The identification of the 2-phenylphthalazin-1(2 H)-one scaffold as a new decorable core skeleton for the design of potent and selective human A 3 adenosine receptor antagonists

Article abstract of DOI:10.1021/jm101328n

Following a molecular simplification approach, we have identified the 2-phenylphthalazin-1(2H)-one (PHTZ) ring system as a new decorable core skeleton for the design of novel hA₃ adenosine receptor (AR) antagonists. Interest for this new series was driven by the structural similarity between the PHTZ skeleton and both the 2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (TQX) and the 4-carboxamido-quinazoline (QZ) scaffolds extensively investigated in our previously reported studies. Our attention was focused at position 4 of the phthalazine nucleus where different amido and ureido moieties were introduced (compounds 2-20). Some of the new PHTZ compounds showed high hA₃ AR affinity and selectivity, the 2,5-dimethoxyphenylphthalazin-1(2H)-one 18 being the most potent and selective hA₃ AR antagonist among this series (K_i = 0.776 nM; hA₁/hA₃ and hA _2A/hA₃ > 12000). Molecular docking studies on the PHTZ derivatives revealed for these compounds a binding mode similar to that of the previously reported TQX and QZ series, as was expected from the simplification approach.

Full text of DOI:10.1021/jm101328n

ARTICLE
pubs.acs.org/jmc
The Identification of the 2-Phenylphthalazin-1(2H)-one Scaffold as a
New Decorable Core Skeleton for the Design of Potent and Selective
Human A3 Adenosine Receptor Antagonists
Daniela Poli,† Daniela Catarzi,*,† Vittoria Colotta,† Flavia Varano,† Guido Filacchioni,† Simona Daniele,‡
Letizia Trincavelli,‡ Claudia Martini,‡ Silvia Paoletta,§ and Stefano Moro*,§
†Dipartimento di Scienze Farmaceutiche, Universitꢀa degli Studi di Firenze, Polo Scientifico, Via U. Schiff, 6-50019 Sesto Fiorentino
(Firenze), Italy
‡Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universitꢀa degli Studi di Pisa, Via Bonanno, 6-50126 Pisa, Italy
§Molecular Modeling Section (MMS), Dipartimento di Scienze Farmaceutiche, Universitꢀa degli Studi di Padova, via Marzolo 5,
I-35131 Padova, Italy
S Supporting Information
b
ABSTRACT: Following a molecular simplification approach, we have identified the
2-phenylphthalazin-1(2H)-one (PHTZ) ring system as a new decorable core
skeleton for the design of novel hA3 adenosine receptor (AR) antagonists. Interest
for this new series was driven by the structural similarity between the PHTZ
skeleton and both the 2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-1-one (TQX) and the
4-carboxamido-quinazoline (QZ) scaffolds extensively investigated in our pre-
viously reported studies. Our attention was focused at position 4 of the phthalazine
nucleus where different amido and ureido moieties were introduced (compounds
2-20). Some of the new PHTZ compounds showed high hA3 AR affinity and
selectivity, the 2,5-dimethoxyphenylphthalazin-1(2H)-one 18 being the most
potent and selective hA3 AR antagonist among this series (Ki = 0.776 nM; hA1/
hA3 and hA2A/hA3 > 12000). Molecular docking studies on the PHTZ derivatives
revealed for these compounds a binding mode similar to that of the previously
reported TQX and QZ series, as was expected from the simplification approach.
’ INTRODUCTION
an opposite manner (through inhibition and stimulation, re-
spectively), the global effect of the fine-tuner adenosine is inhibitory.
The A2B and A3 subtypes are low affinity ARs and might be
outstanding in pathological states. In fact, it is well-known that
under physiological conditions, A3 receptors do not have relevant
effects on neurotransmission, while no data are available on the role
of the A2B subtype.6,7
A large body of experimental evidence has pointed out the A1
and A2A receptor-mediated neuroprotective effects. Activation of
A1 AR leads to the minimization of the toxic effect promoted by
excitatory neurotransmitters, while the A2A subtype mediates the
activation of endogenous neuroprotective mechanisms.7,8
More recently, also the involvement of A3 AR in neuroprotec-
tion has been proposed.7,9-12 However, up to now, it is not clear
which is the real role of this receptor in ischemic conditions: The
mixed A3-mediated protective/damaging effect is still enigmatic
and has to be clarified.5,7,9,13 This dichotomic behavior of the A3
The autacoid adenosine plays a pivotal role in a large variety of
physiological and pathophysiological processes both in the
central nervous system (CNS) and in the periphery. Adenosine
is physiologically present in the extracellular fluid and exerts its
effects through activation of four cell surface receptor subtypes
termed A1, A2A, A2B, and A3, which belong to the superfamily of
G protein-coupled receptors (GPCRs).1,2 Adenosine receptors
(ARs) are widely distributed in the body and are expressed with
different density in the various tissues. The classical transduction
intracellular pathways associated with AR stimulation are inhibi-
tion, via Gi/o protein (A1 and A3 subtypes), or activation, via Gs
protein (A2A and A2B receptors), of adenylate cyclase (AC).1
More recently, other second messenger systems, such as phos-
pholipase C3 or potassium4 and calcium channels,4,5 have been
described as relevant for AR signaling.
Under normoxic conditions, adenosine modulates the activity
of the nervous system through stimulation of high affinity A1 and
A2A receptors, which are highly expressed in the brain. Although the
activation of A1 and A2A subtypes influences neurotransmission in
Received: October 14, 2010
Published: March 14, 2011
r
2011 American Chemical Society
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one skeleton and both TQX and QZ scaffolds extensively
investigated in our previously reported studies21,24,25,28
(Figure 1). In this first part of our work, we focused our attention
at position 4 of the phthalazine ring system, where differently
substituted amido and ureido moieties were introduced
(compounds 2-20, Table 1). In contrast, the 2-phenyl-substi-
tuent was held constant. Interestingly, also, an in silico receptor-
driven analysis on all the three (TQX, QZ, and PHTZ) series led
to the identification of converging ligand-receptor binding
requirements, which we consider as essential features for profit-
able hA3 receptor-antagonist recognition.
’ CHEMISTRY
The 4-substituted-2-phenylphthalazin-1(2H)-one derivatives
2-20 and the parent 4-amino compound 1 were prepared
following the synthetic pathway depicted in Scheme 1. By
reacting phthalic anhydride with an excess of phenylhydrazine,
the 4-hydroxy-2-phenylphthalazin-1(2H)-one 2130,31 was ob-
tained, which was treated with POCl3 to yield the corresponding
4-chloro derivative 22.32 Subsequently, reaction of 22 with
hydrazine sulfate in anhydrous hydrazine gave the 4-hydrazino
compound 23,32 which was reduced with hydrogen, in the
Figure 1. Simplification approach: from the TQX series to the QZ
series and to the herein reported 2-phenylphthalazin-1(2H)-one deri-
vatives (PHTZ series). Colors identify important conserved groups in
different series.
Scheme 1a
subtype has been highlighted also in other different pathological
conditions such as cancer and inflammation, where this receptor
seems to mediate contrasting effects.9 Thus, numerous A3 AR
agonists and antagonists have been investigated for their poten-
tial therapeutic applications7,9-12,14-17 and to shed light on
which could be the best choice for a given disease. It is easy to
understand why we have directed our efforts in this intriguing
direction and why, in the past few years, our attention has been
focused on the development of adenosine A3 receptor antago-
nists. Our research has led to the discovery of many tricyclic
compounds belonging to strictly correlated classes of nitrogen-
containing heterocycles and showing high affinity and selectivity
toward adenosine A3 receptor.12,18-27 Besides their remarkable
pharmacodynamic profiles, a favorable spectrum of pharmaco-
kinetic properties as well as the straightforwardness of their
synthetic pathway have to be considered as essential require-
ments for any drug candidate.
Indeed, our previously reported studies confirmed that struc-
tural simplification can represent a drug design strategy to
shorten synthetic routes while keeping or enhancing the biolo-
gical activity of the original candidate.28,29 In particular, we have
already reported that the 2-aryl-1,2,4-triazolo[4,3-a]quinoxalin-
1-one (TQX)18,20,21,24,25 series can be simplified into new easily
synthesizable 4-carboxamido-quinazoline (QZ) derivatives en-
dowed with high affinity and selectivity toward hA3 AR.28 In our
investigations, many other bicyclic heteroaromatic systems, con-
taining those structural features essential to guarantee an efficient
ligand-receptor recognition, have been taken into consideration
as a possible core skeleton for the design of novel hA3 AR
antagonists. Among them, our attention has been caught by the
phthalazin-1(2H)-one (PHTZ) ring system that has not yet been
considered as a suitable scaffold to obtain AR antagonists.
Our interest for this new series of PHTZ analogues was also
driven by the structure similarity between the phthalazin-1(2H)-
a Reagents and Conditions: (a) 10% HCl; (b) POCl3; (c) NH2NH2xH2SO4,
NH2NH2, ethylene glycol; (d) H2, Raney-Nickel, EtOH; (e) RCOCl,
pyridine, THF; (f) RNCO, CH2Cl2 or THF.
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presence of Raney nickel as catalyst, to the corresponding
4-amino-2-phenylphthalazin-1(2H)-one 1 with good yield.
The 4-aroylamino-2-phenylphthalazin-1(2H)-ones 2 and
4-8 were obtained by reacting 1 with the suitable aroyl chloride
inthe presence ofpyridine. Inthe preparation of the 4-acetylamino
compound 2, treatment of 1 with acetyl chloride also gave the
corresponding diacetylamino derivative 3. Reaction of 1 with the
appropriate aryl or alkyl isocyanate produced the targeted 4-ureido
compounds 9-20.
calculated root-mean-square deviation (rmsd) values relative to
the crystallographic pose of ZM241385.29
Consequently, on the basis of the selected docking protocol,
we performed docking simulations to identify the hypothetical
binding modes of the newly synthesized 2-phenylphthalazin-
1(2H)-one derivatives and of two previously reported TQX and
QZ derivatives, inside the hA3 and hA2A ARs.
Finally, to analyze the possible ligand-receptor recognition
mechanism in a more quantitative way, the individual electro-
static (ΔEintel) and hydrophobic (ΔEinthyd) contributions of each
receptor residue to the interaction energy (ΔEint) were calcu-
lated for all of the selected binding poses (see the Experimental
Section for more details).
’ PHARMACOLOGY
The newly 4-substituted phthalazine derivatives 2-20 and the
parent 1 (Table 1) were tested for their ability to displace
[
125I]N6-(4-amino-3-iodobenzyl)-50-(N-methylcarbamoyl)ade-
nosine ([125I]AB-MECA) from cloned hA3 receptor stably
expressed in Chinese hamster ovary (CHO) cells. Subsequently,
all compounds were evaluated for their ability to displace [3H]8-
cyclopentyl-1,3-dipropylxantine ([3H]DPCPX) from cloned hA1
ARs and [3H]50-(N-ethylcarboxamido)adenosine ([3H]NECA)
from cloned hA2A ARs, to establish their A3 versus A1 and versus
A2A selectivity.
’ RESULTS AND DISCUSSION
Examining the binding results reported in Table 1, it appears
that we have identified some new potent and selective adenosine
hA3 receptor antagonists belonging to the 2-phenylphtalazin-
1(2H)-one series. In particular, it has to be noted that com-
pounds 12-14, 18, and 19 bearing a methoxyphenyl- or a
benzyl-substituted ureido moiety at position 4 are those en-
dowed with high affinity and also selectivity toward the hA3
receptor, as they are on the whole unable to bind at all the others
ARs. These preliminary results indicate that the 2-phenylphtha-
lazin-1(2H)-one moiety is a versatile tool for the design of new
potent and selective hA3 AR antagonists. However, it is rather
evident that clear and robust SARs can be difficult to obtain.
To start in, the binding affinity at the hA3 receptor of the
4-amino-2-phenylphtalazin-1(2H)-one 1 (hA3 I = 8%) was very
discouraging in particular because it was not in line with that
of the 4-amino-2-phenyl-1,2,4-triazolo[4,3-a]quinoxalin-1-one
(hA3 Ki = 490 nM)18 or the 2-aminoquinazoline-4-carboxyani-
lide (hA3 Ki = 350 nM)28 belonging to the reference TQX and
QZ series, respectively.
Moreover, some selected compounds (12-14, 18, and 19)
were tested at the hA2B subtype by measuring their inhibitory
effect on NECA-stimulated cyclic adenosine monophosphate
(cAMP) levels in CHO cells stably transfected with the hA2B AR
(Table 2). To evaluate their hA3 AR antagonistic effect, the same
compounds were tested for their ability to counteract the NECA-
mediated inhibition of cAMP accumulation in CHO cells stably
expressing hA3 AR (Table 2).
The binding data of derivatives 1-20 are shown in Table 1
together with those of A (1,2-dihydro-2-phenyl-4-phenylureido-
1,2,4-triazolo[4,3-a]quinoxalin-1-one)18 and B (2-benzoylami-
noquinazoline-4-carboxyanilide),28 selected as reference com-
pounds of the TQX and QZ series, respectively.
Despite this unfavorable starting point and on the basis of the
SARs derived from both TQX and QZ hA3 antagonists, a series of
4-amido- (2-8) and 4-ureido-derivatives (9-20) were synthe-
sized starting from the 4-amino intermediate 1. All of the
4-amido compounds, including alkyl- (2 and 3), aryl- (4-7),
and also arylalkyl-substituted (8), were inactive with the only
exception being the 4-phenylamido derivative 4 that showed a Ki
value of 1100 nM at the hA3 AR. Very interestingly, replacement
of the phenylamido- (4) with the phenylureido moiety (9) at the
4-position provided an appreciable increase of receptor affinity.
In fact, the 4-phenylureido derivative 9 was about 6-fold more
active (hA3 Ki = 178.4 nM) than its amido analogue 4. As
previously described for other ureido-related hA3 antagonists,35
the urea moiety contributes to the observed large differences
among the hA3 binding affinities of these derivatives. Thus, the
presence of a second NH group able to reinforce the hydrogen
bond network within the putative transmembrane (TM) binding
cavity appears to be primarily responsible for the ameliorating
effect of the receptor-antagonist recognition.
Starting from the encouraging hA3 binding data of the
4-phenylureido compound 9, we decided to explore the role of
a substituted phenyl ring at the level of the 4-ureido moiety by
introducing groups with different electronic and lipophilic prop-
erties at position ortho, meta, or para. Insertion of the electron-
withdrawing and lipophilic chloro atom at position para or ortho
produced, respectively, a total loss (compound 10) or a dramatic
reduction (compound 11) of receptor affinity at the hA3 subtype.
’ MOLECULAR MODELING
To explain the observed structure-affinity relationships (SARs)
and the selectivity profile of these new 2-phenylphthalazin-1(2H)-
one derivatives in comparison with the previously reported TQX
and QZ series,21,24,25,28 a receptor-driven molecular modeling
investigation, based on a lately proposed model of the hA3
receptor derived from the crystallographic structure of hA2A
AR, was also performed in this study.
In fact, the recently published crystallographic structure of
hA2A AR, in complex with the high affinity antagonist ZM241385
{4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]triazin-
5-ylamino]ethyl)phenol} (PDB code: 3EML),33 provides a new
alternative template to perform homology modeling of other
GPCRs and in particular of ARs. Therefore, a homology model of
the hA3 receptor based on the crystal structure of the hA2A receptor
was constructed as previously described29,34 (methodological details
are summarized in the Experimental Section).
Then, in the process of selecting a reliable docking protocol to
be employed in the following docking studies of the new PHTZ
derivatives, we evaluated the ability of different docking softwares
to reproduce the crystallographic pose of ZM241385 inside the
binding cavity of hA2A receptor. As reported in the Experimental
Section, among the four different types of programs used to
calibrate our docking protocol, the Gold program was finally
chosen since it showed the best performance with regards to the
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Table 1. Binding Affinity (Ki) of 4-Substituted 2-Phenylphtalazin-1(2H)-one Derivatives 1-20 and of Reference Compounds A
and B, of the TQX and QZ series, at Human A3, A1, and A2A ARs
Ki (nM) or I%a
b
c
d
compd
R4
hA3
hA1
hA2A
1
NH2
8%
0%
0%
24%
45%
2
NHCOCH3
N(COCH3)2
NHCOC6H5
52%
47%
44%
26%
0%
14%
10%
35%
17%
17%
41%
4%
3
4
1100 ( 100
17%
5
NHCOC6H4-4-Cl
6
NHCOC6H4-4-OCH3
NHCO(furan-2-yl)
10%
7
8%
23%
23%
44%
10%
0%
8
NHCOCH2C6H5
28%
9
NHCONHC6H5
178.4 ( 17
13%
42%
9%
10
11
12
13
14
15
16
17
18
19
20
Ae
Bg
NHCONHC6H4-4-Cl
NHCONHC6H4-2-Cl
NHCONHC6H4-4-OCH3
NHCONHC6H4-3-OCH3
NHCONHC6H4-2-OCH3
NHCONHC6H4-4-CH3
NHCONHC6H4-2-CH3
NHCONHC6H3-2,4-OCH3
NHCONHC6H3-2,5-OCH3
NHCONHCH2C6H5
NHCONHCH2C6H4-2-OCH3
49%
3%
60.6 ( 6.2
9.75 ( 0.25
8.9 ( 1
45%
4%
51%
28%
17%
0%
45%
0%
29%
20%
29%
0%
22%
28%
33%
19%
23%
28%
33%
0.776 ( 0.037
29.6 ( 3
274.2 ( 26
276 ( 21
182 ( 10
20%
28%
50.8 ( 4.2f
2300 ( 291f
7%
10%
a The Ki values are means ( SEMs of four separate assays, each performed in triplicate. b Displacement of specific [125I]AB-MECA binding at hA3
receptors expressed in CHO cells or percentage of inhibition (I%) of specific binding at 1 μM. c Displacement of specific [3H]DPCPX at hA1 receptors
expressed in CHO cells or percentage of inhibition (I%) of specific binding at 10 μM concentration. d Displacement of specific [3H]NECA binding at
hA2A receptors expressed in CHO cells or percentage of inhibition (I%) of specific binding at 10 μM concentration. e Ref 18. f Displacement of specific
[3H]CHA and [3H]CGS21680 binding at A1 and A2A receptors, respectively, in bovine brain membranes. g Ref 28.
The strong electron-donating methoxy group was then intro-
duced on the targeted 4-phenylureido moiety leading to the para-
substituted compound 12, which was shown to be highly potent
at the adenosine hA3 receptor subtype (hA3 Ki = 60.6 nM). Next,
the para-methoxy substituent of 12 was moved to position meta
and ortho to evaluate whether this shift could further optimize
the anchoring at the hA3 receptor site. This modification led,
respectively, to compounds 13 and 14, which are two of the most
potent and selective adenosine hA3 receptor antagonists belong-
ing to the new 2-phenylphthalazin-1(2H)-one derivatives re-
ported in this work. Accordingly, we evaluated the effect on hA3
AR affinity of introduction of another methoxy group on the
4-substituent of 14 by synthesizing the 2,4-dimethoxy and 2,5-
dimethoxyphenyl-substituted compounds 17 and 18, respec-
tively. The second methoxy substituent introduced at position
5 of the phenyl ring of 14 contributes positively to adenosine hA3
receptor affinity, which showed an 11-fold increase (compare 18
to 14). In fact, the (2,5-dimethoxyphenylureido)-phthalazin-
1(2H)-ones 18 is the most potent adenosine hA3 receptor
antagonist among this series, with a Ki value of 0.776 nM and
at least 10000-fold selectivity over hA1 and hA2A receptors.
Unexpectedly, the 2,4-dimethoxy-substitued compound 17 as
well as the 4-(4-methylphenyl)- and 4-(2-methylphenyl)-ureido
derivatives 15 and 16 did not show any appreciable binding
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Table 2. Potencies (IC50) at hA2B and hA3 of Some Selected
4-Substituted 2-Phenylphtalazin-1(2H)-one Derivatives
cAMP assays IC50 (nM) or I%
a
b
no.
hA2B
hA3
12
13
14
18
19
0%
57%
16%
0%
28 ( 3
18 ( 2
17 ( 1.6
8.25 ( 0.6
1.15 ( 0.02
34%
a Percentage of inhibition on cAMP experiments in hA2BCHO cells
stimulated by 100 nM NECA at different examined compound concen-
trations (1 nM to 10 μM). b IC50 values represent the means ( SEMs of
three separate experiments in hA3CHO cells, inhibited by 100 nM NECA
at different examined compound concentrations (1 nM to 10 μM).
affinity toward the hA3 AR. Introduction of a benzyl-ureido
moiety at position 4 of the phthalazin-1(2H)-one scaffold led to
compound 19, which revealed to be 6-fold more active than the
homologue 9 at the hA3 receptor (hA3 Ki = 29.6 nM). Finally, the
highly profitable methoxy group was introduced at the ortho
position on the benzylic group of 19, yielding compound 20 that
unexpectedly showed a 9-fold decreased hA3 affinity.
Among the new PHTZ series, compounds 12-14, 18, and 19
possess the highest hA3 affinity and selectivity versus both hA1
and hA2A receptors. To determine also their hA3 versus hA2B
selectivity, we tested these derivatives in cAMP assays, which
evidenced low or null hA2B affinity, being in general ineffective in
inhibiting NECA-stimulated cAMP levels in hA2B CHO cells.
Furthermore, the effect of compounds 12-14, 18, and 19 in
limiting the NECA-inhibited cAMP accumulation in hA3 CHO
cells was determined. Coherently with their high hA3 affinity, all
of the selected PHTZ derivatives proved to be very potent in this
test, showing an antagonistic behavior (Table 2).
Because the new 2-phenylphthalazin-1(2H)-one derivatives
reported in this study were conceived as simplified derivatives of
the previously synthesized 1,2,4-triazolo[4,3-a]quinoxaline-1-
one compounds (TQX series),18,20,21,24,25 molecular docking
studies were performed on all of the PHTZ derivatives
(compounds 1-20) and on the TQX derivative A,18 taken as
reference. Moreover, docking simulations were also performed
on compound B, a quinazoline-4-carboxamide derivative (QZ
series) previously reported.28
Therefore, all of the selected compounds were docked into
the TM binding site of the hA3 AR three-dimensional model, to
identify their hypothetical binding modes at this receptor and to
analyze possible analogies among the different series. In addition,
docking simulations at the hA2A AR were carried out to explain
the hA3 versus hA2A selectivity profile of all of these derivatives.
Analysis of the docking results reveals that all of the studied
compounds (1-20, A, and B) share a binding pose that is
somehow similar in the TM region of the hA3 AR. In fact, at this
receptor, ligand recognition occurs in the upper region of the TM
bundle, and the tricyclic or bicyclic scaffold of the ligands is
surrounded by TMs 3, 5, 6, and 7. Figure 2A shows the
hypothetical binding mode of the reference TQX derivative A
(hA3 AR Ki = 276 nM). This compound is anchored, inside the
binding cleft, by three stabilizing hydrogen-bonding interactions
with the side chain of Asn250 (6.55). These three hydrogen
bonds involve the N5 atom of the TQX nucleus and the two NH
Figure 2. Hypothetical binding modes obtained after docking simula-
tions inside the hA3 AR binding site of (A) compound A, (B) compound
B, and (C) compound 9. Poses are viewed from the membrane side
facing TM6, TM7, and TM1. The view of TM7 is voluntarily omitted.
Side chains of some amino acids important for ligand recognition and
H-bonding interactions are highlighted. Hydrogen atoms are not
displayed.
groups of the 4-ureidic moiety, respectively. The asparagine
residue 6.55, conserved among all of the AR subtypes, was
already found, through mutagenesis studies,36,37 to be important
for ligand binding at both the hA3 and the hA2A ARs. Compound
A also forms hydrophobic interactions with many residues of the
binding cleft including Ala69 (2.61), Val72 (2.64), Leu90 (3.32),
Leu91 (3.33), Phe168 (EL2), Val169 (EL2), Met177 (5.38),
Trp243 (6.48), Leu246 (6.51), Ile249 (6.54), Ile253 (6.58),
Val259 (EL3), Leu264 (7.35), Tyr265 (7.36), and Ile268 (7.39).
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In particular, the planar tricyclic core of the ligand strongly
interacts with Phe168 (EL2) and with the highly conserved
Trp243 (6.48), an important residue for either receptor activa-
tion or for antagonist binding.37
The hypothetical binding mode, at the hA3 AR, of one of the
herein reported 2-phenyl-phthalazin-1(2H)-ones (compound 9,
hA3 AR Ki = 178.4 nM) is displayed in Figure 2C. Molecular
docking simulations show that the new compound 9 is efficiently
accommodated into the TM binding cavity with the 4-phenylur-
eido substituent directed toward the extracellular loop region.
Interestingly, compound 9 maintains all of the crucial interac-
tions already seen for the TQX and QZ derivatives and also a
similar binding pose. This ligand forms three hydrogen-bonding
interactions with the Asn250 (6.55) side chain, involving the N3
atom of the PHTZ nucleus and the two NH groups of the ureidic
moiety, respectively. In addition, the phthalazinone scaffold
forms a π-π stacking interaction with Phe168 (EL2). Other
hydrophobic interactions are established with several residues of
the binding cavity, such as Ala69 (2.61), Leu90 (3.32), Leu91
(3.33), Val169 (EL2), Met177 (5.38), Phe182 (5.43), Ile186
(5.47), Trp243 (6.48), Leu246 (6.51), Ile249 (6.54), Ile253
(6.58), Val259 (EL3), Leu264 (7.35), and Ile268 (7.39). The
described docking pose of compound 9 reflects more or less the
hypothetical binding mode of all of the analyzed 2-phenyl-
phthalazin-1(2H)-one derivatives (compounds 1-20).
As shown in Figure 2B, the hypothetical binding pose of the
QZ derivative B (hA3 AR Ki = 182 nM), obtained after molecular
docking into the three-dimensional model of the hA3 receptor, is
very similar to that of compound A (Figure 2A). In fact, ligand
recognition occurs in the same region of the TM bundle. In
particular, the appended phenyl ring on the 4-carboxyamide
moiety of B is oriented toward TM2, such as the 2-phenyl ring of
the TQX derivative A, and the 2-benzoylamino group of B points
toward the extracellular loop region, such as the 4-phenylureido
moiety of compound A. Moreover, compound B forms two
H-bonds with Asn250 (6.55) and a strong hydrophobic interac-
tion with Phe168 (EL2).
It is worth noting that in compound B the formation of an
intramolecular H-bond between the nitrogen at the 3-position of
the quinazoline system and the NH of the amide moiety at the
4-position leads to the stabilization of a conformer, which
simulates a planar tricycle with similar steric properties to the
original TQX analogue (compound A). The planarity of the QZ
derivative, due to this intramolecular H-bond, seems to increase
complementarity with the hA3 receptor; the key role of this
intramolecular H-bond was already analyzed in previous docking
studies of the QZ derivatives carried out on the rhodopsin-based
homology model of hA3 AR.28
Then, electrostatic and hydrophobic interaction contributions
between compounds 9, A, and B and each amino acid involved in
ligand recognition (Figures 3 and 4, respectively) were calculated
from the hypothetical binding modes inside the hA3 AR dis-
played in Figure 2. Analysis of these data confirms the hypothesis
of an analogous binding mode at the hA3 AR for the TQX, QZ,
and PHTZ derivatives selected in this study.
Figure 3. Electrostatic interaction energy (in kcal/mol) between compounds 9, A, and B and each single amino acid involved in ligand recognition
calculated from the hypothetical binding modes inside the hA3 AR (Figure 2).
Figure 4. Hydrophobic interaction score (in arbitrary hydrophobic unit) between compounds 9, A, and B and each amino acid involved in ligand
recognition calculated from the hypothetical binding modes inside the hA3 AR (Figure 2).
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As shown in Figure 3, it is clear that, from the electrostatic
point of view, one of the most critical residues affecting the
affinity at the hA3 AR seems to be the Asn250 (6.55) that is
responsible for the stabilizing H-bonding interactions with all of
the three ligands. This is supported by the Asn250 electrostatic
contribution of around -20 kcal/mol to the whole interaction
energy of the three ligand-receptor complexes. Moreover, no
significant detrimental electrostatic contributions (positive elec-
trostatic interaction energy) are observed for these complexes.
The hydrophobic interactions mapped in Figure 4 show a
similar pattern for all of the three ligand-receptor complexes. In
particular, the most important hydrophobic contribution is
mediated by Phe168 (EL2), conserved among all ARs, which
strongly interacts with the bicyclic/tricyclic core of the ligands. In
addition, the ligand scaffold is involved in hydrophobic contacts
with Leu90 (3.32), Leu91 (3.33), Trp243 (6.48), Leu246 (6.51),
and Ile268 (7.39), while the phenyl ring appended on the ureido/
amido moiety interacts with Val169 (EL2), Ile253 (6.58), and
Leu264 (7.35).
Some aspects of the SAR of this phthalazine series are very
difficult to rationalize. Surprisingly, some substituents, such as
phenylamido and benzylamido, that positively affect the affinity in
other series of hA3 AR antagonists, showed, in contrast, discoura-
ging results in these new compounds. The herein proposed
binding mode seems to partially explain why compounds bearing
a 4-ureido substituent possess higher affinity at the hA3 AR than
the 4-amido analogues. In fact, the increased affinities of the
4-ureido-derivatives could be due to the formation of an additional
H-bonding interaction with the Asn250 (6.55) carbonyl group.
With regard to the substituents on the phenyl ring appended
on the 4-ureido moiety, the binding data show that both of their
features, electron-donating or electron-withdrawing, and their
position play a crucial role in modulating the affinity at the hA3
AR. Considering the herein proposed binding mode at this
receptor subtype, it is clear that such substituents are located
near the extracellular loop region and can possibly interact with
residues belonging to EL2 and EL3 (see the Supporting Infor-
mation, Figure 1A, for the binding modes of compounds 11, 14,
and 16 at the hA3 AR). Because of the difficult characterization
and the high plasticity of the loop region, it is hard to accurately
predict particular interactions with this part of the ligand and
therefore to explain the observed effects of these substituents.
Some amino acids possibly involved in interactions with the
substituted phenyl ring are Gln167 (EL2), Val169 (EL2),
Met174 (5.35), Ile253 (6.58), Val259 (EL3), and Leu264
(7.35). Calculation of the per residue electrostatic and hydro-
phobic contributions to the interaction energy was performed on
the docking poses of compounds 11, 14, and 16 at the hA3 AR to
analyze possible differences. However, as expected, no significant
differences were seen considering these ligand-receptor com-
plexes (see the Supporting Information, Figure 1B,C), and so, no
clues about the role of these different substituents were obtained.
Further studies are in progress in our laboratory to better define
the extracellular loops conformation that could be responsible for
the interaction with the substituents at the 4-position of these
ligands.
As far as the hA2A AR is concerned, docking simulations
performed for compounds 1-20 revealed no good binding poses
at this receptor subtype, as highlighted by the docking pose of
compound 9 at the hA2A AR displayed in Figure 5A. In fact, at the
hA2A AR, compound 9 resulted to be turned of 180° as compared
to the docking pose of the same compound inside the hA3 AR
binding site, probably due to the presence of a Glu169 and of the
steric hindrance of the substituent at the 4-position. As a
consequence of this flipped orientation, compound 9 was not
able to strongly interact with the critical residues Asn253 (6.55)
or Glu169 (EL2) through H-bonds as instead previously noticed
for the crystallographic binding pose of ZM241385 at the hA2A
AR33 and found in all other docked compounds possessing
antagonist activity at the hA2A AR.38 Indeed, analyzing the per
residue electrostatic and hydrophobic contributions for the
docking pose of compound 9 inside the hA2A AR (Figure 5B,
C, respectively), it is evident the lack of any strong electrostatic
interaction with residues of the binding site and the presence of
only few strong hydrophobic interactions, such as the one with
Phe168. This finding can explain the low or null affinity at the A2A
Figure 5. (A) Hypothetical binding mode obtained after docking simulation inside the hA2A AR binding site of compound 9. The pose is viewed from
the membrane side facing TM1, TM6, and TM7. The view of TM7 is voluntarily omitted. Hydrogen atoms are not displayed. (B) Electrostatic
interaction energy (in kcal/mol) between compound 9 and each single amino acid involved in ligand recognition calculated from the hypothetical
binding mode inside the hA2A AR. (C) Hydrophobic interaction score (in arbitrary hydrophobic unit) between compound 9 and each amino acid
involved in ligand recognition calculated from the hypothetical binding mode inside the hA2A AR.
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AR subtype of all of the compounds (1-20) reported in
this work.
derivative 23 (5.0 mmol) in ethanol (200 mL). The reaction mixture was
hydrogenated in a Parr apparatus at 15 psi for 12 h. After elimination of
the catalyst by filtration, the ethanol solution was evaporated under
reduced pressure. The resulting solid was worked up with a little ethyl
ether and collected by filtration. Yield, 81%; mp 188-200 °C (EtOAc).
1H NMR: 6.31 (s, 2H, NH2), 7.34 (t, 1H, ar, J = 7.18 Hz), 7.47 (t, 2H, ar,
J = 7.75 Hz), 7.64(d, 2H, ar, J = 7.52 Hz), 7.88 (t, 1H, ar, J = 7.18 Hz), 7.95
(t, 1H, ar, J = 7.54 Hz), 8.15 (d, 1H, ar, J = 7.68 Hz), 8.33 (d, 1H, ar, J =
7.68 Hz). IR: 1625, 3214, 3318, 3422. Anal. (C14H11N3O) C, H, N.
Synthesis of 4-Acetylamino-2-phenylphthalazin-1(2H)-one 2 and
4-Diacetylamino-2-phenylphthalazin-1(2H)-one 3. A solution of acet-
yl chloride (1.85 mmol) in anhydrous tetrahydrofuran (5 mL) was
dropwise added (30 min) to a cooled (5 °C) solution of the 4-amino
derivative 1 (1.68 mmol) in anhydrous tetrahydrofuran (10 mL) and
anhydrous pyridine (3.3 mmol). The reaction mixture was stirred at
room temperature for 24 h and then diluted with EtOAc (70 mL). The
resulting solution was washed with water (50 mL ꢀ 3), then with
NaHCO3 solution (2.5%, 30 mL), and finally again with water (50 mL).
Evaporation under reduced pressure of the dried (Na2SO4) ethyl acetate
solution yielded a solid (mixture 3:1 of compounds 2 and 3, 1HNMR
analysis) which was worked up with the minimal amount of diethyl ether
and collected by filtration. Separation of compounds 2 and 3 was
performed by using silica gel column cromatography, eluting system
CH2Cl2/MeOH 9:1. Evaporation of the first eluates afforded compound
3, while the central eluates contained the monoacetyl derivative 2.
Compound 2. Yield, 49%; mp 197-199 °C (2-methoxyethanol). 1H
NMR: 2.16 (s, 3H, CH3), 7.43 (t, 1H, ar, J = 6.96 Hz), 7.53 (t, 2H, ar, J =
7.74 Hz), 7.62 (d, 2H, ar, J = 7.96 Hz), 7.86 (d, 1H, ar, J = 7.76 Hz),
7.92-8.01 (m, 2H, ar), 8.35 (d, 1H, ar, J = 7.68 Hz), 10.35 (s, 1H, NH).
IR: 1672, 3248. Anal. (C16H13N3O2) C, H, N.
In conclusion, molecular docking studies of the newly synthe-
sized 2-phenylphthalazin-1(2H)-ones, performed at the hA3 AR,
revealed for these compounds a binding mode similar to that of
the previously reported TQX and QZ derivatives, as was
expected from the simplification approach. These three classes
of hA3 AR antagonists show analogous interactions with the
binding cavity of the receptor as confirmed by the analysis of the
electrostatic and hydrophobic contributions to the interaction
energy. Further studies are in progress in our laboratories to
better clarify the structural requirements for profitable hA3
receptor-ligand interaction of this class of compounds and to
develop new PHTZ derivatives with higher hA3 AR affinity.
’ EXPERIMENTAL SECTION
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, and N, and the results were
within (0.4% of the theoretical values except where stated otherwise.
The IR spectra were recorded with a Perkin-Elmer Spectrum RX I
spectrometer in Nujol mulls and are expressed in cm-1. The 1H NMR
spectra were obtained with a Bruker Avance 400 MHz instrument. The
chemical shifts are reported in δ (ppm) and are relative to the central
peak of the solvent, which was always DMSO-d6. All of the exchangeable
protons were confirmed by addition of D2O. Used are the following
abbreviations: s, singlet; d, doublet; dd, double doublet; t, triplet; m,
multiplet; br, broad; and ar, aromatic protons.
Compound 3. Yield, 15%; mp 185-186 °C (2-methoxyethanol). 1H
NMR: 2.38 (s, 6H, 2CH3), 7.45 (t, 1H, ar, J = 7.36 Hz), 7.54 (t, 2H, ar, J
= 7.72 Hz), 7.63 (d, 2H, ar, J = 7.72 Hz), 7.92 (d, 1H, ar, J = 7.12 Hz),
7.98 (m, 2H, ar), 8.42 (d, 1H, ar, J = 7.12 Hz). IR: 1673, 1697, 1729.
Anal. (C18H15N3O3) C, H, N.
Synthesis of 4-Hydroxy-2-phenylphthalazin-1(2H)-one 2130. A mix-
ture of phenylhydrazine (81.0 mmol) and phthalic anhydride (67.5
mmol) in 10% HCl (100 mL) was heated at reflux for 9 h. After it was
cooled, the resulting solid was collected by filtration, washed with water,
and recrystallized. Yield, 75%; mp 212-213 °C (EtOH) (p.f. lit.31
213.85 °C). 1H NMR: 7.37 (t, 1H, ar, J = 7.13 Hz), 7.50 (t, 2H, ar, J =
7.50 Hz), 7.64 (d, 2H, ar, J = 7.76 Hz), 7.92-8.04 (m, 3H, ar), 8.31
(d,1H, ar, J = 7.44 Hz), 11.85 (br s, 1H, NH). IR: 1642, 3400-2000.
Anal. (C14H10N2O2) C, H, N.
General Procedure for the Synthesis of 4-Aroylamino-2-phe-
nylphthalazin-1(2H)-ones 4-7 and 4-Phenylacetylamino-2-phe-
nylphthalazin-1(2H)-one 8. A solution of the suitable aroyl chloride
(compounds 4-7) or phenylacetyl chloride (8) (6.0 mmol) in anhy-
drous tetrahydrofuran (5 mL) was dropwise added (30 min) to a cooled
(5 °C) solution of the 4-amino derivative 1 (2.0 mmol) in anhydrous
tetrahydrofuran (20 mL) and anhydrous pyridine (20 mmol). The
reaction mixture was stirred at room temperature until the disappear-
ance of the starting derivative 1 (TLC monitoring, 15-72 h). The
resulting suspension (compounds 5-8) was diluted with water (50 mL)
and extracted with EtOAc (70 mL). The organic layer was washed with
water (50 mL ꢀ 2), then with NaHCO3 solution (2.5%, 50 mL), and
finally again with water (50 mL). Evaporation under reduced pressure of
the dried (Na2SO4) ethyl acetate solution yielded a solid, which was
worked up with the minimal amount of diethyl ether and collected by
filtration. Compounds 6-8 were recrystallized, while compound 5 was
purified by silica gel column chromatography, eluting system CH2Cl2/
EtOAc 8:2, and then recrystallized. For derivative 4, the reaction mixture
was filtered, and the solid was washed with water and recrystallized.
Compound 4. Yield, 39%; mp 201-202 °C (EtOH). 1H NMR: 7.44
(t, 1H, ar, J = 7.34 Hz), 7.52-7.60 (m, 4H, ar), 7.63-7.68 (m, 3H, ar),
7.85 (d, 1H, ar, J = 8.64 Hz) 7.95-8.02 (m, 2H, ar), 8.07 (d, 2H, ar, J =
7.40 Hz), 8.39 (d, 1H, ar, J = 7.12 Hz), 10.91 (s, 1H, NH). IR: 1651,
1670. Anal. (C21H15N3O2) C, H, N.
Synthesis of 4-Chloro-2-phenylphthalazin-1(2H)-one 22. A mixture
of compound 2130 (25.0 mmol) in a large excess of POCl3 (6 mL) was
heated at reflux for 4 h. After it was cooled (0 °C), the resulting solution
was slowly quenched with cold NaOH solution (5M, 100 mL) yielding a
suspension that was stirred at room temperature for 2 h. The resulting
brown solid was collected by filtration and washed with water. Yield,
1
65%; mp 130-131 °C (EtOH) (p.f. lit.32 130 °C EtOH/H2O). H
NMR: 7.45 (t, 1H, ar, J = 7.13 Hz), 7.54 (t, 2H, ar, J = 7.75 Hz), 7.62 (d,
2H, ar, J = 7.72 Hz), 8.02-8.13 (m, 3H, ar), 8.38 (d, 1H, ar, J = 7.77 Hz).
IR: 1675. Anal. (C14H9ClN2O) C, H, N.
Synthesis of 4-Hydrazino-2-phenylphthalazin-1(2H)-one 23. A
mixture of the 4-chloro derivative 2232 (5.65 mmol), hydrazine sulfate
salt (11.3 mmol), and an excess of anhydrous hydrazine (100%, 2.9 mL)
in ethylene glycol (20 mL) was heated at 115 °C for 1 h. After it was
cooled, the reaction mixture was diluted with water (30 mL), and the
resulting solid was collected by filtration. Yield, 63%; mp 190-191 °C
(ethylene glycol/H2O) (p.f. lit.32 190 °C). 1H NMR: 4.13 (s, 2H, NH2),
7.33(t, 1H, ar, J = 7.28 Hz), 7.47(t, 2H, ar, J = 7.68 Hz), 7.81 (d, 2H, ar, J =
7.76 Hz), 7.85-7.95 (m, 2H, ar), 8.13 (d, 1H, ar, J = 7.76 Hz), 8.20 (s, 1H,
NH), 8.33 (d, 1H, ar, J = 7.52 Hz). IR: 1642, 3354. Anal. (C14H12N4O)
C, H, N.
Compound 5. Yield, 27%; mp 190-191 °C (2-methoxyethanol). 1H
NMR: 7.44 (t, 1H, ar, J = 6.96 Hz), 7.54 (t, 2H, ar, J = 8.62 Hz), 7.63-
7.68 (m, 4H, ar), 7.85 (d, 1H, ar, J = 7.44 Hz) 7.94-7.98 (m, 2H, ar),
8.08 (d, 2H, ar, J = 8.56 Hz), 8.39 (d, 1H, ar, J = 7.44 Hz), 11.00 (s, 1H,
NH). IR: 1671, 3072. Anal. (C21H14ClN3O2) C, H, N.
Synthesis of 4-Amino-2-phenylphthalazin-1(2H)-one 1. Raney nick-
el (2400, slurry, in H2O, 24 g) was added to a solution of the hydrazino
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Compound 6. Yield, 55%; mp 203-204 °C (EtOH). 1H NMR: 3.87
(s, 3H, OCH3), 7.10 (d, 2H, ar, J = 8.88 Hz), 7.44 (t, 1H, ar, J = 7.34 Hz),
7.54 (t, 2H, ar, J= 7.74 Hz), 7.64 (d, 2H, ar, J =8.12 Hz), 7.80(d, 1H, ar, J=
8.88 Hz) 7.94-8.01 (m, 2H, ar), 8.05 (d, 2H, ar, J = 8.88 Hz), 8.39 (d,
1H, ar, J = 8.88 Hz), 10.74 (s, 1H, NH). IR: 1670, 3068. Anal.
(C22H17N3O3) C, H, N.
NH), 9.63 (s, 1H, NH). IR: 1668, 1694, 3098, 3143, 3201, 3270. Anal.
(C22H18N4O3) C, H, N.
Compound 15. Yield, 46%; mp 261-263 °C. 1H NMR: 2.25 (s, 3H,
CH3), 7.10 (d, 2H, ar, J = 8.20 Hz), 7.33 (d, 2H, ar, J = 8.36 Hz), 7.43 (t,
1H, ar, J = 7.38 Hz), 7.54 (t, 2H, ar, J = 7.72 Hz), 7.70 (d, 2H, ar, J = 8.12
Hz), 7.94-8.04 (m, 2H, ar), 8.18 (d, 1H, ar, J = 7.88 Hz), 8.39 (d, 1H, ar,
J = 7.84 Hz), 9.26 (s, 1H, NH), 9.54 (s, 1H, NH). IR: 1667, 1688, 3191.
Anal. (C22H18N4O2) C, H, N.
Compound 7. Yield, 71%; mp 193-194 °C (MeOH). 1H NMR: 6.75
(d, 1H, ar, J = 3.36 Hz), 7.42-7.45 (m, 2H, ar), 7.54 (t, 2H, ar, J = 7.66
Hz), 7.64 (d, 2H, ar, J = 8.32 Hz), 7.83 (d, 1H, ar, J = 7.72 Hz), 7.95-
8.01 (m, 3H, ar), 8.39 (d, 1H, ar, J = 7.28 Hz), 10.83 (s, 1H, NH). IR:
1659. Anal. (C19H13N3O3) C, H, N.
Compound 16. Yield, 51%; mp 247-249 °C. 1H NMR: 1.86 (s, 3H,
CH3), 6.96 (t, 1H, ar, J = 7.36 Hz), 7.11-7.17 (m, 2H, ar), 7.45 (t, 1H, ar,
J = 7.32 Hz), 7.54(t, 2H, ar, J = 7.68Hz), 7.65 (d, 2H, ar, J = 7.76 Hz), 7.87
(d, 1H, ar, J = 7.96 Hz), 7.96-8.06 (m, 2H, ar), 8.34 (d, 1H, ar, J = 7.92
Hz), 8.39 (d, 1H, ar, J = 7.24 Hz), 9.39 (s, 1H, NH), 9.60 (s, 1H, NH). IR:
1659, 1681, 3136, 3190. Anal. (C22H18N4O2) C, H, N.
Compound 8. Yield, 40%; mp 194-195 °C (MeOH). 1H NMR: 3.80
(s, 2H, CH2), 7.26-7.29 (m, 1H, ar), 7.33-7.45 (m, 5H, ar), 7.53 (t,
2H, ar, J = 7.66 Hz), 7.61 (d, 2H, ar, J = 7.64 Hz), 7.72 (d, 1H, ar, J = 7.44
Hz), 7.91-7.97 (m, 2H, ar), 8.34 (d, 1H, ar, J = 7.44 Hz), 10.58 (s, 1H,
NH). IR: 1650, 1666, 3242. Anal. (C22H17N3O2) C, H, N.
Compound 19. Yield, 41%; mp 218-219 °C. 1H NMR: 4.36 (d, 2H,
CH2, J = 5.68 Hz), 7.24-7.33 (m, 5H, ar), 7.37-7.40 (m, 1H, ar); 7.45
(t, 2H, ar, J = 7.54 Hz), 7.59 (d, 2H, ar, J = 7.64 Hz), 7.85 (t, 1H, NH, J =
5.54 Hz), 7.92-8.02 (m, 2H, ar), 8.20 (d, 1H, ar, J = 7.76 Hz), 8.36
(d, 1H, ar, J = 7.20 Hz), 9.23 (s, 1H, NH). IR: 1678, 3140, 3258. Anal.
(C22H18N4O2) C, H, N.
General Procedure for the Synthesis of 4-Arylureido-2-phe-
nylphthalazin-1(2H)-ones 9-13, 15, and 16 and 4-Aralkylureido-2-
phenylphthalazin-1(2H)-ones 19 and 20. A mixture of 1 (1.3 mmol)
and an equimolar amount of the suitable aryl or aralkyl isocyanate in
anhydrous methylene chloride (10 mL) was stirred under nitrogen
atmosphere for 2-15 days. For compounds 9, 10, 12, 13, and 19, the
reaction mixture was kept at room temperature, while derivatives 11, 15,
16, and 20 were obtained by heating at 50 °C. Further additions of the
starting isocyanate (0.5-1 equivalent) were performed when com-
pound 1 was still present in the mixture (TLC monitoring) after 48 h
from the beginning of the reaction. The resulting suspension was filtered,
and the solid phase was resuspended in a mixture of cyclohexane/EtOAc
3:7 (80 mL) and kept under stirring for 4 h. The crude product was
collected by filtration, washed many times with cyclohexane/EtOAc 3:7,
and recrystallized from 2-methoxyethanol. It was not possible to recrys-
tallize compound 10 since it resulted unstable upon heating in the
suitable crystallization solvent, that is, DMF. Compound 10 was purified
by repeated washing with hot ethanol. TLC analysis (cyclohexane/
EtOAc 3:7), 1H NMR spectrum, and melting point of crude 10 showed
that it was pure enough to be tested in the binding assays.
Compound 20. Yield, 48%; mp 244-246 °C. 1H NMR: 3.66 (s, 3H,
OCH3), 4.31 (d, 2H, CH2, J = 5.28 Hz), 6.86 (t, 1H, ar, J = 7.44 Hz), 6.96
(d, 1H, ar, J = 8.24 Hz), 7.17-7.27 (m, 2H, ar), 7.38-7.42 (m, 1H, ar),
7.47 (t, 2H, ar, J = 7.66 Hz), 7.60 (d, 2H, ar, J = 7.68 Hz), 7.83 (br s, 1H,
NH), 7.92-8.01 (m, 2H, ar), 8.24 (d, 1H, ar, J = 8.04 Hz), 8.36 (d, 1H,
ar, J = 7.08 Hz), 9.35 (s, 1H, NH). IR: 1657, 1673, 3314. Anal.
(C23H20N4O3) C, H, N.
Synthesis of 4-(2-Methoxyphenyl)ureido-2-phenylphthalazin-
1(2H)-one 14. A mixture of 1 (2.0 mmol) and 2-methoxyphenyl
isocyanate (3.0 mmol) in anhydrous THF (twice-distilled, 30 mL)
was stirred at 50 °C under nitrogen atmosphere for a total time of 30 h.
After 12 h, further isocyanate was portionwise added (0.3 mmol ꢀ 6) at
3 h intervals to the reaction mixture. The resulting suspension was
filtered, and the solid phase was resuspended in a mixture of cyclohex-
ane/EtOAc 3:7 (80 mL) and kept under stirring for 4 h. The crude
product was collected by filtration, washed many times with cyclohex-
ane/EtOAc 3:7, and recrystallized. Yield, 40%; mp 267-268 °C
Compound 9. Yield, 40%; mp >300 °C. 1H NMR: 7.00 (t, 1H, ar, J =
7.20 Hz), 7.29 (t, 2H, ar, J = 7.08 Hz), 7.43-7.47 (m, 3H, ar), 7.54
(t, 2H, ar, J = 7.22 Hz) 7.70 (d, 2H, ar, J = 7.48 Hz), 7.96-8.02 (m, 2H,
ar), 8.16 (d, 1H, ar, J = 7.56 Hz); 8.38 (d, 1H, ar, J = 7.52 Hz), 9.55 (br s,
1H, NH), 9.83 (br s, 1H, NH). IR: 1672, 1697, 3360, 3420, 3495, 3522,
3564. Anal. (C21H16N4O2) C, H, N.
1
(2-methoxyethanol). H NMR: 3.34 (s, 3H, OCH3), 6.88-7.00 (m,
3H, ar), 7.45 (t, 1H, ar, J = 7.13 Hz), 7.56 (t, 2H, ar, J = 7.38 Hz), 7.69 (d,
2H, ar, J = 7.84 Hz), 7.96-8.05 (m, 2H, ar), 8.15 (d, 1H, ar, J = 7.80 Hz),
8.35-8.40 (m, 2H, ar), 9.71 (s, 1H, NH), 9.90 (s, 1H, NH). IR 1655,
1681, 3186. Anal. (C22H18N4O3) C, H, N.
Compound 10. Yield, 76%; mp 262-264 °C (crude). 1H NMR: 7.35
(d, 2H, ar, J = 8.72 Hz), 7.42 (t, 1H, ar, J = 7.34 Hz), 7.49-7.55 (m, 4H,
ar), 7.69 (d, 2H, ar, J = 7.48 Hz), 7.95-8.04 (m, 2H, ar), 8.12 (d, 1H, ar, J =
7.91 Hz);8.40 (d, 1H, ar, J =7.68 Hz), 9.34 (s, 1H, NH), 9.63 (s, 1H, NH).
IR: 1677, 1698, 3067, 3272. Anal. (C21H15ClN4O2) C, H, N.
General
Procedure
for
the
Synthesis
of
4-(2,4-
Dimethoxyphenylureido)- and 4-(2,5-Dimethoxyphenylureido)-2-
phenylphthalazin-1(2H)-ones 17 and 18. A mixture of 1 (0.63 mmol)
and an equimolar amount of the suitable dimethoxyphenyl isocyanate in
anhydrous methylene chloride (7.0 mL) was stirred under nitrogen
atmosphere for 1 day. Then, the suspension was heated at 40-45 °C,
and further isocyanate (0.31 mmol ꢀ 5) was added at 3 day intervals to
the reaction mixture. Heating was maintained for 18 days. The resulting
solid was filtered, washed with a small amount of fresh methylene
chloride, and then recrystallized from 2-methoxyethanol.
Compound 11. Yield, 49%; mp 251-252 °C. 1H NMR: 7.04 (t, 1H,
ar, J = 8.34 Hz), 7.30 (t, 1H, ar, J = 7.78 Hz), 7.38 (d, 1H, ar, J = 7.96 Hz),
7.44-7.57 (m, 1H, ar), 7.54 (t, 2H, ar, J = 7.60 Hz), 7.62 (d, 2H, ar, J =
7.48 Hz), 7.97-8.06 (m, 2H, ar), 8.22 (d, 1H, ar, J = 8.28 Hz), 8.34-
8.40 (m, 2H, ar), 9.87 (s, 1H, NH), 9.91 (s, 1H, NH). IR: 1663, 1680,
3152, 3277. Anal. (C21H15ClN4O2) C, H, N.
Compound 17. Yield, 42%; mp 257-259 °C. 1H NMR: 3.34 (s, 3H,
OCH3), 3.72 (s, 3H, OCH3), 6.48 (dd, 1H, ar, J = 8.84, 2.68 Hz), 6.53 (s,
1H, ar), 7.45 (t, 1H, ar, J = 7.36 Hz), 7.56 (t, 2H, ar, J = 7.76 Hz), 7.68 (d,
2H, ar, J = 8.20 Hz), 7.95-8.05 (m, 3H, ar), 8.36-8.40 (m, 2H, ar), 9.60
(s, 1H, NH), 9.75 (s, 1H, NH). IR: 1660, 3061, 3139, 3186, 3266. Anal.
(C23H20N4O4) C, H, N.
Compound 12. Yield, 45%; mp 259-260 °C. 1H NMR: 3.72 (s, 3H,
OCH3), 6.88 (d, 2H, ar, J = 7.52 Hz), 7.35 (d, 2H, ar, J = 7.56 Hz), 7.43
(t, 1H, ar, J = 7.34 Hz), 7.54 (t, 2H, ar, J = 7.16 Hz), 7.70 (d, 2H, ar, J =
7.92 Hz), 7.94-8.04 (m, 2H, ar), 8.19 (d, 1H, ar, J = 7.96 Hz), 8.39 (d,
1H, ar, J = 7.76 Hz), 9.25 (s, 1H, NH), 9.50 (s, 1H, NH). IR: 1655, 1682,
3097, 3143, 3193, 3270. Anal. (C22H18N4O3) C, H, N.
Compound 18. Yield, 34%; mp 294-296 °C. 1H NMR: 3.32 (s, 3H,
OCH3), 3.68 (s, 3H, OCH3), 6.53 (dd, 1H, ar, J = 8.92, 3.04 Hz), 6.84
(d, 1H, ar, J = 8.92 Hz), 7.45 (t, 1H, ar, J = 7.36 Hz), 7.55 (t, 2H, ar, J =
7.76 Hz), 7.68 (d, 2H, ar, J = 8.12 Hz), 7.85 (s, 1H, ar), 7.95-8.05
(m, 2H, ar), 8.32 (d, 1H, ar, J = 7.80 Hz), 8.39 (d, 1H, ar, J = 7.88 Hz),
Compound 13. Yield, 64%; mp 244-246 °C. 1H NMR: 3.34 (s, 3H,
OCH3), 6.59 (d, 1H, ar, J = 8.24 Hz), 6.99 (d, 1H, ar, J = 8.12 Hz), 7.11
(s, 1H, ar), 7.19 (t, 1H, ar, J = 8.14 Hz), 7.43 (t, 1H, ar, J = 7.38 Hz), 7.54
(t, 2H, ar, J = 7.64 Hz), 7.71 (d, 2H, ar, J = 8.32 Hz), 7.95-8.04 (m, 2H,
ar), 8.17 (d, 1H, ar, J = 7.88 Hz), 8.39 (d, 1H, ar, J = 7.84 Hz), 9.29 (s, 1H,
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Journal of Medicinal Chemistry
ARTICLE
9.72 (s, 1H, NH), 9.79 (s, 1H, NH). IR: 1657, 1681, 3285. Anal.
(C23H20N4O4) C, H, N.
methodology. Partial charges for protein amino acids were calculated
on the basis of the AMBER99 force field.
Pharmacological Assays. Human Cloned A1, A2A, and A3 AR
Binding Assay. Binding experiments at hA1 and hA2A ARs, stably
expressed in CHO cells, were performed as previously described,47
using [3H]DPCPX and [3H]NECA, respectively, as radioligands. Dis-
placement of [125I]AB-MECA from hA3 AR, stably expressed in CHO
cells, was performed as reported in ref 20.
Molecular Modeling. All modeling studies were carried out on a 20
CPU (Intel Core2 Quad CPU 2.40 GHz) Linux cluster. Homology
modeling, energy calculation, and analyses of docking poses were performed
using the Molecular Operating Environment (MOE, version 2009.10)
suite.39 The software package MOPAC (version 7),40 implemented in
MOE suite, was utilized for all quantum mechanical calculations. Docking
simulations were performed using GOLD suite (version 1.3.2).41
Homology Model of the hA3 AR. On the basis of the assumption that
GPCRs share similar TM boundaries and overall topology, a homology
model of the hA3 AR was constructed, as previously reported,29,34 using as
template the recently published crystal structure of hA2A receptor (PDB
code: 3EML).33 The amino acid sequences of TM helices of the hA3
receptor were aligned with those of the template, and then, the hA3 AR
homology model was constructed using the homology modeling protocol
implemented in MOE as detailed in the Supporting Information.
The numbering of the amino acids follows the arbitrary scheme by
Ballesteros and Weinstein. According to this scheme, each amino acid
identifier starts with the helix number, followed by the position relative
to a reference residue among the most conserved amino acid in that
helix. The number 50 is arbitrarily assigned to the reference residue.42
Protein stereochemistry evaluation was then performed by several
tools (Ramachandran plot; backbone bond lengths, angles, and dihedral
plots; clash contacts report; and rotamers strain energy report) im-
plemented in MOE suite.39,43
Molecular Docking of hA3 AR Antagonists. Ligand structures were
built using MOE builder tool, part of the MOE suite,39 and were
subjected to MMFF94x energy minimization until the rms conjugate
gradient was <0.05 kcal mol-1 Å-1. Partial charges for the ligands were
calculated using PM3/ESP methodology.
Four different programs were used to calibrate our docking protocols:
MOE-Dock,39 GOLD,41 Glide,44 and PLANTS.45 In particular, 4-(2-[7-
amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)
phenol (ZM-241385) was redocked into the crystal structure of the
hA2A AR (PDB code: 3EML) with different docking algorithms and
scoring functions, as already described.29,46 Then, rmsd values between
predicted and crystallographic positions of ZM-241385 were calculated
for each of the docking algorithms. The results showed that docking
simulations performed with GOLD gave the lowest rmsd value, the
lowest mean rmsd value, and the highest number of poses with rmsd
value <2.5 Å.29,46
Measurement of cAMP Levels on CHO Cells Transfected with
Human A2B and A3 ARs. Intracellular cAMP levels were measured using
a competitive protein binding method.48 CHO cells (∼60000), stably
expressing hA2B or hA3 ARs, were plated in 24-well plates. After 48 h, the
medium was removed, and the cells were incubated at 37 °C for 15 min
with 0.5 mL di DMEM in the presence of Ro20-1724 [4-(3-butoxy-4-
methoxybenzyl)imidazolidin-2-one] (20 μM) and adenosine deaminase
(1 U/mL). A stock 1 mM solution of the tested compound was prepared
in DMSO, and subsequent dilutions were accomplished in distilled
water. The antagonistic profile of the new compound toward hA2B AR
was evaluated assessing its ability to inhibit 100 nM NECA-mediated
accumulation of cAMP. The antagonistic profile of the new compound
toward hA3 AR was evaluated by assessing its ability to counteract 100
nM NECA-mediated inhibition of cAMP accumulation stimulated by 1
μM forskolin. Cells were incubated in the reaction medium (15 min at
37 °C) with different compound concentrations (1 nM to 10 μM) and
then treated with NECA. The reaction was terminated by removing the
medium and adding 0.4 N HCl. After 30 min, the lysate was neutralized
with 4 N KOH, and the suspension was centrifuged at 800g for 5 min. To
determine cyclic AMP production, the binding protein, prepared from
beef adrenal glands, was incubated with [3H]cAMP (2 nM) in distilled
water, 50 μL of cell lysate, or standard cAMP (0-16 pmol) at 4 °C for
150 min in a total volume of 300 μL. Bound radioactivity was separated
by rapid filtration through GF/C glass fiber filters and washed twice with
4 mL of 50 mM Tris/HCl, pH 7.4. The radioactivity was measured by
liquid scintillation spectrometry.
Data Analysis. The concentration of the tested compounds that
produced 50% inhibition of specific [3H]DPCPX, [3H]NECA, and
[
125I]AB-MECA binding (IC50) was calculated using a nonlinear
regression method implemented by the InPlot program (Graph-Pad,
San Diego, CA) with five concentrations of displacer, each performed
in triplicate. Inhibition constants (Ki) were calculated according to
the Cheng-Prusoff equation.49 The Kd values of [3H]DPCPX,
[3H]NECA, and [125I]AB-MECA in hA1, hA2A, and hA3 ARs in CHO
cell membranes were 3, 30, and 1.4 nM, respectively. IC50 values
obtained in cAMP assays were calculated by nonlinear regression
analysis using the equation for a sigmoid concentration-response curve
(Graph-Pad).
On the basis of the best docking performance, all antagonist
structures were docked into the hypothetical TM binding site of the
hA3 AR model and that of the hA2A AR crystal structure by using the
docking tool of the GOLD suite.41 Searching was conducted within a
user-specified docking sphere, using the Genetic Algorithm protocol and
the GoldScore scoring function. GOLD performed a user-specified
number of independent docking runs (25 in our specific case) and
wrote the resulting conformations and their scores in a molecular
database file. The resulting docked complexes (ligand and side chains
of residues at 4.5 Å from the ligand) were subjected to MMFF94x energy
’ ASSOCIATED CONTENT
S
Supporting Information. Combustion analysis data of
b
the newly synthesized compounds, methodological details about
the hA3 AR homology model construction, hypothetical binding
modes at the hA3 AR of compounds 11, 14, and 16 with their
relative per residue electrostatic interaction energies and hydro-
phobic interaction scores. This material is available free of charge
via the Internet at http://pubs.acs.org.
minimization until the rms conjugate gradient was <1 kcal mol-1 Å-1
.
Partial charges for the ligands were calculated using MOPAC and in
particular using PM3/ESP methodology.
Prediction of antagonist-receptor complex stability (in terms of
corresponding pKi value) and the quantitative analysis for non-
bonded intermolecular interactions (H-bonds, transition metal,
water bridges, hydrophobic, and electrostatic) was calculated and
visualized using several tools implemented in MOE suite.39 Electro-
static and hydrophobic contributions to the binding energy of
individual amino acids have been calculated as implemented
in MOE suite.39 To estimate the electrostatic contributions,
atomic charges for the ligands were calculated using PM3/ESP
’ AUTHOR INFORMATION
Corresponding Author
*(D.C.) Tel: þ39 55 4573722. Fax: þ39 55 4573780. E-mail:
daniela.catarzi@unifi.it. (S.M.) Tel: þ39 049 8275704. Fax: þ39
049 8275366. E-mail: stefano.moro@unipd.it.
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Journal of Medicinal Chemistry
ARTICLE
’ ACKNOWLEDGMENT
(11) Pugliese, A. M.; Coppi, E.; Volpini, R.; Cristalli, G.; Corradetti,
R.; Jeong, L. S.; Jacobson, K. A.; Pedata, F. Role of adenosine A3
receptors on CA1 hippocampal neurotransmission during oxygen-
glucose deprivation episodes of different duration. Biochem. Pharmacol.
2007, 74, 768–779.
(12) Colotta, V.; Catarzi, D.; Varano, F.; Capelli, F.; Lenzi, O.;
Filacchioni, G.; Martini, C.; Trincavelli, L.; Ciampi, O.; Pugliese, A. M.;
Pedata, F.; Schiesaro, A.; Morizzo, E.; Moro, S. New 2-arylpyrazolo[3,4-
c]quinoline derivatives as potent and selective human A3 adenosine
receptor antagonists: Synthesis, pharmacological evaluation, and ligand-
receptor modeling studies. J. Med. Chem. 2007, 50, 4061–4074.
(13) Gessi, S.; Merighi, S.; Varani, K.; Leung, E.; Mac Lennan, S.;
Borea, P. A. The A3 adenosine receptor: An enigmatic player in cell
biology. Pharmacol. Ther. 2008, 117, 123–140.
The synthetic work was supported by a grant of the Italian
Ministry for University and Research, Rome, Italy (MIUR,
PRIN2007: protocol number 20073EWPF9_001). The molec-
ular modeling work coordinated by S.M. has been carried out
with financial support from the University of Padova, Italy, and
the MIUR (PRIN2008: protocol number 200834TC4L_002). S.
M. is also very grateful to Chemical Computing Group for the
scientific and technical partnership.
’ ABBREVIATIONS USED
AR, adenosine receptor; cAMP, cyclic adenosine monopho-
sphate;CGS21680, 2-[4-(2-carboxyethyl)phenethyl]amino-50-
(N-ethylcarboxamido)adenosine; CHA, N6-cyclohexyladenosine;
CHO, Chinese hamster ovary; DPCPX, 8-cyclopentyl-1,3-dipro-
pyl-xanthine; EL2, second extracellular loop; GPCRs, G protein-
coupled receptors; h, human; I-AB-MECA, N6-(4-amino-3-
iodobenzyl)-50-(N-methylcarboxamido)adenosine;MOE,
Molecular Operating Environment; NECA, 50-(N-ethylcarboxa-
mido)adenosine; rmsd, root-mean-square deviation; Ro20-1724,
4-(3-butoxy-4-methoxybenzyl)imidazolidin-2-one;SAR, struc-
ture-affinity relationship; TM, transmembrane; ZM-241385,
4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-
ylamino]ethyl)phenol
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