of three atom bridges which may
bear various substituents to broaden structural diversity.
The scope of this study is limited to a single N,N-
dimethylethylenediamine side chain on the quinone ring
and methyl, ethyl or phenyl substitution at the middle
atom of the bridge to form the five-membered hetero-
cyclic ring.
Benzothiazoledione (10) was prepared according to the
known method9 with minor modification, as shown in
Scheme 1. Commercially available methoxybenzothiaz-
ole 7 was nitrated and reduced to give amine 9, which
was then oxidized to the corresponding quinone 10 with
FremyÕs salt [potassium nitrosodisulfonate, (KO3S)2NO].
Keywords: Heterocyclic quinone; CDC25 phosphatase; Selectivity;
Anti-proliferative effect.
*
Corresponding author. Tel.: +33 1 60 92 20 00; fax: +33 1 69 07 38
02; e-mail: olivier.lavergne@ipsen.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.030

172
O. Lavergne et al. / Bioorg. Med. Chem. Lett. 16 (2006) 171–175
one 14 gave a mixture of regioisomers 3b,b0 and 3c,c0.
O
O
O
H
H
N
N
N
N
S
1H NMR analysis revealed that ethylbenzoxazole 3b,b0
consisted of a 30:1 ratio of two regioisomers as deter-
mined by the integration of the N-H side-chain signals
at 7.39 and 7.22 ppm, while phenylbenzoxazole 3c,c0
consisted of a 1:3.3 mixture of regioisomers from the
peaks at 7.49 and 7.33 ppm.
N
N
Me
O
Cl
O
2a : BN82685
1: NSC663284
Benzimidazoles were prepared according to previously
described procedures,15,16 from dimethoxybenzene 15
by a nitration reduction sequence to give ortho-diamino-
benzene 16 (along with its para-diamino regioisomer), as
shown in Scheme 3. Intermediates were condensed with
orthoesters to give dimethoxybenzimidazoles 17.17 Ben-
zimidazolediones 18 were obtained by direct oxidation
of dimethoxy intermediates with ceric ammonium
nitrate [CAN, Ce(NH4)2 (NO3)6],18 and treated with
N,N-dimethylethylenediamine to give the desired test
compounds. These products are mixtures of tautomers
in rapid equilibrium, represented as 4b,b0 and 4c,c0,
respectively. In each case, only a single signal was
O
N
O
O
N
R
O
R
S
O
2
3
O
O
O
O
N
R
N
N R
N R
N
H
O
O
4
5
6
Figure 1.
1
observed by H NMR for the N H side-chains at 7.13
and 7.22 ppm, respectively.
Substitutionofthemethoxygroup with theN,N-dimethyl-
ethylenediamine provided benzothiazoledione 2a.
Indazolediones 5 were prepared by thermal decomposi-
tion of 3-phenylsydnone (19)19 in the presence of para-
benzoquinone to provide phenylindazoledione (20,
Scheme 4). Upon treatment with N,N-dimethylethylen-
ediamine indazolediones 5b,b0 were obtained as a 4.7:1
mixture of regioisomers, determined by the integration
of the N-H side-chain signals at 7.22 and 7.10 ppm.
The initial route to benzoxazolediones (3) is shown in
Scheme 2 and consisted of the condensation of 2-ami-
no-3-nitrophenol (11) with either carboxylic10,11 acids
or orthoesters12 to give substituted nitrobenzoxazoles
12. A subsequent reduction of the nitro group12 provid-
ed aminobenzoxazoles, such as 13, which were oxidized
with either FremyÕs salt [potassium nitrosodisulfonate,
(KO3S)2NO]13 or BTI [bis(trifluoroacetoxy)iodoben-
zene]14 to the corresponding quinone 14. Addition of
N,N-dimethylethylenediamine to phenylbenzoxalonedi-
As shown in Scheme 5, isoindolediones were obtained
by trapping with para-benzoquinone the azomethine
ylide generated by heating sarcosine (21) in the presence
of formaldehyde.20 The usual treatment with N,N-
dimethylethylenediamine provided isoindoledione 6a.
Z
O
O
H
N
MeO
MeO
MeO
i
iii
iv
N
S
N
S
N
S
N
S
Me
Me
Me
Me
N
O
O
8: Z = NO2
9: Z = NH2
ii
7
10
2a
Scheme 1. Reagents and conditions: (i) conc. HNO3/conc. H2SO4, rt, 2 h, 64%; (ii) H2, Pd/C, EtOH, 94%; (iii) FremyÕs salt (1.8 eq), NaH2PO4
(0.3 M), acetone, 25 °C, 2 h, 78%; (iv) Me2NCH2CH2NH2 (1.5 eq), MeOH, 60 °C, 2 h, 65%.
NO2
Z
O
O
O
O
H
N
i
iii
iv
NH2
OH
N
O
N
O
N
N
O
N
O
b: R = Et
c: R = Ph
R
R
R
+
R
N
N
H
O
O
12: Z = NO2
13: Z = NH2
14b: R = Et
14c: R = Ph
3b: R = Et
3c: R = Ph
3b': R = Et
3c': R = Ph
ii
11
Scheme 2. Reagents and conditions: (i) 12b: EtC(OEt)3 (1 eq), neat, PTSA (cat), 110 °C, 2 h, 80%; 12c: PhC(OMe)3 (2 eq), neat, PTSA (cat), 110 °C,
2 h, 82%; (ii) H2 (8 atm) 10% Pd/C (cat), MeOH, 13b: 93%, 13c: 70%; (iii) BTI (2.2 eq), MeCN/H2O, ꢀ5 °C, 30 min, 14b: 100%, 14c: 42%;
(iv) Me2NCH2CH2NH2 (2 eq), CeCl3–7H2O (1.1 eq), EtOH, rt 1 h, 3b,b0: 37%, 3c,c0: 10%.

O. Lavergne et al. / Bioorg. Med. Chem. Lett. 16 (2006) 171–175
173
OMe
OMe
O
O
OMe
O
O
H
N
ii
iii
iv
i
N
N
N
N
NH2
NH2
N
R
R
R
R
N
H
N
H
N
H
N
H
N
N
H
OMe
OMe
O
O
OMe
18b: R = Et
18c: R = Ph
17b: R = Et
17c: R = Ph
4b: R = Et
4c: R = Ph
4b': R = Et
4c': R = Ph
16
15
Scheme 3. Reagents and conditions: (i) see Ref. 17 (ii) 17b: EtC(OEt)3 (2 eq), neat, PTSA (cat), 110 °C, 1.6 h, 61%; 17c: PhC(OMe)3 (2 eq), neat,
PTSA (cat), 110 °C, 1.1 h, 84%; (iii) CAN (4 eq), AcOEt/H2O, ꢀ5 °C, 45 min, 18b: 17%, 14c: 21%; (iv) Me2NCH2CH2NH2 (1.5 eq), CeCl3-7H2O
(1.1 eq), EtOH, reflux 1 h, 4b,b0: 10%, 4c,c0: 6%.
O
O
O
O
O
O
O
O
H
N
i
ii
N
N
N
N
N+ Ph
N
O
N Ph
N Ph
+
N Ph
+
HO
N
N
H
-
20
19
5c
5c'
Scheme 4. Reagents and conditions: (i) p-benzoquinone (2 eq) xylene, reflux, 3 h, 24%; (ii) Me2NCH2CH2NH2 (1 eq), EtOH, rt 10 min, 14%.
O
O
O
O
O
O
H
N
OH
i
ii
H
N Me
N Me
N Me
+
O
N
21
6a
Scheme 5. Reagents and conditions: (i) p-benzoquinone (1 eq), sarcosine (2 eq), paraformaldehyde (5 eq) toluene, reflux (Dean–Stark), 4.5 h, 3%;
(ii) Me2NCH2CH2NH2 (1.1 eq), EtOH, rt 10 min, 19%.
For a preliminary study, test compounds were assayed as
mixtures of regiosiomers. Subsequently, we have devel-
oped a regioselective method to prepare aminated ben-
zoxazolediones such as 3b or 3b0 as pure compounds,
via pre-established methoxy groups.21 In other instances,
regioselectively aminated heterocyclic quinones have
been obtained via pre-established halogen22 substituents.
The set of quinonoid compounds was also assayed for
the inhibition of proliferation of two human cancer cell
lines, pancreatic carcinoma Mia PaCa-2 and androgen-
independent prostate carcinoma DU145. Transient
tumor growth inhibition was obtained in nude mice
bearing Mia PaCa-2 xenografts,8 and a recent study
by Ozen and Ittmann shows that CDC25C expression
and activity are increased in prostate cancer cells,
including in DU145.23 The resulting IC50 values were
determined with a colorimetric assay based on the
cleavage of tetrazolium salt WST1 by mitochondrial
dehydrogenase in viable cells and are reported in
Table 1. With IC50 values ranging from 0.11 to
0.44 lM, good antiproliferative activities were obtained
with benzothiazole 2a as well as benzoxazolediones
3b,b0 and 3c,c0. Imidazolediones 4b,b0 and 4c,c0 were
found to be only moderately active with IC50 values
higher than 10 lM. No effect on cellular proliferation
could be detected with indazolediones 5c,c0 and isoin-
doledione 6a.
The foregoing quinonoid compounds were evaluated for
their inhibitory activity against recombinant CDC25C
phosphatase, using 3-O-methylfluorescein phosphate
(OMFP) as a substrate.6 The results (50% inhibitory
concentrations, measured in duplicate) are reported in
Table 1. Benzothiazole BN82685 (2a) is a potent inhib-
itor of CDC25C with an IC50 value of 0.15 lM, compa-
rable to 0.25 lM obtained with NSC 663284 (1a), while
menadione used as a benchmark had an IC50 of 19 lM.
Potent inhibition of CDC25C was also observed for
benzoxazolediones 3b,b0 and 3c,c0, with ethyl and phenyl
substitution, respectively. In contrast, imidazolediones
4b,b0 and 4c,c0, indazolediones 5c,c0, and isoindoledione
6a were found to be much less active, with inhibitory
concentrations several orders of magnitude higher than
that of benzothiazoledione 2a.
Within this focused set of heterocyclic quinones, a good
correlation is observed between CDC25C inhibition and
antiproliferative activity. As reported recently for

174
O. Lavergne et al. / Bioorg. Med. Chem. Lett. 16 (2006) 171–175
Table 1.
Compound
Structure
CDC25Ca (lM)
Cell proliferationb (lM)
Mia PaCa-2 DU145
O
O
H
N
N
N
O
1
0.25 0.033
0.15 0.010
2.5 0.095
2.3 0.070
Cl
O
O
H
N
N
N
S
Me
2a
0.18 0.034
0.11 0.0060
O
N
H
3b,3b0
3c,3c0
4b,4b0
4c,4c0
5c,5c0
6a
0.13 0.051
0.23 0.029
12 0.76
17 1.4
0.44 0.035
0.15 0.019
18 7.7
12 0.25
ned
0.22 0.016
Et
N
N
O
O
O
N
O
H
Ph
Et
0.21 0.0020
N
N
O
O
N
H
>10
N
N
N
H
O
O
N
H
Et
>10
N
N
N
H
O
O
N
H
N
Ph
12 0.86
ne
N
N
O
O
H
N
N
N
Me
81 13
ne
ne
O
O
Me
mndc
19 2.9
6.2 0.15
11 0.10
O
a Inhibition of the activity of a maltose binding protein (MBP)-CDC25C recombinant enzyme monitored with 3-O-methylfluorescein phosphate
(OMFP). The 50% inhibitory concentration mean values 0 SEM were calculated from at least two, usually three, independent experiments.
b The 50% inhibitory concentration mean values 0 SEM of tumor cell proliferation were determined with tetrazolium salt WST1 by mitochondrial
dehydrogenases in viable cell experiment. These experiments were performed at least twice with four or eight determinations per tested
concentration.
c mnd: menadione.
d ne: no effect.
thiazole 2a,8 oxazoles 3b,b0 and 3c,c0 are likely to be
pan-CDC25 inhibitors, and their selectivity with respect
to other phosphatases is not known. Nevertheless, var-
ring the heterocyclic part of these quinonoids allows
us to fine-tune their activity and reveal an interesting as-
pect of their selectivity. Related molecules which bear
the scaffolds of imidazolediones such as 4,24–29 indazol-
ediones like 5,30,31 or isoindoledione 632 are reported to
be active against various biological targets and to have
antiproliferative effects, but they do not have the pat-
terns of substitution of the present set and their hetero-
cyclic quinonoid scaffolds alone are not sufficient to
exert potent CDC25 inhibition. Conversely, benzothiaz-
oles and benzoxazoles such as 2a, 3b,b0, and 3c,c0
are among the most potent inhibitors of CDC25
phosphatases reported to date and deserve further inves-
tigation in the hope of providing new routes to treat
cancer patients.
References and notes
1. Pestell, K. E.; Ducruet, A. P.; Wipf, P.; Lazo, J. S.
Oncogene 2000, 19, 6607.
2. Prevost, G.; Brezak Pannetier, M. C.; Goubin, F.;
Mondesert, O.; Galcera Contour, M. O.; Quaranta, M.;
Alby, F.; Lavergne, O.; Ducommun, B. Prog. Cell Cycle
Res. 2003, 5, 225.
3. Kristjansdottir, K.; Rudolph, J. Chem. Biol. 2004, 11, 1043.
4. Ham, S. W.; Carr, B. I. Drug Des. Rev. Online 2004, 1,
123.

O. Lavergne et al. / Bioorg. Med. Chem. Lett. 16 (2006) 171–175
175
5. Contour-Galcera, M. O.; Lavergne, O.; Brezak, M. C.;
Ducommun, B.; Prevost, G. Bioorg. Med. Chem. Lett.
2004, 14, 5809.
6. 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.
7. Lazo, J. S.; Aslan, D. C.; Southwick, E. C.; Cooley, K. A.;
Ducruet, A. P.; Joo, B.; Vogt, A.; Wipf, P. J. Med. Chem.
2002, 44, 4042.
8. Brezak, M. C.; Quaranta, M.; Galcera Contour, M. O.;
Lavergne, O.; Mondesert, O.; Auvray, P.; Kasprzyk, P.
G.; Prevost, G. Mol. Cancer Ther. 2005, 4, 1378.
9. Ryu, C. K.; Kang, H. Y.; Lee, S. K.; Nam, K. A.; Hong,
C. Y.; Ko, W. G.; Lee, B. H. Bioorg. Med. Chem. Lett.
2000, 10, 461.
18. Tohma, H.; Morioka, H.; Harayama, Y.; Hashizume, M.;
Kita, Y. Tetrahedron Lett. 2001, 42, 6899.
19. Thoman, C. J.; Voaden, D. J. Org. Synth. 1965, 45, 96.
20. Schubert-Zsilavecz, M.; Likussar, W.; Gusterhuber, D.;
Michelitsch, A. Monatsh. Chem. 1991, 122, 383.
21. Brehu, L.; Fernandes, A. C.; Lavergne, O. Tetrahedron
Lett. 2005, 46, 1437.
22. Nebois, P.; Sarciron, M. E.; Bibal, B.; Bouammali, B.;
Cherkaoui, O.; Pautet, F.; Petavy, A. F.; Walchshofer, N.;
Fillion, H. Bioorg. Med. Chem. Lett. 2000, 10, 871.
23. Ozen, M.; Ittmann, M. Clin. Cancer Res. 2005, 11, 4701.
24. Garuti, L.; Roberti, M.; Pizzirani, D.; Pession, A.;
Leoncini, E.; Cenci, V.; Hrelia, S. Farmaco 2004, 59, 663.
25. Garuti, L.; Roberti, M.; Malagoli, M.; Rossi, T.; Castelli,
M. Bioorg. Med. Chem. Lett. 2000, 10, 2193.
26. Hong, S. Y.; Chung, K. H.; You, H. J.; Choi, I. H.; Chae,
M. J.; Han, J. Y.; Jung, O. J.; Kang, S. J.; Ryu, C. K.
Bioorg. Med. Chem. Lett. 2004, 14, 3563.
10. Kanaoka, Y.; Hamada, T.; Yonemitsu, O. Chem. Pharm.
Bull. 1970, 18, 587.
11. Terashima, M.; Ishii, M.; Kanaoka, Y. Synthesis 1982, 484.
12. Katritzky, A. R.; Musgrave, R. P.; Rachwal, B.; Zaklika,
C. Heterocycles 1995, 41, 345.
13. Zhou, R.; Skibo, E. B. J. Med. Chem. 1996, 39, 4321.
14. Barret, R.; Daudon, M. Tetrahedron Lett. 1990, 31, 4871.
15. Weinberger, L.; Day, A. R. J. Org. Chem. 1959, 24, 1451.
16. Ryu, C. K.; Song, E. H.; Shim, J. Y.; You, H. J.; Choi, K.
U.; Choi, I. H.; Lee, E. Y.; Chae, M. J. Bioorg. Med.
Chem. Lett. 2003, 13, 17.
27. Singh, A. K.; Lown, J. W. Anticancer Drug Des. 2000, 15,
265.
28. Ahn, C. M.; Tak, J. A.; Choi, S. J. Arch. Pharm. Res.
2000, 23, 288.
29. Xing, C.; Skibo, E. B. Biochemistry 2000, 39, 10770.
30. Tapia, R. A.; Carrasco, C.; Ojeda, S.; Salas, C.; Valder-
rama, J. A.; Morello, A.; Repetto, Y. J. Heterocycl. Chem.
2002, 39, 1093.
31. Singh, R.; Khanna, J. M.; Anand, N. Indian J. Chem. B
1989, 28, 490.
17. Alvarez, F.; Gherardi, A.; Nebois, P.; Sarciron, M. E.;
Petavy, A. F.; Walchshofer, N. Bioorg. Med. Chem. Lett.
2002, 12, 977.
32. Schubert-Zsilavecz, M.; Michelitsch, A.; Likussar, W.
Monatsh. Chem. 1992, 123, 1223.