Synthesis and optimization of an original V-shaped collection of 4-7-disubstituted Pyrido[3,2-d]pyrimidines as CDK5 and DYRK1A inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.04.055

We here report the synthesis and biological evaluation of an original collection of 4,7-disubstituted pyrido[3,2-d]pyrimidines designed as potential kinase inhibitors. The collection was generated from a single starting material, 4,7-dichloropyrido[3,2-d]pyrimidine, which afforded the final compounds after two steps: a sequential or one-pot sequence including selective cross coupling reactions in C-4, followed by the second cross-coupling in C-7. In position C-4, a Suzuki-Miyaura type reaction led to monosubstituted derivatives whereas in position C-7, synthesis was achieved via a Suzuki or a Buchwald type reaction using commercially available or undescribed boron derivatives. The biological activity of the V-shaped family was measured in protein kinase assays. The structure activity relationship (SAR) revealed that some compounds selectively inhibited DYRK1A and CDK5 without affecting GSK3. Docking studies furnished possible explanations that correlate with the SAR data. The most active compound on the two biological targets was 27 which exhibited the following IC ₅₀: 110 nM for CDK5, 24 nM for DYRK1A and only 1.2 μM for GSK3. In the C-7 amino subfamily, the best derivative was indubitably compound 48 which led to a near selective action on DYRK1A and a remarkable IC₅₀ of 60 nM.

Full text of DOI:10.1016/j.ejmech.2014.04.055

European Journal of Medicinal Chemistry 80 (2014) 352e363
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and optimization of an original V-shaped collection of
4-7-disubstituted Pyrido[3,2-d]pyrimidines as CDK5 and DYRK1A
inhibitors
,
,
Oussama Dehbi a b, Abdellatif Tikad a b, Stephane Bourg a, Pascal Bonnet a, Olivier Lozach c,
Laurent Meijer c 1, Mina Aadil b, Mohammed Akssira b, Gérald Guillaumet a
,
,
,
*
,
Sylvain Routier a
*
a Institut de Chimie Organique et Analytique, CNRS UMR 7311, B.P. 6759, 45067 Orléans Cedex 2, France
b Equipe de Chimie Bioorganique & Analytique, URAC 22, Université Hassan II Mohammedia-Casablanca, BP 146, 28800 Mohammedia, Morocco
c C.N.R.S., Protein Phosphorylation & Human Disease, USR3151, Station Biologique, B.P. 74, 29682 Roscoff Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
We here report the synthesis and biological evaluation of an original collection of 4,7-disubstituted
pyrido[3,2-d]pyrimidines designed as potential kinase inhibitors. The collection was generated from a
single starting material, 4,7-dichloropyrido[3,2-d]pyrimidine, which afforded the final compounds after
two steps: a sequential or one-pot sequence including selective cross coupling reactions in C-4, followed
by the second cross-coupling in C-7. In position C-4, a Suzuki-Miyaura type reaction led to mono-
substituted derivatives whereas in position C-7, synthesis was achieved via a Suzuki or a Buchwald type
reaction using commercially available or undescribed boron derivatives. The biological activity of the V-
shaped family was measured in protein kinase assays. The structure activity relationship (SAR) revealed
that some compounds selectively inhibited DYRK1A and CDK5 without affecting GSK3. Docking studies
furnished possible explanations that correlate with the SAR data. The most active compound on the two
biological targets was 27 which exhibited the following IC50: 110 nM for CDK5, 24 nM for DYRK1A and
Received 2 March 2014
Received in revised form
17 April 2014
Accepted 19 April 2014
Available online 22 April 2014
Keywords:
Pyridopyrimidine
CDK5
DYRK1A
Synthesis
SAR
only 1.2
which led to a near selective action on DYRK1A and a remarkable IC50 of 60 nM.
Ó 2014 Elsevier Masson SAS. All rights reserved.
mM for GSK3. In the C-7 amino subfamily, the best derivative was indubitably compound 48
Molecular modelling
1. Introduction
on cyclin-dependent kinase 5 (CDK5), glycogen synthase kinases-3
(GSK3) and dual-specificity tyrosine phosphorylation-regulated ki-
nase 1A (DYRK1A) [3]. These enzymes were chosen for their
involvement in regulation processes including cancer and central
nervous system (CNS) related disorders. Their strong sequence ho-
mology and high ATP site conservation make the design of selective
inhibitors containing a single organic platform challenging.
Protein kinases constitute a large family of structurally related
enzymes that are responsible for the control of a wide variety of
signal transduction processes within the cell. Deregulation of kinase
activities is frequently observed during the development of human
diseases such as cancer, diabetes, Alzheimer’s disease, and inflam-
mation [1]. Consequently, they have been used as targets to identify
pharmacological inhibitors of potential therapeutic interest [2]. Our
laboratory is currently dedicated to the synthesis of novel disease-
relevant kinase inhibitors, and focusses its efforts on drugs acting
CDK5 plays a central role in neuronal migration during the
development of the central nervous system [4]. GSK3b is involved
in embryonic development, protein synthesis, cell proliferation and
differentiation, microtubule dynamics, cell motility and apoptosis
[5]. DYRK1A encodes a member of the dual-specificity tyrosine
phosphorylation-regulated kinases (DYRKs) family. It may play a
significant role in a signalling pathway regulating cell proliferation
and may be involved in brain development [3]. A recent review
established strategies to inhibit this kinase and related the benefits
of the drugs in CNS pathologies [6]. In our opinion it could be
* Corresponding authors. Institut de Chimie Organique et Analytique, Université
d’Orléans, B.P. 6759, 45067 Orléans Cedex 2, France.
E-mail addresses: gerald.guillaumet@univ-orleans.fr (G. Guillaumet), sylvain.
routier@univ-orleans.fr (S. Routier).
1
Present address: ManRos Therapeutics, Centre de Perharidy, 29680 Roscoff,
interesting to possess dual CDK5/DYRK1A or triple CDK5/GSK3b/
France.
http://dx.doi.org/10.1016/j.ejmech.2014.04.055
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

O. Dehbi et al. / European Journal of Medicinal Chemistry 80 (2014) 352e363
353
DYRK1A inhibitors as they may exert synergistic effects in the
treatment of CNS diseases.
presence of NaBH(OAc)3 with the 2-formyl-4-thiophenyl boronic
acid. The crude adducts 28e30 were next used in the Suzuki-
Miyaura cross coupling reactions without any purification to
afford after hydrolysis the final derivatives 31e33 in satisfying
global yields. The main advantage of this straightforward procedure
is that it is achieved without any protective group (Scheme 2).
We previously developed some molecules able to target some of
the above-mentioned kinases in the nanomolar range and showed
that tetracyclic V-shaped pyradine or pyrazine or indole containing
drugs could be used to design dual ATP competitive CDK5 and
DYRK1A inhibitors [7].
Nevertheless series I optimization showed that the only sub-
stituent that was well tolerated on the indole phenyl ring was the
5-hydroxyl group, which generated an unstable quinonimine sys-
tem. We therefore sought to discover a novel scaffold able to afford
us greater insight into SAR towards kinases. We hypothesised that
pharmacophore I could be transformed by a caesura/closure
strategy into a new model of type II. In order to validate this hy-
pothesis, we identified the pyrido[3,2-d]pyrimidine core as a suit-
able solution. Fortunately from a homemade library extraction [8],
we obtained encouraging biological results using 4,7-disubstituted
derivatives of type IIa (Fig. 1).
In this paper we report the first additional synthesis to complete
the IIa library and we present the novel methodologies leading to
derivatives IIbed. A literature survey revealed that the synthesis
and/or biological evaluation of these derivatives had not been
previously described. The full results of kinase assays on CDK5,
2.2. Synthesis of V-shaped molecules of type IIb and IIc
Having the method in hand to carry out cross coupling reactions
in C-7 by displacement of the chlorine atom, an unknown series of
type IIb could then be envisioned to pursue SAR optimization. On
performing a Buchwald type reaction (Scheme 3) with (hetero)ar-
omatic primary amines we found that the best conditions required
microwave irradiation to activate the reaction and the use of the
Pd(PPh3)4/xantphos couple as catalytic system in the presence of
K2CO3 as base. Under these optimized conditions, derivatives 34e
45 were also obtained in fairly good yields (Table 2, entries 1e12).
Fortunately, under similar conditions, amides were successfully
introduced in C-7, leading to type IIc compounds. The only modi-
fications were the amount of catalytic system (only 5 mol % in
Pd(OAc)2) and an increase in the reaction time from 60 min to
70 min.
GSK3b and DYRK1A are provided and SAR is discussed. Complete
docking studies were achieved and confirm the interactions of the
best candidates with the CDK5 and/or DYRK1A kinase binding sites.
In order to give more flexibility to the tri-aromatic V-shaped
structure, we sought to introduce onto the pyrimidine ring a
“kneecap” through a 4-hydroxyaniline moiety. Beginning the syn-
thesis with 1, a simple SNAr was carried out with a near stoichio-
metric amount of 4-hydroxyaniline and led regioselectively to the
C-4 aminated derivative 51 in a 70% yield. As we were interested in
preparing direct analogues of derivatives 2e4 which possess
essential hydroxyl aryl groups in C-7, the residual chlorine atom of
51 was next involved in the previously presented microwave-
activated Suzuki-Miyaura cross coupling reaction. Starting from
each commercially available hydroxylated phenyl boronic acid,
type IId compounds 52e54 were isolated without any degradation
in 66, 60 and 80% yields respectively, despite the presence of
oxidation sensitive functions.
2. Chemistry
2.1. Synthesis of V-shaped molecules of type IIa
We previously reported the synthesis of compounds 2e21
(Table 1) which were obtained by Suzuki cross coupling reactions
[8] from 2,7-dichloropyridine 1. To explore the potency of the series
and to develop efficient SAR, we prepared some additional de-
rivatives. In a novel approach, we carried out the synthesis of 23
using a one pot regioselective C-4 followed by a C-7 cross coupling
procedure. Using first 2-formylthiophen-4-yl and then 4-
hydroxyphenyl boronic acids, derivative 23 was isolated in a 24%
yield. Starting from 24 [8], which was synthesized on a large scale,
and from the two commercially available formylated thiophenyl
boronic acids, derivatives 25 and 26 were isolated in satisfying
yields (68 and 74% respectively) under microwave irradiation after
only 10 min. Reduction of the formyl group of 25 using NaBH4
occurred smoothly in a MeOH/DMF mixture at room temperature
and the primary alcohol 27 was isolated in a 71% yield (Scheme 1).
As direct reductive aminations of aldehyde 25 in the presence of
secondary aliphatic amines failed, we extra-temporally prepared
three undescribed boronic acids by reacting chosen amines in the
3. Protein kinase inhibition assays
All synthesized products were assayed for potential inhibition of
three protein kinases: DYRK1A, CDK5/p25 and GSK-3. Analysis of
the collection 2e22 (Table 1, entries 1e20) shows that the two
CDK5 and DYRK1A enzymes were independently targeted by pyr-
idopyrimidine drugs of type IIa with little effect on GSK3. The bis-
phenol derivatives 2e5 interacted more favourably whereas the
position of the OH groups on Cy1 and Cy2 modulated the kinase
inhibitions. The Cy1-OH group was better positioned in para (1e3 vs
4e5) whereas the Cy2-OH function could be displaced from the
Fig. 1. Scaffold I modifications and envisioned library II.

354
O. Dehbi et al. / European Journal of Medicinal Chemistry 80 (2014) 352e363
Table 1
Full report of the kinase inhibition of the IIa library.
Entry
Compound
Kinase inhibition IC50,
m
M
Entry
Compound
Kinase inhibition IC50,
m
Ma
GSK3
DYRK 1A
CDK5
GSK3
DYRK 1A
CDK5
1
2
2.7
0.09
ꢀ100
15
16
>10
0.55
>10
2
3
4
5
3
4
5
6
2.4
0.11
0.12
0.72
>10
>100
26
16
17
18
19
17
18
19
20
>10
>10
>10
>10
1.8
>10
>10
>10
>10
0.93
0.74
5.2
0.11
00.12
0.13
4
>10
6
7
24
0.97
0.48
>10
>10
>10
ꢀ100
>100
>10
20
21
22
23
24
21
22
23
25
26
>10
>10
0.38
0.31
0.16
0.65
1.2
>10
>10
>10
0.43
3.2
7
8
>10
>10
>10
>10
8
9
0.25
0.027
0.81
9
10
11
>10
10
>10
11
12
12
13
>10
>10
>10
>10
25
26
27
31
0.11
0.024
1.2
5.3
0.43
1.4
0.41
0.62
13
14
15
0.88
0.17
>10
>10
27
28
32
33
1
0.3
0.56
4.7
14
0.15
0.81
0.97
0.16
a
Values are means of three measured values of IC50
.
ortho to the para positions, leading to a clear selectivity for CDK5
active site interaction. Progressive displacement enhanced the
potency towards DYRK1A while the effect on CDK5 was stable
(compounds 2e4). Selectivity toward GSK3 was affected during
these small changes. A decrease in activity was observed using
classic phenol isostery. The formyl groups were the only groups to
be tolerated by the two kinases and derivatives 13 and 14 were
clearly effective as dual DYRK1A and CDK5 inhibitors without any
effect on GSK3. As previously mentioned for the OH group, the
formyl position modulates the activity profile.
The organoleptic properties of the Cy2 group were next modi-
fied using heterocycles instead of phenol. Pyridine, thiophene and
furane skeletons were well tolerated by the CDK5 active site and
derivatives 14 and 15 appeared as strong CDK5/DYRK1A inhibitors.
Substitutions in C-5 thiophene rings which preserved the steric
enhancement embraced the kinase active sites. Increasing the size
of the substitutions by adding secondary amines led to disap-
pointing results despite the high solubility of derivatives 31e33. In
addition, the use of thiophene globally diminished the selectivity
towards GSK3. Compounds 25 and 27 remained the most selective
derivatives and the best V-shaped CDK5 inhibitors with remarkable
IC50 values of 27 and 24 nM, respectively.
Introduction of an NH or amide C-4 or C-7 between the pyr-
idopyrimidine and Cy1 or Cy2 led respectively to derivatives of type
IIb and IId (Table 2) which were used in kinase assays. In series IIb,
despite our efforts and the modulation of Cy1, the only favourable
group was the 4-hydroxyphenyl group. The corresponding methyl
ether was fully inactive whereas liberation of the hydrogen acid led
to 35 (entry 2), which exhibited a sub-micromolar inhibition of
DYRK1A and good selectivity towards CDK5 and GSK3. The

O. Dehbi et al. / European Journal of Medicinal Chemistry 80 (2014) 352e363
355
HO
S
Cl
N
Cl
CHO
N
a
N
N
N
N
1
23
OH
OH
S
Cl
N
R1
b
N
N
R2
N
N
N
24
25 R1 = CHO, R2 = H
26 R1 = H, R2 = CHO
OH
S
c
25
HOCH2
N
N
N
27
Scheme 1. Preparation of some C-2 and C-7 thiophene containing derivatives. Reagents and conditions : a) 2-formyl-4-thiophenyl boronic acid (1.0 eq.), Na2CO3 (2.0 eq.), Pd(PPh3)4
(5mol %), toluene/EtOH 2/1, 100ꢁC, 6 h and then 4-OHC6H4B(OH)2 (1.2 eq.), 100ꢁC, 1 h, 24% ; b) thiophenyl boron derivative (1.2 eq.), K2CO3 (2.0 eq.), Pd(PPh3)4 (5 mol %), toluene/
EtOH 2/1, 150ꢁ, mW, 10 min, 25 68% or 26 74% ; c) NaBH4 (0.5 eq.), MeOH/DMF 1/1, r.t., 16 h, 71%.
heterocyclic derivatives 36e45 were quite inactive on DYRK1A,
except for the oxazole derivative 42 which inhibited DYRK1A with
an IC50 of 2 mM, again indicating the necessary presence of electron
rich fractions in Cy1. Residual activity on the other two kinases was
found but SAR was still clearly observable.
The derivatives 52e54 which represent the IId family (entries
18e20) were inactive on DYRK1A; their best effect was found
against CDK5 in the submicromolar range. In addition, the ac-
tivity of 35 and 54 on GSK3 remained unchanged whereas the
activity values on DYRK1A/CDK5 were reversed.
A novel
Scheme 2. Preparation of thiophene containing compounds 31-33. Reagents and conditions : a) secondary amine (5.0 eq.), NaBH(OAc)3 (2.0 eq.), DME, 60ꢁC, 5 h ; b) crude boronic
acids 28-30 (5.0 eq.), K2CO3 (2.0 eq.), Pd(PPh3)4 (5 mol %), toluene/EtOH 2/1, 150ꢁC, mW, 10 min, 31 55%, 32 50% or 33 52%.
OH
HN
N
a
24
N
N
IIb, 34-45
OH
Y'
Y
N
N
N
b
24
O
N
IIc 46 Y = 3-pyridinyl, Y' = H
47 Y = 4-pyridinyl, Y' = H
48-50 Y,Y' = cyclic lactam
H
R
H
c
Cl
N
N
N
N
d
1
N
N
N
OH
N
OH
51
52-54 R = OH
Scheme 3. Preparation of C-2 or C-7 amino/amido derivatives. Reagents and conditions : a) (Het)ArNH2 (1.2 eq.), Pd(OAc)2 (10 mol %), xantphos (20 mol %), K2CO3 (2.0 eq.), dioxane,
140ꢁC, mW, 60 min, 34-45 57-97 % ; b) idem with amides (1.2 eq.), Pd(OAc)2 (5 mol %), xantphos (5 mol %), 70 min, 46-50 78-93%, c) 4-HOC6H4NH2 (1.05 eq.), dioxane, rflx, 24 h,
60% ; d) hydroxyphenyl boronic acids (1.2 eq.), K2CO3 (2.0 eq.), Pd(PPh3)4 (5 mol %), toluene/EtOH 2/1, 150ꢁC, mW, 10 min, 52 R ¼ 2-OH 66%, 53 R ¼ 3-OH 60% and 54 R ¼ 4-OH 80%.

356
O. Dehbi et al. / European Journal of Medicinal Chemistry 80 (2014) 352e363
Table 2
Full report of the kinase inhibition of the IIb and IIc library.
Entry
Compound (yield)
Kinase inhibition IC50
,
m
Ma
Entry
Compound (yield)
Kinase inhibition IC50
,
m
Ma
DYRK 1A
CDK5
GSK3
DYRK 1A
CDK5
GSK3
1
2
34 (57%)
35 (80%)
>10
1.8
7.3
5.2
11
12
44 (67%)
45 (87%)
>10
>10
>10
>10
0.8
1.4
>10
5.3
3
4
36 (90%)
37 (71%)
>10
>10
5
>10
>10
13
14
46 (89%)
47 (88%)
4.2
>10
>10
>10
>10
>10
>10
5
38 (72%)
>10
>10
>10
15
48 (79%)
0.06
2.2
4.6
6
7
39 (97%)
40 (80%)
>10
>10
>10
>10
16
17
49 (78%)
50 (93%)
5.1
1
>10
>10
>10
>10
4
5.5
8
9
41 (81%)
>10
7.6
>10
18
52 (66%)
>10
3.7
>10
42 (52%)
43 (84%)
2
2.1
2.2
>10
>10
19
20
53 (60%)
54 (80%)
>10
>10
0.71
0.84
4.5
7.1
10
>10
a
Values are means of three measured values of IC50
.
orientation of the compound in the active site due to the 4-
hydroxyaniline moiety may explain the biological results, as
suggested by docking studies.
kinase inhibitors [9]. The key interactions between the hetero-
aromatic scaffold IIa and the hinge region of the protein kinases
take place through hydrogen bond networks. The hydrogen
acceptor atom N-4 of scaffold IIa interacts with the backbone NH of
Leu241 of DYRK1A. The weak acidic hydrogen on position 5 of
scaffold IIa interacts with oxygen of the backbone carbonyl of
Glu239 and Cys83 of DYRK1A and CDK5 respectively. The Cy1
substituent points towards the solvent area while Cy2 binds to the
hydrophobic pocket close to the gatekeeper residue [10]. The hy-
droxyl group of Cy2 hydroxyphenyl found in many compounds is
involved in a hydrogen bond network with catalytic Lys188 of
DYRK1A or Asp307 from the DFG motif (Fig. 2).
The most attractive result of this nitrogen incorporation is
indubitably the fusion of the amide link and Cy1. The activities of
derivatives 48e50 were fully restored on DYRK1A and a great
selectivity was observed, despite values in the micromolar range.
The most active molecule on DYRK1A was 48 which had an IC50 of
60 nM and a 27- and 76- fold selectivity towards CDK5 or GSK3,
respectively. Interestingly, this compound has the highest ligand
efficiency (LE ¼ 0.42) and lipophilic efficiency (LiPE ¼ 4.29)
amongst all the synthesized compounds.
The binding mode of compound 48, the most efficient ligand, is
shown in Fig. 3. The hydroxyl group of Cy2 creates a hydrogen bond
with Lys188 and the oxo-piperidine points to the solvent area.
Interestingly, docking results suggest that the scaffold IId is flipped
180ꢁ in the active site compared to scaffolds IIa, IIb, and IIc when
Cy2 is a hydroxyaniline moiety (compounds 52e54).
4. Molecular modelling
Docking experiments revealed that the binding mode of most of
the compounds is conserved in the two protein kinases DYRK1A
and CDK5, characteristic of the binding mode of typical type I

O. Dehbi et al. / European Journal of Medicinal Chemistry 80 (2014) 352e363
357
Fig. 4. Binding mode of compound 53 in CDK5 active site. Analysis of the hydrogen
bonding interactions and hydrophobic contacts made by compound 53 (green) to
residues in the CDK5 ATP binding pocket. The three-letter amino acid code and residue
number are labeled next to each side chain. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Binding mode of compound 4 in DYRK1A active site. Analysis of the hydrogen
bonding interactions and hydrophobic contacts made by compound 4 (green) to res-
idues in the DYRK1A ATP binding pocket. The three-letter amino acid code and residue
number are labeled next to each side chain. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)
derivatives. Sequential or one-pot sequence including selective
cross coupling reactions in C-4 followed by the second one in C-7
are also achieved with success. In position C-4, a Suzuki-Myaura
type reaction led to monosubstituted derivatives whereas in C-7 a
Suzuki or a Buchwald type reaction achieves the synthesis using
commercially available or unknown boron derivatives. These drug-
like molecules have been tailored as dual selective DYRK1A and
CDK5 kinase inhibitors with an excellent ligand efficiency of 0.4. A
counter-screen on GSK3, a protein kinase belonging to the same
CMGC group, confirms the selectivity profile of these compounds.
Molecular modelling was used to propose specific substituents for
activity and selectivity. The proposed binding mode of the com-
pounds is in good agreement with the structureeactivity rela-
tionship. These molecules are promising scaffolds for targeting
protein kinases involved in the central nervous system.
Fig. 4 shows compound 53 bound to CDK5. Here, the scaffold in
the CDK5 active site is better oriented compared to the other
scaffolds IIb and IIc. The hydrogen acceptor atom N-4 of scaffold IId
interacts with the backbone NH of Cys83 of CDK5 and the Cy1
hydroxyphenyl forms a hydrogen bond with Asp144. The good
selectivity observed towards GSK3b can be attributed to a proline
residue (Pro136) located in the hinge region, preventing backbone
NH from creating a hydrogen bond with the scaffold. This major
difference between the three protein kinases could explain the
overall poor GSK3
b biological activities of the three scaffolds
compared to DYRK1A and CDK5.
5. Conclusion
6. Experimental section
In this paper, we have presented the design and synthesis of
novel 4-7-disubstituted pyrido[3,2-d]pyrimidines scaffolds adopt-
ing a peculiar V-shaped conformation. The collection was gener-
ated from a single starting material, the 4,7-dichloropyrido[3,2-d]
pyrimidine which afford the final compounds after two steps. We
have elaborated the most straightforward routes to access to final
6.1. Chemistry
1H NMR and 13C NMR spectra were recorded on a Bruker DPX
250 MHz or 400 MHz instrument using CDCl3 or DMSO-d6. The
chemical shifts are reported in parts per million (d scale) and all
coupling constant (J) values are in Hertz (Hz). The following ab-
breviations were used to explain the multiplicities: s (singlet),
d (doublet), t (triplet), q (quartet), m (multiplet) and dd (doublet
doublet). Melting points are uncorrected. IR absorption spectra
were obtained on a Perkin Elmer PARAGON 1000 PC and values are
reported in cmꢂ1. HRMS were recorded on a Maxis Bruker instru-
ment. Monitoring of the reactions was performed using silica gel
TLC plates (silica Merck 60 F254). Spots were visualized by UV light
at 254 nm and 356 nm. Column chromatographies were performed
using silica gel 60 (0.063e0.200 mm, Merck). Microwave experi-
ments were performed on a Biotage Initiator apparatus.
6.1.1. 4-(7-(4-Hydroxyphenyl)pyrido[3,2-d]pyrimidin-2-yl)
thiophene-2-carbalde-hyde 23
In an argon degassed solution, 2,7-dichloropyrido[3,2-d]py-
rimidine 22 (200 mg, 0.99 mmol) was dissolved at room temper-
ature in
a toluene/EtOH 2/1 mixture. 2-Formyl-4-thiophene
Fig. 3. Binding mode of compound 48 in DYRK1A active site. Analysis of the hydrogen
bonding interactions and hydrophobic contacts made by compound 48 (green) to
residues in the DYRK1A ATP binding pocket. The three-letter amino acid code and
residue number are labeled next to each side chain. (For interpretation of the refer-
ences to color in this figure legend, the reader is referred to the web version of this
article.)
boronic acid (155 mg, 0.99 mmol, 1.0 eq.), Na2CO3, (210.0 mg,
1.98 mmol, 2.0 eq.) and finally the catalyst Pd(PPh3)4 (58 mg,
0.05 mmol, 0.05 eq.) were successively added prior to placing the
flask in a preheated oil bath at 100 ꢁC. After 6 h of stirring, the
reaction mixture was cooled at room temperature and the 4-

358
O. Dehbi et al. / European Journal of Medicinal Chemistry 80 (2014) 352e363
hydroxyphenyl boronic acid (164.0 mg, 1.18 mmol, 1.2 eq.) was
added with a small additional amount of Pd(PPh3)4 (1.1 mg,
0.05 mmol, 0.01 e). The reaction mixture was heated at 100 ꢁC for
one additional hour and cooled at room temperature. Volatiles
were removed under reduced pressure and the crude material was
purified by flash chromatography on silica gel (CH2Cl2/Et3N 99/1
then CH2Cl2/MeOH 98/2) to afford 23 as a pale brown solid in 24%
yield. Rf: 0.2 (petroleum ether/EtOAc 5/95); mp > 268 ꢁC; IR (ATR
afford 27 as a white solid in 71% yield. Rf: 0.2 (petroleum ether/
EtOAc 5/95); mp > 268 ꢁC; IR (ATR Diamond, KBr, cmꢂ1
3353,
1578, 1452, 1236, 1148, 840, 739; 1H NMR (400 MHz, DMSO-d6)
)
n
d
4.73 (s, 2H, CH2), 5.64 (s, 1H, OH), 6.93 (d, 2H, J ¼ 8.4 Hz, HPh), 7.76
(s, 1H, HTh), 8.36 (s, 1H, HTh), 8.40 (d, 2H, J ¼ 8.4 Hz, HPh), 8.58 (s, 1H,
H8), 9.44 (s, 1H, H6), 9.59 (s, 1H, H4), 10.10 (s, 1H, OH); 13C NMR
(100.6 MHz, DMSO-d6)
d 58 (CH2), 115 (2xCH), 122 (CH), 124 (CH),
127 (Cq), 129 (CH), 130 (2xCH), 135 (Cq),136 (Cq),137 (Cq), 146 (Cq),
148 (Cq), 150 (CH), 160 (Cq), 160 (Cq), 161 (CH); HRMS (EI-MS) :
Diamond, cmꢂ1
NMR (400 MHz, DMSO-d6)
J ¼ 8.6 Hz, HPh), 8.49 (d, 1H, J ¼ 1.8 Hz, H8), 8.76 (d, 1H, J ¼ 1.2 Hz,
)
n
3389, 1696, 1584, 1472, 1228, 1174, 834, 725; 1
H
d
6.95 (d, 2H, J ¼ 8.6 Hz, HPh), 7.86 (d, 2H,
calculated for
C
18H14N3O2S m/z 336.08012 [MþH]þ found
336.08049 [MþH]þ.
H
Th), 9.00 (s, 1H, HTh), 9.40 (d, 1H, J ¼ 1.8 Hz, H6), 9.65 (s, 1H, H4),
9.97 (br, 1H, OH), 10.07 (d, 1H, J ¼ 1.2 Hz, CHO); 13C DEPT NMR
6.1.5. General procedure for the synthesis of crude boronic acids
28e30
(100.6 MHz, DMSO-d6) d 116 (2xCH),129 (2xCH),130 (CH), 137 (CH),
137 (CH), 151 (CH), 161 (CH), 184 (CHO); HRMS (EI-MS): calculated
2-Formyl-4-thiophene boronic acid (100 mg, 0.64 mmol) was
dissolved in DME (10 mL) and the appropriate amine (3.2 mmol,
5.0 eq.) was added, followed by a drop of AcOH. The resulting so-
lution was stirred for 5 min at room temperature. NaBH(OAc)3
(271 mg, 2.0 eq.) was added and the resulting solution was heated
at 60 ꢁC for 5 h. The volatiles were evaporated under reduced
pressure and the crude material was used without further purifi-
cation in the next step.
for C18H12N3O2S 334.06447 [MþH]þ found 344.06435 [MþH]þ.
6.1.2. 7-(4-(4-Hydroxyphenyl)pyrido[3,2-d]pyrimidin-2-yl)
thiophene-3-carbalde-hyde 25
Under argon, in a sealed vial, compound 24 (91 mg, 0.35 mmol)
was dissolved at room temperature in a mixture of toluene/EtOH 2/
1 and 2-formyl-4-thiophene boronic acid (66 mg, 0.42 mmol,
1.2 eq.), K2CO3 (98 mg, 0.70 mmol, 2.0 eq.) and Pd(PPh3)4 (21 mg,
0.017 mmol, 0.05 eq.) were successively added. The mixture was
heated under microwave irradiation at 150 ꢁC for 10 min. After
cooling, the volatiles were removed under pressure and the crude
material was purified by flash chromatography on silica gel
(CH2Cl2/NH3 99/1 then CH2Cl2/MeOH 98/2) to afford 25 as a brown
solid in 74% yield. Rf: 0.2 (petroꢂle1um ether/EtOAc 5/95);
6.1.6. 4-(7-(5-(Piperidin-1-ylmethyl)thiophen-3-yl)pyrido[3,2-d]
pyrimidin-2-yl)phenol 31
Compound 31 was obtained from 24 as described for compound
25 using boronic acid 28. Flash chromatography using silica gel
(CH2Cl2/NH3 99/1 then CH2Cl2/THF 9/1) afforded the attempted
product as a yellowish solid in 55% yield. Rf: 0.2 (petroleum ether/
mp > 268 ꢁC; IR (ATR Diamond, KBr, cm
)
n
3354, 1696, 1569, 1441,
6.93 (d, 2H,
EtOAc 5/95); mp > 268 ꢁC; IR (ATR Diamond, KBr, cmꢂ1
)
n
3334,
1577, 1451, 1225, 1158, 847, 703; 1H NMR (400 MHz, DMSO-d6)
1.39 (s, 2H, HPP),1.51 (s, 4H, HPP), 2.42 (s, 4H, HPP), 3.71 (s, 2H, CH2),
6.92 (d, 2H, J ¼ 8.7 Hz, HPh), 7.75 (s, 1H, HTh), 8.34 (d, 1H, J ¼ 1.2 Hz,
1253, 1139, 845, 697; 1H NMR (400 MHz, DMSO-d6)
d
J ¼ 8.5 Hz, HPh), 8.41 (d, 2H, J ¼ 8.5 Hz, HPh), 8.75 (s, 1H, HTh), 8.86 (s,
d
1H, H8), 9.00 (s, 1H, H6), 9.49 (s, 1H, HTh), 9.63 (s, 1H, H4), 10.02 (s,
1H, OH), 10.10 (s, 1H, CHO); 13C NMR (100.6 MHz, DMSO-d6)
d
115
H
Th), 8.40 (d, 2H, J ¼ 8.7 Hz, HPh), 8.57 (d, 1H, J ¼ 1.7 Hz, H8), 9.42 (d,
(2 ꢃ CH), 127 (Cq), 130 (2 ꢃ CH), 130 (CH), 134 (Cq), 134 (CH), 136
(CH), 137 (Cq), 138 (Cq), 144 (Cq), 146 (Cq), 150 (CH), 160 (Cq), 160
(Cq), 161 (CH), 184 (CHO); HRMS (EI-MS): calculated for
1H, J ¼ 1.7 Hz, H6), 9.57 (s, 1H, H4), 10.12 (s, 1H, OH); 13C NMR
(100.6 MHz, DMSO-d6)
d
23 (CH2), 25 (2 ꢃ CH2), 53 (2 ꢃ CH2), 57
(CH2), 115 (2 ꢃ CH), 124 (CH), 125 (CH), 127 (Cq), 129 (CH), 130
(2 ꢃ CH), 135 (Cq), 136 (Cq), 137 (Cq), 144 (Cq), 146 (Cq), 150 (CH),
160 (Cq), 160 (Cq), 161 (CH); HRMS (EI-MS) : calculated for
C
18H12N3O2S 334.06500 [MþH]þ found 344.06400 [MþH]þ.
6.1.3. 4-(7-(4-Hydroxyphenyl)pyrido[3,2-d]pyrimidin-2-yl)
thiophene-3-carbaldehyde 26
C
23H23N4OS 403.15871 [MþH]þ found 403.15892 [MþH]þ.
Compound 26 was obtained from 24 as described for compound
25 using 3-formyl-4-thiopheneboronic acid. Flash chromatography
using silica gel (CH2Cl2/NH3 99/1 then CH2Cl2/MeOH 98/2) afforded
the attempted product as a dark brown solid in 68% yield. Rf: 0.2
(petroleum ether/EtOAc 5/95); mp 240e242 ꢁC; IR (ATR Diamond,
6.1.7. 4-(7-(5-(Morpholinomethyl)thiophen-3-yl)pyrido[3,2-d]
pyrimidin-2-yl)phenol 32
Compound 32 was obtained from 24 as described for compound
25 using boronic acid 29. Flash chromatography using silica gel
(CH2Cl2/NH3 99/1 then CH2Cl2/THF 9/1) afforded the attempted
product as a brown solid in 50% yield. Rf: 0.2 (petroleum ether/
KBr, cmꢂ1
) n
3087, 1701, 1571, 1438, 1226, 1160, 827, 737; 1H NMR
(400 MHz, DMSO-d6)
d
6.93 (d, 2H, J ¼ 8.5 Hz, HPh), 8.09 (d, 1H,
EtOAc 5/95); mp > 268 ꢁC; IR (ATR Diamond, KBr, cmꢂ1
)
n
3400,
J ¼ 2.7 Hz, HTh), 8.40e8.43 (m, 3H, HPh and HTh), 8.82 (s, 1H,
J ¼ 2.0 Hz, H8), 9.06 (s,1H, J ¼ 2.0 Hz, H6), 9.65 (s,1H, H4), 9.98 (s,1H,
1571, 1443, 1220, 1153, 803, 706; 1H NMR (400 MHz, DMSO-d6)
d
2.45 (t, 4H, J ¼ 4.3 Hz, Hm), 3.60 (t, 4H, J ¼ 4.3 Hz, Hm), 3.74 (s, 2H,
CHO), 10.09 (s, 1H, OH); 13C NMR (100.6 MHz, DMSO-d6)
d
115
CH2), 6.92 (d, 2H, J ¼ 8.7 Hz, HPh), 7.77 (s, 1H, HTh), 8.36 (d, 1H,
J ¼ 0.9 Hz, HTh), 8.40 (d, 2H, J ¼ 8.7 Hz, HPh), 8.56 (d, 1H, J ¼ 1.6 Hz,
(2 ꢃ CH), 127 (Cq), 130 (CH), 130 (2xCH), 134 (CH), 135 (Cq), 136
(Cq), 137 (Cq), 139 (Cq), 142 (CH), 146 (Cq), 152 (CH), 160 (Cq), 160
(Cq), 161 (CH), 186 (CHO); HRMS (EI-MS): calculated for
H8), 9.41 (d, 1H, J ¼ 1.6 Hz, H6), 9.57 (s, 1H, H4), 10.07 (s, 1H, OH); 13
C
NMR (100.6 MHz, DMSO-d6)
d
52 (2 ꢃ CH2), 56 (CH2), 66 (2 ꢃ CH2),
C
18H12N3O2S 334.06500 [MþH]þ found 344.06660 [MþH]þ.
115 (2 ꢃ CH), 125 (CH), 125 (CH), 127 (Cq), 129 (CH), 130 (2 ꢃ CH),
135 (Cq), 136 (Cq), 137 (Cq), 143 (Cq), 146 (Cq), 150 (CH), 160 (Cq),
160 (Cq), 160 (CH); HRMS (EI-MS): calculated for C22H21N4O2S
405.13797 [MþH]þ found 405.13833 [MþH]þ.
6.1.4. 4-[7-(5-Methyl-thiophen-3-yl)-pyrido[3,2-d]pyrimidin-2-yl]-
phenol 27
Compound 25 (203.0 mg, 0.60 mmol) was dissolved in a mixture
MeOH/DMF 1/1 and the resulting solution was stirred for 5 min.
The temperature was lowered to ꢂ10 ꢁC then NaBH4 (0.3 mmol,
0.5 eq.) was added and the reaction mixture was stirred at room
temperature overnight. Volatiles were removed under reduced
pressure and the crude material was purified by flash chromatog-
raphy on silica gel (CH2Cl2/NH3 99/1 then CH2Cl2/MeOH 95/5) to
6.1.8. 4-(7-(5-(4-Methylpiperazin-1-ylmethylthiophen-3-yl)pyrido
[3,2-d]pyrimidin-2-yl)phenol 33
Compound 33 was obtained from 24 as described for compound
25 using boronic acid 30. Flash chromatography using silica gel
(AcOEt/NH3 99/1 then AcOEt/MeOH 95/5) afforded the attempted
product as a yellow solid in 52% yield. Rf: 0.2 (petroleum ether/

O. Dehbi et al. / European Journal of Medicinal Chemistry 80 (2014) 352e363
359
EtOAc 5/95); mp > 268 ꢁC; IR (ATR Diamond, KBr, cmꢂ1
1571, 1462, 1234, 1152, 844, 740; 1H NMR (400 MHz, DMSO-d6)
2.18 (s, 3H, CH3), 2.37 (br s, 8H, HPr), 3.74 (s, 2H, CH2), 6.93 (d, 2H,
J ¼ 8.7 Hz, HPh), 7.78 (d, 1H, J ¼ 0.9 Hz, HTh), 8.37 (d, 1H, J ¼ 0.9 Hz,
)
n
3387,
1H NMR (400 MHz, DMSO-d6)
d
6.90 (d, 2H, J ¼ 8.5 Hz, HPh), 6.97
(dd, 1H, J ¼ 5.4, 6.6 Hz, HP), 7.05 (d, 1H, J ¼ 8.3 Hz, HP), 7.71e7.78 (m,
1H, HP), 8.38 (d, 3H, J ¼ 8.5 Hz, HPh and HP), 8.90 (d, 1H, J ¼ 2.0 Hz,
H8), 9.04 (d, 1H, J ¼ 2.0 Hz, H6), 9.33 (s, 1H, H4), 9.99 (s, 1H, NH),
d
H
Th), 8.41 (d, 2H, J ¼ 8.7 Hz, HPh), 8.59 (d, 1H, J ¼ 1.9 Hz, H8), 9.43 (d,
10.13 (s, 1H, OH); 13C NMR (100.6 MHz, DMSO-d6)
d 112 (CH), 114
1H, J ¼ 1.9 Hz, H6), 9.59 (s, 1H, H4), 10.09 (br s, 1H, OH); 13C NMR
(CH), 115 (2 ꢃ CH), 116 (CH), 128 (Cq), 130 (2 ꢃ CH), 132 (Cq), 137
(CH), 141 (Cq), 145 (CH), 147 (CH), 148 (Cq), 154 (Cq), 159 (CH), 160
(Cq), 160 (Cq); HRMS (EI-MS): calculated for C18H14N5O 316.11980
[MþH]þ found 316.12100 [MþH]þ.
(100.6 MHz, DMSO-d6)
d
46 (CH3), 52 (2 ꢃ CH2), 55 (2 ꢃ CH2), 56
(CH2), 116 (2 ꢃ CH), 125 (CH), 125 (CH), 128 (Cq), 130 (CH), 130
(2 ꢃ CH), 135 (Cq), 136 (Cq), 137 (Cq), 144 (Cq), 147 (Cq), 150 (CH),
160 (Cq), 161 (Cq), 161 (CH); HRMS (EI-MS): calculated for
C
23H24N5OS 418.16961 [MþH]þ found 418.16992 [MþH]þ.
6.1.13. 4-[7-(Pyridin-3-ylamino)-pyrido[3,2-d]pyrimidin-2-yl]-
phenol 37
6.1.9. General procedure A for preparation of compounds IIbec
In a degassed solution of dioxane containing 24 (1.0 mmol),
primary amines (for IIb) or amides for IIc (1.2 eq.), K2CO3 (2.0 eq.)
and xantphos (0.2 eq; for IIb or 0.1 eq. for IIc) was added Pd(OAc)2
(0.1 eq; for IIb or 0.05 eq. for IIc) and the suspension immediately
placed in the microwave cavity. After irradiation (60 min for IIb or
70 min for IIc) at 140 ꢁC, the solution was cooled and volatiles were
removed under reduced pressure and the crude material was pu-
rified by recrystallization or by flash chromatography on silica gel.
Compound 37 was obtained following the general procedure A
using 3-aminopyridine which afforded the attempted derivative
after a purification under flash chromatography on silica gel
(CH2Cl2/NH3 99/1 then CH2Cl2/MeOH 98/2) as a yellowish solid in a
71% yield. Rf: 0.2 (petroleum ether/EtOAc 5/95); mp > 268 ꢁC; IR
(ATR Diamond, KBr, cmꢂ1
1H NMR (400 MHz, DMSO-d6)
) n 3456, 1556, 1451, 1243, 1158, 845, 696;
d
6.87 (d, 2H, J ¼ 8.6 Hz, HPh), 7.42 (dd,
1H, J ¼ 4.7, 8.1 Hz, HP), 7.51 (s,1H, HP), 7.84 (d,1H, J ¼ 8.1 Hz, HP), 8.34
(d, 3H, J ¼ 8.6 Hz, HPh and HP), 8.59 (s,1H, H8), 8.73 (s,1H, H6), 9.29 (s,
1H, H4), 9.55 (s, 1H, NH), 10.00 (s, 1H, OH); 13C NMR (100.6 MHz,
6.1.10. 4-[7-(4-Methoxy-phenylamino)-pyrido[3,2-d]pyrimidin-2-
yl]-phenol 34
DMSO-d6)
d
109 (CH), 115 (2 ꢃ CH), 124 (CH), 126 (CH), 128 (Cq), 130
(2 ꢃ CH), 132 (Cq), 136 (Cq), 142 (CH), 144 (CH), 144 (Cq), 145 (CH),
Compound 34 was obtained following the general procedure A
using paramethoxyaniline which afforded the attempted derivative
after a purification under flash chromatography on silica gel
(CH2Cl2/NH3 99/1 then CH2Cl2/MeOH 98/2) as a reddish solid in a
57% yield. Rf: 0.2 (petroleum ether/EtOAc 5/95); mp 134e136 ꢁC; IR
148 (Cq), 158 (CH), 160 (Cq), 160 (Cq); HRMS (EI-MS): calculated for
C
18H14N5O 316.11980 [MþH]þ found 316.11890 [MþH]þ.
6.1.14. 4-[7-(Pyridin-4-ylamino)-pyrido[3,2-d]pyrimidin-2-yl]-
phenol 38
(ATR Diamond, KBr, cmꢂ1
1H NMR (400 MHz, DMSO-d6)
)
n
3298, 1573, 1449, 1233, 1159, 830, 702;
3.78 (s, 3H, CH3), 6.88 (d, 2H,
Compound 38 was obtained following the general procedure A
using 4-aminopyridine which afforded the attempted derivative
after a recrystallization in methanol as a dark brown solid in a 72%
yield. Rf: 0.2 (petroleum ether/EtOAc 5/95); mp > 268 ꢁC; IR (ATR
d
J ¼ 8.2 Hz, HPh), 7.01 (d, 2H, J ¼ 8.4 Hz, HPh), 7.22 (s, 1H, H8), 7.28 (d,
2H, J ¼ 8.4 Hz, HPh), 8.32 (d, 2H, J ¼ 8.2 Hz, HPh), 8.64 (s, 1H, H6), 9.18
(s, 1H, H4), 9.21 (s, 1H, NH), 9.97 (s, 1H, OH); 13C NMR (100.6 MHz,
Diamond, KBr, cmꢂ1
) n
3414, 1572, 1453, 1290, 1156, 810, 703; 1H
DMSO-d6)
d
55 (CH3), 106 (CH), 114 (2 ꢃ CH), 115 (2 ꢃ CH), 123
NMR (400 MHz, DMSO-d6)
d
6.89 (d, 2H, J ¼ 8.7 Hz, HPh), 7.29 (d, 2H,
(2 ꢃ CH),128 (Cq),129 (2xCH),132 (Cq),132 (Cq),145 (CH), 145 (Cq),
148 (Cq), 155 (Cq), 158 (CH), 160 (Cq), 160 (Cq); HRMS (EI-MS):
calculated for C20H17N4O2 345.13520 [MþH]þ found 345.13600
[MþH]þ.
J ¼ 6.2 Hz, HP), 7.86 (d,1H, J ¼ 2.4 Hz, H8), 7.94 (d,1H, J ¼ 4.9 Hz, NH),
8.38 (d, 2H, J ¼ 8.7 Hz, HPh), 8.43 (d, 2H, J ¼ 6.2 Hz, HP), 8.80 (d, 1H,
J ¼ 2.4 Hz, H6), 9.39 (s, 1H, H4), 9.84 (br s, 1H, OH); 13C NMR
(100.6 MHz, DMSO-d6)
(Cq), 130 (2xCH), 133 (Cq), 141 (Cq), 146 (CH), 147 (Cq), 147 (Cq), 149
d
111 (2 ꢃ CH), 113 (CH), 115 (2 ꢃ CH), 127
6.1.11. 4-[7-(4-Hydroxyphenylamino)-pyrido[3,2-d]pyrimidin-2-
yl]-phenol 35
(Cq), 150 (2 ꢃ CH), 159 (CH), 160 (Cq); HRMS (EI-MS): calculated for
C
18H14N5O 316.11980 [MþH]þ found 316.11990 [MþH]þ.
Compound 35 was obtained following the general procedure A
using parahydroxyaniline which afforded the attempted derivative
after a purification under flash chromatography on silica gel
(CH2Cl2/NH3 99/1 then CH2Cl2/MeOH 98/02) as a light brown solid
in an 80% yield. Rf: 0.2 (petroleum ether/EtOAc 5/95); mp 272 ꢁC; IR
6.1.15. 4-[7-(2-(Methyl)pyridinylamino)-pyrido[3,2-d]pyrimidin-2-
yl]-phenol 39
Compound 39 was obtained following the general procedure A
using 3-amino-2-methylpyridine which afforded the attempted
derivative after a purification under flash chromatography on silica
gel (CH2Cl2/NH3 99/1 then CH2Cl2/MeOH 98/2) as a brown solid in a
97% yield. Rf: 0.2 (petroleum ether/EtOAc 5/95); mp 186e188 ꢁC; IR
(ATR Diamond, KBr, cmꢂ1
1H NMR (400 MHz, DMSO-d6)
J ¼ 2.4 Hz, H8), 7.17 (d, 2H, J ¼ 8.6 Hz, HPh), 8.32 (d, 2H, J ¼ 8.7 Hz,
Ph), 8.62 (d, 1H, J ¼ 2.4 Hz, H6), 9.07 (s, 1H, H4), 9.20 (s, 1H, NH),
9.45 (s, 1H, OH), 9.95 (s, 1H, OH); 13C NMR (100.6 MHz, DMSO-d6)
)
n
3415, 1581, 1434, 1222, 1158, 835, 701;
6.85e6.89 (m, 4H, HPh), 7.15 (d, 1H,
d
H
(ATR Diamond, KBr, cmꢂ1
1H NMR (400 MHz, DMSO-d6)
)
n
3404, 1577, 1459, 1274, 1156, 843, 751;
2.38 (s, 3H, CH3), 6.90 (d, 2H,
d
d
106 (CH), 115 (2xCH), 116 (2 ꢃ CH), 123 (2 ꢃ CH), 128 (Cq), 130
J ¼ 8.6 Hz, HPh), 6.95 (dd,1H, J ¼ 4.4, 7.1 Hz, HP), 7.59 (d,1H, J ¼ 7.1 Hz,
HP), 8.23 (d, 1H, J ¼ 4.4 Hz, HP), 8.38 (d, 2H, J ¼ 8.6 Hz, HPh), 8.82 (s,
1H, NH), 8.85 (d,1H, J ¼ 2.1 Hz, H8), 9.16 (d,1H, J ¼ 2.1 Hz, H6), 9.34 (s,
(2 ꢃ CH), 130 (Cq), 132 (Cq), 145 (CH), 146 (Cq), 148 (Cq), 154 (Cq),
158 (CH), 160 (Cq), 160 (Cq); HRMS (EI-MS): calculated for
C
19H15N4O2 331.11950 [MþH]þ found 331.11830 [MþH]þ.
1H, H4), 10.00 (s, 1H, OH); 13C NMR (100.6 MHz, DMSO-d6)
d 17.1
(CH3), 115 (2 ꢃ CH), 115 (CH), 117 (CH), 120 (Cq), 128 (Cq), 130
(2 ꢃ CH), 133 (Cq), 138 (CH), 142 (Cq), 144 (CH), 146 (CH), 147 (Cq),
152(Cq), 195 (CH), 160 (Cq), 160 (Cq); HRMS (EI-MS): calculated for
6.1.12. 4-[7-(Pyridin-2-ylamino)-pyrido[3,2-d]pyrimidin-2-yl]-
phenol 36
Compound 36 was obtained following the general procedure A
using 2-aminopyridine which afforded the attempted derivative
after a purification under flash chromatography on silica gel
(CH2Cl2/NH3 99/1 then CH2Cl2/MeOH 98/2) as a reddish solid in a
90% yield. Rf: 0.2 (petroleum ether/EtOAc 5/95); mp > 268 ꢁC; IR
C
19H16N5O 330.13550 [MþH]þ found 330.13470 [MþH]þ.
6.1.16. 4-[7-(5-(Cyano)pyridinylamino]-pyrido[3,2-d]pyrimidin-2-
yl]-phenol 40
Compound 40 was obtained following the general procedure A
using 2-amino-5-cyano-pyridine which afforded the attempted
(ATR Diamond, KBr, cmꢂ1
) n 3452, 1576, 1453, 1280, 1166, 805, 703;

360
O. Dehbi et al. / European Journal of Medicinal Chemistry 80 (2014) 352e363
derivative after a purification under flash chromatography on silica
gel (CH2Cl2/MeOH 95/5) as a reddish solid in an 80% yield. Rf: 0.2
(petroleum ether/EtOAc 5/95); mp > 268 ꢁC; IR (ATR Diamond, KBr,
6.1.20. 4-[7-(4-Methyl-thiazol-2-ylamino)-pyrido[3,2-d]pyrimidin-
2-yl]-phenol 44
Compound 44 was obtained following the general procedure A
using 2-amino-4-methyl-1,3-thiazole which afforded the attemp-
ted derivative after a purification under flash chromatography on
silica gel (CH2Cl2/NH3 99/1 then CH2Cl2/MeOH 98/02) as a green
cmꢂ1
) n
3311, 2224, 1573, 1433, 1280, 1153, 828, 700; 1H NMR
(400 MHz, DMSO-d6)
d
6.89 (d, 2H, J ¼ 8.6 Hz, HPh), 7.07 (d, 1H,
J ¼ 8.7 Hz, HP), 8.03 (dd, 1H, J ¼ 2.0, 8.7 Hz, HP), 8.35 (d, 2H,
J ¼ 8.6 Hz, HPh), 8.77 (d, 1H, J ¼ 1.6 Hz, H8), 8.86 (d, 1H, J ¼ 2.0 Hz,
HP), 8.92 (d, 1H, J ¼ 1.6 Hz, H6), 9.34 (s, 1H, H4), 10.01 (s, 1H, OH),
solid in
mp > 268 ꢁC; IR (ATR Diamond, KBr, cmꢂ1
1166, 806, 700; 1H NMR (400 MHz, DMSO-d6)
6.73 (s, 1H, HTz), 6.90 (d, 2H, J ¼ 8.7 Hz, HPh), 8.38 (d, 2H, J ¼ 8.7 Hz,
Ph), 8.80 (d, 1H, J ¼ 1.9 Hz, H8), 8.86 (d, 1H, J ¼ 1.9 Hz, H6), 9.37 (s,
1H, H4), 10.01 (s, 1H, OH), 11.16 (s, 1H, NH); 13C NMR (100.6 MHz,
DMSO-d6) 17 (CH3), 105 (CH), 114 (CH), 115 (2xCH), 128 (Cq), 130
a
67% yield. Rf: 0.2 (petroleum ether/EtOAc 5/95);
3465,1568, 1452, 1201,
2.36 (s, 3H, CH3),
)
n
10.59 (s, 1H, NH); 13C NMR (100.6 MHz, DMSO-d6)
d
100 (Cq), 112
d
(CH), 115 (2 ꢃ CH), 117 (CH), 117 (Cq), 128 (Cq), 130 (2 ꢃ CH), 133
(Cq), 140 (CH), 140 (Cq), 145 (CH), 147 (Cq), 152 (CH), 156 (Cq), 159
(CH), 160 (Cq), 160 (Cq); HRMS (EI-MS): calculated for C19H13N6O
341.11510 [MþH]þ found 341.11540 [MþH]þ.
H
d
(2xCH),133 (Cq),141 (Cq),145 (CH),148 (Cq),149 (Cq),159 (CH), 160
(Cq), 161 (Cq), 161 (Cq); HRMS (EI-MS): calculated for C17H14N5OS
336.09136 [MþH]þ found 336.09130 [MþH]þ.
6.1.17. 4-[7-(Pyrimidin-2-ylamino)-pyrido[3,2-d]pyrimidin-2-yl]-
phenol 41
Compound 41 was obtained following the general procedure A
using 2-aminopyrimidine which afforded the attempted derivative
after a purification under flash chromatography on silica gel
(CH2Cl2/NH3 99/1 then CH2Cl2/NH3 98/2) as a reddish solid in an
81% yield. Rf: 0.2 (petroleum ether/EtOAc 5/95); mp > 268 ꢁC; IR
6.1.21. 4-[7-(Benzothiazol-2-ylamino)-pyrido[3,2-d]pyrimidin-2-
yl]-phenol 45
Compound 45 was obtained following the general procedure A
using 2-aminobenzothioazole which afforded the attempted de-
rivative after a purification under flash chromatography on silica
gel (CH2Cl2/NH3 99/1 then CH2Cl2/MeOH 98/2) as a dark green
solid in an 87% yield. Rf: 0.2 (petroleum ether/EtOAc 5/95);
(ATR Diamond, KBr, cmꢂ1
) n 3405, 1574, 1437, 1266, 1157, 803, 701;
1H NMR (400 MHz, DMSO-d6)
1H, J ¼ 4.6 Hz, HPm), 8.38 (d, 2H, J ¼ 8.4 Hz, HPh), 8.68 (d, 2H,
J ¼ 4.6 Hz, HPm), 8.96 (s, 1H, H8), 9.09 (s, 1H, H6), 9.38 (s, 1H, H4),
10.01 (s, 1H, OH), 10.64 (s, 1H, NH); 13C NMR (100.6 MHz, DMSO-d6)
d
6.90 (d, 2H, J ¼ 8.4 Hz, HPh), 7.08 (t,
mp > 268 ꢁC; IR (ATR Diamond, KBr, cmꢂ1
1159, 845, 722; 1H NMR (400 MHz, DMSO-d6)
J ¼ 8.4 Hz, HPh), 7.28 (t, 1H, J ¼ 7.5 Hz, HbT), 7.44 (t, 1H, J ¼ 7.5 Hz,
)
n
3408,1569,1443,1269,
6.92 (d, 2H,
d
d
114 (CH),115 (2 ꢃ CH),116 (CH),128 (Cq),130 (2xCH),133 (Cq),140
H
bT), 7.84 (d, 1H, J ¼ 7.9 Hz, HbT), 7.92 (d, 1H, J ¼ 7.9 Hz, HbT), 8.41 (d,
(Cq), 145 (CH), 147 (Cq), 158 (2 ꢃ CH), 159 (Cq), 159 (CH), 160 (Cq),
160 (Cq); HRMS (EI-MS): calculated for C17H13N6O 317.11510
[MþH]þ found 317.11420 [MþH]þ.
2H, J ¼ 8.4 Hz, HPh), 8.91 (d,1H, J ¼ 2.0 Hz, H8), 9.11 (d,1H, J ¼ 2.0 Hz,
H6), 9.40 (s, 1H, H4), 10.05 (br s, 1H, OH), 11.46 (br s, 1H, NH); 13C
NMR (100.6 MHz, DMSO-d6)
d
115 (2 ꢃ CH), 116 (CH), 120 (CH), 121
(CH), 123 (CH), 126 (CH), 128 (Cq), 130 (2 ꢃ CH), 130 (Cq), 133 (Cq),
140 (Cq), 144 (CH), 147 (Cq), 151 (Cq), 159 (CH), 160 (Cq), 160 (Cq),
160 (Cq); HRMS (EI-MS): calculated for C20H14N5OS 372.09136
[MþH]þ found 372.09136 [MþH]þ.
6.1.18. 4-[7-(Isoxazol-3-ylamino)-pyrido[3,2-d]pyrimidin-2-yl]-
phenol 42
Compound 42 was obtained following the general procedure A
using 3-amino-1,2-oxazole which afforded the attempted deriva-
tive after a purification under flash chromatography on silica gel
(CH2Cl2/NH3 99/1 then CH2Cl2/MeOH 98/2) as a yellow solid in a
52% yield. Rf: 0.2 (petroleum ether/EtOAc 5/95); mp > 268 ꢁC; IR
6.1.22. N-[2-(4-Hydroxyphenyl)-pyrido[3,2-d]pyrimidin-7-yl]
isonicotinamide 46
Compound 46 was obtained following the general procedure A
using isonicotinamide, which afforded the attempted derivative
after a purification under flash chromatography on silica gel
CH2Cl2/NH3 99/1 then CH2Cl2/MeOH 95/05) as a yellowish solid in
an 89% yield. Rf: 0.2 (petroleum ether/EtOAc 5/95); mp 230e
(ATR Diamond, KBr, cmꢂ1
700; 1H NMR (400 MHz, DMSO-d6)
)
n
3400, 1560, 1459, 1272, 1150, 805,
6.43 (s, 1H, Hiz), 6.89 (d, 2H,
d
J ¼ 8.6 Hz, HPh), 8.39 (d, 3H, J ¼ 8.6 Hz, HPh and Hiz), 8.80e8.83 (m,
2H, H8 and H6), 9.39 (s, 1H, H4), 10.01 (s, 1H, OH), 10.41 (s, 1H, NH);
13C NMR (100.6 MHz, DMSO-d6)
d
98 (CH), 114 (CH), 115 (2 ꢃ CH),
232 ꢁC; IR (ATR Diamond, KBr, cmꢂ1
1156, 846, 697; 1H NMR (400 MHz, DMSO-d6)
J ¼ 8.6 Hz, HPh), 7.94 (d, 2H, J ¼ 5.6 Hz, HP), 8.41 (d, 2H, J ¼ 8.6 Hz,
Ph), 8.85e8.86 (m, 3H, HP and H8), 9.21 (d, 1H, J ¼ 1.9 Hz, H6), 9.53
(s, 1H, H4), 10.06 (s, 1H, OH), 11.25 (s, 1H, NH); 13C NMR (100.6 MHz,
DMSO-d6)
)
n
3406, 1671, 1558, 1447, 1264,
6.92 (d, 2H,
128 (Cq), 130 (2 ꢃ CH), 132 (Cq), 141 (Cq), 144 (CH), 148 (Cq), 159
(CH), 159 (Cq), 159 (CH), 160 (Cq), 160 (Cq); HRMS (EI-MS):
calculated for C16H12N5O2 306.09910 [MþH]þ found 306.09820
[MþH]þ.
d
H
d
115 (2 ꢃ CH), 121 (CH), 121 (2 ꢃ CH), 127 (Cq), 130
6.1.19. 4-[7-(Thiazol-2-ylamino)-pyrido[3,2-d]pyrimidin-2-yl]-
phenol 43
(2xCH), 135 (Cq), 139 (Cq), 140 (Cq), 146 (CH), 147 (Cq), 150 (2xCH),
160 (Cq), 160 (CH), 160 (Cq), 165 (Cq); HRMS (EI-MS): calculated for
Compound 43 was obtained following the general procedure A
using 2-amino-1,3-thiazole which afforded the attempted deriva-
tive after a purification under flash chromatography on silica gel
(CH2Cl2/NH3 99/1 then CH2Cl2/MeOH 98/2) as a green solid in an
84% yield. Rf: 0.2 (petroleum ether/EtOAc 5/95); mp > 268 ꢁC; IR
C
19H14N5O2 344.11470 [MþH]þ found 344.11460 [MþH]þ.
6.1.23. N-[2-(4-Hydroxyphenyl)-pyrido[3,2-d]pyrimidin-7-yl]
nicotinamide 47
Compound 47 was obtained following the general procedure A
using nicotinamide, which afforded the attempted derivative after a
purification under flash chromatography on silica gel (CH2Cl2/NH3
99/1 then CH2Cl2/MeOH 95/05) as a yellowish solid in an 88% yield.
Rf: 0.2 (petroleum ether/EtOAc 5/95); mp 226e228 ꢁC; IR (ATR
(ATR Diamond, KBr, cmꢂ1
1H NMR (400 MHz, DMSO-d6)
1H, J ¼ 3.6 Hz, HTz), 7.49 (d, 1H, J ¼ 3.6 Hz, HTz), 8.38 (d, 2H,
J ¼ 8.7 Hz, HPh), 8.84 (d, 1H, J ¼ 2.0 Hz, H8), 8.91 (d, 1H, J ¼ 2.0 Hz,
H6), 9.73 (s, 1H, H4), 10.04 (s, 1H, OH), 11.35 (s, 1H, NH); 13C NMR
) n 3412, 1566, 1443, 1247, 1122, 840, 697;
d
6.90 (d, 2H, J ¼ 8.7 Hz, HPh), 7.17 (d,
Diamond, KBr, cmꢂ1
1H NMR (400 MHz, DMSO-d6)
(dd, 1H, J ¼ 4.8, 7.7 Hz, HP), 8.41 (d, 3H, J ¼ 8.6 Hz, HPh and HP), 8.83
(d, 1H, J ¼ 4.8 Hz, HP), 8.88 (d, 1H, J ¼ 2.0 Hz, H8), 9.23 (s, 1H, HP),
9.26 (d, 1H, J ¼ 2.0 Hz, H6), 9.52 (s, 1H, H4), 10.10 (br s, 1H, OH), 11.30
) n 3444, 1672, 1553, 1448, 1230, 1157, 807, 701;
(100.6 MHz, DMSO-d6)
d
111 (CH), 114 (CH), 115 (2 ꢃ CH), 128 (Cq),
d
6.93 (d, 2H, J ¼ 8.6 Hz, HPh), 7.62
130 (2 ꢃ CH), 133 (Cq), 139 (CH), 141 (Cq), 144 (CH), 148 (Cq), 159
(CH), 160 (Cq), 160 (Cq), 162 (Cq); HRMS (EI-MS): calculated for
C
16H12N5OS 322.07630 [MþH]þ found 322.07730 [MþH]þ.

O. Dehbi et al. / European Journal of Medicinal Chemistry 80 (2014) 352e363
361
(s, 1H, NH); 13C NMR (100.6 MHz, DMSO-d6)
d
115 (2xCH), 121 (CH),
the crude material was purified by flash chromatography on
silica gel (CH2Cl2/NH3 99/1 then CH2Cl2/MeOH 98/2) to afford 51
as an orange solid in a 60% yield. Rf: 0.2 (petroleum ether/EtOAc
121 (2 ꢃ CH), 127 (Cq),130 (2 ꢃ CH),135 (Cq),139 (Cq),140 (Cq), 146
(CH), 147 (Cq), 150 (2 ꢃ CH), 160 (Cq), 160 (CH), 160 (Cq), 165 (Cq);
HRMS (EI-MS): calculated for C19H14N5O2 344.11470 [MþH]þ found
344.11440 [MþH]þ.
5/95); mp 232e234 ꢁC; IR (ATR Diamond, KBr, cmꢂ1
)
n
3474,
1607, 1445, 1198, 1072, 820, 714; 1H NMR (400 MHz, DMSO-d6)
d
6.73 (d, 2H, J ¼ 8.5 Hz, HPh), 7.67 (d, 2H, J ¼ 8.5 Hz, HPh), 8.13 (d,
6.1.24. 1-[2-(4-Hydroxyphenyl)pyrido[3,2-d]pyrimidin-7-yl]
piperidin-2-one 48
1H, J ¼ 1.5 Hz, H8), 8.63 (d, 1H, J ¼ 1.5 Hz, H6), 9.17 (s, 1H, H4),
9.26 (s, 1H, NH), 9.96 (s, 1H, OH); 13C NMR (100.6 MHz, DMSO-d6)
Compound 48 was obtained following the general procedure A
using piperidin-2-one which afforded the attempted derivative
after a purification under flash chromatography on silica gel
(CH2Cl2/MeOH 98/2) as a yellowish solid in a 79% yield. Rf: 0.2
(petroleum ether/EtOAc 5/95); mp 268e270 ꢁC; IR (ATR Diamond,
d
114 (2 ꢃ CH), 121 (CH), 131 (Cq), 131 (2 ꢃ CH), 134 (Cq), 134
(Cq), 145 (CH), 147 (Cq), 152 (Cq), 157 (Cq), 162 (CH); HRMS (EI-
MS): calculated for C13H10Cl35N4O 273.05430 [MþH]þ found
273.05390 [MþH]þ.
KBr, cmꢂ1
(400 MHz, DMSO-d6)
J ¼ 6.5 Hz, HPP), 3.88 (t, 2H, J ¼ 5.5 Hz, HPP), 6.92 (d, 2H, J ¼ 8.7 Hz,
)
n
3333, 1621, 1570, 1452, 1223, 1157, 807, 700; 1H NMR
6.1.28. (4-Methoxy-phenyl)-[7-(2-methoxy-phenyl)-pyrido[3,2-d]
pyrimidin-2-yl]-amine 52
d
1.89e1.95 (m, 4H, HPP), 2.53 (t, 2H,
Compound 52 was obtained from 51 as described for com-
pound 25 using 2-hydroxyphenyl boronic acid. Flash chromatog-
raphy using silica gel (CH2Cl2/NH3 99/1 then CH2Cl2/MeOH 98/2)
afforded the attempted product as an orange solid in 66% yield. Rf:
0.2 (petroleum ether/EtOAc 5/95); mp 262e264 ꢁC; IR (ATR Dia-
H
Ph), 8.21 (d, 1H, J ¼ 1.9 Hz, H8), 8.40 (d, 2H, J ¼ 8.7 Hz, HPh), 9.04 (d,
1H, J ¼ 1.9 Hz, H6), 9.57 (s, 1H, H4), 10.08 (s, 1H, OH); 13C NMR
(100.6 MHz, DMSO-d6) d 20 (CH2), 22 (CH2), 32 (CH2), 49 (CH2), 115
(2xCH),127 (CH), 127 (Cq),130 (2 ꢃ CH),135 (Cq),144 (Cq),146 (Cq),
150 (CH), 160 (Cq), 160 (Cq), 160 (CH), 170 (Cq); HRMS (EI-MS):
calculated for C18H17N4O2Na 343.11710 [MþH]þ found 343.11700
[MþH]þ.
mond, KBr, cmꢂ1
NMR (400 MHz, DMSO-d6)
7.05 (m, 2H, HPh), 7.26e7.32 (m, 1H, HPh), 7.48 (d, 1H, J ¼ 7.0 Hz,
Ph), 7.71 (d, 2H, J ¼ 8.6 Hz, HPh), 8.05 (s, 1H, H8), 8.87 (s, 1H, H6),
9.11 (s, 1H, H4), 9.24 (s, 1H, NH), 9.77 (s, 1H, OH), 10.00 (s, 1H, OH);
13C NMR (100.6 MHz, DMSO-d6)
)
n
3433, 1602, 1434, 1213, 1105, 800, 725; 1H
d
6.73 (d, 2H, J ¼ 8.6 Hz, HPh), 6.94e
H
6.1.25. 1-[2-(4-Hydroxyphenyl)pyrido[3,2-d]pyrimidin-7-yl]
piperazin-2-one 49
d
114 (2 ꢃ CH), 116 (CH), 119 (CH),
Compound 49 was obtained following the general procedure A
using piperazin-2-one which afforded the attempted derivative
after a purification under flash chromatography on silica gel
(CH2Cl2/NH3 99/1 then CH2Cl2/MeOH 98/2) as a yellowish solid in a
78% yield. Rf: 0.2 (petroleum ether/EtOAc 5/95); mp > 268 ꢁC; IR
120 (CH), 123 (Cq), 130 (CH), 130 (CH), 131 (2xCH), 131 (Cq), 134
(Cq), 139 (Cq), 147 (Cq), 148 (CH), 152 (Cq), 154 (Cq), 157 (Cq), 162
(CH); HRMS (EI-MS): calculated for C19H15N4O2 331.11950 [MþH]þ
found 331.12110 [MþH]þ.
(ATR Diamond, KBr, cmꢂ1
708; 1H NMR (400 MHz, DMSO-d6)
)
n
3495, 1639, 1566, 1448, 1261, 1161, 847,
3.10 (s, 2H, HPr), 3.16 (s, 1H,
6.1.29. (4-Hydroxy-phenyl)-[7-(3-hydroxy-phenyl)-pyrido[3,2-d]
pyrimidin-2-yl]-amine 53
d
NH), 3.52 (s, 2H, HPr), 3.88 (s, 2H, HPr), 6.93 (d, 2H, J ¼ 8.6 Hz, HPh),
8.27 (d, 1H, J ¼ 1.7 Hz, H8), 8.40 (d, 2H, J ¼ 8.6 Hz, HPh), 9.13 (d, 1H,
J ¼ 1.7 Hz, H6), 9.58 (s, 1H, H4), 10.11 (s, 1H, OH); 13C NMR
Compound 53 was obtained from 51 as described for compound
25 using 3-hydroxyphenyl boronic acid. Flash chromatography
using silica gel (CH2Cl2/NH3 99/1 then CH2Cl2/MeOH 98/2) afforded
the attempted product as an orange solid in 60% yield. Rf: 0.2 (pe-
troleum ether/EtOAc 5/95); mp > 268 ꢁC; IR (ATR Diamond, KBr,
(100.6 MHz, DMSO-d6)
d
42 (CH2), 50 (CH2), 50 (CH2), 115 (2 ꢃ CH),
126 (CH), 127 (Cq), 130 (2 ꢃ CH), 135 (Cq), 143 (Cq), 146 (Cq), 150
(CH), 160 (Cq), 160 (CH), 160 (Cq), 168 (CO); HRMS (EI-MS):
calculated for C17H16N5O2 322.13040 [MþH]þ found 322.13060
[MþH]þ.
cmꢂ1
) n
3476, 1595, 1399, 1219, 1170, 887, 794; 1H NMR (400 MHz,
DMSO-d6)
d
6.73 (d, 2H, J ¼ 8.7 Hz, HPh), 6.87e6.91 (m,1H, HPh), 7.23
(s, 1H, HPh), 7.30e7.35 (m, 2H, HPh), 7.72 (d, 2H, J ¼ 8.7 Hz, HPh), 8.08
(d,1H, J ¼ 1.4 Hz, H8), 8.93 (d,1H, J ¼ 1.4 Hz, H6), 9.11 (s, 1H, H4), 9.25
(s, 1H, NH), 9.70 (s, 1H, OH), 9.82 (s, 1H, OH); 13C NMR (100.6 MHz,
6.1.26. 1-[2-(4-Hydroxyphenyl)pyrido[3,2-d]pyrimidin-7-yl]
pyrrolidin-2-one 50
DMSO-d6)
d
114 (CH), 114 (2 ꢃ CH), 116 (CH), 118 (CH), 120 (CH), 129
Compound 50 was obtained following the general procedure A
using pyrrolidin-2-one which afforded the attempted derivative
after a purification under flash chromatography on silica gel
(CH2Cl2/MeOH 99/1) as a pinkish solid in a 93% yield. Rf: 0.2 (pe-
troleum ether/EtOAc 5/95); mp > 268 ꢁC; IR (ATR Diamond, KBr,
(CH), 130 (2 ꢃ CH), 131 (Cq), 135 (Cq), 137 (Cq), 140 (Cq), 145 (CH),
147 (Cq), 152 (Cq), 157 (Cq), 157 (Cq), 162 (CH); HRMS (EI-MS):
calculated for C19H15N4O2 331.11950 [MþH]þ found 331.12000
[MþH]þ.
cmꢂ1
(400 MHz, DMSO-d6)
)
n
3423, 1665, 1571, 1460, 1260, 1156, 806, 701; 1H NMR
2.11e2.19 (m, 2H, HPy), 2.60 (t, 2H, J ¼ 8.0 Hz,
6.1.30. (4-Hydroxy-phenyl)-[7-(4-hydroxy-phenyl)-pyrido[3,2-d]
pyrimidin-2-yl]-amine 54
d
H
Py), 4.03 (t, 2H, J ¼ 7.0 Hz, HPy), 6.92 (d, 2H, J ¼ 8.6 Hz, HPh), 8.30 (s,
Compound 54 was obtained from 51 as described for compound
25 using 4-hydroxyphenyl boronic acid. Flash chromatography
using silica gel (CH2Cl2/NH3 99/1 then CH2Cl2/MeOH 98/2) afforded
the attempted product as an orange solid in 80% yield. Rf: 0.2 (pe-
troleum ether/EtOAc 5/95); mp > 268 ꢁC; IR (ATR Diamond, KBr,
1H, H8), 8.37 (d, 2H, J ¼ 8.6 Hz, HPh), 9.50 (s, 2H, H6 and H4); 13C
NMR (100.6 MHz, DMSO-d6)
d 17 (CH2), 31 (CH2), 47 (CH2), 115
(2 ꢃ CH), 120 (CH), 127 (CH), 129 (2 ꢃ CH), 134 (Cq), 139 (Cq), 144
(Cq), 146 (Cq), 159 (CH), 160 (Cq), 160 (Cq), 174 (CO); HRMS (EI-MS):
calculated for C17H15N4O2 307.11895 [MþH]þ found 307.11904
[MþH]þ.
cmꢂ1
) n
3453, 1599, 1359, 1210, 1171, 819, 719; 1H NMR (400 MHz,
DMSO-d6)
d
6.74 (d, 2H, J ¼ 8.6 Hz, HPh), 6.91 (d, 2H, J ¼ 8.3 Hz, HPh
)
7.69e7.80 (m, 4H, HPh), 8.06 (s, 1H, H8), 8.97 (s, 1H, H6), 9.11 (s, 1H,
6.1.27. 7-Chloro-2-(4-hydroxyphenylamino)pyrido[3,2-d]
pyrimidine 51
In a solution of dioxane containing (200 mg, 1.0 mmol) of
compound 1, (131.0 mg, 1.19 mmol, 1.2 eq.) of 4-hydroxyaniline
was added and the reaction mixture was refluxed for 24 h. The
solution was cooled, dioxane was removed under pressure and
H4), 9.21 (s, 1H, NH), 9.76 (s, 1H, OH), 9.87 (s, 1H, OH); 13C NMR
(100.6 MHz, DMSO-d6)
d
114 (2 ꢃ CH), 116 (2 ꢃ CH), 120 (2 ꢃ CH),
126 (Cq),127 (CH),128 (2xCH),131 (Cq),134 (Cq),139 (Cq),145 (CH),
147 (Cq), 152 (Cq), 157 (Cq), 158 (Cq), 161 (CH); HRMS (EI-MS):
calculated for C19H15N4O2 331.11950 [MþH]þ found 331.11900
[MþH]þ.

362
O. Dehbi et al. / European Journal of Medicinal Chemistry 80 (2014) 352e363
6.2. Kinase preparations and assays
Acknowledgements
Buffer A: 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 25 mM TriseHCl
pH 7.5, 50 g heparin/ml. Buffer C: 60 mM -glycerolphosphate,
This work was supported by grants from EGIDE Volubilis pro-
gram, from the Ligue contre le Cancer (Comités du Grand Ouest)
and the Canceropôle Grand Ouest Strand “Valorisation des produits
de la mer”. This research was also supported by grants from the
Fonds Unique Interministériel (FUI) PHARMASEA project (LM), the
European Union 7th Framework Program (FP7) Micro-Therapy
project (LM), and the Association France-Alzheimer (Finistère)
(LM).
m
b
15 mM p-nitrophenylphosphate, 25 mM Mops (pH 7.2), 5 mM
EGTA, 15 mM MgCl2, 1 mM DTT, 1 mM sodium vanadate, 1.0 mM
phenylphosphate. Kinase activities were assayed in Buffer A or C, at
30 ꢁC, at a final ATP concentration of 15
mM [10]. Blank values were
subtracted and activities expressed in % of the maximal activity, i.e.
in the absence of inhibitors. Controls were performed with
appropriate dilutions of DMSO. CDK5/p25 (human, recombinant)
was prepared as previously described. Its kinase activity was
assayed in buffer C, with 1 mg histone H1/mL, in the presence of
Appendix A. Supplementary data
15
30
m
m
M [
g
-33P] ATP (3000 Ci/mmol; 10 mCi/mL) in a final volume of
L aliquots of super-
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.055.
L. After 30 min incubation at 30 ꢁC, 25
m
natant were spotted onto 2.5 ꢃ 3 cm pieces of Whatman P81
phosphocellulose paper, and 20 s later, the filters were washed five
times (for at least 5 min each time) in a solution of 10 mL phos-
phoric acid/liter of water. The wet filters were counted in the
presence of 1 mL ACS (Amersham) scintillation fluid. GSK-3ab
(porcine brain, native) was assayed, as described for CDK5/p25 but
References
[1] I. Shchemelinin, L. Sefc, E. Necas, Protein kinases, their function and
implication in cancer and other diseases, Folia Biologica (Praha) 52 (2006)
81e101.
[2] P.A. Cole, D. Sondhi, K. Kim, Chemical approaches to the study of protein
tyrosine kinases and their implications for mechanism and inhibitor design,
Pharmacology & Therapeutics 82 (1999) 219e229;
in Buffer
A and using a GSK-3 specific substrate (GS-1:
YRRAAVPPSPSLSRHSSPHQSpEDEEE) (pS stands for phosphorylated
serine). GS-1 was synthesized by Millegen (Labege, France). DYRK1A
(rat, recombinant, expressed in Escherichia coli as a GST fusion
protein) was purified by affinity chromatography on glutathionee
agarose and assayed as described for CDK5/p25 using myelin basic
protein (1 mg/mL) as a substrate [11].
H. Galons, N. Oumata, L. Meijer, Cyclin-dependent kinase inhibitors: a survey
of recent patent literature, Expert Opinion on Therapeutic Patents 20 (2010)
377e404;
H. Galons, N. Oumata, O. Gloulou, L. Meijer, Cyclin-dependent kinase in-
hibitors closer to market launch? Expert Opinion on Therapeutic Patents 23
(2013) 945e963.
[3] C. Neagoie, E. Vedrenne, F. Buron, J.Y. Mérour, S. Rosca, S. Bourg, O. Lozach,
L. Meijer, B. Baldeyrou, A. Lansiaux, S. Routier, Synthesis of chromeno[3,4-b]
indoles as Lamellarin D analogues: a novel DYRK1A inhibitor class, European
Journal of Medicinal Chemistry 49 (2012) 379e396;
6.3. Molecular modelling
F. Pin, F. Buron, F. Saab, L. Colliandre, S. Bourg, F. Schoentgen, R. Le Guével,
C. Guillouzo, S. Routier, Synthesis and biological evaluation of 2,3-bis(het)aryl-
4-azaindole derivatives as protein kinase inhibitors, Medicinal Chemistry
Communications 2 (2011) 899e903;
R. Boulahjar, A. Ouach A, M. Chiurato, S. Bourg, M. Ravache, R. Le Guével,
S. Marionneau, T. Oullier, O. Lozach, L. Meijer, C. Guguen-Guillouzo, S. Lazar,
M. Akssira, Y. Troin, G. Guillaumet, S. Routier, Novel tetrahydropyrido[1,2-a]
isoindolone derivatives (valmerins): potent cyclin-dependent kinase/glycogen
synthase kinase 3 inhibitors with antiproliferative activities and antitumor
effects in human tumor xenografts, Journal of Medicinal Chemistry 55 (2012)
9589e9606.
Hardware and software: molecular modelling studies were
performed with the Schrodinger Molecular Modelling Suite 2012
update 2 [12] where Maestro is the interface piloting the diverse
modules. Glide was used to dock ligands. Analysis and visualization
tasks were performed within MOE software [13]. Calculations were
run on a Linux station: Intel_ Xeon CPU W3670 @ 3.20 GHz.
Structure preparation:
Crystal structures were retrieved from the protein data bank:
[4] G. Paglini, A. Cáceres, The role of the Cdk5-p35 kinase in neuronal develop-
ment, European Journal of Biochemistry 268 (2001) 1528e1533;
M. Mapelli, L. Massimiliano, C. Crovace, M. Seeliger, L.H. Tsai, L. Meijer,
A. Musacchio, Mechanism of CDK5/p25 binding by CDK inhibitors, Journal of
Medicinal Chemistry 48 (2005) 671e679.
[5] R.S. Jope, C.J. Yuskaitis, E. Beurel, Glycogen synthase kinase-3 (GSK3):
inflammation, diseases, and therapeutics, Neurochemical Research 32 (2007)
577e595.
1. DYRK1A with PDB code 2vx3 [14]. Subunit A was conserved.
2. CDK5 with PDB code 3O0G [15]. The most complete subunit A
was conserved. Asparagine 144 was mutated to Aspartate in
DFG motif to respect human sequence.
3. GSK3b with PDB code 1q41 [16]. The most complete subunit B
[6] L.K. Chico, L.J. Van Eldik, D.M. Watterson, Targeting protein kinases in
central nervous system disorders, Nature Reviews Drug Discovery 8 (2009)
892e909;
was conserved again.
Structures were next prepared using the Protein Preparation
Wizard workflow of the Schrodinger Molecular Modelling Suite.
Proteins were preprocessed (hydrogen atoms added, incomplete
residues filled), bond orders and connections of ligands were
manually corrected. An exhaustive sampling was conducted
regarding hydrogen bond assignment and the complex was finally
refined by a minimization stage with a constraint to converge to a
L. Meijer, M. Flajolet, P. Greengard, Pharmacological inhibitors of glycogen
synthase kinase-3, Trends in Pharmacological Sciences 25 (2004) 471e480.
[7] P. Kassis, J. Brzeszcz, V. Bénéteau, O. Lozach, L. Meijer, R. Le Guével,
ꢁ
C. Guillouzo, K. Lewinski, S. Bourg, L. Colliandre, S. Routier, J.Y. Mérour, Syn-
thesis and biological evaluation of new 3-(6-hydroxyindol-2-yl)-5-(phenyl)
pyridine or pyrazine V-Shaped molecules as kinase inhibitors and cytotoxic
agents, European Journal of Medicinal Chemistry 46 (2011) 5416e5434. Smith
B, Medda F, Gokhale V, Dunckley T, Hulme C, Recent advances in the design,
synthesis, and biological evaluation of selective DYRK1A inhibitors: a new
avenue for a disease modifying treatment of Alzheimer’s?, ACS Chemical
Neuroscience. 3 (2012), 857e872.
ꢀ
structure with an RMSD of 0.3 A (OPLS2005 force field), essentially
in order to remove steric clashes. Ligands, other than the one co-
crystallized, were built within Accelrys Draw [17] and were sub-
mitted to Corina [18], a 3D structure generator. Next 3D structures
were submitted to the LigPrep module of the Schrodinger Molec-
ular Modelling Suite in order to take into account tautomerization
and ionization via the Epik module. The resulting structures
became the starting point for docking simulations.
Docking parameters: docking calculations were performed with
extra precision. Ligand flexibility was taken into account and the
option of sampling of ring conformation was activated.
[8] A. Tikad, O. Dehbi, M. Akssira, M. Aadil, S. Massip, J.M. Leger, C. Jarry,
G. Guillaumet, S. Routier, Efficient access to 2,7-diarylated pyrido[3,2-d]py-
rimidines involving regioselective pallado-dehalogenation and Suzuki cross-
coupling reactions, Synthesis 45 (2013) 491e500. S. Routier, G. Guillaumet,
A. Tikad, O. Dehbi, Preparation of Pyrido[3,2-d]pyrimidine Derivatives as In-
hibitors of CDK1, DYRK1A, CDK5 and/or GSK3 kinases, WO 2011135259,
Chem. Abstract 155:637809.
[9] F. Zuccotto, E. Ardini, E. Casale, M. Angiolini, Through the "gatekeeper door":
exploiting the active kinase conformation, Journal of Medicinal Chemistry 53
(2010) 2681e2694.
[10] J. Zhang, P.L. Yang, N.S. Gray, Targeting cancer with small molecule kinase
inhibitors, Nature Reviews Cancer 9 (2009) 28e39.

O. Dehbi et al. / European Journal of Medicinal Chemistry 80 (2014) 352e363
363
[11] T. Tahtouh, J.M. Elkins, P. Filippakopoulos, M. Soundararajan, G. Burgy,
E. Durieu, C. Cochet, R.S. Schmid, D.C. Lo, F. Delhommel, A.E. Oberholzer,
L.H. Pearl, F. Carreaux, J.P. Bazureau, S. Knapp, L. Meijer, Selectivity, cocrystal
structures, and neuroprotective properties of leucettines, a family of protein
kinase inhibitors derived from the marine sponge alkaloid leucettamine B,
Journal of Medicinal Chemistry 55 (2012) 9312e9330.
[12] Schrödinger, LLC, New York, 2012.
[13] Molecular Operating Environment, Version 2013.08, Chemical Computing
Group Inc., Montreal, Quebec, Canada, 2013.
[15] J.S. Ahn, M.L. Radhakrishnan, M. Mapelli, S. Choi, B. Tidor, G.D. Cuny,
A. Musacchio, L. Yeh, S.K. Kosik, Defining Cdk5 ligand chemical space with
small molecule inhibitors of tau phosphorylation, Chemistry & Biology 12
(2005) 811e823.
[16] J.A. Bertrand, S. Thieffine, A. Vulpetti, C. Cristiani, B. Valsasina, S. Knapp,
H.M. Kalisz, M. Flocco, Structural characterization of the GSK-3beta active site
using selective and non-selective ATP-mimetic inhibitors, Journal of Molec-
ular Biology 333 (2003) 393e407.
[17] Accelrys, Inc., Accelrys Draw 4.0, 2011.
[14] M. Soundararajan, A.K. Roos, P. Stavitsky, P. Filippakopoulos, A.N. Kettenbach,
J.V. Olsen, S.A. Gerber, J. Eswaran, S. Knapp, J.M. Elkins, Structures of Down
syndrome kinases, DYRKs, reveal mechanisms of kinase activation and sub-
strate recognition, Structure 21 (2013) 986e996.
[18] J. Sadowski, J. Gasteiger, From atoms and bonds to three-dimensional atomic
coordinates: automatic model builders, Chemical Reviews 93 (1993) 2567e
2581. http://www.molecular-networks.com/products/corina.