3-Aryl-[1,2,4]triazino[4,3-a ]benzimidazol-4(10 H)-one: A novel template for the design of highly selective A2B adenosine receptor antagonists

Article abstract of DOI:10.1021/jm201177b

In an effort to identify novel ligands possessing high affinity and selectivity for the A_2B AR subtype, we further investigated the class of 3-aryl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-ones V, previously disclosed by us as selective A₁ AR antagonists. Preliminary assays on a number of triazinobenzimidazoles derived from our "in-house" collection revealed that all the derivatives selected showed significant affinity at A _2B AR, no affinity at A₃ AR, and various degrees of selectivity toward A₁ and A_2A ARs. Investigation of a new series featuring modified substituents at the 10-position (4′-chlorophenyl or phenylethyl groups), and a chlorine atom at the 7-position (X) of the triazinobenzimidazole nucleus, yielded highly potent and selective A _2B AR antagonists. The presence of a pendant 3-phenyl ring appears to hamper the interaction with A_2A AR, conferring high A _2B/A_2A AR selectivity. Derivative 13 (X = Cl, R = C ₆H₅) is the most potent and selective compound, with an IC₅₀ of 3.10 nM at A_2B AR and no affinity at A ₁, A_2A, and A₃ ARs.

Full text of DOI:10.1021/jm201177b

Article
pubs.acs.org/jmc
3-Aryl-[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one: A Novel
Template for the Design of Highly Selective A2B Adenosine Receptor
Antagonists
Sabrina Taliani,*,† Isabella Pugliesi,† Elisabetta Barresi,† Francesca Simorini,† Silvia Salerno,†
Concettina La Motta,† Anna Maria Marini,† Barbara Cosimelli,‡ Sandro Cosconati,§ Salvatore Di Maro,‡
Luciana Marinelli,‡ Simona Daniele,∥ Maria Letizia Trincavelli,⊥ Giovanni Greco,‡ Ettore Novellino,‡
Claudia Martini,⊥ and Federico Da Settimo†
†Dipartimento di Scienze Farmaceutiche, Universita
̀
di Pisa, Via Bonanno 6, 56126 Pisa, Italy
‡Dipartimento di Chimica Farmaceutica e Tossicologica, Universita
Italy
̀
di Napoli “Federico II”, Via D. Montesano, 49, 80131 Napoli,
§Dipartimento di Scienze Ambientali, Seconda Universita
̀
di Napoli, Via Vivaldi 43, 81100 Caserta, Italy
∥Department of Drug Discovery and Development, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova, Italy
⊥Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Universita
̀
di Pisa, Via Bonanno 6, 56126 Pisa, Italy
S
* Supporting Information
ABSTRACT: In an effort to identify novel ligands possessing high
affinity and selectivity for the A2B AR subtype, we further investigated
the class of 3-aryl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-ones V,
previously disclosed by us as selective A1 AR antagonists. Preliminary
assays on a number of triazinobenzimidazoles derived from our “in-
house” collection revealed that all the derivatives selected showed
significant affinity at A2B AR, no affinity at A3 AR, and various degrees
of selectivity toward A1 and A2A ARs. Investigation of a new series
featuring modified substituents at the 10-position (4′-chlorophenyl or
phenylethyl groups), and a chlorine atom at the 7-position (X) of the
triazinobenzimidazole nucleus, yielded highly potent and selective A2B
AR antagonists. The presence of a pendant 3-phenyl ring appears to
hamper the interaction with A2A AR, conferring high A2B/A2A AR
selectivity. Derivative 13 (X = Cl, R = C6H5) is the most potent and selective compound, with an IC50 of 3.10 nM at A2B AR and
no affinity at A1, A2A, and A3 ARs.
affinity A1 and A2A ARs. However, adenosine normally increases
under conditions of hypoxia, ischemia, or high metabolism that
typically occur in pathological or stressful situations and can
grow to very high micromolar concentrations and thus activate
the low affinity A2B and A3 AR subtypes.
A2B AR activation implies stimulation of adenylate cyclase
and activation of phospholipase C through coupling to Gs and
Gq/11 proteins, respectively, producing an increase in intra-
cellular cAMP and calcium ion levels.7 A2B AR subtype is highly
conserved among species (i.e., human (h) A2B AR shares 86−
87% amino acid sequence homology with the rat and mouse
ARs)5 and is primarily expressed in the gastrointestinal tract,
bladder, lung, and on mast cells but also in the eye, adipose
tissues, brain, kidney, liver, and other tissues.3
INTRODUCTION
■
Adenosine is an endogenous purine nucleoside that plays a key
role in numerous important physiological functions through
interactions with specific cell-surface G-protein-coupled
receptors (GPCRs), which are classified into four subtypes,
namely A1, A2A, A2B, and A3 adenosine receptors (ARs).1−3
Responses to activation of ARs are mediated by different
secondary messenger systems, such as adenylate cyclase,
calcium or potassium channels (A1 AR), phospholipase C
(A1, A2B, and A3 ARs), and phospholipase D (A3 AR).4
A2B ARs have been generally defined as “low-affinity ARs”
due to their low affinity not only for the endogenous ligand but
also for several typical agonists, such as 5′-(N-
ethylcarboxamide)adenosine (NECA), N6-(R)-phenylisopropy-
ladenosine (R-PIA), and 2-{[4-(2-carboxyethyl)phenylethyl]-
amino}-5′-N-ethylcarboxamidoadenosine (CGS21680), in con-
trast with other AR subtypes.5,6 Under physiological conditions,
extracellular adenosine reaches a concentration of approx-
imately 100 nM and is thus able to interact only with the high-
Numerous studies have shown that A2B ARs play a crucial
role in the regulation of a wide range of physiopathological
Received: August 3, 2011
Published: January 18, 2012
© 2012 American Chemical Society
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Chart 1. Structure of Known and Newly Synthesized A2B AR Antagonists
Table 1. Affinities at Human A1, A2A, A2B, and A3 ARs of Derivatives 1−14
a,
b
a,
c
a,
d
a,e
no.
X
R
Ar
hA1 Ki (nM)
hA2A Ki (nM)
hA2B IC50 (nM)
hA3 Ki (nM)
f
f
f
1
2
3
4
5
6
7
8
9
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
H
C6H5
728 38
>10000
33.0 3.3
>10000
1.40 0.21
15.3 1.9
58.0 3.4
20.4 3.3
15.0 2.4
3200 120
2.42 0.16
2.11 0.04
54.3 6.9
10.0 1.35
2.50 0.77
5.34 0.81
3.10 0.10
7850 1570
>1000
>1000
>1000
>1000
>1000
>1000
>1000
702 69
>1000
>1000
328 30
>1000
>1000
>1000
>1000
H
fur-2-yl
C6H5
25.0 2.4
62.0 4.0
211 21
CH3
CH3
C6H5
C6H5
fur-2-yl
C6H5
2608 261
>10000
83.0
6
fur-2-yl
C6H5
>10000
1357 120
>10000
3277 154
>10000
C6H4-4-Cl
C6H4-4-Cl
CH2C6H5
CH2C6H5
(CH2)2C6H5
(CH2)2C6H5
C6H5
fur-2-yl
C6H5
50.6 5.1
>10000
f
299 16
>10000
10
11
12
13
14
fur-2-yl
C6H5
156 14
>10000
>10000
fur-2-yl
C6H5
>10000
15.8 1.7
>10000
>10000
C6H5
fur-2-yl
>10000
>10000
DPCPX
0.5 0.03
337 28
NECA
14
4
16
3
73
5
CI-IBMECA
890 61
401 25
0.22 0.02
a
The data are expressed as means
SEM derived from an iterative curve-fitting procedure (Prism program, GraphPad, San Diego, CA).
c
b
Displacement of specific [3H]DPCPX binding in membranes obtained from hA1 AR stably expressed in CHO cells. Displacement of specific
d
[3H]NECA binding in membranes obtained from hA2A AR stably expressed in CHO cells. Inhibition of 100 nM NECA-mediated cAMP
e
f
accumulation Displacement of specific [125I]AB-MECA binding in membranes obtained from hA3 AR stably expressed in CHO cells. Data for hA1,
hA2A, and hA3 taken from ref 29.
events. They are involved in the genesis of inflammatory
processes, mediating the release of several inflammatory
cytokines from mast cells, airway and bronchial epithelial
cells, fibroblasts, smooth cells, intestinal epithelial cells, and
monocytes.8,9 In addition, a role in angiogenesis induction,10,11
glucose metabolism,12 and growth and development of some
tumors13 has been suggested. In view of these findings, high-
affinity and selective A2B AR antagonists are needed as valuable
tools to fully establish their therapeutic potential for the
treatment of inflammatory diseases, such as asthma14−16 and
colitis,17−19 and angiogenic diseases such as diabetic retinop-
athy and cancer.20
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Nevertheless, A2B ARs are the most poorly characterized of
the four ARs subtypes from a pharmacological point of view
due to their general low affinity toward the prototypic standard
ligands that are commonly utilized to characterize ARs. In
particular, the scarce medicinal chemistry knowledge on the
structural requirements crucial for high A2B AR affinity and
selectivity raised wide-ranging difficulty in the study of this
receptor. However, a number of high-affinity A2B AR
antagonists containing different heterocyclic cores have been
described which showed various degree of binding selectivity
with respect to the other subtypes,21,22 including deazax-
anthines I,23 pyrrolopyrimidines II,24 pyrazolotriazolopyrimi-
dines III,25 and 2-aminopyrimidines IV26,27 (Chart 1).
Over the past decade, we have focused our attention on ARs,
disclosing several classes of selective A1 AR antagonists,
including the 3-aryl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-
ones,28,29 the N-alkyl and N-acyl-(7-substituted-2-
phenylimidazo[1,2-a][1,3,5]triazin-4-yl)amines,29,30 the 2-
(benzimidazol-2-yl)quinoxalines,31 and selective A3 AR antag-
onists, such as the 5-amino-2-phenyl[1,2,3]triazolo[1,2-a]-
[1,2,4]benzotriazines,32 the 4-amino-6-hydroxy-2-mercaptopyr-
imidines,33 and N2-substituted-pyrazolo[3,4-d]pyrimidines.34
In an effort to identify novel ligands possessing high affinity
and selectivity for the A2B AR subtype, we further investigated
the class of 3-aryl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-
ones V by virtue of their structural similarity with the
pyrazolotriazolopyrimidine class III. Both series possess a
central aromatic tricyclic nitrogen-containing core (6,5,6 for the
triazinobenzimidazoles V and 5,6,5 for the pyrazolotriazolo-
benzotriazines III) featuring mostly lipophilic substituents and
groups able to engage in hydrogen bonds with the receptor
protein.
Scheme 1. Synthesis of Triazinobenzimidazole Derivatives 7,
8, and 11−14
a
a
Reagents: (A) heating at 30−40 °C above the melting point; (B)
glacial acetic acid, reflux.
We preliminarily assayed a number of triazinobenzimidazole
derivatives derived from our “in-house” collection (compounds
1−6, 9, and 10) to define their binding profiles at human A1,
A2A, A2B, and A3 ARs (Table 1). All the derivatives, with the
exception of 6, showed high affinity at A2B AR (IC50 values in
the low nanomolar range) with various degrees of selectivity
toward A1 and A2A ARs. These data represented the rationale
for the investigation of a series of new derivatives from this class
(compounds 7, 8, and 11−14), featuring modified substituents
at the 10-position (4′-chlorophenyl or phenylethyl groups) and
a chlorine atom at the 7-position (X) of the triazinobenzimi-
dazole nucleus, to obtain potent and selective A2B AR
antagonists.
The present paper describes the synthesis, the biological
evaluation, and the molecular modeling studies of a number of
triazinobenzimidazole derivatives 1−14 as A2B AR antagonists.
Furthermore, the efficacy of the compounds 1, 6−8, 11, 13,
14 in an in vitro model of experimental colitis was evaluated by
assessing their ability to promote the survival of human
epithelial colorectal adenocarcinoma cells (Caco-2 cells).35
oxo-2-phenylacetic acid, 2-furan-2-yl-2-oxoacetic acid) were
allowed to react in refluxing ethanol. In agreement with our
previous findings,28,36 obtaining the target tricyclic compounds
7, 8, and 11−14 reasonably proceeded through the formation
of the intermediate 2-(benzimidazol-2-ylhydrazono)-2-substi-
tuted-acetic acids. In fact, in the reaction with the 2-furan-2-yl-
2-oxoacetic acid, the acid intermediates 21−23 were isolated
(Scheme 1) and purified by suspension in hot methanol (yield
65−80%). The best yields (62−67%) in the cyclization process
of acids 21−23 to the desired derivatives 8, 12, and 14 were
obtained using one of the following methods: (A) heating the
acid 21 at a temperature 30−40 °C above its melting point for
10 min or (B) refluxing the acids 22 and 23 for 1 h in glacial
acetic acid (Scheme 1). In the reaction of 2-hydrazinobenzi-
midazoles 18−20 with 2-oxo-2-phenylacetic acid in refluxing
ethanol, it was not possible to isolate the intermediate
hydrazono derivatives, but the target products 7, 11, and 13
were directly obtained (yield 60−65%, Scheme 1).
The 2-chloro-1-(4-chlorophenyl)-1H-benzimidazole 15 and
2,5-dichloro-1-phenyl-1H-benzimidazole 17 were easily pre-
pared in two steps starting from the commercially available N-
(4-chlorophenyl)-1,2-phenylendiamine 24 and the 4-chloro-N-
phenyl-1,2-phenylendiamine 25,37 respectively (Scheme 2).
These last two compounds were quantitatively cyclocondensed
through a carbonylation reaction that employed triphosgene
(bis-trichloromethyl carbonate)28 to yield the 1-aryl-1,3-
dihydro-2H-benzimidazol-2-ones 26 and 27 (yield 96−97%).
The corresponding 2-chloro derivatives 15 and 17 were
obtained by reaction of 26 and 27 with phenylphosphonic
dichloride (PhPOCl2) at 170 °C (yield 60−66%).28
CHEMISTRY
■
Compounds 1−6 and 9−10 were synthesized as previously
reported.28,36
The general synthetic pathway yielding the novel triazino-
benzimidazole derivatives 7, 8, and 11−14 is outlined in
Scheme 1, and it proceeded with the conversion of the 2-
chlorobenzimidazoles 15−17 into the corresponding 2-
hydrazones 18−20 by reaction with hydrazine hydrate at 180
°C in a Pyrex-capped tube (yield 92−93%). Subsequently,
derivatives 18−20 and the appropriate aryloxoacetic acid (2-
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reduced A2B/A2A selectivity. Reasonably, this small five-
membered furyl ring might be able to engage positive
interactions with an area in the A2A AR site that is not feasible
for the phenyl group. In contrast, the A2B AR antagonist
binding site could be characterized by lipophilic areas well
tolerating both the 2-furyl and phenyl rings.
Scheme 2. Synthesis of 2-Chlorobenzimidazole Derivatives
15 and 17
Stating these considerations, an in-depth analysis of the data
reported in Table 1 shows that the affinity and selectivity
profile for the ARs within the 3-phenyl and 3-(2-furyl)
subseries strictly depends on the substitution at N10.
The 3-(2-furyl)-N10-unsubstituted compound 2 shows
comparable affinity at A1, A2A, and A2B ARs, probably due to
the absence of groups able to discriminate between the different
binding sites. Insertion at N10 of the small lipophilic methyl (4),
as well as of the phenyl ring (6) or benzyl group (10), lowers
affinity at both A1 and A2A ARs, suggesting the disruption of an
H-bond involving the NH at position 10 or the absence of
efficacious lipophilic interactions between these substituents
and specific areas of the binding sites. This effect becomes even
more evident when a chlorine atom is placed at the 7-position
of 6, yielding derivative 14, completely inactive at A1 and A2A
ARs. Conversely, substitution at N10 with 4-chlorophenyl (8) or
phenylethyl (12) moieties determine a loss in A1 affinity, with a
substantial maintenance of the ability to bind A2A AR,
producing quite potent/nonselective A2A/A2B antagonists (8
Ki (hA2A) 50.6 nM, IC50 (hA2B) 2.11 nM; 12 Ki (hA2A) 15.8
nM, IC50 (hA2B) 5.34 nM).
Concerning the subset of 3-phenyl-substituted derivatives,
the lack of substitution at N10 in compound 1 afforded a very
good A2B/A1 selectivity, with 1 as an excellent performing
derivative in terms of A2B affinity and selectivity (Ki (hA1) 728
nM, Ki (hA2A) > 10000 nM, IC50 (hA2B) 1.40 nM, Ki (hA3) >
1000 nM). Insertion of a methyl, phenyl, or benzyl group (3, 5,
and 9, respectively) generally increases A1 AR affinity and
maintains A2B AR affinity in the nanomolar range, thus reducing
A2B/A1 selectivity. Insertion of a chlorine at the 4-position of
the N10-phenyl ring of 5 (7), as well as homologation of the
N10-benzyl group of 9 to a 2-phenylethyl chain (11), causes a
loss in A1 AR affinity while maintaining high A2B AR affinity (7
Ki (hA1) 1357 nM, IC50 (hA2B) 2.42 nM; 11 Ki (hA1) > 10000
nM, IC50 (hA2B) 2.50 nM). Taken together, these data suggest
that the lipophilic area hosting the N10-substituent in the A2B
AR is more tolerant with respect to the corresponding pocket
in the A1 AR binding site.
Finally, insertion of a chlorine at the 7-position of the
triazinobenzimidazole nucleus of 5 produces derivative 13,
which is the most potent and selective of all the compounds
investigated, with a IC50 of 3.10 nM at A2B AR and no activity at
A1, A2A, and A3 ARs. The same substitution pattern on the furyl
derivative 6 yields derivative 14, showing no activity at all AR
subtypes.
Compounds 1, 7, 8, 11, and 13 were selected and tested in
vitro for their ability to modulate the survival of Caco-2 cells.
A2B AR is the subtype predominantly expressed in these
cultured human epithelia cells widely used as an in vitro model
of experimental colitis. Cell treatment for 24 h with the
nonselective AR agonist, NECA (100 nM), induced a
significant decrease in cell viability with respect to untreated
control cells. To evaluate if NECA-induced toxicity could be
reversed by A2B AR antagonists, Caco-2 cells were incubated
with different concentrations of 1, 7, 8, 11, and 13 (ranging
from 0.1 to 50 nM), either in the absence or presence of 100
nM NECA for 24 h. The results show that the compounds
The preparation of the N1-phenylethyl compound 16 was
performed by alkylation of the commercially available 2-
chlorobenzimidazole with 2-bromoethylbenzene in the pres-
ence of sodium hydride, in accordance with a reported
procedure.38
BIOLOGY
■
The affinities of the new compounds toward human A1, A2A,
and A3 ARs were evaluated by competition experiments
assessing their respective abilities to displace [3H]8-cyclo-
pentyl-1,3-dipropylxanthine ([3H]DPCPX), [3H]5′-N-ethylcar-
boxamideadenosine ([3H]NECA), or [125I]4-aminobenzyl-5′-
N-methylcarboxamidoadenosine ([125I]AB-MECA) binding
from transfected CHO cells. The experiments were performed
as described elsewhere.29 Binding affinity at human A2B AR was
evaluated in functional assays by measuring the compound’s
effect on NECA-mediated cAMP accumulation in transfected
CHO cells. Finally, selected compounds (1, 6−8, 11, 13, and
14) were evaluated in vitro for their ability to modulate the
survival of Caco-2 cells by the 3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
(MTS) assay.
RESULTS AND DISCUSSION
■
The binding data of the triazinobenzimidazole derivatives 1−14
at the human A1, A2A, A2B, and A3 ARs are summarized in Table
1, together with those of DPCPX, NECA, and Cl-IBMECA
reported as reference standards.
From a general point of view, all the reported compounds,
with the exception of 6 and 14, showed affinity at the A2B AR
subtype in the nanomolar range (1.40−54.3 nM) with almost
complete selectivity toward the A3 AR and different degrees of
selectivity with regard to the A1 and A2A AR subtypes.
Interestingly, the affinity at the A2B AR is similar for all the
derivatives, independent of the nature of the substitution at the
3-, 7-, or 10-positions of the triazinobenzimidazole nucleus. On
the contrary, these substitutions seem to significantly modulate
the affinity toward the other receptor subtypes, with a
consequent influence on selectivity, in an interdependent and
not additive way.
In particular, for the major SAR, we can draw concerns on
the effect of the aryl group at the 3-position of the
triazinobenzimidazole nucleus on A2A AR binding ability. The
presence of a pendant 3-phenyl ring seems to be a crucial
requirement to gain A2B AR/A2A AR selectivity, as this phenyl
hampers the interaction with A2A AR; all the derivatives
featuring such a substitution were completely inactive at this
latter subtype. In fact, a number of compounds bearing a 2-furyl
ring at the 3-position show significant A2A AR affinity with Ki
values in the nanomolar range (2 Ki (hA2A) 33 nM, 8 Ki (hA2A)
50.6 nM, 10 Ki (hA2A) 156 nM, 12 Ki (hA2A) 15.8 nM) and
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Figure 1. Effect of A2B AR antagonists on Caco-2 cell survival. Cells were treated for 24 h with 100 nM NECA in the absence or in the presence of
different compound concentrations. Following incubation, cell survival was quantified by MTS assay as described in Experimental Section. Data are
expressed as percentage of control value (set to 100%) and represent the mean SEM of three different experiments. *** P < 0.001 vs control; # P
< 0.05, ## P < 0.01, ### P < 0.001 vs NECA alone.
Figure 2. Binding conformation of 1 (a,b) and 2 (c,d) in A2A AR (a,c) and A2B AR (b,d) as calculated by Glide. The receptor is represented as
orange (A2A AR) and green (A2B AR) sticks and ribbons. The ligand is represented as cyan sticks, while H-bonds are represented as blue dashed
lines. For sake of clarity, only interacting residues are shown.
alone were unable to induce any significant effect on cell
viability (data not shown). In the presence of NECA, all the A2B
AR antagonists significantly reversed NECA-induced cell death
at all tested concentrations (Figure 1); these effects were
detectable at nanomolar compound concentrations, comparable
to IC50 values of each compound toward A2B ARs. To confirm
that the observed cellular effects were derived from the selective
A2B AR antagonism, the same experiments were performed
using compounds 6 and 14 as negative controls. The data
depicted in Figure 1 show that these compounds were able to
revert NECA-induced cell death only at the highest
concentration used based on their low affinity for A2B AR
subtype. The results suggest that these A2B AR antagonists act
as pro-survival agents in protecting Caco-2 cells by death
induced by the AR agonist NECA and may provide the basis for
the development of potential adjuvant agents in the treatment
of colitis.
compound ZM241385.39 This study by Stevens and co-workers
demonstrated that the A2A AR structure shares an overall seven
transmembrane (TM) helix architecture similar to that of the
rhodopsin and adrenergic receptors, with small differences
residing in the positions and orientations of the helices.
Conversely, larger structural differences were recorded in the
extracellular loops and, most of all, in the ligand orientation.
Indeed, if compared with the β-adrenergic ligands and retinal,
ZM241385 in the A2A AR occupies a significantly different
position in the transmembrane (TM) bundle, being almost
parallel to the receptor axis. This orientation allows the bicyclic
triazolotriazine core of the antagonist to establish favorable
interactions with the F168, I274, N253, and E169 residues.
The above-described structure was used to obtain a
homology model of the hA2B ARs. The sequence alignment
between the receptors as well as the positions of the disulfide
bridges we obtained are consistent with what has already been
suggested by Moro and co-workers (see Figure S1 in
Supporting Information).40 Then, the structures of the
modeled receptors were used to dock the highly affine and
selective 3-phenyl derivative 1 and its fur-2-yl analogue 2, which
is instead unselective for the four ARs. These calculations were
obtained with the Glide program of the Schrodinger package,
MOLECULAR MODELING
■
To better understand the reasons behind the selective binding
of the newly discovered AR ligands, molecular modeling studies
were undertaken starting from the recently published 2.6 Å
crystal structure of A2A AR (PDB code 3EML) in complex with
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which has already been used to successfully dock a series of ARs
antagonists.41 Interestingly, in the inspected compounds, the
simple substitution of a furyl ring (2) with a phenyl one (1)
completely abolishes the affinity for the A2A AR while
maintaining low nanomolar affinities at the A2B AR. Thus, as
a first step, we decided to analyze the docking results obtained
in these two receptors. Unexpectedly, in the Glide results, the
same well-defined binding pose was found for the four
dockings. As shown in Figure 2, compounds 1 and 2 are
both capable of placing their triazinobenzimidazole ring on the
outer portion of the A2A and A2B ARs, surrounded by TMs III,
V, VI, VII helices, and ELII.
Such a position allows H-bond interactions with the N253
and N254 of the A2A and A2B ARs, respectively. These
interactions seem to be critical for antagonist affinity, as
underscored by X-ray crystallography39 and mutagenesis data
demonstrating that the sole substitution of the aforementioned
asparagine with an alanine results in the complete loss of
antagonist radioligands binding.42 Moreover, compounds 1 and
2, with their imidazole NH group, H-bond with E169(A2A) and
E174(A2B). These favorable contacts anchor the phenyl and
furyl rings of 1 and 2 in a hydrophobic cleft (herein referred as
L1) made up by V84, L85, M177, W246, P248, L249, and H250
in the A2AAR and by V85, L86, M182, W247, P249, V250, and
H251 in the A2BAR, respectively. Previous studies39,43,44 and
the very recent pioneering work by Stevens and co-workers45
have revealed several residues lining the L1 pocket to be
responsible for A2A AR activation. In particular, comparison
between the agonist- and antagonist-bound X-ray structures
have revealed that W246 and H250 respectively experience 1.9
and ∼1.8 Å movements to adapt to the bound ligand. These
modest local rearrangements trigger major movements in the
receptor helical bundle. Thus, the favorable van der Waals
contacts between the ligand furyl and phenyl rings and these
residues might prevent the structural rearrangements respon-
sible for the activation, constraining the receptors in an inactive
state. Additionally, in the four calculated complexes, the
triazinobenzimidazolone scaffold is stably anchored to the
receptor through a π-stacking interaction with F168(A2A) and
F173(A2B) of ELII.
In contrast to compounds 1 and 2, compounds 3−14 bear a
substituent at the imidazole nitrogen, and such a feature
prevents these ligands from adopting the same binding pose
observed for the unsubstituted analogues. In fact, docking of
compound 11 in A2B AR resulted in a new binding pose where
the phenyl ring is still placed in the L1 pocket, whereas the
triazinobenzimidazole scaffold H-bonds N254 through its
carbonyl oxygen in position 4 (Figure 3). This change in the
heterocyclic orientation prevents the steric clash with
E174(A2B) and orients the phenylethyl pendant chain toward
a rather lipophilic cleft, made up by I67, A64, F63, and V85, at
the crevice between TMII and TMIII. By featuring the
interaction with the critical N254 and L1 pocket, the different
binding pose would still allow 11 to be as equipotent and
selective as 1.
Figure 3. Binding conformation of 11 in the A2BAR as calculated by
Glide. The receptor is represented as green sticks and ribbons. The
ligand is represented as cyan sticks, while H-bonds are represented as
blue dashed lines. For sake of clarity, only interacting residues are
shown.
L1 pocket in the two receptors (A2A and A2B ARs) might deeply
influence the dynamical stabilization of the aforementioned
binding modes, thus explaining the recorded selectivity profiles.
Interestingly, the same conclusions have also been drawn by
other authors.46,47
CONCLUSIONS
■
A novel class of high affinity and selectivity A2B AR ligands
featuring the 3-aryl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-
one as the core scaffold has been developed. The majority of
the compounds show good to high A2B AR affinity and are
completely inactive and moderately active at A3 and A1 ARs,
respectively, whereas the A2B AR/A2A AR selectivity is strictly
dependent on the aryl group at the 3-position of the central
core. Specifically, as rationalized by molecular modeling studies,
the presence of a pendant 3-phenyl ring appears to hamper the
interaction with A2A AR, conferring high A2B/A2A AR selectivity.
Finally, selected compounds 1, 7, 8, 11, and 13, when tested
in vitro in a widely used model of experimental colitis,
significantly reversed NECA-induced cell death at nanomolar
compound concentrations relative to their A2B AR IC50 values,
acting as pro-survival agents and providing the basis for the
development of potential adjuvant agents in the treatment of
colitis.
EXPERIMENTAL SECTION
Chemistry. Melting points were determined using a Reichert
Kofler hot-stage apparatus and are uncorrected. Infrared spectra were
■
̈
recorded with a Nicolet/Avatar FT-IR spectrometer in Nujol mulls.
Routine nuclear magnetic resonance spectra were recorded in DMSO-
d6 solution on a Varian Gemini 200 spectrometer operating at 200
MHz. Evaporation was performed in vacuo (rotary evaporator).
Analytical TLC was carried out on Merck 0.2 mm precoated silica gel
aluminum sheets (60 F-254). Combustion analyses on target
compounds were performed by our Analytical Laboratory in Pisa.
All compounds showed ≥95% purity.
Unfortunately, the above-described theoretical results some-
how disagree with the observed experimental data suggesting a
high binding of 1 at the A2B AR, whereas no binding was
detected at the A2A AR. To overcome the intrinsic limitations of
docking algorithms (i.e., absence of receptor flexibility),
preliminary molecular dynamics simulations were performed
(see Supporting Information for details). These findings
allowed us to speculate that the residue composition of the
2-Chlorobenzimidazole, 2-phenylethyl bromide, N-(4-chlorophen-
yl)-1,2-phenylendiamine 24, triphosgene (bis-trichloromethyl carbo-
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nate), and phenylphosphonic dichloride (PhPOCl2) were from Sigma-
Aldrich. The following compounds were prepared in accordance with
reported procedures: 3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-4-
(10H)-one 1,36 3-(2-furyl)[1,2,4]triazino[4,3-a]benzimidazol-4-
(10H)-one 2,36 10-methyl-3-phenyl[1,2,4]triazino[4,3-a]-
benzimidazol-4(10H)-one 3,36 3-(2-furyl)-10-methyl[1,2,4]triazino-
[4,3-a]benzimidazol-4(10H)-one 4,36 3,10-diphenyl[1,2,4]tri-
azino[4,3-a]benzimidazol-4(10H)-one 5,28 3-(2-furyl)-10-phenyl-
[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one 6,28 10-benzyl-3-
phenyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one 9,28 10-benzyl-
3-(2-furyl)[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one 10,28 2-
chloro-1-(2-phenylethyl)benzimidazole 16,38,48 and 4-chloro-N-phe-
nyl-1,2-phenylendiamine 25.37
General Procedure for the Synthesis of 3-Phenyl-5-substituted-
10-(substituted-phenyl)[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-
ones 7, 11, and 13. A solution of the appropriate 2-hydrazinobenzi-
midazole 18−20 (4 mmol) and phenyloxoacetic acid (0.660 g, 4.4
mmol) in 10 mL of absolute ethanol was refluxed for 5 h (TLC
analysis). After cooling, the precipitate which formed was collected
and purified by crystallization from the appropriate solvent.
stirring and with exclusion of moisture. After cooling, the excess
reagent was decomposed by addition of ice and water; the obtained
aqueous suspension was neutralized with conc NH4OH, after which
the desired products 15 and 17 were isolated directly in a pure state by
filtration.
2-Chloro-1-(4-chlorophenyl)-1H-benzimidazole 15. Yield 66%;
mp: 128−130 °C (lit.49 mp = 132−133 °C). IR (nujol, cm−1):
1
1590, 1500, 1030, 700. H NMR (DMSO-d6, ppm): 7.19−7.22 (m,
1H), 7.29−7.34 (m, 2H), 7.64−7.76 (m, 5H). Anal. Calcd for C, H, N.
2,5-Dichloro-1-phenyl-1H-benzimidazole 17. Yield 60%; mp:
105−107 °C. IR (nujol, cm−1): 1590, 1500, 1050, 760. 1H NMR
(DMSO-d6, ppm): 7.15−7.40 (AB part of ABX system, 2H), 7.63−
7.66 (m, 5H), 7.81−7.83 (X part of ABX system, 1H). Anal. Calcd for
C, H, N.
General Procedure for the Synthesis of 1-Substituted-1,3-
dihydro-2H-benzimidazol-2-one hydrazones 18−20. The appropri-
ate substituted-2-chlorobenzimidazole 15−17 (2.7 mmol) was heated
at 180 °C in a Pyrex capped tube with 1.0 mL of hydrazine
monohydrate for 5 h. After the mixture was cooled, a white solid
separated, which was collected and resulted sufficiently pure to be used
in the next reaction without further purification.
10-(4-Chlorophenyl)-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-
4(10H)-one 7. Yield 65%; mp = 225−227 °C (EtOH). IR (nujol,
1-(4-Chlorophenyl)-1,3-dihydro-2H-benzimidazol-2-one hydra-
1
cm−1): 1680, 1600, 1300, 1150, 760. H NMR (DMSO-d6, ppm):
zone 18. Yield 92%; mp = 115−117 °C. IR (nujol, cm−1): 3250−
1
7.43−7.83 (m, 10H), 8.19−8.24 (AA′ part of AA′BB′C system, 2H),
8.61 (pd, 1H). Anal. Calcd for C, H, N.
2700, 1650, 1610, 1090, 830. H NMR (DMSO-d6, ppm): 6.94−7.08
(m, 3H), 7.33−7.68 (m, 5H). Anal. Calcd for C, H, N.
3-Phenyl-10-(2-phenylethyl)[1,2,4]triazino[4,3-a]benzimidazol-4-
(10H)-one 11. Yield 60%; mp = 228−230 °C (DMF). IR (nujol,
cm−1): 1680, 1570, 1460, 1150, 750. 1H NMR (DMSO-d6, ppm): 3.20
(t, 2H, J = 7.3 Hz), 4.65 (t, 2H, J = 7.3 Hz), 7.16−7.33 (m, 5H), 7.38−
7.68 (m, 6H), 8.18−8.24 (AA′ part of AA′BB′C system, 2H), 8.48 (pd,
1H). Anal. Calcd for C, H, N.
7-Chloro-3,10-diphenyl[1,2,4]triazino[4,3-a]benzimidazol-4-
(10H)-one 13. Yield 65%; mp = 248−250 °C (EtOH). IR (nujol,
cm−1): 1680, 1590, 1550, 1280, 1170, 770. 1H NMR (DMSO-d6,
ppm): 7.45−7.53 (m, 4H), 7.64−7.80 (m, 6H), 8.12−8.22 (AA′ part
of AA′BB′C system, 2H), 8.57 ppm (ps, 1H). Anal. Calcd for C, H, N.
General Procedure for the Synthesis of 3-(2-Furyl)-10-(substi-
tuted-phenyl)[1,2,4]triazino[4,3-a]benzimidazol-4(10H)ones 8, 12,
and 14. The appropriate (1,5-disubstituted-benzimidazol-2-
ylhydrazono)substituted-acetic acids 21−23 were cyclized by means
of the following methods.
Method A. The acid derivative 21 (2 mmol) was heated at a
temperature 30−40 °C above its melting point for 10 min, so that
thermal cyclization occurred. After cooling, product 8 was purified by
recrystallization from EtOH.
Method B. A suspension of the acid derivatives 22, 23 (2 mmol) in
20 mL of glacial acetic acid was refluxed for 1 h. The solution obtained
was evaporated to dryness, and the oily residue was purified by
recrystallization from EtOH.
1-(2-Phenylethyl)-1,3-dihydro-2H-benzimidazol-2-one hydra-
zone 19. Yield 92%; mp = 99−101 °C. IR (nujol, cm−1): 3500−
1
2650, 1620, 1600, 1560, 1140, 740. H NMR (DMSO-d6, ppm): 3.12
(t, 2H, J = 7.3 Hz), 4.50 (t, 2H, J = 7.3 Hz), 7.03−7.46 (m, 7H), 7.60−
7.64 (m, 2H). Anal. Calcd for C, H, N.
5-Chloro-1-phenyl-1,3-dihydro-2H-benzimidazol-2-one hydra-
zone 20. Yield 93%; mp = 109−111 °C. IR (nujol, cm−1): 3340−
1
2875, 1620, 1300, 1160, 790. H NMR (DMSO-d6, ppm): 6.87−7.12
(m, 3H), 7.35−7.68 (m, 5H). Anal. Calcd for C, H, N.
General Procedure for the Synthesis of (1,5-Disubstituted-
benzimidazol-2-ylhydrazono)-(2-furyl)acetic Acids 21−23. A sol-
ution of the appropriate benzimidazol-2-one hydrazones 18−20 (4
mmol) and the 2-furyloxoacetic acid (0.616 g, 4.4 mmol) in 10 mL of
absolute ethanol was refluxed for 2 h. After cooling, the precipitate
which formed was collected to give the substituted (benzimidazol-2-
ylhydrazono)acetic acids 21−23 which were purified by suspension in
hot methanol.
{[1-(4-Chlorophenyl)-1,3-dihydro-2H-benzimidazol-2-ylidene]-
hydrazono}-(2-furyl)acetic Acid 21. Yield 76%; mp = 128−131 °C. IR
(nujol, cm−1): 3500−2800, 1600, 1380, 1020, 720. 1H NMR (DMSO-
d6, ppm): 6.61 (dd, 1H, J = 3.4, J = 1.7 Hz), 7.09−7.18 (m, 4H), 7.23−
7.46 (m, 1H), 7.73−7.79 (m, 5H), 12.60 (bs, 1H). Anal. Calcd for C,
H, N.
2-Furyl-{[1-(2-phenylethyl)-1,3-dihydro-2H-benzimidazol-2-
ylidene]hydrazono}acetic Acid 22. Yield 65%; mp = 202−204 °C. IR
10-(4-Chlorophenyl)-3-(2-furyl)[1,2,4]triazino[4,3-a]-
1
(nujol, cm−1): 3200−2800, 1650, 1620, 1280, 1140, 740. H NMR
benzimidazol-4(10H)-one 8. Method A. Yield 67%; mp = 233−235
1
°C. IR (nujol, cm−1): 1685, 1600, 1560, 1260, 1100, 760. H NMR
(DMSO-d6, ppm): 3.06 (t, 2H, J = 7.6 Hz), 4.29 (t, 2H, J = 7.6 Hz),
6.61 (dd, 1H, J = 3.3, J = 1.8 Hz), 7.16−7.49 (m, 10H), 7.77−7.79 (m,
1H), 12.34 (bs, 1H). Anal. Calcd for C, H, N.
(DMSO-d6, ppm): 6.72 (dd, 1H, J = 3.4, J = 1.8 Hz), 7.47−7.68 (m,
5H), 7.81−7.85 (m, 3H), 7.92 (dd, 1H, J = 1.8, J = 0.9 Hz), 8.59 (pd,
1H). Anal. Calcd for C, H, N.
3-(2-Furyl)-10-(2-phenylethyl)[1,2,4]triazino[4,3-a]benzimidazol-
4(10H)-one 12. Method B. Yield 62%; mp = 190−192 °C. IR (nujol,
cm−1): 1675, 1560, 1480, 1150, 750. 1H NMR (DMSO-d6, ppm): 3.19
(t, 2H, J = 7.3 Hz), 4.65 (t, 2H, J = 7.3 Hz), 6.69−6.72 (m, 1H), 7.14−
7.30 (m, 5H), 7.37−7.45 (m, 2H), 7.53−7.67 (m, 2H), 7.90−7.91 (m,
1H), 8.47 (pd, 1H). Anal. Calcd for C, H, N.
7-Chloro-3-(2-furyl)-10-phenyl[1,2,4]triazino[4,3-a]benzimidazol-
4(10H)-one 14. Method B. Yield 66%; mp = 252−254 °C. IR (nujol,
cm−1): 1680, 1570, 1520, 1280, 1190, 770. 1H NMR (DMSO-d6,
ppm): 6.76 (dd, 1H, J = 3.5, J = 1.8 Hz), 7.44−7.50 (m, 3H), 7.60−
7.68 (m, 5H), 7.97−7.98 (m, 1H), 8.57 (ps, 1H). Anal. Calcd for C, H,
N.
General Procedure for the Synthesis of 2-Chloro-5-substituted-1-
(4-substituted-phenyl)benzimidazole 15 and 17. A mixture of the
appropriate benzimidazole derivative 26 and 27 (6.0 mmol) and 14.0
mL of PhPOCl2 was heated at 170 °C for 19 h (TLC analysis), under
(5-Chloro-1-phenyl-1,3-dihydro-2H-benzimidazol-2-
ylidenehydrazono)(2-furyl)acetic Acid 23. Yield 80%; mp = 186−189
1
°C. IR (nujol, cm−1): 3500−2500, 1660, 1590, 1305, 1120, 790. H
NMR (DMSO-d6, ppm): 6.60−6.62 (m, 1H), 7.06−7.19 (m, 3H),
7.33−7.41 (m, 1H), 7.57−7.81 (m, 5H), 12.27 (bs, 1H). Anal. Calcd
for C, H, N.
General Procedure for the Synthesis of 5-Substituted-1-(4-
substituted-phenyl)benzimidazol-2-ones 26 and 27. Triphosgene
(bis-trichloromethylcarbonate) (1.48 g, 5.0 mmol) was added
portionwise, under a nitrogen atmosphere, to an ice-cooled solution
of the appropriate 1,2-phenylendiamine 24, 25 (5.0 mmol) in 47 mL
of anhydrous THF. Then a solution of triethylamine (1.7 mL, 12
mmol) in 13 mL of the same solvent was added dropwise, and the
reaction mixture was maintained under stirring at 0 °C for 1 h and at
room temperature until the disappearance of the starting material (24
h, TLC analysis). The suspension obtained was filtered and the solvent
evaporated at reduced pressure, yielding the crude benzimidazolone
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derivative 26 and 27, which was purified by crystallization from
toluene.
1-(4-Chlorophenyl)-1,3-dihydro-2H-benzimidazol-2-one 26. Yield
98%; mp: 211−212 °C (lit.50 mp = 206−208 °C). Anal. Calcd for C,
H, N.
5-Chloro-1-phenyl-1,3-dihydro-2H-benzimidazol-2-one 27. Yield
97%; mp: 228−230 °C (lit.51 mp = 222−223 °C). Anal. Calcd for C,
H, N.
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.
Effect of A2B AR Antagonists on Caco-2 Cell Survival. Caco-2 cells
were maintained in DMEM medium supplemented with 10% fetal
bovine serum, 2 mM L-glutamine, and penicillin/streptomycin in
humidified atmosphere (5% CO2) at 37 °C. Cells were seeded in 96-
well microplates (5000 cells/well); the day after, cells were incubated
with 100 nM NECA, in the absence or in the presence of A2B AR
antagonists, for 24 h. The A2B AR antagonists were added for 5 min
prior to addition of NECA in order to determine the inhibition of
NECA-mediated cell toxicity. Following incubation times, cell viability
was determined using the MTS assay according to manufacturer’s
instruction. The dehydrogenase activity in active mitochondria reduces
MTS to the soluble formazan product. The absorbance of formazan at
490 nM was measured in a colorimetric assay with an automated plate
reader.
Data Analysis. Within an experiment, each condition was assayed
in duplicate or triplicate, and each experiment was performed at least
three times. The results were calculated by subtracting the mean
background from the values obtained from each test condition and
were expressed as the percentage of the control (untreated cells).
Student’s t-test was used to evaluate whether differences between the
experimental groups and the control were statistically significant.
Computational Methods. Homology Modeling. hA2A AR
structure was downloaded from the Protein Data Bank (PDB
code 3EML), and hydrogens were added to the protein and
ligand (ZM 241385) using the MOLPROBITY server.55 Then,
Biology. Adenosine Receptor Binding Assay. Materials.
[3H]DPCPX, [3H]NECA, and [125I]AB-MECA were obtained
from DuPont-NEN (Boston, MA). ADA was from Sigma
Chemical Co. (St. Louis, MO). All other reagents were from
standard commercial sources and of the highest commercially
available grade. CHO cells stably expressing human A1, A2A, and
A3 ARs were kindly supplied by Prof. K. N. Klotz, Wurzburg
University, Germany.52
Human A1 Adenosine Receptors. Aliquots of membranes (50 μg
proteins) obtained from A1 CHO cells were incubated at 25 °C for
180 min in 500 μL of T1 buffer (50 mM Tris-HCl, 2 mM MgCl2, 2
units/mL ADA, pH 7.4) containing [3H]DPCPX (3 nM) and six
different concentrations of the newly synthesized compounds.
Nonspecific binding was determined in the presence of 50 μM R-
PIA.29 The dissociation constant (Kd) of [3H]DPCPX in A1 CHO cell
membranes was 3 nM.
Human A2A Adenosine Receptors. Aliquots of cell membranes (80
μg) were incubated at 25 °C for 90 min in 500 μL of T2 buffer (50
mM Tris-HCl, 2 mM MgCl2, 2 units/mL ADA, pH 7.4) in the
presence of 30 nM of [3H]NECA and six different concentrations of
the newly synthesized compounds. Nonspecific binding was
determined in the presence of 100 μM NECA.29 The dissociation
constant (Kd) of [3H]NECA in A2A CHO cell membranes was 30 nM.
Human A3 Adenosine Receptors. Aliquots of cell membranes (40
μg) were incubated at 25 °C for 90 min in 100 μL of T3 buffer (50
mM Tris-HCl, 10 mM MgCl2, 1 mM EDTA, 2 units/mL ADA, pH
7.4) in the presence of 1.4 nM [125I]ABMECA and six different
concentrations of the newly synthesized compounds. Nonspecific
binding was determined in the presence of 50 μM R-PIA.29 The
dissociation constant (Kd) of [125I]AB-MECA in A3 CHO cell
membranes was 1.4 nM.
All compounds were routinely dissolved in DMSO and diluted with
assay buffer to the final concentration, where the amount of DMSO
never exceeded 2%. Percentage inhibition values of specific radio-
labeled ligand binding at 1−10 μM concentration are means SEM of
at least three determinations. For compound IC50 determination, at
least six different ligand concentrations were used. IC50 values,
computer-generated using a nonlinear regression formula on a
computer program (Graph-Pad, San Diego, CA), were converted to
Ki values, based on the Kd values of radioligands in the different tissues
and using the Cheng and Prusoff equation.53 Ki values are means
SEM of at least three determinations.
Measurement of Cyclic AMP Levels on hA2B CHO Cells.
Intracellular cyclic AMP (cAMP) levels were measured using a
competitive protein binding method.54 CHO cells, expressing
recombinant hA2B ARs, were harvested by trypsinization. After
centrifugation and resuspension in medium, cells (∼48000) were
plated in 24-well plates in 0.5 mL of medium. After 48 h, the medium
was removed, and the cells were incubated at 37 °C for 15 min with
0.5 mL of Dulbecco’s Modified Eagle Medium (DMEM) in the
presence of adenosine deaminase (1U/mL) and the phosphodiesterase
inhibitor Ro20-1724 (20 μM). The antagonism profile of the new
compounds toward A2B AR was evaluated by assessing their ability to
inhibit 100 nM NECA-mediated accumulation of cAMP. Cells were
incubated in the reaction medium (15 min at 37 °C) with different
concentrations of the target compound (1 nM to 10 μM) and then
were treated with NECA. The reaction was terminated by the removal
of the medium and the addition of 0.4 N HCl. After 30 min, lysates
were neutralized with 4 N KOH, and the suspension was centrifuged
at 800g for 5 min. For the determination of cAMP production, bovine
adrenal cAMP binding protein was incubated with [3H]cAMP (2 nM)
and 50 μL of cell lysate or cAMP standard (0−16 pmol) at 0 °C for
the homology model of the hA AR was built using Schrodinger
̈
2B
Prime56 software accessible through the Maestro interface.57
Unless otherwise stated, default parameters were used
throughout. The sequences of the hA1 (P30542), hA2B
(P29275), and hA3 (P33765) ARs were aligned against the
sequence of hA2A AR (P29274). Alignment of anchor residues
within each TM domain were attained according to the
sequence alignment suggested by Moro and co-workers (see
Figure S1 in Supporting Information).40
During the homology model building, Prime keeps the backbone
rigid for the cases in which the backbone does not need to be
reconstructed due to gaps in the alignment. The loop refinements
were carried out using Prime with default parameter settings, if not
mentioned otherwise. Prior to refinement, the protein structures were
subjected to a protein preparation step to reorientate side chain
hydroxyl groups and alleviate potential steric clashes. The
implemented loop modeling protocol consists of several steps.58
First, large numbers of loops are created by a conformational search in
dihedral angle space. Clustering of loop conformations and side chain
optimization is being used to select representative solutions. On the
basis of the user parameters, a limited number of structures is then
processed using complete energy minimization. The top ranked
solution in terms of Prime energy was considered best. The short
loops (ELI and ELIII) were refined using default sampling rates in the
initial step, while the extended, highest sampling rate was chosen for
the larger amino acid loops (ELII). Side chains were unfrozen within
7.5 Å of the corresponding loop, and the energy cutoff was set to 10
kcal.
Molecular Docking. The three-dimensional (3D) structures of
compounds 1, 2, and 11 were drawn using the Builder tool and
generated with Ligprep module. The Glide program of the
Schrodinger package was used to dock these compounds 1, 2, and
11 to the hA2A and hA2B ARs structures. The receptor grid generation
were performed for the box with a center in the putative binding site.
The size of the box was determined automatically. The extra precision
mode (XP) of Glide was used for the docking. The ligand scaling
factor was set to 1.0. The geometry of the ligand binding site of the
complex between the selected ligands and the four receptors was then
optimized. The binding site was defined as the ligand and all amino
acid residues located within 8 Å from the ligand. All the receptor
residues located within 2 Å from the binding site were used as a shell.
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The following parameters of energy minimization were used:
OPLS2005 force field; water was used as an implicit solvent; a
maximum of 5000 iterations of the Polak−Ribier conjugate gradient
minimization method was used with a convergence threshold of 0.01
kJ 3 mol−1 Å−1.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Additional molecular modeling details and figures, analytical
data of compounds 7, 8, 11−15, 17−23, 26, and 27. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*Phone: (+39)0502219547. Fax: (+39)0502219605. E-mail:
taliani@farm.unipi.it.
ACKNOWLEDGMENTS
■
This work was financially supported by MIUR (PRIN 2008).
We thank Prof. Karl-Norbert Klotz for his generous gift of
transfected CHO cells expressing the human A1, A2A, and A3
receptors.
ABBREVIATIONS USED
■
AR, adenosine receptor; AB-MECA, 4-aminobenzyl-5′-N-
methyl-carboxamidoadenosine; CNS, central nervous system;
DMEM, Dulbecco’s Modified Eagle Medium; DPCPX, 8-
cyclopentyl-1,3-dipropylxanthine; GPCRs, G-protein coupled
receptors; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxyme-
thoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; NECA, 5′-(N-
ethylcarboxamide)adenosine; R-PIA, N6-(R)-phenylisopropyla-
denosine; SEM, standard error of mean
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