Pyrazolo[1,5-A]quinazoline scaffold as 5-deaza analogue of pyrazolo[5,1-c][1,2,4]benzotriazine system: Synthesis of new derivatives, biological activity on GABA A receptor subtype and molecular dynamic study

Article abstract of DOI:10.3109/14756366.2015.1014475

To investigate the binding affinity of GABA^A receptor subtype, new pyrazolo [1,5-A]quinazolines were designed, synthesized, and in vitro evaluated. These compounds, 5-deaza analogues of pyrazolo[5,1-c][1,2,4]benzotriazine derivatives which were already studied in our research group, permit us to evaluate the relevance of the nitrogen or the oxygen atom at 5-position of the tricyclic scaffold. Molecular dynamic study was done on a set of the new and known ligands to rationalize and to explain the lack of affinity on the 4-or 5-substituted new derivative. In fact, from biological results, it can be found that the only 5-unsubstituted new derivative, compound 15, has receptor recognition (Kⁱ = 834.7 nM).

Full text of DOI:10.3109/14756366.2015.1014475

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–10
2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1014475
!
RESEARCH ARTICLE
Pyrazolo[1,5-a]quinazoline scaffold as 5-deaza analogue of
pyrazolo[5,1-c][1,2,4]benzotriazine system: synthesis of new
derivatives, biological activity on GABAA receptor subtype and
molecular dynamic study
Gabriella Guerrini1, Giovanna Ciciani1, Samuele Ciattini2, Letizia Crocetti1, Simona Daniele3, Claudia Martini3,
Fabrizio Melani1, Claudia Vergelli1, and Maria Paola Giovannoni1
1
2
Dipartimento NEUROFARBA, Sezione Farmaceutica e Nutraceutica, Universita degli Studi di Firenze, Firenze, Italy, Dipartimento di Chimica,
`
3
Centro di Cristallografia, Universita degli Studi di Firenze, Firenze, Italy, and Dipartimento di Farmacia, Universita degli Studi di Pisa, Pisa, Italy
`
`
Abstract
Keywords
To investigate the binding affinity of GABAA receptor subtype, new pyrazolo [1,5-a]quinazolines
were designed, synthesized, and in vitro evaluated. These compounds, 5-deaza analogues
of pyrazolo[5,1-c][1,2,4]benzotriazine derivatives which were already studied in our research
group, permit us to evaluate the relevance of the nitrogen or the oxygen atom at 5-position of
the tricyclic scaffold. Molecular dynamic study was done on a set of the new and known ligands
to rationalize and to explain the lack of affinity on the 4- or 5-substituted new derivative. In fact,
from biological results, it can be found that the only 5-unsubstituted new derivative,
compound 15, has receptor recognition (Ki ¼ 834.7 nM).
Fused tricyclic system, GABAA subtype
receptor affinity, molecular modeling
study, pyrazolobenzotriazine,
pyrazoloquinazoline
History
Received 12 December 2014
Revised 22 January 2015
Accepted 22 January 2015
Published online 20 March 2015
Introduction
as non-camptothecin Topoisomerase 1 (Top1) inhibitors5, while
they have never been studied earlier as GABAA receptor ligands.
Only tricyclic systems, containing pyrazole, triazole, and imid-
azole rings, which are condensed differently with quinoxaline,
Fused tricyclic systems occur frequently in Medicinal Chemistry
and are an attractive starting point for the design of ligands.
Among the heteroatoms containing tricyclic rigid templates, the
pyrazolo[5,1-c][1,2,4]benzotriazine represents a versatile system
that let us to obtain selective subtype ligands to g-aminobutyric
acid type A receptor (GABAA-R)1,2. In these compounds, we have
reported a structure–activity relationship (SAR) and elaborated, in
our laboratory, a ligand-based pharmacophoric model3 to confirm
the essential interaction points for the recognition of binding and
the key-areas for modulation of the affinity. From these findings,
we have obtained ligands with subnanomolar affinity as dual
functional modulators (promnemonic and anxyolitic) and anthy-
quinazoline and ftalazine, are reported for this study6–9
.
Thus, we synthesized the pyrazolo [1,5-a]quinazoline deriva-
tives 3 and/or 4 and/or 5 which were substituted to enhance our
SAR knowledge; moreover, a molecular dynamic study (MDS)
and a principal component analysis (PCA) were performed to
evaluate the requirements of GABAA receptor in the interaction
with the ligands.
Materials and methods
peralgesic compounds on GABAA-R1,2
.
Melting points were determined in open capillary tubes on a
Bu¨chi apparatus (Sigma, St. Louis, MO) and were uncorrected. IR
spectra were recorded (in KBr pellets in nujol mulls) on Perkin-
Elmer 1420 spectrophotometer (Perkin-Elmer, Waltham, MA).
1H-NMR spectra were recorded with an Advance 400 Instrument
(Bruker BiospinVersion 002 with SGU, Bruker AXS Inc.,
Madison, WI). Chemical shifts are reported in ꢀ ppm using the
solvent as an internal standard. Extracted samples were dried over
Na2SO4 and the solvent was removed under reduced pressure.
Merk F-254 commercial plates (Merk-Gruppe, Darmstadt,
Germany) were used for analytical thin layer chromatography
(TLC) to follow the reaction course. Silica gel 60 (70–230 mesh,
Merck, Darmstadt, Germany) was used for column
As part of our lead optimization program, in the present work,
we report the synthesize of new series of pyrazolo[1,5-a]
quinazolines (A) as 5-deaza-analogues of the pyrazolo[5,1-c]
[1,2,4]benzotriazine system (B), Scheme 1.
Compounds containing scaffold A are reported in the recent
literature as negative allosteric modulators of metabotropic
glutamate receptors 2 and 3 (dual mGlu2/mGlu3 NAMs)4 and
Address for correspondence: Gabriella Guerrini, Dipartimento
`
NEUROFARBA, Sezione Farmaceutica e Nutraceutica, Universita degli
Studi di Firenze, Via Ugo Schiff 6, 50019 Sesto Fiorentino, Firenze, Italy.
E-mail: gabriella.guerrini@unifi.it

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G. Guerrini et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
(A)
(B)
procedure is reported:
a
solution of benzylic alcohol
N
N
(0.21 mmoles) and tBuOK (0.21 mmoles) in anhydrous DMF
(3.0 mL) was stirred for 1 h to permit the formation of the
benzyloxy anion. Then, 0.14 mmoles were added to compound 3
and the reaction was refluxed for 2 h. After cooling, the addition
of water gave a precipitate that was filtered and then the
precipitate was recrystallized by methoxyethanol.
N
N
N
N
N
Scheme
c][1,2,4]benzotriazine (B).
1. Pyrazolo[1,5-a]quinazoline
(A)
and
pyrazolo[5,1-
Cream crystals: TLC eluent: toluene/ethyl acetate/acetic acid
8:2:1 v/v/v. 1H-NMR (DMSO-d6): ꢀ 8.44 (s, 1H, H-2), 8.36
(d, 1H, H-9), 8.23 (d, 1H, H-6), 8.07 (t, 1H, H-7), 7.69 (m, 3H,
H-8, H-20 and H-60 phenyl), 7.42 (m, 3H, H-30, H-40 and H-50
phenyl), 5.73 (s, 2H, CH2), 4.32 (q, 2H, CH2), 1.36 (t, 3H, CH3).
IR cmꢀ1: 1704 (COO). Anal Calcd for C20H17N3O3: C 69.15; H
4.93; N 12.10. Found: C 69.32; H 4.82; N 13.21.
chromatography. Microanalyses were performed with a Perkin-
Elmer elemental analyzer (Perkin-Elmer, Waltham, MA) for C, H,
and N. Results within 0.4% of the theoretical materials were
commercially available. Experimental data of more representative
compounds are reported.
Ethyl 4,5-dihydropyrazolo[1,5-a]quinazoline-3-carboxylate (14)
Chemistry
To a suspension of compound 3 (0.300 mmol) in 3.5 mL of THF
anhydrous, 25 mg of Pd/C 10%, 1.0 mmol of ammonium formate,
and 10 mL of methanol were added. The reaction was maintained
at reflux temperature, and it was monitored by TLC (toluene/ethyl
acetate/methanol 8:2:1.5 v/v as eluent), and after 2.5 h the starting
material disappeared. The filtration of catalyst and the next
evaporation of the solution gave a residue that was recovered by
i-propyl ether. Off-white crystals: yield: 52%. 1H-NMR (DMSO):
ꢀ 7.71 (d, 1H, H-2), 7.55 (d, 1H, H-9), 7.36 (t, 1H, H-8), 7.22
(d, 1H, H-6), 7.20 (bs, 1H, NH exchange), 7.19 (t, 1H, H-7), 4.53
3-Iodopyrazolo[1,5-a]quinazolin-5(4H)-one (1c)
A solution of 1b (0.050 g, 0.27 mmol) in dichloromethane (5 mL)
was added to 0.40 mmol of iodine monochloride. The suspension
was refluxed for 1 h and monitored by TLC. The final suspension
was filtered and the precipitate was recovered. Cream crystals;
yield 87%; TLC eluent: toluene/ethyl acetate/acetic acid 8:2:1
1
v/v/v. H-NMR (DMSO): ꢀ 12.26 (bs, 1H, NH exchange), 8.15
(d, 1H, H-9), 8.07 (d, 1H, H-6), 7.90 (t, 1H, H-7), 7.86 (s, 1H,
H-2), 7.51 (t, 1H, H-8). IR cmꢀ1: 3228 (NH), 1678 (C¼O). Anal
Calcd for C10H6IN3O: C 38.61; H 1.94; N 16.51. Found: C 38.48;
H 1.79; N 16.39.
(s, 2H, CH2NH); 4.19 (q, 2H, CH2); 1.26 (t, 3H, CH3). IR cmꢀ1
:
3337 (NH), 1692 (C¼O). Anal Calcd for C13H13N3O2: C 64.19;
H 5.39; N 17.27. Found: C 64.36; H 5.27; N 17.31.
General procedure for the synthesis of compounds 2a–c and 2e
Ethyl pyrazolo[1,5-a]quinazoline-3-carboxylate (15)
To promote the deprotonation, the suitable starting material (1a–c
and 1e) and tBuOK (0.76 mmoles) were suspended in anhydrous
DMF (5 mL) and stirred for 1 h at room temperature; then benzyl
chloride (0.57 mmoles) was added and the mixture was refluxed
for 2 h. The reaction was monitored by TLC (toluene/ethyl
acetate/acetic acid 8:2:1 v/v/v as eluent) until the disappearance
of the starting material. After cooling and addition of water, the
formed precipitate was filtered and recrystallized by a suitable
solvent.
An excess of Pd/C (10%, 100 mg) was added to a solution of
compound 14 (0.350 mmol) in 5 mL of toluene. The mixture was
refluxed for 1 h followed by the filtration of catalyst and the
solvent was evaporated. A yellow residue, recovered by i-propyl
ether, was obtained. Yield: 50%. 1H-NMR (CDCl3): ꢀ 9.21 (s, 1H,
H-5); 8.55 (m, 2H, H-2, and H-9), 8.07 (d, 1H, H-6), 8.01 (t, 1H,
H-8), 7.67 (t, 1H, H-7), 4.49 (q, 2H, CH2); 1.46 (t, 3H, CH3). IR
cmꢀ1: 1692 (C¼O). Anal. Calc. for C13H11N3O2: C 64.72; H
4.60; N 17.42. Found: C 64.59; H 4.69; N 17.31.
Ethyl 4-benzylpyrazolo[1,5-a]quinazolin-5(4H)-one-3-carboxy-
late (2a)
Radioligand binding assay
[3H]Ro15-1788 (specific activity of 78.8 Ci/mmol) was obtained
from Perkin-Elmer (Waltham, MA). All other chemicals were of
reagent grade and were obtained from commercial suppliers.
Bovine cerebral cortex membranes were prepared as previously
described. The membrane preparations were diluted with 50 mM
Tris-citrate buffer pH 7.4, and used in the binding assay. Protein
concentration was assayed using the method of Lowry. [3H]Ro
15-1788 binding studies were performed as previously reported10.
At least six different concentrations of each compound were used.
The data of n ¼ 5 experiments carried out in triplicate were
analyzed by means of an iterative curve-fitting procedure
(program Prism, GraphPad, San Diego, CA), which provided
IC50, Ki, and SEM values for the tested compounds, the Ki values
were calculated from the Cheng and Prusoff equation.
Cream crystals; yield: 89%. 1H-NMR (DMSO): 8.21 (s, 1H, H-2),
8.18 (m, 2H, H-9, H-6), 7.96 (t, 1H, H-7), 7.60 (t, 1H, H-8), 7.23
(m, 3H, H-3, H-4, and H-5 benzyl), 7.08 (d, 2H, H-2, and H-6
benzyl), 5.88 (s, 2H, CH2), 4.11 (q, 2H, CH2), 1.43 (t, 3H, CH3).
IR cmꢀ1: 1707, 1684 (CO). Anal Calcd for C20H17N3O3: C 69.15;
H 4.93; N 12.10. Found: C 69.27; H 4.97; N 12.21.
General procedure for the synthesis of compounds 4–6
To the crude tosyl derivate (1a0, 1c0 and 1d0) in anhydrous DMF
(3.0 mL), 0.6 mmoles of benzyl alcohol and 0.6 mmoles of tBuOK
were added and the reaction was maintained at reflux temperature
for 3 h. After cooling, water was added to the solution and the
precipitate was filtered and purified by recrystallization.
Compound 4 was obtained using either the corresponding
5-tosyl- or 5-chloro derivatives as the starting material. The two
methods of synthesis are reported below.
Docking calculation
The poses of ligands within the interaction site were searched
with AUTODOCK11 and only the amino acids that are in a range
Ethyl 5-benzyloxypyrazolo[1,5-a]quinazoline 3-carboxylate (4)
˚
of 15 A from the center of interaction site in the a/g interface are
considered to simplify the calculation.
The title compound was obtained either from tosyl derivative
(1a0) following the above mentioned method, yield 90%, or from
5-chloro derivative (3) but in low yield (35%). The followed
The search area of poses, established by autogrid, was
delineated by a cubic grid of 40 points (with a distance of

DOI: 10.3109/14756366.2015.1014475
Pyrazolo[1,5-a]quinazoline scaffold
3
˚
0.375 A) for each coordinate. The genetic algorithm LGA In general, the binding free energies in the condensed phase
(Lamarckian Genetic Algorithm), implemented in AUTODOCK can be calculated according to the following equations.
À
Á
4, was used to search most of the probable ligand poses. The
principal conditions imposed by LGA are the following: 150 was
the number of individuals in population; 250 000 was the
maximum number of energy evaluations; 27 000 was the
maximum number of generations, 100 was the search realized
by LGA, and the 100 were obtained poses which are collected in
clusters with a similarity of 2 rms. From clusters, three poses are
picked up: two poses belonging to the most abundant cluster and
to the cluster with the best docking energy, while the third belongs
to cluster with the second better docking energy. These three
DGbind ¼ Gcomplex ꢀ Gligand þ Gpeptide chains
G ¼ Egas þ Gsolve ꢀ TS
Egas ¼ Ebond þ Eangle þ Etorsion þ EvdW þ Eele
Gsolve ¼ GPB þ GSASA
where Gcomplex, Gligand, and Gpeptide chains are the free energies of
the complex, the molecule, and the CD, respectively. Egas is the
standard force field energy that consists of strain energies (Ebond
Eangle, and Etorsion). The solvation free energy (Gsolve) is further
divided into a polar component (GPB) and a non-polar one
(GSASA). The polar component was calculated by using the
PBSA program in AMBER 9.014, and in this work, the dielectric
constant was set to 1 in solute and 80 in solvent. The non-polar
component was determined by DGnonpol ¼ gSASA + ꢁ, in which
SASA is the solvent-accessible surface area determined with
MOLSURF19. In our calculations, the values for ꢂ and ꢁ were set
,
˚
poses and the selected amino acids (within a range of 15 A)
constitute the starting complex conformation to molecular
dynamic simulation.
Molecular dynamics method
The structures of ligands were built using the Discovery Studio
3.5 Visualizer free program (http://accelrys.com). The ligands’
partial atomic charges were derived by using AM1-BCC12
implemented in the ANTECHAMBER suite13. Energy minimiza-
tions and molecular dynamic simulations (MD) were carried out
by using the SANDER module of AMBER 914 with the GAFF15
force field.
2
˚
to 0.0072 kcal/mol A and 0 kcal/mol, respectively. The contribu-
tion of entropy (TS) to binding free energies via normal mode
analyses is not evaluated as they usually have large error bars and
require long simulation times.
Statistical analysis
For the dynamic simulation in water with periodic boundary
condition, initially the entire system was subjected to energy
minimization in two stages to remove bad contacts between the
complex and the solvent molecules. First, the water molecules
were minimized by holding the peptide chains fixed with a
The histograms were performed with Microsoft Excel (vers.
2013). The PCA was analyzed with the Statgraphics software for
Windows (SAS Inc., Cary, NC).
2
˚
harmonic constraint of strength 2.0 kcal/(mol A ). Second, the Results and discussion
entire system was minimized without restriction. The first stage
was performed using the steepest descent minimization of 500
Chemistry
steps followed by a conjugate gradient minimization of 1000 steps All compounds described here are listed in Table S1 and the
and the second stage was performed by using the steepest descent synthetic strategies are depicted in Chart S1. The synthesis of
minimization of 1000 steps followed by a conjugate gradient the pyrazolo[1,5-a]quinazoline system, in a first attempt and the
minimization of 5000 steps.
To solvate the part of interaction site released by the ligand, aldehyde,
condensation of the 4-substituted-5-aminopyrazole with salicylic
two-component condensation are considered.
a
the system was soaked in a truncated octahedral periodic The easily available starting materials, 4-ethoxycarbonyl- or
box of TIP3P16 water molecules. The distance between the 4-ciano-5-aminopyrazole and salicylic aldehyde, were reacted
edges of the water box and the closest atom of the solutes was in dehydrating conditions using a Dean–Stark apparatus. The
˚
at least 2 A.
intermediate Schiff base, rapidly formed, would have to cyclize
The system was then heated from 0 to 300 K in 5 ps and giving the desired pyrazolo[1,5-a]quinazoline nucleus through a
equilibrated at 300 K for another 5 ps by holding the peptide synthetic strategy already used in our laboratory to synthesize the
chains fixed with a harmonic constraint of a strength of 2.0 kcal/ pyrazolo[5,1-c][1,2,4]benzotriazine derivatives20. In contrast,
2
˚
(mol A ). After the minimization and heating, 100 ps of dynamic using other dehydrating methods (sublimation, high-boiling
simulations by holding the peptide chains weakly fixed with a solvent), the reaction did not give good results and the aldimmine
2
harmonic constraint of a strength of 0.01 kcal/(mol A ) were intermediate (base Schiff) was always recovered. Therefore, the
˚
performed at a constant temperature of 300 K and a constant synthesis of pyrazolo[1,5-a]quinazoline compounds was obtained
starting from the well-known pyrazolo[1,5-a]quinazoline-5(4H)-
pressure of 1 atm.
During the minimization and MD simulations, the particle one derivatives 1a–b and 1d–e (Table S1) and these derivatives
mesh Ewald method17 was employed to treat the long-range were synthesized following reported methods21–24. The new
electrostatic interactions in a periodic boundary condition. The 3-iodiopyrazolo[1,5-a]quinazoline-5(4H)-one, 1c, was obtained
Langevin dynamics with a collision frequency of 1.0 psꢀ1 was by reacting 1b with N-iodiosuccinimide (NIS).
applied to control the temperature of the system. The SHAKE
In an attempt to synthesize the 5-benzyloxy derivatives,
method18 was used to constrain hydrogen atoms. The time step for starting products (1a–c and 1e) were reacted in anhydrous DMF,
all MD simulations was 2 fs, with a direct-space, non-bonded K2CO3, and benzyl chloride following a reported method for
cutoff of 9 A. Initial velocities were assigned from a Maxwellian analogue compounds25. Unexpectedly, the reaction gave always
˚
distribution at the initial temperature.
the 4-N-benzyl derivatives (2a, 2b, and 2e), even using different
In this study, the binding free energies were calculated bases such as NaH and t-BuOK. Only starting compound 1c
by using the MM-PBSA method as implemented in the gave a mixture of the two regio-isomers 4-N-benzyl- (2c) and
AMBER package. 100 snapshots were extracted from the MD 5-O-benzyl derivative (5) in a 1:1 ratio. This fact is probably due
simulation, and the binding free energies were calculated between to a major steric hindrance of iodine atom in the 3-position that
the guest and the CD. The average binding free energies were addressed the alkylation also in position 5 (Scheme 2). On the
calculated for every five snapshot getting 20 values of energy for regioisomer 2a, a crystallographic analysis was performed to
MD trajectory.
confirm the structure (Figure S1).

4
G. Guerrini et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Comp
1a
R
Scheme 2. Reagent and conditions: (i) DMF,
K2CO3, benzyl chloride (for 1c, a mixture of
2c and 5 were obtained).
COOEt
1b
H
I
N
N
1c
R
1e
CONH
NH
2
O
1a, 1b, 1e
i
1c
i
N
N
R
N
CH Ph
2
O
N
N
Comp
2a
R
N
N
I
I
COOEt
H
+
N
N
2b
CH Ph
2
OCH Ph
2
O
2e
CONH
2
5
2c
To obtain the desired benzyloxy derivatives, it was necessary brain membrane and was expressed as Ki value only for those
to insert a suitable leaving group like a chlorine or a tosyl group at compounds which inhibit radioligand binding by more than 80%
C-5 position of the pyrazolo[1,5-a]quinazoline system. The at fixed concentrations of 10 mM. Unexpectedly, the arylalkylation
desired tosyl derivatives (1a0, 1c0, and 1d0) were obtained in at position 4- or 5- gave compounds that were unable to inhibit
good yield and pure enough for the next reaction by the simple radioligand binding, while the only derivative that shows binding
treatment of the lactam (1a, 1c, and 1d) with tosyl chloride in affinity in a nanomolar range was the ethyl pyrazolo[1,5-
DCM, in the presence of NEt3. These intermediates were reacted a]quinazoline 3-carboxylate (15) with Ki ¼ 834.7 nM.
in anhydrous DMF, t-BuOK, and benzyl alcohol to give the
desired 5-benzyloxyderivatives (4–6).
To explain that our compounds were not able to bind with
the receptor protein, a molecular dynamic study and a PCA
When the starting materials 1a, 1c, and 1d were treated were performed using, as ‘‘positive control’’, some literature
with POCl3 to obtain the 5-chloroderivatives, only the ethyl compounds: pyrazolo[5,1-c]quinazolines6, 1,2,4-triazolo[1,5-a]
5-chloropyrazolo[1,5-a]quinazoline-3-carboxylate26 3 was recov- quinoxalines7, triazoloquinazolindiones9, triazolo[1,5-a]quinazo-
ered with good yield. Compound 3 was used to obtain the lines8,
5-aryl(heteroaryl)methylamino derivatives (7–12) by treating phtalazines29.
with an excess amount of suitable amine in DMF, as depicted To achieve our aim, compounds 1a, 1b, 1d, 6, 7, and 13 and
imidazo[2,1-a]phtalazines28,
and
triazolo[3,4-a]
in Scheme 3. To evaluate the relevance of hydrogen bond the positive references (16–21) were docked in a hGABAA
interaction with receptor protein in molecular dynamic study, receptor protein model, obtained by homology-modelling from
compound 7 was hydrolyzed with the corresponding 3-carboxylic acetylcholine-binding protein (AChBP)30. Since the ‘‘benzodi-
acid (13).
azepine binding site’’, or better said GABAA subtype receptor, is
Finally, the 5-unsubstituted 3-ethoxycarbonylpyrazolo[1,5- located between the alpha and the gamma subunits, the amino
a]quinazoline (15) was obtain starting from compound 3 by acids considered in this study belong to these two subunits.
reduction with ammonium formate, 10% Pd/C in ethanol. Compounds were grouped based on good affinity value (16–18,
Unexpectedly, the reduction gave the 4,5-dihydro derivative (14) Ki range 0.62–74 nM), low affinity value (19–21, Ki range 243–
and it was not possible to stop the reaction at the first step of 570 nM), and inactive (1a, 1b, 1d, 6, 7, and 13), Scheme 5.
reduction (5-H derivative) as reported in the literature27. Thus, the
In particular, the occurrence of hydrogen bonds and Van der
4,5-dihydro derivative (14) was dehydrogenated to the desired Walls interactions between the ligand and the amino acids of
compound (15) by an excess amount of 10% Pd/C in toluene at receptor protein was evaluated. The ligand–receptor binding
refluxed temperature (Scheme 4).
energy was calculated by AMBER 914 on three ligand–receptor
complex conformations that were selected by AUTODOCK11. On
the complex conformation that showed the better docking energy,
the occurrence of hydrogen bonds and Van der Waals interactions
Experimental binding and molecular dynamic study
The Bz site/GABAA-R binding affinity of newly synthesized between ligand and receptor protein was evaluated; the results of
compounds was evaluated by their ability to displace these interactions were plotted and the involved amino acids (of
[3H]flumazenil (Ro15-1788) from its specific binding in bovine alpha1 and gamma subunits) stand out.

DOI: 10.3109/14756366.2015.1014475
Pyrazolo[1,5-a]quinazoline scaffold
5
N
Scheme 3. (i) Tosylchloride, NEt3, and DCM.
(ii) DMF, t-BuOK, and benzyl alcohol. (iii)
POCl3, DIPEA. (iv) DMF and excess of
suitable Ar(Het)NH2. (v) Sodium hydroxide
10% solution, then HCl.
N
N
1a
iii
COOEt
N
R
N
NH
3
Cl
O
1a, 1c, 1d
iv
N
Comp
1a
R
N
R
i
COOEt
N
N
1c
I
N
COOEt
O
1d
Ph
N
SO2
NHCH2R
1a’, 1c’, 1d’
Comp.
R
CH3
7
Ph
ii
8
2-OMePh
2-fur
2-Py
3-Py
4-Py
9
v
10
11
12
N
N
R
N
OCH2Ph
N
Comp.
R
N
COOH
4
5
6
COOEt
N
I
NHCH2Ph
Ph
13
N
N
Scheme 4. Reagent and conditions: (i) EtOH,
Pd/C 10%, and HCOONH4; (ii) toluene and
Pd/C 10% reflux.
COOEt
N
3
Cl
i
N
N
N
COOEt
N
COOEt
NH
N
H
ii
H
H
14
15

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G. Guerrini et al.
The data-set
J Enzyme Inhib Med Chem, Early Online: 1–10
N
N
N
N
N
N
N
H C
3
O
N
N
H
O
N
H
O
N
H
O
16 Ki = 59 nM
17 Ki = 74 nM
18 Ki = 0.62 nM
COOEt
N
N
N
N
N
Ph
N
COOEt
N
N
N
N
N
O
CH
3
NH
Ph
Ph
19 Ki = 740 nM
20 Ki = 570 nM
21 Ki = 243 nM
N
N
N
N
N
N
COOEt
H
N
N
H
H
N
H
O
O
O
1a
1b
1d
N
N
N
N
N
N
COOH
COOEt
N
N
N
O
Ph
HN
HN
Ph
Ph
6
7
13
Scheme 5. The data-set.
In Figure 1(a) and (b), the histograms represent the average
The frequencies of interaction with the alpha/gamma subunit
frequency of contacts (expressed in %) measured during the amino acids that show greater changes have been used for PCA for
dynamic simulation of the ligand–receptor complex (hydrogen the overall data set of 12 compounds, Figure 2. The first two
bond, Figure 1a and Van der Waals interactions, Figure 1b). The principal components of the variables (% of frequencies contacts
distribution of average occurrence of interactions is represented average) group together well enough the three types of ligands
by black, grey, and white columns, respectively, for compounds (inactive, black square; low affinity value, grey square; good
with absent (1a, 1b, 1d, 6, 7, and 13), low (19–21), and good affinity value, white square). From the PCA score’s plot, it is
affinity values (16–18). In particular, Figure 1(a) shows ligands observed that the most of the affine compounds (white square,
which have major probability of hydrogen bond interactions in the 16–18, Ki range 0.62–74 nM) engage hydrogen bond interaction
following amino acids: the aY160 (Tyr 160), aS206 (Ser206), and through the NH proton, with an oxygen atom of gT142 carbonyl
gT142 (Thr142). The three ‘‘families’’ of ligands show different amidic group. For example, in Figure 3, the binding pocket and
probabilities of the interaction with amino acids aY160 and the relative orientation of ligand are depicted in compound 17;
gT142 unlike aS206 to be engaged, for which all ligands should hydrogen bond interaction, involving gT142, is represented by
have the same probability of hydrogen bond interaction. For Van blue solid line. For the Van der Waals interaction, aV203 and
der Waals interactions, the greater variation of interactions is aY210 are amino acids involved that engage hydrophobic
evidenced with amino acids aA161 (Ala161), aV203 (Val203), interactions with the phenyl group at position 2 of compounds
aY210 (Tyr210), gM81 (Met81), and gY141 (Tyr141).
(pink dashed line) through the methyl groups and the phenyl ring.

DOI: 10.3109/14756366.2015.1014475
Pyrazolo[1,5-a]quinazoline scaffold
7
Figure 1. (a) Average frequency contacts (expressed in %) of hydrogen bond interaction measured during the dynamic simulation of ligand-receptor
complex. White columns, affine ligands; grey columns, low affine ligands; black columns, inactive ligands. (b) Average frequency contacts (expressed
in %) of Van der Waals interaction measured during the dynamic simulation of ligand–receptor complex. White columns, affine ligands; grey columns,
low affine ligands; black columns, inactive ligands.
The inactive compounds have low probability of interaction
with gT142, aV203, and aY210 amino acids as they lack a
hydrogen bond donor group (like NH) and a lipophilic group (like
phenyl) at a proper distance. In fact, as shown in Figure 4, ligand
1b shows Van der Waals interactions (pink dashed lines) with
different amino acids with respect to the active ligands, in
particular gM81, gY141, and aA161 and it does not engage any
hydrogen bond interaction with gT142.
In low-affinity compounds (19–21, Ki range 243–570 nM), the
only interactions with receptor protein are those of Van der Waals
with aV203 and aY210 and a hydrogen bond interaction with
aY160. The latter is an amino acid for which interactions only for
low affinity and inactive compounds are evidenced. In Figure 5,
binding pocket in compound 21 is depicted; the Van der Waals
interaction (pink dashed line) and the hydrogen bond interaction
(blue solid line) are also evidenced.
Figure 2. Principal components analysis. The ‘‘biplot’’ shows the value
of the first two components of the original variables. The original
variables considered are the average frequency (expressed in %) of
hydrogen bonds (hb) and van der Waals (vdw) interactions between the
The aim of this work was to synthesize new derivatives with
ligand and the amino acids gT142, aY160, aY210, aV203, gM81, gY141,
and aA161. The squares represent the ligands (white, affine; grey, low
pyrazolo[5,1-c][1,2,4]benzotriazine, to evaluate their biological
affine; black, inactive), the lines represent the component weights of the
original variables.
Conclusion
pyrazolo[1,5-a]quinazoline scaffold, as 5-deaza analogue of
activity on GABAA receptor subtype. With disappointing, the 4-
or 5-substitutions on this new system gave compounds lacking
recognition of receptor protein and, then to rationalize these

8
G. Guerrini et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Figure 3. Interaction between affine compound 17 and receptor protein. Hydrogen bond interaction: blue solid line; Van der Waals interaction: pink
dashed line.
Figure 4. Interaction between affine compound 1b and receptor protein. In particular, only Van der Waals interactions are evidenced, pink dashed line
and they lack the hydrogen bond interactions.
results, a small library of the new and known compounds was necessary modification for binding affinity: in fact, compound 15
properly chosen for a MDS. This study shows the importance of shows receptor recognition in nanomolar range. Based on these
hydrogen bond interaction of ligand with gT142 of receptor findings, further studies are in progress to investigate the
protein, to enhance the binding affinity.
pyrazolo[1,5-a]quinazoline system with suitable substituents in
On the contrary, the unsaturation between 4 and 5 positions position 3 and/or 8, positions in the 5-azaanalogue system
with achievement of the aromatization of the system was a (pyrazolobenzotriazine) are proved to be compelling.

DOI: 10.3109/14756366.2015.1014475
Pyrazolo[1,5-a]quinazoline scaffold
9
Figure 5. Interaction between affine compound 21 and receptor protein. In particular, Van der Waals interactions, pink dashed line, and the hydrogen
bond interaction with aY160 are evidenced.
¨
9. Nilsson J, Gidlof R, Nielsen EØ, et al. Triazoloquinazolinediones as
Declaration of interest
novel high affinity ligands for the benzodiazepine site of GABA(A)
receptors. Bioorg Med Chem 2011;19:111–21.
10. Costanzo A, Guerrini G, Ciciani G, et al. Benzodiazepine receptor
ligands. 4: synthesis and pharmacological evaluation of 3-hetero-
aryl-8-chloropyrazolo[5,1-c][1,2,4]benzotriazine 5-oxides. J Med
Chem 1999;42:2218–26.
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
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AutoDockTools4: automated docking with selective receptor flexi-
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Supplementary material available online
Supplementary Table S1 and Figure S1.