Novel 9-oxo-thiazolo[5,4-f]quinazoline-2-carbonitrile derivatives as dual cyclin-dependent kinase 1 (CDK1)/glycogen synthase kinase-3 (GSK-3) inhibitors: Synthesis, biological evaluation and molecular modeling studies

Article abstract of DOI:10.1016/j.ejmech.2007.09.020

Continuous efforts in microwave-assisted synthesis and the structure-activity relationships' (SARs) studies of novel modified 9-oxo-thiazolo[5,4-f]quinazoline-2-carbonitriles, allowed identification of new amidine and imidate derivatives as potent and dual CDK1/GSK-3 inhibitors. Combination of lead optimization and molecular modeling studies allowed identification of a dual CDK1/GSK-3 inhibitor (compound 13d) with submicromolar values.

Full text of DOI:10.1016/j.ejmech.2007.09.020

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1469e1477
http://www.elsevier.com/locate/ejmech
Original article
Novel 9-oxo-thiazolo[5,4-f]quinazoline-2-carbonitrile derivatives
as dual cyclin-dependent kinase 1 (CDK1)/glycogen synthase
kinase-3 (GSK-3) inhibitors: Synthesis, biological evaluation and
molecular modeling studies
a,
b
b
c
c
*
´
´
´
´
´
Cedric Loge , Alexandra Testard , Valerie Thiery , Olivier Lozach , Melina Blairvacq ,
b,**
Jean-Michel Robert a, Laurent Meijer c, Thierry Besson
a Universite´ de Nantes, Nantes Atlantique Universite´s, Biomole´cules et Cibles The´rapeutiques, De´partement de Pharmacochimie,
BioCiT, UPRES EA 1155, UFR Sciences Pharmaceutiques, 1 rue Gaston Veil, F-44035 Nantes Cedex 1, France
b Laboratoire de Biotechnologies et de Chimie Bio-organique, FRE CNRS 2766, UFR Sciences Fondamentales et Sciences pour l’Inge´nieur,
Universite´ de La Rochelle, Baˆtiment Marie Curie, 17042 La Rochelle, France
c Cell Cycle Group, UMR7150 & UPS2682, Station Biologique, B. P. 74, 29682 Roscoff Cedex, Bretagne, France
Received 26 June 2007; received in revised form 13 September 2007; accepted 18 September 2007
Available online 29 September 2007
Abstract
Continuous efforts in microwave-assisted synthesis and the structureeactivity relationships’ (SARs) studies of novel modified 9-oxo-thia-
zolo[5,4-f]quinazoline-2-carbonitriles, allowed identification of new amidine and imidate derivatives as potent and dual CDK1/GSK-3 inhibitors.
Combination of lead optimization and molecular modeling studies allowed identification of a dual CDK1/GSK-3 inhibitor (compound 13d) with
submicromolar values.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: CDK1 inhibitor; GSK-3 inhibitor; Molecular modeling; Microwave-assisted chemistry; Quinazolinones
1. Introduction
become a major area for therapeutic intervention and a variety
of kinase inhibitor pharmacophores has been described. Most
kinase inhibitor molecules currently developed are targeted at
the ATP-binding site, an ubiquitous domain in nature, and
mimic mainly the H-bonding motif of the ATP aminopyrimi-
dine ring. Among the 518 human kinases [1], two classes
have been particularly explored: the cyclin-dependent kinases
(CDKs) which are involved in regulating the cell division cy-
cle, apoptosis, neuronal cell physiology, pain signaling, tran-
scription, RNA splicing and insulin release, among other
activities [2,3] and the glycogen synthase kinase-3 (GSK-3)
which is a family of kinases involved in cell cycle control, in-
sulin action, apoptosis, neuronal cell death and developmental
regulation, among other processes [4,5]. Both families of
kinases are implicated in various human diseases such as can-
cers, Alzheimer’s disease, diabetes and therefore both have
Protein kinases have a fundamental role in signal transduc-
tion pathways, and aberrant kinase activity has been observed
in many diseases. In recent years, kinase inhibition has
Abbreviations: CDK, cyclin-dependent kinase; GSK-3, glycogen synthase
kinase-3; ATP, adenosine triphosphate; MEPs, molecular electrostatic poten-
tials; MW, microwave; AMP-PNP, 50-adenylyl-imidodiphosphate; pdb code,
RCSB Protein Data Bank.
* Corresponding author. Tel.: þ33 (0) 240411108; fax: þ33 (0) 240412876.
** Corresponding author. Present address: UMR CNRS 6014, Laboratoire de
´
´
Chimie Pharmaceutique, UFR Medecine-Pharmacie, Universite de Rouen, 22
Boulevard Gambetta, 76183 Rouen Cedex 1, France. Tel.: þ33 (0) 235148399;
fax: þ33 (0) 235148423.
´
E-mail addresses: cedric.loge@univ-nantes.fr (C. Loge), thierry.besson@
univ-rouen.fr (T. Besson).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.09.020

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C. Loge´ et al. / European Journal of Medicinal Chemistry 43 (2008) 1469e1477
Table 1
Kinase inhibition values (IC50 in mM) for compounds 12e15 (NT ¼ not tested)
been extensively used as targets to identify small molecular
weight pharmacological inhibitors of potential therapeutic in-
terest. A recent mini-review also shows that CDK/GSK-3 in-
hibitors may emerge as effective therapeutic agents for
proliferative renal diseases, furthering the prospect that these
inhibitors may emerge as effective therapeutic agents in the
near future [6]. Over the past decades, several groups have
identified and characterized a fair number of potent CDK in-
hibitors (olomoucine, roscovitine, purvalanols, indirubins,
aloisines, hymenialdisine, etc.). Many of these molecules are
derived from marine organisms. The most advanced com-
pound, the purine analogue (R)-roscovitine (CYC-202),
a CDK2 selective inhibitor, is currently in phase 2 clinical tri-
als against non-small cell lung cancer, breast cancer and vari-
ous B-cell malignancies, in phase 1 against various kidney
inflammations (glomerulonephritis), in pre-clinical, animal
testing against Alzheimer’s disease and stroke. Interestingly,
many inhibitors are in fact dual inhibitors of CDKs and
GSK-3, due mainly to the high degree of homology
(w86%) between the ATP-binding site of the two kinases.
In an attempt to identify a potent and selective GSK-3 in-
hibitor focused on 2,8-substituted 9-oxo-thiazoloquinazoline-
2-carbonitriles, we unexpectedly discovered a compound,
bearing an N-isopropyl side chain on the N-8 nitrogen and
an amidine function with a bulky N,N-dimethylethylenedi-
amine group at the C-2 position of the thiazole moiety. This
compound showed submicromolar IC50 against GSK-3 but
also a moderate activity against CDKs (Table 1, entry 15c)
[7]. This article describes our continued efforts in the struc-
tureeactivity relationships’ (SARs) studies and toward the
identification of new amidine and imidate derivatives as potent
and dual CDK1/GSK-3 inhibitors.
Compounds
CDK1/cyclin B
CDK5/p25
GSK-3a/b
12a
12b
50
4.3
NT
NT
NT
>100
4
2.1
0.64
0.15
0.17
0.3
12c
12d
1.4
1.3
12e
13a
1.4
13
43
0.15
0.26
0.28
0.13
7.2
13b
13c
5.2
2.4
NT
NT
>100
NT
>10
NT
NT
>10
4
13d
14a
0.15
>100
>10
17
14b
14c
2.4
1.3
15a
15b
>100
9
10
9.4
0.52
0.56
130
15c
(R)-Roscovitine
0.45
0.16
from commercially available 5-nitroanthranilic acid. The multi-
step synthesis was mainly performed under microwaves taking
in account of our experience in this domain [8e10]. The exper-
imental conditions and the yields of the different steps are de-
scribed in Scheme 1. The 6-nitro-3H-quinazolin-4-one 1 was
synthesized as we previously described by microwave heating,
at atmospheric pressure, of 5-nitroanthranilic acid with 5 equiv
of formamide (150 ꢀC) [11]. Selective N-alkylation in position 3
of the quinazolin-4-one ring was performed at atmospheric
pressure by treatment of 1 with sodium hydride and ethyl or
isopropyl iodide as alkylating agents. Reduction of the 3-
substituted-6-nitroquinazolin-4-ones 2 and 3 led to the 6-amino
derivatives 4 and 5 by using ammonium formate for catalytic
transfer hydrogenation in ethanol. Compounds 4 and 5 were
brominated in the presence of bromine in acetic acid, to give
the ortho brominated imines 6 and 7. The latest were con-
densed, under microwaves, with 4,5-dichloro-1,2,3-dithiazolium
chloride (Appel’s salt) [12] in dichloromethane and in a sealed
tube. Addition of pyridine led to the desired imino-1,2,3-
dithiazoloquinazolinones 8 and 9. Products 10 and 11 were ob-
tained by microwave-assisted thermolysis consisting of a rapid
2. Chemistry
2.1. Synthesis of the thiazoloquinazolinone core
The expected 9-oxo-thiazolo[4,5-f]quinazoline-2-carbonitrile
derivatives (10 and 11) (Scheme 1) were obtained in six steps
O
O
O
O2N
R
O2N
H2N
R
N
NH
a
N
b
N
N
N
2, R = Et (98%)
3, R = iPr (90%)
4, R = Et (95%)
5, R = iPr (85%)
NC
Br
O
N
Br
O
N
O
N
Cl
S
H2N
R
N
R
N
R
N
N
c
d
e
N
N
S
S
6, R = Et (93%)
7, R = iPr (83%)
8, R = Et (45%)
9, R = iPr (78%)
10, R = Et (93%)
11, R = iPr (71%)
Scheme 1. Reaction conditions: (a) alkyl iodide, NaH, DMF, (MW) 140 ꢀC, 5 min; (b) HCO2NH4, Pd/C, EtOH, (MW) 80 ꢀC; 15 min; (c) Br2, AcOH, rt, 2 h; (d)
4,5-dichloro-1,2,3-dithiazolium chloride, pyridine, CH2Cl2, (MW) 80 ꢀC, 4 min; (e) CuI, pyridine, (MW) 160 ꢀC, 1 min.

C. Loge´ et al. / European Journal of Medicinal Chemistry 43 (2008) 1469e1477
1471
heating of the starting imines (1 min at 160 ꢀC in a sealed
tube), in the presence of cuprous iodide in pyridine. Technol-
ogy used for heating, time of reactions and yields are given in
Scheme 1.
(IC50 values of 10 mM and 0.56 mM, respectively) (Table 1),
we first decided to examine the activities of another bulky sub-
stituent at the R position of the amidine function. Replacement
of the N,N-dimethylethylenediamine group by a benzyl group
gave compound 15b with comparable potency (IC50 values of
9 mM and 0.52 mM, respectively) leading to the assumption
that these molecules interact with the ATP-binding site of
both enzymes in a very similar manner. In a parallel study,
we found that substitution of the amidine function of 15b by
an imidate analogue 13d considerably improves the efficacy
of this compound to reach submicromolar activities in both
CDK1 and GSK-3 (IC50 values of 0.15 mM and 0.13 mM, re-
spectively). Taken together, these results seem to imply the
role of both electrostatic and steric parameters at this level
of interactions. In order to confirm the observed structureeac-
tivity relationships, we decided to investigate structure modi-
fications on thiazoloquinazolinone nucleus either by
introducing N-ethyl or N-isopropyl side chains on the N-8 ni-
trogen or by varying the substitution of the imidate side chain.
A small number of compounds (12aee;13aec) was thus syn-
thesized and their potency in the purified enzymes was mea-
2.2. Synthesis of the 8-substituted thiazoloquinazolinones
It is known that cyano group in position 2 of benzothiazole
ring is very reactive and that its transformation into imidate,
imidazoline, amidine and decyanated derivative can be easily
realized [12]. Imidates 12aee and 13aed were, respectively,
obtained in good yields from derivatives 10 and 11 by treat-
ment with various alcohols (ethanol, isopropanol, butanol, or
benzyl alcohols) in the presence of 1 equiv of NaOH 2.5 N
(Scheme 2). The condensation of thiazoloquinazolinone-2-car-
bonitriles 10 and 11 with the commercially available appropri-
ate amines in various solvents (e.g. ethanol, THF) was studied
to give the desired amidines 14aec and 15aec (Scheme 2).
Thus, employing microwave-assisted organic synthesis al-
lowed us to establish efficient conditions for the preparation
of N-substituted thiazoloquinazolinones. Our results are all
confirming that it is possible to achieve better yields and
cleaner reactions by performing a majority of the reactions un-
der microwave irradiation instead of using the purely thermal
processes.
sured. Compounds 12cee and 13c bearing
a bulky
substituent at the R position of the imidate function (butyl
for 12c and 13c, benzyl for 12d and phenyl ethyl for 12e)
maintained a high inhibitory activity against GSK-3 (IC50
ranging from 0.15 to 0.30 mM) and were also able to inhibit
CDK1, although a 10-fold drop in potency was found, com-
pared to the lead compound 13d (IC50 ranging from 1.30 to
2.40 mM). In contrast, compounds 12a and 13a with a smaller
ethyl group led to weaker inhibitors of CDK1 (IC50 values of
50.0 mM and 13.0 mM, respectively). From a steric point of
view, this result is in accordance with the previous observation
3. Results and discussion
3.1. Lead optimization
With the thiazolo[5,4-f]quinazoline-2-carboxamidine 15c,
as a moderate dual CDK1/GSK-3 lead molecule in hand
NH
NH
H
O
NC
N
N
O
N
R
S
O
N
S
O
N
R
S
N
N
N
N
N
b
a
14a, R = iPr (50%)
10
12a, R = Et (64%)
14b, R = CH2-Ph (50%)
12b, R= iPr (55%)
14c, R = CH2-CH2-N(CH3)2 (50%)
12c, R = Bu (67%)
12d, R = CH2-Ph (90%)
12e, R = CH2-CH2-Ph (90%)
NH
NH
H
O
R
NC
N
N
O
S
O
N
S
O
N
R
S
b
a
N
N
N
N
N
N
13a, R = Et (44%)
13b, R = iPr (40%)
13c, R = Bu (90%)
13d, R = CH2-Ph (90%)
11
15a, R = iPr (68%)
15b, R = CH2-Ph (53%)
15c, R = CH2-CH2-N(CH3)2 (40%)
Scheme 2. Reaction conditions: (a) NaOH, alcohol, rt, 15 min; (b) amine, THF, (MW) 80 ꢀC, 30 min.

1472
C. Loge´ et al. / European Journal of Medicinal Chemistry 43 (2008) 1469e1477
that bulky substituents have a favorable effect on a dual
CDK1/GSK-3 inhibitory activity.
suggests that this residue plays a significant role in the potency
of the compounds because such polar interactions are usually
strong. Second, we speculated that it may also play a role in
the dual CDK1/GSK-3 inhibitory activity displayed by these
compounds since Lys85 (Lys33 in CDK1) is a residue con-
served in all kinase enzymes and functionally important in ki-
nase catalysis [1]. Therefore, considering the conserved ranges
in CDK1/GSK-3 activities obtained for these molecules, we
can hypothesize that an identical interaction could take place
in the two enzymes.
In addition, for an explanation of the observed differences
in activity between imidates 12d, 13d and amidine analogues
14b, 15b, which adopted a same orientation in the cavity of
GSK-3b (not shown), molecular electrostatic potentials
(MEPs) were calculated and projected onto the electron den-
sity surface of the inhibitors (in their docked conformations).
Knowledge of MEPs is critically important when molecular
interactions are to be studied. The results of these calculations
are depicted in Fig. 2. Obviously, the molecular shape is rather
similar. However, the MEP of inhibitor 15b is quite electro-
positive near the NH of its amidine function (red area indi-
cated by an arrow), in contrast to the imidate analogue 13d,
which is having a relatively more electronegative potential
(blue region). It is probable that this charge distribution could
be affecting more intensely the binding within the ATP cavity
of CDK1 than in GSK-3b since amidine 15b exhibited a dra-
matic decrease of CDK1 inhibitory potency (IC50 ¼ 9 mM) as
compared to imidate 13d (IC50 ¼ 0.15 mM at CDK1). Taking
into account that the solvent effect was not considered, we
cannot discard the possibility of an unfavorable desolvation
with water during the binding process.
3.2. Molecular modeling studies
In attempts to understand the observed structureeactivity
relationships (SARs), we have carried out molecular modeling
studies using automated docking procedure into the ATP-bind-
ing site of GSK-3b in complex with the non-hydrolysable ATP
˚
analogue AMP-PNP at 1.80 A resolution (pdb code: 1J1B)
[13], following a flexible ligand/rigid receptor docking proto-
col, as described in Section 5.
The binding pose found by GOLD for imidate inhibitors
12cee and 13c,d is shown in Fig. 1. As can be seen, the dock-
ing program positioned these molecules in such a way that the
quinazoline nitrogen N-6 makes hydrogen bond with the back-
bone NH of residue Val135 in the hinge segment. This region
of protein kinases makes critical H-bonding contacts to the
vast majority of inhibitory molecules that have been published
to date. At least, one H-bond is formed in practically all
known kinase ligand structures in the Protein Data Bank
[14]. Interestingly, due to the bulkiness of the substituents at
the R position, these compounds may not fit into the hydro-
phobic backpocket of GSK-3b (a region which is not con-
served and used to gain affinity as well as selectivity [15]
but may form polar interactions between the imine nitrogen
atom and the side-chain amino group of Lys85. This interac-
tion drew our attention for two reasons. First, the model
4. Conclusion
In conclusion the rapid and efficient synthesis of novel se-
ries of 9-oxo-thiazolo[5,4-f]quinazoline-2-carbonitrile deriva-
tives was performed and optimized under microwave
irradiation. Most of the amidine and imidate derivatives are
potent and dual CDK1/GSK-3 inhibitors. Among these mole-
cules, compound 13d exhibits an efficient capacity to inhibit
both CDK1 and GSK-3 (IC50 values of 0.15 mM and
0.13 mM, respectively). This work confirms that this family
constitutes a promising scaffold from which more potent in-
hibitors could be designed.
5. Experimental
5.1. Chemistry
Commercial reagents were used as received without addi-
tional purification. Melting points were determined using a Ko-
¨
fler melting point apparatus and are uncorrected. IR spectra
were recorded on a PerkineElmer Paragon 1000PC instru-
ment. 1H and 13C NMR were recorded on a JEOL NMR
LA400 (400 MHz) spectrometer (Centre Commun d’Analyses,
´
Universite de la Rochelle); chemical shifts (d) are reported in
parts per million (ppm) downfield from tetramethylsilane
Fig. 1. Proposed binding mode for imidate inhibitors 12cee and 13c,d into the
ATP-binding site of GSK-3b starting from 1J1B (PDB code). The active site
pocket is highlighted in cyan (MOLCAD surface; program SYBYL 7.2).
The hydrogen bonds are indicated as yellow dotted lines. For interpretation
of the references to color in the text, the reader is referred to the web version
of this article.

C. Loge´ et al. / European Journal of Medicinal Chemistry 43 (2008) 1469e1477
1473
Fig. 2. Visualization of the molecular electrostatic potentials (MEPs) of 13d and 15b displayed onto electron density surfaces. Blue areas represent negative elec-
trostatic potentials; red areas represent positive values. (The calculations have been performed using program MOLCAD.) Arrows indicate noteworthy differences
in the charge distribution of the two congeners. For interpretation of the references to color in the text, the reader is referred to the web version of this article.
(TMS) which was used as an internal standard. Coupling con-
stants J are given in hertz. The mass spectra (HRMS) were re-
corded on a Varian MAT311 spectrometer in the «Centre
speed rotation, irradiation monitored by PC computer, infrared
measurement and continuous feedback temperature control.
Experiments may be performed at atmospheric pressure or
in a sealed tube in pressure-rated reaction tubes with continu-
ous pressure measurement.
´
Regional de Mesures Physiques de l’Ouest» (CRMPO), Uni-
versite de Rennes. Column chromatography was performed
´
by using Merck silica gel (70e230 mesh) at medium pressure.
Light petroleum refers to the fraction boiling point 40e60 ꢀC.
Other solvents were used without purification. Analytical thin
layer chromatography (TLC) was performed on Merck Kiesel-
gel 60 F254 aluminium backed plates. Focused microwave
irradiations were carried out with a Smith-SynthesizerÒ (Per-
sonal Chemistry AB now Biotage) or a CEM DiscoverÒ fo-
cused microwave reactor (300 W, 2450 MHz, monomode
system). The Smith-SynthetizerÒ was a single mode cavity,
producing controlled irradiation at 2450 MHz. Reaction tem-
perature and pressure were determined using the built-in, on-
line IR and pressure sensors. Microwave-assisted reactions
were performed in sealed Smith process vials (0.5e5 mL, total
volume 10 mL) under air with magnetic stirring. The software
algorithm regulates the microwave output power so that the se-
lected maximum temperature was maintained for the desired
reaction/irradiation time. After the irradiation period, the reac-
tion vessel was cooled rapidly to ambient temperature by com-
pressed air (gas-jet cooling). The minimal reaction times were
determined by performing sequential series of identical reac-
tions at constant temperature and with continuous heating,
but with different irradiation times. Completion of the reaction
was estimated by TLC after each individual heating period.
The DiscoverÒ focused microwave reactor (300 W,
2450 MHz, monomode system) has in situ magnetic variable
Spectral data for compound 1 is consistent with assigned
structure as previously described by Alexandre et al. [11].
5.1.1. Synthesis of N-substituted quinazolinones 2 and 3
Alkylating agent (6 mmol) was added dropwise to a stirred
suspension of quinazolinone 1 (5 mmol) and sodium hydride
(6 mmol; 60% dispersion in mineral oil) in DMF (3 mL).
The mixture was irradiated for 5 min in a sealed tube. The ir-
radiation was programmed to obtain a constant temperature
(140 ꢀC). The solvent was removed under reduced pressure,
and the residue was hydrolyzed with water and extracted
with ethyl acetate. The organic layers dried over magnesium
sulfate were evaporated in vacuo. The product was obtained
by purification by column chromatography with dichlorome-
thaneeethyl acetate (90/10) as eluent.
5.1.1.1. 3-Ethyl-6-nitro-3H-quinazolin-4-one (2). Obtained in
98% yield as a yellow solid (mp ¼ 156 ꢀC). Data supporting
its chemical structure are reported in Ref. [10].
5.1.1.2. 3-Isopropyl-6-nitro-3H-quinazolin-4-one (3). Ob-
tained in 90% yield as a yellow solid (mp ¼ 166 ꢀC), IR
(KBr) nmax/cmꢁ1 3093, 2979, 1912, 1668, 1621, 1568, 1530,
1
1467, 1340, 1276, 1176, 1139, 1062, 854, 752, 696, 627; H
NMR d (400 MHz, CDCl3) 1.53 (d, 6H, J ¼ 6.8 Hz, 2CH3),

1474
C. Loge´ et al. / European Journal of Medicinal Chemistry 43 (2008) 1469e1477
5.16e5.23 (m, 1H, CH), 7.83 (d, 1H, J ¼ 8.8 Hz, H8), 8.25 (s,
1H, H2), 8.54 (dd, 1H, J ¼ 2.9 Hz, J ¼ 8.8 Hz, H7), 9.18 (d,
1H, J ¼ 2.9 Hz, H5); 13C NMR d (100 MHz, CDCl3) 21.95,
46.93, 122.14, 123.64, 128.20, 129.02, 145.97, 146.61,
151.63, 159.64; MS (EI) Mþ: 233.
d (100 MHz, CDCl3) 21.99, 45.95, 103.75, 120.29, 121.85,
127.74, 140.78, 141.98, 144.51, 158.99; MS (EI) Mþ: 282.
5.1.4. General procedure for the synthesis of imino-1,2,3-
dithiazoles 8 and 9
A suspension of the bromo amine 6 or 7 (2.5 mmol), 4,5-di-
chloro-1,2,3-dithiazolium chloride (2.75 mmol) in dichloro-
methane (4 mL) was irradiated for 4 min in a sealed tube.
The irradiation was programmed to obtain a constant temper-
ature (80 ꢀC). After cooling at room temperature, pyridine
(5.5 mmol) was added. The resulting solution was dissolved
in ethyl acetate, washed with water, dried and concentrated un-
der reduced pressure. The crude residue was purified by col-
umn chromatography (dichloromethaneeethyl acetate: 90/
10) to afford the expected compound 8 or 9.
5.1.2. Synthesis of quinazolinones 4 and 5
A stirred mixture of 1 mmol of 2 or 3, 5 mmol of ammo-
nium formate and a catalytic amount of 10% palladium char-
coal in 20 mL of ethanol was irradiated for 15 min. The
irradiation was programmed to obtain a constant temperature
(80 ꢀC) with a maximal power output of 40 W. The catalyst
was removed by filtration. The resulting filtrate was dissolved
in ethyl acetate, washed with water, dried and concentrated un-
der reduced pressure. The amines 4 or 5 were obtained without
further purification.
5.1.4.1. 5-Bromo-6-[(4-chloro-5H-1,2,3-dithiazol-5-ylidene)-
amino]-3-ethyl-quinazolin-3H-4-one 8. Obtained in 45% yield
as an orange solid (mp ¼ 182 ꢀC). Data supporting its chemi-
cal structure are reported in Ref. [10].
5.1.2.1. 6-Amino-3-ethyl-3H-quinazolin-4-one 4. Obtained in
95% yield as a white solid (mp ¼ 168 ꢀC). Data supporting
its chemical structure are reported in Ref. [10].
5.1.4.2. 5-Bromo-6-[(4-chloro-5H-1,2,3-dithiazol-5-ylidene)-
amino]-3-isopropyl-quinazolin-3H-4-one 9. Obtained in 78%
yield as an orange solid (mp ¼ 210 ꢀC), IR (KBr) nmax/cmꢁ1
2978, 1664, 1596, 1533, 1457, 1373, 1254, 1156, 940, 864,
5.1.2.2. 6-Amino-3-isopropyl-3H-quinazolin-4-one 5. Ob-
tained in 85% yield as a white solid (mp ¼ 138 ꢀC), IR
(KBr) nmax/cmꢁ1 3221, 2977, 2368, 1652, 1496, 1460, 1352,
1316, 1276, 1252, 1176, 1096, 880, 749, 554; 1H NMR
d (400 MHz, CDCl3 þ D2O) 1.47 (d, 6H, J ¼ 6.8 Hz, 2CH3),
5.16e5.23 (m, 1H, CH), 7.10 (dd, 1H, J ¼ 2.4 Hz,
J ¼ 8.8 Hz, H7), 7.49 (d, 1H, J ¼ 2.4 Hz, H5), 7.53 (d, 1H,
J ¼ 8.8 Hz, H8), 7.96 (s, 1H, H2); 13C NMR d (100 MHz,
CDCl3) 21.96, 45.77, 108.89, 122.84, 122.91, 128.37,
140.18, 140.22, 145.86, 160.47; MS (EI) Mþ: 203.
1
813, 764, 663. H NMR d (400 MHz, CDCl3) 1.50 (d, 6H,
J ¼ 6.8 Hz, 2CH3), 5.16e5.23 (m, 1H, CH), 7.39 (d, 1H,
J ¼ 8.8 Hz, H7), 7.75 (d, 1H, J ¼ 8.8 Hz, H8), 8.12 (s, 1H,
H2); 13C NMR d (100 MHz, CDCl3) 22.00, 46.43, 113.07,
121.20, 124.13, 129.12, 143.61, 147.25, 147.40, 150.90,
158.95, 162.16. MS (EI) Mþ: 416.
5.1.5. Synthesis of thiazolo[5,4-f]quinazoline-2-carbonitriles
10 and 11
5.1.3. Synthesis of brominated quinazolinones 6 and 7
A solution of bromine (5.6 mmol) in dichloromethane
(5 mL) was added dropwise, under an inert atmosphere, to
a solution of amines 4 or 5 (5.6 mmol), in acetic acid
(30 mL). After 2 h of stirring at room temperature, the solvent
was removed in vacuo. Then the mixture was dissolved in
ethyl acetate and washed with sodium thiosulfate solution
(20 mL). The solvent was removed in vacuo and the crude
residue purified by column chromatography (dichloromethanee
ethyl acetate: 90/10) to afford the expected compound
6 or 7.
A suspension of imine 8 or 9 (1 mmol), copper iodide
(2 mmol) in pyridine (4 mL) was irradiated for 1 min in
a sealed tube. The irradiation was programmed to obtain a con-
stant temperature (160 ꢀC). After cooling, the mixture was dis-
solved in ethyl acetate, washed with sodium thiosulfate
solution (20 mL). The solvent was removed in vacuo and the
crude residue purified by column chromatography (dichloro-
methaneeethyl acetate: 80/20) to afford the expected
compound.
5.1.5.1. 8-Ethyl-9-oxo-8,9-dihydro-thiazolo[5,4-f]quinazoline-
2-carbonitrile 10. Obtained in 93% yield as a white solid
(mp > 260 ꢀC). Data supporting its chemical structure are re-
ported in Ref. [10].
5.1.3.1. 6-Amino-5-bromo-3-ethyl-3H-quinazolin-4-one 6. Ob-
tained in 93% yield as a red solid (mp ¼ 140 ꢀC). Data sup-
porting its chemical structure are reported in Ref. [10].
5.1.3.2. 6-Amino-5-bromo-3-isopropyl-3H-quinazolin-4-one
7. Obtained in 83% yield as a red solid (mp ¼ 140 ꢀC), IR
(KBr) nmax/cmꢁ1 3469, 3354, 3158, 2978, 1665, 1619, 1537,
5.1.5.2. 8-Isopropyl-9-oxo-8,9-dihydro-thiazolo[5,4-f]quinazo-
line-2-carbonitrile 11. Obtained in 71% yield as a white solid
(mp > 260 ꢀC), IR (KBr) nmax/cmꢁ1 2973, 2236, 1674, 1580,
1496, 1357, 1276, 1247, 1174, 839, 581; 1H NMR
d (400 MHz, CDCl3) 1.59 (d, 6H, J ¼ 6.8 Hz, 2CH3),
5.24e5.31 (m, 1H, CH), 8.00 (d, 1H, J ¼ 8.8 Hz, H4), 8.36
(s, 1H, H7), 8.53 (d, 1H, J ¼ 8.8 Hz, H5); 13C NMR
1
1482, 1414, 1339, 1236, 1096, 936, 828, 632, 548; H NMR
d (400 MHz, CDCl3 þ D2O) 1.46 (d, 6H, J ¼ 6.8 Hz, 2CH3),
5.17e5.24 (m, 1H, CH), 7.18 (d, 1H, J ¼ 8.8 Hz, H7), 7.50
(d, 1H, J ¼ 8.8 Hz, H8), 7.96 (s, 1H, H2); 13C NMR

C. Loge´ et al. / European Journal of Medicinal Chemistry 43 (2008) 1469e1477
1475
d (100 MHz, CDCl3) 22.18, 46.52, 114.59, 126.39, 129.41,
143.73, 147.07, 159.28, 176.49. MS (EI) Mþ: 270.
14.97, 35.06, 42.58, 67.77, 116.82, 126.54, 126.76, 126.52,
129.09, 129.81, 133.14, 138.03, 146.37, 147.82, 151.80,
159.57, 161.23, 162.06. Found [MeC8H8]þ: 274.0517,
C20H18N4O2S requires 274.0524.
5.1.6.2.2. 8-Isopropyl-9-oxo-8,9-dihydro-thiazolo[5,4-f]qui-
nazoline-2-carboximidic acid ethyl ester 13a. Obtained in
5.1.6. Methods for the synthesis of 2-substituted thiazolo-
[5,4-f]quinazolinones
5.1.6.1. Synthesis of imidates. A stirred mixture of carbonitrile
10 or 11 (0.5 mmol) and 2.5 N NaOH (0.5 mmol) in ethanol
(5 mL) under argon was stirred at room temperature for
15 min. The resulting precipitate was collected, washed with
water and dried to give the imidate 12 or 13 as a white crys-
talline powder.
5.1.6.1.1. 8-Ethyl-9-oxo-8,9-dihydro-6-thiazolo[5,4-f]qui-
nazoline-2-carboximidic acid ethyl ester 12a. Obtained in
64% yield as a white solid (mp ¼ 232 ꢀC). Data supporting
its chemical structure are reported in Ref. [10].
44% yield as a white solid (mp ¼ 240 ꢀC), IR (KBr) nmax
1
1072, 832, 692, 511; H NMR d (400 MHz, CDCl3) 1.49 (t,
/
cmꢁ1 2985, 2322, 1674, 1586, 1500, 1353, 1284, 1120,
3H, J ¼ 6.8 Hz, CH3), 1.58(d, 6H, J ¼ 6.8 Hz, 2CH3), 4.51
(q, 2H, J ¼ 6.8 Hz, CH2), 5.23e5.30 (m, 1H, CH), 7.90 (d,
1H, J ¼ 8.8 Hz, H4), 8.29 (s, 1H, H7), 8.44 (d, 1H,
J ¼ 8.8 Hz, H5), 8.96 (s, 1H, NH). Found Mþ: 316.0974,
C15H16N4O2S requires 316.0994.
5.1.6.2.3. 8-Isopropyl-9-oxo-8,9-dihydro-thiazolo[5,4-f]qui-
nazoline-2-carboximidic acid isopropyl ester 13b. Obtained in
5.1.6.1.2. 8-Ethyl-9-oxo-8,9-dihydro-thiazolo[5,4-f]quina-
zoline-2-carboximidic acid isopropyl ester 12b. Obtained in
40% yield as a white solid (mp ¼ 140 ꢀC), IR (KBr) nmax
/
cmꢁ1 3278, 2924, 2852, 1655, 1587, 1496, 1452, 1353,
1323, 1317, 1280, 1248, 1160, 110, 1072, 832; 1H NMR
d (400 MHz, CDCl3) 1.46 (d, 6H, J ¼ 6.4 Hz, 2CH3), 1.57
(d, 6H, J ¼ 6.8 Hz, 2CH3), 5.22e5.28 (m, 1H, CH), 5.37e
5.40 (m, 1H, CH), 7.89 (d, 1H, J ¼ 8.8 Hz, H4), 8.29 (s, 1H,
H7), 8.45 (d, 1H, J ¼ 8.8 Hz, H5), 8.95 (s, 1H, NH). Found
Mþ: 330.1147, C16H18N4O2S requires 330.1150.
55% yield as a white solid (mp ¼ 210 ꢀC), IR (KBr) nmax
/
cmꢁ1 3267, 2984, 1662, 1586, 1497, 1366, 1350, 1337,
1317, 1260, 1144, 1053, 896; 1H NMR d (400 MHz,
CDCl3 þ D2O) 1.46 (d, 6H, J ¼ 6.3 Hz, 2CH3), 1.51 (t, 3H,
J ¼ 7.3 Hz, CH3), 4.21 (q, 2H, J ¼ 7.3 Hz, CH2), 5.37e5.40
(m, 1H, CH), 7.90 (d, 1H, J ¼ 8.8 Hz, H4), 8.23 (s, 1H, H7),
8.45 (d, 1H, J ¼ 8.8 Hz, H5). Found Mþ: 316.0974,
C15H16N4O2S requires 316.0994.
5.1.6.2.4. 8-Isopropyl-9-oxo-8,9-dihydro-thiazolo[5,4-f]qui-
nazoline-2-carboximidic acid butyl ester 13c. Obtained in
5.1.6.1.3. 8-Ethyl-9-oxo-8,9-dihydro-thiazolo[5,4-f]quina-
zoline-2-carboximidic acid butyl ester 12c. Obtained in 67%
yield as a white solid (mp ¼ 160 ꢀC), IR (KBr) nmax/cmꢁ1
3268, 2937, 2877, 1660, 1586, 1498, 1451, 1370, 1350,
90% yield as a white solid (mp ¼ 164 ꢀC), IR (KBr) nmax
/
cmꢁ1 3274, 2924, 2853, 1656, 1583, 1499, 1354, 1317,
1277, 1248, 1132, 118, 1071, 839; 1H NMR d (400 MHz,
CDCl3) 1.02 (t, 3H, J ¼ 7.2 Hz, CH3), 1.54e1.58 (m, 2H,
CH3), 1.58 (d, 6H, J ¼ 6.8 Hz, 2CH3), 1.83e1.87 (m, 2H,
CH2), 4.43e4.46 (m, 2H, CH2), 5.26e5.30 (m, 1H, CH),
7.90 (d, 1H, J ¼ 8.8 Hz, H4), 8.30 (s, 1H, H7), 8.45 (d, 1H,
J ¼ 8.8 Hz, H5), 8.94 (s, 1H, NH). Found Mþ: 344.1316,
C17H20N4O2S requires 344.1307.
1
1323, 1256, 1131, 1066; H NMR d (400 MHz, CDCl3) 1.02
(t, 3H, J ¼ 7.4 Hz, CH3), 1.51 (t, 2H, J ¼ 7.6 Hz, CH3), 1.57
(q, 2H, J ¼ 7.6 Hz, CH2), 1.81e1.88 (m, 2H, CH2), 4.21 (q,
2H, J ¼ 7.4 Hz, CH2), 4.51 (t, 2H, J ¼ 7.6 Hz, CH2), 7.91 (d,
1H, J ¼ 8.8 Hz, H4), 8.23 (s, 1H, H7), 8.46 (d, 1H,
J ¼ 8.8 Hz, H5), 8.96 (s, 1H, NH). Found Mþ: 330.1143
C16H18N4O2S requires 330.1150.
5.1.6.2.5. 8-Isopropyl-9-oxo-8,9-dihydro-thiazolo[5,4-f]qui-
nazoline-2-carboximidic acid benzyl ester 13d. Obtained in
90% yield as a white solid (mp ¼ 214 ꢀC), IR (KBr) nmax
/
5.1.6.2. 8-Ethyl-9-oxo-8,9-dihydro-thiazolo[5,4-f]quinazoline-
2-carboximidic acid benzyl ester 12d. Obtained in 90% yield
as a white solid (mp ¼ 210 ꢀC), IR (KBr) nmax/cmꢁ1 3290,
2971, 1663, 1585, 1497, 1337, 1262, 1149, 1118, 1081, 835,
cmꢁ1 3207, 2930, 1660, 1584, 1498, 1454, 1354, 1277,
1251, 1141, 1112, 1054, 830, 751, 696; 1H NMR
d (400 MHz, CDCl3) 1.57 (d, 6H, J ¼ 7.0 Hz, 2CH3), 5.23e
5.30 (m, 1H, CH), 5.52 (s, 2H, CH2), 7.37e7.54 (m, 5H,
Har), 7.91 (d, 1H, J ¼ 8.8 Hz, H4), 8.30 (s, 1H, H7), 8.46 (d,
1H, J ¼ 8.8 Hz, H5), 9.13 (s, 1H, NH). Found Mþ:
378.1137, C20H17N4O2S requires 378.1150.
1
731; H NMR d (400 MHz, CDCl3) 1.50 (t, 3H, J ¼ 7.2 Hz,
CH3), 4.20 (q, 2H, J ¼ 7.2 Hz, CH2), 5.51 (s, 2H, CH2),
7.37e7.54 (m, 5H, Har), 7.90 (d, 1H, J ¼ 8.8 Hz, H4), 8.22
(s, 1H, H7), 8.45 (d, 1H, J ¼ 8.8 Hz, H5), 9.13 (s, 1H, NH).
Found Mþ: 364.1000, C19H15N4O2S requires 364.0994.
5.1.6.2.1. 8-Ethyl-9-oxo-8,9-dihydro-thiazolo[5,4-f]quina-
zoline-2-carboximidic acid phenyl ethyl ester 12e. Obtained
5.1.6.3. Synthesis of amidines. A stirred mixture of carboni-
trile 10 or 11 (1 mmol) and amine (5 mmol) in dry THF
(10 mL) under argon was irradiated in a sealed tube for
30 min. The irradiation was programmed to obtain a constant
temperature (80 ꢀC). The mixture was dissolved in dichloro-
methane, washed with water. The solvent was removed in va-
cuo and the crude residue purified by column chromatography
(dichloromethaneemethanol: 90/10) to afford the amidine 14
or 15.
in 90% yield as a white solid (mp ¼ 200 ꢀC), IR (KBr) nmax
/
cmꢁ1 3207, 2930, 1660, 1584, 1498, 1454, 1354, 1277,
1251, 1141, 1112, 1054, 830, 751, 696; 1H NMR
d (400 MHz, CDCl3) 1.52 (t, 3H, J ¼ 7.2 Hz, CH3), 3.19 (t,
2H, J ¼ 6.8 Hz, CH2), 4.22 (q, 2H, J ¼ 7.2 Hz, CH2), 4.66 (t,
2H, J ¼ 6.8 Hz, CH2), 7.32e7.38 (m, 5H, Har), 7.89 (d, 1H,
J ¼ 8.8 Hz, H4), 8.23 (s, 1H, H7), 8.44 (d, 1H, J ¼ 8.8 Hz,
H5), 8.99 (s, 1H, NH); 13C NMR d (100 MHz, CDCl3)
5.1.6.3.1.
N-Isopropyl-8-ethyl-9-oxo-8,9-dihydrothiazolo
[5,4-f]quinazoline-2-carboxamidine 14a. Obtained in 50%

1476
C. Loge´ et al. / European Journal of Medicinal Chemistry 43 (2008) 1469e1477
yield as white solid (mp ¼ 197 ꢀC), IR (KBr) nmax/cmꢁ1 3394,
3278, 2965, 2932, 1653, 1619, 1587, 1522, 1451, 1365, 1184,
1159, 828; 1H NMR d (400 MHz, CDCl3 þ D2O) 1.34 (d, 6H,
J ¼ 6.4 Hz, 2CH3), 1.51 (t, 3H, J ¼ 7.4 Hz, CH3), 3.87e3.90
(m, 1H, CH), 4.20 (q, 2H, CH2), 7.85 (d, 1H, J ¼ 8.8 Hz,
H4), 8.20 (s, 1H, H7), 8.36 (d, 1H, J ¼ 8.8 Hz, H5). Found
[M]þ: 315.1169, C15H17N5OS requires 315.1154.
ATP and appropriate protein substrates (histone H1 for
CDKs, GS-1 peptide for GSK-3). IC50 values were determined
from doseeresponse curves and are provided in Table 1.
5.3. Molecular modeling
5.3.1. Docking studies
5.1.6.3.2. N-Benzyl-8-ethyl-9-oxo-8,9-dihydrothiazolo[5,4-
f]quinazoline-2-carboxamidine 14b. Obtained in 50% yield
as white solid (mp ¼ 128 ꢀC), IR (KBr) nmax/cmꢁ1 3447,
3324, 2932, 1646, 1588, 1502, 1450, 1344, 1261, 1165,
Molecular modeling studies were performed with the com-
mercially available SYBYL 7.2 software package running on
a Silicon Graphics Octane workstation [19]. The three-dimen-
sional structure of all compounds were built from a standard
fragments library, and their geometry was subsequently opti-
mized using the Tripos force field [20] including the electro-
static term calculated from Gasteiger and Huckel atomic
¨
charges. Powell’s method available in Maximin2 procedure
was used for energy minimization until the gradient value
1
1106, 967, 823, 742; H NMR d (400 MHz, CDCl3 þ D2O)
1.50 (t, 3H, J ¼ 7.2 Hz, CH3), 4.20 (q, 2H, CH2), 4.58 (s,
2H, CH2), 7.31e7.40 (m, 5H, CHar), 7.85 (d, 1H,
J ¼ 8.8 Hz, H4), 8.20 (s, 1H, H7), 8.36 (d, 1H, J ¼ 8.8 Hz,
H5). Found [M]þ: 363.1144, C19H17N5OS requires 363.1154.
5.1.6.3.3. N-(2-Dimethylamino-ethyl)-8-ethyl-9-oxo-8,9-di-
˚
was smaller than 0.001 kcal/mol A. Atomic coordinates for
the GSK-3b in complex with AMP-PNP have been deposited
˚
in the Protein Data Bank with resolution of 1.80 A (PDB code:
hydrothiazolo[5,4-f]quinazoline-2-carboxamidine
14c. Ob-
tained in 50% yield as a white solid (mp ¼ 144 ꢀC). Data
supporting its chemical structure are reported in Ref. [10].
5.1.6.3.4. N-Isopropyl-8-isopropyl-9-oxo-8,9-dihydro[1,3]-
thiazolo[5,4-f]quinazoline-2-carboxamidine 15a. Obtained in
1J1B) [13] and was used as the target structure for docking.
The original ligand as well as the water molecules were re-
moved from the coordinates set. Flexible docking of mole-
cules into the ATP-binding site was performed using GOLD
software [21]. For each compound, the most stable docking
model was selected according to the best scored conformation
predicted by the GoldScore [21] and X-Score [22] scoring
functions. The complexes were energy-minimized using Po-
well’s method available in Maximin2 procedure with the Tri-
pos force field and a dielectric constant of 4.0 until the
68% yield as white solid (mp ¼ 217 ꢀC), IR (KBr) nmax
/
cmꢁ1 3389, 3276, 2979, 2925, 1649, 1621, 1584, 1519,
1453, 1349, 1174, 827, 808; 1H NMR d (400 MHz,
CDCl3 þ D2O) 1.38 (d, 6H, J ¼ 6.4 Hz, 2CH3), 1.57 (d, 6H,
J ¼ 7.2 Hz, 2CH3), 4.06e4.08 (m, 1H, CH), 5.26e5.31 (m,
1H, CH), 7.88 (d, 1H, J ¼ 8.8 Hz, H4), 8.29 (s, 1H, H7), 8.38
(d, 1H, J ¼ 8.8 Hz, H5). Found [M]þ: 329.1295,
C16H19N5OS requires 329.1310.
˚
gradient value reached 0.1 kcal/mol A.
5.1.6.3.5.
thiazolo[5,4-f]quinazoline-2-carboxamidine 15b. Obtained in
53% yield as white solid (mp > 240 ꢀC), IR (KBr) nmax
N-Benzyl-8-isopopyl-9-oxo-8,9-dihydro[1,3]
5.3.2. Molecular electrostatics potentials (MEPs)
/
Molecular electrostatic potentials (MEPs) for the most ac-
tive compound imidate 13d and its amidine analogue 15b
(in their docked conformations) were displayed using the pro-
gram MOLCAD in SYBYL 7.2. Partial atomic charges were
computed with the quantum chemistry package program MO-
PAC version 6.0 [23] using the semi-empirical molecular or-
bital method AM1. Partial charges were derived using
Mulliken population analysis. Electrostatic potential was map-
ped onto the electron density surface for both inhibitors.
cmꢁ1 3456, 3317, 3176, 2981, 1651, 1584, 1495, 1451,
1347, 1279, 1174, 1092, 824, 738, 697; 1H NMR
d (400 MHz, DMSO-d6 þ D2O) 1.50 (d, 6H, J ¼ 7.2 Hz,
2CH3), 3.15 (s, 2H, CH2), 5.04e5.08 (m, 1H, CH), 7.24e
7.47 (m, 5H, Har), 7.83 (d, 1H, J ¼ 8.8 Hz, H4), 8.42 (d, 1H,
J ¼ 8.8 Hz, H5), 8.59 (s, 1H, H7). Found [M]þ: 377.1330,
C20H19N5OS requires 377.1310.
5.1.6.3.6. N-(2-Dimethylamino-ethyl)-8-isopopyl-9-oxo-8,9-
dihydro[1,3]thiazolo[5,4-f]quinazoline-2-carboxamidine 15c.
Obtained in 40% yield as a white solid (mp ¼ 148 ꢀC), IR
(KBr) nmax/cmꢁ1 2978, 2380, 2323, 1656, 1588, 1348, 1281,
Acknowledgements
1021, 831, 732, 634, 540; 1H NMR
d (400 MHz,
´
We thank the ‘‘Comites de Charente et de Charente-Maritime
CDCl3 þ D2O) 1.55 (d, 6H, J ¼ 6.8 Hz, 2CH3), 2.50 (s, 6H,
2CH3), 2.89 (s, 2H, CH2), 3.60 (s, 2H, CH2), 5.27e5.30 (m,
1H, CH), 7.84 (d, 1H, J ¼ 8.8 Hz, H4), 8.26 (s, 1H, H7),
8.36 (d, 1H, J ¼ 8.8 Hz, H5). Found [MeC4H8N]þ:
288.0943, C17H22N6OSeC4H8N requires 288.0919.
de la Ligue Nationale Contre le Cancer’’ for financial support.
The work presented here was also supported by a grant from
the EEC (FP6-2002-Life Sciences & Health, PRO-KINASE
´
Research Project) (L.M.) and the Canceropole Grand-Ouest.
ˆ
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