New pyrido[3,4-g]quinazoline derivatives as CLK1 and DYRK1A inhibitors: synthesis, biological evaluation and binding mode analysis

Article abstract of DOI:10.1016/j.ejmech.2019.01.052

Cdc2-like kinase 1 (CLK1) and dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) are involved in the regulation of alternative pre-mRNA splicing. Dysregulation of this process has been linked to cancer progression and neurodegenerative diseases, making CLK1 and DYRK1A important therapeutic targets. Here we describe the synthesis of new pyrido[3,4-g]quinazoline derivatives and the evaluation of the inhibitory potencies of these compounds toward CDK5, CK1, GSK3, CLK1 and DYRK1A. Introduction of aminoalkylamino groups at the 2-position resulted in several compounds with low nanomolar affinity and selective inhibition of CLK1 and/or DYRK1A. Their evaluation on several immortalized or cancerous cell lines showed varying degree of cell viability reduction. Co-crystal structures of CLK1 with two of the most potent compounds revealed two alternative binding modes of the pyrido[3,4-g]quinazoline scaffold that can be exploited for future inhibitor design.

Full text of DOI:10.1016/j.ejmech.2019.01.052

Accepted Manuscript
New pyrido[3,4-g]quinazoline derivatives as CLK1 and DYRK1A inhibitors: synthesis,
biological evaluation and binding mode analysis
Helmi Tazarki, Wael Zeinyeh, Yannick J. Esvan, Stefan Knapp, Deep Chatterjee,
Martin Schröder, Andreas C. Joerger, Jameleddine Khiari, Béatrice Josselin, Blandine
Baratte, Stéphane Bach, Sandrine Ruchaud, Fabrice Anizon, Francis Giraud, Pascale
Moreau
PII:
S0223-5234(19)30056-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.052
EJMECH 11059
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 October 2018
Revised Date: 18 January 2019
Accepted Date: 18 January 2019
Please cite this article as: H. Tazarki, W. Zeinyeh, Y.J. Esvan, S. Knapp, D. Chatterjee, M. Schröder,
A.C. Joerger, J. Khiari, Bé. Josselin, B. Baratte, Sté. Bach, S. Ruchaud, F. Anizon, F. Giraud, P.
Moreau, New pyrido[3,4-g]quinazoline derivatives as CLK1 and DYRK1A inhibitors: synthesis, biological
evaluation and binding mode analysis, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.01.052.
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ACCEPTED MANUSCRIPT
New pyrido[3,4-g]quinazoline derivatives as CLK1 and DYRK1A inhibitors: synthesis,
biological evaluation and binding mode analysis
Helmi Tazarki‡,§, Wael Zeinyeh‡, Yannick J. Esvan‡, Stefan Knappǁ,#, Deep Chatterjeeǁ,#, Martin
Schröderǁ,#, Andreas C. Joergerǁ,#, Jameleddine Khiari§, Béatrice Josselin†, Blandine Baratte†,
Stéphane Bach†, Sandrine Ruchaud†, Fabrice Anizon‡, Francis Giraud ‡,*, Pascale Moreau‡,*
‡
Université Clermont Auvergne, CNRS, SIGMA Clermont, ICCF, F-63000 Clermont-Ferrand,
France.
§ Carthage University, Laboratory of Organic and Analytical Chemistry (ISEFC), Tunis, Tunisia.
ǁ Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe University, Max-von-Laue-Str. 9,
60438, Frankfurt am Main, Germany.
#
Buchmann Institute for Molecular Life Sciences and Structural Genomics Consortium (SGC),
Max-von-Laue-Str. 15, 60438, Frankfurt am Main, Germany.
†
Sorbonne Université, CNRS, Plateforme de Criblage KISSf (Kinase Inhibitor Specialized
Screening Facility), Protein Phosphorylation and Human Diseases Unit, Station Biologique, Place
Georges Teissier, F-29688 Roscoff, France.
* Corresponding authors
(PM) Tel: +33 (0) 4 73 40 79 63. E-mail: pascale.moreau@uca.fr
(FG) Tel: +33 (0) 4 73 40 71 27. E-mail: francis.giraud@uca.fr
1

ACCEPTED MANUSCRIPT
Abstract
Cdc2-like kinase 1 (CLK1) and dual specificity tyrosine phosphorylation-regulated kinase 1A
(DYRK1A) are involved in the regulation of alternative pre-mRNA splicing. Dysregulation of this
process has been linked to cancer progression and neurodegenerative diseases, making CLK1 and
DYRK1A important therapeutic targets. Here we describe the synthesis of new pyrido[3,4-
g]quinazoline derivatives and the evaluation of the inhibitory potencies of these compounds toward
CDK5, CK1, GSK3, CLK1 and DYRK1A. Introduction of aminoalkylamino groups at the 2-
position resulted in several compounds with low nanomolar affinity and selective inhibition of
CLK1 and/or DYRK1A. Their evaluation on several immortalized or cancerous cell lines showed
varying degree of cell viability reduction. Co-crystal structures of CLK1 with two of the most
potent compounds revealed two alternative binding modes of the pyrido[3,4-g]quinazoline scaffold
that can be exploited for future inhibitor design.
Graphical abstract
NHCH3
N
NH(CH2)2NMe2
N
NH(CH2)3NMePiperazin-1
N N
N
N
O2N
O2N
O2N
N
N
N
9m
IC50 CLK1 = 18 nM
9a
IC50 CLK1 = 62 nM
9j
IC50 CLK1 = 133 nM
9m bound to CLK1 (PDB ID: 6Q8P)
2

ACCEPTED MANUSCRIPT
Introduction
Protein kinases are important cellular targets for the development of novel chemical probes and
drugs in many therapeutic areas, including cancer, neurodegenerative disorders, inflammation or
pain therapy. To date, more than five hundred protein kinases have been identified in the human
kinome. All of them share the same co-factor (ATP), and the ATP binding pocket shows high
structural conservation in most of them. Developing kinase inhibitors that display selectivity within
the human kinome, therefore, remains a major challenge [1]. However, as shown by FDA approved
drugs, especially in oncology, absolute selectivity for a single kinase is not always needed [2]. As
part of our ongoing efforts toward the development of potent and selective kinase inhibitors, we
recently described pyrido[3,4-g]quinazolines as inhibitors of Cdc2-like kinases (CLK1) and dual
specificity tyrosine phosphorylation-regulated kinases (DYRK1A) [3]. CLK1 and DYRK1A are
involved in the regulation of alternative pre-mRNA splicing via SR-protein phosphorylation, and
dysfunction of this tightly regulated process is linked to the progression of cancer,
neurodegenerative diseases, and viral infections [4,5]. As reported in recent reviews, various
chemical scaffolds could lead to potent DYRK1A and/or CLK1 inhibitors [6-9]. We carried out a
structure-activity relationship (SAR) study around the tricyclic pyridoquinazoline scaffold. Previous
results demonstrated that the substitution by nitro/amino groups at the 10-position is essential to
target CLK1 and/or DYRK1A kinases [3]. The introduction of alkyl/aryl substituents at the 5-
position led to a change in the kinase inhibition profile, as 5-substituted derivatives exhibited
improved potencies toward CDK5/GSK3, while CLK1/DYRK1A inhibition was impaired (Figure
1A) [10]. Since modification of this heteroaromatic scaffold resulted in a major change in activity
and selectivity, we decided to further extend the SAR studies on the pyrido[3,4-g]quinazoline
scaffold by varying the substitution at the 2-position (Figure 1A).
3

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Figure 1. Design strategy for the optimization of pyrido[3,4-g]quinazoline-based kinase inhibitors.
A) Structures of pyrido[3,4-g]quinazolines identified as CLK1/DYRK1A protein kinase inhibitors,
highlighting earlier SAR results (blue and red) [3,10] and the optimization strategy employed in this
work. B) Structure of the lead compound (R = NH2, R’ = H) bound to the ATP binding site of
CLK1 (PDB ID: 5J1V). Hydrogen bonds between the compound and CLK1 are shown as dashed
lines.
Results and discussion
Synthesis of compound library
Analysis of the binding mode of the best CLK1/DYRK1A inhibitor of the pyrido[3,4-
g]quinazolines series showed that the aminopyrimidine moiety forms two hydrogen-bonds with the
backbone amine and carbonyl group of Leu244 upon binding to CLK1 (Figure 1B). Additionally,
the pyridine nitrogen atom is hydrogen-bonded to the Lys191 side chain [3]. Our aim was to extend
4

ACCEPTED MANUSCRIPT
this lead compound by adding a series of aminoalkylamino groups at the 2-position to figure out if
this alters the potency or selectivity of the compounds. To confirm the importance of the amino
group at the pyrimidine moiety 2-position in the interaction with the targeted kinases, we first
synthesized presumed inactive analogues 2 and 3 missing the 2-amino function, using benzamidine
hydrochloride salt. Compound 2 was obtained in 22% yield by reacting isoquinoline 1, which was
prepared according to a previously reported procedure [3], with benzamidine hydrochloride salt.
The reduction of the nitro group by catalytic hydrogenation led quantitatively to 3 (Scheme 1).
Cl
O2N
CHO
a
N
N
N
N
b
O2N
H2N
N
N
N
1
2, 22%
3, quant.
O
O
c
d
H2N(CH2)nNR2
N(CH2)nBr
N(CH2)nNR2
7b–f, h–l
Method A, B or C
O
O
4 n = 2
5 n = 3
6b–f, h–l
(CH2)nNR2
HN
HN
e
H2N(CH2)nNR2
NH2.H2SO4
7a–l
8a–l
Scheme 1. Synthesis of compounds 2, 3 and diversely substituted guanidines 8a-l. Reagents and
conditions: (a) Benzamidine HCl, DMA (b) H2, Pd/C, CH2Cl2/MeOH 1:1 (c) Method A: 4 or 5,
5

ACCEPTED MANUSCRIPT
DIPEA, amine, DMF; Method B: 4 or 5, K2CO3, amine, CH3CN; Method C: 4 or 5, amine, toluene
(d) H2NNH2.H2O, EtOH (e) O-methylisourea bisulfate salt, H2O.
We then condensed 1 with diversely substituted guanidines in order to modify the substituent at
position 2. The preparation of guanidines that were not commercially available started from
phthalimides 4 or 5, which were reacted with various amines to give 6b-f, h-l in 40% to quantitative
yields (Scheme 1). This step was performed according to published procedures (method A [11],
method B [12], and method C [13]). After hydrazinolysis, amines 7b-f, h-l were obtained in
acceptable yields (43% to quantitative). Reaction of commercially available (7a, 7g) or prepared
amines (7b-f, h-l) with O-methylisourea bisulfate [14] led to the desired guanidines 8a-l in
acceptable to good yields (42% to 88%).
Next, different N-substituted-aminopyrimidines 9a-m were obtained by reaction between
isoquinoline 1 and the guanidine bisulfates. Reactions were performed using K2CO3 as a base in
DMA [15] under conventional heating conditions. Finally, the nitro derivatives were reduced to
their amino analogues 10a-m under hydrogen atmosphere to further extend the compound library
(Scheme 2).
NHR
N
NHR
N
N
Cl
N
H2N
O2N
CHO
O2N
a
b
N
N
N
1
9a–m
10a–m
Scheme 2. Synthesis of compounds 9a-m and 10a-m. Reagents and conditions: (a) Guanidine
hemisulfate 8a-m (8m commercially available as chlorhydrate salt), K2CO3, DMA (b) H2, Pd/C,
CH2Cl2 or CH2Cl2/MeOH (8:2).
Kinase inhibition assays
6

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The inhibitory potency toward CDK5/p25, CLK1, DYRK1A, CK1δ/ε and GSK-3α/β of nitro
derivatives 2, 9a-m, amino analogues 3, 10a-m, references 9 (R = H) and 10 (R = H) was evaluated
(Table 1).
Table 1. Kinase inhibition assays (% residual kinase activity).
Kinase inhibition (% residual activity and IC50values)1
Cpds
CDK5
CLK1
DYRK1A
10 µM
CK1
GSK3
10 µM
62
1 µM
100
92
10 µM
17
1 µM
71
1 µM
100
80
10 µM
57
1 µM
100
62
10 µM
100
1 µM
100
85
2
38
41
3
Ref 9 (R=H)
9a
65
32
68
51
40
(> 104 nM)
(69 nM)
(62 nM)
(620 nM)
(780 nM)
100
(2.2 103 nM)
52
100
5
22
17
89
36
21
70
9b
9c
9d
9e
9f
79
69
77
85
83
64
50
60
100
100
84
8
5
0
1
7
3
4
3
46
36
32
42
27
27
31
14
47
20
6
88
73
32
52
83
84
72
68
78
75
38
37
73
59
59
38
91
93
63
81
92
86
87
100
26
43
22
1
74
88
69
39
81
80
92
76
96
26
29
31
23
10
100
100
100
100
33
24
24
15
9g
9h
9i
(120 nM)
(133 nM)
(125 nM)
9j
48
53
100
100
8
7
22
16
3
6
54
56
32
34
92
2
59
76
9k
100
24
9l
50
38
100
73
5
2
37
5
6
87
39
37
45
100
98
24
51
78
70
9m
13
(18 nM)
(41 nM)
Ref 10 (R = H)
(4.8 103 nM)
(28 nM)
(5.8 103 nM)
(9.1 103 nM)
10a
10b
86
40
96
97
21
3
51
31
32
6
60
49
82
60
78
91
48
34
58
73
10c
10d
10e
10
55
76
52
88
95
4
4
27
21
44
1
17
34
70
52
77
100
74
24
22
24
76
44
43
(197 nM)*
19
(120 nM)
16 37
2
(210 nM)
11
7

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10f
51
34
100
77
4
1
33
14
7
1
54
17
61
61
90
96
29
10
62
36
10g
(120 nM)
(160 nM)
(290 nM)
(117 nM)
(120 nM)
(80 nM)
(23 nM)
(20 nM)
10h
10i
10j
20
23
15
68
67
49
2
4
6
21
20
8
2
6
1
24
5
45
24
30
68
53
61
22
6
61
41
35
4
4
10k
10l
62
44
100
86
4
4
30
26
4
1
26
25
48
23
76
69
30
23
51
50
(90 nM)
3
33
1
9
1
27
60
85
40
79
10m
(274 nM)*
1IC50 values were determined when the residual kinase activity was ≤ 25% at a compound concentration of 1
µM (given in parentheses). IC50 values for references 9 and 10 (R = H) as previously reported [3]. Kinase
activities were assayed in triplicate in the presence of 15 µM ATP. Typically, the standard deviation of
32
single data points was below 10%. All assays were performed using a P radioassay in the presence of 15
µM ATP (method A), except for the determination of the two IC50 values marked with an asterisk that was
carried out using the ADP-Glo assay in the presence of 10 µM ATP (method B).
CLK1 and DYRK1A were generally the most strongly inhibited kinases (Table 1). A similar
inhibition profile was already observed for the lead compound of this series [3]. As expected,
compounds 2 and 3, lacking the amino group at the 2-position of the pyrimidine moiety, did not
exhibit any significant inhibitory effect toward the kinases tested. These results are in accordance
with the binding mode of the lead compound for this series in the co-crystal structure with CLK1
(Figure 1B), showing that the amino group forms a hydrogen bond with Leu244 in the hinge region.
All N-2 substituted compounds of the nitro (9a-9m) or amino (10a-10m) series were active toward
CLK1 with residual kinase activities ≤ 50% when tested at 1 µM, indicating that the
aminoalkylamino groups at the 2-position did not cause major steric hindrance within the CLK1
pocket. DYRK1A inhibition, however, was generally less effective, in particular for the nitro series,
with only 5 derivatives (9d, 9e, 9j, 9k, 9m) leading to residual kinase activities around or < 50%
when tested at 1 µM. Thus, several nitro derivatives, particularly 9a, 9i-9k, and 9m, exhibited a
better activity toward CLK1. The best selectivity profiles for CLK1 over DYRK1A were found for
9a bearing a dimethylaminoethyl group and 9m bearing a methyl group, with IC50 values against
CLK1 of 62 nM and 18 nM, respectively.
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Amino derivatives 10a-10m displayed inhibitory potencies toward both CLK1 and DYRK1A in the
micro/submicromolar range, with the notable exception of compounds 10i and 10j. Those two
compounds with either a 3-(morpholin-1-yl)propyl) group (10i) or a 3-(N-methylpiperazin-1-
yl)propyl group (10j) were particularly active toward DYRK1A, with IC50 values of 23 nM and 20
nM, respectively, and a more than 5-fold selectivity for DYRK1A over CLK1 (12.6 for 10i, 5.9 for
10j). In comparison, amino analogue reference (10, R = H, Scheme 2) exhibited lower selectivity
with only 1.5 times better activity toward DYRK1A [3]. These results demonstrated an
improvement of selectivity in the amino series.
Biological evaluation in cancer cells
As CLK1 and DYRK1A upregulation plays a key role in various cancer types, the compounds
exhibiting the best kinase inhibitory potencies (9a, 9i, 9j, 9m, 10d, 10g, 10i, 10j, 10l) were
evaluated in hTERT-immortalized retinal pigment epithelial (RPE1) cells and four cancer cell lines,
HCT116 (colon carcinoma), MDA-MB231 (breast adenocarcinoma), SH-SY5Y (neuroblastoma),
and U-2 OS (bone osteosarcoma). Cellular activities are given as % cell viability at a compound
concentration of 2 µM compared with control DMSO treated cells (Table 2). Staurosporine was
used as a positive control. Treatment with many compounds resulted in less than 25% of viable
cells after 48 h in comparison to DMSO treated cells, both with the non-tumor and tumor cell lines.
Indeed, all compounds were active toward the cell lines tested and induced viability reductions of
more than 50%, except compounds 9i, 9m and 10d on SH-SY5Y cells and for 10l on hTERT-RPE1
and SH-SY5Y cells. The reference compound (10, R = H, Scheme 2) as well as three new 10-amino
derivatives, 10g, 10i and 10j, were particularly active toward all five cell lines, including hTERT-
RPE1, with only 0% to 15% of remaining cellular viability. This lack of selectivity suggests that
these amino analogues are too toxic to be considered as potential drug candidates.
Table 2. Effects of pyrido[3,4-g]quinazoline-based kinase inhibitors on the viability of cancer and
non-cancer cells.
hTERT-RPE1 HCT116 MDA-MB231
Viability
SH-SY5Y
U-2 OS
Viability
(%)
Compound
Viability1 (%)
Viability (%) Viability (%)
(%)
0.2 ± 0.4
29.2 ± 0.7
31 ± 3
1.3 ± 0.0
13.3 ± 0.8
16.8 ± 0.7
9.7 ± 0.5
43.4 ± 0.4
9.4 ± 0.6
39 ± 1
0.9 ± 0.3
42.7 ± 0.4
54 ± 2
0.4 ± 0.1
3.3 ± 0.8
27 ± 1
Ref 10 (R = H)
9a
9i
25.8 ± 0.3
11.7 ± 0.9
36 ± 2
2.5 ± 0.7
9j
9

ACCEPTED MANUSCRIPT19.5 ± 0.6
36 ± 1
19.4 ± 0.1
0 ± 0
22 ± 1
6.1 ± 0.9
0 ± 0
41.0 ± 0.6
21.7 ± 0.4
0.4 ± 0.1
15 ± 1
52 ± 2
62 ± 3
9m
13 ± 1
0 ± 0
10d
0.0 ± 0.0
3.6 ± 0.9
0.1 ± 0.1
92.2 ± 0.3
9.4 ± 0.1
10g
0.0 ± 0.1
0 ± 0
0.8 ± 0.1
0 ± 0
6.4 ± 0.9
0.1 ± 0.1
33 ± 2
10i
10j
3.6 ± 0.8
22 ± 1
52 ± 2
21 ± 6
27 ± 2
10l
Staurosporine
9.1 ± 0.8
3.9 ± 0.5
0.4 ± 0.4
1Cellular viability (%) at a compound concentration of 2 µM after 48 hours compared with untreated cells.
Cell viability was assayed in triplicate, and mean value ± SD are given.
Most interesting compounds, 9a and 9j, were slightly more potent in killing U-2 OS cancer cells
than hTERT-RPE1 cells, showing that, compared to 10 (R = H), selectivity could be enhanced by
introducing alkyl or aminoalkyl groups on the amine group at the 2-position of the pyrido[3,4-
g]quinazoline scaffold. However, a more detailed study of this series cellular effect would be of
valuable interest to evaluate its potential, as well as additional structural modifications to further
enhance the selectivity of this compound class for cancer cells.
Structural analysis of the binding mode of compounds 9m and 10i in complex with CLK1
To provide insights into the binding modes of our 2-amino-substituted pyrido[3,4-g]quinazolines,
we determined the crystal structures of 9m and 10i in complex with CLK1 at 3.0 and 2.3 Å
resolution, respectively. Interestingly, for compound 10i bearing an amino group at the 10-position
and a morpholinopropylamino substituent at the 2-position, we observed two overlapping
alternative binding modes of this highly symmetrical molecule where the central ring system is
tightly packed between a series of hydrophobic side chains, including Val175, Phe241, Leu295, and
Val324 (Figure 2A/B). In both orientations, a nitrogen atom of the pyrido[3,4-g]quinazoline
scaffold forms a hydrogen bond with the backbone nitrogen of Leu244 in the hinge region, and a
nitrogen in the distal ring forms a water-mediated hydrogen bond with the Glu206 side chain in the
back pocket. Details of this interaction network in the back pocket with corresponding distances are
shown in Figure 2C. The secondary amino group introduced at C-2 of the tricycle interacts with
either the backbone oxygen of Leu244 (as seen for the lead compound) or points in the direction of
the Lys191-Glu206 salt bridge, but without forming additional electrostatic interactions with the
latter. There was no clear electron density to unambiguously model the morpholino group in both
cases, indicating that it is very flexible and does not interact with CLK1 in a defined orientation.
This may explain why this compound is less potent against CLK1 than against DYRK1A.
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The CLK1-9m complex was solved in a different crystal form with three molecules in the
asymmetric unit. There was a preferred binding mode of the inhibitor, with the 2-aminomethyl
substituent facing the Lys191-Glu206 salt bridge and packing against the aromatic ring of Phe172
(Figure 2D). In this orientation, the pyridine nitrogen forms a hydrogen bond with the Leu244
backbone nitrogen in the hinge region (3.0 Å distance). The presence of a minor second
conformation, however, cannot be ruled out. The distance between the Lys191 side chain amine and
the exocyclic nitrogen of 9m is 3.2-3.6 Å, and the distance to the proximal ring nitrogen of the
pyrimidine is 3.7-3.9 Å, just outside the range for hydrogen bonding. It is likely that the compound
also forms a water-mediated hydrogen bond with Glu206 and the backbone nitrogen of Asp325 (as
seen in the CLK1 complex with 10i), which could not be unambiguously modelled due to the low
resolution of the data set. Overall, this arrangement enables favorable electrostatic interactions in
the back pocket and might be further stabilized via the adjacent side chain carboxylate of Asp325,
which is within 4 Å distance of the exocyclic nitrogen. To assess the contribution of the different
side chains or structural waters to stabilizing this binding mode in more detail, a high-resolution
structure would be needed.
Interestingly, the lead compound that formed the starting point for this SAR study adopts a flipped
orientation where the 2-amino group forms a hydrogen bond with the backbone oxygen of Leu244,
and the pyridine nitrogen interacts with the Lys191 side chain (Figure 1B) [3]. Taken together,
comparison of the new CLK1-inhibitor complexes with the binding mode of the parent molecule
indicates that the pyrido[3,4-g]quinazoline scaffold can bind in two alternative orientations,
depending on the nature of the ring substituents, which can be exploited for the design of inhibitors
with improved potency and selectivity.
11

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Figure 2. Structures of 2-substituted pyrido[3,4-g]quinazolines bound to CLK1. (A-B) Crystal
structure of the CLK1-10i complex. Compound 10i bound in two alternative orientations shown as
yellow (A) and green (B) stick models, respectively. Parts of the inhibitor that were not clearly
resolved in the crystal structure are shown as transparent sticks. The inset shows a 2Fo-Fc electron
density map for the modeled inhibitor at a contour level of 1.0 σ. Hydrogen bonds between the
inhibitor and the CLK1 hinge region and a water-mediated contact with Glu206 are highlighted
with dashed lines. (C) Close-up view of the water-mediated hydrogen bond in the back-pocket
region of the CLK1-10i complex. Relevant distances for electrostatic interactions are highlighted as
green dashed lines, and the corresponding values are given in Å. For clarity, only one conformer is
shown. Distances to the pyrimidine ring nitrogen of the second conformer are given in parentheses.
(D) Crystal structure of the CLK1-9m complex (chain B). The inset shows a 2Fo-Fc electron density
map for the modeled inhibitor at a contour level of 1.4 σ. The hydrogen bond of the inhibitor with
12

ACCEPTED MANUSCRIPT
the hinge region is highlighted as a green dashed line. To confirm a potential water-mediated
hydrogen bond of one of the pyrimidine nitrogens, as observed in the CLK1-10i complex, a higher
resolution than the 3.0 Å of the current structure would be needed.
Conclusion
A new series of pyrido[3,4-g]quinazolines with diverse substitutions at the 2-position and either an
amino or a nitro group at the 10-position was synthesized and evaluated against five protein kinases
(CDK5/p25, CLK1, DYRK1A, CK1δ/ε, and GSK-3α/β). The results demonstrated that the
aminopyrimidine part is essential to the kinase inhibitory potency of this series (compounds 2 and
3). The most strongly inhibited kinases were CLK1 and DYRK1A, with a better selectivity toward
CLK1 for 10-nitro derivatives (9a, 9m), whereas 10-amino derivatives were more selective toward
DYRK1A (10i, 10j). Evaluation of the cellular activities of the most active compounds revealed
that compounds 9a and 9j are slightly more active toward U-2 OS cancer cells, compared with a
non-cancer cell line (hTERT-RPE1). Interestingly, structural analysis of the binding modes of two
of the most potent binders showed that they can adopt alternative binding modes in the ATP-
binding pocket of CLK1, depending on the substitution pattern of the heteroaromatic scaffold.
Taken together our data provide a framework for the development of potent and selective
CLK1/DYRK1A inhibitors for potential treatment of cancers and neurodegenerative diseases.
Experimental section
4.1 Chemistry
4.1.1. General
Starting materials were obtained from commercial suppliers and used without further purification.
Solvents were distilled prior to use. IR spectra were recorded on a Shimadzu FTIR-8400S
spectrometer ( in cm-1). NMR spectra, performed on a Bruker AVANCE 400 (1H: 400 MHz, 13C:
100 MHz), are reported in ppm using the solvent residual peak as an internal standard; the
following abbreviations are used: singlet (s), doublet (d), triplet (t), quadruplet (q), quintet (quint),
doublet of doublet (dd), multiplet (m), broad signal (br s). High resolution mass spectra (ESI+) were
determined on a high-resolution Waters Micro Q-Tof apparatus (UCA-Partner, Université Clermont
Auvergne, Clermont-Ferrand, France). Chromatographic purifications were performed by column
chromatography using 40–63 µm silica gel. Reactions were monitored by TLC using fluorescent
silica gel plates (60 F254 from Merck). Melting points were measured on a Stuart SMP3 apparatus
and are uncorrected. The purity of key compounds 9a, 9j, 9m, 10i and 10j was established to be >
13

ACCEPTED MANUSCRIPT
95% by HPLC analysis using a Hitachi liquid chromatograph (Oven 5310, 30 °C; Pump 5160;
DAD detector 5430) and a C18 Acclaim column (4.6 mm x 250 mm, 5 µm, 120 Å). Detection
wavelength was 240 nm for nitro derivative/280 nm for amino analogues, and flow rate 0.5
mL/min. Gradient elution used (A) water/0.1% TFA; (B) acetonitrile: 95:5 A/B for 5 min then 95:5
A/B to 5:95 A/B in 25 min and then 5:95 A/B for 10 min.
4.1.2 10-Nitro-2-phenylpyrido[3,4-g]quinazoline (2)
A suspension of compound 1 (50 mg, 0.21 mmol) and benzamidine hydrochloride (66 mg, 0.42
mmol) in DMA (2 mL) was degassed with argon for 30 min then heated at 75 °C (oil bath) for a
total time of 2 h. After completion of the reaction, EtOAc was added. The resulting slurry was
filtered on a pad of Celite and washed with EtOAc. The organic layer was washed with water and
brine, dried (MgSO4) and the volatiles were removed under reduced pressure. The residue was
purified twice by flash chromatography using firstly EtOAc/cyclohexane (7:3) and secondly
CH2Cl2/AcOEt (1:1), yielding compound 2 (14 mg, 0.046 mmol, 22%) as a light brown-yellow
powder. Mp > 260 °C; Rf = 0.2 (EtOAc/Cyclohexane 7:3). IR (ATR): 1627, 1594, 1565, 1525,
1
1425, 1381, 1293, 1271, 1148 cm-1. H NMR (400 MHz, DMSO-d6) δ 7.60 – 7.71 (3H, m), 7.89
(1H, d, J = 6.3 Hz), 8.56 – 8.63 (2H, m), 8.78 (1H, d, J = 6.3 Hz), 9.53 (1H, s), 9.89 (1H, d, J = 1.1
13
Hz), 10.23 (1H, s); C NMR (100 MHz, DMSO-d6): Not recorded due to low solubility. HRMS
(ESI+) calcd for C17H11N4O2 (M+H)+ 303.0876, found 303.0879.
4.1.3 2-Phenylpyrido[3,4-g]quinazolin-10-amine (3)
To a solution of 11 mg (0.036 mmol) of compound 2 in 4 mL of CH2Cl2/MeOH 1:1 at room
temperature was added 2 mg of Pd/C. The suspension was stirred under 1 atm of H2 in the dark at
room temperature overnight. After filtration over Celite in a Pasteur pipette, washing with CH2Cl2
and concentration, compound 3 (9.7 mg, 0.036 mmol, quant. yield) was isolated without further
purification as a dark red powder. Mp: degradation; Rf = 0.25 (EtOAc/Cyclohexane 7:3). IR (ATR):
1
3420-2602, 1623, 1591, 1541, 1405, 1362 cm-1. H NMR (400 MHz, DMSO-d6) δ 7.39 (2H, br s,
NH2), 7.55-7.60 (3H, m), 7.99 (1H, s), 8.28 (1H, d, J = 6.4 Hz), 8.38 (1H, d, J = 6.4 Hz), 8.78 (2H,
13
dd, J = 9.6 Hz, J = 1.6 Hz), 9.43 (1H, s), 9.81 (1H, s); C NMR (100 MHz, DMSO-d6) δ 111.1,
115.9 (CHarom), 128.3 (2CHarom), 128.7 (2CHarom), 130.6, 139.8, 154.8, 163.4 (CHarom), 114.9,
122.4, 127.5, 132.3, 137.5, 141.6, 156.1 (Carom). HRMS (ESI+) calcd for C17H13N4 (M+H)+
273.1135, found 273.1135.
14

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4.1.4 2-(2-N,N-Diethylaminoethyl)-1H-isoindole-1,3(2H)-dione (6b): prepared according to method
C [13] in 98% yield as an orange yellow solid. Mp: 44-45 °C; Rf = 0.1 (EtOAc/cyclohexane 1:2). IR
1
(ATR): 2967, 2809, 1706, 1434, 1386 cm-1. H NMR (400 MHz, DMSO-d6) δ 0.86 (6H, t, J = 7.2
Hz), 2.44 (4H, q, J = 7.2 Hz), 2.59 (2H, t, J = 6.8 Hz), 3.63 (2H, t, J = 6.8 Hz), 7.81-7.88 (4H, m);
13C NMR (100 MHz, DMSO-d6) δ 12.0 (2CH3), 35.8 (CH2), 46.6 (2CH2), 49.5 (CH2), 123.0
(2CHarom), 134.4 (2CHarom), 131.6 (2Carom), 167.9 (2CO).
4.1.5 2-(2-Morpholin-4-ylethyl)-1H-isoindole-1,3(2H)-dione (6c): prepared according to method A
[11] in 80% yield as a beige solid. Mp: 139-140 °C; Rf = 0.15 (EtOAc/cyclohexane 1:2). IR (ATR):
1
2792, 1703, 1436, 1395 cm-1. H and 13C NMR spectra were in accordance with published data
[11].
4.1.6 2-(2-(N-Methylpiperazin-4-yl)ethyl)-1H-isoindole-1,3(2H)-dione (6d): prepared according to
method B [12] in 73% yield as a yellow oil. Rf = 0.1 (EtOAc/cyclohexane 1:2). IR (ATR): 2946,
1
2796, 1702, 1437, 1400 cm-1. H NMR (400 MHz, DMSO-d6) δ 2.09 (3H, s), 2.20-2.25 (4H, m),
13
2.27-2.40 (4H, m), 2.51 (2H, t, J = 5.2 Hz), 3.68 (2H, t, J = 5.2 Hz), 7.81-7.89 (4H, m). C NMR
(100 MHz, DMSO-d6) δ 45.7 (CH3), 35.0 (CH2), 52.5 (2CH2), 54.7 (2CH2), 55.1 (CH2), 123.0
(2CHarom), 134.4 (2CHarom), 131.6 (2Carom), 167.8 (2CO).
4.1.7 2-(2-Piperidinylethyl)-1H-isoindole-1,3(2H)-dione (6e): prepared according to method B [12]
in a quantitative yield as a brown solid. Mp: 75-76 °C; Rf = 0.1 (EtOAc/cyclohexane 1:2). IR
1
(ATR): 2945, 1704, 1436, 1396 cm-1. H NMR (400 MHz, DMSO-d6) δ 1.31-1.42 (6H, m), 2.34-
13
2.37 (4H, m), 2.47 (2H, t, J = 5.2 Hz), 3.68 (2H, t, J = 5.2 Hz), 7.81-7.89 (4H, m); C NMR (100
MHz, DMSO-d6) δ 23.9 (CH2), 25.6 (2CH2), 35.1 (CH2), 53.9 (2CH2), 55.8 (CH2), 123.0 (2CHarom),
134.4 (2CHarom), 131.6 (2Carom), 167.8 (2CO).
4.1.8 2-(2-Pyrrolidinylethyl)-1H-isoindole-1,3(2H)-dione (6f): prepared according to method C [13]
in 40% yield as a pale yellow solid. Mp: 101-102 °C; Rf = 0.3 (CH2Cl2/EtOH/Et3N 9:1:1). IR
1
(ATR): 2976, 2792, 1702, 1442, 1390 cm-1. H NMR (400 MHz, DMSO-d6) δ 1.61-1.64 (4H, m),
13
2.44-2.48 (4H, m), 2.63 (2H, t, J = 6.8 Hz), 3.68 (2H, t, J = 6.8 Hz), 7.82-7.87 (4H, m); C NMR
(100 MHz, DMSO-d6) δ 23.1 (2CH2), 36.7, 53.2 (CH2), 53.5 (2CH2), 123.0 (2CHarom), 134.4
(2CHarom), 131.6 (2Carom), 167.8 (2CO).
15

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4.1.9 2-(3-Diethylaminopropyl)-1H-isoindole-1,3(2H)-dione (6h): prepared according to method C
1
[13] in 93% yield. Mp and H NMR data were in accordance with published data [16]. IR (ATR):
2968, 2805, 1705, 1394, 1366 cm-1. 13C NMR (100 MHz, DMSO-d6) δ 11.5 (2CH3), 25.4, 36.1
(CH2), 46.1 (2CH2), 50.0 (CH2), 122.9 (2CHarom), 134.3 (2CHarom), 131.7 (2Carom), 167.9 (2CO).
4.1.10 2-(3-Morpholinylpropyl)-1H-isoindole-1,3(2H)-dione (6i): prepared according to method A
[11] in 95% yield as a brown oil. Rf = 0.1 (EtOAc/cyclohexane 1:1). IR (ATR): 2954, 2813, 1702,
1395, 1361 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 1.73 (2H, quint, J = 6.8 Hz), 2.21-2.23 (4H, m),
13
2.30 (2H, t, J = 6.8 Hz), 3.32-3.41 (4H, m), 3.64 (2H, t, J = 6.8 Hz), 7.82-7.88 (4H, m); C NMR
(100 MHz, DMSO-d6) δ 24.0, 36.2 (CH2), 53.2 (2CH2), 56.0 (CH2), 66.0 (2CH2), 122.9 (2CHarom),
134.2 (2CHarom), 131.8 (2Carom), 168.0 (2CO).
4.1.11 2-(3-(N-Methylpiperazin-4-yl)propyl)-1H-isoindole-1,3(2H)-dione (6j): prepared according
to method B [12] in 81% yield as a brown solid. Mp: 62-63 °C. Rf = 0.1 (EtOAc/cyclohexane 1:1).
IR (ATR): 2933, 2805, 1706, 1392, 1359 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 1.72 (2H, quint, J
= 6.8 Hz), 1.97 (3H, s), 1.98-2.32 (8H, m), 2.29 (2H, t, J = 6.4 Hz), 3.63 (2H, t, J = 6.8 Hz), 7.82-
13
7.88 (4H, m); C NMR (100 MHz, DMSO-d6): 45.6 (CH3), 24.1, 36.4 (CH2), 52.6 (2CH2), 54.4
(2CH2), 55.7 (CH2), 123.0 (2CHarom), 134.2 (2CHarom), 132.0 (2Carom), 168.0 (2CO).
4.1.12 2-(3-Piperidinylpropyl)-1H-isoindole-1,3(2H)-dione (6k): prepared according to method A
1
13
[11] in 82% yield. Mp, H and C NMR were in accordance with published data [17]. Rf = 0.1
(EtOAc/cyclohexane 1:1). IR (ATR): 2932, 2771, 1697, 1395 cm-1.
4.1.13 2-(3-Pyrrolidinylpropyl)-1H-isoindole-1,3(2H)-dione (6l): prepared according to method B
[12] in quantitative yield as a brown oil. Rf = 0.1 (EtOAc/cyclohexane 1:1). IR (ATR): 2959, 2792,
1
1706, 1394, 1366 cm-1. H NMR (400 MHz, DMSO-d6) δ 1.48-1.52 (4H, m), 1.73 (2H, quint, J =
6.8 Hz), 2.28-2.35 (4H, m), 2.40 (2H, t, J = 6.8 Hz), 3.63 (2H, t, J = 6.8 Hz), 7.81-7.88 (4H, m); 13C
NMR (100 MHz, DMSO-d6) δ 22.9 (2CH2), 26.7, 36.2, 53.2 (CH2), 53.4 (2CH2), 122.8 (2CHarom),
134.2 (2CHarom), 131.8 (2Carom), 168.0 (2CO).
General procedures for the preparation of compounds 7b-f and 7h-l.
To a solution of compounds 6b-l (9.15 mmol) in 30 mL EtOH was added hydrazine monohydrate
(36.6 mmol; 4 eq.) and the mixture was vigorously stirred under reflux for 24 h. The white solid
16

ACCEPTED MANUSCRIPT
was filtered off and washed with EtOH. Filtrate was concentrated under vacuum to dryness and
taken up with EtOAc. The solid residue was filtered again and filtrate was concentrated in vacuo to
afford the desired products 7b-7l. NH2 signals are missing for all compounds in 1H NMR spectra.
4.1.14 2-(N,N-(Diethylamino))ethanamine (7b): brown orange oil obtained in quantitative yield. IR
1
(ATR): 3368-2500, 1445, 1371, 1068 cm-1. H NMR (400 MHz, DMSO-d6) δ 0.93 (6H, t, J = 7.2
13
Hz), 2.34 (2H, t, J = 6.8 Hz), 2.43 (4H, q, J = 7.2 Hz), 2.53 (2H, t, J = 6.8 Hz); C NMR (100
MHz, DMSO-d6) δ 11.9 (2CH3), 39.7 (CH2), 46.7 (2CH2), 56.0 (CH2).
4.1.15 2-Morpholin-4-ylethanamine (7c): orange oil obtained in 95% yield. 1H, 13C NMR and mass
spectra were in accordance with published data [11]. IR (ATR): 3568-2579, 1456, 1274, 1113 cm-1.
4.1.16 2-(N-methylpiperazin-1-yl)ethanamine (7d): orange oil obtained in 56% yield. NMR spectra
were in accordance with published data [18,19]. IR (ATR): 3631-2394, 1462, 1286, 1148 cm-1.
4.1.17 2-(Piperidin-1-yl)ethanamine (7e): orange oil obtained in 43% yield. IR (ATR): 3566-2500,
1
1443, 1307, 1098 cm-1. H NMR (400 MHz, DMSO-d6) δ 1.33-1.38 (2H, m), 1.44-1.51 (4H, m),
2.22 (2H, t, J = 6.4 Hz), 2.24-2.32 (4H, m), 2.58 (2H, t, J = 6.8 Hz); 13C NMR (100 MHz, DMSO-
d6) δ 24.2 (CH2), 25.6 (2CH2), 38.8 (CH2), 54.3 (2CH2), 61.9 (CH2).
4.1.18 2-(Pyrrolidin-1-yl)ethanamine (7f): orange oil obtained in 82% yield. IR (ATR): 3579-2500,
1
1462, 1305, 1141 cm-1. H NMR (400 MHz, DMSO-d6) δ 1.63-1.67 (4H, m), 2.37 (2H, t, J = 7.2
13
Hz), 2.36-2.41 (4H, m), 2.59 (2H, t, J = 7.2 Hz); C NMR (100 MHz, DMSO-d6) δ 23.1 (2CH2),
40.7 (CH2), 53.7 (2CH2), 59.2 (CH2).
4.1.19 3-(N,N-Diethylamino)propanamine (7h): yellow oil obtained in 73% yield. IR (ATR): 3395-
2710, 1467, 1381, 1202, 1069 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 0.92 (6H, t, J = 7.2 Hz), 1.42
(2H, quint, J = 7.2 Hz), 2.36 (2H, t, J = 7.2 Hz), 2.41 (4H, t, J = 7.2 Hz), 2.52 (2H, t, J = 6.8 Hz);
13C NMR (100 MHz, DMSO-d6) δ 11.8 (2CH3), 30.8, 40.1 (CH2), 46.3 (2CH2), 50.2 (CH2).
4.1.20 3-(Morpholin-4-yl)propanamine (7i): brown oil obtained in 86% yield. IR (ATR): 3579-
2618, 1448, 1307, 1271, 1113 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 1.47 (2H, quint, J = 7.2 Hz),
17

ACCEPTED MANUSCRIPT
13
2.27 (2H, t, J = 7.2 Hz), 2.25-2.32 (4H, m), 2.53 (2H, t, J = 7.2 Hz), 3.55 (4H, t, J = 4.4 Hz); C
NMR (100 MHz, DMSO-d6) δ 53.5 (3CH2), 56.2 (CH2), 66.3 (3CH2).
4.1.21 3-(N-Methylpiperazin-1-yl)propanamine (7j): brown oil obtained in 86% yield. IR (ATR):
3461-2395, 1448, 1283, 1163 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 1.45 (2H, quint, J = 5.6 Hz),
13
2.13 (3H, s), 2.23-2.45 (8H, m), 2.26 (2H, t, J = 5.6 Hz), 2.52 (2H, t, J = 5.2 Hz); C NMR (100
MHz, DMSO-d6) δ 45.8 (CH3), 30.3, 40.0 (CH2), 52.8 (2CH2), 54.8 (2CH2), 55.8 (CH2).
4.1.22 3-(Piperidin-1-yl)propanamine (7k): light brown oil obtained in 92% yield. Data in
1
accordance with H and 13C NMR spectra already described [17]. IR (ATR): 3457-2511, 1561,
1468, 1442, 1304, 1156 cm-1.
4.1.23 3-(Pyrrolidin-1-yl)propanamine (7l): brown orange oil obtained in 51% yield. IR (ATR):
3500-2579, 1591, 1460, 1350, 1141 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 1.48 (2H, quint, J = 7.2
Hz), 1.60-1.70 (4H, m), 2.30-2.43 (6H, m), 2.54 (2H, t, J = 7.2 Hz); 13C NMR (100 MHz, DMSO-
d6) δ 23.0 (2CH2), 32.5, 40.1, 53.6 (CH2), 53.7 (2CH2).
Synthetic procedures for the preparation of compounds 8a-l.
To a solution of primary amines 7a-l (2 eq) in water (0.5 mol/L) was added O-methylisourea
bisulfate (1 eq). Solution was stirred at 100 °C for 24 h. After concentration to dryness, ethanol was
added and the residue was sonicated until apparition of a precipitate. In some difficult cases, the
suspension in ethanol was stored overnight in a freezer and triturated with cold ethanol to get a
powder. Addition of a few drops of diethylether was also occasionally needed to induce
precipitation. The solids 8a-l were collected by filtration, washed with ethanol and dried overnight
under vacuum. Due to the hygroscopicity of isolated solids, no melting points were determined for
these series. NH signals are missing for all compounds in 1H NMR spectra.
4.1.24 1-(2-(N,N-Dimethylamino)ethyl)guanidine (8a): obtained as a white powder in 73% yield. IR
(ATR): 3500-2344, 1694, 1637, 1480, 1061 cm-1.1H NMR (400 MHz, CD3OD/D2O 9:1) δ 2.92
(6H, s), 3.36 (2H, t, J = 6.0 Hz), 3.66 (2H, t, J = 6.0 Hz); 13C NMR (100 MHz, CD3OD/D2O 9:1) δ
44.0 (2CH3), 37.5, 56.7 (CH2), 158.3 (C).
18

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4.1.25 1-(2-(N,N-Diethylamino)ethyl)guanidine (8b): obtained as a white powder in 58% yield. IR
1
(ATR): 3434-2342, 1620, 1396, 1028 cm-1. H NMR (400 MHz, CD3OD/D2O 9:1) δ 1.33 (6H, t, J
= 7.2 Hz), 3.23 (4H, q, J = 7.2 Hz), 3.33 (2H, t, J = 6.4 Hz), 3.67 (2H, t, J = 6.4 Hz); 13C NMR (100
MHz, CD3OD/D2O 9:1) δ 9.0 (2CH3), 37.6 (2CH2), 38.5 (CH2), 51.7 (CH2), 158.7 (C).
4.1.26 1-(2-(Morpholin-1-yl)ethyl)guanidine (8c): obtained as a beige powder in 45% yield. IR
1
(ATR): 3552-2368, 1408, 1019 cm-1. H NMR (400 MHz, CD3OD/D2O 9:1) δ 2.75-2.85 (4H, m),
13
2.93 (2H, t, J = 6.4 Hz), 3.22 (2H, t, J = 6.4 Hz), 3.83 (4H, t, J = 4.8 Hz); C NMR (100 MHz,
CD3OD/D2O 9:1) δ 36.4 (CH2), 53.9 (2CH2), 55.5 (CH2), 66.9 (2CH2), 158.4 (C).
4.1.27 1-(2-(N-Methylpiperazin-1-yl)ethyl)guanidine (8d): obtained as a white powder in 73% yield.
IR (ATR): 3447-2197, 1682, 1624, 1458, 1038 cm-1. 1H NMR (400 MHz, CD3OD/D2O 9:1) δ 2.73-
2.79 (4H, m), 2.87 (3H, s), 2.86-2.93 (4H, m), 3.31-3.38 (4H, m); 13C NMR (100 MHz,
CD3OD/D2O 9:1) δ 44.0 (CH3), 39.5 (2CH2), 50.7 (CH2), 54.5 (2CH2), 56.3 (CH2), 158.6 (C).
4.1.28 1-(2-(Piperidin-1-yl)ethyl)guanidine (8e): obtained as a white powder in 69% yield. IR
1
(ATR): 3553-2237, 1687, 1626, 1457, 1038 cm-1. H NMR (400 MHz, CD3OD/D2O 9:1) δ 2.00-
2.42 (6H, m), 2.84-3.75 (4H, m), 3.36 (4H, t, J = 6.4 Hz); 13C NMR (100 MHz, CD3OD/D2O 9:1) δ
23.9 (2CH2), 26.1, 39.4, 53.2 (CH2), 55.1 (2CH2), 158.3 (C).
4.1.29 1-(2-(Pyrrolidin-1-yl)ethyl)guanidine (8f): obtained as a white powder in 66% yield. IR
(ATR): 3553-2237, 1633, 1407, 1034 cm-1. 1H NMR (400 MHz, CD3OD/D2O 9:1) δ 2.10-2.13 (4H,
13
m), 3.31-3.44 (4H, m), 3.55 (2H, t, J = 6.4 Hz), 3.66 (2H, t, J = 6.0 Hz); C NMR (100 MHz,
CD3OD/D2O 9:1) δ 23.9 (2CH2), 38.6 (CH2), 49.2 (2CH2), 55.4 (CH2), 158.5 (C).
4.1.30 1-(3-(N,N-Dimethylamino)propyl)guanidine (8g): obtained as a white powder in 88% yield.
IR (ATR): 3605-2395, 1678, 1629, 1485, 1075 cm-1. 1H NMR (400 MHz, CD3OD/D2O 9:1) δ 2.06
(2H, quint, J = 8.0 Hz), 2.89 (6H, s), 3.20 (2H, t, J = 8.0 Hz), 3.32 (2H, t, under solvent signal); 13C
NMR (100 MHz, CD3OD/D2O 9:1) δ 43.7 (2CH3), 24.8, 39.2, 55.9 (CH2), 158.1 (C).
4.1.31 1-(3-(N,N-Diethylamino)propyl)guanidine (8h): obtained as a white powder in 62% yield. IR
1
(ATR): 3498-2395, 1689, 1637, 1483, 1038 cm-1. H NMR (400 MHz, CD3OD/D2O 9:1) δ 1.31
(6H, t, J = 7.2 Hz), 1.98-2.08 (2H, m), 3.15-3.26 (2H, m), 3.22 (4H, q, J = 7.2 Hz), 3.28-3.33 (2H,
19

ACCEPTED MANUSCRIPT
m); 13C NMR (100 MHz, CD3OD/D2O 9:1) δ 9.0 (2CH3), 24.3, 39.4 (CH2), 48.1 (2CH2), 50.2
(CH2), 158.2 (C).
4.1.32 1-(3-(Morpholin-1-yl)propyl)guanidine (8i): obtained as a beige powder in 42% yield. IR
1
(ATR): 3605-2184, 1684, 1633, 1480, 1042 cm-1. H NMR (400 MHz, CD3OD/D2O 9:1) δ 2.00-
2.18 (2H, m), 3.18-3.55 (8H, m), 3.89-4.07 (4H, m); 13C NMR (100 MHz, CD3OD/D2O 9:1) δ 23.8,
39.3 (CH2), 52.9 (2CH2), 55.3 (CH2), 64.9 (2CH2), 158.3 (C).
4.1.33 1-(3-(N-Methylpiperazin-1-yl)propyl)guanidine (8j): obtained as a white powder in 54%
yield. IR (ATR): 3433-2574, 1685, 1641, 1474, 1027 cm-1. 1H NMR (400 MHz, CD3OD/D2O 9:1) δ
1.86 (2H, quint, J = 7.2 Hz), 2.64-2.72 (2H, m), 2.75 (3H, s), 2.74-3.32 (8H, m), 3.25 (2H, t, J = 7.2
Hz); 13C NMR (100 MHz, CD3OD/D2O 9:1) δ 44.1 (CH3), 25.8 (CH2), 39.9 (2CH2), 51.0, 53.8
(CH2), 54.8 (2CH2), 158.2 (C).
4.1.34 1-(3-(Piperidin-1-yl)propyl)guanidine (8k): obtained as a white powder in 83% yield. IR
1
(ATR): 3316-2737, 1680, 1629, 1389, 1028 cm-1. H NMR (400 MHz, CD3OD/D2O 9:1) δ 1.71-
2.00 (6H, m), 2.07 (2H, quint, J = 7.2 Hz), 2.84-2.96 (2H, m), 3.12-3.17 (2H, m), 3.29 (2H, t, J =
7.2 Hz), 3.46-3.59 (2H, m); 13C NMR (100 MHz, CD3OD/D2O 9:1) δ 22.4 (CH2), 23.9 (2CH2),
24.2, 39.4 (CH2), 54.3 (2CH2), 55.0 (CH2), 158.1 (C).
4.1.35 1-(3-(Pyrrolidin-1-yl)propyl)guanidine (8l): obtained as a white powder in 74% yield. IR
1
(ATR): 3552-2118, 1674, 1632, 1479, 1027 cm-1. H NMR (400 MHz, CD3OD/D2O 9:1) δ 1.45-
1.80 (2H, m), 1.60-2.34 (4H, m), 2.69-3.78 (6H, m), 3.69 (2H, t, J = 6.4 Hz); 13C NMR (100 MHz,
CD3OD/D2O 9:1) δ 22.5 (CH2), 23.8 (2CH2), 37.0 (CH2), 54.7 (2CH2), 56.1 (CH2), 158.4 (C).
Synthetic procedure for the preparation of compounds 9a-m.
To a solution of previously described compound 1 (50 mg, 0.211 mmol, 1.1 eq) in 0.8 mL of N,N-
dimethylacetamide were successively added the guanidinium salts 8a-m (1 eq) and potassium
carbonate (87 mg, 0.633 mmol, 3.3 eq). The suspension was heated at 70 °C for 25 to 150 min.
Reaction mixture was filtered over Celite and washed with EtOAc. After evaporation of the
solvents, the crude residue was purified by column chromatography using a mixture of CH2Cl2/NH3
7N solution in MeOH from 98:2 to 96:4.
For each compound, two rotamers are observed by NMR but only the major one is described.
20

ACCEPTED MANUSCRIPT
4.1.36 N-(2-(N,N-Dimethylamino)ethyl)-10-nitropyrido[3,4-g]quinazolin-2-amine (9a): 90 min
reaction time, obtained as an orange yellow powder in 14% yield. Mp: 224-225 °C; Rf = 0.35
(CH2Cl2/NH3 7N solution in MeOH 96:4). IR (ATR): 3266-2359, 1581, 1521, 1342, 1186 cm-1. 1H
NMR (400 MHz, DMSO-d6) δ 2.22 (6H, s), 2.45-2.55 (2H, m, under solvent signal), 3.46-3.52 (2H,
m), 7.57 (1H, d, J = 6.0 Hz), 8.51 (1H, t, J = 6.4 Hz, NH), 8.58 (1H, d, J = 6.4 Hz), 8.98 (1H, s),
9.535 (1H, s), 9.543 (1H, s); 13C NMR (100 MHz, DMSO-d6) δ 44.8 (2CH3), 38.5, 57.1 (CH2),
112.3, 134.3, 146.3, 155.0, 165.1 (CHarom), 119.9, 122.0, 128.7, 136.5, 141.3, 159.5 (Carom). HRMS
(ESI+) calcd for C15H17N6O2 (M+H)+ 313.1413, found 313.1405. HPLC: purity > 96%, λ = 240 nm,
tR = 18.5 min.
4.1.37 N-(2-(N,N-Diethylamino)ethyl)-10-nitropyrido[3,4-g]quinazolin-2-amine (9b): 25 min
reaction time, obtained as an orange yellow powder in 14% yield. Mp: 134-135 °C; Rf = 0.35
(CH2Cl2/NH3 7N solution in MeOH 96:4). IR (ATR): 3447-2342, 1584, 1525, 1355 cm-1. 1H NMR
(400 MHz, DMSO-d6) δ 0.97 (6H, t, J = 6,8 Hz), 2.40-2.78 (6H, m), 3.40-3.47 (2H, m), 7.56 (1H,
d, J = 6.4 Hz), 8.49 (1H, t, J = 6.4 Hz, NH), 8.58 (1H, d, J = 6.4 Hz), 8.98 (1H, s), 9.53 (1H, s), 9.54
13
(1H, s); C NMR (100 MHz, DMSO-d6) δ 11.9 (2CH3), 1CH2 under solvent signal, 46.8 (2CH2),
50.6 (CH2), 112.3, 134.2, 146.2, 155.0, 165.0 (CHarom), 119.9, 122.0, 128.7, 136.5, 141.4, 159.5
(Carom). HRMS (ESI+) calcd for C17H21N6O2 (M+H)+ 341.1721, found 341.1712.
4.1.38 N-(2-Morpholinoethyl)-10-nitropyrido[3,4-g]quinazolin-2-amine (9c): 140 min reaction
time, obtained as an orange powder in 12% yield. Mp: 211-212 °C; Rf = 0.25 (acetone). IR (ATR):
1
3595-2543, 1584, 1527, 1361, 1304, 1113 cm-1. H NMR (400 MHz, DMSO-d6) δ 2.41-2.58 (6H,
m), 3.50 (2H, t, J = 6.4 Hz), 3.54 (4H, t, J = 4.4 Hz), 7.57 (1H, d, J = 6.4 Hz), 8.52 (1H, t, J = 5.6
Hz, NH), 8.58 (1H, d, J = 6.0 Hz), 8.98 (1H, s), 9.537 (1H, s), 9.543 (1H, s); 13C NMR (100 MHz,
DMSO-d6) δ 37.9 (CH2), 53.3 (2CH2), 56.7 (CH2), 66.2 (2CH2), 112.3, 134.3, 146.3, 155.0, 165.1
(CHarom), 119.9, 122.0, 128.7, 136.5, 141.4, 159.5 (Carom). HRMS (ESI+) calcd for C17H19N6O3
(M+H)+ 355.1513, found 355.1508.
4.1.39 N-(2-(N-Methylpiperazin-1-yl)ethyl)-10-nitropyrido[3,4-g]quinazolin-2-amine (9d): 45 min
reaction time, obtained as an orange yellow powder in 15% yield. Mp: 177-178 °C; Rf = 0.20
(CH2Cl2/NH3 7N solution in MeOH 96:4). IR (ATR): 3355-2158, 1583, 1523, 1347, 1153 cm-1. 1H
NMR (400 MHz, DMSO-d6) δ 2.12 (3H, s), 2.20-2.55 (8H, m), 2.54 (2H, t, J = 7.2 Hz), 3.46-3.52
21

ACCEPTED MANUSCRIPT
(2H, m), 7.57 (1H, d, J = 6.0 Hz), 8.48 (1H, t, J = 6.0 Hz, NH), 8.58 (1H, d, J = 6.0 Hz), 8.98 (1H,
s), 9.53 (1H, s), 9.54 (1H, s); 13C NMR (100 MHz, DMSO-d6) δ 45.7 (CH3), 38.3 (CH2), 52.6
(2CH2), 54.7 (2CH2), 56.2 (CH2), 112.4, 134.3, 146.3, 155.0, 165.1 (CHarom), 119.9, 122.0, 128.7,
136.5, 141.4, 159.5 (Carom). HRMS (ESI+) calcd for C18H22N7O2 (M+H)+ 368.1830, found
368.1826.
4.1.40 N-(2-(Piperidin-1-yl)ethyl)-10-nitropyrido[3,4-g]quinazolin-2-amine (9e): 45 min reaction
time, obtained as an orange powder in 30% yield. Mp: 125-126 °C; Rf = 0.20 (CH2Cl2/NH3 7N
solution in MeOH 96:4). IR (ATR): 3421-2355, 1583, 1525, 1342, 1123 cm-1. 1H NMR (400 MHz,
DMSO-d6) δ 1.34-1.38 (2H, m), 1.41-1.50 (4H, m), 2.25-2.60 (6H, m), 3.46-3.52 (2H, m), 7.57
(1H, d, J = 6.0 Hz), 8.48 (1H, t, J = 6.0 Hz, NH), 8.58 (1H, d, J = 6.0 Hz), 8.98 (1H, s), 9.53 (1H,
s), 9.54 (1H, s); 13C NMR (100 MHz, DMSO-d6) δ 24.0 (CH2), 25.6 (2CH2), 53.6 (CH2), 54.1
(2CH2), 56.9 (CH2), 112.3, 134.2, 146.3, 155.0, 165.0 (CHarom), 119.9, 122.0, 128.8, 135.4, 141.4,
159.5 (Carom). HRMS (ESI+) calcd for C18H21N6O2 (M+H)+ 353.1721, found 353.1729.
4.1.41 N-(2-(Pyrrolidin-1-yl)ethyl)-10-nitropyrido[3,4-g]quinazolin-2-amine (9f): 25 min reaction
time, obtained as an orange powder in 15% yield. Mp: 158-159 °C; Rf = 0.20 (CH2Cl2/NH3 7N
1
solution in MeOH 96:4).IR (ATR): 3434-2342, 1583, 1524, 1346, 1151 cm-1. H NMR (400 MHz,
DMSO-d6) δ 1.60-1.75 (4H, m), 2.40-2.60 (6H, m, under solvent signal), 3.46-3.54 (2H, m), 7.57
(1H, d, J = 6.4 Hz), 8.55 (1H, t, J = 6.8 Hz, NH), 8.58 (1H, d, J = 6.0 Hz), 8.98 (1H, s), 9.536 (1H,
s), 9.544 (1H, s); 13C NMR (100 MHz, DMSO-d6) δ 23.2 (2CH2), 1 CH2 under solvent signal, 53.6
(2CH2), 54.0 (CH2), 112.3, 134.3, 146.3, 155.0, 165.1 (CHarom), 119.9, 122.0, 128.8, 136.5, 141.4,
159.5 (Carom). HRMS (ESI+) calcd for C17H19N6O2 (M+H)+ 339.1564, found 339.1557.
4.1.42 N-(3-(N,N-Dimethylamino)propyl)-10-nitropyrido[3,4-g]quinazolin-2-amine (9g): 45 min
reaction time, obtained as an orange yellow powder in 16% yield. Rf = 0.15 (CH2Cl2/NH3 7N
solution in MeOH 97:3). Mp: 136-137 °C; IR (ATR): 3375-2453, 1586, 1521, 1355, 1294 cm-1. 1H
NMR (400 MHz, DMSO-d6) δ 1.74 (2H, quint, J = 7.2 Hz), 2.14 (6H, s), 2.29 (2H, t, J = 7.2 Hz),
3.35-3.40 (2H, m), 7.56 (1H, d, J = 6.8 Hz), 8.57 (1H, d, J = 6.4 Hz), 8.65 (1H, t, J = 5.6 Hz, NH),
8.97 (1H, s), 9.52 (1H, s), 9.54 (1H, s); 13C NMR (100 MHz, DMSO-d6) δ 45.1 (2CH3), 26.0, 56.8
(CH2), 112.3, 134.1, 146.2, 154.9, 164.9 (CHarom), 119.9, 122.0, 128.7, 136.5, 141.4, 159.4 (Carom).
The missing CH2 was under the solvent signal. HRMS (ESI+) calcd for C16H19N6O2 (M+H)+
327.1569, found 327.1540.
22

ACCEPTED MANUSCRIPT
4.1.43 N-(3-(N,N-Diethylamino)propyl)-10-nitropyrido[3,4-g]quinazolin-2-amine (9h): 45 min
reaction time, obtained as a ochre yellow powder in 19% yield. Mp: 151-152 °C; Rf = 0.20
(CH2Cl2/NH3 7N solution in MeOH 96:4). IR (ATR): 3316-2355, 1584, 1526, 1355, 1205 cm-1. 1H
NMR (400 MHz, DMSO-d6) δ 0.95 (6H, t, J = 7.2 Hz), 1.73 (2H, quint, J = 7.2 Hz), 2.40-2.61 (6H,
m), 3.35.3.42 (2H, m), 7.56 (1H, d, J = 6.4 Hz), 8.57 (1H, d, J = 6.0 Hz), 8.68 (1H, t, J = 6.0 Hz,
NH), 8.97 (1H, s), 9.52 (1H, s), 9.54 (1H, s); 13C NMR (100 MHz, DMSO-d6) δ 11.6 (2CH3), 25.5
(CH2), 46.20 (2CH2), 46.24 (CH2), 50.1 (CH2), 112.3, 134.1, 146.2, 155.0, 165.0 (CHarom), 119.9,
121.9, 128.7, 136.5, 141.4, 159.4 (Carom). HRMS (ESI+) calcd for C18H23N6O2 (M+H)+ 355.1877,
found 355.1881.
4.1.44 N-(3-Morpholinopropyl)-10-nitropyrido[3,4-g]quinazolin-2-amine (9i): 45 min reaction
time, obtained as an orange yellow powder in 24% yield. Mp: 209-210 °C; Rf = 0.20 (CH2Cl2/NH3
1
7N solution in MeOH 96:4). IR (ATR): 3289-2342, 1583, 1530, 1357, 1144 cm-1. H NMR (400
MHz, DMSO-d6) δ 1.77 (2H, quint, J = 6.0 Hz), 2.30-2.40 (6H, m), 3.39-3.44 (2H, m), 3.54-3.59
(4H, m), 7.56 (1H, d, J = 4.8 Hz), 8.57 (1H, d, J = 4.8 Hz), 8.65 (1H, t, J = 4.8 Hz, NH), 8.97 (1H,
13
s), 9.52 (1H, s), 9.54 (1H, s); C NMR (100 MHz, DMSO-d6) δ 24.9, 39.4 (CH2), 53.2 (2CH2),
55.9 (CH2), 66.3 (2CH2), 112.3, 134.1, 146.3, 155.0, 165.0 (CHarom), 119.9, 121.9, 128.7, 136.5,
141.4, 159.5 (Carom). HRMS (ESI+) calcd for C18H21N6O3 (M+H)+ 369.1670, found 369.1667.
4.1.45 N-(3-(N-Methylpiperazin-1-yl)propyl)-10-nitropyrido[3,4-g]quinazolin-2-amine (9j): 60 min
reaction time, obtained as an orange yellow powder in 17% yield. Mp: 192-193 °C; Rf = 0.20
(CH2Cl2/NH3 7N solution in MeOH 96:4). IR (ATR): 3289-2342, 1584, 1524, 1345, 1141 cm-1. 1H
NMR (400 MHz, DMSO-d6) δ 1.75 (2H, quint, J = 5.2 Hz), 2.13 (2H, t, J = 5.2 Hz), 2.16 (3H, s),
2.17-2.60 (8H, m), 3.37-3.44 (2H, m), 7.57 (1H, d, J = 5.2 Hz), 8.57 (1H, d, J = 4.8 Hz), 8.64 (1H,
t, J = 4.8 Hz, NH), 8.97 (1H, s), 9.52 (1H, s), 9.54 (1H, s); 13C NMR (100 MHz, DMSO-d6) δ 45.7
(CH3), 25.3 (CH2), 1 CH2 under solvent signal, 52.6 (2CH2), 54.8 (2CH2), 55.5 (CH2), 112.3, 134.1,
146.2, 155.0, 165.0 (CHarom), 119.9, 121.9, 128.7, 136.5, 141.4, 159.4 (Carom). HRMS (ESI+) calcd
for C19H24N7O2 (M+H)+ 382.1986, found 382.1987. HPLC: purity > 95%, λ = 240 nm, tR = 18.3
min.
4.1.46 N-(3-(Piperidin-1-yl)propyl)-10-nitropyrido[3,4-g]quinazolin-2-amine (9k): 45 min reaction
time, obtained as a brown orange powder in 22% yield. Mp: 185-186 °C; Rf = 0.20 (CH2Cl2/NH3
23

ACCEPTED MANUSCRIPT
1
7N solution in MeOH 97:3). IR (ATR): 3289-2316, 1587, 1530, 1359, 1152 cm-1. H NMR (400
MHz, DMSO-d6) δ 1.34-1.52 (6H, m), 1.76 (2H, quint, J = 6.8 Hz), 2.31-2.36 (6H, m), 3.36-3.42
(2H, m), 7.56 (1H, d, J = 6.0 Hz), 8.57 (1H, d, J = 6.0 Hz), 8.67 (1H, t, J = 5.2 Hz, NH), 8.97 (1H,
13
s), 9.52 (1H, s), 9.53 (1H, s); C NMR (100 MHz, DMSO-d6) δ 24.1, 25.2 (CH2), 25.6 (2CH2),
1CH2 under solvent signal, 54.0 (2CH2), 56.3 (CH2), 112.3, 134.1, 146.3, 155.0, 165.0 (CHarom),
119.9, 121.9, 128.7, 136.5, 141.4, 159.4 (Carom). HRMS (ESI+) calcd for C19H23N6O2 (M+H)+
367.1877, found 367.1882.
4.1.47 N-(3-(Pyrrolidin-1-yl)propyl)-10-nitropyrido[3,4-g]quinazolin-2-amine (9l): 45 min reaction
time, obtained as an orange yellow powder in 15% yield. Mp: 172-173 °C; Rf = 0.20 (CH2Cl2/NH3
1
7N solution in MeOH 97:3). IR (ATR): 3368-2342, 1583, 1525, 1349, 1147 cm-1. H NMR (400
MHz, DMSO-d6) δ 1.68-1.72 (4H, m), 1.78 (2H, quint, J = 6.8 Hz), 2.40-2.52 (6H, m), 3.38-3.45
(2H, m), 7.56 (1H, d, J = 6.0 Hz), 8.57 (1H, d, J = 6.4 Hz), 8.65 (1H, t, J = 5.2 Hz, NH), 8.97 (1H,
s), 9.52 (1H, s), 9.53 (1H, s); 13C NMR (100 MHz, DMSO-d6) δ 23.1 (2CH2), 27.3, 39.6, 53.4
(CH2), 53.6 (2CH2), 112.3, 134.2, 146.3, 155.0, 165.0 (CHarom), 119.9, 121.9, 128.7, 136.5, 141.4,
159.5 (Carom). HRMS (ESI+) calcd for C18H21N6O2 (M+H)+ 353.1721, found 353.1722.
4.1.48 N-Methyl-10-nitropyrido[3,4-g]quinazolin-2-amine (9m): 45 min reaction time, obtained as
a yellow powder in 12% yield. Mp: 224-225 °C; Rf = 0.45 (CH2Cl2/NH3 7N solution in MeOH
97:3). IR (ATR): 3344-2502, 1586, 1508, 1382, 1351, 1295 cm-1. 1H NMR (400 MHz, DMSO-d6) δ
2.93 (3H, d, J = 4.8 Hz), 7.57 (1H, d, J = 6.4 Hz), 8.51-8.56 (1H, m, NH), 8.59 (1H, d, J = 6.4 Hz),
8.99 (1H, s), 9.53 (1H, s), 9.55 (1H, s); 13C NMR (100 MHz, DMSO-d6) δ 27.9 (CH3), 112.3, 134.2,
146.3, 155.0, 164.9 (CHarom), 119.9, 122.0, 128.8, 136.6, 141.4, 159.9 (Carom). HRMS (ESI+) calcd
for C12H10N5O2 (M+H)+ 256.0834, found 256.0815. HPLC: purity > 98%, λ = 240 nm, tR = 18.9
min.
Synthetic procedure for the preparation of compounds 10a-m.
To a solution of nitro derivatives 9a-m in anhydrous CH2Cl2 (0.04 mol/L) was added 6 mg/mmol of
10% Pd/C at room temperature. The suspension was stirred in the dark under 1 atm of H2 until
completion of the reaction (4 to 40 hours). The reaction mixture was filtered over Celite in a Pasteur
pipette, washed with CH2Cl2 and evaporated. The product was not purified. Two rotamers were
observed by NMR, but only the major one is described.
24

ACCEPTED MANUSCRIPT
4.1.49 N2-(2-(N,N-Dimethylamino)ethyl)pyrido[3,4-g]quinazoline-2,10-diamine (10a): 18
h
reaction time, obtained as a red oil in 65% yield. Rf = 0.35 (CH2Cl2/NH3 7N solution in MeOH
96:4). IR (ATR): 3500-2407, 1607, 1575, 1372, 1291 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 2.30
(6H, s), 2.45-2.65 (2H, m, under solvent signal), 3.60-3.65 (2H, m), 6.31 (2H, br s, NH2), 7.40 (1H,
br s, NH), 7.84 (1H, s), 7.99 (1H, d, J = 6.0 Hz), 8.22 (1H, d, J = 6.0 Hz), 9.24 (1H, s), 9.33 (1H, s);
13C NMR (100 MHz, DMSO-d6) δ 45.0 (2CH3), 29.0, 38.6 (CH2), 113.5, 115.3, 139.5, 154.7, 164.4
(CHarom), 119.1, 121.3, 124.4, 136.1, 142.5, 157.2 (Carom). HRMS (ESI+) calcd for C15H19N6
(M+H)+ 283.1666, found 283.1665.
4.1.50 N2-(2-(N,N-Diethylamino)ethyl)pyrido[3,4-g]quinazoline-2,10-diamine (10b): 24 h reaction
time, obtained as a red oil in 82% yield. Rf = 0.15 (CH2Cl2/NH3 7N solution in MeOH 96:4). IR
(ATR): 3531-2189, 1606, 1574, 1372, 1028 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 1.00 (6H, t, J =
7.2 Hz), 2.57 (4H, q, J = 7.2 Hz), 2.62-2.71 (2H, m), 3.50-3.57 (2H, m), 6.28 (2H, br s, NH2), 7.39
(1H, br s, NH), 7.83 (1H, s), 7.96 (1H, d, J = 6.0 Hz), 8.22 (1H, d, J = 6.4 Hz), 9.22 (1H, s), 9.31
13
(1H, s); C NMR (100 MHz, DMSO-d6) δ 11.9 (2CH3), 1 CH2 under solvent signal, 46.7 (2CH2),
50.5 (CH2), 113.7, 115.3, 139.6, 154.7, 164.4 (CHarom), 119.2, 121.3, 124.4, 136.0, 142.3, 159.5
(Carom). HRMS (ESI+) calcd for C17H23N6 (M+H)+ 311.1979, found 311.1979.
4.1.51 N2-(2-(Morpholin-1-yl)ethyl)pyrido[3,4-g]quinazoline-2,10-diamine (10c): 24 h reaction
time, obtained as a red oil in 59% yield. Rf = 0.20 (CH2Cl2/NH3 7N solution in MeOH 96:4). IR
(ATR): 3465-2449, 1606, 1575, 1372, 1112 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 2.50-2.62 (6H,
m), 3.56-3.65 (6H, m), 6.30 (2H, br s, NH2), 7.42 (1H, br s, NH), 7.83 (1H, s), 7.99 (1H, d, J = 6.4
13
Hz), 8.21 (1H, d, J = 6.4 Hz), 9.22 (1H, s), 9.32 (1H, s); C NMR (100 MHz, DMSO-d6) δ 24.4,
38.0 (CH2), 53.4 (2CH2), 66.2 (2CH2), 113.6, 115.3, 139.5, 154.7, 164.9 (CHarom), 119.1, 121.2,
124.4, 136.1, 142.2, 159.7 (Carom). HRMS (ESI+) calcd for C17H21N6O (M+H)+ 325.1771, found
325.1770.
4.1.52 N2-(2-(N-Methylpiperazin-1-yl)ethyl)pyrido[3,4-g]quinazoline-2,10-diamine (10d): 24 h
reaction time, obtained as a red oil in 64% yield. Rf = 0.15 (CH2Cl2/NH3 7N solution in MeOH
96:4). IR (ATR): 3552-2598, 1608, 1576, 1374, 1147 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 2.14
(3H, s), 2.25-2.60 (10H, m), 3.56-3.62 (2H, m), 6.30 (2H, br s, NH2), 7.38 (1H, br s, NH), 7.82 (1H,
13
s), 7.99 (1H, d, J = 7.2 Hz), 8.21 (1H, d, J = 7.2 Hz), 9.22 (1H, s), 9.32 (1H, s); C NMR (100
MHz, DMSO-d6) δ 45.7 (CH3), 38.4 (CH2), 52.7 (2CH2), 54.7 (2CH2), 56.3 (CH2), 113.6, 115.4,
25

ACCEPTED MANUSCRIPT
139.6, 154.7, 164.7 (CHarom), 120.0, 121.3, 124.4, 136.1, 142.2, 159.6 (Carom). HRMS (ESI+) calcd
for C18H24N7 (M+H)+ 338.2088, found 338.2087.
4.1.53 N2-(2-(Piperidin-1-yl)ethyl)pyrido[3,4-g]quinazoline-2,10-diamine (10e): 24 h reaction
time, obtained as a red oil in 44% yield. Rf = 0.15 (CH2Cl2/NH3 7N solution in MeOH 97:3). IR
1
(ATR): 3482-2362, 1607, 1574, 1303, 1128 cm-1. H NMR (400 MHz, DMSO-d6): 1.34-1.62 (6H,
m), 2.25-2.60 (6H, m), 3.56-3.65 (2H, m), 6.31 (2H, br s, NH2), 7.37 (1H, br s, NH), 7.83 (1H, s),
8.00 (1H, d, J = 5.6 Hz), 8.22 (1H, d, J = 5.6 Hz), 9.23 (1H, s), 9.33 (1H, s); 13C NMR (100 MHz,
DMSO-d6): 23.7 (CH2), 25.4 (2CH2), 38.2 (CH2), 1CH2 under solvent signal, 54.1 (2CH2), 113.6,
115.4, 139.6, 154.7, 164.6 (CHarom), 119.2, 121.3, 124.5, 136.2, 142.3, 159.0 (Carom). HRMS (ESI+)
calcd for C18H23N6 (M+H)+ 323.1979, found 323.1978.
4.1.54 N2-(2-(Pyrrolidin-1-yl)ethyl)pyrido[3,4-g]quinazoline-2,10-diamine (10f): 24 h reaction
time, obtained as a red oil in 98% yield. Rf = 0.15 (CH2Cl2/NH3 7N solution in MeOH 96:4). IR
(ATR): 3531-2189, 1606, 1575, 1365, 1029 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 1.74-1.94 (4H,
m), 2.40-2.75 (6H, m), 3.68-3.85 (2H, m), 6.41 (2H, br s, NH2), 7.66 (1H, br s, NH), 7.85 (1H, s),
8.03 (1H, d, J = 6.4 Hz), 8.24 (1H, d, J = 6.0 Hz), 9.25 (1H, s), 9.37 (1H, s); 13C NMR (100 MHz,
DMSO-d6) δ 22.7 (2CH2), 29.3, 46.2 (CH2), 53.3 (2CH2), 113.3, 115.4, 139.5, 154.7, 164.7
(CHarom), 119.4, 121.2, 124.7, 136.6, 142.8, 159.9 (Carom). HRMS (ESI+) calcd for C17H21N6
(M+H)+ 309.1822, found 309.1823.
4.1.55 N2-(3-(N,N-Dimethylamino)propyl)pyrido[3,4-g]quinazoline-2,10-diamine (10g): 40 h
reaction time, obtained as a red oil in quantitative yield. Rf = 0.15 (CH2Cl2/NH3 7N solution in
MeOH 97:3). IR (ATR): 3527-2437, 1607, 1574, 1361 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 1.87
(2H, quint, J = 7.2 Hz), 2.29-2.54 (2H, m), 2.39 (6H, s), 3.50-3.57 (2H, m), 6.33 (2H, br s, NH2),
7.71 (1H, br s, NH), 7.83 (1H, s), 8.00 (1H, d, J = 6.4 Hz), 8.22 (1H, d, J = 6.4 Hz), 9.23 (1H, s),
13
9.33 (1H, s); C NMR (100 MHz, DMSO-d6) δ 43.9 (2CH3), 21.1, 38.7, 56.1 (CH2), 113.6, 115.4,
139.5, 154.7, 164.4 (CHarom), 119.1, 121.2, 124.4, 136.2, 142.4, 159.5 (Carom). HRMS (ESI+) calcd
for C16H21N6 (M+H)+ 297.1822, found 297.1823.
4.1.56 N2-(3-(N,N-Diethylamino)propyl)pyrido[3,4-g]quinazoline-2,10-diamine (10h): 24
h
reaction time, obtained as a red oil 86% in yield. Rf = 0.10 (CH2Cl2/NH3 7N solution in MeOH
1
96:4). IR (ATR): 3515-2131, 1606, 1574, 1359, 1198 cm-1. H NMR (400 MHz, DMSO-d6): 1.00
26

ACCEPTED MANUSCRIPT
(6H, t, J = 7.2 Hz), 1.78 (2H, quint, J = 7.2 Hz), 2.50-2.57 (6H, m), 3.47-3.57 (2H, m), 6.29 (2H, br
s, NH2), 7.71 (1H, br s, NH), 7.82 (1H, s), 7.99 (1H, d, J = 6.0 Hz), 8.21 (1H, d, J = 6.0 Hz), 9.22
(1H, s), 9.32 (1H, s); 13C NMR (100 MHz, DMSO-d6): 11.7 (2CH3), 25.9 (CH2), 46.3 (2CH2), 50.5
(CH2), 113.6, 115.3, 139.5, 154.7, 164.3 (CHarom), 119.1, 121.2, 124.4, 136.0, 144.7, 157.4 (Carom).
The missing CH2 was under the solvent signal. HRMS (ESI+) calcd for C18H25N6 (M+H)+
325.2135, found 325.2138.
4.1.57 N2-(3-(Morpholin-1-yl)propyl)pyrido[3,4-g]quinazoline-2,10-diamine (10i): 4 h reaction
time, obtained as a red oil in 51% yield. Rf = 0.40 (CH2Cl2/MeOH 9:1) IR (ATR): 3622-2164, 1607,
1575, 1362, 1291, 1111 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 1.81 (2H, quint, J = 4.4 Hz), 2.30-
2.55 (6H, m), 3.50-3.54 (2H, m), 3.59-3.62 (4H, m), 6.27 (2H, br s, NH2), 7.63 (1H, br s, NH), 7.83
(1H, s), 7.99 (1H, d, J = 4.8 Hz), 8.22 (1H, d, J = 4.8 Hz), 9.23 (1H, s), 9.32 (1H, s); 13C NMR (100
MHz, DMSO-d6) δ 25.4, 38.5 (CH2), 53.4 (2CH2), 56.2 (CH2), 66.2 (2CH2), 113.6, 115.4, 139.5,
154.7, 164.3 (CHarom), 119.1, 121.3, 124.4, 134.7, 136.0, 157.4 (Carom). HRMS (ESI+) calcd for
C18H23N6O (M+H)+ 339.1928, found 339.1927. HPLC: purity > 96%, λ = 280 nm, tR = 18.1 min.
4.1.58 N2-(3-(N-Methylpiperazin-1-yl)propyl)pyrido[3,4-g]quinazoline-2,10-diamine (10j): 18 h
reaction time, obtained as a red oil in 56% yield. Rf = 0.10 (CH2Cl2/NH3 7N solution in MeOH
96:4). IR (ATR): 3540-2536, 1608, 1577, 1372, 1284, 1152 cm-1. 1H NMR (400 MHz, DMSO-d6) δ
1.79 (2H, quint, J = 5.6 Hz), 2.16 (3H, s), 2.30-2.56 (8H, m), 2.50-2.53 (2H, m), 3.47-3.54 (2H, m),
6.26 (2H, br s, NH2), 7.65 (1H, br s, NH), 7.82 (1H, s), 7.98 (1H, d, J = 5.2 Hz), 8.21 (1H, d, J = 5.2
13
Hz), 9.22 (1H, s), 9.31 (1H, s); C NMR (100 MHz, DMSO-d6) δ 45.8 (CH3), 25.7 (CH2), 52.7
(2CH2), 54.8 (2CH2), 55.9 (CH2), 113.6, 115.4, 139.5, 154.7, 164.3 (CHarom), 119.1, 121.3, 124.4,
134.7, 136.0, 157.4 (Carom). The missing CH2 was under the solvent signal. HRMS (ESI+) calcd for
C19H26N7 (M+H)+ 352.2244, found 352.2247. HPLC: purity > 96%, λ = 280 nm, tR = 17.9 min.
4.1.59 N2-(3-(Piperidin-1-yl)propyl)pyrido[3,4-g]quinazoline-2,10-diamine (10k): 40 h reaction
time, obtained as a red oil in 18% yield. Rf = 0.20 (CH2Cl2/NH3 7N solution in MeOH 97:3). IR
1
(ATR): 3478-2358, 1607, 1574, 1350 cm-1. H NMR (400 MHz, DMSO-d6): 1.33-1.42 (2H, m),
1.45-1.56 (4H, m), 1.79 (2H, quint, J = 6.8 Hz), 2.25-2.43 (6H, m), 3.44-3.57 (2H, m), 6.26 (2H, br
s, NH2), 7.68 (1H, br s, NH), 7.82 (1H, s), 7.98 (1H, d, J = 6.4 Hz), 8.21 (1H, d, J = 6.4 Hz), 9.22
13
(1H, s), 9.31 (1H, s); C NMR (100 MHz, DMSO-d6): 24.0 (CH2), 25.4 (2CH2), 42.5 (CH2), 54.0
(2CH2), 56.5 (CH2), 113.6, 115.3, 139.5, 154.7, 165.1 (CHarom), 119.1, 121.6, 124.4, 136.0, 141.7,
27

ACCEPTED MANUSCRIPT
160.5 (Carom). The missing CH2 was under the solvent signal. HRMS (ESI+) calcd for C19H25N6
(M+H)+ 337.2135, found 337.2136.
4.1.60 N2-(3-(Pyrrolidin-1-yl)propyl)pyrido[3,4-g]quinazoline-2,10-diamine (10l): 18 h reaction
time, obtained as a red oil in 63% yield. Rf = 0.15 (CH2Cl2/NH3 7N solution in MeOH 97:3). IR
1
(ATR): 3482-2288, 1606, 1574, 1361 cm-1. H NMR (400 MHz, DMSO-d6) δ 1.68-1.76 (4H, m),
1.84 (2H, quint, J = 7.2 Hz), 2.42-2.65 (6H, m), 3.48-3.56 (2H, m), 6.28 (2H, br s, NH2), 7.66 (1H,
br s, NH), 7.82 (1H, s), 7.99 (1H, d, J = 5.8 Hz), 8.21 (1H, d, J = 6.0 Hz), 9.22 (1H, s), 9.31 (1H, s);
13C NMR (100 MHz, DMSO-d6) δ 22.1 (CH2), 22.9 (2CH2), 29.0, 31.3 (CH2), 53.5 (2CH2), 113.6,
115.4, 139.5, 154.7, 164.4 (CHarom), 119.1, 121.3, 124.4, 136.1, 142.3, 160.2 (Carom). HRMS (ESI+)
calcd for C18H23N6 (M+H)+ 323.1979, found 323.1983.
4.1.61 N2-Methylpyrido[3,4-g]quinazoline-2,10-diamine (10m): due to poor solubility in CH2Cl2,
compound 9m was dissolved in CH2Cl2/MeOH 8:2 before reaction for 24 h. Compound 10m was
obtained as a red powder in 58% yield. Mp: degradation. Rf = 0.20 (CH2Cl2/MeOH 9:1). IR (ATR):
3506-2408, 1579, 1375 cm-1. 1H NMR (400 MHz, DMSO-d6) δ 3.00 (3H, d, J = 5.2 Hz), 6.33 (2H,
br s, NH2), 7.83 (1H, s), 8.00 (1H, d, J = 6.4 Hz), 8.15 (1H, br s, NH), 8.22 (1H, d, J = 6.4 Hz), 9.23
13
(1H, s), 9.31 (1H, s); C NMR (100 MHz, DMSO-d6) δ 28.0 (CH3), 113.5, 115.4, 139.5, 154.7,
164.6 (CHarom), 119.4, 121.7, 124.4, 136.2, 142.5, 160.1 (Carom). HRMS (ESI+) calcd for C12H12N5
(M+H)+ 226.1087, found 226.1089.
4.2 In vitro kinase inhibition assays
4.2.1. Method A (33P radioassay)
Kinase activities were assayed in appropriate buffer for each kinase, with either protein or peptide
as substrate in the presence of 15 µM [γ-33P] ATP (3,000 Ci/mmol; 10 mCi/ml) in a final volume of
30 µl following the assay described in [20]. Controls were performed with appropriate dilutions of
dimethylsulfoxide. Full-length kinases are used unless specified. Peptide substrates were obtained
from ProteoGenix (Schiltigheim, France).
HsCDK5/p25 (human, recombinant, expressed in bacteria) was assayed on 0.8 µg/µl of histone H1
as substrate. SscCK1δ/ε (Sus scrofa domesticus, casein kinase 1δ/ε, affinity purified from porcine
brain) was assayed on 0.022 µg/µl of the following peptide: RRKHAAIGSpAYSITA as specific
substrate. HsCDK5/p25 and SscCK1δ/ε were tested in the following buffer: 60 mM β-
28

ACCEPTED MANUSCRIPT
glycerophosphate, 30 mM p-nitrophenyl-phosphate, 25 mM MOPS (pH 7), 5 mM EGTA, 15 mM
MgCl2, 1 mM DTT, 0.1 mM sodium orthovanadate.
SscGSK-3 α/β (Sus scrofa domesticus, glycogen synthase kinase-3, affinity purified from porcine
brain) was assayed on 0.01 µg/µl of GS-1 peptide, a GSK-3-selective substrate
(YRRAAVPPSPSLSRHSSPHQSpEDEEE, “Sp” stands for phosphorylated serine). RnDYRK1A-
kd (Rattus norvegicus, kinase domain aa 1 to 499, expressed in bacteria, DNA vector kindly
provided by Dr. W. Becker, Aachen, Germany) was assayed on 0.033 µg/µl of the following
peptide: KKISGRLSPIMTEQ as substrate. MmCLK1 (from Mus musculus, recombinant, expressed
in bacteria) was assayed on 0.027 µg/µl of the following peptide: GRSRSRSRSRSR as substrate.
SscGSK-3 α/β, RnDYRK1A and MmCLK1 were tested in the following buffer: 10 mM MgCl2, 1
mM EGTA, 1 mM DTT, 25 mM Tris-HCl pH 7.5, 50 µg/ml heparin, 0.15 mg/ml of BSA, 0.23
mg/ml of DTT.
4.2.2. Method B (ADP-glo)
Kinase assays were carried out following ADP-Glo protocol (Promega). Briefly, reactions
were carried out in a final volume of 5 µl, at 10 µM ATP, for 30 min at 30 °C. The luminescent
signal emitted was measured using an Envision luminometer (PerkinElmer, Waltham, MA) and
expressed in Relative Light Unit (RLU).
4.3 In vitro cellular antiproliferative assays
HCT116 cells were cultured in McCoy’s 5a medium, SH-SY5Y, MDA-MB231 and U-2 OS
cells were cultured in Dulbecco's modified Eagle's medium (DMEM), hTERT RPE1 were cultured
in DMEM:F12 medium. All media were supplemented with 10% fetal calf serum, 2 mM L-
glutamine, 50 IU penicillin and streptomycin. Cell viability was assayed using CellTiter96 AQueous
from PROMEGA according to manufacturer’s instructions.
4.4 Protein crystallography
Recombinant CLK1 was expressed and purified as described previously [21]. Two different crystal
forms of the apo protein were obtained at 4 °C using the sitting drop vapor diffusion technique by
mixing equal volumes of protein solution (7-8 mg/ml protein in 30 mM Hepes, pH 7.5, 300 mM
NaCl, 50 mM arginine/glutamine mix 1:1, 0.5 mM TCEP, and 1% v/v glycerol) and the appropriate
reservoir solution. Crystal form A grew with a reservoir solution of 25% (w/v) PEG 6000, and 0.1
M bicine, pH 9.0; and crystal form B with a reservoir solution of 29% (v/v) 1,2 propanediol, 0.08 M
29