Computer-aided molecular design of pyrazolotriazines targeting glycogen synthase kinase 3

Article abstract of DOI:10.1080/14756366.2018.1530223

Numerous studies have highlighted the implications of the glycogen synthase kinase 3 (GSK-3) in several processes associated with Alzheimer’s disease (AD). Therefore, GSK-3 has become a crucial therapeutic target for the treatment of this neurodegenerative disorder. Hereby, we report the design and multistep synthesis of ethyl 4-oxo-pyrazolo[4,3-d][1–3]triazine-7-carboxylates and their biological evaluation as GSK-3 inhibitors. Molecular modelling studies allow us to develop this new scaffold optimising the chemical structure. Potential binding mode determination in the enzyme and the analysis of the key features in the catalytic site are also described. Furthermore, the ability of pyrazolotriazinones to cross the blood–brain barrier (BBB) was evaluated by passive diffusion and those who showed great GSK-3 inhibition and permeation to the central nervous system (CNS) showed neuroprotective properties against tau hyperphosphorylation in a cell-based model. These new brain permeable pyrazolotriazinones may be used for key in vivo studies and may be considered as new leads for further optimisation for the treatment of AD.

Full text of DOI:10.1080/14756366.2018.1530223

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Computer-aided molecular design of
pyrazolotriazines targeting glycogen synthase
kinase 3
M. Lourdes Sciú, Victor Sebastián-Pérez, Loreto Martinez-Gonzalez, Rocio
Benitez, Daniel I. Perez, Concepción Pérez, Nuria E. Campillo, Ana Martinez &
E. Laura Moyano
To cite this article: M. Lourdes Sciú, Victor Sebastián-Pérez, Loreto Martinez-Gonzalez,
Rocio Benitez, Daniel I. Perez, Concepción Pérez, Nuria E. Campillo, Ana Martinez & E. Laura
Moyano (2019) Computer-aided molecular design of pyrazolotriazines targeting glycogen
synthase kinase 3, Journal of Enzyme Inhibition and Medicinal Chemistry, 34:1, 87-96, DOI:
10.1080/14756366.2018.1530223
To link to this article: https://doi.org/10.1080/14756366.2018.1530223
© 2018 The Author(s). Published by Informa
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2018, VOL. 34, NO. 1, 87–96
https://doi.org/10.1080/14756366.2018.1530223
RESEARCH PAPER
Computer-aided molecular design of pyrazolotriazines targeting glycogen
synthase kinase 3
a
ꢀa,b
ꢀ
ꢀ
, Loreto Martinez-Gonzaleza, Rocio Beniteza, Daniel I. Pereza
and E. Laura Moyanob
,
ꢀ
ꢀ
M. Lourdes Sciu , Victor Sebastian-Perez
c
ꢀ
ꢀ
Concepcion Perez
, Nuria E. Campilloa , Ana Martineza
a
ꢀ
Department of Chemical and Physical Biology, Centro de Investigaciones Biologicas (CIB, CSIC) Ramiro de Maeztu, Madrid, Spain;
b
c
ꢀ
ꢀ
INFIQC- Department of Organic Chemistry, School of Chemical Sciences, National University of Cordoba, Cordoba, Argentine; Instituto de
ꢀ
ꢀ
Quımica Medica (IQM, CSIC), Madrid, Spain
ABSTRACT
ARTICLE HISTORY
Received 3 May 2018
Revised 15 August 2018
Accepted 17 September 2018
Numerous studies have highlighted the implications of the glycogen synthase kinase 3 (GSK-3) in several
processes associated with Alzheimer’s disease (AD). Therefore, GSK-3 has become a crucial therapeutic tar-
get for the treatment of this neurodegenerative disorder. Hereby, we report the design and multistep syn-
thesis of ethyl 4-oxo-pyrazolo[4,3-d][1–3]triazine-7-carboxylates and their biological evaluation as GSK-3
inhibitors. Molecular modelling studies allow us to develop this new scaffold optimising the chemical
structure. Potential binding mode determination in the enzyme and the analysis of the key features in the
catalytic site are also described. Furthermore, the ability of pyrazolotriazinones to cross the blood–brain
barrier (BBB) was evaluated by passive diffusion and those who showed great GSK-3 inhibition and perme-
ation to the central nervous system (CNS) showed neuroprotective properties against tau hyperphosphory-
lation in a cell-based model. These new brain permeable pyrazolotriazinones may be used for key in vivo
studies and may be considered as new leads for further optimisation for the treatment of AD.
KEYWORDS
GSK-3 inhibitors;
pyrazolotriazinones;
Alzheimer’s disease;
drug design
1. Introduction
one of the main targets studied for the treatment of the neurode-
generative disease. Over the last few years, different heterocyclic
compounds have been described as GSK-3 inhibitors including
natural products as manzamine alkaloids7 and palinurine8, but a
great number of chemical substances come from synthetic
approaches. Among them, pyrazol-5-ones9, pyrazolo-fused deriva-
Alzheimer’s disease (AD) is a neurodegenerative disorder charac-
terised by memory loss and progressive impairment in cognitive
functions. To date, no cure has been found that stops or reverses
the underlying progression of the disease1. Although the cause of
AD remains unknown, three main hypotheses have been devel-
oped that define the neuropathological profile of this disease. AD
is characterised by three key structural changes in the brain such
as neuronal loss, extracellular b-amyloid plaques accumulations,
and intracellular neurofibrillary tangles (NFT) deposits formation
by hyperphosphorylation of tau protein2.
tives10–14
,
pyrazines15, thiadiazolidinones16,17, triazinones18, are
good examples. Some of them contain privileged scaffolds, as the
pyrazole nucleus, and others can be classified as purine analogues,
since they are structurally related to purine due to their fused five
and six-membered nitrogen heterocycles19.
Purine analogues have been widely studied in drug design due
to the action of purine nucleotides like ATP, GTP, cAMP, cGMP,
NAD, FAD, as co-factors, substrates or mediators in the functioning
of many proteins19,20. In fact, structurally purine related com-
pounds have shown multiple biological activities, such as antiviral,
Protein kinases are key regulator of cellular functions since
they direct the activity, localisation, and overall function of many
proteins by adding phosphate groups to protein substrates. As
protein kinases are involved in the regulation of almost every cel-
lular process, they have become the main target of study for the
treatment of several diseases. The glycogen synthase kinase 3
(GSK-3) is a serine/threonine kinase involved in the regulation of
glycogen synthesis and modulation of other intracellular proc-
esses. Over-activation of GSK-3 is implicated in several processes
associated with AD, such as tau hyperphosphorylation, increase of
b-amyloid plaques production, microglia activation, neurofibrillary
antibacterial, anti-inflammatory activity, among others20–22
.
Following our efforts to attain purine analogues of biological
interest, hereby we describe the evaluation of pyrazolo[3,4-
d][1–3]triazin-4-one as potential GSK-3 inhibitors and docking
studies to determine the binding mode to the enzyme.
Subsequently, molecular modelling was used to guide the chem-
ical optimisation of these compounds. New ethyl 4-oxo-pyra-
tangle formation, and cell apoptosis3,4
Moreover, a full etiopathological hypothesis for AD has been
proposed based on GSK-3 dysfunction5,6. Therefore, this kinase is computational tools, synthesised from accessible precursors and
.
zolo[4,3-d][1–3]triazine-7-carboxylates
were
conceived
by
ꢀ
CONTACT E. Laura Moyano
Haya de la Torre y Medina Allende, X5000HUA Cordoba, Argentine; Ana Martinez
lauramoy99@gmail.com
INFIQC- Department of Organic Chemistry, School of Chemical Sciences, National University of Cordoba,
ana.martinez@csic.es Department of Chemical and Physical Biology, Centro
ꢀ
ꢀ
de Investigaciones Biologicas (CIB, CSIC) Ramiro de Maeztu, 9, 28040 Madrid, Spain
ꢀ
These authors contributed equally to this publication.
Supplemental Data for this article can be accessed here.
ß 2018 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,
distribution, and reproduction in any medium, provided the original work is properly cited.

ꢀ
M. L. SCIU ET AL.
88
evaluated as GSK-3 inhibitors. Their ability to cross the blood–- electrostatic energy and ligand efficiency. Finally, best-docked
brain barrier by passive diffusion was also evaluated to consider clusters (within the default 2.0 Å RMSD) according to the binding
them as potential drug candidates for the treatment of AD.
energies and relative population provided by Autodock were ana-
lysed by visual inspection.
Analysis of the receptor-ligand complex models was based on
energetic score, population of the conformations in the different
clusters and interactions. Interactions considered were hydrogen
bonds, aromatic and hydrophobic ones, all were predicted
with Maestro.
2. Materials and methods
2.1. Molecular modelling
2.1.1. Protein preparation
The protein 4NM523 was retrieved from the protein data bank and
was prepared using Protein Preparation Wizard of Schrodinger
Suite24. 4NM5 was selected because it is crystallized with a purine
derivative, a similar scaffold to the compounds described here.
Also, present a good resolution and present no gaps comparing
with other structures. The protein structure was checked to assign
correct bond orders, hydrogen atoms were added, and waters
were deleted beyond 5 Å of heteroatoms groups. The hydrogen
bonds were optimised and assigned and the protonation states of
all the residues were optimised using a physiological pH of 7.3. All
the waters molecules were removed and finally, an energy
restrained minimisation was carried out using default constraint of
0.3 Å RMSD and OPLS 2005 force field25.
2.1.5. Hotspot analysis
After preparation of the protein as described above, the hotspot
maps were calculated using the protocol developed by the group
of Prof. Blundell28. To generate fragment hotspots, atomic hot-
spots are first calculated. SuperStar calculated the atomic hot-
spots. Three maps were generated: hydrophobic ꢂ aromatic CH
probe, donor ꢂ uncharged NH probe, acceptor ꢂ carbonyl
O
probe. SuperStar uses the LIGSITE32 algorithm to detect cavities,
and in the absence of a starting coordinate or residue from which
to grow the cavity, LIGSITE runs on the whole protein.
It gives each grid point a buriedness score between zero (com-
pletely solvent exposed) and seven (completely buried). SuperStar
then provided atomic propensities for cavities that contained grid
points with a LIGSITE score of five or above. To find areas where
high interaction propensity coincides with buried pockets, the
SuperStar maps are weighted by the LIGSITE score for each grid
point. The weighted SuperStar maps show propensity throughout
the protein, but in order to find fragment hotspots, the atomic
propensities were sampled with molecular probes. These probes
were toluene for a polar region, aniline for donors region and a
carbonyl for acceptors region.
2.1.2. Docking
Automated docking was used to assess the appropriate binding
orientations and conformations of the ligand molecules with dif-
ferent protein inhibitors. A Lamarckian genetic algorithm method
implemented in the programme AutoDock 4.2 was employed26,27
;
to carry out the docking simulations AutoDock suite was used. For
docking calculations, Gasteiger charges were added and the rotat-
able bonds were set by AutoDock tools (ADT) and all torsions
could rotate for the ligand. The ligand was treated as a flexible
molecule while the protein was treated as rigid. Polar hydrogen
atoms were added and Gasteiger charges were assigned to the
protein, using ADT and AD4 atom type was assigned. The modi-
fied structures obtained were converted in PDBQT format in ADT
for AutoDock calculations.
2.2. Chemistry
2.2.1. General chemical methods
All reagents were obtained from commercial sources and used
without further purification. Proton (1H) and Carbon (13C) NMR
spectra were recorded on a 400 MHz Bruker spectrometer (1H at
400.16 MHz and 13 C at 100.9 MHz) at ambient temperature.
Solutions were typically prepared in either deuterochloroform
(CDCl3), deuteroacetone ((CD3)2CO), deuterated acetonitrile
(CD3CN) or deuterated dimethylsulphoxide ((CD3)2SO) with chem-
ical shifts referenced to deuterated solvent as an internal standard.
1H NMR data are reported indicating the chemical shift (d), the
multiplicity (s, singlet; d, doublet; t, triplet; q, quartette; m, multi-
plet; br, broad; dd, doublet of doublets, etc.), the coupling con-
stant (J) in Hz and the integration (e.g. 1H). High-resolution mass
spectra (HRMS) were recorded on a LC-MS Bruker Daltonics Micro
TOFF QII with electrospray ionisation (ESI) and Q-TOF detection.
2.1.3. Validation
To validate the docking protocol, 1Q3W was retrieved from pdb.
This pdb was selected because it contains alsterpaullone, one of
the most potent known inhibitors of GSK3. Alsterpaullone and
pdb protein structure were prepared as described above. Grid was
centred in Val135 using a grid box size of 50 ꢁ 50 ꢁ 50 points
with a grid-point spacing of 0.375 Å. The grid maps were gener-
ated by Autogrid programme.
2.1.4. Evaluation
For the compounds evaluated the grid was centred in Val135, as
it has been proved to be an important residue in the protein-lig-
and recognition. The grid maps were generated by Autogrid pro-
gramme. In all the cases grid maps with a grid box size of
50 ꢁ 50 ꢁ 50 Å3 and a grid-point spacing of 0.375 Å were used.
The docking protocol for all the compounds consisted of 200
independent Genetic Algorithm (GA) runs per ligand, population
size of 150, maximum number of evaluation 2,500,000, maximum
number of 27,000 generation, mutation rate of 0.02 and a cross-
over rate of 0.8 were used for this study. The docking results for a
2.2.2. General procedure for the synthesis of N-aryl-hydra-
zones (7a–h)
To an ice-cold solution of the aniline 6 (1.0 mmol) and 37% aq.
HCl (1.1 ml, 13.4 mmol) in water (5 ml mmolꢂ1) was added drop-
wise 1 M aq. NaNO2 (1 ml, 1.0 mmol). The mixture was stirred for
30 min and then added dropwise to a solution of malononitrile
(0.09 ml, 1.5 mmol) and sodium acetate trihydrate (4.22 g,
31.0 mmol) in water (8.5 ml mmolꢂ1 of aniline) with continuous
given macromolecule-ligand pair mainly comprised of the inter- stirring and cooling to 0 ꢃC. After 2 h, the insoluble hydrazone was
molecular interaction energies including inhibition constant, isolated by filtration and washed with cold water. The desired
hydrogen bond interaction energy, van der Waals forces, hydrazone was used without further purification in the next step.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
89
2.2.3. General procedure for the preparation of ethyl 4-amino-3- ring) cmꢂ1. HRMS calculated for C13H11BrN4O2Hþ: 335.01381,
found: 335.01368.
cyano-1H-pyrazole-5-carboxylates (8a–h)
mixture of hydrazone (1.0 mmol), potassium carbonate
Ethyl 4-amino-3-cyano-1–(4-iodophenyl)-1H-pyrazole-5-carboxyl-
ate (8g): This compound was obtained as brown solid in a 55%
yield, m.p. 135–138 ꢃC. 1H NMR (400 MHz, (CD3)2CO, 25 ꢃC):
d ¼ 7.95–7.86 (m, 2H), 7.37–7.31 (m, 2H), 5.70 (s, 2H), 4.24 (q,
J ¼ 7.1 Hz, 2H), 1.17 (t, J ¼ 7.1 Hz, 3H) ppm. 13 C NMR (101 MHz,
CD3CN, 25 ꢃC): d ¼ 159.8, 143.6, 141.2, 139.4, 138.7 (2C), 128.9 (2C),
115.2, 113.6, 95.0, 62.1, 14.3 ppm. IR (AgBr): t ¼ 3450, 3361
(NH), 2233 (CN), 1700 (C¼O), 1640, 1489 (C¼C and C¼N ring)
A
7
(0.138 g, 1.0 mmol), and ethyl bromoacetate (0.45 g, 2.7 mmol) in
dimethylformamide (3 ml mmolꢂ1) was heated at 90 ꢃC for 5 to
7 h. After the reaction was finished, the mixture was poured in a
mix of ice and water and kept at 4 ꢃC for 4–5 h. The residue
obtained was isolated by filtration and washed with cold water.
The resulting solid was used without further purification for com-
pounds 8a–c and 8g. Instead, the solid obtained for compounds
8d–f and 8h was recrystallized from ethanol.
cmꢂ1
.
HRMS calculated for C13H11IN4O2Hþ: 382.99995,
Ethyl 4-amino-3-cyano-1-phenyl-1H-pyrazole-5-carboxylate (8a):
This compound [CAS 58838–10-1] was obtained as a pale orange
solid in 39% yield, m.p. 113–116 ꢃC. 1H NMR (400 MHz, CD3CN,
25 ꢃC): d ¼ 7.56–7.33 (m, 5H), 5.15 (s, 2H), 4.18 (q, J ¼ 7.1 Hz, 2H),
found: 382.999978.
Ethyl
4-amino-3-cyano-1–(4-nitrophenyl)-1H-pyrazole-5-carb-
oxylate (8h): This compound was obtained as an orange solid in a
18% yield. 1H NMR (400 MHz, (CD3)2SO, 25 ꢃC): d ¼ 8.33 (d,
J ¼ 8.8 Hz, 2H), 7.81 (d, J ¼ 8.8 Hz, 2H), 6.16 (s, 2H), 4.19 (q,
J ¼ 6.9 Hz, 2H), 1.13 (t, J ¼ 7.1 Hz, 3H) ppm. 13 C NMR (101 MHz,
CD3CN, 25 ꢃC): d ¼ 159.8, 148.8, 145.8, 143.9, 127.9 (2C), 125.9,
125.0 (2C), 116.3, 113.4, 62.3, 14.3 ppm.
13
1.11 (t, J ¼ 7.1 Hz, 3H) ppm. C NMR (101 MHz, (CD3)2CO, 25 ꢃC):
d ¼ 159.7, 143.8, 141.5, 129.4 (2C), 126.9 (2C), 118.1, 114.7, 113.5,
61.5, 14.2 ppm.
Ethyl
4-amino-3-cyano-1-(p-tolyl)-1H-pyrazole-5-carboxylate
(8b): This compound [CAS 1237748–26-3] was obtained as an
orange solid in 38% yield, m.p. 122–124 ꢃC. 1H NMR (400 MHz,
(CD3)2CO, 25 ꢃC): d ¼ 7.42–7.23 (m, 4H), 5.65 (s, 2H), 4.21 (q,
J ¼ 7.1 Hz, 2H), 2.41 (s, 3H), 1.14 (t, J ¼ 7.1 Hz, 3H) ppm. 13 C NMR
(101 MHz, CD3CN, 25 ꢃC): d ¼ 159.9, 143.4, 140.5, 139.0, 131.1,
130.0 (2C), 126.8 (2C), 114.5, 113.8, 61.9, 21.23 14.3 ppm.
2.2.4. General procedure for the synthesis of 4-oxo-4,6-dihydro-
3H-pyrazolo[4,3-d][1–3]triazine-7-carboxylates (5a–c,e–g)
Aqueous NaNO2 (1 ml, 1.8 mmol) was added to a stirred and
cooled solution (0–5 ꢃC) of pyrazole 8 (1.0 mmol) in a mixture of
HCl: AcOH (3:1, 20 ml) over a period of 10 min. The reaction mix-
ture was allowed to warm at room temperature and was stirred
for 20 h. The precipitate of ethyl-pyrazolotriazine-carboxylate
obtained was filtered off and then diluted with water (20 ml).
After that, the solution was extracted with dichloromethane
(3 ꢁ 20 ml) and the organic phase was dried with anhydrous
MgSO4. The resulting solution was concentrated to dryness, and
the solid was then subject to chromatographic column separation
with dichloromethane and dichloromethane/ethyl acetate in differ-
ent proportions.
Ethyl
4-amino-3-cyano-1–(4-methoxyphenyl)-1H-pyrazole-5-
carboxylate (8c): This compound was obtained as an orange solid
1
in 42% yield, m.p. 124–126 ꢃC. H NMR (400 MHz, (CD3)2CO, 25 ꢃC):
d ¼ 7.45–7.37 (m, 2H), 7.07–7.01 (m, 2H), 5.62 (s, 2H), 4.21 (q,
J ¼ 7.1 Hz, 2H), 3.88 (s, 3H), 1.14 (t, J ¼ 7.1 Hz, 3H) ppm. 13 C NMR
(101 MHz, CD3CN, 25 ꢃC): d ¼ 161.2, 159.9, 143.4, 134.5, 128.3 (2C),
115.9, 114.6 (2C), 114.6, 114.3, 113.9, 61.9, 56.4, 14.3 ppm. IR
(AgBr): t ¼ 3471, 3359 (NH), 2230 (CN), 1721 (C¼O), 1617, 1514
(C¼C and C¼N ring) cmꢂ1. HRMS calculated for C14H14N4O3Hþ:
287.11387, found: 287.11996.
Ethyl 4-amino-3-cyano-1–(4-fluorophenyl)-1H-pyrazole-5-carb-
oxylate (8d): This compound was obtained as a pale brown solid
Ethyl
4-oxo-6-phenyl-4,6-dihydro-3H-pyrazolo[4,3-d][1–3]tria-
1
in 49% yield, m.p. 118–124 ꢃC. H NMR (400 MHz, CDCl3, 25 ꢃC):
zine-7-carboxylate (5a): This compound was obtained as a pale
yellow solid in a 35% yield, m.p. 191.5–193 ꢃC. 1H NMR (400 MHz,
(CD3)2CO, 25 ꢃC): d ¼ 13.90 (s, 1H), 7.77–7.54 (m, 5H), 4.38 (q,
J ¼ 7.1 Hz, 2H), 1.27 (t, J ¼ 7.1 Hz, 3H) ppm. 13 C NMR (100 MHz,
(CD3)2CO, 25 ꢃC): d ¼ 158.2, 152.9, 140.8, 137.4, 135.6, 131.0, 130.6,
129.9 (2C), 126.8 (2C), 63.0, 14.2 ppm. IR (AgBr): t ¼ 1723 (C¼O),
1496 (C¼N ring) cmꢂ1. HRMS calculated for C13H11N5O3Naþ:
308.0754; found: 308.0760.
d ¼ 7.42–7.30 (m, 2H), 7.21–7.10 (m, 2H), 4.80 (s, 2H), 4.23 (q,
J ¼ 7.1 Hz, 2H), 1.17 (t, J ¼ 7.1 Hz, 3H) ppm. 13 C NMR (101 MHz,
(CD3)2CO), 25 ꢃC) d ¼ 163.6 (d, J ¼ 246.9 Hz), 159.6, 143.7, 137.8,
129.2 (d, J ¼ 9.1 Hz, 2C), 118.3, 116.1 (d, J ¼ 23.4 Hz, 2C), 114.7,
113.4, 61.6, 14.2 ppm. IR (AgBr): t ¼ 3475, 3369 (NH), 2229 (CN),
1721 (C¼O), 1616, 1511 (C¼C and C¼N ring) cmꢂ1. HRMS calcu-
lated for C13H11FN4O2Hþ: 275.09388, found: 275.09379.
Ethyl
4-oxo-6-(p-tolyl)-4,6-dihydro-3H-pyrazolo[4,3-d][1–3]tria-
Ethyl 4-amino-1–(4-chlorophenyl)-3-cyano-1H-pyrazole-5-carb-
oxylate (8e): This compound was obtained as brown solid in a
zine-7-carboxylate (5b): This compound was obtained as a yel-
low solid in 38% yield, m.p. 193–196 ꢃC. 1H NMR (400 MHz,
(CD3)2SO, 25 ꢃC): d ¼ 14.99 (s, 1H), 7.52 (d, J ¼ 8.4 Hz, 2H), 7.39
(d, J ¼ 8.1 Hz, 2H), 4.33 (q, J ¼ 7.1 Hz, 2H), 2.43 (s, 3H), 1.21 (t,
J ¼ 7.1 Hz, 3H) ppm. 13 C NMR (100 MHz, (CD3)2SO, 25 ꢃC):
d ¼ 157.1, 152.3, 140.1, 137.0, 136.1, 134.0, 129.4 (2C), 128.8,
125.7 (2C), 62.1, 20.8, 13.8 ppm. IR (AgBr): t ¼ 1706, 1725 (C¼O),
1511 (C¼N ring) cmꢂ1. HRMS calculated for C14H13N5O3Naþ:
322.0911; found: 322.0906.
1
52% yield, m.p. 128–133 ꢃC. H NMR (400 MHz, CD3CN, 25 ꢃC):
d ¼ 7.49 (d, J ¼ 8.8 Hz, 2H), 7.41 (d, J ¼ 8.8 Hz, 2H), 5.17 (s, 2H),
4.20 (q, J ¼ 7.1 Hz, 2H), 1.14 (t, J ¼ 7.1 Hz, 3H) ppm. 13 C NMR
(101 MHz, CD3CN, 25 ꢃC): d ¼ 159.8, 143.6, 140.1, 135.5, 129.6 (2C),
128.8, 128.6 (2C), 115.1, 113.6, 62.1, 14.3 ppm. IR (AgBr): t ¼ 3424,
3309 (NH), 2230 (CN), 1724 (C¼O), 1627, 1498 (C¼C and C¼N
ring) cmꢂ1. HRMS calculated for C13H11ClN4O2Hþ: 291.06433,
found: 291.06441.
Ethyl 6–(4-methoxyphenyl)-4-oxo-4,6-dihydro-3H-pyrazolo[4,3-
d][1–3]triazine-7-carboxylate (5c): This compound was obtained as
a light brown solid in 28% yield, m.p. 194 ꢃC (dec.). 1H NMR
(400 MHz, CDCl3, 25 ꢃC): d ¼ 11.55 (s, 1H), 7.47 (d, J ¼ 8.9 Hz, 2H),
7.03 (d, J ¼ 8.9 Hz, 2H), 4.47 (q, J ¼ 7.1 Hz, 2H), 3.90 (s, 3H), 1.37
(t, J ¼ 7.1 Hz, 3H) ppm. 13 C NMR (101 MHz, CDCl3, 25 ꢃC):
Ethyl 4-amino-1–(4-bromophenyl)-3-cyano-1H-pyrazole-5-carb-
oxylate (8f): This compound was obtained as a brown solid in a
1
40% yield, m.p. 132–134 ꢃC. H NMR (400 MHz, CDCl3, 25 ꢃC):
d ¼ 7.59 (d, J ¼ 8.7 Hz, 2H), 7.27 (d, J ¼ 8.7 Hz, 2H), 4.81 (s, 2H),
4.26 (q, J ¼ 7.1 Hz, 2H), 1.20 (t, J ¼ 7.1 Hz, 3H) ppm. 13 C NMR
(101 MHz, CD3CN, 25 ꢃC): d ¼ 159.8, 143.6, 140.6, 133.2, 132.7 (2C),
128.9 (2C), 123.5, 120.5, 113.6, 62.1, 14.3 ppm. IR (AgBr): t ¼ 3426, d ¼ 161.2, 158.8, 157.7, 151.9, 134.4, 132.2, 127.1 (2C), 114.3 (2C),
3309 (NH), 2230 (CN), 1723 (C¼O), 1627, 1490 (C¼C and C¼N 109.9, 63.0, 55.8, 29.9 ppm. IR (AgBr): t ¼ 1714, 1725 (C¼O), 1512

ꢀ
M. L. SCIU ET AL.
90
(C¼N ring) cmꢂ1. HRMS calculated for C14H13N5O4Naþ: 338.0806; Kinase Assay. Kinetic experiments varying both ATP (from 1 to
found: 338.0863.
50 lM) and the inhibitor (at 0.5 and 1 lM) concentrations were
performed, while the concentration of GS-2 was kept constant
at 12.5 lM.
Ethyl
6–(4-chlorophenyl)-4-oxo-4,6-dihydro-3H-pyrazolo[4,3-
d][1–3] triazine-7-carboxylate (5e): This compound was obtained
as a pale yellow solid in a 31% yield, m.p. 198 ꢃC (dec.). 1H NMR
(400 MHz, CD3CN, 25 ꢃC): d ¼ 12.70 (s, 1H), 7.59 (m, 4H), 4.38 (q,
J ¼ 7.1 Hz, 2H), 1.26 (t, J ¼ 7.1 Hz, 3H) ppm. 13 C NMR (101 MHz,
2.3.3. In vitro parallel artificial membrane permeability
CD3CN, 25 ꢃC): d ¼ 158.3, 153.1, 139.4, 137.6, 136.7, 135.8, 130.8, assay (PAMPA)
130.1 (2C), 128.7 (2C), 63.6, 14.2 ppm. IR (AgBr): t ¼ 1723, 1710
Prediction of the brain penetration was evaluated using a parallel
artificial membrane permeability assay (PAMPA). Ten commercial
drugs, phosphate buffer saline solution at pH 7.4 (PBS), ethanol,
and dodecane were purchased from Sigma Aldrich, Across organ-
ics and Fluka. The porcine polar brain lipid (PBL) (catalogue no.
141101) was from Avanti Polar Lipids. The donor plate was a 96-
well filtrate plate (Multiscreen IP Sterile Plate PDVF membrane,
pore size is 0.45 lM, catalogue no. MAIPS4510), and the acceptor
plate was an indented 96-well plate (Multiscreen, catalogue no.
MAMCS9610), both from Millipore. Filter PDVF membrane units
(diameter 30 mm, pore size 0.45 lm) from Symta were used to fil-
ter the samples. A 96-well plate UV, (Thermoscientific, Varioskan
Lux multimode microplate reader) was used for the UV measure-
ments. Test compounds [(3ꢂ5 mg of caffeine, enoxacine, hydro-
cortisone, desipramine, ofloxacine, piroxicam, and testosterone),
(12 mg of promazine), and 25 mg of verapamil and atenolol] were
dissolved in ethanol (1000 lL). Then 100 lL of this compound
stock solution was taken, and 1400 lL of ethanol and 3500 lL of
PBS pH 7.4 buffer were added to reach 30% of ethanol concentra-
tion in the experiment. These solutions were filtered. The acceptor
96-well microplate was filled with 180 lL of PBS:ethanol (70:30).
The donor 96-well plate was coated with 4 lL of porcine brain
lipid in dodecane (20 mg mLꢂ1), and after 5 min, 180 lL of each
compound solution was added. Then 1ꢂ2 mg of every compound
to be determined for their ability to pass the brain barrier were
dissolved in 1500 lL of ethanol and 3500 lL of PBS pH 7.4 buffer,
filtered, and then added to the donor 96-well plate. Then the
donor plate was carefully put on the acceptor plate to form a
“sandwich”, which was left undisturbed for 4 h at 25 ꢃC. During
this time, the compounds diffused from the donor plate through
the brain lipid membrane into the acceptor plate. After incuba-
tion, the donor plate was removed. The concentration of com-
pounds and commercial drugs in the acceptor and the donor
wells was determined by UV plate reader. Every sample was ana-
lysed at three to five wavelengths, in three wells and in three
independent runs. Results are given as the mean [standard devi-
ation (SD)], and the average of the three runs is reported. Ten
quality control compounds (previously mentioned) of known BBB
permeability were included in each experiment to validate the
analysis set.
(C¼O), 1496
(C¼N ring) cmꢂ1
.
HRMS calculated for
C13H10ClN5O3Naþ: 342.0364; found: 342.0374.
Ethyl 6–(4-bromophenyl)-4-oxo-4,6-dihydro-3H-pyrazolo[4,3-d]
[1–3]triazine-7-carboxylate (5f): This compound was obtained as a
yellow solid in 36% yield, m.p. 195 ꢃC (dec.). 1H NMR (400 MHz,
(CD3)2CO, 25 ꢃC): d ¼ 13.93 (s, 1H), 7.83 (d, J ¼ 8.8 Hz, 2H), 7.67 (d,
J ¼ 8.8 Hz, 2H), 4.41 (q, J ¼ 7.1 Hz, 2H), 1.30 (t, J ¼ 7.1 Hz, 3H)
13
ppm. C NMR (101 MHz, (CD3)2CO, 25 ꢃC): d ¼ 158.2, 152.8, 140.0,
137.4, 135.8, 133.0 (2C), 130.6, 129.0 (2C), 124.6, 63.1, 14.3 ppm. IR
(AgBr): t ¼ 1710 broad signal (C¼O), 1490 (C¼N ring) cmꢂ1. HRMS
calculated for C13H10BrN5O3Naþ: 385.9859; found: 385.9868.
Ethyl
6–(4-iodophenyl)-4-oxo-4,6-dihydro-3H-pyrazolo[4,3-d]
[1–3]triazine-7-carboxylate (5g): This compound was obtained as a
yellow solid in 33% yield. m.p. 198 ꢃC (dec.). 1H NMR (400 MHz,
(CD3)2CO, 25 ꢃC): d ¼ 13.90 (s, 1H), 7.77–7.54 (m, 4H), 4.38 (q,
J ¼ 7.1 Hz, 2H), 1.27 (t, J ¼ 7.1 Hz, 3H) ppm. 13 C NMR (101 MHz,
(CD3)2CO, 25 ꢃC): d ¼ 158.2, 152.8, 140.7, 139.1 (2C), 137.4, 135.8,
130.6, 128.9 (2C), 96.3, 63.1, 14.3 ppm. IR (AgBr): t ¼ 1723 broad
signal (C¼O), 1490 (C¼N ring) cmꢂ1
. HRMS calculated for
C13H10IN5O3Naþ: 433.9721; found: 433.9710.
2.3. Biology
2.3.1. Inhibition of GSK-3b
Human recombinant GSK-3b and the pre-phosphorylated polypep-
tide substrate were purchased from Millipore (Millipore Iberica
ꢀ
SAU). Kinase-Glo Luminescent Kinase Assay was obtained from
Promega (Promega Biotech Ibeꢀrica, SL)29. ATP and all other
reagents were from Sigma Aldrich (St. Louis, MO). Assay buffer
contained 50 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM EGTA, and
15 mM magnesium acetate.
The method of Baki et al. was followed to analyse the inhib-
ition of GSK-3b30. Kinase-Glo assays were performed in assay buf-
fer using black 96-well plates. In a typical assay, 10 lL (10 lM) of
test compound (dissolved in dimethyl sulfoxide [DMSO] at 1 mM
concentration and diluted in advance in assay buffer to the
desired concentration) and 10 lL (20 ng) of enzyme were added
to each well followed by 20 lL of assay buffer containing 25 lM
substrate and 1 lM ATP. The final DMSO concentration in the
reaction mixture did not exceed 1%. After 30 min incubation at
30 ꢃC, the enzymatic reaction was stopped with 40 lL of Kinase-
Glo reagent. Glow-type luminescence was recorded after 10 min
2.3.4. Okadaic acid-induced tau hyperphosphorylation cell model
Human SH-SY5Y cells were grown in DMEM supplemented with
10% FBS and 1% penicillin/streptomycin at 37 ꢃC and 5% CO2 in
an incubator. SH-SY5Y cells were seeded onto 96-well plate at
60.000 cells per well. 48 h later, cells were pre-incubated with the
compounds at the desired concentration for 1 h and after that
time okadaic acid (OA) (Sigma Aldrich, catalogue no: 09381) was
added at a concentration of 30 nM and incubated for another
24 h. Afterwards, cells were incubated with 0.5 mg mLꢂ1 MTT solu-
tion for at least 4 h at 37 ꢃC and 5% CO2. Then culture media was
removed and the formazan crystals attached to the bottom of the
using
a
FLUOstar Optima (BMG Labtechnologies GmbH,
Offenburg, Germany) multimode reader. The activity is propor-
tional to the difference of the total and consumed ATP. The
inhibitory activities were calculated based on maximal activities
measured in the absence of inhibitor. The IC50 was defined as the
concentration of each compound that reduces a 50% the enzym-
atic activity with respect to that without inhibitors.
2.3.2. Kinetic studies on GSK-3b
To investigate the inhibitory mechanism of compounds 5 on GSK-
3b, several kinetic experiments were performed using the ADP-Glo plate were dissolved with 200 mL of DMSO. Finally, UV-absorbance

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
91
was measured at 595 nM in a microplate reader (Varioskan Flash
Microplate reader, Thermo Scientific).
3. Results and discussion
Following with our efforts to develop effective and selective of
GSK-3 inhibitors, we assayed the potential inhibitory effect of a
family of pyrazolo[3,4-d][1–3]triazin-4-ones 4 previously synthes-
ised in our group (Scheme 1)31.
This family did not show any GSK-3 inhibition at a fixed com-
pound concentration of 10 lM. To redesign this heterocyclic family
of compounds, docking studies were performed to identify struc-
tural clues involved in the kinase-small molecule binding. First, a
validation protocol was carried out using alsterpaullone as a refer-
ence inhibitor32. This inhibitor has been crystallized in a complex
with the enzyme and the 3D-coordinates are available in the pro-
tein data bank 1Q3W. To validate the docking protocol here used,
we performed docking studies of the ligand in the ATP-binding
site and analysed the best poses found by the programme.
Comparing the position of alsterpaullone in the available crystal
structure and the docking poses found (Figure 1), it can be con-
cluded that both conformations are very similar, and the interac-
tions found in both cases are the same. These results support the
docking protocol developed here.
Figure 1. Superimposition of the crystal structure of Alsterpaullone ligand in
complex with GSK-3, 1Q3W (blue) and the best pose resulting from docking stud-
ies (grey).
Once the protocol was validated, docking studies for com-
pounds 4a–g were performed. The results were analysed based
on the binding energy, the population of the conformations in
each cluster and the main interactions between the pyrazolotriazi-
nones and the enzyme. Both, electrostatic and hydrophobic inter-
actions were analysed.
According to the results, all compounds showed a similar bind-
ing mode in the catalytic region of GSK-3. As an example, the
docking pose obtained for compound 4a is shown in Figure 2(a).
Among the main interactions analysed, two hydrogen bonds were
observed between the triazin-4-one moiety and residues Asp133
and Val135. Also, a negative interaction was found because of the
orientation of the phenyl ring towards the residue Arg141 due to
steric impediment. Moreover, this negative interaction was
increased for compounds with a substituent in the para position
of the phenyl group, such as compound 4e, as it is shown in
Figure 2(b). This interaction could explain why compounds 4a–g
turned out to be inactive in GSK-3.
Following, different molecular modelling studies were per-
formed to analyse the enzyme cavity and design new compounds
for the potential inhibition of GSK-3.
To study the enzyme cavity, a hotspot analysis was per-
formed28. This method samples atomic hotspots with simple
molecular probes to produce fragment hotspot maps. These maps
specifically highlight fragment-binding sites and their correspond-
ing pharmacophores. For ligand-bound structures, they provide
Figure 2. Binding mode for inactive compound 4a (a) and 4e (b) in GSK-3 show-
ing two relevant H-bonds with nearby residues Asp133 and Val135 and negative
interaction with Arg141, due to steric impediment.
Scheme 1. Synthesis of pyrazolo[3,4-d][1–3]triazin-4-ones 4a–g.

ꢀ
M. L. SCIU ET AL.
92
Figure 4. New design scaffold for potential GSK-3 inhibition.
Figure 5. Binding mode for compound 5b (R¼CH3) in GSK-3 showing three rele-
vant H-bonds with nearby residues.
found by the hotspots studies that end near residues Lys85
and Asp200.
Figure 3. (a) Hotspot analysis of the binding site of the enzyme. (b) Polar regions
in the protein cavity that could act as H-bond donor (blue) or H-bond
acceptor (red).
Bearing in mind experimental and docking studies of inactive
compounds 4a–g together with the hotspot analysis; new GSK-3
potential inhibitors were designed. Since triazin-4-one scaffold
interacts favourable with the enzyme cavity, this moiety remains
unaltered. Instead, we decided to modify the type of ring fused
between the pyrazole and triazine rings, to direct the phenyl sub-
stituent towards the hydrophobic region and try to occupy the
entire catalytic pocket. Also, a carboethoxy substituent was intro-
duced in the pyrazole ring as a polar group to attempt a favour-
able interaction with Arg141.
The newly designed scaffold is shown in Figure 4. Docking
studies were performed to evaluate the binding mode with the
enzyme, varying the substituent in the para position of the phenyl
group. The docking protocol previously described was used, and
both, hydrophobic and electrostatic interactions were analysed.
The substituents in the phenyl group were chosen considering
commercially available starting materials.
an intuitive visual guide within the binding site, directing medi-
cinal chemists where to make modifications in the molecule to
improve potency.
The analysis generates protein hotspots, which are regions
within enzyme pockets that might be critical to contribute to the
binding mode of the ligand. Identification of hotspots and their
specific interactions can be used to evaluate the ligand ability of a
pocket and suggest which interactions fragments and larger
ligands will be needed to make a stronger binding.
With this study, a colour map of the main interactions can be
obtained that could be important for the activity in the enzyme.
In this sense, we can identify either novel inhibitors or explain the
differences in activities between ligands considering critical
protein-ligand recognition features.
As a result, all compounds showed a similar binding mode in
the catalytic region of GSK-3, shown in Figure 5 for compound 5b
(R¼CH3). Among the main interactions analysed, the two hydro-
gen bonds between triazine moiety and residues Asp133 and
Val135 remain unaltered. Additionally, a new H-bond was formed
between the carboethoxy group in C-7 and residue Arg141.
Afterwards, docking results were combined with the hotspot
analysis. The superimposition of the polar regions of the enzyme
pocket and the docking pose obtained for compound 5b are
shown in Figure 6. The three H-bonds matches exactly with the
hotspot prediction. Moreover, phenyl group was oriented towards
the hydrophobic region and was able to occupy a larger area of
the catalytic pocket. This analysis suggested that both, pyrazolo-
Because of the analysis, a surfaces map, shown in Figure 3(a),
was obtained, where optimal interactions could be determined
depending on the different colours. Thus, the yellow region indi-
cates favourable hydrophobic interactions (Figure 3(a)), while red
and blue indicate favourable polar interactions as shown in
Figure 3(b).
Considering this analysis, the two hydrogen bonds between 4a
and nearby residues Asp133 and Val135 in the binding site seems
relevant, since they match with the hotspot maps. Also, phenyl
ring should be removed from the molecule in position N7 to
avoid negative interactions and a polar group might be added in
this position capable of interact favourable with Arg141. This
modification might allow a hydrogen-bond acceptor in the mol-
ecule with the Arg141. Moreover, phenyl ring could be re-oriented triazinone scaffold and the carboethoxy group in C-7, could be
in the molecule towards the hydrophobic region in the pocket considered as chemical key features for the inhibition of GSK-3.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
93
With the aim to synthesise compounds ethyl 4-oxo-4,6-dihy- due to the electronegativity of the substituents in the phenyl ring
dro-3H-pyrazolo[4,3-d][1–3]triazine-7-carboxylates 5, multistep that could destabilise or avoid the diazonium salt formation. It is
a
route was planned using para-substitutes anilines as starting
reagents. Initially, the synthesis of N-aryl-hydrazones (7a–h) was
carried out using commercially available anilines in a simple
protocol previously described33. Sodium nitrite in aqueous hydro-
chloric acid was used for the diazotisation of anilines 6a–h.
Followed by the diazonium ion reacted with malononitrile to give
the N-aryl-hydrazones (Scheme 2). In all cases, dicyano-hydrazones
7a–h were obtained in excellent yields (Table 1).
Afterwards, the syntheses of ethyl 4-amino-3-cyano-1H-pyra-
zole-5-carboxylates 8a–h were performed. As it is described in lit-
erature33–36, a methylene moiety is needed for the formation of
pyrazole ring closure. In this approach, ethyl bromoacetate was
used under basic conditions (K2CO3) in DMF at 90 ꢃC for 5–7 h to
afford the corresponding pyrazole (Scheme 2) with low to good
yield (Table 1). Pyrazoles 8a and 8b are described in literature35,
while compounds 8c–h are all new compounds and were fully
characterised in this work.
known that the stability and reactivity of pyrazole-derived diazo-
nium salts are strongly dependent on the nature of its substitu-
ents in N-137. The structure of the 3H-pyrazolotriazinones
tautomer was confirmed by NMR experiments agreeing with previ-
ous results31. As an example, the analysis of 13 C and 2 D spectra
for compound 5b is shown in Figure 7. These experiments
allowed the assignation of both carbonyl carbons, one from the
triazine ring and the other from the carboethoxy group. Also, a
correlation between H-3 and the carbonyl carbon from the triazine
could be established that confirmed the presences of the tauto-
mer 3H.
Ethyl pyrazolotriazinones carboxylates 5a–c, e–g were eval-
uated as potential GSK-3 inhibitors with the luminescent assay
previously used30. As a result, derivative compound 5 acted as
GSK-3 inhibitors at a fixed compound concentration of 10 lM.
Thus, the concentration at which the 50% of inhibition (IC50) is
produced was calculated for compounds 5a–c, e–g, obtaining low
micromolar-submicromolar values (Table 2).
Finally, pyrazoles 8a–h were subjected to diazotisation condi-
tions to prepare novel 4-oxo-pyrazolo[4,3-d][1–3]triazine-7-carbox-
ylates, using the protocol previously described for aminopyrazoles
331, as shown in Scheme 2.
Table 1. Synthesis of hydrazones 7a–h, pyrazoles 8a–h and pyrazolotriazinones
5a–c, e–g.
Yieldb
Yields of pyrazolotriazinones 5 (Table 1) were not as good as
expected due to difficulties in the purification from the reaction
mixture. In the case of diazotisation of compounds 8d and 8h,
the corresponding pyrazolotriazines were not obtained, probably
R
Comp.
Yielda
Comp.
Yield
Comp.
H
7a
7b
7c
7d
7e
7f
99 %
95 %
92 %
86 %
99 %
98 %
94 %
96 %
8a
8b
8c
8d
8e
8f
39 %
38 %
42 %
49 %
52 %
40 %
55 %
18 %
5a
5b
5c
5d
5e
5f
35 %
38 %
28 %
–
31 %
36 %
33 %
–
CH3
OCH3
F
Cl
Br
I
7g
7h
8g
8h
5g
5h
NO2
aIsolated product; bPurified product.
Figure 6. Superimposition of binding mode for compound 5b (R¼CH3) with
Figure 7. HMBC and 13C assignation of carbons (in ppm) and correlations for
compound 5b.
polar regions predicted by the hotspot analysis in GSK-3 cavity.
Scheme 2. Synthesis of hydrazones 7a–h and pyrazoles 8a–h, precursor of the pyrazolotriazinones 5, previously designed. (i) aq. NaNO2 [1.0 mmol], HCl [13.4 mmol] ,
0 ꢃC, 30min; (ii) CH2(CN)2 [1.5 mmol], NaOAc.3H2O [31.0 mmol], H2O [8.5 ml], 0 ꢃC, 2 h; (iii) Br-CH2-CO2Et [2.7 mmol], K2CO3 [1.0 mmol], DMF [3ml], 90 ꢃC, 5–7 h; (iv)
NaNO3 [1.8 mmol], HCl:AcOH 3:1 [20ml], 0–5 ꢃC to r.t., 20 h.

ꢀ
94
M. L. SCIU ET AL.
Table 2. Biological inhibition of GSK-3 of ethyl 4-oxo-pyr-
azolo[4,3-d][1,2,3]triazine-7-carboxylates 5.
Table 3. Permeability (Pe 10ꢂ6 cm sꢂ1) in the PAMPA-BBB assay for 10 commer-
cial drugs (used in the experiment validation) and ethyl pyrazolo[4,3-d][1,2,3]tria-
zine-7-carboxylates with their predictive penetration in the CNS.
R
Comp.
GSK-3 IC50 (lM)a
Compound
Pe bibli. (10ꢂ6 cm sꢂ1 a
)
Pe (10ꢂ6 cm sꢂ1 b
)
BBB prediction
H
5a
5b
5c
5e
5f
4.0 0.2
0.9 0.1
1.7 0.3
3.3 0.3
1.4 0.4
0.8 0.2
CH3
OCH3
Cl
Br
I
Atenolol
Caffeine
Desipramine
Enoxacine
Hydrocortisone
Ofloxacine
Piroxicam
Promazine
Testosterone
Verapamil
5a
0.8
1.3
12
0.9
1.9
0.8
2.5
8.8
17
16
–
–
–
0.6 0.4
1.1 0.4
10.4 0.1
1.1 0.6
0.9 0.3
0.5 0.5
0.5 0.1
8.4 0.3
12.5 2.5
11.9 0.1
1.1 0.6
2.3 0.3
1.6 0.5
2.9 0.7
3.20 0.1
5.0 1.0
–
–
–
–
–
–
–
–
–
5g
aIC50 values are reported as a mean value of three inde-
pendent determinations.
–
CNSꢂ
CNSþ/ꢂ
CNSþ/ꢂ
CNSþ/ꢂ
CNSþ
CNSþ
5b
5c
5e
5f
–
–
–
5g
aReference38
;
bData are the mean SD of three independent experiments.
150
100
50
0
**
Figure 8. Kinetic data determined for 5b suggesting an ATP-competi-
tive behaviour.
****
*
*
To identify the type of inhibition of pyrazolotriazinones 5 on
GSK-3, kinetic experiments were carried out. In that case, com-
pound 5b was evaluated at two different concentrations of 1 lM
and 0.5 lM varying ATP concentration, while GS2 concentration
remains constant. Comparable results were obtained for all com-
pounds. As an example, the data obtained for compound 5b is
represented in a Lineweaver–Burk graph shown in Figure 8. This
graphic suggests that pyrazolotriazinones acted as ATP-competi-
tive inhibitors, indicating that they competed with the ATP in their
binding mode to the enzyme.
g
5f
5b
5
Control
OA 30nM
TDZD8 10uM
The experimental data indicating that these compounds act as
ATP-competitive inhibitors was consistent with docking studies,
suggesting the binding mode in the catalytic region of GSK-3. In
order to assess pyrazolotriazinones 5a-c, e-g as potential new
drug candidates for the treatment of Alzheimer’s disease, their
ability to penetrate the blood–brain barrier (BBB) was evaluated.
For this purpose, a permeability assay based on parallel artificial
membrane was used (PAMPA methodology)38. This procedure is a
high throughput technique that allows the prediction of perme-
ability evaluation of organic compounds using a porcine brain
lipid that emulates the blood–brain barrier.
Treatment
Figure 9. OA-induced neurodegeneration cell model. Effects of treatment with
5b, 5f and 5e. SH-SY5Y cells were treated with the GSK-3 inhibitors at 10 mM for
1 h and subsequently with OA (30nM). The % of cell viability was measured by
ꢀ
ꢀꢀ
p < .001,
the MTT test. Results Mean SEM of three experiments, p < .01,
ꢀꢀꢀ ꢀꢀꢀꢀ
p < .0001, p < .00001.
Furthermore, to confirm cellular activity of our GSK-3 inhibitors,
we check their anti-tau profile by exploring their rescue
potential in the okadaic acid (OA)-induced neurodegeneration cell
model, in comparison with the well know GSK-3 inhibitor named
as TDZD850. OA represents the most robust way to induce PHF-
like tau hyperphosphorylation. Thus, OA-treated cell lines and
primary neuronal cultures have been used as established
The PAMPA assay was used to predict the in vitro permeability
(Pe) of compounds 5a–c, e–g and evaluate their brain penetration
by passive diffusion. An assay validation was made comparing the
reported permeability values of commercial drugs with the experi-
mental data obtained employing this methodology. A good correl-
ation between experimental-described values was obtained Pe
(exp) ¼ 0.7857(bibl) – 0.0816 (R2 ¼ 0.972) (Figure 1SI). From this
equation, and following the pattern established in the literature
for BBB permeation prediction, we could classify compounds as
cellular
models
of
.
hyperphosphorylated
tau-induced
neurodegeneration39,40
We studied the effect of pyrazolotriazinones 5f, 5 g, and also
5b. We select this last compound based on its submicromolar
activity in GSK-3 together with the potential penetration in BBB.
We used a fixed concentration of 10 mM in a human neuroblast-
oma cell line (SH-SY5Y) treated with OA at 30 nM (Figure 9). As
expected, OA induced a decrease in cell viability higher than 70%.
Importantly, the three pyrazolotriazinones assayed were able to
increase the cell viability measured by the MTT test to the same
CNS þ when they present
a
permeability >3.1 ꢁ 10ꢂ6 cm sꢂ1
.
Considering these results, we can determine that compounds 5f
and 5g would be able to cross the BBB by passive permeation
(Table 3) while some others such as 5b–c and 5e are in the limit
of a positive prediction.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
95
level than the standard TDZD-8 does, probably due to the
decrease on tau phosphorylation by GSK-3 inhibition.
Based on all results, ethyl 4-oxo-pirazolo[4,3-d][1–3]triazine-7-
carboxylates 5f and 5g, which show low micromolar-submicro-
molar values of GSK-3 inhibition, were predicted to be able to
pass the BBB, and show a neuroprotective profile against tau
hyperphosphorylation in cell-based models could be considered
as potential candidates for further studies for the treatment
of AD.
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4. Conclusions
In the first part of this study, pyrazolo[3,4-d][1–3]triazin-4-ones
compounds (4a–g) were explored in their capacity to
inhibit GSK-3. Although these compounds did not show inhibi-
tory activity at a fixed concentration of 10 lM, docking studies
were performed to determine their binding mode to
the enzyme.
Secondly, based on the results obtained from computational
studies such as docking and hotspot analysis, a new scaffold of
pyrazolo[4,3-d][1–3]triazin-4-ones 5 was designed as potential
GSK-3 inhibitors. Docking studies were performed to describe
binding mode of 5 to the enzyme. Three hydrogen bonds were
observed between each compound and residues Asp133, Val135,
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ꢀ
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boxylates were prepared using a simple multistep protocol. These
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submicromolar range, validating the computational calculations.
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fusion was also evaluated using an in vitro model and the kinase
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Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
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M. L. Sciu acknowledges the receipt of a scholarship from the
15. Berg S, Bergh M, Hellberg S, et al. Discovery of novel potent
and highly selective glycogen synthase kinase-3b (GSK3b)
inhibitors for Alzheimer’s disease: design, synthesis, and
characterization of pyrazines. J Med Chem 2012;55:9107–19.
16. Martinez A, Alonso M, Castro A, et al. First non-ATP competi-
tive glycogen synthase kinase 3 b (GSK-3b) inhibitors: thia-
diazolidinones (TDZD) as potential drugs for the treatment
of Alzheimer’s disease. J Med Chem 2002;45:1292–9.
National Research Council of Argentina (CONICET). Funding from
MINECO (grant no. SAF2016-76693-R to A.M.) and MEC (FPU15/
1465 to VSP.), CONICET and SECYT-UNC are also acknowledged.
ORCID
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Victor Sebastian-Perez
Daniel I. Perez
http://orcid.org/0000-0002-8248-4496
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http://orcid.org/0000-0003-1774-4471
http://orcid.org/0000-0001-7183-4035
http://orcid.org/0000-0002-9948-2665
http://orcid.org/0000-0002-2707-8110
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ase b (GSK-3b) and phosphodiesterase 7 (PDE7) inhibitors:
determination of blood-brain barrier penetration and
Concepcion Perez
Nuria E. Campillo
Ana Martinez

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