Synthesis, structure-affinity relationships, and molecular modeling studies of novel pyrazolo[3,4-c]quinoline derivatives as adenosine receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2011.05.001

This paper reports the study of new 2-phenyl- and 2-methylpyrazolo[3,4-c] quinolin-4-ones (series A) and 4-amines (series B), designed as adenosine receptor (AR) antagonists. The synthesized compounds bear at the 6-position various groups, with different

Full text of DOI:10.1016/j.bmc.2011.05.001

Bioorganic & Medicinal Chemistry 19 (2011) 3757–3768
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, structure–affinity relationships, and molecular modeling studies
of novel pyrazolo[3,4-c]quinoline derivatives as adenosine receptor antagonists
Ombretta Lenzi a, Vittoria Colotta a, , Daniela Catarzi a, Flavia Varano a, Lucia Squarcialupi a,
⇑
Guido Filacchioni a, Katia Varani b, Fabrizio Vincenzi b, Pier Andrea Borea b, Diego Dal Ben c,
Catia Lambertucci c, Gloria Cristalli c
a Dipartimento di Scienze Farmaceutiche, Laboratorio di Progettazione, Sintesi e Studio di Eterocicli Biologicamente Attivi, Universita’ di Firenze, Polo Scientifico, Via Ugo Schiff 6,
50019 Sesto Fiorentino, FI, Italy
b Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Università di Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy
c School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Via S. Agostino 1, 62032 Camerino (MC), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper reports the study of new 2-phenyl- and 2-methylpyrazolo[3,4-c]quinolin-4-ones (series A)
and 4-amines (series B), designed as adenosine receptor (AR) antagonists. The synthesized compounds
bear at the 6-position various groups, with different lipophilicity and steric hindrance, that were thought
to increase human A1 and A2A AR affinities and selectivities, with respect to those of the parent 6-unsub-
stituted compounds. In series A, this modification was not tolerated since it reduced AR affinity, while in
series B it shifted the binding towards the hA1 subtype. To rationalize the observed structure–affinity
relationships, molecular docking studies at A2AAR-based homology models of the A1 and A3 ARs and at
the A2AAR crystal structure were carried out.
Received 21 February 2011
Revised 28 April 2011
Accepted 1 May 2011
Available online 6 May 2011
Keywords:
G protein-coupled receptors
Adenosine receptor antagonists
Pyrazoloquinolines
Ó 2011 Elsevier Ltd. All rights reserved.
Tricyclic heteroaromatic systems
Ligand-receptor modeling studies
1. Introduction
ARs are abundant within the central nervous system, with high
levels being expressed in many regions of the brain, such as
Adenosine is an ubiquitous nucleoside which affects a wide
range of physiopathological processes by activation of G protein-
coupled receptors, currently subdivided into four subtypes: A1,
cerebral and cerebellar cortex, hippocampus, thalamus, and limbic
region. A1 ARs are localized at post-synaptic level and also on pre-
synaptic terminals; activation of these latter A1 ARs inhibits gluta-
matergic and cholinergic transmission. Thus, A1 AR blockade can
increase the release of acetylcholine and glutamate in brain areas
which are important for cognitive and emotional functions. Coher-
ently, A1 AR antagonists ameliorate cognitive deficits due to cholin-
ergic hypofunction and have potential for the treatment of
neurological disorders, such as dementia or anxiety.6 Also A2A
ARs are expressed through the brain, showing the highest levels
in the striatum, nucleus accumbens, and olfactory tubercle, regions
which are rich in dopamine. A2AAR inactivation in the brain expli-
cates a neuroprotective effect, attributed to the release inhibition
of glutamate and proinflammatory cytokines from microglia. A2A
receptor antagonists improve dopamine transmission, thus being
effective in the treatment of Parkinson’s disease (PD).7 Recently,
also the usefulness of dual A2A/A1 receptor antagonists in the treat-
ment of PD has been highlighted because these compounds not
only improve motor disabilities (A2AAR) but also reduce the cogni-
tive impairment (A1AR) associated with the disease.8,9
A
2A, A2B, and A3. Each receptor subtype mediates distinct pharma-
cological actions through coupling to various secondary effectors.
Adenylate cyclase can be considered the principal effector system,
common to all ARs. It can be either inhibited (A1 and A3) or stimu-
lated (A2A and A2B) thus decreasing or increasing, respectively,
cAMP production.1,2 Other second messenger signaling pathways
have also been described.1–3 The A1 receptor activates phospholi-
pase C, leading to increased production of inositol 1,4,5-triphos-
phate and Ca2+ mobilization; the A1 subtype is also known to
modulate several types of K+ and Ca2+ channels that are, respec-
tively, activated or inactivated. The A2AAR is recognized to activate
protein kinase C, KATP and Ca2+ channels. Both A1 and A2A ARs reg-
ulate the extracellular signal-regulated kinases (ERK) 1/2, mem-
bers of the mitogen-activated protein kinase (MAPK) family that
play an important role in cell differentiation, survival, proliferation
and death.1,4,5 ARs exhibit different tissue/cell distribution.1,3 A1
⇑
On the basis of the interesting potential therapeutic
applications, numerous classes of AR antagonists, with different
Corresponding author. Tel.: +39 055 4573731; fax: +39 055 4573780.
E-mail address: vittoria.colotta@unifi.it (V. Colotta).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.001

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O. Lenzi et al. / Bioorg. Med. Chem. 19 (2011) 3757–3768
heterocyclic structures, have been developed and intensive struc-
ture–affinity relationship studies have been performed.10–15
Also in our laboratory much research has been devoted to the
study of AR antagonists, belonging to various classes of heteroaro-
matic tricyclic or bicyclic derivatives.16–25 One of the tricyclic
series recently investigated is represented by the 2-arylpyrazol-
o[3,4-c]quinolines, both 4-oxo and 4-amino substituted (series A
and B, respectively, Fig. 1).17,21,23
chain in a suitable position shifted the affinity towards A1 and/or
A2A ARs.26–28 The choice of synthesizing analogues of 1A and 1B,
lacking X substituent on the 2-phenyl moiety, was made because
the presence of X groups might have been disadvantageous for the
binding at the A2AAR, as well as for A1 or A2A AR selectivity. Finally,
the effect of replacing the 2-phenyl moiety with the smaller and less
lipophilic 2-methyl group was investigated in both series A and B
(compounds 12–15, Fig. 2).
The 2-phenyl derivatives 1A and 1B21 can be considered the par-
ent compounds of the two series: the 4-oxo derivative 1A shows
nanomolar affinities for human (h) A3 (Ki = 30.8 nM) and A1
(Ki = 203 nM) ARs and a scarce binding activity at the hA2A AR
2. Chemistry
The target pyrazolo[3,4-c]quinoline derivatives 1–15 were
synthesized as depicted in Schemes 1–3. Scheme 1 shows the
pathways followed to obtain the 2-phenylpyrazoloquinolin-4-
one derivatives 1–6. A mixture of the commercially available
7-benzyloxyindole 16 or the suitably synthesized 7-R indoles
17–19,29–31 ethoxalyl chloride and anhydrous pyridine was
refluxed for two days to give the corresponding 7-R-3-ethoxaly-
lindoles 20–2132,33 and 22–23. Reaction of 20–21 with phen-
ylhydrazine hydrochloride in refluxing absolute ethanol and a
few drops of glacial acetic acid afforded the desired 6-substi-
tuted pyrazoloquinolin-4-one derivatives 1–2. Instead, the 7-ben-
(I% = 45, at 10 lM); instead, the 4-amino derivative 1B binds the
A2AAR with high affinity (Ki = 91 nM) and is about five- to six-fold
less active at A1AR (Ki = 659 nM) and A3AR (Ki = 551 nM) receptors
(Table 1). Introduction of a small substituent on the meta- or para-
position of the 2-phenyl ring of 1A (X = Me, OMe) gave non-selective
A1/A3 antagonists since it ameliorated both A1AR (Ki = 12–176 nM)
and A3AR affinity (Ki = 3–30 nM); instead, the A2AAR binding remain
unsatisfactory (I% = 25–50 at 10 l
M).21 Also in series B, the X
substituents increased both A1 AR (Ki = 21–45 nM) and A3 AR
(Ki = 90–228 nM) affinity of the parent compound 1B, thus leading
to non-selective antagonists. On the contrary, the A2A AR affinity is
decreased by the presence of X, being the 2-aryl derivatives B from
three- to ten-fold less active at the A2AAR than the parent compound
1B.21 Based on these results, we planned the synthesis of new pyraz-
olo[3,4-c]quinolines with the aim of obtaining A1AR selective antag-
onists or dual A1/A2A antagonists, due to their interesting
therapeutic applications in the neurological disorders cited above.
Thus, we decided to introduce at the 6-position (R) of both com-
pounds 1A and 1B various groups with different steric hindrance
and lipophilicity (compounds 1–11, Fig. 2). In particular, the pres-
ence of 6-R bulky and lipophilic groups, such as bromine or arylalkyl
moieties, was thought to enhance the A1AR and A2AAR affinities
since literature data on several tricyclic AR antagonists of similar
size and shape to our 4-amino series B, indicate that an arylalkyl
zhydryloxyindole 22, under the same conditions, yielded
a
mixture (about 1:1) of the corresponding 6-benzhydryloxy-pyr-
azoloquinoline 3 and of the 6-hydroxy derivative 5, which was
also prepared by debenzylation of compound 1. Catalytic reduc-
tion of compounds 4 gave the 6-amino derivative 6.
The 2-phenylpyrazoloquinolin-4-amino derivatives 7–11 were
prepared as outlined in Scheme 2. The 4-oxo derivatives 1–2 and
4 were refluxed in a mixture of phosphorus pentachloride/phos-
phorus oxychloride to give the corresponding 4-chloro derivative
24–26. Compounds 24–25 were transformed into the 4-amino
substituted compounds 7–8 with ammonia. Catalytic hydrogena-
tion of derivative 7 yielded the corresponding 6-hydroxy substi-
tuted compound 9 that was reacted with phenethyl bromide to
obtain the 6-phenethyloxy-substituted derivative 10. When the
4-cloro derivative 26 was heated with ammonia, both the 4-amino
derivative 27 and the 4,6-diamino-2-phenylpyrazoloquinoline 28
O
NH2
N
X
N
X
HN
N
N
N
O
O
OEt
O
N
HN
N
Ph
Ph
a
b
R
Series A
Series B
X= H, 3-Me, 4-Me
3-OMe, 4-OMe
N
H
N
H
1A X= H
1B X= H
R
R
1-4
c
16-19
20-23
Figure 1. Previously reported 2-arylpyrazolo[3,4-c]quinolin-4-ones (series A) and
4-amines (series B).
d
O
O
N
N
HN
HN
N
N
Ph
HO
O
H2N
NH2
N
N
HN
N
N R2
N R2
R
R
5
6
R
Series A
Series B
1, 16, 20
2, 17, 21
3, 18, 22
4, 19, 23
OCH2Ph
Br
OCH(Ph)2
R= Br, O(CH2)1-2Ph, OCH(Ph)2, NHCOOCH2Ph,
NHCH2Ph, OH, NH2
1-6
R2= Ph
7-11 R2= Ph
NHCOOCH2Ph
14-15 R2= CH3
12-13 R2= CH3
Scheme 1. Reagents and conditions. (a) EtOCO–COCl, anhydrous Et2O, reflux; (b)
PhNHNH2ÁHCl, glacial AcOH, absolute EtOH, reflux; (c) 48% HBr, AcOH, reflux; (d)
H2, Pd/C, DMF, 45 psi.
Figure 2. Herein reported 6-substituted 2-phenylpyrazolo[3,4-c]quinoline deriva-
tives 1–11 and 2-methylpyrazolo[3,4-c]quinolines 12–15.

O. Lenzi et al. / Bioorg. Med. Chem. 19 (2011) 3757–3768
3759
NH2
Cl
O
N
N
N
N
N
HN
N Ph
N Ph
N Ph
R
b
a
R
R
7 R= OCH2Ph
8 R= Br
9 R= OH
24-26
c
1-2, 4
b
d
NH2
NH2
NH2
N
N
N
N
N
N
e
N Ph
N Ph
N Ph
R
O
Ph
N
Ph
10
27 R= NHCOOCH2Ph
28 R= NH2
29
f
NH2
N
N
H
N
N Ph
Ph
R
1, 24
2, 25
4, 26
OCH2Ph
Br
NHCOOCH2Ph
11
Scheme 2. Reagents and conditions. (a) PCl5/POCl3, reflux; (b) NH3(g), absolute EtOH, T = 120 °C, sealed tube; (c) H2, 10% Pd/C, 35 psi; (d) phenethyl bromide, K2CO3, 2-
butanone, reflux; (e) PhCHO, anhydrous ZnCl2, anhydrous THF, reflux; (f) NaBH4, anhydrous MeOH, reflux.
3. Pharmacology
O
O
OEt
O
N
HN
The synthesized compounds 1–15 were tested for their ability
to displace specific [3H]DPCPX, [3H]ZM241385 and [125I]AB-MECA
binding from cloned hA1, hA2A and hA3 receptors, respectively,
stably expressed in CHO cells. Compounds 1–15 were also tested
at the hA2B subtype by measuring their inhibitory effects on
NECA-stimulated cAMP levels in CHO cells stably transfected with
the hA2B receptor. The results of binding experiments and cAMP as-
says are reported in Table 1, where hA1, hA2A and hA3 affinities of
the parent compounds 1A and 1B are also included as reference
data.
N CH3
a
R
N
H
R
20 R= OCH2Ph
30 R= H
12 R= OCH2Ph
13 R= H
b
NH2
Cl
N
N
N
N
N CH3
N CH3
R
c
R
4. Results and discussion
14 R= OCH2Ph
15 R= H
31 R= OCH2Ph
32 R= H
4.1. Structure–affinity relationships
Scheme 3. Reagents and conditions. (a) CH3NHNH2ÁHCl, glacial AcOH, absolute
EtOH, reflux; (b) PCl5/POCl3, reflux; (c) NH3(g), absolute EtOH, T = 110 °C, sealed
tube.
The binding results indicate that the aim of the paper has been
partially satisfied. In fact, most of the newly synthesized 6-substi-
tuted 2-phenylpyrazolo[3,4-c]quinolin-4-amines (series B) showed
A1 receptor affinity and selectivity (derivative 7–9, and 11).
However, only one compound, the 2-methyl-6-benzyloxy derivative
14, possesses affinity towards both A1 and A2A receptors. Moreover,
it binds at the A3 receptor subtype with comparable affinity. Quite
unexpectedly, none of the 6-substituted 2-phenylpyrazolo
[3,4-c]quinolin-4-one derivatives 1–6 (series A) possesses affinities
towards ARs, whatever the nature of the 6 substituent. In fact, either
lipophilic or hindering groups such as the benzyloxy, benzhydryl-
oxy, benzyloxycarbamoyl or bromine (compounds 1–4) or small
hydrophilic substituents, such as hydroxyl or amino (compounds 5
and 6), exerted a deleterious effect since they reduced the A1 and
A3 receptor affinities of the parent compound 1A, as well as the
A2A receptor binding. As stated above, the 6-substituted 2-phen-
ylpyrazolo[3,4-c]quinolin-4-amino derivatives 7–11 turned out
were obtained. Reaction of this latter with benzaldehyde in reflux-
ing anhydrous tetrahydrofuran, and in the presence of anhydrous
zinc chloride, afforded the Schiff’s base 29 which was reduced with
sodium borohydride to give the 6-benzylamino derivative 11. The
2-methylpyrazolo[3,4-c]quinolin-4-ones 1234 and 13 (Scheme 3)
were synthesized starting from the suitable 3-ethoxalylindoles
2032 or 3035 which were reacted with methylhydrazine hydrochlo-
ride in boiling absolute ethanol and a few drops of glacial acetic
acid to give the desired 4-oxo derivatives 12–13. These compounds
were reacted with boiling phosphorus pentachloride/phosphorus
oxychloride to give the corresponding 4-chloro derivatives 31–32
which were transformed into the target 2-methylpyrazolo[3,4-
c]quinolin-4-amines 14 and 1536,37 with ammonia.
selective A1 AR antagonists. Indeed, they are inactive at the A2A
,

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O. Lenzi et al. / Bioorg. Med. Chem. 19 (2011) 3757–3768
Table 1
Binding affinity (Ki) at hA1, hA2A and hA3 ARs and potencies (IC50) at hA2B
.
O
NH2
O
NH2
N
N
N
N
HN
N
HN
N
N
Ph
N
Ph
N
CH3
N CH3
R
R
R
R
1A, 1-6
1B, 7-11
12-13
14-15
R
Binding experiments Ki (nM) or I%
cAMP assays I%
b
c
d
e
hA1
hA2A
hA3
hA2B
1Af
1
2
3
4
H
203 12
20%
8%
6%
3%
43%
1%
17%
1%
1%
5%
30.8 2.6
33%
16%
6%
14%
16%
18%
551 34
20%
29%
20%
28%
1%
600 58
12%
Ntg
2%
5%
7%
5%
4%
5%
Ntg
5%
2%
4%
5%
3%
3%
2%
4%
5%
OCH2Ph
Br
OCHPh2
NHCOOCH2Ph
OH
NH2
H
OCH2Ph
Br
OH
O(CH2)2Ph
NHCH2Ph
OCH2Ph
H
OCH2Ph
H
5
6
1%
8%
1%
1Bf
7
659 43
140 12
300 28
100 12
40%
455 47
2%
2%
91 7.3
16%
35%
19%
11%
1%
1%
1%
750 70
26%
8
9
10
11
12
13
14
15
970 80
20%
500 47
18%
a
Ki values are means SEM of four separate assays each performed in duplicate. Percentage of inhibition (I%) are determined at 1
compounds.
l
M concentration of the tested
b
Displacement of specific [3H]DPCPX competition binding to hA1CHO cells.
c
d
e
f
Displacement of specific [3H]ZM241385 competition binding to hA2ACHO cells.
Displacement of specific [125I]AB-MECA competition binding to hA3CHO cells.
Percentage of inhibition on cAMP experiments in hA2BCHO cells, stimulated by 200 nM NECA, at 1
l
M concentration of the examined compounds.
Ref. 17: I% at the A2A receptor was determined at 10
Nt: Not tested.
lM concentration of compound 1A.
g
A2B and A3 receptors while they showed hA1 receptor affinities in the
nanomolar range (Ki = 100–455 nM), with the only exception being
the 6-phenethyloxy-substituted derivative 10 that, at 1 lM concen-
tration, inhibited the radioligand binding by only 40%.
duced complementarity with the receptor recognition sites. Simi-
larly, the A3 affinity of the 6-benzyloxy-4-oxo derivative 12 was
annulled by removal of the 6-substituent (compound 13). The roles
of the 2- and 6-substituents and of the 4-oxo or amino function
were investigated with molecular modeling studies.
In general, the substituents introduced at the 6-position of the
parent compound 1B enhanced the hA1 affinity, the best being
the 6-hydroxy group (compound 9); unexpectedly, the presence
of a 6-substituent, either lipophilic or hydrophilic, dramatically de-
creased the A2A receptor binding of the parent derivative 1B. These
findings led us to presume that the lack of A2A affinity of the new
2-phenyl derivatives, and in particular of the 4-amino substituted
one, could be due to the great volume of the molecules which
might hinder their accommodation in the receptor binding site.
Thus, to reduce the steric hindrance, the 2-phenyl ring of the 6-
benzyloxy derivatives 1 and 7 was replaced with a methyl group
to afford derivatives 12 and 14, respectively. The binding data of
2-methyl-4-oxo derivative 12 resembled those of the correspond-
ing 2-phenyl-4-oxo substituted compounds 1, with the exception
of the A3 affinity that was significantly higher (Ki = 600 nM).
Even more interesting results were obtained for the 2-methyl-
4-amino derivative 14 that turned out to be a non selective AR
antagonist. In fact it displayed comparable affinity for the A1, A2A
and A3 ARs, while it was inactive at the A2B subtype. Therefore,
replacement of the phenyl ring of compound 7 with the smaller
methyl group (compound 14) made the hA2A affinity appear, as
well as the A3 one, while it reduced the binding at the A1 receptor.
Removal of the 6-benzyloxy substituent from the 2-methyl-4-ami-
no derivative 14, to give compound 15, caused a drastic drop of A1,
4.2. Molecular modeling studies
To define the structural features at the base of the different
binding affinities of the new derivatives, a molecular docking anal-
ysis was performed on homology models of hA1 and hA3 ARs,
developed by using the recently published crystal structure of
the hA2A AR in complex with the ZM241385 antagonist (Fig. 3) as
template,38 and on the hA2AAR crystal structure itself. In particular,
docking analysis at the A1 AR was aimed at rationalizing the effect
of the different 2- and 6-substituents on the affinity for this AR
subtype, while comparative docking analysis at the three AR
subtypes was performed for compounds 1A and 1B to evaluate
the impact of the different chemical functions in the 4-position
on the AR binding. The hA2A AR crystal structure allows improve-
ment of the accuracy of AR homology models, due to the high res-
NH2
HO
N
N
O
N
N
N
H
N
ZM241385
A2A and A3 affinity that can be ascribed to the loss of interaction of
the benzyl moiety with the receptor pocket or, in general, to a re-
Figure 3. High affinity hA2A AR antagonist ZM241385.

O. Lenzi et al. / Bioorg. Med. Chem. 19 (2011) 3757–3768
3761
idue conservation in the primary sequences of the AR subtypes,
which share a sequence identity of ꢀ57% within the transmem-
brane (TM) domains.39 The residues located within the seven TM
domains in the upper part of ARs, corresponding to the ligand
binding site, are conserved with an average identity of 71%.40 Fur-
thermore, the hA2A AR crystal structure is solved in complex with
the high affinity antagonist ZM241385, hence presenting a cavity
suitable as binding site for docking analysis. The obtained A1 and
A3 AR homology models were checked by using the Protein Geom-
etry Monitor application within MOE, which provides a variety of
stereochemical measurements for inspection of the structural
quality in a given protein, such as backbone bond lengths, angles
with respect to the X-ray data. As displayed in Figure 4, the docking
method showed good ability in reproducing the ZM241385 binding
mode observed in the experimental data. In particular, the RMSD of
the two conformations was calculated as 1.4547 Å, with the best
matching being given by the 2-(furan-2-yl)-[1,2,4]triazolo[1,5-
a][1,3,5]triazin-7-amine moiety (0.8551 Å) and the highest devia-
tion being given by a slightly different orientation of the 4-(2-ami-
noethyl)phenol group (2.1143 Å).
Considering the general binding mode of both 4-oxo and 4-amino
substituted pyrazoloquinoline derivatives in the A2AAR, the pyrazolo
[3,4-c]pyridine moiety is located approximately in correspondence
with the [1,2,4]triazolo[1,5-a][1,3,5]triazine scaffold of the co-crys-
tallized ZM241385 antagonist. The docking conformations present
the 2-substituent pointed towards the central transmembrane core
and located in a mainly hydrophobic subpocket in proximity of
Val84, Leu85, Met177, Trp246, and Leu249. Analogue binding motif
of the pyrazoloquinoline derivatives was observed in A1 (Fig. 5) and
A3 AR subtypes in which the hydrophobic subpockets that accommo-
date the 2-substituent are surrounded by Val87, Leu88, Met180,
Trp247, Leu250 (A1 AR), and Leu90 Leu91, Met177, Trp243 and
Leu246 (A3AR). In all three ARs, the 6-substituent of the new com-
pounds is located at the entrance of the binding site. Some of the
ZM241385-A2AAR interactions are reproduced by the synthesised
and dihedrals, Ramachandran
u–w dihedral plots, and side chain
rotamer and nonbonded contact quality.
A1, A2A, and A3 AR structures were then used as target for the
docking analysis of synthesised derivatives. Due to the presence
in the template crystal structure of some water molecules playing
a relevant role in hA2AAR-ZM241385 interaction, it was decided to
reintroduce these water molecules in the hAR binding sites for the
post-docking energy minimization stage, to verify possible roles of
solvent molecules in stabilizing compound binding and to compare
these roles to the one played for hA2AR-ZM241385 interaction. All
ligand structures were optimized using RHF/AM1 semi-empirical
calculations and the software package MOPAC41 implemented in
MOE was utilized for these calculations. The compounds were then
docked into the binding site of the ARs by using the MOE Dock tool.
Top-score docking poses of each compound were subjected to en-
ergy minimization and then rescored using two available methods
implemented in MOE: the London dG scoring function that esti-
mates the free energy of binding of the ligand from a given pose;
and Affinity dG Scoring that estimates the enthalpic contribution
to the free energy of binding. Additional scoring was performed
using the dock-pKi predictor that uses the MOE scoring.svl script
to estimate for each ligand a pKi value, which is described by the
H-bonds, transition metal interactions, and hydrophobic interac-
tions energy. For each compound, the top-score docking pose,
according to at least two out of three scoring functions, was se-
lected for final ligand-target interaction analysis.
derivatives, such as the
p-stacking interaction between the
compound scaffold and the Phe168 residue within EL2 segment. An
analogous interaction was engaged within the A1 (Phe171) and A3
(Phe168) ARs.
On the contrary, the double polar interaction of Asparagine 6.55
amide group (Asn253) with both the NH2 group and the N1 atom of
ZM241385 was not observed in the pyrazoloquinoline derivatives.
In this series, the possible interaction of Asn 250 with both the
4-substituent and N3 atom is actually reduced to the unique inter-
action with the 4-group, due to the steric hindrance of 2-phenyl
The docking methodology was firstly tested by comparing the
predicted binding mode of ZM241385 within the A2AAR pocket
Figure 4. Validation of docking methodology by using the crystal structure of
A
2AAR-ZM241385 complex. The X-ray binding mode of ZM241385 is taken as
reference and displayed with ‘stick style’ in dark color. The predicted conformation
of the same compound (after docking-minimization-rescoring processes) is shown
with ‘ball & stick style’ in light color.
Figure 5. Homology model of A1AR and binding mode of compound 9.

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O. Lenzi et al. / Bioorg. Med. Chem. 19 (2011) 3757–3768
ring that obstructs the second interaction. Similarly, in the binding
mode of the pirazoloquinolines at A1 and A3 receptors only the 4-
substituent seems to interact with the asparagine amide group
(Asn254 and Asn250, respectively). To reduce the steric hindrance
of the 2-moiety, the 2-phenyl ring (compounds 1A, 1B, 1 and 7)
was replaced with the smaller 2-methyl group (compounds
12–15). This modification did not ameliorated the A2A binding,
the only exception being the 6-benzyloxy derivative 14 which
showed an increased A2A AR affinity (Ki = 750 nM), with respect
to that corresponding 2-phenyl substituted derivative 7 (I% = 16).
Replacement of the 2-phenyl moiety with the 2-methyl group
was also detrimental for A1 affinity. An analogous effect was al-
ready observed for 8-substituted adenine derivatives when tested
at both human and rat A1AR.42 Given the high level of conservation
of the above indicated hydrophobic subpocket among ARs, the
opposite effect of 2-phenyl and 2-methyl substituents for AR affin-
ity could be explained by hypothesizing slightly different volumes
of the subpocket itself in the AR subtypes. As a consequence, the
different effect of the 2-substituents on AR affinity could be related
to their steric impact and to the different occupancy of the sub-
pocket itself and not to specific ligand-receptor interactions.
Still considering the 4-group of the pyrazoloquinoline scaffold,
docking conformations of these derivatives at A1 and A2A ARs pres-
ent this group inserted between the above cited Asparagine 6.55
and a Glutamate residue conserved in both subtypes (Glu172 and
Glu169, respectively, in A1 and A2A ARs). In the A2AAR crystal struc-
ture, the carbonyl group of Asparagine amide and the carboxy
function of Glutamate seem to both play a role as H-bond acceptor
for the free amino group of ZM241385. This role is played also in
the case of the 4-amino substituted pyrazoloquinolines and the
importance of this interaction is particularly evident for binding
to the A2A AR. In fact, the only two compounds (1B and 14) present-
ing nanomolar affinity, for this AR subtype, are substituted in the
4-position with a free amino group working as double H-bond do-
nor. On the contrary, the presence of this pharmacophoric feature
is not critical for the binding to the A1 AR, as the pyrazoloquinolin-
4-one derivative 1A is endowed with higher affinity at this sub-
type, with respect to the 4-amino substituted analogue 1B. The dif-
ference between the affinities of 1A and 1B is even more
pronounced at the A3 AR in which the Glutamate residue in EL2,
conserved in A1 (Glu172) and A2A (Glu169), is replaced by a Valine
(Val169). Hence the presence of a double H-bond donor group in
the compound structure is not required (compare A3AR affinity
of compound 1A to that of 1B, Table 1) if not detrimental.43 To
interpret how the 4-oxo group of derivative 1A can interact better
than the amino group of 1B with the Asparagine residue 6.55 of A1
and A3 ARs, the possible existence of the 4-oxo compound 1A in the
4-enol form was evaluated. This hypothesis ensued from
previously reported study on A3AR antagonists belonging to the
4-oxo-substituted 1,2,4-triazolo[1,5-a]quinoxaline series.19 This
study suggested a possible ‘tautomeric’ selection made by the
receptor binding site, with the keto form predicted as energetically
more stable in solution and the enol form as fitting the binding site
even better than the keto one.19
Figure 6. Binding modes of compound 1A within A3AR. EL2 is partially hidden for
clarity. Receptor residues in ligand proximity are displayed.
binding modes of the enol form at the A3 AR, suggesting that both
ways of binding for this tautomer can occur at the A3 AR. This data
could explain the good A3 AR affinity of compound 1A.
Concerning the 6-substituent, it is positioned in proximity of
A1AR residues presenting a mainly polar/hydrophilic profile, such
as Glu170 and Glu172 in EL2 segment and Thr270 and Tyr271 in
TM7 domain. The corresponding residues in the
A2AAR are
Leu167 and Glu169 in EL2 segment and Met270 and Tyr271 in
TM7 domain, while in the A3AR this subsection is defined by
Gln170 and Val169 in EL2 segment and Leu264 and Tyr265 in
TM7 domain. As a consequence, the chemical-physical profile of
this region in A2A and A3 ARs is markedly less hydrophilic than that
of the corresponding A1 AR area. Thus, the presence of 6-substitu-
ents gives an increasing or decreasing contribution to AR affinity
related to their chemical profile. In this sense, while 6-unsubstitut-
ed compound 1B is endowed with higher affinity for the A2AAR,
with respect to the A1 and A3 ARs, its 6-hydroxy substituted
analogue 9 presents an increased affinity for A1 AR and a significant
loss of binding to the other AR subtypes (Fig. 7).
Enthalpies of formation of compound 1A in keto and enol forms
confirm the higher (ꢀ20 kcal/mol) stability of the first tautomer.
Docking analysis of both forms at the three AR models shows that
their binding modes are comparable to the one of 4-amino substi-
tuted compounds. Figure 6A shows the docking conformation of
the enol tautomer of 1A in the A3 AR. Furthermore, the enol tauto-
mer is observed to bind the three ARs also with a mirror orienta-
tion (Fig. 6B), that is with the 2-substituent located at the
entrance of the binding site and the 6-position pointing towards
the TM core region. In both cases, the enol polar hydrogen points
towards the carbonyl group of Asparagine 6.55. Each scoring func-
tion used in our docking study presents comparable scores for both
Additional considerations can be made regarding the length and
the nature of the 6-substituent. In particular, the nanomolar A1 AR
affinity of compound 7 (R6 = OCH2Ph) and the strong decrease of
binding of its higher homolog 10 are explained by comparison of

O. Lenzi et al. / Bioorg. Med. Chem. 19 (2011) 3757–3768
3763
compared to those of the parent compound 1B. Concerning the A2A
receptor, only the 2-methylpyrazoloquinolin-4-amine 14, bearing
the 6-benzyloxy moiety, showed some A2A binding affinity. Docking
results at A1, A2A, and A3 AR binding sites highlighted the role of
non-conserved receptor residues in the different interactions with
the compound substituents whose effects on the AR affinity have
been thus clarified. In conclusion, this study has provided new
insights about the structural requirements of the AR antagonists
belonging to the pyrazolo[3,4-c]quinoline series.
6. Experimental section
6.1. Chemistry
Silica gel plates (Merck F254) and silica gel 60 (Merck, 70–230
mesh) were used for analytical and column chromatography,
respectively. All melting points were determined on a Gallenkamp
melting point apparatus. Microanalyses were performed with a
Perkin-Elmer 260 elemental analyzer for C, H, N, and the results
were within 0.4% of the theoretic values, unless otherwise stated.
The IR spectra were recorded with a Perkin-Elmer Spectrum RX I
spectrometer in Nujol mulls and are expressed in cmÀ1. The 1H
NMR spectra were obtained with a Brucker Avance 400 MHz
instrument. The chemical shifts are reported in d (ppm) and are rel-
ative to the central peak of the solvent which was DMSO-d6 or
CDCl3. The following abbreviations are used: s = singlet, d = dou-
blet, t = triplet, m = multiplet, br = broad, and ar = aromatic pro-
tons. The mass spectra (MS) were acquired in positive mode over
100:600 m/z range using a Varian 1200L triple quadrupole mass
spectrometer equipped with electrospray ionization (ESI) source.
6.1.1. General procedure for the synthesis of ethyl 7-
substituted-3-indolylglyoxylates 20–23
Ethyl oxalyl chloride (2.24 mmol) was added to a solution of the
commercially available 7-benzyloxyindole 16 and the suitably syn-
thesized 7-R-indole derivatives 17–1929–31 (2.24 mmol) in anhy-
drous tetrahydrofuran (15 mL) and pyridine (4.48 mmol). The
mixture was refluxed for 48 h, then the solid filtered off. The
mother liquor was diluted with diethyl ether (about 20 mL) and
the organic phase was washed twice with water (30 mL), anhydri-
fied over Na2SO4 and evaporated to dryness at reduce pressure. The
oily residue was taken up with cyclohexane (about 5 mL) to give a
solid which was collected by filtration and recrystallized. Upon this
treatment the 7-benzhydryloxy derivative 22 did not solidify, nev-
ertheless the crude oil was pure enough to be used for the next
step.
Figure 7. A and B: detailed view of binding mode of compound 9 within A1AR and
compound 1B within 2AAR, respectively. EL2 is partially hidden for clarity.
Receptor residues in ligand proximity are displayed.
A
their docking conformations. The excessive length of the 6-substi-
tuent of compound 10 makes the phenyl ring being placed too ex-
posed to the external of the receptor, and hence to the solvent,
with a possible detrimental effect to receptor binding. On the other
hand, comparison of binding modes and conformation energies of
compounds 7 and 11 (R6 = NHCH2Ph) suggests that the presence of
an amino group directly conjugated to the tricyclic scaffold
provides more rigidity to the 6-substituent than an ether oxygen.
As a consequence, while both derivatives are able to bind the A1
AR, the slightly higher flexibility of the 6-substituent in compound
7 allows this molecule to fit the binding cavity with a more stable
conformation than its analogue 11.
6.1.1.1. Ethyl (7-benzyloxy-1H-indol-3-yl)glyoxylate (20)32
. Yield:
77%; mp 166–167 °C (cyclohexane/EtOAc). 1H NMR (CDCl3) 1.46 (t,
3H, CH3, J = 7.1 Hz), 4.43 (q, 2H, CH2, J = 7.1 Hz), 5.23 (s, 2H, CH2),
6.89 (d, 1H, ar, J = 7.9 Hz), 7.29 (m, 1H, ar), 7.40–7.50 (m, 5H, ar), 8.05
(d, 1H, ar, J = 7.9 Hz), 8.43 (s, 1H, H-2), 9.05 (br s, 1H, NH). Anal. Calcd
for C19H17NO4: C, 70.58; H, 5.30; N, 4.33. Found: C, 70.31; H, 5.69; N,
4.01.
5. Conclusion
6.1.1.2. Ethyl (7-bromo-1H-indol-3-yl)glyoxylate (21)33
. Yield:
70%; mp 151–153 °C (cyclohexane/EtOAc); 1H NMR (CDCl3) 1.46
(t, 3H, CH3, J = 7.1 Hz), 4.42 (q, 2H, CH2, J = 7.1 Hz), 7.24–7.26 (m,
1H, ar), 7.51 (d, 1H, ar, J = 7.8 Hz), 8.42 (d, 1H, ar, J = 8.0 Hz), 8.57
(s, 1H, H-2), 9.09 (br s, 1H, NH). Anal. Calcd for C12H10BrNO3: C,
48.67; H, 3.40; N, 4.73. Found: C, 48.93; H, 3.73; N, 4.47.
The present work describes new 2-phenyl- and 2-methylpyrazolo
[3,4-c]quinolin-4-ones (series A) and 4-amines (series B), bearing at
the 6-position various groups with different lipophilicity and steric
bulk. The 6-substituent was introduced with the aim of shifting affin-
ities towards the A1 and A2A receptor subtypes. In series A, this mod-
ification wasdetrimentalbecauseitannulledthe affinity oftheparent
compound 1A. On the contrary, in series B it shifted affinity toward
the A1 receptor subtype. Indeed, most of the 2-phenylpyrazoloquino-
lin-4-amines showed an increased A1 receptor affinity and selectivity,
6.1.1.3.
Ethyl
(7-benzhydryloxy-1H-indol-3-yl)glyoxylate
(22). Yield: 78%; 1H NMR (CDCl3) 1.44 (t, 3H, CH3, J = 7.2 Hz),
4.42 (q, 2H, CH2, J = 7.2 Hz), 6.41 (s, 1H, CH), 6.71 (d, 1H, ar,

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O. Lenzi et al. / Bioorg. Med. Chem. 19 (2011) 3757–3768
J = 7.9 Hz), 7.14 (t, 1H, ar, J = 8.0 Hz), 7.31–7.45 (m, 10H, ar), 8.00
(d, 1H, ar, J = 8.0 Hz), 8.43 (s, 1H, H-2), 9.15 (br s, 1H, NH). ESI-
MS m/z calcd for C25H21NO4: 399.2; found 400.2 [MÀH]+
refluxed for 15 min. The mixture was concentrated under reduced
pressure and the residue solution (about 5 mL) was cooled at room
temperature and diluted with water (15 mL). The solid was col-
lected, washed with water and recrystallized. Yield 90%; mp
297–298 °C (EtOH). 1H NMR (DMSO-d6) 6.88 (d, 1H, ar,
J = 7.9 Hz), 7.07 (t, 1H, ar, J = 7.8 Hz), 7.43–7.50 (m, 2H, ar), 7.63
(t, 2H, ar, J = 7.9 Hz), 8.05 (d, 2H, ar, J = 8.3 Hz), 9.45 (s, 1H, H-1),
9.99 (br s, 1H, OH or NH), 10. 29 (br s, 1H, OH or NH); IR 1654,
3368. Anal. Calcd for C16H11N3O2: C, 69.31; H, 4.00; N, 15.15.
Found: C, 69.04; H, 4.32; N, 15.37.
6.1.1.4. Ethyl (7-benzyloxycarbonylamino-1H-indol-3-yl)gly-
oxylate (23). Yield: 55%; mp 211–213 °C. 1H NMR (CDCl3) 1.45
(t, 3H, CH3, J = 7.2 Hz), 4.43 (q, 2H, CH2, J = 7.2 Hz), 5.28 (s, 2H,
CH2), 6.80 (d, 1H, ar, J = 7.6 Hz), 7.22–7.28 (m, 2H, ar), 7.38–7.46
(m, 5H, ar), 8.29 (d, 1H, ar, J = 7.9 Hz), 8.46 (s, 1H, H-2), 11.08 (br
s, 1H, NH). Anal. Calcd for C20H18N2O5: C, 65.25; H, 4.95; N, 7.65.
Found: C, 65.25; H, 5.18; N, 7.92.
6.1.4. Synthesis of 6-amino-2,5-dihydro-2-phenyl-4H-
6.1.2. General procedure for the synthesis of 6-substituted 2,5-
dihydro-2-phenyl-4H-pyrazolo[3,4-c]quinolin-4-ones 1–4
A suspension of phenylhydrazine hydrochloride (2.8 mmol) and
the suitable ethyl 3-indolylglyoxylate 20–23 (1.4 mmol) in abso-
lute ethanol (15 mL) and glacial acetic acid (2–3 drops) was heated
at reflux until the disappearance of the starting material (TLC mon-
itoring, 4–6 h). The mixture was cooled at room temperature and
the solid was collected, washed with water and recrystallized.
For compound 22, no solid was obtained, thus the reaction mixture
was worked up as follows. After cooling at room temperature, the
solvent was evaporated under reduced pressure and the residue ta-
ken up with dichloromethane (about 70 mL). The solid was filtered
off and the solvent evaporated to yield an oily residue which solid-
ified after treatment with cyclohexane/diisopropyl ether. The
crude solid was a mixture of the 6-benzylhydryloxy derivative 3
and of the 6-hydroxy compound 5, which were separated by col-
umn chromatography (eluent cyclohexane/EtOAc/MeOH, 6:4:0.5).
pyrazolo[3,4-c]quinolin-4-one (6)
Compound 4 (0.66 mmol) was dissolved in hot DMF (about
20 mL), then 10% Pd/C (30 mg) was added to the solution. The mix-
ture was hydrogenated in a Parr apparatus, at 45 psi for 12 h. The
catalyst was filtered off, the solution diluted with water (about
40 mL) and extracted with EtOAc (50 mL Â 3 times). The organic
phases were anhydrified (Na2SO4) and the solvent evaporated un-
der reduced pressure. The residue was treated with water (a few
mL) to yield a solid which was collected, washed with water and
diethyl ether. Yield 82%; mp >300 °C (DMF). 1H NMR (DMSO-d6)
5.52 (s, 2H, NH2), 6.73 (d, 1H, ar, J = 7.7 Hz), 6.97 (t, 1H, ar,
J = 7.7 Hz), 7.23 (d, 1H, ar, J = 7.7 Hz), 7.43–7.48 (m, 1H, ar), 7.63
(t, 2H, ar, J = 7.6 Hz), 8.04 (d, 2H, ar, J = 7.6 Hz), 9.42 (s, 1H, H-1),
10.49 (br s, 1H, NH); IR 1677, 3193, 3379. Anal. Calcd for
C16H12N4O: C, 69.55; H, 4.38; N, 20.28. Found: C, 69.80; H, 4.12;
N, 20.52.
6.1.5. General procedure for the synthesis of 6-substituted 4-
chloro-2-phenyl-2H-pyrazolo[3,4-c]quinolines 24–26
6.1.2.1.
6-Benzyloxy-2,5-dihydro-2-phenyl-4H-pyrazolo[3,4-
c]quinolin-4-one (1). Yield: 40%; mp 229–230 °C (acetonitrile).
1H NMR (DMSO-d6) 5.33 (s, 2H, CH2), 7.14–7.20 (m, 2H, ar),
7.34–7.39 (m, 1H, ar), 7.47–7.64 (m, 8H, ar), 8.06 (d, 2H, ar,
J = 7.9 Hz), 9.49 (s, 1H, H-1), 10.25 (s, 1H, NH); IR 1703. Anal. Calcd
for C23H17N3O2: C, 75.19; H, 4.66; N, 11.44. Found: C, 75.38; H,
4.92; N, 11.15.
A mixture of the 4-oxo derivatives 1, 2, and 4 (6.8 mmol) and
phosphorous pentachloride (2.04 mmol) in phosphorous oxychlo-
ride (8 mL) was heated at reflux until the disappearance of the
starting material (TLC monitoring, 15 min to 2 h). Evaporation of
the excess of phosphorous oxychloride under reduced pressure
gave an oil which after treatment with cold water (50 mL) yielded
a solid that was collected, washed with water and dried. The 4-
chloro derivatives 24–26 were unstable, thus they could not be
re-crystallized. Nevertheless they were pure enough to be charac-
terized (1H NMR) and used without further purification.
6.1.2.2. 6-Bromo-2,5-dihydro-2-phenyl-4H-pyrazolo[3,4-c]quin-
olin-4-one (2). Yield: 30%; mp 286–288 °C (2-ethoxyethanol). 1H
NMR (DMSO-d6) 7.23 (t, 1H, ar, J = 7.9 Hz), 7.50 (t, 1H, ar,
J = 7.4 Hz), 7.58–7.65 (m, 2H, ar), 7.73 (d, 1H, ar, J = 7.8 Hz), 8.02–
8.05 (m, 3H, ar), 9.58 (s, 1H, H-1), 9.98 (s, 1H, NH). Anal. Calcd
for C16H10BrN3O: C, 56.49; H, 2.96; N,12.35. Found: C, 56.13; H,
2.68; N, 12.01.
6.1.5.1. 6-Benzyloxy-4-chloro-2-phenyl-2H-pyrazolo[3,4-c]quino-
line (24). Yield 90%; 1H NMR (DMSO-d6) 5.35 (s, 2H, CH2), 7.29–
7.46 (m, 4H, ar), 7.53–7.69 (m, 6H, ar), 7.87 (d, 1H, ar, J = 7.8 Hz),
8.13 (d, 2H, ar, J = 7.9 Hz), 9.82 (s, 1H, H-1). ESI-MS m/z calcd for
C
23H16ClN3O: 385.1; found 386.1 [MÀH]+.
6.1.2.3.
6-Benzhydryloxy-2,5-dihydro-2-phenyl-4H-pyrazol-
o[3,4-c]quinolin-4-one (3). Yield: 35%; mp 267–270 °C (acetoni-
trile). 1H NMR (DMSO-d6) 6.16 (br s, 1H, CH), 6.67 (d, 1H, ar,
J = 8.1 Hz), 7.12–7.28 (m, 12H, ar), 7.46 (t, 1H, ar, J = 7.5 Hz), 7.62
(t, 2H, ar, J = 7.8 Hz), 8.05 (d, 2H, J = 7.8 Hz), 9.38 (br s, 1H, H-1);
IR 1660. Anal. Calcd for C29H21N3O2: C, 78.54; H, 4.77; N, 9.47.
Found: C, 78.30; H, 4.46; N, 9.68.
6.1.5.2. 6-Bromo-4-chloro-2-phenyl-2H-pyrazolo[3,4-c]quinoline
(25). Yield 98%; 1H NMR (DMSO-d6) 7.57–7.69 (m, 4H, ar), 8.02
(d, 1H, ar, J = 7.4 Hz), 8.14 (d, 2H, ar, J = 7.4 Hz), 8.35 (d, 1H, ar,
J = 6.5 Hz), 9.92 (s, 1H, H-1). ESI-MS m/z calcd for C16H9BrClN3:
356.9; found 357.9 [MÀH]+.
6.1.2.4. 6-Benzyloxycarbonylamino-2,5-dihydro-2-phenyl-4H-
pyrazolo[3,4-c]quinolin-4-one (4). Yield: 38%; mp >300 °C
(DMF). 1H NMR (DMSO-d6) 5.19 (s, 2H, CH2), 7.25 (t, 1H, ar,
J = 7.8 Hz), 7.36–7.62 (m, 9H, ar), 7.65 (d, 1H, ar, J = 7.2 Hz), 8.05
(d, 2H, ar, J = 7.9 Hz), 9.33 (br s, 1H, NH), 9.52 (s, 1H, H-1), 10.65
(br s, 1H, NH); IR 1730, 3250. Anal. Calcd for C24H18N4O3: C,
70.23; H, 4.42; N, 13.65. Found: C, 69.98; H, 4.74; N, 13.81.
6.1.5.3. 6-Benzyloxycarbonylamino-4-chloro-2-phenyl-2H-pyr-
azolo[3,4-c]quinoline (26). Yield 90%; 1H NMR (DMSO-d6) 5.27
(s, 2H, CH2), 7.37–7.73 (m, 9H, ar), 8.01 (d, 1H, ar, J = 7.8 Hz),
8.14 (d, 2H, ar, J = 7.7 Hz), 8.20 (d, 1H, ar, J = 7.9 Hz), 9.15 (s, 1H,
NH), 9.87 (s, 1H, H-1). ESI-MS m/z calcd for C24H17ClN4O2: 428.1;
found 429.1 [MÀH]+.
6.1.6. General procedure for the synthesis of 6-substituted 2-
phenyl-2H-pyrazolo[3,2-c]quinolin-4-amines 7–8, 27–28
A mixture of the suitable 4-chloro derivative 24–26 (4.7 mmol)
in absolute ethanol saturated with ammonia (15 mL) was heated
6.1.3. Synthesis of 6-hydroxy-2,5-dihydro-2-phenyl-4H-
pyrazolo[3,4-c]quinolin-4-one (5)
A suspension of compound 1 (2.72 mmol) in glacial acetic acid
(10 mL) and 48% aqueous hydrobromic acid solution (10 mL) was

O. Lenzi et al. / Bioorg. Med. Chem. 19 (2011) 3757–3768
3765
overnight at 120 °C in a sealed tube. The solid that precipitated
upon cooling was collected, washed with water and recrystallized.
Reaction of the 4-chloroderivative 26 afforded a mixture of the cor-
responding 4-amino derivative 27 and of the 6,4-diamino-substi-
tuted compound 28 which were separated by column
chromatography (eluent cyclohexane/EtOAc/MeOH, 6:3.5:0.5).
Compound 28 was not recrystallized, due to its scarce quantities.
Thus, its melting point was not determined.
washed with water and recrystallized. Yield 30%; mp 228–230 °C
(2-butanone); 1H NMR (DMSO-d6) 3.14 (t, 2H, CH2, J = 7.3 Hz),
4.31 (t, 2H, CH2, J = 7.3 Hz), 6.90 (br s, 2H, NH2), 7.00 (d, 1H, ar,
J = 7.9 Hz), 7.14 (t, 1H, ar, J = 8.1 Hz), 7.20–7.66 (m, 9H, ar), 8.10
(d, 2H, ar, J = 8.1 Hz), 9.47 (s, 1H, H-1). Anal. Calcd for C24H20N4O:
C, 75.77; H, 5.30; N, 14.73. Found: C, 75.39; H, 5.62; N, 14.91.
6.1.9. Synthesis of 6-benzylamino-2-phenyl-2H-pyrazolo[3,4-
c]quinolin-4-amine (11)
A mixture of the 4,6-diammino derivative 28 (0.36 mmol),
anhydrous zinc chloride (0.73 mmol), benzaldehyde (0.43 mmol)
in anhydrous tetrahydrofuran (10 mL) was heated at reflux for
3 h. The suspension was cooled at room temperature and the solid
collected by filtration, washed with water and then with diethyl
ether. The crude Schiff base 29 was directly used for the next step.
NaBH4 (0.33 mmol) was added to a boiling suspension of 29
(0.16 mmol) in anhydrous methanol (7 mL). After 30 min, the solu-
tion was cooled at room temperature, diluted with water (5 mL)
and stirred for 5 min. The solid was collected, washed with water
and recrystallized. Yield 98%; mp 173–175 °C (MeOH/EtOH); 1H
NMR (DMSO-d6) 4.46 (d, 2H, CH2, J = 5.9 Hz), 6.28 (t, 1H, NH,
J = 5.9 Hz), 6.52 (d, 1H, ar, J = 7.5 Hz), 6.82 (s, 2H, NH2), 7.00 (t,
1H, ar, J = 7.7 Hz), 7.23–7.49 (m, 7H, ar), 7.63 (t, 2H, ar,
J = 7.6 Hz), 8.11 (d, 2H, J = 8.0 Hz), 9.42 (s, 1H, H-1). Anal. Calcd
for C23H19N5: C, 75.59; H, 5.24; N, 19.16. Found: C, 75.32; H,
5.49; N, 18.96.
6.1.6.1. 6-Benzyloxy-2-phenyl-2H-pyrazolo[3,4-c]quinolin-4-
amine (7). Yield 76%; mp 211–212 °C (MeOH/2-ethoxyethanol);
1H NMR (DMSO-d6) 5.26 (s, 2H, CH2), 6.94 (br s, 2H, NH2), 7.03
(d, 1H, ar, J = 7.1 Hz), 7.11–7.15 (m, 1H, ar), 7.33–7.53 (m, 6H, ar),
7.60–7.66 (m, 3H, ar), 8.11 (d, 2H, ar, J = 7.1 Hz), 9.47 (s, 1H, H-
1); IR 1630, 3265, 3477. Anal. Calcd for C23H18N4O: C, 75.39; H,
4.95; N, 15.29. Found: C, 75.62; H, 5.23; N, 15.52.
6.1.6.2. 6-Bromo-2-phenyl-2H-pyrazolo[3,4-c]quinolin-4-amine
(8). Yield 65%; mp 233–235 °C (2-methoxymethanol); 1H NMR
(DMSO-d6) 7.13 (t, 1H, ar, J = 7.1 Hz), 7.26 (br s, 2H, NH2), 7.65 (t,
2H, ar, J = 7.9 Hz), 7.72 (t, 1H, ar, J = 6.9 Hz), 8.11 (d, 1H, ar,
J = 7.9 Hz), 8.31 (d, 2H, ar, J = 7.9 Hz), 9.56 (s, 1H, H-1); IR 1631,
3294, 3474. Anal. Calcd for C16H11BrN4: C, 56.66; H, 3.27; N,
16.52. Found: C, 56.40; H, 3.42; N, 16.85.
6.1.6.3. 6-Benzyloxycarbonylamino-2-phenyl-2H-pyrazolo[3,4-
c]quinolin-4-amine (27). Yield 7%; mp 167–169 °C (cyclohex-
ane/EtOAc); 1H NMR (DMSO-d6) 5.23 (s, 2H, CH2), 7.20–7.32 (m,
3H, 1 ar + NH2), 7.38–7.49 (m, 6H, ar), 7.51–7.69 (m, 3H, ar),
8.08–8.12 (m, 3H, ar), 9.15 (s, 1H, H-1), 9.51 (s, 1H, NH). Anal. Calcd
for C24H19N5O2: C, 70.40; H, 4.68; N, 17.10. Found: C, 70.57; H,
4.45; N, 16.93.
6.1.10. General procedure for the synthesis of 6-substituted 2,5-
dihydro-2-methyl-4H-pyrazolo[3,4-c]quinolin-4-ones 12–13
The title compounds were prepared by reacting the suitable
ethyl 3-indolylglyoxylate 2032 or 3035 (1.4 mmol) with meth-
ylhydrazine hydrochloride (2.8 mmol) in absolute ethanol
(15 mL) and glacial acetic acid (2–3 drops). The mixture was
heated at reflux for 24 h, then the solvent evaporated under re-
duced pressure. The residue was treated with water (about 20–
30 mL) and the solid obtained was collected by filtration, washed
with water and recrystallized.
6.1.6.4.
(28). Yield 20%; 1H NMR (DMSO-d6) 5.33 (br s, 2H, NH2 at the
6-position), 6.68–6.71 (m, 3H, ar + NH2), 6.97 (t, 1H, ar,
2-Phenyl-2H-pyrazolo[3,4-c]quinolin-4,6-diamine
1
6.1.10.1. 6-Benzyloxy-2,5-dihydro-2-methyl-4H-pyrazolo[3,4-
c]quinolin-4-one (12). Yield 85%; mp 178–180 °C (MeOH); 1H
NMR (DMSO-d6) 4.12 (s, 3H, CH3), 5.31 (s, 2H, CH2), 7.08–7.13
(m, 2H, ar), 7.31–7.46 (m, 4H, ar), 7.59 (m, 2H, ar, J = 7.32 Hz),
8.64 (s, 1H, H-1), 9.98 (br s, 1H, NH); IR 1665. Anal. Calcd for
J = 7.7 Hz), 7.21 (d, 1H, ar, J = 7.5 Hz), 7.47–7.65 (m, 3H, ar), 8.11
(d, 2H, ar, J = 8.0 Hz), 9.39 (s, 1H, ar). ESI-MS m/z calcd for
C
16H13N5: 275.1; found 276.1 [MÀH]+.
6.1.7. Synthesis of 6-hydroxy-2-phenyl-2H-pyrazolo[3,4-
c]quinolin-4-amine (9)
C
18H15N3O2: C, 70.81; H, 4.95; N, 13.76. Found: C, 70.56; H, 4.62;
N, 13.49.
10% Pd/C (80 mg) was added to a solution of compound 7
(2.3 mmol) in DMF (15 mL). The mixture was hydrogenated in a
Parr apparatus for 3 h, at 35 psi, then the catalyst was filtered
off. The solution was diluted with water (about 40 mL) to yield a
solid which was collected by filtration and washed with water.
The crude solid was purified by column chromatography (eluent
cyclohexane/EtOAc/MeOH, 5:4.5:0.5). Yield 45%; mp 183–185 °C
(acetonitrile); 1H NMR (DMSO-d6) 6.87 (d, 1H, ar, J = 7.8 Hz), 7.01
(br s, 2H, NH2), 7.10 (t, 1H, ar, J = 7.8 Hz), 7.47–7.51 (m, 2H, ar),
7.62–7.66 (m, 2H, ar), 8.12 (d, 2H, ar, J = 7.6 Hz), 8.23 (br s, 1H,
OH), 9.47 (s, 1H, H-1); IR 1620, 3348, 3461. Anal. Calcd for
6.1.10.2. 2,5-Dihydro-2-methyl-4H-pyrazolo[3,4-c]quinolin-4-
one (13)34
. Yield 40%; mp 284–286 °C (acetonitrile), lit 285–
287 °C (MeOH); 1H NMR (DMSO-d6) 4.12 (s, 3H, CH3), 7.15–7.19
(m, 1H, ar), 7.31–7.33 (m, 2H, ar), 7.86 (d, 1H, ar), 8.63 (s, 1H, H-
1), 11.31 (br s, 1H, NH); IR 1665. Anal. Calcd for C11H9N3O: C,
66.32; H, 4.55; N, 21.09. Found: C, 66.01; H, 4.83; N, 21.37.
6.1.11. Synthesis of 6-benzyloxy-4-chloro-2-methyl-2H-
pyrazolo[3,4-c]quinoline (31)
A mixture of the 4-oxo derivatives 12 (6.8 mmol) and phospho-
rous pentachloride (2.04 mmol) in phosphorous oxychloride
(8 mL) was heated at reflux for 3 h. Evaporation under reduced pres-
sureoftheexcessof phosphorousoxychloride gaveanoilwhichafter
treatment with cold water (50 mL) yielded a solid which was col-
lected, washed with water and dried. The 4-chloro derivative 31
was unstable, nevertheless it was pure enough to be characterized
(1H NMR) and used without further purification. Yield 85%; 1H
NMR (DMSO-d6) 4.29 (s, 3H, CH3), 5.33 (s, 2H, CH2), 7.24–7.56 (m,
7H, ar), 7.77 (d, 1H, ar, J = 7.6 Hz), 9.01 (s, 1H, H-1). ESI-MS m/z calcd
for C18H14ClN3O: 323.1; found 324.1 [MÀH]+.
C16H12N4O: C, 69.55; H, 4.38; N, 20.28. Found: C, 69.86; H, 4.07;
N, 20.56.
6.1.8. Synthesis of 6-phenethoxy-2-phenyl-2H-pyrazolo[3,4-
c]quinolin-4-amine (10)
A mixture of compound 9 (0.4 mmol), potassium carbonate
(1.08 mmol), phenethyl bromide (1.08 mmol) in 2-butanone
(10 mL) was heated at reflux for 48 h. After cooling at room tem-
perature, the solid was filtered off and the solution concentrated
to small volume (4–5 mL). The precipitated solid was collected,

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O. Lenzi et al. / Bioorg. Med. Chem. 19 (2011) 3757–3768
6.1.12. Synthesis of 2-methyl-2H-pyrazolo[3,4-c]quinolin-4-
amine (15)36,37
elled, all hydrogen atoms were added, and the protein coordinates
were then minimized with MOE using the AMBER99 force field.
The minimizations were performed by 1000 steps of steepest des-
cent followed by conjugate gradient minimization until the RMS
A mixture of the 4-oxo derivatives 13 (6.8 mmol) and phospho-
rous pentachloride (2.04 mmol) in phosphorous oxychloride
(8 mL) was heated at reflux for 3 h. The oily residue, obtained from
evaporation under reduced pressure of the excess of phosphorous
oxychloride, was taken up with cyclohexane (40 mL) which was
then evaporated under reduced pressure. This procedure was re-
peated again twice and the tarry residue was immediately used
for the next step. The 1H NMR (DMSO-d6) spectrum of this residue
is not reported since it showed the presence of a mixture of com-
pounds. Thus the proton signals of the 4-chloro derivative 32 could
not be unequivocally assigned. The tarry residue was taken up with
absolute ethanol saturated with ammonia (15 mL) and the mixture
heated at 110 °C for 12 h in a sealed tube. After cooling at room
temperature, the solid was collected by filtration and washed with
water. The 4-amino derivative 15 was purified by column chroma-
tography (eluent CH2Cl2/MeOH, 9.5:0.5). Yield 16% (with respect to
the starting 4-oxo derivative 32); mp 210–212 °C; 1H NMR (DMSO-
d6) 4.25 (s, 3H, CH3), 6.74 (s, 2H, NH2), 7.17 (t, 1H, ar, J = 7.6 Hz),
7.31 (t, 1H, ar, J = 7.7 Hz), 7.47 (d, 1H, ar, J = 8.1 Hz), 7.89 (d, 1H,
ar, J = 7.7 Hz), 8.67 (s, 1H, H-1). Anal. Calcd for C11H10N4: C,
66.65; H, 5.08; N, 28.26. Found: C, 66.38; H, 5.32; N, 28.54.
gradient of the potential energy was less than 0.05 kJ molÀ1 ÅÀ1
.
Reliability and quality of these models were checked using the Pro-
tein Geometry Monitor application within MOE, which provides a
variety of stereochemical measurements for inspection of the
structural quality in a given protein, like backbone bond lengths,
angles and dihedrals, Ramachandran
u–w dihedral plots, and side
chain rotamer and nonbonded contact quality.
6.2.3. Molecular docking analysis
All compound structures were docked into the A1, A2A, and A3
AR binding site using the MOE Dock tool. This method is divided
into a number of stages: Conformational Analysis of ligands. The
algorithm generated conformations from a single 3D conformation
by conducting a systematic search. In this way, all combinations of
angles were created for each ligand. Placement. A collection of
poses was generated from the pool of ligand conformations using
Alpha Triangle placement method. Poses were generated by super-
position of ligand atom triplets and triplet points in the receptor
binding site. The receptor site points are alpha sphere centres
which represent locations of tight packing. At each iteration a ran-
dom conformation was selected, a random triplet of ligand atoms
and a random triplet of alpha sphere centres were used to deter-
mine the pose. Scoring. Poses generated by the placement method-
ology were scored using two available methods implemented in
MOE, the London dG scoring function which estimates the free en-
ergy of binding of the ligand from a given pose, and Affinity dG
Scoring which estimates the enthalpic contribution to the free en-
ergy of binding. The top 30 poses for each ligand were output in a
MOE database.
6.1.13. Synthesis of 6-benzyloxy-2-methyl-2H-pyrazolo[3,4-
c]quinolin-4-amine (14)
A mixture of the 4-chloro derivative 31 (1.2 mmol) in absolute
ethanol saturated with ammonia (15 mL) was heated overnight
at 120 °C in a sealed tube. The solid that precipitated upon cooling
was collected, washed with water and purified by column chroma-
tography (eluent cyclohexane/EtOAc/MeOH, 2:6:2). Yield 55%; mp
239–241 °C (2-methoxyethanol); 1H NMR (DMSO-d6) 4.18 (s, 3H,
CH3), 5.24 (s, 2H, CH2), 6.74 (br s, 2H, NH2), 6.97 (d, 1H, ar,
J = 7.4 Hz), 7.06 (t, 1H, ar, J = 7.9 Hz), 7.31–7.48 (m, 3H, ar), 7.51–
7.53 (m, 3H, ar), 8.65 (s, 1H, H-1); IR 1641, 3271, 3479. Anal. Calcd
for C18H16N4O: C, 71.04; H, 5.30; N, 18.41. Found: C, 70.82; H, 5.61;
N, 18.26.
6.2.4. Post docking analysis
The five top-score docking poses of each compound were then
subjected to energy minimization AMBER99 force field energy
minimization until the RMS gradient of the potential energy was
less than 0.05 kJ molÀ1 ÅÀ1. Receptor residues within 6 Å distance
from the ligand were left free to move, while the remaining recep-
tor coordinates were kept fixed. In this phase, water molecules
originally present in the hA2AAR were recovered and subjected to
energy minimization together with the ligands. AMBER99 partial
charges of receptor and water molecules and MOPAC output partial
charges of ligands were utilized. Once the compound-binding site
energy minimization was completed, receptor coordinates were
fixed and a second energy minimization stage was performed leav-
ing free to move only compound atoms. MMFF9446–52 force field
was applied. For each compound, the minimized docking poses
at A1AR were then rescored using London dG and Affinity dG scoring
functions and the dock-pKi predictor. The latter tool allows esti-
mating the pKi for each ligand using the ‘scoring.svl’ script retriev-
able at the SVL exchange service (Chemical Computing Group, Inc.
SVL exchange: http://svl.chemcomp.com). The algorithm is based
on an empirical scoring function consisting of a directional hydro-
gen-bonding term, a directional hydrophobic interaction term, and
an entropic term (ligand rotatable bonds immobilized in binding).
For each compound, the top-score docking pose according to at
least two out of three scoring functions was selected for final li-
gand-target interaction analysis.
6.2. Computational methodologies
All molecular modeling studies were performed on a 2 CPU (PIV
2.0–3.0 GHz) Linux PC. Homology modeling, energy minimization,
and docking studies were carried out using Molecular Operating
Environment (MOE, version 2009.10) suite.44 All ligand structures
were optimized using RHF/AM1 semiempirical calculations and
the software package MOPAC implemented in MOE was utilized
for these calculations.41
6.2.1. Human A2AAR crystal structure refinement
The recently solved X-ray crystal structure of the hA2AAR in
complex with ZM241385 was retrieved from the RCSB Protein Data
Bank (pdb code: 3EML; 2.6-Å resolution).38 All hydrogen atoms
were added, and the protein coordinates were then minimized
with MOE using the AMBER99 force field.45 The minimizations
were performed by 1000 steps of steepest descent followed by con-
jugate gradient minimization until the RMS gradient of the poten-
tial energy was less than 0.05 kJ molÀ1 ÅÀ1
.
6.2.2. Homology modeling of the human A1 and A3 ARs
Homology models of the hA1 and hA3 ARs were built using the
crystal structure of the hA2AAR as template. The alignment of the
AR primary sequences was built within MOE. The boundaries iden-
tified from the X-ray crystal structure of hA2AAR were applied for
the corresponding sequences of the TM helices of the hA1 and
hA3 AR. The missing loop domains were built by the loop search
method implemented in MOE. Once the heavy atoms were mod-
6.3. Pharmacology
6.3.1. Human cloned A1, A2A and A3 AR binding assay
All synthesized compounds were tested to evaluate their
affinity at hA1, hA2A and hA3 adenosine receptors. Displacement

O. Lenzi et al. / Bioorg. Med. Chem. 19 (2011) 3757–3768
3767
experiments of [3H]DPCPX (1 nM) to hA1 CHO membranes (50
lg
References and notes
of protein/assay) and at least six to eight different concentra-
tions of antagonists for 120 min at 25 °C in 50 mM Tris–HCl buf-
fer pH 7.4 were performed.53 Non specific binding was
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determined in the presence of 1
binding). Binding of [3H]ZM-241385 (1 nM) to hA2ACHO mem-
branes (50 g of protein/assay) was performed using 50 mM
lM of DPCPX (610% of the total
l
Tris–HCl buffer, 10 mM MgCl2 pH 7.4 and at least six to eight
different concentrations of antagonists studied for an incubation
time of 60 min at 4 °C.54 Non specific binding was determined in
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[
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ing in the presence of 1 lM AB-MECA and was about 20% of to-
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filtering the assay mixture through Whatman GF/B glass fiber fil-
ters using a Brandel cell harvester. The filter bound radioactivity
was counted by Scintillation Counter Packard Tri Carb 2810 TR
with an efficiency of 62%.
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6.3.2. Measurement of cyclic AMP levels in CHO cells transfected
with hA2B
CHO cells transfected with hA2B subtype were washed with
phosphate-buffered saline, diluted tripsine and centrifuged for
10 min at 200 g. The pellet containing the CHO cells (1 Â 106
cells/assay) was suspended in 0.5 ml of incubation mixture
(mM): NaCl 15, KCl 0.27, NaH2PO4 0.037, MgSO4 0.1, CaCl2 0.1,
Hepes 0.01, MgCl2 1, glucose 0.5, pH 7.4 at 37 °C, 2 IU/ml aden-
osine deaminase and 4-(3-butoxy-4-methoxybenzyl)-2-imidazo-
lidinone (Ro 20–1724) as phosphodiesterase inhibitor and
preincubated for 10 min in a shaking bath at 37 °C. The potency
of antagonists to A2B ARs was determined by antagonism of
NECA (200 nM)-induced stimulation of cyclic AMP levels. The
reaction was terminated by the addition of cold 6% trichloroace-
tic acid (TCA). The TCA suspension was centrifuged at 2000g for
10 min at 4 °C and the supernatant was extracted four times
with water saturated diethyl ether. The final aqueous solution
was tested for cyclic AMP levels by a competition protein bind-
ing assay. Samples of cyclic AMP standard (0–10 pmol) were
added to each test tube containing [3H] cyclic AMP and the incu-
bation buffer (trizma base 0.1 M, aminophylline 8.0 mM, 2-
mercaptoethanol 6.0 mM, pH 7.4). The binding protein, previ-
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incubated at 4 °C for 150 min, and after the addition of charcoal
was centrifuged at 2000g for 10 min. The clear supernatant was
counted in a Scintillation Counter Packard Tri Carb 2810 TR with
an efficiency of 62%.56
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