Synthesis and preliminary structure-activity relationship study of 2-aryl-2H-pyrazolo[4,3-c]quinolin-3-ones as potential checkpoint kinase 1 (Chk1) inhibitors

Article abstract of DOI:10.1080/14756366.2017.1404592

The serine-threonine checkpoint kinase 1 (Chk1) plays a critical role in the cell cycle arrest in response to DNA damage. In the last decade, Chk1 inhibitors have emerged as a novel therapeutic strategy to potentiate the anti-tumour efficacy of cytotoxic chemotherapeutic agents. In the search for new Chk1 inhibitors, a congeneric series of 2-aryl-2 H-pyrazolo[4,3-c]quinolin-3-one (PQ) was evaluated by in-vitro and in-silico approaches for the first time. A total of 30 PQ structures were synthesised in good to excellent yields using conventional or microwave heating, highlighting that 14 of them are new chemical entities. Noteworthy, in this preliminary study two compounds 4e₂ and 4h₂ have shown a modest but significant reduction in the basal activity of the Chk1 kinase. Starting from these preliminary results, we have designed the second generation of analogous in this class and further studies are in progress in our laboratories.

Full text of DOI:10.1080/14756366.2017.1404592

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Synthesis and preliminary structure-activity
relationship study of 2-aryl-2H-pyrazolo[4,3-
c]quinolin-3-ones as potential checkpoint kinase 1
(Chk1) inhibitors
Ivana Malvacio, Alberto Cuzzolin, Mattia Sturlese, D. Mariano A. Vera, E.
Laura Moyano & Stefano Moro
To cite this article: Ivana Malvacio, Alberto Cuzzolin, Mattia Sturlese, D. Mariano A. Vera, E.
Laura Moyano & Stefano Moro (2018) Synthesis and preliminary structure-activity relationship
study of 2-aryl-2H-pyrazolo[4,3-c]quinolin-3-ones as potential checkpoint kinase 1 (Chk1)
inhibitors, Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 171-183, DOI:
10.1080/14756366.2017.1404592
To link to this article: https://doi.org/10.1080/14756366.2017.1404592
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Date: 07 December 2017, At: 07:50

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY, 2017
VOL. 33, NO. 1, 171–183
https://doi.org/10.1080/14756366.2017.1404592
RESEARCH PAPER
Synthesis and preliminary structure-activity relationship study of
2-aryl-2H-pyrazolo[4,3-c]quinolin-3-ones as potential checkpoint kinase 1
(Chk1) inhibitors
Ivana Malvacioa,b, Alberto Cuzzolinb, Mattia Sturleseb , D. Mariano A. Verac, E. Laura Moyanoa and Stefano Morob
b
aDepartment of Organic Chemistry, INFIQC, School of Chemical Sciences, National University of Cordoba, Cordoba, Argentina; Molecular
c
ꢀ
Modeling Section (MMS), Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Padova, via Marzolo, Padova, Italy; Department of
Chemistry, QUIAMM-INBIOTEC, School of Exact and Natural Sciences, National University of Mar del Plata, Mar del Plata, Buenos Aires, Argentina
ABSTRACT
ARTICLE HISTORY
Received 21 September 2017
Revised 8 November 2017
Accepted 8 November 2017
The serine-threonine checkpoint kinase 1 (Chk1) plays a critical role in the cell cycle arrest in response to
DNA damage. In the last decade, Chk1 inhibitors have emerged as a novel therapeutic strategy to potenti-
ate the anti-tumour efficacy of cytotoxic chemotherapeutic agents. In the search for new Chk1 inhibitors, a
congeneric series of 2-aryl-2 H-pyrazolo[4,3-c]quinolin-3-one (PQ) was evaluated by in-vitro and in-silico
approaches for the first time. A total of 30 PQ structures were synthesised in good to excellent yields using
conventional or microwave heating, highlighting that 14 of them are new chemical entities. Noteworthy,
in this preliminary study two compounds 4e2 and 4h2 have shown a modest but significant reduction in
the basal activity of the Chk1 kinase. Starting from these preliminary results, we have designed the second
generation of analogous in this class and further studies are in progress in our laboratories.
KEYWORDS
2-Aryl-2H-pyrazolo[4,3-c]qui-
nolin-3-one (PQ); serine-
threonine checkpoint kinase
1 (Chk1); molecular
docking; molecular
dynamics (MD); supervised
molecular dynamics (SuMD)
Introduction
of anticancer drugs by enhancing the apoptosis in tumour cells
with a defective p53 response12–15. The first Chk1 inhibitor to
enter phase I and II clinical trials against a wide range of
tumour types was UCN-01 (7-hydroxystaurosporine)16,17. Many
Chk1 inhibitors have subsequently been developed and subjected
The pyrazolo[4,3-c]quinolone nucleus has a broad variety of
pharmacological activity, making it an attractive structure for syn-
thesis in recent years. Such heterocyclic system has been incorpo-
rated in ligands of the c-aminobutyric acid type A receptor
(GABAA) or benzodiazepine receptor. One of the most interesting
properties of these ligands is the change of the intrinsic activity
caused by small structural modifications. Their activity can shift
from agonist (anxiolytic, hypnotic, anticonvulsant), through antag-
onist (without pharmacological response) to inverse agonist (pro-
to phase I trials like AZD776218, PF-47773619 and SCH90077620,21
.
Unfortunately, many of these clinical studies have been aban-
doned due to the low selectivity and the incidence of side effects
of such compounds15,22
.
In the search for new potent and selective Chk1 inhibitors, a
congeneric series of 2-aryl-2 H-pyrazolo[4,3-c]quinolin-3-ones (PQs)
was studied, for the first time. We present here the synthesis of a
PQ library, containing 14 novel chemical entities, and the applica-
tion of molecular modelling as supporting methodology to obtain
preliminary data. Particularly, a hybrid approach based on dock-
ing/molecular dynamics (MD) simulations were implemented for
the interpretation of the structure-activity relationship. For this
convulsant and anxiogenic)1–4
.
The pyrazolo[4,3-c]-quinolone
have also been identified as cyclooxygenase-2 (COX-2) inhibitors
which can reduce pain and inflammation5, as phosphodiesterase-4
inhibitors with antiasthmatic properties6, as well as Topoisomerase
II inhibitors, which results in a high level of cytotoxicity and antitu-
mour activity7,8
.
During the last decade, heterocycles sharing a common pattern
with the previously studied pyrazoloquinolones have emerged as
checkpoint kinase 1 (Chk1) inhibitors9–11. The serine-threonine pro-
tein kinase Chk1 is a key mediator in response to DNA damage
which, along with the tumour suppressor protein p53, is required
for cell cycle arrest and activation of DNA repair before progress-
ing into mitosis. However, many tumour cells rely only on Chk1
checkpoint because of mutations in p53. Therefore, the inhibition
of Chk1 represents a strategy to increase the therapeutic efficacy
purpose,
EC50 ¼ 510 nM) was considered as reference compound since it
has a similar PQ motif (Scheme 1)10,11
a
co-crystal Chk1 inhibitor (YEX, IC50 ¼ 0.1 nM, and
.
In this preliminary study two compounds 4e2 and 4h2 have
shown a modest, but encouraging, reduction in the basal activity
of the Chk1 kinase. Starting from these encouraging results, we
have designed the second generation of analogous in this class
and further studies are in progress in our laboratories.
CONTACT Stefano Moro
Universita degli Studi di Padova, via Marzolo 5, Padova 35131, Italy
lauramoy99@gmail.com, stefano.moro@unipd.it
Molecular Modeling Section (MMS), Dipartimento di Scienze Farmaceutiche,
ꢀ
Supplemental data for this article can be accessed here.
ß 2017 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distri-
bution, and reproduction in any medium, provided the original work is properly cited.

172
I. MALVACIO ET AL.
temperature of the process was kept between 130–140 ꢁC, for
1–3.5 h as it is specified in Table 1. Finally, compounds 4b2, 4d1,
4e1, 4f1, and 4h2–3 were purified by recrystallisation from
EtOH:H2O (96:4) and 4f2–4, 4g4, and 4h4 were purified by column
chromatography using CHCl3: EtOH (9:1) as elution solvents. To
purify 4e3–4 both methodologies were used, whereas 4a1–2, 4a4
and 4c2 were obtained pure.
Materials and methods
General procedure for the conventional synthesis of PQ
For the conventional preparation of PQs, 0.15 g (0.44–0.69 mmol)
of quinolones (1a–h) were mixed with thionyl chloride in excess
(1 ml) at reflux temperature, for 1–2 h, to give the corresponding
chloroquinolines (2a–h) in excellent yields (>95%). The excess of
thionyl chloride was removed by evaporation and co-evaporation
with dichloromethane (3 ꢀ 10 ml). The final products (4a1–2, 4a4,
4b2, 4c2, 4d1, 4e1, 4e3–4, 4f1–4, 4g4, 4h2–4) were obtained by
reaction between 2a and h with a 20% excess of the appropriated
hydrazines 31–4: phenylhydrazine monohydrochloride, o-fluorophe-
nylhydrazine monohydrochloride, p-fluorophenylhydrazine mono-
hydrochloride and p-methoxyphenylhydrazine monohydrochloride.
Hydrochloride salts were neutralised with equimolar amounts of
triethylamine (TEA) before the addition of chloroquinolines. N,N-
dimethylformamide (DMF) was used as solvent and the
General procedure for the microwave assisted synthesis of PQ
A CEM Discovery monomode equipment was used for the prepar-
ation of PQs applying MW heating in closed vessels. A mixture of
0.15 g (0.44–0.69 mmol) of quinolones (1a–e, g–h) and thionyl
chloride in excess (1 ml) was irradiated with a dynamic power
method at 85 ꢁC, for 1–4 min, to give the chloroquinolines (2a–e,
g–h) in excellent yields (>95%). The thionyl chloride was removed
in the same way as previously described for the conventional syn-
thesis. The final products (4a3, 4b1, 4b3–4, 4c1, 4c3–4, 4d2–4, 4e2,
4g1, 4h1) were obtained by reaction between 2a–e, g–h with a
20% excess of the appropriated hydrazine hydrochlorides 31–4
,
previously neutralised with TEA, using 2 ml of DMF as solvent. The
mixture of reactants was irradiated with a fix power method
(300 W) at 140 10 ꢁC, for 1–5 min, as it is specified in Table 1.
Finally, compounds 4b3, 4c3–4, 4d2–3, and 4e2 were purified by
recrystallisation from EtOH:H2O (96:4); compounds 4b4, 4d4, 4g1
and 4h1 were purified by column chromatography using CHCl3:
EtOH (9:1) as elution solvent, whereas compounds 4a3, 4b1, and
4c1 were obtained pure.
Characterisation of products
2-Phenyl-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-one (4a1): yellow solid;
m.p.: 328–331 ꢁC23; 1 H-NMR (400 MHz, DMSO-d6): d (ppm) 7.16
Scheme 1. Reference structure (YEX) and PQs series studied.
Table 1. Reaction times and yields for the synthesis of PQs 4a–h1–4
PQ
R1
R2
R3
X
Y
Heating method
Step1 (min)
Step2 (min)
Yield (%)b
4a1
4a2a
4a3
4a4
4b1a
4b2
4b3
4b4
4c1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
H
H
H
H
H
H
CF3
CF3
CF3
CF3
H
F
H
H
H
F
H
H
H
F
H
H
H
F
H
H
H
F
H
H
H
F
H
H
H
H
H
F
H
H
F
OCH3
H
H
F
OCH3
H
H
F
OCH3
H
H
F
OCH3
H
H
F
OCH3
H
H
F
OCH3
H
OCH3
H
H
F
C
C
MW
C
MW
C
MW
MW
MW
C
MW
MW
C
MW
MW
MW
C
MW
C
C
C
C
C
C
MW
C
MW
C
C
60
60
1
60
1
60
1
1
1
60
1
1
120
2
120
90
5
120
1
210
1
1
4
180
5
4
150
2
70
78
90
63
88
H
CH3
CH3
CH3
CH3
OCH3
OCH3
OCH3
OCH3
Cl
Cl
Cl
Cl
H
H
H
H
Br
Br
Br
Br
I
79 (63)
78 (60)
75 (69)
73
4c2a
4c3a
4c4
73
70 (42)
64 (38)
55 (36)
67 (40)
59 (31)
92 (56)
89 (71)
61 (35)
87 (40)
85 (41)
92 (73)
91 (66)
90 (76)
90 (56)
56 (27)
53 (23)
57 (15)
87 (55)
95 (74)
72 (43)
4d1a
4d2a
4d3
4d4
4e1a
4e2
4e3
4e4
4f1a
4f2
2
2
120
2
2
2
150
2
120
120
90
90
90
90
4
120
3
120
120
120
90
90
60
210
180
150
5
120
5
120
120
150
4f3
4f4
a
4g1a
4g4a
4h1a
4h2a
4h3a
4h4
I
F
F
F
H
H
F
OCH3
C
aNew chemical entities.
bYield calculated from ethyl-quinolin-4-one-3-carboxylate moles. In brackets the yield of purified product.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
173
(t, 1 H, J ¼ 7.3 Hz); 7.44 (t, 2 H, J ¼ 8.0 Hz); 7.54 (t, 1 H, J ¼ 7.2 Hz); 126.83; 128.77; 129.06; 129.61; 138.18; 143.55; 155.14; 157.55;
157.64; 161.64.
7.66 (t, 1 H, J ¼ 7.7 Hz); 7.80 (t, 1 H, J ¼ 6.4 Hz); 8.22 (d, 3 H,
2-(4-Fluorophenyl)-8-methoxy-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-
J ¼ 8.4 Hz); 8.66 (s, 1 H).
one (4c3): yellow solid; m.p.: >300 ꢁC; 1 H-NMR (400 MHz, DMSO-
2-(2-Fluorophenyl)-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-one
(4a2):
yellow solid; m.p.: not informed24; 1 H-NMR (400 MHz, DMSO-d6): d
(ppm) 7.28–7.47 (m, 3 H); 7.50–7.60 (m, 2 H); 7.63–7.74 (m, 2 H);
8.13 (d, 1 H, J ¼ 7.9 Hz); 8.72 (s, 1 H); 12.80 (s, 1 H).
d6): d (ppm) 3.92 (s, 3 H); 7.26–7.31 (m, 3 H); 7.58 (d, 1 H,
J ¼ 2.7 Hz); 7.68 (d, 1 H, J ¼ 9.0 Hz); 8.23–8.26 (m, 2 H); 8.69 (d, 1 H,
J ¼ 6.6 Hz); 12.87 (d, 1 H, J ¼ 6.3 Hz); 13 C-NMR (400 MHz, DMSO-d6):
d (ppm) 55.69; 102.57; 105.02; 115.17; 115.39; 119.74; 119.97;
120.40; 120.49; 121.32; 129.77; 136.67; 138.05; 143.01; 157.41;
157.60; 159.80; 161.49.
2-(4-Methoxyphenyl)-8-methoxy-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-
one (4c4): yellow solid; m.p.: >300 ꢁC; 1 H-NMR (400 MHz, DMSO-d6):
d (ppm) 3.78 (s, 3 H); 3.92 (s, 3 H); 7.01 (d, 2 H, J ¼ 9.2 Hz); 7.29 (dd,
1 H, J ¼ 2.6 and 9.0 Hz); 7.57 (d, 1 H, J ¼ 2.7 Hz); 7.68 (d, 1 H,
J ¼ 8.9 Hz); 8.10 (d, 2 H, J ¼ 9.0 Hz); 8.63 (s, 1 H); 13 C-NMR (400 MHz,
DMSO-d6): d (ppm) 55.71; 56.13; 103.02; 105.70; 114.25; 119.90;
120.53; 120.95; 121.73; 130.19; 134.15; 138.10; 143.02; 156.36;
157.98; 161.62.
2-(4-Fluorophenyl)-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-one
(4a3):
yellow solid; m.p.: >300 ꢁC; 1 H-NMR (400 MHz, DMSO-d6): d (ppm)
7.29 (t, 2 H, J ¼ 9.0 Hz); 7.56 (t, 1 H, J ¼ 8.1 Hz); 7.66–7.73 (m, 2 H);
8.20–8.25 (m, 3 H); 8.74 (s, 1 H); 12.88 (s, 1 H); 13 C-NMR (400 MHz,
DMSO-d6): d (ppm) 106.50; 115.64; 115.86; 119.14; 120.03; 120.92;
122.58; 126.93; 130.65; 135.96; 137.10; 139.90; 143.51; 157.90;
160.30; 161.93.
2-(4-Methoxyphenyl)-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-one (4a4):
yellow solid; m.p.: 268–270 ꢁC23; 1 H-NMR (400 MHz, DMSO-d6): d
(ppm) 3.78 (s, 3 H); 7.02 (d, 2 H, J ¼ 9.2 Hz); 7.55 (t, 1 H, J ¼ 8.0 Hz);
7.63–7.73 (m, 2 H); 8.08 (d, 2 H, J ¼ 9.2 Hz); 8.21 (d, 1 H, J ¼ 7.9 Hz);
8.70 (d, 1 H, J ¼ 5.1 Hz); 12.80 (s, 1 H).
8-Chloro-2-phenyl-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-one (4d1): yel-
low solid; m.p.: 396–400 ꢁC25; 1 H-NMR (400 MHz, DMSO-d6): d
(ppm) 7.21 (t, 1 H, J ¼ 7.4 Hz); 7.48 (t, 2 H, J ¼ 7.7 Hz); 7.69 (d, 1 H,
J ¼ 8.8 Hz); 7.85 (dd, 1 H, J ¼ 2.3 and 8.8 Hz); 8.23 (d, 2 H, J ¼ 8.0 Hz);
8.35 (d, 1 H, J ¼ 2.0 Hz); 8.79 (s, 1 H).
8-Methyl-2-phenyl-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-one
(4b1):
yellow solid; m.p.: 364–366 ꢁC25; 1 H-NMR (400 MHz, DMSO-d6): d
(ppm) 2.48 (s, 3 H); 7.17 (t, 1 H, J ¼ 7.3 Hz); 7.44 (t, 2 H, J ¼ 7.6 Hz);
7.50 (dd, 1 H, J ¼ 1.8 and 8.7 Hz); 7.62 (d, 1 H, J¼ 8.4 Hz); 8.03 (s,
1 H); 8.22 (d, 2 H, J ¼ 8.2 Hz); 8.68 (s, 1 H); 12.77 (s, 1 H); 13 C-NMR
(400 MHz, DMSO-d6): d (ppm) 21.34; 106.38; 119.10; 119.88; 122.07;
124.39; 129.13; 131.87; 133.96; 136.73; 139.29; 140.64; 143.41;
162.16.
2-(2-Fluorophenyl)-8-chloro-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-one
(4d2): yellow solid; m.p.: >320 ꢁC; 1 H-NMR (400 MHz, DMSO-d6): d
(ppm) 7.39–7.56 (m, 3 H); 7.66 (t, 1 H, J ¼ 7.8 Hz); 7.81 (d, 2 H,
J ¼ 1.7 Hz); 8.16 (d, 1 H, J ¼ 1.8 Hz); 8.85 (s, 1 H); 12.48 (s, 1 H); 13 C-
NMR (400 MHz, DMSO-d6): d (ppm) 105.21; 117.00; 120.61; 121.49;
122.09; 125.03; 129.00; 129.59; 130.60; 131.09; 134.59; 140.26;
143.01; 155.50; 158.00; 161.91.
2-(2-Fluorophenyl)-8-methyl-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-one
(4b2): yellow solid; m.p.: >300 ꢁC; 1 H-NMR (400 MHz, DMSO-d6): d
(ppm) 2.45 (s, 3 H); 7.29–7.45 (m, 3 H); 7.49 (dd, 1 H, J ¼ 1.5 and
8.3 Hz); 7.55–7.62 (m, 2 H); 7.93 (s, 1 H); 8.68 (d, 1 H, J ¼ 6.3 Hz);
12.74 (d, 1 H, J ¼ 5.3 Hz); 13 C-NMR (400 MHz, DMSO-d6): d (ppm)
20.85; 104.27; 116.32; 116.52; 118.77; 119.34; 121.50; 124.50;
128.46; 128.78; 128.85; 131.27; 133.31; 136.18; 139.01; 143.46;
154.97; 157.47; 161.59.
2-(4-Fluorophenyl)-8-methyl-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-one
(4b3): yellow solid; m.p.: not informed24; 1 H-NMR (400 MHz, DMSO-
d6): d (ppm) 2.48 (s, 3 H); 7.28 (t, 2 H, J ¼ 8.8 Hz); 7.50 (dd, 1 H,
J ¼ 1.8 and 8.3 Hz); 7.62 (d, 1 H, J ¼ 8.6 Hz); 8.02 (s, 1 H); 8.22–8.25
(m, 2 H); 8.71 (d, 1 H, J ¼ 6.5 Hz); 12.82 (d, 1 H, J ¼ 6.8 Hz); 13 C-NMR
(400 MHz, DMSO-d6): d (ppm) 20.86; 105.72; 115.18; 115.40; 118.60;
119.44; 120.37; 121.57; 131.44; 133.47; 136.30; 136.64; 138.97;
142.96; 157.37; 159.77; 161.48.
2-(4-Fluorophenyl)-8-chloro-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-one
(4d3): yellow solid; m.p.: >320 ꢁC; 1 H-NMR (400 MHz, DMSO-d6): d
(ppm) 7.38 (t, 2 H, J ¼ 8.8 Hz); 7.75 (d, 1 H, J ¼ 8.7 Hz); 7.78–7.83 (m,
2 H); 8.29–8.33 (m, 2 H); 8.87 (s, 1 H); 13 C-NMR (400 MHz, DMSO-d6):
d (ppm) 106.67; 115.70; 115.92; 120.46; 120.93; 121.01; 121.61;
122.23; 130.71; 131.18; 134.78; 136.92; 140.27; 142.52; 158.02;
160.41; 161.76.
2-(4-Methoxyphenyl)-8-chloro-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-
one (4d4): yellow solid; m.p.: 326–328 ꢁC26; 1 H-NMR (400 MHz,
DMSO-d6): d (ppm) 3.87 (s, 1 H); 7.11 (d, 2 H, J ¼ 9.2 Hz); 7.77–7.86
(m, 3 H); 8.16 (d, 2 H, J ¼ 9.0 Hz); 8.81 (s, 1 H).
7,9-Dichloro-2-phenyl-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-one (4e1):
yellow solid; m.p.: >310 ꢁC27; 1 H-NMR (400 MHz, DMSO-d6):
d
(ppm) 7.19 (t, 1 H, J ¼ 7.2 Hz); 7.45 (t, 2 H, J ¼ 7.5 Hz); 7.70 (d, 1 H,
J ¼ 1.8 Hz); 7.75 (d, 1 H, J ¼ 1.8 Hz); 8.21 (d, 2 H, J ¼ 7.5 Hz); 8.77 (s,
1 H); 12.77 (s, 1 H); 13 C-NMR (400 MHz, DMSO-d6): d (ppm) 107.79;
116.03; 117.65; 118.61; 124.34; 127.34; 128.78; 131.44; 133.58;
138.11; 139.74; 139.81; 141.18; 160.94.
7,9-Dichloro-2–(2-fluorophenyl)-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-
one (4e2): yellow solid; m.p.: >310 ꢁC; 1 H-NMR (400 MHz, DMSO-
d6): d (ppm) 7.39–7.58 (m, 3 H); 7.66 (td, 1 H, J ¼ 1.5 and 7.8 Hz);
7.76 (d, 1 H, J ¼ 1.8); 7.81 (d, 1 H, J ¼ 1.8 Hz); 8.85 (s, 1 H); 13 C-NMR
(400 MHz, DMSO-d6): d (ppm) 105.35; 116.81; 117.01; 119.22;
120.96; 122.26; 124.54; 125.03; 128.98; 129.58; 133.30; 134.95;
140.34; 142.85; 155.49; 157.99; 161.92.
7,9-Dichloro-2–(4-fluorophenyl)-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-
one (4e3): yellow solid; m.p.: >320 ꢁC27; 1 H-NMR (400 MHz, DMSO-
d6): d (ppm) 7.30 (t, 2 H, J ¼ 9.0 Hz); 7.68 (s, 1 H); 7.75 (s, 1 H); 8.22
(m, 2 H); 8.78 (s, 1 H); 12.88 (s, 1 H); 13 C-NMR (400 MHz, DMSO-d6):
d (ppm) 107.59; 115.28; 115.50; 115.94; 117.64; 120.34; 127.32;
131.39; 133.55; 136.29; 138.05; 139.77; 141.14; 157.59; 159.99;
160.69.
2-(4-Methoxyphenyl)-8-methyl-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-
one (4b4): yellow solid; m.p.: not informed24; 1 H-NMR (400 MHz,
DMSO-d6): d (ppm) 2.48 (s, 3 H); 3.78 (s, 3 H); 7.01 (d, 2 H,
J ¼ 9.3 Hz); 7.49 (dd, 1 H, J ¼ 1.8 and 8.5 Hz); 7.61 (d, 1 H, J ¼ 8.3 Hz);
8.01 (s, 1 H); 8.09 (d, 2 H, J ¼ 8.3 Hz); 8.65 (s, 1 H); 12.72 (s, 1 H);
13 C-NMR (400 MHz, DMSO-d6):
d (ppm) 20.87; 55.23; 105.96;
113.80; 118.68; 119.38; 120.28; 121.52; 131.22; 133.38; 133.65;
136.15; 138.61; 142.47; 155.83; 161.10.
8-Methoxy-2-phenyl-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-one (4c1):
yellow solid; m.p.: 317–320 ꢁC25; 1 H-NMR (400 MHz, DMSO-d6): d
(ppm) 3.92 (s, 3 H); 7.17 (t, 1 H, J ¼ 7.4 Hz); 7.30 (dd, 1 H, J ¼ 2.8 and
9.1 Hz); 7.44 (t, 2 H, J ¼ 8.0 Hz); 7.59 (d, 1 H, J ¼ 2.9 Hz); 7.67 (d, 1 H,
J ¼ 9.0 Hz); 8.23 (d, 2 H, J ¼ 7.6 Hz); 8.66 (s, 1 H); 12.82 (s, 1 H).
2-(2-Fluorophenyl)-8-methoxy-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-
one (4c2): yellow solid; m.p.: >300 ꢁC3; 1 H-NMR (400 MHz, DMSO-
d6): d (ppm) 3.90 (s, 3 H); 7.28 (dd, 1 H, J ¼ 3.0 and 9.3 Hz);
7.32–7.45 (m, 3 H); 7.49 (d, 1 H, J ¼ 3.0 Hz); 7.58 (td, 1 H, J ¼ 1.5 and
7.9 Hz); 7.67 (d, 1 H, J ¼ 3.0 Hz); 8.67 (d, 1 H, J ¼ 6.5 Hz); 12.80 (d,
1 H, J ¼ 6.3 Hz); 13 C-NMR (400 MHz, DMSO-d6): d (ppm) 55.66;
7,9-Dichloro-2–(4-methoxyphenyl)-2 H-pyrazolo[4,3-c]quinolin-3
102.46; 103.55; 116.32; 116.51; 119.62; 120.14; 121.25; 124.55; (5 H)-one (4e4): yellow solid; m.p.: >320 ꢁC27; 1 H-NMR (400 MHz,

174
I. MALVACIO ET AL.
DMSO-d6): d (ppm) 3.78 (s, 3 H); 7.03 (d, 2 H, J ¼ 9.0 Hz); 7.67 (d, (s, 1 H); 13 C-NMR (400 MHz, DMSO-d6): d (ppm) 106.42; 109.84;
1 H, J ¼ 1.8 Hz); 7.73 (d, 1 H, J ¼ 1.8 Hz); 8.09 (d, 2 H, J ¼ 8.8 Hz); 8.74
110.06; 115.70; 115.92; 119.97; 121.10; 123.89; 132.19; 136.64;
140.86; 141.99; 158.16; 160.56; 161.60.
(s, 1 H); 12.83 (s, 1 H); 13 C-NMR (400 MHz, DMSO-d6): d (ppm)
55.26; 107.80; 113.91; 116.07; 117.73; 120.31; 127.22; 131.30;
133.31; 138.15; 139.63; 140.72; 156.12; 160.38.
8-Fluoro-2-(4-methoxyphenyl)-7-(trifluoromethyl)-2 H-pyrazolo[4,3-c]
quinolin-3 (5 H)-one (4h4): yellow solid; m.p.: >300 ꢁC; 1 H-NMR
(400 MHz, DMSO-d6): d (ppm) 3.79 (s, 3 H); 7.03 (d, 2 H, J ¼ 8.9 Hz);
8.03–8.13 (m, 3 H); 8.18 (d, 1 H, J ¼ 10.6 Hz); 8.87 (s, 1 H).
8-Bromo-2-phenyl-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-one (4f1): yel-
low solid; m.p.: >305 ꢁC27; 1 H-NMR (400 MHz, DMSO-d6): d (ppm)
7.18 (t, 1 H, J ¼ 7.5 Hz); 7.45 (t, 2 H, J ¼ 7.7 Hz); 7.66 (d, 1 H,
J ¼ 8.8 Hz); 7.83 (dd, 1 H, J ¼ 2.3 and 8.8 Hz); 8.21 (d, 2 H, J ¼ 8.0 Hz);
8.31 (d, 1 H, J ¼ 2.0 Hz); 8.76 (s, 1 H); 12.89 (s, 1 H); 13 C-NMR
(400 MHz, DMSO-d6): d (ppm) 106.49; 118.6; 118.68; 120.35; 121.80;
124.18; 128.68; 132.91; 134.56; 139.66; 139.93; 141.80; 161.47.
8-Bromo-2-(2-fluorophenyl)-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-one
(4f2): yellow solid; m.p.: >320 ꢁC; 1 H-NMR (400 MHz, DMSO-d6): d
(ppm) 7.31–7.47 (m, 3 H); 7.58 (td, 1 H, J ¼ 1.6 and 7.7 Hz); 7.66
(d, 1 H, J ¼ 8.8 Hz); 7.82 (dd, 1 H, J ¼ 2.2 and 8.8 Hz); 8.20 (d, 1 H,
J ¼ 2.2 Hz); 8.76 (s, 1 H); 12.87 (s, 1 H); 13 C-NMR (400 MHz, DMSO-
d6): d (ppm) 105.34; 116.81; 117.01; 119.21; 120.97; 122.28; 124.54;
124.99; 128.99; 129.57; 133.29; 134.98; 140.37; 142.85; 155.49;
157.98; 161.91.
8-Bromo-2-(4-fluorophenyl)-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-one
(4f3): yellow solid; m.p.: >365 ꢁC27; 1 H-NMR (400 MHz, DMSO-d6): d
(ppm) 7.29 (t, 2 H, J ¼ 9.0 Hz); 7.67 (d, 1 H, J ¼ 8.8 Hz); 7.84 (dd, 1 H,
J ¼ 2.3 and 8.8 Hz); 8.21–8.25 (m, 2 H); 8.31 (d, 1 H, J ¼ 2.2 Hz); 8.79
(d, 1 H, J ¼ 6.3 Hz); 12.94 (d, 1 H, J ¼ 5.0 Hz); 13 C-NMR (400 MHz,
DMSO-d6): d (ppm) 106.32; 115.25; 115.47; 118.88; 120.51; 121.85;
124.20; 132.99; 134.55; 136.41; 139.86; 141.85; 157.53; 159.96;
161.29.
8-Bromo-2-(4-methoxyphenyl)-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-
one (4f4): yellow solid; m.p.: >305 ꢁC27; 1 H-NMR (400 MHz, DMSO-
d6): d (ppm) 3.78 (s, 3 H); 7.01 (d, 2 H, J ¼ 9.0 Hz); 7.65 (d, 1 H,
J ¼ 8.8 Hz); 7.81 (dd, 1 H, J ¼ 2.0 and 8.8 Hz); 8.08 (d, 2 H, J ¼ 9.0 Hz);
8.28 (d, 1 H, J ¼ 2.0 Hz); 8.75 (s, 1 H); 12.87 (s, 1 H); 13 C-NMR
(400 MHz, DMSO-d6): d (ppm) 55.26; 106.54; 113.85; 118.75; 120.43;
121.88; 124.13; 132.74; 133.42; 134.55; 139.59; 141.39; 156.02;
160.94.
8-Iodo-2-phenyl-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-one (4g1): yel-
low solid; m.p.: >310 ꢁC; 1 H-NMR (400 MHz, DMSO-d6): d (ppm)
7.12 (d, 3 H, J ¼ 7.3 Hz); 7.46 (d, 1 H, J ¼ 8.9 Hz); 7.82 (d, 1 H,
J ¼ 8.7 Hz); 7.93 (dd, 2 H, J ¼ 1.7 and 8.6 Hz); 8.47 (d, 1 H, J ¼ 1.5 Hz);
9.08 (s, 1 H).
8-Iodo-2-(4-methoxyphenyl)-2 H-pyrazolo[4,3-c]quinolin-3(5 H)-one
(4g4): yellow solid; m.p.: >310 ꢁC; 1 H-NMR (400 MHz, DMSO-d6): d
(ppm) 3.77 (s, 3 H); 7.00 (d, 2 H, J ¼ 9.0 Hz); 7.49 (d, 1 H, J ¼ 8.5 Hz);
7.94 (dd, 1 H, J ¼ 1.9 and 8.7 Hz); 8.07 (d, 2 H, J ¼ 9.3 Hz); 8.47 (d,
1 H, J ¼ 2.0 Hz); 8.72 (s, 1 H); 12.83 (s, 1 H); 13 C-NMR (400 MHz,
DMSO-d6): d (ppm) 55.72; 91.85; 107.18; 114.31; 121.04; 122.08;
130.68; 133.89; 135.29; 138.72; 139.91; 141.56; 156.49; 161.43.
8-Fluoro-2-phenyl-7-(trifluoromethyl)-2 H-pyrazolo[4,3-c]quinolin-
3(5 H)-one (4h1): yellow solid; m.p.: >300 ꢁC; 1 H-NMR (400 MHz,
DMSO-d6): d (ppm) 7.13 (d, 2 H, J ¼ 7.2 Hz); 7.23–7.26 (m, 1 H); 7.30
(t, 2 H, J ¼ 7.6 Hz); 8.28 (s, 1 H); 8.45 (d, 1 H, J ¼ 7.1 Hz); 9.19 (s, 1 H).
8-Fluoro-2–(2-fluorophenyl)-7-(trifluoromethyl)-2 H-pyrazolo[4,3-c]
quinolin-3 (5 H)-one (4h2): yellow solid; m.p.: >300 ꢁC; 1 H-NMR
(400 MHz, DMSO-d6): d (ppm) 7.34 (td, 1 H, J ¼ 2.0 and 7.7 Hz); 7.40
(td, 1 H, J ¼ 1.4 and 9.3 Hz); 7.44–7.50 (m, 1 H); 7.58 (td, 1 H, J ¼ 1.7
and 7.7); 8.10 (m, 1 H); 8.13 (s, 1 H); 8.88 (s, 1 H); 13.0 (s, 1 H); 13 C-
NMR (400 MHz, DMSO-d6): d (ppm) 104.98; 109.87; 110.09; 116.84;
117.03; 119.96; 124.26; 125.06; 129.10; 129.91; 132.21; 141.09;
142.62; 155.56; 158.05; 161.85.
Protein kinase assay
The inhibitory activity of PQ was carried out by ProQinase com-
pany (Freiburg, Germany). A radiometric protein kinase assay
R
(
33PanQinaseV Activity Assay) was used for measuring the kinase
activity of the Chk1 protein kinase. All kinase assays were per-
formed in 96-well FlashPlatesTM from Perkin Elmer (Boston, MA) in
a 50 ll reaction volume. The reaction cocktail was pipetted in
four steps in the following order: 10 ll of non-radioactive ATP
aqueous solution (1 lM), 25 ll of assay buffer/[c-33 P]-ATP mixture,
5 ll of the test sample in 10% DMSO and 10 ll of the enzyme
(25 ng/50 lL)/substrate (2 lg/50 lL) mixture. The assay for Chk1
protein kinases contained 70 mM HEPES-NaOH pH 7.5, 3 mM
MgCl2, 3 mM MnCl2, 3 lM Na-orthovanadate, 1.2 mM DTT, ATP
(variable amounts, corresponding to the apparent ATP-Km of the
respective kinase, [c-33 P]-ATP (approx. 8 ꢀ 105 cpm per well). The
final DMSO concentration in all reaction cocktails (including high
and low controls) was 1%.
All protein kinases provided by ProQinase were expressed in
Sf9 insect cells or in Escherichia coli as recombinant GST-fusion
proteins or His-tagged proteins, either as full-length or enzymati-
cally active fragments. All kinases were produced from human
cDNAs. Kinases were purified by either GSH-affinity chromatog-
raphy or immobilised metal. Affinity tags were removed from a
number of kinases during purification. The purity of the protein
kinases was examined by SDS-PAGE/Coomassie staining, the iden-
tity was checked by mass spectroscopy.
The reaction cocktails were incubated at 30 ꢁC for 60 min. The
reaction was stopped with 50 ll of H3PO4 (2% v/v), plates were
aspirated and washed two times with 200 ll of aqueous NaCl
(0.9% w/v). Incorporation of 33Pi (counting of “cpm”) was deter-
mined with a microplate scintillation counter (Microbeta, Wallac).
All assays were performed with a BeckmanCoulter Biomek 2000/SL
robotic system.
For each assay, the median value of the cpm of three wells with
complete reaction cocktails, but without kinase, was defined as
“low control” (n ¼ 3). This value reflects unspecific binding of radio-
activity to the plate in the absence of protein kinase but in the
presence of the substrate. In addition, for each assay the median
value of the cpm of three other wells with the complete reaction
cocktail, but without any compound, was taken as the “high con-
trol”, i.e. full activity in the absence of any inhibitor (n ¼ 3). The dif-
ference between high and low control was taken as 100% of the
kinase activity. As part of the data evaluation, the low control value
was subtracted from the high control value as well as from their
corresponding “compound values”. The inhibition percentage for
each compound was calculated by using the following formula:
ð
cpm of compound ꢂ low control
ðhigh control ꢂ low controlÞ
Þ
ð
Þ
Res: activity % ¼
ꢀ 100
ð
Þ
Inhibition % ¼ 100% ꢂ Res: activity ð%Þ
8-Fluoro-2-(4-fluorophenyl)-7-(trifluoromethyl)-2 H-pyrazolo[4,3-c]
Computational simulations
quinolin-3 (5 H)-one (4h3): yellow solid; m.p.: >300 ꢁC; 1 H-NMR
(400 MHz, DMSO-d6): d (ppm) 7.19 (t, 2 H, J ¼ 8.9 Hz); 8.08 (d, 1 H, General. All computations were performed on a hybrid CPU/GPU
J ¼ 6.27 Hz); 8.14 (s, 1 H); 8.16–8.20 (m, 2 H); 8.87 (s, 1 H); 13.02 cluster. In particular, molecular docking simulations have been

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
175
carried out using 8 Intel Xeon E5620 CPU cluster, whereas MD sim- (50 ps) followed by 500,000 steps of NPT (1 ns) simulation, using 2
ulations were carried out with the ACEMD engine on a GPU cluster
equipped with four NVIDIA GTX 580, two NVIDIA GTX 680, three
NVIDIA GTX 780, and four NVIDIA GTX 980. The GOLD 5.2 suite28
was used for docking runs and a combination of protocols based
on AMBER1429/general Amber force field (GAFF)30 was adopted
for MD simulations. The Molecular Operating Environment (MOE,
version 2014.09) suite31 and VMD 1.932 were used for the visualisa-
tion analysis.
fs as the time step. For the first cycle, a harmonic positional
restraint of 1 kcal molꢂ1 Åꢂ2 was applied on protein and ligand
atoms, whereas for the second cycle, the restraint was applied
only to the protein backbone. The pressure was maintained at
1 atm using a Berendsen barostat44. The Langevin thermostat was
set with a low damping constant of 1psꢂ145. Bond lengths involv-
ing hydrogen atoms were constrained using the M-SHAKE algo-
rithm46. The MD productive runs were conducted in an NPT
ensemble. Long-range Coulomb interactions were handled using
the particle mesh Ewald summation method setting the mesh
spacing to 1.0 Å47.
SuMD is a command line tool written in Python, TCL, and Bash
that operates the supervision of MD trajectories according to the
algorithm that has been previously described by Cuzzolin et al.48
Three replicas were carried out for each ligand using the above-
mentioned methodology. The production step of this kind of sim-
ulations depends on the time required for the ligand to reach the
binding site (ꢄ18–36 ns). On the contrary, the production step for
post-docking MD was 100 ns.
Protein preparation. The protein crystal structure PDB code
1ZYS33 was retrieved from the RCSB PDB database34
.
Crystallisation solvent and ions were removed, whereas hydrogen
atoms were added, and appropriate ionisation states were
assigned with the “Protonate-3D” tool35, as implemented in MOE
2014.09 suite31. Missing residues were modelled by the default
homology modelling protocol implemented in the MOE protein
preparation tool. Non-natural N-terminal and C-terminal were
capped to mimic the previous residue. Then, the structure was
subjected to energy minimisation with Amber99 force field29, by
keeping the heavy atoms fixed at their positions. Finally, co-crys-
tallised ligand and water molecules were removed and protein
atoms partial charges computed with the Amber99 force field29
.
Ligands preparation. Co-crystallised ligand YEX was extracted
from the corresponding crystallographic complex (PDB code
2YEX)11, whereas all PQ were created with MOE 2014.0931.
Hydrogen atoms were added to YEX, the protonation state (pH:
7.4) were assigned and they were subjected to a minimisation
with MMFF94 force field36. For docking simulations, partial charges
on ligands atoms were computed on the basis of the PM3/ESP
semiempirical Hamiltonian37,38. Particularly, for post-docking MD
simulations the ligands 4h2 and YEX were kept in their docking
and crystallographic pose, respectively; whereas for supervised
molecular dynamics (supervised molecular dynamics (SuMD) simu-
lations, the ligands were subjected to two energy minimisation
steps with MOPAC201239 using PM6 method40 and Gaussian0941
Results and discussion
Synthesis of PQ
A set of 2-aryl-2 H-pyrazolo[4,3-c]quinolin-3-ones (PQs) was syn-
thesised from ethyl-quinolin-4-one-3-carboxylates using a protocol
previously published by our research group49. The synthesis was
carried out by using conventional (C) or microwave (MW) heating
27
ꢁ
upon convenience. The Lopez-Rivilli methodology was used for
the former, whereas an analogous procedure was optimised for
the MW-assisted synthesis of such compounds (Scheme 2). Table 1
summarises the conditions applied as well as the yield obtained
for each compound.
Generally, the chlorination step (Step1) was quantitative for all
the reactions (>95%), meaning that the pyrazolone ring formation
(Step2) had more influence in the global yields of the reaction
(15–90%). Even if the results obtained using MW heating were
comparable to those where the synthesis was performed with con-
ventional heating, the advantage of the first one was the reduc-
tion of reaction times from hours to minutes. To the best of our
knowledge, the MW-assisted methodology was applied for the first
time in the synthesis of such compounds. Moreover, it is import-
ant to highlight that 14 of the synthesised structures are new
molecular entities.
ꢃ
(HF/6–31 G ). Then, ligand parameters were derived with GAFF(30)
as implemented in Ambertools201429 by using antechamber and
paramcheck tools. RESP partial charges were calculated with
Gaussian 0941 following the procedure suggested by antechamber.
Only for SuMD simulations, ligands were placed at ꢄ30 Å from the
mass centre of Chk1 ATP-binding site.
Docking simulations. Ligands were docked into the orthosteric
binding site of the Chk1 protein with GOLD 5.2 suite28 using the
GoldScore scoring function. The binding cavity centre was defined
by the YEX ligand mass centre in the X-ray structure and the
radius of such cavity set to 20 Å. For each docking run, 20 confor-
mations were generated with an RMSD threshold 1.0 Å.
To estimate the electrostatic contributions to the binding
energy of individual amino acids in the “interaction energy finger-
prints” (IEFs) analysis, atomic charges for the ligands were com-
puted on the basis of the PM3/ESP methodology, whereas partial
charges for the protein amino acids were computed with the
AMBER99 force field. The hydrophobic contributions have been
calculated by using the directional hydrophobic interaction term
Docking simulations
As mentioned earlier, the synthesised PQs share a similar scaffold
with some recently published Chk1 inhibitors9,11,50. Encouraged by
the low IC50 value of these reference structures (980–0.1 nM), we
decided to give our compounds a chance to be tested as Chk1
inhibitors.
In order to select the most appropriate analogous to perform
the biological assay, docking simulations of the synthesised PQs
were carried out using the scoring function goldscore, implemented
in GOLD Suite 5.228, and the Chk1 crystal structure 1ZYS33,51, which
were previously validated by using DockBench 1.052.
based on contact surfaces as implemented in the MOE31
MD simulations solvated system setup and equilibration.
.
–
Protein–ligand complexes were assembled with the tleap tool
using AMBER14SB42 as the force field for the protein. The systems
were explicitly solvated by a cubic water box using TIP3P as the
water model43. To neutralise the total charge, Naþ/Clꢂ counterions
were added to a final salt concentration of 0.154 M. The systems
were energy minimised by 2500 steps with the conjugate-gradient
method and then, they were subjected to two consecutive cycles
Although there are three tautomeric forms possible for such
PQs, only the most stable tautomer in solution (keto tautomer
shown in Scheme 1) was considered for the docking study53. The
selection of the most favourable pose of each compound investi-
of equilibration steps. Both cycles consisted of 25,000 steps of NVE gated was carried out by computing the total electrostatic and

176
I. MALVACIO ET AL.
Scheme 2. Synthesis of 2-aryl-2 H-pyrazolo[4,3-c]quinolin-3-ones from ethyl-quinolin-4-one-3-carboxylates.
Figure 1. IEFs of 4a3, 4d2, 4e1–3, 4h2 and reference YEX. The key residues involved in the binding are displayed (x values) along with the electrostatic interaction
energy (left) and the hydrophobic interaction score (right).
hydrophobic contributions of each pose to the interaction energy share a common binding mode (Figure 2(A)), the electrostatic
interaction with Cys87 is more favourable than Glu85 (Figure 1,
left panel). In addition, the amide nitrogen of Cys87 establishes a
hydrogen bond with the carbonyl oxygen of pyrazolone ring. Such
interaction is also observed with the triazolone ring of the refer-
ence compound YEX, although the binding mode, and therefore,
the hydrogen bond interaction profile is different (Figure 2(B)).
The X-ray crystal structure of YEX shows three hydrogen bonds:
the above-described between the carbonyl oxygen of triazolone
and Cys87, another one between the pyrrole nitrogen and Cys87
and the third one between the triazolone nitrogen and the amide
carbonyl of residue Glu85.
(IE). Moreover, the hydrogen bond interactions with Chk1 binding
site residues and the IE patterns, displayed as 3 D colour maps,
were also considered for the selection (data not shown).
Once the most favourable pose of each compound was identi-
fied, the ligand–receptor interaction was analyzed in a more quan-
titative manner. That is, we calculated the individual electrostatic
and hydrophobic contributions to the IE of each receptor residue
involved in the binding with the ligand. The analysis of these con-
tributions has been reported as IEFs, showing the key residues
involved in the binding with the considered ligands along with a
quantitative estimate of the occurring interactions (Video S1).
Figure 1 displays the IEFs for the six PQs with the most favourable
interactions with Chk1 binding site residues, as well as a compari-
son with the reference inhibitor YEX.
Chk1 inhibitory activity
The most important residues involved in the hydrophobic inter-
actions between the Chk1 binding site and the studied ligands are
Leu15, Val23, Tyr86, and Leu137. Particularly, compound 4h2 also
shows a strong interaction with Tyr20 (Figure 1, right panel).
Regarding the electrostatic interactions, Glu85 and Cys87 are the
residues with the higher contribution, being also the most fre-
quently involved residues in hydrogen bond interactions with the
In order to have a first idea about the kinase inhibition profile of
the studied PQs, a Chk1 protein kinase assay at one concentration
(1 lM) was carried out for a dozen of compounds. At the same
time, MD simulations were performed in order to understand the
importance of the binding interactions profile at the molecular
level.
For the kinase inhibition test, we selected not only the struc-
kinase inhibitors54–57. For the case of the investigated PQs, which tures shown in Figure 1 but also some analogous compounds

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
177
Figure 2. (A) Binding mode of PQs into the Chk1. (B) Comparison of the ligands binding modes between PQ 4h2 (grey) and reference YEX (black). The hydrogen bond
interactions are shown as dashed lines.
respectively. The ligand RMSD plot shows that the docking pose
Table 2. Residual activity and inhibition percentages of Chk1 protein kinase
of compound 4h2 is not stable since its coordinates are not
retained during the simulation (Video S2). The mentioned ligand
undergoes through two major changes in its orientation until
reach a more stable conformation. The first orientation is retained
for about 11 ns (RMSD ꢄ0.8 Å) and the second one is maintained
until the end of the simulation with an average RMSD ꢄ4 Å
(Figure 3, blue curve). The dashed line in Figure 3 shows the
cumulative sum of the total IE values for each frame of the time.
Hence, changes in the observed trend highlight how the variation
of ligand conformation/position affects the IE. Therefore, the
change in the slope of the cumulative ligand ꢂ protein IE plot
(Figure 3, blue dashed line) suggests that the first position
explored by 4h2 is energetically more favourable than the last
one. On the other hand, YEX does not suffer large conformational
changes (Video S3). Figure 3 shows that the reference ligand
remains stable in its initial crystallographic position (RMSD oscillat-
ing around ꢄ1 Å) with constant IE since no changes are observed
in the slope of the cumulative IE plot (red dashed line).
In particular, it is noteworthy to mention that the IE of YEX
with the protein is much more favourable than the corresponding
for PQ 4h2, as it is apparent from the comparison of both cumula-
tive IE plots (see Figure 3, dashed lines).
The higher stability of the YEX-Chk1 complex suggests a lower
dissociation rate constant (koff) for this compound than for 4h2.
However, through the MD simulations performed it is not possible
to get information about the binding recognition event, which is
related to the association rate constant (kon). Therefore, in order to
complete the qualitative thermodynamic analysis, the recently
implemented methodology SuMD48 was used to compare the
ligand–protein recognition pathway of both compounds.
induced by PQs.
PQ
R1
R2
R3
X
Y
Residual activity (%)a Inhibition (%)
4a3
4c3
4d2
4d3
4d4
4e1
4e2
4e3
4e4
4f3
H
H
H
H
H
Cl
Cl
Cl
Cl
H
H
H
H
H
H
H
H
H
H
F
H
H
H
F
H
H
H
F
F
F
H
F
OCH3
H
H
F
97
121
96
110
124
109
89
117
123
123
91
3
0
4
0
0
0
11
0
0
0
OCH3
Cl
Cl
Cl
H
H
H
H
Br
F
H
Cl
Cl
Cl
Cl
H
OCH3
F
H
F
4h2
4h3
CF3
CF3
9
F
H
98
2
aMean value of two independent analysis.
(maintaining the quinolone substitution) with a different type of
substituents in the phenylpyrazole ring (i.e. 4d3–4, 4e4, and 4h3).
Moreover, two derivatives which shown unfavourable energy inter-
actions into the binding site were included (i.e. 4c3 and 4f3). Such
selection was made with the aim to correlate the potential inhibi-
tory activity of the investigated PQs according to their substitu-
ents. Unfortunately, the profiling of the tested compounds (4a3,
4c3, 4d2–4, 4e1, 4e3–4, 4f3, 4h3) against Chk1 protein kinase
showed no significant inhibitory potency at an assay concentration
of 1 lM with the exception of the two compounds 4e2 and 4h2
which show a modest but significant (approximately 10%) reduc-
tion in the basal activity of the kinase (Table 2).
MD simulations
Thus, these seemingly disappointing results offer a great oppor-
tunity to strengthen scientific investigation in this novel class of
potential Chk1 inhibitors. Starting from the biological activity
observed for the two compounds 4e2 and 4h2, we decided to
extent the molecular modelling simulations in order to rationalise
the experimental results. We propose here to take advantage of
MD simulations to figure out the possible causes of their low
activity as Chk1 inhibitors, which could be useful for the design of
new candidates.
In the starting geometry, 4h2 was placed at a distance of 30 Å
from the binding site. As depicted in Figure 4(A) and shown in
Video S4, the first interaction between the ligand and the protein
is established after 1.5 ns of productive trajectory and is mediated
by Glu17, Gly18, and Ala19. Such residues move along with the
ligand making new interactions with Glu91, Glu134, and Asp148,
where it resides for about 2.7 ns. In fact, the ligand RMSD plot
(Figure 4(B)) records stable values in the 2 ꢂ 4.7 ns time lapse.
Afterward, guided by Lys132, Glu134, and Asp148, 4h2 is oriented
As a first attempt, we investigated the stability of the ligand–- to the binding site remaining there for about 3 ns (second plateau
protein complexes through MD simulations of 4h2 (selected from Figure 4(B)). The IE with the protein in this site is about ꢂ55 kcal/
previous docking simulations) and YEX (reference inhibitor)10,11 mol (Figure 4(C) at dcmL-R ¼ 8 Å). Approximately after 9 ns of simu-
starting from its docking and crystallographic coordinates, lation the ligand moves toward the orthosteric site, where Leu15

178
I. MALVACIO ET AL.
Figure 3. RMSD of 4h2 (blue continuous line) and YEX (red continuous line) with respect to its mass centre; and the cumulative sum of the total IE values for each
frame against the time for 4h2 (blue dashed line) and YEX (red dashed line).
and Glu91 are the key residues that induce the ligand to find its the loss of the hydrogen bond with Glu85 respect to the crystallo-
final conformation. Consistently, the RMSD plot presents another
plateau in the time range of 9 ꢂ 20 ns (Figure 4(B)) that corre-
sponds to the swarm of dots in the IE landscape at dcmL-R ¼ 4 Å
and around –50 and –70 kcal/mol (Figure 4(C)). During the last
10 ns of simulation, 4h2 interacts with Val23, Lys38, Leu 84, Glu85,
Tyr86, Cys87, Glu91, Leu137, Ser147, and Asp148. The ligand–pro-
tein recognition map (Figure 4(A)) highlights that the above-men-
tioned residues are those establishing the greatest number of
contacts, whereas the Pollicino analysis summarises the ligand rec-
ognition pathway (Figure 4(D)). Finally, it is important to mention
that the final SuMD pose is different from that suggested by the
docking simulation (RMSD ꢄ7 Å, Figure 4(B)) so that the hydrogen
bond with Cys87 observed in the latter is not present in SuMD
pose which instead interacts with Asp148 (Figure 4(E)).
graphic structure (Figure 5(E)).
Whereas traditional MD simulations revealed an important dif-
ference in the stability of the complexes, SuMD analysis suggests
that the kon of both compounds are not as different as their koff
.
In other words, the PQ nucleus decoration would affect the dis-
sociation but not the binding process. These observations indicate
that the scaffold modification based on the final state interactions
would result in new active compounds without affecting the bind-
ing process.
Docking simulations of newly proposed compounds
Interestingly, the reference compound YEX is strongly trapped in
the binding cavity since it retains three hydrogen bonds through-
out the MD simulation. As it was described above, the triazolone
ring is oriented in a way that allows the formation of two hydro-
gen bonds interactions: one between its carbonyl group and the
amide nitrogen of residue Cys87, and the other between the tri-
azole NH group and the carbonyl oxygen of residue Glu85. In add-
ition to those interactions, the pyrrole ring present in YEX is a key
portion of the formation of an additional hydrogen bond with the
carbonyl of residue Cys87 (Figure 2(B)). It is important to highlight
that the last mentioned moiety is essential for the inhibitory activ-
ity of YEX, as it was demonstrated by Lv et al.57
With these observations in mind, we propose some chemical
modifications to perform on the PQ nucleus with the aim to
improve the affinity of such compounds for Chk1 kinase. First, the
removal of phenyl group from pyrazolone ring was applied to
increase the possibilities of the N-H pyrazolone to establish hydro-
gen bonds (either as donor or acceptor) as it is present in YEX
structure. Moreover, the addition of pyrrole ring to the quinolone
scaffold and a combination of both alternatives were also taken
into account (Scheme 3).
On the other hand, the first interaction of the reference com-
pound YEX with Chk1 is mediated by residues Glu97, Pro98, and
Asp99 (Figure 5(A)). However, the ligand is immediately stabilised
by nearby residues Asp94, Arg95, and Ile96 until 2 ns of productive
trajectory (first plateau, Figure 5(B)). The latter three residues along
with Gly89 and Gly90 induce a shift in the ligand position that pla-
ces it toward the orthosteric site. Mediated by the same key resi-
dues (Glu91 and Leu15) found in the previous case, YEX reaches
the binding site in less than 4 ns (second plateau, Figure 5(B)). The
ligand–protein recognition map (Figure 5(A)) shows the ligand
main contacts with residues Leu15, Gly16, Glu17, Val23, Lys38,
Leu84, Glu85, Cys87, Glu91, Leu137, and Ser147 during the rest of
SuMD simulation (Video S5). Meanwhile, the IE landscape shows
that there is a unique zone explored by the ligand that corre-
sponds to the highly populated region at dcmLꢂR ¼ 3 Å and
around –40 and –60 kcal/mol (Figure 5(C)). The same conclusion
arises from Pollicino analysis (Figure 5(D)) where the ligand path-
way converges quickly in the binding site. Once YEX reaches the
binding site, that pose is maintained until the end of the simula-
tion (17.5 ns), with the exception of conformational changes occur-
ring to the pyrrole ring, able to establish a hydrogen bond with
the side chain of Glu91 (Figure 5(E)). Although the final SuMD
pose seems to be close to the structure of the complex obtained
by X-ray crystallography, the RMSD between them is ꢄ5 Å (Figure
The electrostatic and hydrophobic contributions to the IE of
the newly proposed compounds are displayed in Figure 6 as IEFs
(Video S6).
A first glance at the docking poses of the new derivatives
5(B)) as they are rotated about 100ꢁ. Such conformation implies revealed that the exclusion of phenyl ring improves the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
179
Figure 4. PQ 4h2 recognition pathway: (A) ligandꢂ protein recognition map, (B) ligandꢂ RMSD, (C) IE landscape, (D) Pollicino analysis (M1, M2, M3, and M4, indicate
clusters) and (E) hydrogen bond interactions (dashed lines) of final SuMD pose in comparison with the pose suggested by docking simulation.
electrostatic interaction with Glu85 but decreases the hydrophobic it was mentioned above for YEX (Figure 7). Furthermore, the
interactions with some of the receptor residues involved in the additional hydrogen bond interaction with Glu85 would not be
binding with the ligand. On the other hand, the simple addition possible when the pyrazolone moiety is substituted with a phenyl
of the pyrrole ring, by maintaining the phenyl group in the pyra- group which in that case, makes the ligand place with a different
zolone (compound 10), does not show any improvement in the binding mode (Figure 2).
electrostatic contribution (Figure 6). Finally, the best results arose
As is suggested in Figures 6 and 7, compound 8 has demon-
from compound 8, which shares with YEX a common positioning strated not only an improvement in the electrostatic contribution
of the pyrazolone ring. It is worth recalling that such orientation to the IE with Cys87 but also an enhancement of the hydrogen
allows the formation of hydrogen bonds with Cys87 and Glu85 as bond interactions, essential for the inhibitory activity as some

180
I. MALVACIO ET AL.
Figure 5. Reference compound YEX recognition pathway: (A) ligand ꢂ protein recognition map, (B) ligandꢂ RMSD, (C) IE landscape, (D) Pollicino analysis (M1, M2, M3,
M4, and M5 indicate clusters) and (E) hydrogen bond interactions (dashed lines) of final SuMD pose in comparison with the crystallographic pose.
pharmacophore models propose56,58. Taking all these factors into 90%, independently of the methodology used. However, the reac-
tion times were considerably reduced when MW heating was
applied.
account, a new library of PQs is being synthesised considering the
modifications proposed and they will be tested as Chk1 inhibitors
in the next future.
For the first time, the selected PQs were tested as Chk1 inhibi-
tors. Therefore, the implementation of molecular modelling, espe-
cially a hybrid approach based on docking and MD simulations,
was critical for the understanding of the ligand–protein interac-
tions at molecular level and, to generate a possible hypothesis to
the experimentally observed inactivity of these compounds.
Starting from docking simulations, a difference in the binding
Conclusions
A library of 2-aryl-2 H-pyrazolo[4,3-c]quinolin-3-ones, containing 14
new chemical structures, was synthesised using conventional or
MW heating. The yields after purification were ranged from 15 to mode was identified for the studied compounds with respect to

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
181
the reference inhibitor YEX. Whereas, the PQs make only one
hydrogen bond interaction with Cys87, the reference YEX has two
interactions with Cys87 and one with Glu85. This hydrogen bond
network of YEX is retained during the MD simulation, suggesting
that a more stable complex with the protein is formed compared
with 4h2, which changed its conformation as soon as simulation
starts. Moreover, the use of SuMD offered a precious insight into
the ligand–protein recognition pathway. From the comparative
analysis of both simulations, it is worthy to remark the role of resi-
dues Leu15 and Glu91 in the ligand entrance to the binding site.
The SuMD analysis suggests that the entrance path is weakly
dependent on the chemical substitution of the ligand. Thus, ligand
affinity could be improved by changing the scaffold decoration
without affecting the recognition pathway. Indeed, the results of
Scheme 3. New proposed compounds 5–10.
Figure 6. IEFs of new proposed compounds 5–10. The interactions observed for 4h2 and YEX are also included for comparison.
Figure 7. The binding mode of YEX (black) and new proposed compound 8 (grey) into the Chk1. Hydrogen bond interactions are shown as dashed lines.

182
I. MALVACIO ET AL.
MD simulations guided us to propose new structures by taking
into account the hydrogen bond arrangement observed for YEX.
It is important to mention that one promising compound
emerged from this study. The new candidate presents not only an
improvement in the electrostatic contribution to the IE with Cys87
but also an enhancement of the hydrogen bond interactions,
essential for the inhibitory activity.
Finally, we want to emphasise the importance of getting useful
information from an apparently disappoint result, which could be
the beginning for further hits identification. Besides, this article
illustrates the relevance of using molecular modelling as part of
an iterative cycle of design, synthesis, biological evaluation,
hypothesis generation and leader optimisation in the discovery of
new potential drug candidates.
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Disclosure statement
No potential conflict of interest was reported by the authors.
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Funding
The authors acknowledge the financial support from the National
Scientific and Technical Research Council of Argentina (CONICET).
I. Malvacio acknowledges the PhD fellowship from CONICET and
the research fellowship BEC.AR from Argentine government.
Molecular Modeling Section (MMS) is very grateful to Chemical
Computing Group, Acellera and OpenEye for the long and fruitful
collaborations.
17. Zhao B, Bower MJ, McDevitt PJ, et al. Structural basis for
ORCID
Chk1 inhibition by UCN-01.
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Biol Chem 2002;277:
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Mattia Sturlese
http://orcid.org/0000-0003-3944-0313
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