Thiazolo[5,4-f]quinazolin-9-ones, inhibitors of glycogen synthase kinase-3

Article abstract of DOI:10.1016/j.bmcl.2006.04.006

In an effort to identify new protein kinase inhibitors with increased potency and selectivity, we have developed the microwave-assisted synthesis of thiazolo[5,4-f]quinazolin-9-ones. The effects of eighteen derivatives on CDK1/cyclin B, CDK5/p25, and GSK-

Full text of DOI:10.1016/j.bmcl.2006.04.006

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3419–3423
Thiazolo[5,4-f]quinazolin-9-ones, inhibitors of glycogen
synthase kinase-3
a
b
b
b
c
´
´
´
Alexandra Testard, Cedric Loge, Benoˆıt Leger, Jean-Michel Robert, Olivier Lozach,
c
c
a
a,
*
´
´
´
Melina Blairvacq, Laurent Meijer, Valerie Thiery and Thierry Besson
aLaboratoire de Biotechnologies et de Chimie Bio-organique, FRE CNRS 2766, UFR Sciences Fondamentales et Sciences
ˆ
pour l’Inge´nieur, Universite´ de La Rochelle, Batiment Marie Curie, 17042 La Rochelle, France
bUniversite´ de Nantes, Nantes Atlantique Universite´s, Biomole´cules et Cibles The´rapeutiques, De´partement de Pharmacochimie,
BioCiT-EA 1155, UFR Sciences Pharmaceutiques, 1 rue Gaston Veil, Nantes F-44000, France
cCell Cycle Group, UMR7150 and UPS2682, Station Biologique, B. P. 74, 29682 Roscoff Cedex, Bretagne, France
Received 10 March 2006; revised 3 April 2006; accepted 3 April 2006
Available online 27 April 2006
Abstract—In an effort to identify new protein kinase inhibitors with increased potency and selectivity, we have developed the micro-
wave-assisted synthesis of thiazolo[5,4-f]quinazolin-9-ones. The effects of eighteen derivatives on CDK1/cyclin B, CDK5/p25, and
GSK-3 were investigated. Several turned out to inhibit GSK-3 in the micromolar range. Molecular modeling studies suggest that the
most selective GSK-3 inhibitors 7a–d bind into the ATP-binding site through a key hydrogen bond interaction with Val135 and
target the specific hydrophobic backpocket of the enzyme.
Ó 2006 Elsevier Ltd. All rights reserved.
Cyclin-dependent kinases (CDKs) constitute a family of
highly conserved protein kinases involved in regulating
the cell division cycle, apoptosis, numerous neuronal
functions, insulin release, and transcription. Glycogen
synthase kinase-3 (GSK-3) is a family of kinases in-
volved in cell cycle control, insulin action, apoptosis,
neuronal cell death, and developmental regulation. Both
families of kinases are implicated in various human dis-
eases such as cancers, Alzheimer’s disease, and diabetes,
and therefore both have been extensively used as targets
to identify small molecular weight pharmacological
inhibitors of potential therapeutic interest.1–4 More than
100 CDK inhibitors and 40 GSK-3 inhibitors have been
identified;2–4 most of which act by competing with ATP
binding at the catalytic site of the kinase. Among the
numerous inhibitors described, the most studied mem-
bers contain a purine (e.g., olomoucine I and roscovitine
II) or an oxindole ring (e.g., oxindole 91 III) (Fig. 1).
While investigating the chemical interest of 4,5-dichloro-
1,2,3-dithiazolium chloride (Appel’s salt)5–7 and
its derivatives, we recently described the microwave-
assisted multi-step synthesis of novel thiazoloquinazoli-
nones8–10 (IV and V, Fig. 1) which can be considered, at
first glance, as hybrid molecules between the purines and
the oxindoles mentioned above. After testing the effects
of these molecules on CDKs and GSK-3,10 it was ob-
served that isomer (V) is completely inactive whatever
substituent is present on the angular structure. Although
most synthesized quinazolinones exhibited a moderate
to potent GSK-3 inhibitory activity (IC50 ranging from
1.3 to 60 lM), one derivative (molecule 7a, Scheme 1),
analogue of IV, exerted a selective inhibition toward
GSK-3 (IC50 = 4.2 lM). This compound bears, at C-2
of the thiazoloquinazolinone, an amidine function
incorporated into an imidazoline ring. Considering this
product as a possible lead compound, we focused our
efforts on the synthesis of various thiazoloquinazolinon-
es, which possess an amidine function on C-2 of the
thiazole moiety and various alkyl or aryl groups on
N-8 of the quinazoline part of the molecule.
Abbreviations: CDK, cyclin-dependent kinase; GSK-3, glycogen
synthase kinase-3; ATP, adenosine triphosphate; MW, microwave;
DMF, N,N-dimethylformamide; THF, tetrahydrofuran; AMP-PNP,
50-adenylyl-imidodiphosphate; pdb code, RCSB Protein Data Bank.
Keywords: Glycogen synthase kinase-3 inhibitors; Quinazolines;
Microwave-assisted chemistry; Molecular modeling.
In this article, we report the benefits associated with the
microwave methodology10,11 for the preparation of
these new products and their effects on CDKs and
GSK-3. Docking studies targeting the ATP-binding site
*
Corresponding
+33(0)546458247; e-mail: tbesson@univ-lr.fr
author.
Tel.:
+33(0)546458255;
fax:
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.006

3420
A. Testard et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3419–3423
H
N
N
SO2
HN
N
HN
N
N
N
N
N
N
N
S
N
H
OH
OH
N
N
N
H
H
O
N
H
I
II
III
olomoucine
roscovitine
oxindole 91
O
9
NC
N
NH
O
S
1
8
3
6
N
9
NH
3
N
1
8
6
S
N
NC
IV
V
Figure 1. Structure of olomoucine (I), roscovitine (II), oxindole 91 (III), 8H-thiazolo[5,4-f]quinazolin-9-one (IV), and 7H-thiazolo[4,5-h]quinazolin-
6-one (V).
O
N
O
O
R1
R1
O2N
O2N
H2N
a
b
NH
N
N
N
N
1
2a-g
3a-g
N
S
NC
Br
O
S
Cl
R1
Br
O
N
O
S
H2N
c
R1
d
e
N
R1
N
N
N
N
N
N
4-a-g
5a-g
6a-g
f
g
H
R2
S
a : R1 = CH2-CH3
b : R1 = CH(CH3)2
c : R1 = CH2-CH2-CH3
d : R1 = CH2-CH(CH3)2
e : R1 = (CH2)2-CH(CH3)2
f : R1 = CH2Ph
g : R1 = CH2Ph-(4-MeO)
N
N
N
HN
H
O
N
O
N
S
R1
N
R1
N
N
N
8a-b : R2 = CH(CH3)2
9a-b : R2 = (CH2)2-N(CH3)2
7a-g
Scheme 1. Reagents and conditions: (a) alkyl iodide and benzyl chloride, NaH, DMF, MW, 30 min; (b) HCO2NH4, Pd/C, EtOH, MW, 80 °C,
30 min; (c) Br2, AcOH, rt, 2 h; (d) 4,5-dichloro-1,2,3-dithiazolium chloride, pyridine, CH2Cl2, rt, 1 h; (e) CuI, pyridine, MW, 115 °C, 15 min; (f and g)
alkylamine or diamine, THF, MW, 80 °C, 30 min.
of a GSK-3b crystal structure were also performed to
provide a hypothesis on the possible binding mode of
this new class of inhibitors.
or benzyl chloride derivatives). Using ammonium
formate for catalytic transfer hydrogenation in ethanol,
reduction of the N-alkylated 7-nitroquinazolin-4-ones
(2a–g) led to the amino derivatives (3a–g) in good yields
(83–90%) (Scheme 1). Bromination of compounds (3a–
g), in the presence of bromine in acetic acid, gave the
ortho-brominated imines (4a–g) (yields: 70–87%). These
compounds were condensed with 4,5-dichloro-1,2,3-
dithiazolium chloride in dichloromethane at room
temperature, followed by addition of pyridine, to give
the intermediate imino-1,2,3-dithiazoloquinazolinones
(5a–g) (yields: 60–78%). Final fusion of the thiazole ring
onto the quinazoline moiety was realized by a thermolysis
The pharmaceutical interest of the unsubstituted mole-
cule IV (Fig. 1) has been judged limited and we demon-
strated that N-alkylation of the nude skeleton was not
possible in the conditions tested. Then, we decided to per-
form an N-alkylation of the quinazolinone 1 before fusing
the thiazole ring. Selective N-alkylation in position 3 of
the quinazolin-4-one skeleton was realized in various
yields (44–50%) by treatment of 7-nitroquinazolin-4-one
1, with sodium hydride and alkylation agents (alkyl iodide

A. Testard et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3419–3423
3421
procedure consisting of heating the imines (5a–g) at
115 °C, in the presence of cuprous iodide, in pyridine.
The expected compounds (6a–g) were obtained in yields
superior to 60%. The reactive cyano group of the
thiazolo-2-carbonitrile ring was efficiently transformed
into various amidines (7, 8, and 9) by treatment, under
microwave irradiation, of compounds 6a–g with ethylene
diamine (products 7a–g), isopropylamine (products 8a,b)
or N,N-dimethylethylenediamine (products 9a,b). The
basic side chain present on the latest compounds might
provide cationic molecules leading to better water
solubility and impacting on their biological properties.
to have a real influence on the activity of this family. A
bulky chain substitution (e.g., N-isopentyl or N-benzyl
derivatives 7e–g) results in a lack of activity, whilst a
short chain of 2 or 3 carbons leads to compounds (7a–c)
with similar GSK-3 potency. Interestingly, replacement
of the imidazoline ring of 7a,b by a less rigid amidine
function gives compounds 8a,b and 9a,b which differ in
their activities according to the substituent at the R2
position. Therefore, introduction of
a bulky N,
N-dimethylethylenediamine group leads to compounds
9a,b which show an increased inhibitory activity toward
both GSK-3 and CDK1, while a smaller isopropyl group
8a,b enhances selectivity toward GSK-3 versus CDK1, as
previously observed for compounds 7a–d.
Our experience in microwave-assisted chemistry of het-
erocycles11 encouraged us to establish an efficient mul-
ti-step (five or six steps) synthesis of the 2,8-substituted
thiazolo[5,4-f]quinazolin-9-one derivatives 6, 7, 8, and
9 (Scheme 1). In all cases, besides resulting in good to
excellent yields, our method offers much faster reactions
compared to earlier published procedures at atmospher-
ic pressure.
In silico docking15 gave the structures shown in Figure 2.
The most selective GSK-3 inhibitors 7a–d share a similar
docking mode and make a critical hydrogen bond be-
tween the quinazoline nitrogen N-6 of the molecules
and the backbone NH of Val135. According to previous
studies,20,21 all ATP-competitive kinase inhibitors bind
in the adenine-binding region and interact with the hinge
domain via hydrogen bond interactions. Due to the
lengthened structure of these inhibitors, the rigid imidaz-
oline ring targets the hydrophobic backpocket. This
region, which is not occupied by ATP, displays structural
diversity between members of the kinase family and con-
tains unique residues which could be used to increase
selectivity.21–23 Access to this region is controlled by
the so-called ‘gatekeeper residue’ Leu132, which is
replaced by Phe80 in CDK1. Due to the bulkiness of
the Phe80 side chain, these compounds may not fit into
the hydrophobic pocket, inducing unfavorable contacts
in CDK1. Such hypothesis has already been proposed
in previous works.24–26
Thiazolo[5,4-f]quinazolin-9-one derivatives (6, 7, 8, and
9) were tested against three protein kinases, CDK1/cy-
clin B, CDK5/p25, and GSK-3a/b.12–14 All assays were
run in the presence of 15 lM ATP and appropriate pro-
tein substrates (histone H1 for CDKs, GS-1 peptide for
GSK-3). IC50 values were determined from dose–
response curves and are provided in Table 1.
Most synthesized quinazolinones exhibit a moderate to
potent GSK-3 inhibitory activity with IC50 ranging from
0.56 to 50 lM (Table 1). As we previously observed in
our preliminary work,10 the selectivity for GSK-3 versus
CDKs is enhanced for amidine derivatives (7a–d and
8a,b), compared to their cyano precursors (6a–d). The
most active derivatives (7a–d) possess an amidine func-
tion incorporated into an imidazoline ring. It is also
noticeable that N-substitution by an alkyl group seems
Additionally, imidazoline compounds 7e–g bearing a
bulky chain at the R1 position would be expected to
Table 1. Kinase inhibitiona values (IC50 in lM) for compounds 6–9
Compound
CDK1/cyclin B
CDK5/p25
GSK-3a/b
6a
6b
6c
6d
6e
6f
12
>10
>100
>100
>100
NT
27
>10
>100
>10
>100
>10
NT
6.2
>10
6.5
10
50
>10
>10
4.2
6g
7a
7b
7c
7d
7e
7f
>10
>100
>100
>100
85
>100
>100
>100
>100
>10
>10
NT
1.6
1.6
4.6
>10
NT
>10
>10
>10
7.2
7g
8a
8b
9a
9b
>10
>100
>100
17
NT
NT
9.4
1.3
NT
Figure 2. Illustration of the possible binding mode of compounds 7a–d
into the ATP-binding site of GSK-3b (based on pdb code: 1UV5),
suggested by the molecular docking studies. The active site pocket is
highlighted in cyan (MOLCAD surface; program Sybyl 7.0). The
hydrogen bonds are indicated as yellow dotted lines.
10
4
0.56
a Kinase inhibition experiments were carried out as described previ-
ously.12–14 The final ATP-concentration in the test was 15 lM (NT,
not tested).

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A. Testard et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3419–3423
encounter unfavorable steric interactions with some res-
idues in GSK-3. In order to substantiate this hypothesis,
we compared our model with another crystal structure
of GSK-3b in complex with the non-hydrolyzable ATP
analogue AMP-PNP (pdb code: 1J1B).17 The three-di-
mensional structures were superimposed using the a-car-
bon of residues in the ATP pocket (Fig. 3). Interestingly,
the main difference is the side-chain guanidine group of
Arg141 which is oriented into a different direction and is
closer to the R1 position of our imidazoline compounds,
resulting in a possible steric clash with encumbering
substituents.
served for imidazoline compounds 7a–d. In particular,
the isopropyl group at the R2 position is oriented into
the hydrophobic backpocket. However, this part of the
binding site appears to accommodate only a relatively
small substituent since replacement by a bulky N,N-
dimethylethylenediamine group leads to compounds
9a,b which are more potent ATP-competitive inhibitors
of GSK-3 (IC50 values of 1.30 and 0.56 lM, respective-
ly), but present also a moderate inhibition for CDK1
(IC50 values of 17.0 lM and 10.0 lM, respectively). A
possible change in binding conformation may occur
and this will be the object of future computational
studies.
Finally, the binding mode of less rigid amidine com-
pounds 8a,b derived from the best docking pose is illus-
trated in Figure 4. According to this model, these
compounds may form interactions similar to those ob-
In conclusion, this work has uncovered a family of 2,8-
substituted thiazoloquinazolinones from which selective
micromolar GSK-3 inhibitors could be identified (e.g.,
7a–d and 8a,b) and has shown how the known hydro-
phobic backpocket subsite of the enzyme can be exploit-
ed to gain affinity as well as selectivity. In two instances,
the most GSK-3 active compounds exhibit a moderate
inhibitory activity on CDK1 (e.g., 9a and 9b). These
two molecules suggest that it might be possible to obtain
a combined inhibition of GSK-3 and CDKs. In order
to improve our knowledge on this series, new amidine
and/or imidate derivatives, with various R1 and R2
substituents, are currently being evaluated. This family
constitutes an interesting scaffold from which more
potent inhibitors could be designed, helped by molecular
modeling studies.
Acknowledgments
´
We thank the ‘Comite de Charente et de Charente-Mar-
itime de la Ligue Nationale Contre le Cancer’ for finan-
cial support. The work presented here was also
supported by a grant from the EEC (FP6-2002-Life
Sciences and Health, PRO-KINASE Research Project)
(L.M.) and the Canceropole Grand-Ouest. A.T. is
Figure 3. Superimposition of two GSK-3b crystal structures (orange,
pdb code: 1UV5 and cyan, pdb code: 1J1B).
´
´
thankful to the ‘Communaute d’Agglomeration de la
Ville de La Rochelle’ for a research fellowship.
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¨
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˚
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