9-Cyano-1-azapaullone (Cazpaullone), a Glycogen Synthase Kinase-3 (GSK-3) inhibitor activating pancreatic β cell protection and replication

Article abstract of DOI:10.1021/jm701582f

Recently, the serine/threonine kinase glycogen synthase kinase-3 (GSK-3) emerged as a regulator of pancreatic β cell growth and survival. On the basis of the previous observation that GSK-3 inhibitors like 1-azakenpaullone promote β cell protection and re

Full text of DOI:10.1021/jm701582f

2196
J. Med. Chem. 2008, 51, 2196–2207
9-Cyano-1-azapaullone (Cazpaullone), a Glycogen Synthase Kinase-3 (GSK-3) Inhibitor
Activating Pancreatic ꢀ Cell Protection and Replication
Hendrik Stukenbrock,§, Rainer Mussmann,#, Marcus Geese,# Yoan Ferandin,† Olivier Lozach,† Thomas Lemcke,‡
Simone Kegel,# Alexander Lomow,# Ulrike Burk,# Cord Dohrmann,# Laurent Meijer,† Matthias Austen,# and Conrad Kunick*,§
Institut für Pharmazeutische Chemie, Technische UniVersität Braunschweig, BeethoVenstrasse 55, 38106 Braunschweig, Germany, DeVeloGen AG,
Marie-Curie-Strasse 7, 37079 Göttingen, Germany, Protein Phosphorylation & Human Disease Group, CNRS, Station Biologique, Place Georges
Teissier, B.P. 74, 29682 Roscoff, France, and Institut für Pharmazie, UniVersität Hamburg, Bundesstrasse 45, 20146 Hamburg, Germany
ReceiVed December 17, 2007
Recently, the serine/threonine kinase glycogen synthase kinase-3 (GSK-3) emerged as a regulator of pancreatic
ꢀ cell growth and survival. On the basis of the previous observation that GSK-3 inhibitors like
1-azakenpaullone promote ꢀ cell protection and replication, paullone derivatives were synthesized including
1-aza-, 2-aza-, and 12-oxapaullone scaffolds. In enzymatic assays distinct 1-azapaullones were found to
exhibit selective GSK-3 inhibitory activity. Within the series of 1-azapaullones, three derivatives stimulated
INS-1E ꢀ cell replication and protected INS-1E cells against glucolipotoxicity induced cell death. Cazpaullone
(9-cyano-1-azapaullone), the most active compound in the protection assays, also stimulated the replication
of primary ꢀ cells in isolated rat islets. Furthermore, cazpaullone showed a pronounced transient stimulation
of the mRNA expression of the ꢀ cell transcription factor Pax4, an important regulator of ꢀ cell development
and growth. These features distinguish cazpaullone as a unique starting point for the development of ꢀ cell
regenerative agents which might be useful in the treatment of diabetes.
Introduction
findings in preclinical as well as clinical studies with ꢀ cell
growth or protecting factors have substantiated the regenerative
approach. For instance, the combination treatment consisting
of the epidermal growth factor (EGF)a and the peptide hormone
gastrin, which stimulates the expansion of insulin producing ꢀ
cell mass,10 demonstrated efficacy in animal models of
diabetes.11,12 Lately, the combination treatment of EGF and
gastrin has entered clinical development.13 Likewise, in a recent
clinical study an IL-1 receptor antagonist significantly improved
the glycemic control of type 2 diabetes patients, most likely
through a direct anti-inflammatory and protective effect on ꢀ
cells.14
Another potential drug target for ꢀ cell regenerative ap-
proaches is the glycogen synthase kinase-3 (GSK-3), a recently
identified regulator of ꢀ cell mass.15–17 GSK-3 is a constitutively
active serine/threonine kinase occurring in two forms, GSK-
3R and GSK-3ꢀ.18 The kinase is widely expressed and plays a
central regulatory role in intracellular signaling pathways such
as the Wnt- and the phosphoinositide 3-kinase (PI3K)/Akt
pathways, which are most important for ꢀ cell function.19,20
Consistent with these findings, the inactivation of GSK-3 in ꢀ
cells through RNA interference or small molecular inhibitors
was found to protect ꢀ cells against experimentally induced cell
death.15–17 Moreover, the suppression of GSK-3 enzymatic
activity stimulated the replication of primary rat ꢀ cells.16 Within
the past decade, elucidation of the manifold roles of GSK-3 in
Diabetes mellitus has become a health and economic burden
in recent years, in particular for Western industrial nations.1
Future prospects are alarming as the prevalence rises. Estima-
tions speak of 300 million adults worldwide suffering from
diabetes by 2025.2 Though a number of new antidiabetic drugs
have entered the market within the past decade, many patients
still have inadequately controlled blood sugar levels.3 Appar-
ently, current pharmacological treatments are symptomatic and
do not cure the disease. Over time many patients with diabetes
develop devastating secondary complications especially affecting
the heart, eyes, and kidneys.4,5 In view of this situation the
pharmaceutical industry and academic research groups have
undertaken enormous efforts to identify new drug targets and
to develop new therapeutic options to face the emerging threat
of widespread diabetes.
One promising concept proposes that the preservation and
expansion of ꢀ cell mass by pharmacological means will
improve the blood sugar control in patients with type 1 or type
2 diabetes. In type 1 diabetes almost all ꢀ cells are destroyed
by an autoimmune reaction.6,7 In type 2 diabetes, ꢀ cells fail to
compensate for the increased insulin demand caused by insulin
resistance in the liver and muscle. ꢀ cell failure is characterized
by a secretory dysfunction and a ꢀ cell loss of about 40% in
glucose intolerant individuals and lean type 2 diabetes patients
and to about 60% in obese type 2 diabetes patients compared
to the respective nondiabetic control subjects. It appears that
the relative decrease in ꢀ cell mass in patients with type 2
diabetes results from an increased ꢀ cell apoptosis rate.8,9 Recent
a Abbreviations: ATP, adenosine triphosphate; AUC, area under the
curve; 6BIO, 6-bromoindirubin-3′-oxime; BrdU, bromodeoxyuridine; BSA,
bovine serum albumin; CDK, cyclin dependent kinase; CK1, casein kinase
1; DAD, diode array detector; DCM, dichloromethane; DMEM, Dulbecco’s
modified Eagle medium; DMF, N,N-dimethylformamide; DMSO, dimethyl
sulfoxide; dppf, 1,1′-bis(diphenylphosphino)ferrocene; EGF, epidermal
growth factor; ER, endoplasmic reticulum; EtOH, ethanol; GSK-3ꢀ, gly-
cogen synthase kinase-3ꢀ; HPLC, high-performance liquid chromatography;
IL-1, interleukin 1; IRS1, insulin receptor substrate 1; NCW, near-critical
water; qRT-PCR, quantitative real time polymerase chain reaction; siRNA,
small interfering ribonucleic acid; TFA, trifluoroacetic acid; TLC, thin layer
chromatography.
* To whom correspondence should be addressed. Phone: +49-531-391-
2754. Fax: +49-531-391-2799. E-mail: c.kunick@tu-braunschweig.de.
§
Technische Universität Braunschweig.
These authors contributed equally.
DeveloGen AG.
CNRS, Station Biologique.
Universität Hamburg.
#
†
‡
10.1021/jm701582f CCC: $40.75
2008 American Chemical Society
Published on Web 03/18/2008

9-Cyano-1-azapaullone
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2197
Figure 3. 1-Azakenpaullone (2a, blue), 2-azakenpaullone (13a, yellow),
and 12-oxakenpaullone (24b, red) aligned by FlexX in the GSK-3ꢀ ATP
binding site (generated from the PDB file 1Q3W.pdb).34
For a rational structure-based design of improved 1-azaken-
paullone congeners, we performed a docking study based on
the X-ray structure of an alsterpaullone/GSK-3ꢀ complex
published recently.34 In GSK-3ꢀ, alsterpaullone is adjusted to
the ATP binding pocket by two hydrogen bonds between the
lactam group and Val135 of the hinge area. Moreover, the
lactam carbonyl oxygen is connected to the backbone carbonyl
group of Asp133 by a water molecule (W5) bridge. Another
hydrogen bond emanating from the side chain amino group of
Lys85 makes contact with the nitro group of the ligand. The
indole NH makes a fifth hydrogen bond to a water molecule
(W2), which itself is hydrogen-bound to Glu185 and another
water molecule (W1). The latter bridges a gap to Thr138
positioned at the edge of the ribose binding pocket34 (Figure
2). The model derived by docking of 1-azakenpaullone to the
ATP binding pocket showed a very similar pose of the ligand
in GSK-3ꢀ, with the main difference being the missing hydrogen
bond to Lys85 (data not shown). Various considerations based
on this model provided ideas for a rational structure modification
of 1-azakenpaullone. First, the substituent in the 9-position was
varied systematically in order to investigate the importance for
the hydrogen bond to Lys85. One of the compounds prepared
for this purpose was the 9-cyano-1-azapaullone (2b), which,
similar to alsterpaullone (1b), is able to realize the hydrogen
bond to Lys85 (Figure 2).
Figure 1. Structures of established GSK-3 inhibitors: kenpaullone (1a),
alsterpaullone (1b), 1-azakenpaullone (2a), CHIR99021 (3), and 6-
bromoindirubin-3′-oxime (6BIO, 4).
Since the nitrogen in position 1 seems to be responsible for
the selectivity of 1-AKP for GSK-3 versus CDKs, we prepared
a second series of congeners (13a-f) in which this nitrogen is
shifted to the 2-position. Docking studies showed that 2-aza-
paullones probably are slightly differently aligned within the
ATP binding site compared to the 1-aza congeners (Figure 3).
In a third group of compounds (24a-c), the indole nitrogen of
the original (non-aza) paullone scaffold was substituted for
oxygen. This modification was considered because in the ATP
binding pocket of CDK1/cyclin B, the paullones use the indole
NH as hydrogen bond donor aiming the side chain carboxyl
function of Asp86, of which a homologue is missing in GSK-
3. We speculated that in GSK-3 the depicted water molecule
W2 could be reoriented to serve as a hydrogen bond donor
enabling a bridge to the benzofuran oxygen of 24. This should
make the resulting [1]benzofuro[3,2-d][1]benzazepin-6-ones 24
compatible with the ATP binding site of GSK-3 but not with
the corresponding site of CDKs, resulting in improved GSK-3
selectivity. Results of docking studies suggested that because
of similar molecular shapes, the 12-oxapaullones 24 should fit
into the ATP binding site as well as the 1-azapaullones 2
(Figure 3).
Figure 2. Alsterpaullone (1b; light-brown, hydrogen atoms omitted)
and 9-cyano-1-azakenpaullone (2b, atom color code) bound to the ATP
binding site of GSK-3ꢀ. Hydrogen bonds involving 2b are indicated
as green dashed lines. The pose of alsterpaullone is based on PDB file
1Q3W.pdb,34 and the pose of 2b was generated using the FlexX module
in Sybyl.
cellular functions made the enzyme interesting as a putative
drug target for several diseases.21–24
A variety of small molecules inhibiting GSK-3 have been
published during the past decade, including paullones like
kenpaullone (1a) and alsterpaullone (1b),25,26 indirubins like
6-bromoindirubin-3′-oxime (6BIO, 4),27–29 and maleimides
(Figure 1).30–32 While both 1a and 1b also inhibit cyclin-
dependent kinases (CDKs), the structurally related 1-azaken-
paullone (2a) displays a 100-fold selectivity for GSK-3 versus
CDK1/cyclin B.33
Considering the beneficial activity of 2a in previous inves-
tigations, a project was initiated for the development of paullone
derivatives with enhanced ꢀ-cell protective and proliferation
stimulating properties. For this purpose, we first were interested
in the design of azakenpaullone congeners with improved
GSK-3 inhibitory potency and selectivity. The most promising
entities then were to be tested for antiapoptotic and proliferation
stimulating activity in cell based models.
Also, with the aim of designing GSK-3 selective inhibitors,
we replaced, in both the 1-aza and in the 2-aza series of
compounds, the hydrogen bound to the indole nitrogen by
aliphatic or aromatic substituents (15a-e and 16a-c, respec-
tively). These side chains were intended to displace the water

2198 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Scheme 1. Synthesis of Cyclic Ketones 9a-ca
Stukenbrock et al.
a (i) EtOH, concentrated H2SO4, 100 °C, 90 h; (ii) ethylsuccinyl chloride, CaCO3, toluene; (iii) KH, toluene, DMF, -10 °C f 60 °C; (iv) Br2, HOAc,
H2O, H2SO4; (v) DMF, H2O, N2, 150 °C.
Scheme 2. Synthesis of 1-Azapaullones 2 and 2-Azapaullones
Scheme 3. Synthesis of N-Substituted 1-Azapaullones 15 and
2-Azapaullones 16a
13a
a (i) (1) HOAc, NaOAc, 70 °C, (2) concentrated H2SO4, HOAc, 90 °C
(refers to compounds 13a,c-f); (ii) (1) HOAc, NaOAc, 70 °C, (2) EtOH,
reflux (refers to compound 2l); (iii) HOAc, NaOAc, 70 °C; (iv) Ph2O, reflux,
N2 (refers to compounds 2b-m and 13b,c); (v) H2O, MW, 175-215 °C,
0.5 h (refers to compounds 2a,b,d,g and 13a); (vi) DMF, MW, 230 °C
(refers to compound 2c). For R1 refer to Table 1.
a (i) HOAc, NaOAc, 70 °C. For R2 refer to Table 1.
molecule W2 from the ATP binding pocket in GSK-3, a
mechanism that was considered not to be applicable to the
binding pocket of CDKs, where the corresponding water
molecule is missing.
cyclic ketone 9 (Scheme 1)35 and a subsequent Fischer indole
ring closure for the annulation of the indole system (Scheme
2).36 The preparation of the cyclic ketones 9a-c started with
an appropriate aminopyridine carboxylic acid 5a or 5b which
was esterified by prolonged reflux in ethanol catalyzed by
sulfuric acid.37 3-Aminopicolinic acid ethyl ester 6a was
converted to the corresponding 6-bromo derivative 6c by
reaction with bromine in acetic acid.38 The esters 6a-c were
acylated with ethylsuccinyl chloride, respectively, to yield the
amides 7a-c. Subsequent treatment with potassium hydride in
a toluene/DMF mixture induced a Dieckmann ring closure
reaction leading to the enolized cyclic ꢀ oxocarboxylic esters
8a-c. When these esters were heated in wet DMF, the seven-
membered cyclic ketones 9a-c were obtained by a dealkoxy-
carbonylation reaction.
Besides 2j, two further 1-azapaullones 2n and 2o were
synthesized to probe the suitability of positions 2 and 11 for
halogen atom substitution. In the case of retained GSK-3
inhibitory activity and selectivity, these structures would be
useful as intermediates for the attachment of solubilizing side
chains by palladium catalyzed reactions. Eventually, the paullone
2,9-dicarbonitrile 30 was designed to study whether the electron-
poor pyridine ring of the azapaullones 2 and 13 could be
mimicked by a benzene ring bearing a strongly electron-
withdrawing substituent.
Chemistry
The new 1-aza- and 2-azapaullones were synthesized by a
general method that has been published for the preparation of
1-azakenpaullone, comprising the synthesis of a seven-member
For the indole ring closure, various modifications of the
Fischer reaction were employed (Scheme 2). The 2-azapaullones
13a,c-f were obtained by a one-pot procedure omitting isolation

9-Cyano-1-azapaullone
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2199
Scheme 4. Synthesis of 12-Oxapaullones 24 a
a (i) CuCl, pyridine, 1,2-dichloroethane, molecular sieves, room temp; (ii) (1) MeOH, CHCl3, hydrazine, room temp, (2) EtOH, HCl; (iii) HOAc, NaOAc,
70 °C; (iv) HCOOH, H3PO4, 60 °C. For R1 refer to Table 1.
of the corresponding phenylhydrazone intermediate. For the
reaction, the cyclic ketone 9b was reacted with an appropriate
phenylhydrazine consecutively in acetic acid and an acetic acid/
sulfuric acid mixture. In contrast, for the synthesis of the
1-azapaullones 2b-l and the 2-azapaullone 13b the thermal
Fischer indolization method proved to be advantageous. Thus,
the crude phenylhydrazone 10 or 11 prepared from 9a or 9b
were refluxed in diphenyl ether for 2 h to furnish the indolization
products in poor to moderate yields. In the case of the 9-donor-
substituted 1-azapaullones 2k and 2l, the thermal Fischer
indolization occurred at an unexpected low temperature in
boiling ethanol. This observation was made upon the attempted
crystallization of the corresponding phenylhydrazones from
ethanol, which instead of purified precursors yielded the desired
indole products. To further improve the performance of the
thermal Fischer indolization in paullone synthesis, an alternative
method was developed in which the phenylhydrazone precursors
were heated in the sealed vessel of a monomode microwave
reactor in near-critical water (NCW) as reaction medium.39,40
Application of this procedure for the preparation of 2b and 2d
increased the yield from 18% to 36% and from 17% to 51%,
respectively. In a similar manner 1-azakenpaullone 2a was
obtained employing the microwave assisted method in 73%
yield. The 9-hydroxy-1-azapaullone 2m was prepared by boron
tribromide ether cleavage41 in dichloromethane from the cor-
responding methoxy derivative 2k.
The azapaullones 15a-d and 16a-c substituted at the indole
nitrogen were easily accessible by heating the cyclic ketones
9a,b with 1-aryl- or 1-alkyl-substituted phenylhydrazines 14 in
glacial acetic acid, even in the absence of an additional acidic
catalyst. The novel pentacyclic ring system 15e was obtained
from 9a and 1-aminoindoline (17) in a similar reaction
(Scheme 3).
For the preparation of the novel 12-oxapaullones 24a-c, the
aryloxyamines 21 were needed as building blocks. While the
unsubstituted derivative 21a was commercially available, the cor-
responding para-substituted analogues 21b,c were prepared by
a copper-mediated coupling of suitable boronic acids 19 and
N-hydroxyphthalimide at room temperature.42 Hydrazinolysis
of the resulting intermediates 20b,c furnished the desired
aryloxyamines which were converted to the corresponding
hydrochlorides.42 The latter were reacted with the cyclic ketone
2235 in acetic acid to afford the O-aryloximes 23a-c which
were subsequently heated in a mixture of formic and phosphoric
acid yielding the 12-oxapaullones 24a-c (Scheme 4).43
The syntheses of the three special paullone derivatives 2n,
2o, and 30 are summarized in Scheme 5. Treatment of 4-amino-
3-iodobenzonitrile 25 with sodium nitrite and hydrochloric acid
gave a diazonium salt solution, which was not isolated but added
to a cold tin chloride solution yielding the hitherto unknown
4-hydrazino-3-iodobenzonitrile hydrochloride 26.44 Reaction of
26 with the cyclic ketone 9a in acetic acid furnished the
phenylhydrazone 10n. The subsequent thermal Fischer indoliza-
tion procedure mentioned above led to the 11-iodo-1-aza-
paullone-9-carbonitrile 2n. A similar sequential procedure
involving reaction of ketone 9c and 4-hydrazinobenzonitrile
hydrochloride 27, isolation, and thermal indolization of the
phenylhydrazone 10o was employed for the preparation of
the 2-bromo-substituted 1-azapaullone-9-carbonitrile 2o. Pal-
ladium catalyzed reaction of the iodoarene 2845 with zinc
cyanide in DMF in the presence of 1,1′-bis(diphenylphos-
phino)ferrocene (dppf) as ligand gave the 2,5-dioxo-2,3,4,5-
tetrahydro-1H-1benzazepine-7-carbonitrile 29.46 The acid cata-
lyzed one-pot procedure with acetic acid/sulfuric acid at 70 °C
proved to be suitable for the indolization of 29 with the
4-hydrazinobenzonitrile hydrochloride 27, furnishing the paullone-
2,9-dicarbonitrile 30.
Results and Discussion
Kinase Inhibition. Since the aim of the project was the
identification of potent and selective new GSK-3 inhibitors in
the paullone series, all new paullone derivatives were tested on
a set of three protein kinases: first, GSK-3R/ꢀ as the main target
enzyme of the new structures; second, CDK5/p25, a protein
kinase somewhat structurally related to GSK-3; third, casein
kinase 1 (CK1), a serine/threonine kinase structurally less related
but like GSK-3 involved in the Wnt signaling pathway.47 CDK5
inhibition has recently been shown to stimulate insulin secretion
under high glucose concentration conditions.48,49 Distinct

2200 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Stukenbrock et al.
Scheme 5. Synthesis of 1-Azapaullones 2n,o and Paullone-2,9-dicarbonitrile 30a
a (i) (1) NaNO2, HCl, -10 °C, (2) SnCl2, HCl, -10 °C; (ii) HOAc, NaOAc, 70 °C; (iii) Ph2O, reflux, N2; (iv) Zn(CN)2, Pd2(dba)3, dppf, DMF, N2, 120
°C; (v) (1) HOAc, NaOAc, 70 °C, (2) HOAc, concentrated H2SO4, 70 °C.
derivatives from all series were further checked for inhibition
of CDK1/cyclin B, a protein kinase involved in regulating the
cell cycle G2/M transition and very similar to CDK5/p25. The
antiproliferative activity of paullones like 1a and 1b, which
exhibit antiproliferative activity for tumor cell lines, is at least
partially assigned to CDK1 inhibition.36,50 For the objectives
of the investigation reported here, antiproliferative activity
resulting from CDK1 inhibition is explicitly undesirable.
The in vitro test results (Table 1) revealed a strong depen-
dence of the kinase inhibitory properties on subtle structural
changes. None of the new entities inhibited CK1 with IC50
values below 10 µM. Regarding GSK-3 inhibition, the exchange
of the 9-bromo substituent in 1-azakenpaullone for either a cyano
(in 2b) or a trifluoromethyl group (in 2c) resulted in improved
potency. Later experiments showed a strong ꢀ cell protection
and proliferation stimulation especially by 9-cyano-1-aza-
paullone 2b. We will refer to 2b in the further text using the
name “cazpaullone”. Compared to the parent 1-azakenpaullone
2a, the trifluoromethyl derivative 2c was more selective versus
CDK1. Besides cazpaullone (2b) and 2c, the other derivatives
of the 1-aza series were less potent than 2a. Within the series
2, the relationship between the electronic parameters of 9-sub-
stituents and the kinase inhibitory properties described for
paullones of the carba analogue series was reproduced: electron-
donating substituents (e.g., in 2k, 2l, 2m, and also 2f, if the
carboxylate anion form is assumed) showed poor activity, while
high inhibitory activity was found with structures bearing
electron withdrawing groups in the 9-position (2b-e,i). Intro-
duction of a second substituent into the 11-position was
unfavorable, as the comparison of 9-monosubstituted compounds
(2b,d) and 9,11-disubstituted analogues (2n,j) revealed. The
compounds 13a-e of the 2-aza series showed retained GSK-3
inhibitory activity. Again, compound 13f with the electron
donating substituent in the 9-position was clearly less active.
All congeners of the 2-aza series lacked the selectivity shown
by the 1-aza analogues. For example, the 9-cyano-2-azapaullone
13b inhibits GSK-3R/ꢀ, CDK1/cyclin B, and CDK5/p25 with
IC50 values in the two-digit nanomolar range. Noteworthy, alkyl
substitutions at the indole nitrogens have different consequences
in the 1-aza- and 2-aza-series. While in the 1-aza-series the
substitution at the indole nitrogen more or less abrogates the
GSK-3 inhibitory activity (entries 15a-d), a similar substitution
in the 2-aza series produces derivatives that still are submicro-
molar GSK-3 inhibitors, showing even some selectivity versus
CDK5/p25 (entries 16a-c). The novel pentacyclic paullone
derivative 15e constitutes an exception, being the only submi-
cromolar GSK-3R/ꢀ inhibitor in the series of 12-substituted
1-azapaullones. In the 1-aza series, introduction of a 2-bromo

9-Cyano-1-azapaullone
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2201
Table 1. Inhibition of GSK-3R/ꢀ, CDK5/p25, and CDK1/Cyclin B by
The 12-oxapaullone derivatives 24a-c failed to show any
kinase inhibitory activity in the set of the four kinases used in
this study. This observation disproves our assumption that the
ring oxygen of the benzofurane structure could act as a hydrogen
bond acceptor for the water molecule W2 that usually accepts
the hydrogen bond from the paullone indole NH.
In conclusion, testing of the novel paullone derivatives in
kinase assays revealed that only congeners of the 1-aza-series
2 and 12-substituted derivatives of the 2-aza-series 16 exhibited
GSK-3 inhibition with sufficient selectivity. While the 12-
unsubstituted 2-azapaullones 13 and the paullone 2,9-dicarbo-
nitrile 30 showed potent GSK-3 inhibition, the selectivity against
CDKs appeared insufficient for further studies. Both the 12-
substituted 1-azapaullones 15 and the 12-oxapaullones 24 failed
to exhibit a noteworthy kinase inhibitory activity. We therefore
decided to carry out cell-based assays directed to investigate
ꢀ-cell protection and proliferation with compounds of the series
2. 12-Substituted derivatives of the 2-aza-series 16 also appear
to be useful GSK-3 inhibitors and will be the subject of future
studies.
Paullone Derivatives
IC50 (µM)
CDK1/
compd
X
Y
R1
R2 GSK-3R/ꢀ CDK5/p25 cyclin B
1aa CH
1ba CH
2ab CH
CH 9-Br
CH 9-NO2
0.023
0.004
0.018
0.008
0.008
0.080
0.063
100
0.8
0.80
0.025
1.8
0.85
0.040
4.2
0.3
10
3
>10
30
100
9
0.4
0.035
2.0
0.5
1.65
4.5
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
9-Br
9-CN
9-CF3
9-F
9-Cl
9-COOH
8,10-di-Cl
9-H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Et
Bn
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
13a
13b
13c
13d
13e
13f
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
C-Br
N
>10
2.2
>30
7
9-I
6
1
>10
2
9,11-di-F
9-OCH3
9-CH3
9-OH
9-CN, 11-I
9-CN
>10
0.8
4
0.13
1.40
0.12
3.8
0.052
0.021
0.013
0.051
0.018
0.39
1
>10
5
2.0
10
0.18
8
4
0.41
10
0.11
0.031
0.19
1.2
0.12
0.2
>10
>10
>10
>10
>10
8.0
3.8
>10
CH 9-Br
CH 9-CN
CH 9-CF3
CH 9-F
CH 9-Cl
CH 9-OCH3
N
N
N
N
0.032
0.12
0.26
0.21
0.4
Results of Cell-Based Assays. Confirmation of the Intra-
cellular Inhibition of GSK-3 by Paullones. SH-SY5Y neu-
roblastoma cells were exposed to various concentrations of
kenpaullone (1a) or 2b or 2c in the presence of a constant level
of MG132 (an inhibitor of the proteasome that prevented the
rapid degradation of ꢀ-catenin once phosphorylated by GSK-
3). The level of GSK-3-phosphorylated ꢀ-catenin, estimated by
an ELISA assay, revealed a dose-dependent inhibition of GSK-3
selective phosphorylation sites on ꢀ-catenin, demonstrating that
these compounds are able to inhibit GSK-3 in a cellular context
(Figure 4).
N
15a CH
15b CH
15c CH
15d CH
15e
N
N
N
N
9-H
9-H
9-H
9-H
3
13
2.1
Ph >10
0.41
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
16a
16b
16c
N
N
N
CH 9-H
CH 9-H
CH 9-H
CH 9-H
CH 9-Br
CH 9-Cl
Me
Et
Bn
0.40
0.8
0.41
24a CH
24b CH
24c CH
>10
>10
>10
>10
>10
>10
Selection of Biologically Active GSK-3 Inhibitors of the
1-Azapaullone Series. We used INS-1E ꢀ cells, a rat insulinoma
cell line widely used to study ꢀ cell functions,51 in combination
with a standard viability assay to rapidly identify azapaullone-
derived GSK-3 inhibitors with ꢀ cell protective potential.
1-Azakenpaullone (2a) served as a positive control because this
substance has previously demonstrated robust antiapoptotic
effects on INS-1E cells.16 We found that the four new
azapaullones 2b, 2c, 2d, and 2i protected INS-1E cell viability
against a toxic glucose/palmitate mixture (parts B, C, D, and E
of Figure 5, respectively). In contrast, other congeners of the
1-aza series showed only minor rescuing effects (2e, 2h, 2l,
2m) or behaved neutrally in this assay (2g, 2k) (data not shown).
Cazpaullone (2b) was the most potent compound exhibiting
protective activity at concentrations as low as 0.3 µM and with
maximal activity at 2 µM (Figure 5B). Significantly higher levels
of 1-azakenpaullone (2a) or the other azapaullone compounds
were needed to achieve effects comparable to cazpaullone.
Above 1 µM, however, treatment with cazpaullone slightly
suppressed the metabolic activity of otherwise untreated INS-
1E cells (Figure 5B), which indicates a narrow concentration
range in which cazpaullone confers beneficial effects to ꢀ cells.
The viability assay turned out to be very informative regarding
the biological activity of the tested compounds. Also, the assay
is simple, cost effective, and adjustable to a large scale
throughput format for the screening of chemical libraries.
30
C-CN CH 9-CN
H
0.028
0.73
0.12
a Data taken from ref 26 (Leost et al., 2000). b Data taken from ref 33
(Kunick et al., 2004).
Figure 4. Intracellular inhibition of GSK-3 by kenpaullone (1a) and
compounds 2b and 2c. SH-SY5Y neuroblastome cells were exposed
to various concentrations of kenpaullone (1a), cazpaullone (2b), or 2c
in the presence of a constant level of MG132. The level of GSK-3-
phosphorylated ꢀ-catenin was estimated by ELISA and was expressed
as a percentage of phosphorylated ꢀ-catenin in untreated control cells.
substituent produced a spectacular drop in the kinase inhibitory
activity. Compared to the 2-unsubstituted cazpaullone (2b), the
2-bromo-cazpaullone 2o is approximately 500-fold less active
as a GSK-3R/ꢀ inhibitor. The 2,9-dicyanopaullone 30 exhibited
an activity pattern similar to analogues of the 2-azapaullone
series, inhibiting GSK-3R/ꢀ in the two-digit and the CDKs in
the three-digit nanomolar concentration range.
We next tested if the newly identified azapaullone GSK-3
inhibitors 2b, 2c, 2d are able to inhibit ꢀ cell apoptosis induced
by high levels of glucose and palmitate (Figure 6). INS-1E
apoptosis was monitored using a DNA fragmentation assay and
a caspase activity assay. The antiapoptotic effects of 1-azak-
enpaullone 2a were found to be comparable to the activity
reported previously.16 2c and 2d turned out to be as active as

2202 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Stukenbrock et al.
Figure 5. Identification of biological active 1-azapaullone GSK-3 kinase inhibitors. (A) 1-Azakenpaullone 2a and the new azapaullone GSK-3
inhibitors (B) 2b, (C) 2c, (D) 2d, and (E) 2i were found to strongly improve the metabolic activity or viability of INS-1E ꢀ cells treated with toxic
levels of palmitate (0.3 mM palmitate coupled to BSA) and glucose (25 mM) for 24 h (gray lines and filled gray circles). The viability is an indirect
measure of the mitochondrial energy production. The effects of the tested compounds on INS-1E cells not exposed to the toxic glucose/palmitate
mixture are also shown (black line and filled black diamonds). Shown are representative experiments of at least three independently performed
studies. Given values are the mean values of measured data from at least four wells. Values show fold change relative to control ( SD. Asterisks
indicate statistically significant differences (Student’s t test): (/) P < 0.05 versus INS-1E cells treated with a mixture of high palmitate and glucose
in the absence of the indicated inhibitor.
1-azakenpaullone (2a) and moderately suppressed INS-1E cell
death. Cazpaullone (2b) more strongly protected INS-1E cells
against glucolipotoxicity, decreasing the level of apoptosis at 2
µM by more than 50%. In conclusion, our findings substantiate
earlier observations that the inactivation of GSK-3 protects INS-
1E ꢀ cells against glucolipotoxicity. New azapaullone GSK-3
inhibitors have been identified that are able to promote the
survival of INS-1E cells. Cazpaullone was found to be the most
potent of the new compounds.
New Azapaullone GSK-3 Inhibitors Stimulate the Re-
plication of Pancreatic ꢀ Cells in Vitro. 1-Azakenpaullone
has previously been shown to activate the replication of INS-
1E cells as well as of primary ꢀ cells. Likewise, the new
azapaullone GSK-3 inhibitors 2b, 2c, and 2d promoted INS-
1E cell proliferation in a dose dependent manner (Figure
7A-D). Again, cazpaullone (2b) was active at a lower
concentration (0.1 µM) than the other compounds and reached
maximal activity at 1-2 µM.
A similar result for cazpaullone (2b) was observed in the
replication experiment with primary pancreatic rat ꢀ cells. In
subsequent experiments the effects of cazpaullone on replication
of primary ꢀ cells were compared to those of CHIR99021 (3),
a highly specific and well characterized small molecular wheight
GSK-3 inhibitor.52,53 For these experiments cazpaullone was
selected because of its superior activity in INS-1E cells.
CHIR99021 (3, 5 µM) activated replication of rat ꢀ cells about
2.5-fold, confirming previous observations.16 At 0.5 µM caz-
paullone (2b) stimulated primary ꢀ cell replication by about
2.5-fold (Figure 8). At 2 µM, however, no positive effect on ꢀ
cell replication was observed. At higher compound concentra-
tions, off-target effects probably occur that neutralize the
proliferative effects resulting from GSK-3 inactivation. In
summary, we have identified three new azapaullone GSK-3
inhibitors (cazpaullone (2b), 2c, and 2d) promoting INS-1E cell
replication. For cazpaullone (2b) this proliferative effect was
confirmed with primary rat ꢀ cells.
scription Factor Pax4. Through quantitative real-time PCR
(qRT-PCR) we found that the above-described ꢀ cell protective
effects of 1-azakenpaullone (2a) and cazpaullone (2b) are
associated with the ability to activate the expression of the ꢀ
cell specific transcription factor Pax4 in INS-1E cells and
primary rat ꢀ cells (Table 2). Pax4 is essential for ꢀ cell
development during embryogenesis, and its overexpression has
been demonstrated to promote the survival and replication of
mature rat and human ꢀ cells.54 Also, Pax4 protects INS-1E
cells from apoptosis55 because a ∼50% reduction in Pax4
expression level mediated by RNA interference resulted in an
increased rate of apoptosis. Interestingly, the prominent anti-
apoptotic effects of cazpaullone correlate with a comparatively
strong activation of Pax4 mRNA expression in INS-1E cells. It
is therefore tempting to speculate that the up-regulation of Pax4
mRNA expression contributes to the observed cytoprotective
effects of cazpaullone and 1-azakenpaullone and may explain
the superior biological activity of cazpaullone compared to other
equally or even more potent GSK-3 inhibitors such as CHIR99021
(3) or compound 2c. Alsterpaullone (1b), a paullone with strong
antitumor activity known to potently inhibit GSK-3, CDKs, and
a number of other kinases, was also found to stimulate Pax4
mRNA expression in INS-1E cells. In contrast, specific GSK-3
inhibitors including CHIR99021 (3), 6BIO (4), and the new
azapaullones 2c and 2d did not activate Pax4 mRNA expression
in ꢀ cells (Table 2A). This indicates that GSK-3 inhibition is
not sufficient to activate Pax4 transcription in ꢀ cells. 1-Aza-
kenpaullone, cazpaullone. and alsterpaullone probably interact
with one or more yet unknown targets besides GSK-3 that are
involved in the regulation of Pax4 expression.
Conclusion
Here, we report the design, the synthesis, and the selection
of new paullone GSK-3 inhibitors with ꢀ cell regenera-
tive capabilities. Cazpaullone appeared to be the most active
1-azapaullone in a number of ꢀ cell assays. Compared to other
GSK-3 inhibitors, cazpaullone exhibited stronger cytoprotective
effects on ꢀ cells. Also, cazpaullone, 1-azakenpaullone, and
1-Azakenpaullone (2a) and Cazpaullone (2b) Tran-
siently Activate the Expression of the Pancreatic Tran-

9-Cyano-1-azapaullone
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2203
Figure 6. 1-Azapaullone GSK-3 inhibitors 2a-2d protect INS-1E cells against glucolipotoxicity induced cell death. As in the experiments presented
in Figure 5, cell death of INS-1E cells was induced by treatment of cells with toxic concentrations of glucose and palmitate (Gluc/Pal) for 24 h.
Apoptosis was monitored by assaying the level of cytosolic DNA fragments (DNA fragment) and the activity of caspases versus untreated cells
(Co.). At low levels, apoptosis is detectable with these methods in untreated INS-1E cells (Co.). The combination of high glucose and palmitate
strongly stimulated the respective apoptotic processes in INS-1E cells. Cells were incubated 1 h before the addition of the toxic mixture with the
indicated amounts of the test agents. Compound concentrations are indicated in micromolar. Shown are representative experiments of at least three
independently performed studies. Given values are mean values of measured data of at least four wells. Values show fold change relative to control
( SD. Asterisks indicate statistically significant differences (Student’s t test): (/) P < 0.05 versus INS-1E cells treated with a mixture of high
palmitate and glucose in the absence of the indicated inhibitor.
alsterpaullone, but not any other tested GSK-3 inhibitor, were
found to transiently activate the expression of the ꢀ cell
transcription factor Pax4. The observed increase in Pax4 mRNA
levels is remarkable because the ectopic expression of murine
Pax4 in human and rat islets induced ꢀ cell replication and
conferred resistance against cytokine-induced apoptosis.54 Thus,
the inhibition of GSK-3 in parallel with the activation of Pax4
may in a complementary fashion deliver strong survival as well
as growth signals to the investigated rat insulin producing ꢀ
cells.
Further experiments with human islets will help to evaluate
the therapeutic potential of Pax4 activating GSK-3 inhibitors.
Notably, recent research results with human ꢀ cells in culture
point to an inherent difference between human and rodent ꢀ
cells with regard to the regulation of cell replication. Basically,
factors able to stimulate replication of human ꢀ cells in culture
do not robustly work on human islets.56,57 While little is known
about the regulation of human ꢀ cell mass in vivo, the
observations with cultured human islets may at least in part
reflect the situation in the intact human pancreas. One possibility
is that human ꢀ cells require the stimulation of multiple
pathways at the same time for the induction of proliferation. If
so, agents like the Pax4 activating azapaullones targeting more
than one key regulator of ꢀ cell survival and growth may be a
superior way to expand human ꢀ cell mass in patients with
diabetes. At the moment it is unclear how cazpaullone stimulates
Pax4 expression and which targets are involved. However, the
understanding of the mechanism will be important for further
systematic development of Pax4 activating azapaullone GSK-3
inhibitors for ꢀ cell regeneration. Also, the Pax4 activating
paullones may represent valuable tools for the dissection of Pax4
regulating pathways. Cazpaullone was selected among a series

2204 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Stukenbrock et al.
regenerative agents and identified cazpaullone, a GSK-3 inhibi-
tor with strong cytoprotective and mitogenic activity in ꢀ cells.
Experimental Section
Kinase Assays. The kinase inhibition assays were performed as
described previously for GSK-3ꢀ, CDK5/p25, CDK1/cyclin B, and
CK1 (ref 58 and Supporting Information). Tests were carried out
in triplicate, and the final ATP concentration was 15 µM. The
typical standard error for IC50 values in the kinase assays was <5%.
Cell-Based Assays. Culture of INS-1E-cells, isolation of rat
islets, viability assays, caspase activity assays, DNA fragmentation
assays, in vitro ꢀ cell proliferation assays, BrdU labeling and
detection assays, and quantitative real-time PCR were performed
as described recently.16
ꢀ-Catenin Phosphorylation in SH-SY5Y Human Neuro-
blastoma Cells. Nearly confluent SH-SY5Y human neuroblastoma
cells were grown in 96-well plates in DMEM (supplemented with
10% fetal calf serum from Invitrogen and antibiotics penicillin-
streptomycin from Lonza). Cells were co-treated with tested
compounds and 2 µM MG132 (to allow accumulation of phospho-
ꢀ-catenin). Final DMSO concentration did not exceed 1%. Cells
were then subjected to an ELISA assay using antibodies directed
against Ser33/Ser37/Thr41-phosphorylated (1:1000) obtained from
Cell Signaling Technology. Results are expressed in percentage of
maximal ꢀ-catenin phosphorylation, i.e., in untreated cells exposed
to MG132 only as positive control (100% phosphorylation).
Docking. Docking was performed using the FlexX59 interface
in Sybyl.60 Before docking, the compounds were preminimized,
partial atomic charges were calculated, and they were minimized
again. Minimization was done by employing the Tripos force field61
(Powell conjugate gradient, convergence criterion 0.005 kcal/(mol
Å), 1000 iterations), and the charges were calculated using MOPAC
6.062 applying the AM1 Hamiltonian. FlexX calculated 30 docking
solutions for each compound. The poses depicted in Figure 3
represent the docking solutions with the lowest FlexX total scores.
Information of the X-ray crystal structure of alsterpaullone coc-
rystallized in the GSK-3 (1Q3W)34 was retrieved from the PDB.
To define the active site, a FlexX receptor description file was
created, which refers to all atoms of the protein within a distance
of 6.5 Å from an atom of alsterpaullone at its crystalline position.
Synthetic Chemistry. The monomode microwave device was a
CEM Discover focused microwave synthesis system with Chem-
Driver software. Melting points (mp) were determined on an electric
variable heater (Barnstead Electrothermal IA 9100) and were not
corrected. IR spectra were recorded as KBr disks on a Thermo
Figure 7. Stimulation of INS-1E ꢀ cell replication by azapaullone
GSK-3 inhibitors. INS-1E cells were treated with the test compounds
1-azakenpaullone (2a), 2b, 2c, and 2d at indicated concentrations for
24 h. Cell replication was determined by BrdU incorporation. The figure
illustrates the relative increase in incorporated BrdU in INS-1E cells
(“Fold increase BrdU”). Results are presented as fold change relative
to control ( SD. Figure shows representative experiments of two
independently performed studies. Given values are mean values of
measured data of at least four wells. Asterisks indicate statistically
significant differences (Student’s t test): (/) P < 0.05 versus INS-1E
cells not treated with the indicated inhibitor.
1
Nicolet FT-IR 200. H NMR spectra and 13C NMR spectra were
recorded on the following instruments: Bruker Avance DRX-400
and Bruker Avance II-600. The solvent was DMSO-d6 if not stated
otherwise, and the internal standard was tetramethylsilane. Signals
are in ppm (δ scale). Elemental analyses were conducted on a CE
Instruments FlashEA 1112 elemental analyzer (Thermo Quest).
Mass spectra were recorded on a double-focused sector field mass
spectrometer Finnigan-MAT 90. Accurate measurements were
conducted according to the peak match method using perfluoro-
kerosene (PFK) as an internal mass reference. (EI)-MS ionization
energy was 70 eV. TLC parameters are as follows: Polygram Sil
G/UV254, Macherey-Nagel, 40 mm × 80 mm, visualization by UV
illumination (254 nm). Column chromatography parameters are as
follows: silica gel 60 (Merck), column width 2 cm, column height
10 cm unless stated otherwise. HPLC parameters are as follows:
Elite LaChrom (Merck/Hitachi), pump L-2130, autosampler L-2200,
diode array detector L-2450, organizer box L-2000, column Merck
LiChroCART 125-4, LiChrosphere 100, RP 18, 5 µm; flow rate
1.000 mL/min, isocratic, volume of injection 10 µL, detection
(DAD) at 254 and 280 nm, AUC % method, time of detection 15
min, net retention time (ts), dead time (tm) related to DMSO.
Preparation of HPLC eluents involved H2O + TFA (pH 2.11/pH
2.55), and water was adjusted to pH 2.11/2.55 by addition of
trifluoroacetic acid. Buffer at pH 2.30 consisted of 980 mL of water/
20 mL of triethylamine/242 mg of sodium hydroxide. Adjustment
Figure 8. 2b stimulates replication of primary ꢀ cells in isolated rat
islets. Replicating ꢀ cells in isolated rat islets were identified by double
immunofluorescence staining (A) using antibodies against C-peptide
(green) and antibodies recognizing the nuclear proliferation marker Ki-
67 (yellow). After incubation with 2b and the positive control
CHIR99021 (3) for 72 h, islets were disaggregated and the resultant
cell suspension was spotted on microscope slides before staining with
antibodies and analysis. Islets not treated with any factor were included
as controls (“Co.”). Part B shows the percentage of Ki-67 expressing
cells of all C-peptide positive cells (% Ki-67+/C-peptide+ cells), which
corresponds to the fraction of replicating ꢀ cells. Part B illustrates a
representative experiment of three independent studies, and each data
point includes about 20.000 C-peptide positive cells. Error bars are
(SD. Asterisks indicate statistically significant differences (Student’s
t test): (/) P < 0.05; (//) P < 0.01 versus control.
of new and rationally designed paullone derivatives using an
innovative technological platform consisting of a sequence of
biochemical and cellular assays. This platform has proven to
be suitable for the detection, characterization, and evaluation
of potential ꢀ cell regenerative agents. In summary, we have
outlined an approach to the development of innovative ꢀ cell

9-Cyano-1-azapaullone
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2205
Table 2. Activation of Pax4 RNA Expression in INS-1E Cells (A) and Primary Rat Islets (B)a
(A) In INS-1E Cells
relative gene expression analyzed by qRT-PCR
Pax4
CypB
compd
4 h
24 h
4 h
24 h
Co.
1.00 ( 0.15
3.38 ( 0.34*
1.87 ( 0.14*
7.1 ( 0.4*
4.87 ( 1.06*
0.72 ( 0.22
0.97 ( 0.12
0.78 ( 0.24
1.02 ( 0.1
1.08 ( 0.47
4.31 ( 0.12
0.8 ( 0.16
1.00 ( 0.25
0.56 ( 0.23*
0.61 ( 0.3
0.45 ( 0.22*
0.76 ( 0.31
0.81 ( 0.12*
1.00 ( 0.11
1.04 ( 0.12
1.22 ( 0.1
1.3 ( 0.14
1.11 ( 0.32
1.2 ( 0.21
1.14 ( 0.23
1.31 ( 0.48
1.04 ( 0.1
1.00 ( 0.32
1.12 ( 0.18
1.14 ( 0.22
1.1 ( 0.2
1.42 ( 0.51
1.3 ( 0.32
0.97 ( 0.16
1.12 ( 0.03
1.2 ( 0.2
Act-A (1 nM)
1-AKP (2a) (10 µM)
alsterpaullone (1b) (12.5 µM)
cazpaullone (2b) (1 µM)
2c (10 µM)
2d (10 µM)
CHIR99021 (3) (10 µM)
6BIO (4) (1µM)
(B) In Rat Islets
relative gene expression analyzed by qRT-PCR 4h
compd
Pax4
CypB
Co.
1.00 ( 0.1
2.8 ( 0.1*
2.4 ( 0.4*
1.00 ( 0.12
1.00 ( 0.35
0.73 ( 0.15
1-AKP (2a, 10 µM)
cazpaullone (2b, 2 µM)
a Expression of Pax4 and cyclophilin B (CypB) genes was analyzed using quantitative RT-PCR (qRT-PCR). Results are presented as fold change relative
to the vehicle treated control (Co.). Values are the average of triplicates ( SD. The expression level of the respective gene in vehicle treated controls was
defined to be 1. Test agents were activin-A (Act-A), 1-azakenpaullone (1-AKP, 2a), cazpaullone 2b, CHIR99021 (3), 6-bromoindirubin-3′-oxime (6BIO, 4),
and 2d. These agents were applied to INS-1E cells (A) or primary rat islets (B) at the indicated concentrations (µM) for 4 h or 24 h. Section A shows
representative data, and results were confirmed by at least two independent experiments. Section B shows representative data of four similar experiments.
Asterisks indicate statistically significant differences (Student’s t test): (/) P < 0.05 versus control.
to pH 2.30 was done by addition of sulfuric acid. Preparative HPLC
parameters are as follows: Merck LaPrep, LaPrep P110 preparative
HPLC pump, Knauer injection loop 5 mL, LaPrep P216 fraction
collector, LaPrep P311 spectrophotometer, self-packing device
Merck NW 25 with a column tube 125 mm, inner diameter 25 mm,
silica gel Merck LiChrospher 100 RP-18, 12 µm, flow rate 40 mL/
min, detection at 254 nm. The following compounds were prepared
according to literature methods: 20b,c and 21b,c,42 22,35 28,45 and
7a, 8a, 9a, 10a.33 Synthetic procedures for the following compounds
are available in the Supporting Information: 6a-c, 7b,c, 8b,c, 9b,c,
10b-j, 10n-o, 11a-c, 2a,c-o, 13b-f, 15a-e, 16a-c, 23a-c,
24a,c, 26, 29, 30.
General Procedure A for the Synthesis of the Phenylhy-
drazones 10b-j,n-o and 11a-c. 5H-Pyrido[3,2-b]azepine-6,9
(7H,8H)-dione (9a) (1.0 mmol) or 3,4-dihydropyrido[4,3-b]azepine-
2,5-dione (9b) (1.0 mmol), respectively, and an appropriate
substituted phenylhydrazine (1.5 mmol) (respectively, an appropriate
substituted phenylhydrazine hydrochloride (1.1 mmol) and sodium
acetate (1.1 mmol)) were suspended in glacial acetic acid (10 mL)
and stirred for 15–80 min at 70 °C. After cooling to room
temperature, the mixture was poured into a 5% aqueous sodium
acetate solution. The precipitate was filtered off with suction and
washed with 5% aqueous sodium acetate solution and water. The
phenylhydrazones 10 and 11 were used after a single crystallization
from ethanol for the following synthetic procedures without further
purification.
to the mixture, and stirring was continued at 90 °C for 2–5 h. After
cooling to room temperature, the mixture was poured into a 5%
aqueous sodium acetate solution. The precipitate was filtered off
with suction and washed with 5% aqueous sodium acetate solution
and water. Crystallization was carried out from ethanol.
General Procedure D for the Synthesis of the Azapaul-
lones 2a,b,d,f. An appropriate hydrazone derivative (0.1 mmol),
obtained by general procedure A, was heated in water (1 mL). The
reaction was conducted in a sealed microwave reaction vessel
employing the following conditions: ramp time 5 min, reaction time
30 min, reaction temperature 175–215 °C. After the mixture was
cooled to room temperature, the precipitate was filtered off and
washed with petroleum ether and water.
General Procedure E for the Synthesis of the N-12-Substi-
tuted Azapaullones 15a-e and 16c. 5H-Pyrido[3,2-b]azepine-
6,9(7H,8H)-dione (9a) (1.0 mmol) or 3,4-dihydro-pyrido[4,3-
b]azepine-2,5-dione (9b) (1.0 mmol) and an appropriate substituted
phenylhydrazine (1.5 mmol) (i.e., an appropriate substituted phe-
nylhydrazine hydrochloride (1.1 mmol) and sodium acetate (1.1
mmol)) were suspended in glacial acetic acid (10 mL) and stirred
for 0.5–2 h at 70 °C. After cooling to room temperature, the mixture
was poured into a 5% aqueous sodium acetate solution. The
precipitate was filtered off with suction and washed with 5%
aqueous sodium acetate solution and water. The material was
purified by crystallization from ethanol.
General Procedure F for the Synthesis of the Oximes 23a-c.
1H-[1]Benzazepine-2,5(7H,8H)-dione (22) (0.50 mmol) and an
appropriate O-arylhydroxylamine hydrochloride (0.55 mmol) and
sodium acetate (0.55 mmol) were suspended in glacial acetic acid
(10 mL) and stirred for 1–3 h at 70 °C. After cooling to room
temperature, the mixture was poured into a 5% aqueous sodium
acetate solution. The precipitate was filtered off with suction and
was then washed with 5% aqueous sodium acetate solution and
water. The material was purified by crystallization from ethanol.
General Procedure B for the Synthesis of the Azapaullones
2b-j,o and 13b,c. An appropriate hydrazine derivative (1.0 mmol),
obtained by general procedure A, was refluxed in diphenyl ether
(80 mL) under nitrogen for 2 h. The mixture was allowed to cool
to room temperature. n-Hexane (100 mL) was added, and the
forming precipitate was separated by filtration and washed with
petroleum ether. Crystallization was carried out from ethanol.
General Procedure C for the Synthesis of the Azapaul-
lones 13a,c-f. 3,4-Dihydropyrido[4,3-b]azepine-2,5-dione (9b) (1.0
mmol) and an appropriate substituted phenylhydrazine (1.5 mmol)
(i.e., an appropriate substituted phenylhydrazine hydrochloride (1.1
mmol) and sodium acetate (1.1 mmol)) were suspended in glacial
acetic acid (10 mL), stirred for 1 h at 70 °C, and cooled to room
temperature. A few drops of concentrated sulfuric acid were added
General Procedure G for the Synthesis of the 12-Oxa-
paullones 24a-c. An appropriate oxime derivative (0.30 mmol),
obtained by general procedure F, was stirred in 5 mL of formic
acid (96%) and 0.5 mL of phosphoric acid (85%) at 60 °C. After
20–60 min a gray precipitate appeared. After cooling to room

2206 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Stukenbrock et al.
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temperature, the mixture was poured into water (20 mL). The
precipitate was separated by filtration and washed with petrol ether
and water.
6-Oxo-5,6,7,12-tetrahydropyrido[3′,2′:2,3]azepino[4,5-b]indole-
9-carbonitrile (Cazpaullone) (2b). Preparation of 2b was either
accomplished according to general procedure B or D. Preparation
according to general procedure B from 10b (260 mg, 0.89 mmol)
yielded 18% of a brown powder, mp >330 °C. 1H NMR δ 3.76 (s,
2H, azepine-CH2), 7.46 (dd, 1H, 8.2/4.5 Hz, ArH), 7.53 (dd, 1H,
8.5/1.5 Hz, ArH), 7.62 (dd, 1H, 8.5/0.5 Hz, ArH), 7.65 (dd, 1H,
8.2/1.4 Hz, ArH), 8.38 (s, 1H, ArH), 8.51 (dd, 1H, 4.5/1.4 Hz,
ArH), 10.34 (s, 1H, NH), 12.30 (s, 1H, NH); (C16H10N4O) HRMS
(EI) (m/z) [M+] calcd 274.0855, found 274.0174. Preparation
according to general procedure D from 10b (29 mg, 0.10 mmol)
(13) A Study in Type 2 Diabetic Patients with Repeated Doses of E1 in
Combination with G1; Identifier NCT00239187; U.S. National Institute
of Health, 2007; www.clinicaltrials.gov.
1
yielded 36% of a brown powder. H NMR data were consistent
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B.; Mandrup-Poulsen, T.; Donath, M. Interleukin-1-receptor antagonist
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A.; Burk, U.; Onichtchouk, D.; Dohrmann, C.; Austen, M. Inhibition
of GSK3 promotes replication and survival of pancreatic beta cells.
J. Biol. Chem. 2007, 282, 12030–12037.
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partly mediated by reduced insulin signaling through phosphatidyli-
nositol 3-kinase/Akt and increased glycogen synthase kinase-3beta in
mouse insulinoma cells. Diabetes 2005, 54, 968–975.
with the data obtained for 2b prepared by general procedure B.
9-Bromo-7,12-dihydropyrido[4′,3′:2,3]azepino[4,5-b]indol-
6(5H)-one (13a). Preparation according to general procedure C from
9b (56 mg, 0.32 mmol), (4-bromophenyl)hydrazine hydrochloride
(78.1 mg, 0.35 mmol), and sodium acetate (28.6 mg, 0.35 mmol)
1
afforded 20% of a gray solid, mp >330 °C. H NMR δ 3.66 (s,
2H, azepine-CH2), 7.19 (d, 1H, 5.5 Hz, ArH), 7.31 (dd, 1H, 8.6/
1.9 Hz, ArH), 7.42 (d, 1H, 8.6 Hz, ArH), 7.96 (d, 1H, 1.8 Hz,
ArH), 8.45 (d, 1H, 5.5 Hz, ArH), 8.92 (s, 1H, ArH), 10.53 (s, 1H,
NH), 11.96 (s, 1H, NH); (C15H10BrN3O) HRMS (EI) (m/z) [M+]
calcd 327.0007, found 326.9990.
9-Bromo-5,7-dihydro-6H-[1]benzofuro[3,2-d][1]benzazepin-
6-one (24b). Preparation according to general procedure G from
23b (104 mg, 0.30 mmol) yielded 38% of a gray solid, mp >330
°C. 1H NMR (DMSO-d6 + TFA, 600 MHz) δ 3.69 (s, 2H, azepine-
CH2), 7.30–7.34 (m, 2H, ArH), 7.46–7.49 (m, 1H, ArH), 7.54 (dd,
1H, 8.7/2.0 Hz, ArH), 7.65 (d, 1H, 8.6 Hz, ArH), 7.87 (dd, 1H,
7.8/1.4 Hz, ArH), 8.12 (d, 1H, 2.0 Hz, ArH), 10.41 (s, 1H, NH).
Anal. (C16H10BrNO2) C, H, N.
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(19) Elghazi, L.; Rachdi, L.; Weiss, A.; Cras-Méneur, C.; Bernal-Mizrachi,
E. Regulation of beta-cell mass and function by the Akt/protein kinase
B signalling pathway. Diabetes Obes. Metab. 2007, 9 (Suppl. 2), 147–
57.
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Taketo, M.; Nusse, R.; Hebrok, M.; Kim, S. Wnt signaling regulates
pancreatic beta cell proliferation. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 6247–6252.
Acknowledgment. We thank Dr. Ursula Hoffmann for
critical reading of the manuscript. The work was supported by
a grant from the German Ministry of Education and Research
(BMBF, BioProfil “Funktionelle Genomanalyse”, Grant
0313348A, to DeveloGen AG).
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(GSK-3): a kinase with exeptional therapeutic potential. Annu. Rep.
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potential. Nat. ReV. Drug DiscoVery 2004, 3, 479–487.
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signalling. Expert Opin. Ther. Targets 2006, 10, 429–444.
(24) Meijer, L.; Flajolet, M.; Greengard, P. Pharmacological inhibitors of
glycogen synthase kinase-3. Trends Pharmacol. Sci. 2004, 25, 471–
480.
(25) Kunick, C.; Lauenroth, K.; Wieking, K.; Xie, X.; Schultz, C.; Gussio,
R.; Zaharevitz, D.; Leost, M.; Meijer, L.; Weber, A.; Jorgensen, F. S.;
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for CDK1, CDK5, and GSK-3 inhibition by paullones. J. Med. Chem.
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(26) Leost, M.; Schultz, C.; Link, A.; Wu, Y.-Z.; Biernat, J.; Mandelkow,
E.-M.; Bibb, J. A.; Snyder, G. L.; Greengard, P.; Zaharevitz, D. W.;
Gussio, R.; Senderowicz, A. M.; Sausville, E. A.; Kunick, C.; Meijer,
L. Paullones are potent inhibitors of glycogen synthase kinase-3ꢀ and
cyclin-dependent kinase 5/p25. Eur. J. Biochem. 2000, 267, 5983–
5994.
Supporting Information Available: Details for the synthesis
of 6a-c, 7b,c, 8b,c, 9b,c, 10b-j,n-o, 11a-c, 2a,c-o, 13b-f,
15a-e, 16a-c, 23a-c, 24a,c, 26, 29, 30, spectroscopic data, HPLC
purity data, and data from elemental analyses. This material is
available free of charge via the Internet at http://pubs.acs.org.
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