1-Azakenpaullone is a selective inhibitor of glycogen synthase kinase-3β

Article abstract of DOI:10.1016/j.bmcl.2003.10.062

Kenpaullone derivatives with a modified parent ring system were synthesized in order to develop kinase inhibitors with enhanced selectivity. Among the novel structures, 1-azakenpaullone was found to act as a selective GSK-3β versus CDK1 inhibitor. The charge distribution within the 1-azakenpaullone molecule is discussed as a possible explanation for the enhanced GSK-3β selectivity of 1-azakenpaullone compared to other paullone derivatives.

Full text of DOI:10.1016/j.bmcl.2003.10.062

Bioorganic & Medicinal Chemistry Letters 14 (2004) 413–416
1-Azakenpaullone is a selective inhibitor of glycogen
synthase kinase-3ꢀ
Conrad Kunick,a,* Kathrin Lauenroth,b Maryse Leost,c Laurent Meijerc
and Thomas Lemckeb
aInstitut fur Pharmazeutische Chemie, Technische Universitat Braunschweig, Beethovenstraße 55, 38106Braunschweig, Germany
¨
¨
bInstitut fur Pharmazie, Abteilung fur Pharmazeutische Chemie, Universitat Hamburg, Bundesstraße 45, 20146Hamburg, Germany
¨
¨
¨
cCentre National de la Recherche Scientifique, Station Biologique, BP 74, 29682 Roscoff, France
Received 29 April 2003; revised 13 October 2003; accepted 28 October 2003
Abstract—Kenpaullone derivatives with a modified parent ring system were synthesized in order to develop kinase inhibitors with
enhanced selectivity. Among the novel structures, 1-azakenpaullone was found to act as a selective GSK-3b versus CDK1 inhibitor.
The charge distribution within the 1-azakenpaullone molecule is discussed as a possible explanation for the enhanced GSK-3b
selectivity of 1-azakenpaullone compared to other paullone derivatives.
# 2003 Elsevier Ltd. All rights reserved.
Glycogen synthase kinase-3 (GSK-3) is a serine/threonine
kinase which is ubiquitously expressed in mammalian
tissues and interferes with manifold physiological pro-
cesses.1 Two isoforms of GSK-3 exist, designated GSK-
3a and GSK-3b, which share a high homology at their
catalytic site.2 Among the proteins which are regulated by
phosphorylation through GSK-3 are transcription fac-
tors, translation initiation factors, and microtubule-sta-
bilizing proteins. Through the interaction with these
targets, GSK-3 is involved in cell differentiation, cellular
growth and proliferation, apoptosis control, inflamma-
tion and mechanisms involved in neuronal function.1,3,4
Moreover, GSK-3 is an important factor in the regula-
tion of glucose metabolism being one of the intracellular
links downstream the insulin receptor.5 The inactivation
of glycogen synthase by phosphorylation through GSK-
3 results in decreased glycogen synthesis. Thus, GSK-3
inhibitors have been suggested as potential antidiabetic
drugs.4,6 The discovery of structural diverse GSK-3
inhibitor classes has been described recently, for exam-
ple pyridyl-oxadiazoles,7 thiadiazolidinones,8 and male-
imides.9,10 The maleimides SB-216763 and SB-415286
have been shown to stimulate glycogen synthesis in
human liver cells.9 Current advances in the search for
GSK-3 inhibitors have been reviewed.11 Since GSK-3 is
phylogenetically most closely related to the cyclin-
dependent kinases (CDKs),4 it is not surprising that at
least four ATP-competitive kinase inhibitor classes,
which extensively have been evaluated for kinase selec-
tivity, were found to act on both GSK-3 and distinct
CDKs: hymenialdisine (1),12 the indirubins [e.g., 5-iodo-
indirubin-30-monoxime (2)],13 the aloisines [e.g., aloisine
B (3)],14 and the paullones, a family of 7,12-dihydro-
indolo[3,2-d][1]benzazepin-6(5H)-ones.15
* Corresponding author. Fax: +49-531-391-2799; e-mail: kunick@
chemie.uni-hamburg.de
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.10.062

414
C. Kunick et al. / Bioorg. Med. Chem. Lett. 14 (2004) 413–416
In a recent comparison of commercially available kinase
inhibitors, kenpaullone (4) in combination with lithium
as complementary reagent was suggested for investi-
gations towards the biological effects through GSK-3
inhibition.16 Although it has been shown that kenpaul-
lone (4) exhibits only poor activity on other kinases, its
remaining CDK-inhibitory property is still a concern.
Even though a large number of paullone derivatives
with different substituents on the parent ring system
have been synthesized and evaluated, a paullone with
both high potency and GSK-3 versus CDK selectivity
has not been identified so far. Hence, for a rational
design of a paullone with higher GSK-3 selectivity,
kenpaullone derivatives with a modified heterocyclic
b]azepine-5,8(6H,7H)-dione 517 and 4-bromophenylhy-
drazine catalyzed by sulfuric acid was carried out. The
4-aza-derivative 10 of kenpaullone was synthesized start-
ing from 2-aminopyridine-3-carboxylic acid ethyl ester
7,18 which was cyclized by means of succinic acid diethyl
ester in the presence of sodium hydride. The resulting
enolized b-oxocarboxylic ester 8 was then heated in wet
DMSO to yield the cyclic ketone 9, which afforded 10 by
an acid-catalyzed Fischer ring closure (Scheme 1).
The sequence leading to the 1-aza-analogue 16 is out-
lined in Scheme 2. 3-Aminopyridine-2-carboxylic acid
ethyl ester 11 was acylated by ethyl succinyl chloride.
Dieckmann cyclization of the resulting amide 12 by
means of potassium hydride afforded the enolized
b-oxocarboxylic ester 13, which was subsequently dealk-
oxycarbonylated by heating in wet DMF. Because the
classical Fischer indolization gave unsatisfactory yields
when applied to the ketone 14, the 4-bromophenylhy-
drazone 15 was prepared, which upon refluxing in
diphenyl ether under neutral conditions afforded the
1-azakenpaullone 16 by a thermal indolization reaction.
basic
structure,
namely
8-bromo-6,11-dihydro-
thieno[30,20:2,3]azepino[4,5-b]indol-5(4H)-one (6), 4-aza-
kenpaullone (10), and 1-azakenpaullone (16), were
synthesized. These derivatives show a molecular shape
similar to kenpaullone, but due to different charge dis-
tribution throughout the molecules a potential to distin-
guish between the ATP cavities of CDKs and GSK-3 was
regarded as possible.
For the preparation of the thieno analogue 6 of ken-
paullone, a Fischer indole synthesis with 4H-thieno[3,2-
For a biological evaluation the inhibition of the three
kinases CDK1/cyclin B, CDK5/p25 and GSK-3b by the
new paullone analogues was determined. The CDK1/
cyclin B complex [also known as M-phase promoting
Scheme 1. (i) (1) 4-Bromophenylhydrazine HCl, NaOAc, HOAc,
70 ꢀC; (2) HOAc, concd H2SO4, 70 ꢀC; (26%); (ii) succinic acid diethyl
ester, NaH, toluene, 90 ꢀC, N2 (39%); (iii) DMSO, H2O, 150 ꢀC, N2
(81%); (iv) (1) 4-bromophenylhydrazine HCl, NaOAc, HOAc, 70 ꢀC;
(2) HOAc, concd H2SO4, 70 ꢀC; (43%); mps: 6: >330 ꢀC; 8: 212 ꢀC; 9:
221 ꢀC; 10: >330 ꢀC.
Scheme 2. (i) (1) Ethyl succinyl chloride, toluene, pyridine, reflux, 2 h
(81%); (ii) KH, toluene, DMF, 70 ꢀC, N2 (43%); (iii) DMF, H2O,
150 ꢀC, N2 (95%); (iv) 4-bromophenylhydrazine HCl, NaOAc, HOAc,
70 ꢀC (75%); (v) diphenyl ether, reflux, N2 (81%); mps: 12: 68 ꢀC; 13:
221 ꢀC; 14: 185.5 ꢀC, 16: >330 ꢀC.

C. Kunick et al. / Bioorg. Med. Chem. Lett. 14 (2004) 413–416
415
factor (MPF)] is an important regulator of the cell cycle,
in which it is required for transition from the G2 to the
M-stage.19 The CDK5/p25 complex is found as a factor
of pathological relevance in brain cells of Alzheimer’s
disease patients, where it leads to hyperphosphorylation
of the tau protein.20,21 In Table 1, the IC50 values of 6,
10 and 16 are given compared with the results for potent
GSK-3b inhibitors, which were also evaluated for
CDK1 and CDK5 inhibition in our test system before,
namely hymenialdisine (1), 5-iodo-indirubin-30-monox-
ime (2), aloisine B (3) and kenpaullone (4). The selec-
tivity was expressed as the ratio of the IC50 values of
CDK1/cyclin B and GSK-3b. The three new paullone
derivatives exhibited different behavior in the kinase
testing. The thieno analogue 6 showed similar properties
to kenpaullone, that is a considerable CDK1, CDK5
and GSK-3b inhibitory activity and a moderate selec-
tivity for GSK-3. While the 4-azakenpaullone 10 is a
poor inhibitor for the CDKs and GSK-3b, the 1-aza-
derivative 16 only lost its CDK-inhibitory activity.
Because the GSK-3b inhibitory potency of the parent
compound kenpaullone is conserved in 1-azapaullone
16, it constitutes a novel potent GSK-3b inhibitor with
a noteworthy selectivity versus CDKs, expressed by a
GSK-3/CDK1 selectivity index of 111 (see Fig. 1).
For an explanation of the observed differences between
kenpaullone and the three kenpaullone congeners 6, 10
and 16, the structures were constructed in the SYBYL
Figure 2. Charge distribution on the surface of the kenpaullone
molecule 4 and its congeners 6, 10, and 16. Charge is indicated by
color: negative: red; positive: blue, neutral: green. Arrows indicate
noteworthy differences in the charge distribution of the congeners with
respect to kenpaullone.
Figure 1. Inhibition of CDK1/cyclin B (filled circles) and GSK3-b
(open squares) by 1-azakenpaullone (16). Vertical bars indicate stan-
dard error of the mean (SEM).
Table 1. Selectivity of kinase inhibition by the new paullone analogues 6, 10, 16 compared to dual GSK-3b/CDK inhibitors from the literature
Entry
IC50 (mM)a
CDK5/p25
Selectivity-index
GSK-3-CDK1b
Ref
CDK1/cyclin B
GSK-3b
1
2
3
4
6
10
16
Hymenialdisine
5-Iodo-indirubin-30-monoxime
Aloisine B
0.022
0.025
0.85
0.4
0.6
8.4
0.028
0.020
13.0
0.85
4.0
12
4.2
0.010
0.009
0.75
0.023
0.12
2.2
2.8
1.1
17.4
5.0
12
13
14
15
Kenpaullone
Thieno analogue of kenpaullone
4-Azakenpaullone
1-Azakenpaullone
6.01.4
0.018
2.0
111
a The kinase inhibition experiments were carried out as described before.15 The final ATP-concentration in the test was 15 mM.
b Selectivity-index GSK-3-CDK1=IC50 CDK1/IC50 GSK-3b.

416
C. Kunick et al. / Bioorg. Med. Chem. Lett. 14 (2004) 413–416
modeling package using the Tripos force field, employing
the previously published structure of kenpaullone22 as
template. The structures were then energy minimized by
a quantum mechanical method (ab initio Hartree-Fock
3-21G*) using the SPARTAN program.23 Subsequently,
the electrostatic potentials were calculated and projected
on the van-der-Waals surfaces of the molecules. The
results of these calculations are depicted in Figure 2.
3. Ali, A.; Hoeflich, K. P.; Woodgett, J. R. Chem. Rev. 2001,
101, 2527.
4. Frame, S.; Cohen, P. Biochem. J. 2001, 359, 1.
5. Cohen, P. Phil. Trans. R. Soc. Lond. B 1999, 354, 485.
6. Wagman, A. S.; Nuss, J. M. Curr. Pharm. Des. 2001, 7,
417.
7. Naerum, L.; Norskov-Lauritsen, L.; Olesen, P. H. Bioorg.
Med. Chem. Lett. 2002, 12, 1525.
8. Martinez, A.; Alonso, M.; Castro, A.; Perez, C.; Moreno,
F. J. Med. Chem. 2002, 45, 1292.
Obviously, the molecular shape of all four molecules is
rather similar. Moreover, the charge distribution within
the thieno analogue 6 fits very well the picture of ken-
paullone, an observation which is reflected in the similar
inhibitory activity of the compounds towards CDKs
and GSK-3b. However, 4-azakenpaullone (10) exhibits
a center of negative charge at position 4 (colored red in
the molecule image and indicated by an arrow) in contrast
to its parent compound. Preliminary docking studies
with 10 in a homology model of CDK122 showed that the
negative partial charge at position 4 leads to an unfa-
vorable electrostatic repulsion to the backbone carbonyl
oxygen atom of the amino acid in position 84 in the
CDK1 ATP-binding cavity. In contrast, 1-azaken-
paullone (16) shows a concentration of negative charge
around the 1-position (colored red in the molecule
image and indicated by an arrow). It can be speculated
that this charge distribution disturbs the binding within
the ATP cavity of CDKs only, but not the binding to
GSK-3b. In order to find out why 16 can distinguish
between the binding pockets of CDKs and GSK-3b,
detailed docking studies will be carried out in the future.
9. Coghlan, M. P.; Culbert, A. A.; Cross, D. A. E.; Cor-
coran, S. L.; Yates, J. D.; Pearce, N. J.; Rausch, O. L.;
Murphy, G. J.; Carter, P. S.; Cox, L. R.; Mills, D.;
Brown, M. J.; Haigh, D.; Ward, R. W.; Smith, D. G.;
Murray, K. J.; Reith, A. D.; Holder, J. Chem. Biol. 2000,
7, 793.
10. Smith, D. G.; Buffet, M.; Fenwick, A. E.; Haigh, D.; Ife,
R. J.; Saunders, M.; Slingsby, B. P.; Stacey, R.; Ward,
R. W. Bioorg. Med. Chem. Lett. 2001, 11, 635.
11. Martinez, A.; Castro, A.; Dorronsoro, I.; Alonso, M.
Med. Res. Rev. 2002, 22, 373.
12. Meijer, L.; Thunnissen, A.-M. W. H.; White, A. W.;
Garnier, M.; Nikolic, M.; Tsai, L.-H.; Walter, J.; Clever-
ley, K. E.; Salinas, P. C.; Wu, Y.-Z.; Biernat, J.; Man-
delkow, E. M.; Kim, S.-H.; Pettit, G. R. Chem. Biol. 2000,
7, 51.
13. Leclerc, S.; Garnier, M.; Hoessel, R.; Marko, D.; Bibb,
J. A.; Snyder, G. L.; Greengard, P.; Biernat, J.; Wu, Y.-
Z.; Mandelkow, E.-M.; Eisenbrand, G.; Meijer, L. J. Biol.
Chem. 2001, 276, 251.
14. Mettey, Y.; Gompel, M.; Thomas, V.; Garnier, M.; Leost,
M.; Ceballos-Picot, I.; Noble, M.; Endicott, J.; Vierfond,
J.-M.; Meijer, L. J. Med. Chem. 2003, 46, 222.
15. Leost, M.; Schultz, C.; Link, A.; Wu, Y.-Z.; Biernat, J.;
Mandelkow, E.-M.; Bibb, J. A.; Snyder, G. L.; Green-
gard, P.; Zaharevitz, D. W.; Gussio, R.; Senderowicz,
A. M.; Sausville, E. A.; Kunick, C.; Meijer, L. Eur. J.
Biochem. 2000, 267, 5983.
Acknowledgements
16. Bain, J.; McLauchlan, H.; Elliott, M.; Cohen, P. Biochem.
J. 2003, 371 (Pt 1), 199.
The generous support by Prof. Geffken and the Uni-
versity of Hamburg is gratefully acknowledged. This
research was also supported by a grant from the ‘Asso-
ciation pour la Recherche sur le Cancer’ (ARC 5343)
(L.M.) and a grant (‘Molecules & Cibles Therapeu-
tiques’) from the ‘Ministere de la Recherche/INSERM/
CNRS’ (L.M.).
17. Kunick, C. Arch. Pharm. (Weinheim) 1992, 325, 297.
18. Fox, H. H. J. Org. Chem. 1952, 17, 547.
19. Morgan, D. O. Ann. Rev. Cell Dev. Biol. 1997, 13, 261.
20. Dhavan, R.; Tsai, L.-H. Nature Rev. Mol. Cell Biol. 2001,
2, 749.
21. Tseng, H. C.; Zhou, Y.; Shen, Y.; Tsai, L. H. FEBS Lett.
2002, 523, 58.
22. Gussio, R.; Zaharevitz, D. W.; McGrath, C. F.; Patta-
biraman, N.; Kellogg, G. E.; Schultz, C.; Link, A.;
Kunick, C.; Leost, M.; Meijer, L.; Sausville, E. A. Anti-
Cancer Drug Des. 2000, 15, 53.
References and notes
1. Doble, B. W.; Woodgett, J. R. J. Cell Sci. 2003, 116, 1175.
2. Woodgett, J. R. EMBO J. 1990, 9, 2431.
23. SPARTAN, Vers. 5.0.1; Wavefunction Inc.: Irvine, USA,
1997.