Design and synthesis of N-alkyl oxindolylidene acetic acids as a new class of potent Cdc25A inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.04.027

The oxindolylidene acetic acids having long N-alkyl chains exhibited strong inhibitory activity toward dual specificity phosphatase Cdc25A.

Full text of DOI:10.1016/j.bmcl.2008.04.027

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3350–3353
Design and synthesis of N-alkyl oxindolylidene acetic acids
as a new class of potent Cdc25A inhibitors
Rumiko Shimazawa,*,ꢀ Masami Kuriyama and Ryuichi Shirai*
Faculty of Pharmaceutical Sciences, Doshisha Women’s College of Liberal Arts, Kodo, Kyotanabe, Kyoto 610-0395, Japan
Received 11 March 2008; revised 4 April 2008; accepted 10 April 2008
Available online 15 April 2008
Abstract—The oxindolylidene acetic acids having long N-alkyl chains exhibited strong inhibitory activity toward dual specificity
phosphatase Cdc25A.
Ó 2008 Elsevier Ltd. All rights reserved.
Cdc25 phosphatases, dual specificity enzymes, which
can dephosphorylate both phospho-Ser/Thr and phos-
pho-Tyr residues, are essential regulators by dephospho-
rylation of Cdk/cyclin complexes. The Cdc25
homologues, Cdc25A, Cdc25B, and Cdc25C, are en-
coded by the human genome.1 Cdc25A is responsible
for regulating the G1-S cell cycle transition,2 while
Cdc25B and Cdc25C regulate the G2-M cell cycle tran-
sition.3 Cdc25A and B also have oncogenic properties.4
They are transcriptional targets of the c-Myc oncogene5
and overexpressed in many human tumors.6
nolide residue (hydrophilic substructure) of 1 serves as a
surrogate phosphate, and that the octahydronaphtha-
lene framework and side chain (hydrophobic substruc-
ture) occupy a hydrophobic binding pocket when the
molecule binds Cdc25A.9 Through biochemical evalua-
tion of synthetic dysidiolide and its analogs, it was
found that some unnatural diastereomers were more po-
tent inhibitors of Cdc25 than dysidiolide itself.10 There-
fore, the introduction of some hydrophilic residues into
hydrophobic framework might generate a new class of
potent inhibitors. In previous reports, we demonstrated
that perhydroindan framework, which is available from
vitamin D3 via Grundmann’s ketone, is useful to con-
struct a hydrophobic substructure of novel Cdc25A
inhibitors (2–4).11
Because of their important role in cell cycle regulation
and their correlation with a wide variety of cancers,
Cdc25A has been one of the attractive targets for drug
development.7 Although great efforts to find effective
Cdc25A inhibitors have been reported, most structures
developed so far are quinonoid-based compounds,7
and an efficient strategy to design nonquinone inhibitors
is in the process of being developed.7a,8
In this letter, we describe the design, synthesis, and bio-
logical activity of N-alkyl oxindolylidene acetic acids 5
as Cdc25A inhibitors (Fig. 1). The introduction of N-
heterocyclic frameworks as a linker module between
hydrophobic and hydrophilic substructures may afford
a novel class of potent Cdc25A inhibitors.
We thought that Cdc25A inhibitors could be created by
an appropriate combination of hydrophilic and hydro-
phobic moieties on the basis of the structure of dysidio-
lide (1), which was the first natural inhibitor of
Cdc25A.9 It has been suggested that the c-hydroxybute-
For initial approach to design a new Cdc25A inhibitor,
we introduced a long alkyl chain, dodecanyl group, as a
hydrophobic framework. N-Dodecanyl substituted
derivatives with different hydrophilic motifs were syn-
thesized as shown in Scheme 1.
Keywords: Cdc25A; Dysidiolide; Inhibitor.
*
Corresponding authors. Tel.: +81 22 717 7122; fax: +81 22 717 7104
(R.S.); e-mail addresses: shimazawa@m.tains.tohoku.ac.jp; rshirai@
dwc.doshisha. ac.jp
N-Dodecanyl isatin 7d was obtained through alkylation
of isatin 6 using sodium hydride. The unsaturated ester
8d was prepared by the Horner–Wadsworth–Emmons
reaction.12 This reaction was completely stereoselective,
the formation of the Z-isomer consistently not being
ꢀ
Present address: Innovation of New Biomedical Engineering Center,
Tohoku University, Seiryomachi, Aoba-ku, Sendai, Miyagi 980-8574,
Japan.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.027

R. Shimazawa et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3350–3353
3351
hydrophobic
R
R
N
N
O
O
∗
∗
HO
( )n
∗
HO
HO
HO
OH
HO
H2O3PO
O
O
(E)-5
O
(Z)-5
HO
hydrophilic
dysidiolide (1)
2
3
4
O
(n=1,2)
O
O
O
Figure 1.
C12H25
N
C12H25
N
C12H25
N
H
H
N
N
i
ii
iii
O
O
O
O
O
O
OCH3
8d
OH
HO
7d
6
5d
(E)-
12
O
O
O
v
iv
vi
C12H25
N
C12H25
N
C12H25
N
C12H25
N
iii
O
O
O
NOH
OCH3
OH
11
HO
9
10
13
O
O
O
Scheme 1. Reagents and conditions: (i) C12H25I, NaH, DMF, 88%; (ii) (CH3O)2POCH2CO2CH3, NaH, THF, 78%; (iii) NaOH, MeOH–H2O or
EtOH–H2O, (E)-5d (56%), 11 (81%); (iv) H2NOHÆHCl, CH3CO2Na, MeOH, 73%; (v) NaBH4, EtOH, 92%; (vi) C12H25I, NaH, DMF, 98%.
observed. The hydrolysis of the unsaturated ester 8d
proceeded quickly to oxindolylidene acetic acid (E)-5d
as the E-isomer.12 Oxime 9 was synthesized by conden-
sation of 7d with hydroxylamine. The oxindole acetic
acid 11 was obtained by the borohydride reduction of
8d and the hydrolysis of 10. N-Alkylation of 12 gave
N-dodecanyl indole acetic acid 13.
N-alkyl group of oxindolylidene acetic acid 5 to investi-
gate the effect of hydrophobic substructures.
As shown in Scheme 2, we synthesized compounds (E)-
5a–h in the same way as the compound (E)-5d in Scheme
1. The isomeric acids (E)-5a–f were thermally converted
to (Z)-5a–f.12 Heating the E-isomers at 85–120 °C gave
a glassy mixture of the E-isomers and the Z-isomers,
which were easily isolated by column chromatogra-
phy.14–17
The synthesized compounds (E)-5d, 7d, 8d, 9, 11, and 13
were tested for Cdc25A-inhibitory activity in an assay
system utilizing the dephosphorylation of O-methylfluo-
rescein monophosphate (Table 1).13 The carboxylic acid
derivative 2, a potent Cdc25A inhibitor, was employed
as a positive reference compound.11a The compound
(E)-5d showed the strongest Cdc25A-inhibitory activity
in the investigated compounds, and hence we changed
Cdc25A-inhibitory activity of the compounds (E)-5a–h
and (Z)-5a–f is shown in Table 2.
The strength of the inhibitory activity depended on the
length of the alkyl chains at the N position of an indo-
line ring. The compounds bearing the longer hydropho-
bic chains showed the stronger inhibitory activity. The
acids having N-dodecanyl or above length N-alkyl
group ((E)-5d–f and (Z)-5d–f) showed higher inhibition
than the positive reference compound 2. Their inhibitory
activities were not much different between the E-isomers
and the Z-isomers. The substitution of phenylpropyl
((E)-5g) or ether ((E)-5h) group for alkyl groups greatly
decreased the inhibition.
Table 1. Cdc25A inhibition assay results for compounds 2, (E)-5d, 7d,
8d, 9,11, and 13
Compound
Cdc25A inhibition IC50, lM (SD)
2
12( 4)
(E)-5d
7d
8d
9
2.6( 0.4)
15( 2)
37( 3)
33( 2)
50( 8)
8.0( 0.4)
In conclusion, we designed and synthesized novel N-al-
kyl oxindolylidene acetic acids ((E)-5d–f and (Z)-5d–f)
having high-Cdc25A-inhibitory activity. These findings
11
13

3352
R. Shimazawa et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3350–3353
R
N
R
N
R
N
R
N
H
N
i
ii
iii, iv
O
O
O
O
O
+
O
O
OCH3
OH
HO
O
8
O
(E)-5
O
(Z)-5
7
6
a : R=C6H13
b : R=C8H17
c : R=C10H21
d : R=C12H25
e : R=C14H29
f : R=C16H33
7a (87%)
7b (93%)
7c (99%)
7d (88%)
7e (87%)
7f (89%)
7g (92%)
7h (78%)
8a (69%)
8b (70%)
8c (83%)
8d (78%)
8e (67%)
8f (83%)
8g (68%)
8h (60%)
(E)-5a (46%)
(E)-5b (8%)
(E)-5c (14%)
(E)-5d (14%)
(E)-5e (25%)
(E)-5f (15%)
(Z)-5a (11%)
(Z)-5b (13%)
(Z)-5c (10%)
(Z)-5d (15%)
(Z)-5e (21%)
(Z)-5f (16%)
(E)-5g (41%)a
(E)-5h (36%)a,b
g : R=(CH2)3Ph
h : R=(CH2CH2O)3CH2CH3
Scheme 2. Reagents and conditions: (i) R–I (a– d, f) or R–Br (e, g, h), NaH, DMF; (ii) (CH3O)2POCH2CO2CH3, NaH, THF; (iii) NaOH,
MeOH–H2O or EtOH–H2O; (iv) neat, 85–120 °C; aobtained through the reagents and condition (iii) without (iv). bE/Z=4:1.
Richardson, H.; Russell, P. Proc. Natl. Acad. Sci. U.S.A.
1990, 87, 5139.
Table 2. Cdc25A inhibition assay results for compounds (E)-5 and
(Z)-5
˚
2. Mailand, N.; Falck, J.; Lukas, C.; Syljuasen, R. G.;
Cdc25A inhibition IC50, lM (SD)
Compound
Welcker, M.; Bartek, J.; Lukas, J. Science 2000, 288, 1425.
3. Bulavin, D. V.; Higashimoto, Y.; Popoff, I. J.; Gaarde, W.
A.; Basrur, V.; Potapova, O.; Appell, E.; Fornace, A. J.,
Jr. Nature 2001, 411, 102.
4. Galaktionov, K.; Lee, A. K.; Eckstein, J.; Draetta, G.;
Meckler, J.; Loda, M.; Beach, D. Science 1995, 269, 1575.
5. Galaktionov, K.; Chen, X.; Beach, D. Nature 1996, 382,
511.
R
N
R
N
O
O
OH HO
O
O
6. See the following reviews: (a) Boutros, R.; Dozier, C.;
Ducommun, B. Curr. Opin. Cell Biol. 2006, 18, 185; (b)
Kristjansdottir, K.; Rudolph, J. Chem. Biol. 2004, 11,
1043.
(E)-5
(Z)-5
a: R = C6H13
b: R = C8H17
>100
>100
39( 4)
78( 13)
13( 0.1)
2.6( 0.4)
2.3( 0.2)
1.9( 0.1)
>100
c: R = C10H21
d: R = C12H25
12( 0.3)
2.9( 0.3)
1.7( 0.0)
1.6( 0.2)
—
7. See the following reviews of Cdc25s inhibitors and the
references therein: (a) Contour-Galcera, M.-O.; Sidhu, A.;
Prevost, G.; Bigg, D.; Ducommun, B. Pharmacol. Ther.
2007, 115, 1; (b) Bialy, L.; Waldmann, H. Angew. Chem.,
Int. Ed. 2005, 44, 3814; (c) Lyon, M. A.; Ducruet, A. P.;
Wipf, P.; Lazo, J. S. Nat. Rev. Drug Discov. 2002, 1, 961 ;
(d) Carnero, A. Br. J. Cancer 2002, 87, 129; (e) Eckstein, J.
W. Invest. New Drugs 2000, 18, 149; (f) Pestell, K. E.;
Ducruet, A. P.; Wipf, P. Oncogene 2000, 19, 6607.
8. Docking studies for Cdc25B inhibitors: (a) Lavecchia, A.;
Cosconati, S.; Limongelli, V.; Novellino, E. ChemMed-
Chem 2006, 1, 540; (b) Montes, M.; Braud, E.; Miteva, M.
A.; Goddard, M.-L.; Mondesert, O.; Kolb, S.; Brun, M.-P.;
Ducommun, B.; Garbay, C.; Villoutreix, B. O. J. Chem.
Inf. Model 2008, 48, 157.
e: R = C14H29
f: R = C16H33
g: R = (CH2)3Ph
h: R = (CH2CH2O)3 CH2CH3
>100a
—
a The mixture of E- and Z-form (E/Z = 4/1).
on the structure–activity relationship should be helpful
for the design of novel Cdc25A inhibitors. We would
like to investigate isoform selectivity, because Cdc25B
and C inhibitory activities of those compounds have
not been tested. Design and synthesis of further isatin
analogs as candidate for potent inhibitors of Cdc25 fam-
ily members are in progress.
9. Gunesekera, S. P.; McCarthy, P. J.; Kelly-Borges, M.;
Lobkovsky, E.; Clardy, J. J. Am. Chem. Soc. 1996, 118,
8759.
10. (a) Takahashi, M.; Dodo, K.; Sugimoto, Y.; Aoyagi, Y.;
Yamada, Y.; Hashimoto, Y.; Shirai, R. Bioorg. Med.
Chem. Lett. 2000, 10, 615; (b) Brohm, D.; Metzger, S.;
Bhargava, A.; Muller, O.; Lieb, F.; Waldmann, H. Angew.
Chem., Int. Ed. 2002, 41, 307; (c) Brohm, D.; Philipe, N.;
Metzger, S.; Bhargava, A.; Muller, O.; Lieb, F.; Wald-
mann, H. J. Am. Chem. Soc. 2002, 124, 13171.
Acknowledgments
This work was supported by Health Labour Science Re-
search Grants and Individual Research Grants of the
Doshisha Women’s College of Liberal Arts.
11. (a) Dodo, K.; Takahashi, M.; Yamada, Y.; Sugimoto, Y.;
Hashimoto, Y.; Shirai, R. Bioorg. Med. Chem. Lett. 2000,
10, 615; (b) Shimazawa, R.; Suzuki, T.; Dodo, K.; Shirai,
R. Bioorg. Med. Chem. Lett. 2004, 14, 3291; (c) Shimaz-
awa, R.; Gochomori, M.; Shirai, R. Bioorg. Med. Chem.
Lett. 2004, 14, 4339.
References and notes
1. (a) Galaktionov, K.; Beach, D. Cell 1991, 67, 1181; (b)
Nagata, A.; Igarashi, M.; Jinno, S.; Suto, K.; Okayama,
H. New Biol. 1991, 3, 959; (c) Sadhu, K. S.; Reed, I.;
12. Autrey, R. L.; Tahk, F. C. Tetrahedron 1967, 23, 901.

R. Shimazawa et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3350–3353
3353
13. Cdc25A phosphatase assay. Catalytic domain proteins of
human Cdc25A were purchased from BIOMOL (Product
Number SE-364). Phosphatase activity of Cdc25A was
assayed in 100 lL of buffer containing 50 mM Tris–HCl
(pH 8.0), 100 mM NaCl, and 1 mM dithiothreitol, with
40 lM O-methylfluorescein monophosphate as the sub-
strate, using 96-well microtiter plate. The IC50 values were
means of two–five experiments, standard deviation is given
in parentheses.
14. Typical procedure for the synthesis of the oxindolylidene
acetic acids (E)-5d and (Z)-5d from 8d. (E)-Methyl 2-(1-
dodecyl-2-oxoindolin-3-ylidene)acetate 8d (487 mg) was
hydrolyzed at rt in 10 min with 0.4 M NaOH in EtOH–
water (3:2). To the solution, 10% HCl, water, and diethyl
ether were added. The acidified aqueous layer was
extracted with diethyl ether, and the combined organic
layers were washed with brine. They were dried over
Na2SO4 and concentrated to give the crude oxindolylidene
acetic acid as an orange solid. The crude acid was heated
at 100 °C for 2 h. The obtained glassy mixture was
separated by silica gel column chromatography (CH2Cl2/
AcOEt =20:1–1:1). The Z-isomer ((Z)-5d) and the E-
isomer ((E)-5d) were recrystallized from hexane and
hexane–AcOEt, respectively. The products (Z)-5d and
(E)-5d were obtained as yellow needles of mp 92–93 °C
(70 mg, 15% from 8d) and as red needles of mp 111–112
°C (64 mg, 14% from 8d), respectively.
15. The ether derivatives (E)-5h were obtained as the mixture
(E/Z = 4:1) through the same hydrolysis condition. Com-
pound (E)-5h was a red thick oil and could not be isolated
by recrystallization.
1
16. The following is the H NMR spectra of the oxindolylid-
ene acetic acids (E)-5d and (Z)-5d. (E)-5d (CDCl3): 0.88
(3H, t, J = 7.0 Hz, CH3), 1.25–1.37 (18H, m, CH2), 1.68
(2H, pentet, J = 7.2 Hz, NCH2CH2), 3.72 (2H, t, J = 7.3
Hz, NCH2), 6.81 (1H, d, J = 7.8 Hz, H-7), 6.94 (1H, s,
@CH), 7.06 (1H, dt, J = 0.9, 7.7 Hz, H-5), 7.37 (1H, dt,
J = 1.2, 7.7 Hz, H-6), 8.51 (1H, d, J = 7.8 Hz, H-4); (Z)-5d
(CDCl3): 0.88 (3H, t, J = 7.0 Hz, CH3), 1.26–1.38 (18H,
m, CH2), 1.73 (2H, pentet, J = 7.2 Hz, NCH2CH2), 3.79
(2H, t, J = 7.4 Hz, NCH2), 6.92 (1H, d, J = 7.9 Hz, H-7),
6.95 (1H, s, @CH), 7.16 (1H, dt, J = 0.8, 7.6 Hz, H-5), 7.44
(1H, dt, J = 1.1, 7.8 Hz, H-6), 7.52 (1H, d J = 7.5 Hz, H-
4), 14.8 (1H, s, COOH).
17. The structure of oxindolylidene acetic acid E-isomer and
Z-isomer was determined by 1H NMR. The farther
downfield shift is observed the shift of the hydrogen at
4-position in the E-isomers (e.g., (E)-5d: 8.51 ppm) than
the Z-isomers (e.g., (Z)-5d: 7.52 ppm), because the hydro-
gen at 4-position in the E-isomers is in proximity to a
1
carbonyl group. Additionally, H NMR spectrum of the
carboxylic proton in the Z-isomers shows a sharp singlet
peak (e.g., (Z)-5d: 14.8 ppm) by chelation of the proton in
a seven-membered ring. See Ref. 12.