Synthesis, biological evaluation and molecular modelling studies of 4-anilinoquinazoline derivatives as protein kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.01.044

A series of novel 4-anilinoquinazoline derivatives (3a-3j) has been synthesized and evaluated as potential inhibitors for protein kinases implicated in Alzheimer's disease. Among all the synthesized compounds, compound 3e (N-(3,4-dimethoxyphenyl)-6,7-dimethoxyquinazolin-4-amine) exhibited the most potent inhibitory activity against CLK1 and GSK-3α/β kinase with IC₅₀ values of 1.5 μM and 3 μM, respectively. Docking studies were performed to elucidate the binding mode of the compounds to the active site of CLK1 and GSK-3β. The results of our study suggest that compound 3e may serve as a valuable template for the design and development of dual inhibitors of CLK1 and GSK-3α/β enzymes with potential therapeutic application in Alzheimer's disease.

Full text of DOI:10.1016/j.bmc.2014.01.044

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Bioorganic & Medicinal Chemistry 22 (2014) 1909–1915
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, biological evaluation and molecular modelling studies
of 4-anilinoquinazoline derivatives as protein kinase inhibitors
Digambar Kumar Waiker a, , Chandrabose Karthikeyan a, , Vasanthanathan Poongavanam b,
Jacob Kongsted b, Olivier Lozach c, Laurent Meijer c,d, Piyush Trivedi a,
⇑
a School of Pharmaceutical Sciences, Rajiv Gandhi Proudyogiki Vishwavidyalaya, Airport Bypass Road, Gandhi Nagar, Bhopal (MP) 462036, India
b Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense M, Denmark
c Protein Phosphorylation and Human Disease Group, USR3151, Station Biologique, B.P. 74, 29682 Roscoff cedex, Bretagne, France
d ManRos Therapeutics, Centre de Perharidy, 29680 Roscoff, Bretagne, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 4-anilinoquinazoline derivatives (3a–3j) has been synthesized and evaluated as
potential inhibitors for protein kinases implicated in Alzheimer’s disease. Among all the synthesized
compounds, compound 3e (N-(3,4-dimethoxyphenyl)-6,7-dimethoxyquinazolin-4-amine) exhibited the
Received 17 December 2013
Revised 22 January 2014
Accepted 23 January 2014
Available online 31 January 2014
most potent inhibitory activity against CLK1 and GSK-3
M, respectively. Docking studies were performed to elucidate the binding mode of the compounds
to the active site of CLK1 and GSK-3b. The results of our study suggest that compound 3e may serve as
a/b kinase with IC50 values of 1.5 lM and
3
l
Keywords:
a valuable template for the design and development of dual inhibitors of CLK1 and GSK-3
with potential therapeutic application in Alzheimer’s disease.
a/b enzymes
Anilino quinazolines
Protein kinase inhibitor
CLK1 kinase
Ó 2014 Elsevier Ltd. All rights reserved.
Docking studies
1. Introduction
In AD, aberrant phosphorylation of tau, an alternatively spliced
microtubule-binding protein, is believed to contribute to neurode-
generation.6 Hyperphosphorylation of tau leads to loss of normal
tau functioning and attenuates the stability of neuronal microtu-
bules.7,8 In addition, tau hyperphosphorylation is also associated
with aggregation of the protein into neurofibrillary tangles, contrib-
uting to neurofibrillary degeneration, neuronal death, and demen-
tia severity.8,9 A plethora of evidence recorded in the literature
suggests that targeting tau phosphorylation through inhibition of
protein kinases could represent a valid therapeutic approach to
reduce tau aggregation and associated neuronal death in AD and
The significant increase in the number of incidence of
Alzheimer’s disease (AD) especially in developed and developing
countries is a matter of serious concern. It is predicted that 35
million people are affected by dementia, mainly AD, worldwide
and that it will be tripled by the year 2050.1 Current treatments
for AD provide only modest symptomatic relief and there is
currently a great need for ‘disease modifying’ agents that either
slow the course of the disease or fully prevent or delay the disease
in susceptible individuals.
Protein phosphorylation is the most common post-translational
mechanism used by cells to regulate enzymes and structural
proteins. The process is controlled by more than 500 protein
kinases and 80 protein phosphatases.2 Since protein phosphoryla-
tion is significant for maintenance and regulation of many cellular
and physiological processes, abnormal phosphorylation turns out
to be a cause or consequence of numerous human diseases such
as cancer, diabetes, inflammation, and neurodegenerative dis-
eases.3–5 Hence, pharmacological inhibitors of kinases have become
a major interest in drug discovery.
other neurodegenerative ‘taupathies’.1,10 CDK5/p25, CK1d/
GSK-3 /b, DYRK1A, and CLK1 are involved in the two key molecular
e,
a
features of AD, production of amyloid-b peptides (extracellular
plaques) and hyperphosphorylation of the microtubule-binding
protein tau (intracellular neurofibrillary tangles).11–13 Conse-
quently, small molecule inhibitors acting on these kinases could
be of great therapeutic value in AD and related taupathies.
The quinazoline scaffold has been extensively studied for their
many pharmacological properties,14 which includes anti-cancer,15
anti-inflammatory,16–18 anti-bacterial,19,20 analgesic,16,18 anti-
viral,21 anti-tubercular,22 anti-malarial,23 anti-hypertensive,24
anti-obesity,25 anti-diabetic26 activities. Importantly, 4-anilinoqui-
nazolines have emerged as a versatile template for inhibition of a
diverse range of protein kinases.27 This pharmacophore is present
⇑
Corresponding author. Tel.: +91 755 2678883; fax: +91 755 2742001.
E-mail address: piyush.trivedi@rgtu.net (P. Trivedi).
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2014.01.044
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

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D. K. Waiker et al. / Bioorg. Med. Chem. 22 (2014) 1909–1915
Table 1
in approximately 80% of all ATP-competitive kinases inhibitors that
have received approval for treatment of cancer.28 The spectacular
therapeutic and financial success of 4-anilinoquinazoline based ki-
nase inhibitors prompted us investigate their utility as a lead for
Structural data and kinase inhibitory activity of 6,7-dimethoxy-N-phenylquinazolin-
4-amines
R
development of potent inhibitors of CDK5/p25, CK1d/e, GSK-3a/b,
HN
DYRK1A, and CLK1 enzymes. With this purview, the present work
describes synthesis and evaluation of new diversely substituted
4-anilinoquinazolines for inhibitory potency against a panel of five
H3CO
N
H3CO
N
protein kinases (CDK5/p25, CK1d/e, GSK-3a/b, DYRK1A, and CLK1).
(3a-3j)
2. Results and discussion
2.1. Chemistry
Compound
R
IC50 (lM)
CDK5 CK1 CLK1 DYRK1A
rat
GSK-3a/
b
3a
3b
3c
3d
3e
H
3-CF3
>10
>10
>10
>10
>10
>10 P10 >10
>10
>10
>10
>10
3
The 6,7-dimethoxy-N-phenylquinazolin-4-amines (3a–3j) were
synthesized in excellent yields by the reaction of 4-chloro-6,7-
dimethoxy quinazoline with aniline substrates under the condi-
tions illustrated in Scheme 1.29 The structures of the synthesized
compounds were assigned based on their elemental analysis and
spectral data. For example 1H NMR of 3b shows a singlet for the
NH proton at d 11.50 and –OCH3 protons at d = 4.05 and 4.09 (3H
for each) and the aromatic protons were observed as three singlets
at d = 7.40, 8.86, 8.36 (1H for each singlet) and two doublets at
d = 7.59 and 8.08 (with 1H and 2H) and a triplet at d = 7.67 (with
1H) respectively. Similarly, IR Spectra of 3b shows characteristic
peaks at 3340 (NH stretching), 3250 (C@N stretching), 3075 (Ar
C–H stretching), 1637 (Ar –C–C– stretching), 1280 (–O–CH3
stretch), 1068 (–O– stretching), 784 (–CF3 stretching). The Molec-
ular ion peak (M+H) for compound 3b was observed at m/z 282.2 in
the mass spectra.
>10 >10
>10 >10
>10
>10
3,4-Dimethyl
4-COCH3
3,4-
Dimethoxy
4-OCH3
4-NHCOCH3
3-Cl, 4-F
4-CF3
>10 P10 >10
>10 1.5
P10
3f
3g
3h
3i
>10
>10
>10
>10
>10
>10 >10
>10 >10
>10 7.6
>10 >10
>10 >10
>10
>10
>10
>10
>10
0.52
>10
>10
>10
>10
>10
0.005
3j
4-OCF3
—
6BIOa
0.083 1.9
2.1
a
Positive control.
moiety shows less than 5
(IC50 = 1.5 M) and GSK-3 /b (IC50 = 3
compound 3h with 3-fluoro and 4-chloro substitution in the aryl
ring exhibited a 5 fold reduced inhibition on CLK1 (IC50 = 7.6 M)
and no inhibition on GSK-3 /b.
l
M inhibition towards both CLK1
l
a
lM) enzymes. Surprisingly,
l
a
2.2. Biological activity
2.3. Molecular modeling studies
The synthesized 6,7-dimethoxy-N-phenylquinazolin-4-amines
(3a–3j) were tested for their potential inhibitory effect on five dif-
Molecular modeling studies were performed in order to validate
the kinase inhibitory profile shown by 4-anilinoquinazolines and
provide better insight into the binding mode of this family of com-
pounds to the CLK1 and GSK-3b kinases. To this end, compounds
3e and 3h were docked into the CLK1 and GSK-3b ATP binding site.
The crystallographic structures of the CLK1 enzyme in complex
with debromohymenialdisine, DBHD, (PDB ID: 1Z57) and GSK-3b
in complex with 5-aryl-4-carboxamide-1,3-oxazoles (PDB ID:
4AFJ) were used as protein models for the InducedFit docking
(IFD) experiments, which take into account both receptor and li-
gand flexibility during the docking.
In order to determine whether a docking scheme is applicable
to a given system, initially, the crystal structure-based binding
pose observed for the bound ligand should be reproduced by re-
docking. To this end DBHD was redocked into the receptor with a
root-mean-square-deviation of 0.30 Å. In addition to the bound li-
gand (DBHD), hymenialdisine (HD, a known CLK1 inhibitor and a
bromo derivative of DBHD) was also considered in the IFD experi-
ments in order to compare the predicted binding mode with that of
the synthesized compounds (e.g., 3e and 3h). The active site of hu-
man CLK1 has previously been well characterized as being narrow
and mainly formed by the backbone of residues PHE172, LYS191,
PHE241, GLU242, LEU243, LEU244, ASN293, VAL324 and ASP325.
The protein bound water molecules also play a key role in stabiliz-
ing the protein–inhibitor complex. First, we investigated the bind-
ing mode of hymenialdisine using IFD. It is clear from Figure 2A
that the HD binding mode is similar to that of DBHD and that
the nitrogen atoms of HD are involved in hydrogen bonding with
the active site water molecules and side chains of LEU244,
GLU242, ASP325 and LYS191, as reported.30 It has previously been
reported that HD interact through two main-chain hydrogen bonds
ferent kinases namely CDK5/p25 (CDK5/p25), CK1d/
e (casein ki-
nase 1), GSK-3 /b (Glycogen Synthase Kinase 3 /b), DYRK1A
a
a
(dual-specificity, tyrosine phosphorylation regulated kinase) and
CLK1 (cdc2-like kinase 1). All compounds were first tested at a final
concentration of 10
tion were considered as inactive (IC50 >10
playing more than 50% inhibition at 10 M were next tested over
a wide range of concentrations (usually 0.01–10 M) and IC50 val-
lM. Compounds showing less than 50% inhibi-
lM). Compounds dis-
l
l
ues were determined from the dose response curves (Sigma-Plot).
The results of the in vitro kinase assay are summarized in Table 1.
The results of kinase inhibitory assays demonstrated that none
of the synthesized anilino quinazolines showed any inhibitory
activity against CDK5/p25, DYRK1A and CK1d/e at the maximum
concentration tested (10 M). The 4-anilinoquinazolines appeared
l
to be most effective towards CLK1 as four compounds (3a, 3b, 3e
and 3h) showed inhibitory activity on the enzyme. Two among
the 10 anilinoquinazolines synthesized (compounds 3e and 3h)
showed significant inhibitory potency against CLK1 at less than
10 lM concentrations (Fig. 1). It is noteworthy that compound 3e
bearing 3, 4 dimethoxy substitution on the aryl ring of the aniline
R
Cl
HN
H3CO
H3CO
(i)
H3CO
H3CO
N
R
N
N
N
NH2
(3a-j)
Reagents and conditions: (i) Isopropanol, 900C, 2-3 hrs
Scheme 1. Synthesis of 6,7-dimethoxy-N-phenylquinazolin-4-amines (3a–3j).

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D. K. Waiker et al. / Bioorg. Med. Chem. 22 (2014) 1909–1915
1911
Figure 1. Kinase activity (%) of average max activity for compound 3e and 3h.
with the GLU242, LEU243 and LEU244 residues and one hydrogen
bond through a water molecule. A similar interaction pattern be-
tween the protein–water–ligand was observed from the IFD exper-
iment (–OLigand–LEU244–H2O–NHLigand), this observation being in
good agreement with the previously reported binding mode of
HD and DBH (see the protein–ligand interaction diagram provided
in the Supplementary information section).
GLU292, ASN 293 in the kinase. The anilino ‘NH’ group of com-
pound 3e formed a hydrogen bond with LEU241 and the quinazo-
line nitrogen (N3) formed a water mediated hydrogen bond with
LEU167 of the kinase. The oxygen of the dimethoxy substitution
on the anilino phenyl ring also formed hydrogen bonding with
the GLU292 and ASN293 residues through a network of water mol-
ecules. Overall, compared to 3h, compound 3e strongly binds with
residues through various hydrogen-bonding network. This obser-
vation is also reflected from the IFD scores: 3e (ꢀ12.76), 3h
(ꢀ10.7) and HD (ꢀ10.92).
Compounds 3e (IC50 = 1.5 lM) and 3h (IC50 = 7.5 lM) were
shown to be strong inhibitors against human CLK1 in our screening
experiments. To investigate a more realistic binding mode of these
compounds and how well these compounds are ranked according
to the docking fitness function in respect to inhibition, we ran
IFD experiments and analyzed the various docking poses. As shown
in Figure 2B and C, both compounds bind to the ATP binding site of
the kinase through hydrogen bonding and extensive hydrophobic
interactions. The quinazoline ring of both compounds 3e and 3h
is placed above the hinge region of the kinase facilitating hydrogen
bonding between the quinazoline nitrogen (N1) and LEU241 which
is deemed to be important for inhibitor potency. Compound 3e
exhibited four hydrogen bonds with amino acid residues LEU241,
Among the synthesized compounds, only 3h is active against
GSK-3
activity pattern of compound 3h and 3e (active against CLK1 and
inactive against GSK-3 /b) in terms of its binding mode in GSK-
a/b with inhibitory potency of 3 lM. To differentiate the
a
3b, we performed IFD experiments. From the binding pose analysis
(Fig. 3A), as expected, both compounds show different interaction
patterns with counter partner although both molecules share the
common scaffold. The amino group between two aromatic systems
of compound 3h is strongly interacting with the backbone carbonyl
group of VAL135 (distance 2.7 Å) as previously observed for

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D. K. Waiker et al. / Bioorg. Med. Chem. 22 (2014) 1909–1915
Figure 2. Comparison of the binding mode of bound ligand (A) with the active compounds 3e (B), 3h (C) in the CLK1 active site. Important residues are highlighted (cyan
stick) including bound waters (red sphere). On the right panel a schematic diagram of the protein–ligand interaction is shown for the bound ligand and the active compounds
3e and 3h.
pyridine derivatives for GSK-3b.31,32 This backbone interaction
facilitates a -cation interaction between ARG141 and the phenyl
establishes hydrogen bonding with phenolic hydroxyl group on
TYR 134.
p
ring of 3h, which was not observed for other compounds from this
family for example, 3e. The close proximity (distance 2.8 Å) of the
quinazoline ring nitrogen (N3) to amido ‘NH’ of VAL135 suggests
the likelihood of hydrogen bonding interaction between them.
Moreover, the 3 methoxy group on the anilino phenyl ring also
On the other hand, the binding pose of 3e is slightly different
compared to 3h, meaning that the whole molecule is slightly down
posed (Fig. 3B). Moreover the NH (4-amino) flipped to the other
side of VAL135 (with a distance of 5.7 Å), which may abolish
the GSK-3b inhibitory activity. In addition, no significant polar