Probing distal regions of the A2B adenosine receptor by quantitative structure-activity relationship modeling of known and novel agonists

Article abstract of DOI:10.1021/jm701442d

The binding modes at the A_2B adenosine receptor (AR) of 72 derivatives of adenosine and its 5′-N-methyluronamide with diverse substitutions at the 2 and N⁶ positions were studied using a molecular modeling approach. The compounds in their receptor-docked conformations were used to build CoMFA and CoMSIA quantitative structure-activity relationship models. Various parameters, including different types of atomic charges, were examined. The best statistical parameters were obtained with a joint CoMFA and CoMSIA model: R² = 0.960, Q ² = 0,676, SEE = 0.175, F = 158, and R²_test = 0.782 for an independent test set containing 18 compounds. On the basis of the modeling results, four novel adenosine analogues, having elongated or bulky substitutions at N⁶ position and/or 2 position, were synthesized and evaluated biologically. All of the proposed compounds were potent, full agonists at the A_2B AR in adenylate cyclase studies. Thus, in support of the modeling, bulky substitutions at both positions did not prevent A_2B AR activation, which predicts separate regions for docking of these moieties.

Full text of DOI:10.1021/jm701442d

2088
J. Med. Chem. 2008, 51, 2088–2099
Probing Distal Regions of the A2B Adenosine Receptor by Quantitative Structure-Activity
Relationship Modeling of Known and Novel Agonists
Andrei A. Ivanov, Ben Wang, Athena M. Klutz, Vincent L. Chen, Zhan-Guo Gao, and Kenneth A. Jacobson*
Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and DigestiVe and Kidney Diseases,
National Institutes of Health, Bethesda, Maryland 20892
ReceiVed NoVember 15, 2007
The binding modes at the A2B adenosine receptor (AR) of 72 derivatives of adenosine and its
5′-N-methyluronamide with diverse substitutions at the 2 and N6 positions were studied using a molecular
modeling approach. The compounds in their receptor-docked conformations were used to build CoMFA
and CoMSIA quantitative structure–activity relationship models. Various parameters, including different
types of atomic charges, were examined. The best statistical parameters were obtained with a joint CoMFA
and CoMSIA model: R2 ) 0.960, Q2 ) 0.676, SEE ) 0.175, F ) 158, and R2test ) 0.782 for an independent
test set containing 18 compounds. On the basis of the modeling results, four novel adenosine analogues,
having elongated or bulky substitutions at N6 position and/or 2 position, were synthesized and evaluated
biologically. All of the proposed compounds were potent, full agonists at the A2B AR in adenylate cyclase
studies. Thus, in support of the modeling, bulky substitutions at both positions did not prevent A2B AR
activation, which predicts separate regions for docking of these moieties.
Introduction
properties of known ligands and in virtual screening.17,18 QSAR
models are useful to explore the contribution of specific
functional groups and moieties on the ligand to the overall
biological activity. The comparative molecular field analysis
(CoMFA) and comparative molecular similarity indices analysis
(CoMSIA) are two of the most effective and descriptive methods
of QSAR-modeling.19 CoMFA/CoMSIA in conjunction with
receptor docking has been used to study the binding of agonists20
and antagonists21,22 at the human A3 AR. CoMFA has also been
used to study ligand recognition at other ARs.23,24
Recently, novel adenosine derivatives substituted at the N6
and/or 5′ positions or at the C2 position were reported to be
potent and/or selective A2B AR agonists (Chart 1).16,25,26 In the
present study we have synthesized new analogues in which
several of these affinity- (or selectivity-) enhancing substituents
have been recombined in order to probe the compatibility of
multiple extended groups in receptor docking and activation.
Previous studies have reported that certain combinations of
otherwise affinity-enhancing substitutions at the N6 and C2
positions were incompatible in binding to a given AR
subtype.16,27,28 Here, the molecular docking at the A2B AR of
both previously reported and novel derivatives of adenosine and
the potent, nonselective agonist 5′-N-ethylcarboxamidoadenosine
(NECA, 1) combined with the CoMFA and CoMSIA modeling
was used to characterize the distal regions of the receptor
involved in ligand recognition.
The A2B adenosine receptor (AR)a is one of the four known
subtypes of ARs: A1, A2A, A2B, and A3. These receptors are
members of the rhodopsin family of G-protein-coupled receptors
(GPCRs).1–3 A2B AR agonists are of interest in the treatment
of diseases such as cardiac ischemia and diabetes.4,5
Like other members of the class A GPCR family, ARs are
transmembrane proteins containing the typical seven transmem-
brane R-helical domains (TM I–VII) as well as extracellular
(EL) and intracellular (IL) loops. X-ray crystallographic data
are available for only a few GPCRs, i.e., rhodopsin and the
ꢀ2-adrenergic receptor; the experimental three-dimensional
structures of the ARs are unknown. Therefore, the homology
modeling approach is commonly used to study the structure and
ligand–receptor interactions of various GPCRs and other
proteins.6–9 Several rhodopsin-based molecular models of the
A1, A2A, A2B, and A3 ARs have been proposed.10–15 The SAR
(structure–activity relationship) of adenosine derivatives acting
as A2B AR agonists has been studied qualitatively (representative
reference structures in Chart 1). Recently we have published a
model of the A2B AR complex with its potent agonist MRS3997
4.16
Since molecular models can provide information about
ligand–receptor interactions, quantitative SAR (QSAR) statistical
models are widely used to evaluate the activity or other
* To whom correspondence should be addressed. Phone: (301) 496-9024.
Fax: (301) 480-8422. E-mail: kajacobs@helix.nih.gov.
Results and Discussion
a AR, adenosine receptor; CoMFA, comparative molecular field analysis;
CoMSIA, comparative molecular similarity indices analysis; CPA, N6-
cyclopentyladenosine; EL, extracellular loop; GPCR, G-protein-coupled
receptor; HENECA, 2-(1-(E)-hexenyl)adenosine-5′-N-ethyluronamide;
MCMM, Monte Carlo multiple minimum; NECA, 5′-N-ethylcarboxami-
doadenosine; (S)-PHPAdo, (S)-2-[(3-hydroxy-3-phenyl)propyn-1-yl]adenos-
ine; (S)-PHPNECA, (S)-2-[(3-hydroxy-3-phenyl)propyn-1-yl]-5′-N-ethyl-
carboxamidoadenosine; PLS, partial least squares; HBD, hydrogen bond
donor; HBA hydrogen bond acceptor; RP, relative potency; DS, data set;
QSAR; quantitative structure–activity relationship; TM, transmembrane
helical domain; PyBop, benzotriazol-1-yloxytripyrrolidinophosphonium
hexafluorophosphate; DIPEA, diisopropylethylamine; DMF, N,N-dimeth-
ylformamide; HRMS, high-resolution mass spectrometry; TEA, triethyl-
amine; TLC, thin layer chromatography.
A2B Agonists Binding Mode. For a long time within the
superfamily of GPCRs, an X-ray structure was available for
rhodopsin only.29,30 Very recently Kobilka and co-workers
reported the first X-ray structure of the ꢀ2-adrenergic receptor.53
However, the structure of other GPCRs and their ligand–receptor
complexes can be studied with the rhodopsin-based homology
modeling approach.31,32 In this paper, our previously published
molecular model of the human A2B AR15,16 was used to study
the binding modes of various well-known and recently proposed
agonists of the A2B AR. The A2B AR model was built in
10.1021/jm701442d This article not subject to U.S. Copyright. Published 2008 by the American Chemical Society
Published on Web 03/06/2008

Distal Regions of A2B Adenosine Receptor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2089
Chart 1. Structures of Known AR Agonists That Were Used as Reference Compounds at the A2B ARa
a RP: NECA related potency. RP ) pEC50(comp)i – pEC50(NECA)i, where i represents the same reference publication.
Figure 1. (a) Overlay of all docked ligands at the A2B AR. (b) Structural alignment of the ligands extracted from the ligand–receptor models. The
structure of the A2B AR is colored by residue position: N-terminus and TM1 in orange, TM2 in ochre, TM3 in yellow, TM4 in green, TM5 in cyan,
TM6 in blue, TM7 and C-terminus in purple.
homology with the X-ray structure of bovine rhodopsin and
contains all seven transmembrane R-helices, as well as the
intracellular and the extracellular hydrophilic loops.
adenosine and 1) were studied with the Monte Carlo multiple
minimum (MCMM) conformational search analysis applied to the
ligand docked to the binding site of the receptor. The structures of
all ligands used in the study with their experimental and predicted
activities are given in the Supporting Information. MacroModel
software was used for automated docking.33
Adenine and Ribose Moieties in the Putative Receptor
Binding Site. Prior to the building of the CoMFA and CoMSIA
quantitative structure–activity relationship models, the receptor-
docked conformations of the nucleoside ligands were calculated.
Unfortunately, the X-ray structure of rhodopsin that serves as
the template for our GPCR models was obtained for its ground
state only. For this reason, there is an opinion that rhodopsin-
based homology modeling of GPCRs is more applicable for
studying antagonist than agonist binding modes. On the other hand
successful docking studies of agonists were reported for various
rhodopsin-based models of GPCRs. It was demonstrated that
differences in the binding site configuration in the active and
inactive states of the receptor can be taken into account using
protein-flexible docking or conformational search analysis.6,12,14
In this study the putative binding modes of various agonists (1-6,
11-76) of the A2B AR (2- and N6-substituted analogues of
The results obtained indicated that the adenosine moiety of
all studied ligands had a similar position and orientation inside
the putative binding site. In particular, the N6-amino group of
the ligands was oriented toward TM V, and position 2 of the
adenine ring was oriented toward EL2. The 5′ position of the
ribose ring and the N6 substituent, when present, were oriented
toward the intracellular part of the receptor (Figure 1).
A schematic representation of the ligand binding mode at
the putative nucleoside binding site of the human A2B AR is
shown in Figure 2. In agreement with the published data of
molecular modeling and site-directed mutagenesis of the AR
family,12,14–16,34 the 2′- and 3′-hydroxyl groups of the docked
nucleosides could form H-bonds with the conserved His2807.43
.

2090 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
IVanoV et al.
Figure 2. Schematic representation of the ligand binding mode obtained after the docking studies. The binding mode of the 5′-OH derivative 9 is
shown as an example. The green arrows correspond to the putative H-bonds. The critical residues involved in interactions with a ligand are colored
in blue. For 5′-uronamide derivatives, the carbonyl oxygen atom was H-bonded to Ser2797.42 (not shown).
In addition, Thr893.36 was involved in H-bonding with the
oxygen atom of the ribose ring and the 5′-hydroxyl group of
adenosine analogues. The 5′-NH-group of 5′-N-alkylcarboxa-
midoadenosine derivatives also formed an H-bond with
Thr893.36, while the oxygen atom of the 5′-amido group was
H-bonded to Ser2797.42. The side chains of Asn1865.42 and
Asn2546.55 were observed in proximity to the adenine ring of
the ligands. Also, Trp2476.48 and His2516.52 were located near
the N6 and 7 positions of the adenine ring, in agreement with
previous studies.12 On the basis of the data of site-directed
mutagenesis of other ARs, all of these conserved residues were
reported to be critical for ligand binding.34
its ether oxygen atom was involved in H-bonding. However,
the backbone oxygen atoms of Cys1905.46 and Val1915.47 were
located in proximity to these groups. The terminal aromatic
ring of the N6 substituent was favorably located in the pocket
formed by several hydrophobic residues including Pro1945.50
,
Leu1955.51, Met1985.54, Phe2436.44, and Ala2446.45
.
On the basis ofthe models obtained, the arylcarbonyl oxygen
atom of N6-(arylcarbonyl)hydrazino derivatives, such as 5, can
form a H-bond with His2516.52. In addition, it was found that
the oxygen atom of the furan ring of such compounds could
form a weak H-bond with the NH-group of the indole ring of
Trp2476.48
.
Detailed Interactions of N6- and 2-Substituted Deriva-
tives of Adenosine and 1. The binding modes of various N6-
substituted analogues of adenosine and 1 at the A2B AR were
studied using MCMM calculations. As already mentioned, the
N6 position of the ligands was oriented toward TM V. In
addition, the N6-amino group appeared in proximity to the side
chain of Asn2546.55, with which it likely could form an H-bond.
This observation confirmed the previously published results of
molecular modeling of ARs.15,16 However, extended groups at
the N6 position, in particular N6-arylcarbamoylmethoxyphenyl
or N6-(arylcarbonyl)hydrazino groups, present in recently
reported A2B AR agonists,25,26 were found to be oriented toward
the intracellular region of the receptor. Interestingly, previous
modeling studies indicated that similar groups at the 8 position
of some xanthine antagonists of the A2B AR could be oriented
in the opposite direction, i.e., toward the extracellular region.10,11
In this study the MCMM calculations performed for such
agonists were initiated from various starting points with various
randomly generated initial orientations of the N6 substituent.
The results obtained indicated that the orientation of this group
toward the intracellular region was the most favorable energeti-
cally. Moreover, in those cases when the opposite orientation
of the long N6 substituent was observed, critical ligand–receptor
interactions were lost. In particular, in such cases the 2′- and
3′-hydroxyl groups were not involved in H-bonding with
His2807.43, and the H-bonds with Thr893.36 were lost.
In summary, in our docking hypothesis the substituent at the
2 position was oriented toward the extracellular loops and the
N6 substituent toward TM V, in the direction of the cytosolic
side of the receptor. The binding modes obtained for known
2-substituted derivatives of adenosine and 1 such as 2, 4, 16,
18–26, 28, 33–47 were the same as we reported previously.15,16
QSAR Models. Statistical models of quantitative structure–
activity or structure–property relationships (QSAR/QSPR) are
widely used to evaluate computationally various properties of
chemical structures.35–37 In particular, such methods are useful
to evaluate and predict the ligand potency or activity. Numerous
approaches to build QSAR models have been introduced.38–42
Most of these approaches are based on multiple linear-regression
analysis or on partial least-squares (PLS) analysis. They use a
number of independent variables, i.e., descriptors calculated
from the molecular structure, to obtain a reasonable correlation
with a dependent variable, the studied biological property.
In the present study, CoMFA and CoMSIA implemented in
Sybyl 7.343 were utilized to build statistically reliable “structure-
potency” models for agonists of the human A2B AR. Both
CoMFA and CoMSIA approaches are based on the PLS
analysis.44,45 To build the CoMFA or CoMSIA model, an
alignment of the studied compounds is needed. Since the initial
model of the A2B AR used for the ligand docking had the same
coordinates for all compounds, a reasonable and receptor-related
alignment was obtained automatically after the docking studies
(Figure 2). The relative potency (RP) with respect to 1 was
calculated for each compound as described in the Experimental
In the models obtained for N6-arylcarbamoylmethoxyphenyl
derivatives such as 6, neither the amido group of this chain nor

Distal Regions of A2B Adenosine Receptor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2091
Table 1. Statistical Parameters of the CoMFA and CoMSIA Modelsa
lattice space ) 2 Å
lattice space ) 1 Å
MNDO charge type
GH charge type
AM1 charge type
PM3 charge type
CoMFA
MNDO charge type
R2 ) 0.233
Q2 ) 0.538
SEE ) 0.233
F ) 87
R2 ) 0.914
Q2 ) 0.585
SEE ) 0.252
F ) 102
R2 ) 0.705
Q2 ) 0.579
SEE ) 0.245
F ) 91
R2 ) 0.705
Q2 ) 0.492
SEE ) 0.458
F ) 40
R2 ) 0.952
Q2 ) 0.525
SEE ) 0.193
F ) 129
C ) 7
C ) 5
C ) 6
C ) 3
C ) 7
CoMSIA,b R ) 0.30
R2 ) 0.782
Q2 ) 0.475
SEE ) 0.398
F ) 44
R2 ) 0.919
Q2 ) 0.605
SEE ) 0.250
F ) 75
R2 ) 0.917
Q2 ) 0.612
SEE ) 0.253
F ) 73
R2 ) 0.927
Q2 ) 0.571
SEE ) 0.237
F ) 84
R2 ) 0.915
Q2 ) 0.589
SEE ) 0.253
F ) 84
C ) 4
C ) 7
C ) 7
C ) 7
C ) 6
CoMFA and CoMSIA,b R ) 0.30
Model 1
R2 ) 0.676
Q2 ) 0.474
SEE ) 0.480
F ) 35
R2 ) 0.946
Q2 ) 0.598
SEE ) 0.205
F ) 114
R2 ) 0.933
Q2 ) 0.636
SEE ) 0.227
F ) 92
R2 ) 0.958
Q2 ) 0.649
SEE ) 0.182
F ) 128
R2 ) 0.963
Q2 ) 0.668
SEE ) 0.171
F ) 146
C ) 3
C ) 7
C ) 7
C ) 8
C ) 8
R2test ) 0.714
CoMFA and CoMSIA,c R ) 0.30
Model 2
R2 ) 0.952
Q2 ) 0.691
R2test ) 0.759
SEE ) 0.192
F ) 131
C ) 7
CoMFA and CoMSIA,c R ) 0.50
Model 3
R2 ) 0.960
Q2 ) 0.676
R2test ) 0.782
SEE ) 0.175
F ) 158
C ) 7
a GH, Gasteiger-Hückel; SEE, standard error of estimation; C, PLS components. The training set consisted of compounds 1–6, 12, 13, 15–17, 20–22, 24–26,
30–35, 37, 38, 40–48, 50–52, 54–56, 58, 60–62, 64–66, 68–70, 72–74, 76 (Supporting Information). b Steric, electrostatic, H-bond donor and acceptor CoMSIA
regions were used; R, attenuation factor. c Steric, electrostatic, H-bond donor and acceptor, and hydrophobic CoMSIA regions were used; R, attenuation factor.
Section, and it was used as the dependent variable in QSAR
models. Then the steric and electrostatic CoMFA contours as
well as electrostatic, H-bond donor (HBD) and acceptor (HBA),
and hydrophobic CoMSIA contours were calculated with Sybyl
7.3.
from the data, the joint CoMFA/CoMSIA model allowed a better
correlation and error of estimation in comparison with individual
CoMFA and CoMSIA models. It is known that in some cases
the quality of a CoMFA/CoMSIA model can be significantly
improved using a decreased value of the lattice spacing. To
examine this possibility, the joint CoMFA/CoMSIA model
derived for the MNDO-charged compounds was rebuilt with a
lattice spacing of 1 Å. This model (model 1) was characterized
by the following improved statistical parameters: Q2 ) 0.668,
R2 ) 0.963, SEE ) 0.171, F ) 146, C ) 8. The value of the
regression coefficient obtained for the independent test set of
18 compounds (indicated in Table S2 of Supporting Information)
Individual CoMFA and CoMSIA and joint CoMFA/CoMSIA
models were built for the training set of 54 randomly selected
A2B AR agonists, for which data in the stimulation of adenylate
cyclase has been reported (Table 1 and Tables S1 and S2 of
Supporting Information). The four different types of partial
atomic charges, namely, Gasteiger-Hückel, AM1, PM3, and
MNDO, were used. Initially the lattice spacing of 2 Å and only
electrostatic, HBA, and HBD but not hydrophobic CoMSIA
contours were calculated. The results obtained for a succession
of models are summarized in Table 1. The best CoMFA model
was derived for the data set (DS) of compounds with AM1
partial charges (Q2 )0.585, R2 ) 0.914, standard error of
estimation (SEE) ) 0.252, F ) 102; PLS components (C) )
5). In contrast, the best statistical parameters of the CoMSIA
models were obtained for the compounds with the PM3 atomic
charges (Q2 ) 0.612, R2 ) 0.917, SEE ) 0.253, F ) 73, C )
7). Both AM1 and PM3 partial atomic charges provided
reasonable statistical parameters of the joint CoMFA/CoMSIA
models. However, the best results were obtained for the set of
compounds with the MNDO charges assigned (Q2 ) 0.649, R2
) 0.958, SEE ) 0.182, F ) 128, C ) 8). Also, as follows
was as high as R2 ) 0.714.
test
Then with the aim of improving the model quality, the
hydrophobic CoMSIA contours were taken into account to
provide model 2. The lattice spacing of 1 Å and MNDO partial
atomic charges were used. The optimal number of PLS
components was found to be C ) 7. The addition of the
hydrophobic CoMSIA regions resulted in slightly decreased
values of the regression coefficient (R2 ) 0.952) and Fisher’s
criterion (F ) 131). Also, the value of standard error of
estimation was increased (SEE ) 0.192). However, the value
of leave-one-out cross-validated correlation coefficient (Q2 )
0.691) was found to be higher than in model 1, demonstrating
the better stability of model 2. Moreover, the regression
coefficient obtained for the independent test set of compounds

2092 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
IVanoV et al.
cyclopentyl, N6-isopropyl, or N6-ethyl derivatives. However,
because of the presence of more potent N6-(arylcarbonyl)hy-
drazino substituted derivatives of 1, the favorable bulky group
area iv appeared just below the unfavorable yellow region iii
(Figure 4A). The green area v in the lower part of Figure 4A
corresponds to the terminal aryl ring of N6-arylcarbamoyl-
methoxyphenyl derivatives, namely, 6, 58, 60–62, 64–66.
In contrast to N6-arylcarbamoylmethoxyphenyl-substituted
derivatives, benzyl- or cyclohexyl-substituted compounds (such
as 70, 72, 76) are generally less potent at the A2B AR. For this
reason, the area vi occupied by those groups was recognized
by the model as unfavorable (yellow) for the bulky groups.
CoMFA Electrostatic Regions. The 2-chloro substituted
derivatives of adenosine and 1 (e.g., pEC50 (51) ) -2.322,
pEC50 (73) ) -1.629) are less potent at the A2B AR than the
corresponding compounds unsubstituted at the 2 position (e.g.,
pEC50 (5) ) -1.914, pEC50 (73) ) -0.863). Since a chlorine
atom has a negative partial atomic charge, more positively
charged groups were predicted to be favorable near position 2
of the adenine ring (Figure 4B, region i).
In contrast, the red region ii, which is favorable for the less
negatively charged groups, was derived in the upper part of
Figure 4B. This red area corresponds to the hydroxyl group of
the 2-(3-hydroxy-3-phenyl) propyn-1-yl moiety of (S)-PHPN-
ECA 2 and (S)-PHPAdo 33. Also, the region iii of the favorable
position of more negatively charged atoms appeared just below
the N6-amino group of the studied compounds. This area
corresponds to the carbonyl oxygen atom and the oxygen atom
of the furanyl ring of the N6-(arylcarbonyl)hydrazino derivatives
such as 5.
In addition, positively charged groups were predicted to be
favorable in proximity to the distal NH group of the N6-
arylcarbamoylmethoxyphenyl derivatives such as 6 (Figure 4B,
region iv). Additional negatively charged groups were predicted
to be favorable in proximity to the oxygen atom of the
arylcarbamoyl moiety in this series (region v).
CoMSIA Regions. The CoMSIA steric and electrostatic
contours were found to be nearly identical to the corresponding
CoMFA contours. Since these contours were discussed above,
the following section will concern the CoMSIA H-bond donor
and acceptor contours as well as the CoMSIA hydrophobic
contours.
Figure 3. Correlation plots obtained for the training (A) and test (B)
sets of compounds with the CoMFA/CoMSIA model 3.
was also higher in model 2 (R2test ) 0.759) than in model 1 (R2
) 0.714). As a final step of the model refinement, model 2 was
rebuilt with the attenuation factor (R) increased from its standard
value of 0.30 to 0.50. The higher value of the attenuation factor
provided more detailed and localized CoMSIA contours.45
Model 3 was obtained and characterized by the following
statistical parameters: R2 ) 0.960, Q2 ) 0.676, SEE ) 0.176,
F ) 158, C ) 7, R2 ) 0.782. The higher value of the
test
CoMSIA H-Bond Donor and Acceptor Regions. To take
into account the role of H-bond donor and acceptor groups of
the compounds, the corresponding CoMSIA contours were
calculated. The CoMSIA acceptor contour corresponds to the
H-bond donating groups of the receptor, and similarly, the
CoMSIA donor contour describes the regions where the H-bond
acceptor groups of the receptor should be located.
As shown in Figure 5A, H-bond donating groups of the
receptor were predicted to be favorable in proximity to the
oxygen atoms of the hydroxyl group of the 2-(3-hydroxy-3-
phenyl)propyn-1-yl moiety of (S)-PHPNECA 2 and (S)-PHPAdo
33 (orange region i). A second region (ii) favorable for receptor
H-bond donating groups appeared between the ribose oxygen
atom and the 5′-OH group of the adenosine derivatives.
Since the morpholino-substituted analogue 68 is one of the
weak compounds in the series of N6-arylcarbamoylmethoxy-
phenyl derivatives, the oxygen atom of the morpholine ring was
recognized as a disfavored feature by the model. For this reason,
the location of the receptor H-bond donating groups was
predicted to be unfavorable near that oxygen atom (red region
iii in the bottom part of Figure 5A). A second red region (iv) in
the bottom part of the Figure 5A was derived from the presence
attenuation factor provided a further improvement of the model
statistics, namely, a higher value of the regression coefficient
for the test set, a lower value of SEE, and optimal values of R2
and Q2 coefficients. Also the F criterion was increased. As
shown in Figure 3, all points on the correlation plot fit well
with the ideal line for both training and test sets of compounds,
and no outliers were observed. Since the statistical parameters
of model 3 were found to be reasonable, a further refinement
of the model quality was not performed.
CoMFA Regions. CoMFA Steric Regions. In agreement
with experimentally determined ligand potencies at the A2B AR,
bulky groups were predicted to be favorable around the indolyl
ring of 2-(3-(indolyl)ethyloxy) derivatives such as MRS3997 4
(Figure 4A, region i). In contrast, the existence of bulky groups
between the indole ring and the 2 position of the adenine ring
was predicted to be less favorable (Figure 4A, region ii). Those
conditions are reflected in the experimental data, particularly
with the low potency of 2-chloro substituted derivatives and
compounds with an alkyl chain at the 2 position. The unfavor-
able region for bulky groups (yellow region iii) was also
observed near the N6 position of the ligands. This region
corresponds to weak N6 substituted ligands, for example, N6-

Distal Regions of A2B Adenosine Receptor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2093
Figure 4. Contour maps of the CoMFA regions are shown around (S)-PHPNECA 2 (carbon atoms colored in green), MRS3997 4 (carbon atoms
colored in magenta), and ligand 6 (carbon atoms colored in white) superimposed in their conformations obtained after the docking studies; regions
i and ii surround the 2 substituent. (A) Contour maps of the CoMFA steric regions (green, favored; yellow, disfavored). Regions iii–vi surround the
N6 substituent, and regions i and ii are located around the C2 substituent. (B) Contour maps of the CoMFA electrostatic regions. Blue regions are
favorable for more positively charged groups; red regions are favorable for less positively charged groups. Polyhedral regions labeled with Roman
numerals are discussed in the text. Regions i and ii surround the C2 substituent, and regions iii–v surround the N6 substituent.
Figure 5. (A) Contour maps of the CoMSIA H-bond donor and acceptor regions are shown around (S)-PHPNECA 2 (carbon atoms colored in
green), CPA 3 (carbon atoms colored in yellow), and ligand 68 (carbon atoms colored in white): cyan plots, regions favored for the receptor
H-bond acceptor groups; purple, regions disfavored for the receptor H-bond acceptor groups; orange, regions favored for the receptor H-bond
donating groups; red, regions disfavored for the receptor H-bond donating groups. Region i appeared in proximity to the hydroxyl group of the
2-(3-hydroxy-3-phenyl)propyn-1-yl moiety of (S)-PHPNECA 2 and (S)-PHPAdo 33. Region ii is located between the ribose oxygen atom and the
5′-OH group of the adenosine derivatives. Regions iii–v and vii–xi surround the N6 substituent. The group of regions vi and xii surround the 2′-
and 3′-hydroxyl groups of the ribose moiety. (B) Contour maps of the CoMSIA hydrophobic regions (yellow, favored; white, disfavored) are
shown around (S)-PHPNECA 2 (carbon atoms colored in green), MRS3997 4 (carbon atoms colored in magenta), and ligand 6 (carbon atoms
colored in white) superimposed in their receptor-docked conformations. Polyhedral regions labeled with Roman numerals are discussed in the text.
Region i corresponds to the 5′-position. Region ii is located in proximity to the 2′- and 3′-hydroxyl groups of the ribose moiety. Regions iii–vi
surround the N6 substituent, and regions vii–x appear around the 2 substituent.
of N6-arylcarbamoylmethoxyphenyl derivatives containing meth-
oxy groups at positions 3, 4, and 5 of the phenyl ring. These
compounds are weaker than unsubstituted compound 6 (Chart
1) or 4-phenyl substituted derivatives. For this reason, the model
considered methoxy groups at those positions of the phenyl ring
as unfavorable modifications. The red contour appearing near

2094 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
IVanoV et al.
the N6-phenyl ring (Figure 5A, region v) reflects the sulfo group
of N6-(4-sulfophenyl)adenosine (SPA) 32, which also binds
weakly at the A2B AR.
potency of compounds with more hydrophilic methoxy groups
in comparison with the unsubstituted phenyl ring or 4-halo-
genophenyl substituted derivatives.
One favored region (vii) and one disfavored region (viii)
appeared in the upper part of Figure 5B. The favored (yellow)
region vii corresponds to the aromatic rings of 2-(3-(indolyl)ethyloxy)
substituted adenosine derivatives (e.g., 4), and to the phenyl
ring of PHPNECA and PHPAdo analogues such as 2 and 34.
In contrast, the disfavored (white) region viii was located at
the position of the long alkyl chain of 2-hexynyl substituted
adenosine derivatives (38, 40, 41) and 2-(1-(E)-hexenyl)ad-
enosine-5′-N-ethyluronamide (HENECA) 45. Also, a disfavored
hydrophobic region appeared in proximity to the hydroxyl group
of the 2-(3-hydroxy-3-phenyl)propyn-1-yl moiety of (S)-PHP-
NECA 2 and (S)-PHPAdo 34. In addition, the area occupied
by the oxygen atom of the ethyloxy group of 2-(3-(indolyl)
ethyloxy) substituted analogues (e.g., 4, 16, 20, 22) was
recognized as a disfavored region (x) for hydrophobic groups,
while a favored region appeared near position 2 of PHPNECA
and PHPAdo analogues (Figure 5B).
Several regions that were unfavorable for the occurrence of
H-bond donating groups on the receptor appeared around the
ribose and adenine rings of the ligands (vi). However, these
regions do not correspond to any unique structural features of
individual ligands. It was found that, in general, the adenosine
moieties of small nucleosides that were weak ligands of the
A2B AR, namely, N6-cyclopentyladenosine (CPA) 3, 32, and
some other derivatives, were located deeper inside the receptor
than the corresponding moiety of more potent, larger ligands,
for example, compound 1 or 6. The positions of the functional
groups of the ribose moiety varied in the models obtained for
various compounds. These differences were taken into account
and resulted in the corresponding region vi (Figure 5A).
For similar reasons, multiple regions that were unfavorable
for H-bond acceptors on the receptor were derived in proximity
to the ribose ring of the ligands.
In addition, as shown in Figure 5A, the position of the N6-
amino group was found to be different for various ligands. In
particular, the N6 position of the adenine ring of 3 had the same
position as adenine N1 of compound 6. For this reason, regions
where H-bond acceptor groups on the receptor are predicted to
decrease the potency appeared in proximity to the N6 and 1
positions of the ligands (regions vii, viii).
Novel A2B AR Agonists. The analysis of the ligand binding
modes obtained after the MCMM calculations were performed for
the known A2B AR agonists indicated that the substituents at the
N6- and 2 positions were located in two distinct pockets of the
binding site. As already discussed, the groups at the N6 position
were oriented toward the intracellular region. In contrast, the
substituents at position 2 were oriented toward the extracellular
loops of the receptor. At the same time, the adenosine moiety had
almost the same position inside the receptor for all of the studied
ligands. These observations made it possible to propose that the
ligands containing both extended groups at the N6 position25,26 and
a bulky substituent at position 2, namely, a 2-(3-(indolyl)ethyloxy)
group as occurs in 4,16 could also fit the binding site well. The
findings stand in contrast to previous models that suggest an overlay
between bulky substituents at the 2 and N6 positions.27 We have
found that in the present A2B AR model these groups occupy
distinct regions inside the binding site.
With the aim to test the above hypothesis, four novel
adenosine derivatives 7–10 were designed and synthesized as
potential agonists of the A2B AR (Table 2). The synthetic routes
are shown in Schemes 1 and 2. These structures were first
docked to the A2B AR model with the protocol used for all other
studied ligands. All four compounds fit the binding site well
and established similar ligand–receptor interactions as the
corresponding individually substituted compounds (Figure 2).
The RP of these four novel agonists was predicted using the
CoMFA/CoMSIA model 3. The results obtained are summarized
in Table 2. In general, all compounds were predicted to be potent.
As expected, compound 9, the combination of the two most potent
ligands 6 and 4, was predicted as the most potent agonist. For
similar reasons, a high potency was predicted for 10.
The experimental values of EC50 demonstrated that in
agreement with the docking studies all compounds bound to
the A2B receptor. Also, the experimentally measured potency
of these ligands indicated that all of them are potent agonists
of the A2B AR. Ligand 10 was found to be weaker than the
other three novel compounds (EC50 ) 965 nM). However, the
selectivity of compounds 7–10 for the A2B AR was low in
comparison to the human A1 and A2A ARs (Table 2).
Thus, in the present study, the computational design of four
novel agonists of the A2B receptor was performed on the basis
of the molecular and statistical modeling of the A2B AR and its
known ligands. These compounds were successfully synthesized
and biologically evaluated at the human A2B AR. The results
Interestingly, it was observed after the docking studies that
the phenylcarbamoyl moiety of the less potent N6-arylcarbam-
oylmethoxybenzyl derivatives, namely, 70 included in the
training set, appeared slightly deeper inside the receptor than
this moiety of more potent N6-arylcarbamoylmethoxyphenyl
derivatives (e.g., 6). This difference in the ligand binding modes
was characterized by two polyhedra in the CoMSIA model that
appeared near the NH group of those compounds. The purple
area ix, unfavorable for H-bond acceptor groups on the receptor,
was located deeper than the area x that was favorable for such
groups (cyan). The regions xi and xii, favoring H-bond acceptors
on the receptor, were predicted to be near the 2′- and 3′-hydroxyl
groups and the N6-amino group of the potent ligands of the A2B
AR.
CoMSIA Hydrophobic Regions. Not surprisingly, the
calculated CoMSIA hydrophobic contours are in a good
agreement with the experimentally observed potencies of the
studied compounds and their binding modes proposed with the
molecular modeling (Figure 5B). Region i, unfavorable for
hydrophobic groups on the receptor, appeared in proximity to
the 5′ region, i.e., consisting of a hydroxyl group for derivatives
of adenosine or an NH group for 1. Also, the presence of
hydrophobic groups in positions occupied by 2′- and 3′-hydroxyl
groups was predicted to be unfavorable (region ii). In addition,
hydrophobic groups at the N6 position were predicted to be
unfavorable (region iii). These findings are in agreement with
experimental values of EC50 of N6-methyl- (e.g., 29, 39, 46),
N6-ethyl (e.g., 35, 36, 40), N6-isopropyl (e.g., 44, 47) derivatives,
CPA 3, and N6-phenyladenosine 30. The small white area iv
located below the N6 position in Figure 5B was placed between
the aromatic ring of N6-benzyladenosine 31 and the aromatic
ring of compound 5 and its derivatives.
As shown in Figure 5B, the hydrophilic oxygen atom at
position 4 of the phenyl ring of compound 6 was recognized
by the model, and the corresponding hydrophobic disfavored
region v appeared at that position.
The favored hydrophobic region vi appeared near the aromatic
ring of the arylcarbamoyl moiety of 6, indicating a lower

Distal Regions of A2B Adenosine Receptor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2095
Table 2. Experimental and Predicted Potencies of the Novel Agonists in Activation of the Human A2B AR and Experimental Binding Affinities
Measured at Two Other ARs (EC50 and Ki Values in nM)
a Stimulation of adenylate cyclase measured in CHO cells stably expressing the human A2B AR. RP: NECA 1 related potency. Compounds 7–10 appear
to be full agonists compared to 1 at the A2B AR (Supporting Information). b Using model 3 (Table 1). c Radioligand binding determined in CHO cell
membranes (human A1 AR) or in HEK293 cell membranes (human A2AAR), as described in Experimental Section. Ki ( SEM values unless otherwise noted.
d Percent displacement at 10 µM.
Scheme 1. Synthetic Routes to the Novel 6-Hydrazinopurine-9-riboside Derivativesa
a Reagents: (i) hydrazine; (ii) furoic acid, PyBop, DIPEA, DMF.
revealed that all of the proposed compounds are moderately
potent but nonselective agonists of the A2B AR.
2 and N6 positions occupy distinct and nonoverlapping subre-
gions within the putative A2B AR binding site. Furthermore,
these groups were oriented in opposite directions inside the
binding pocket. The similarity and differences in the binding
modes of the studied compounds were analyzed on the basis of
the ligand–receptor models obtained.
Conclusions
We utilized a molecular modeling approach to study putative
binding modes of various N6- and 2-substituted derivatives of
adenosine and its 5′-N-methyluronamide 1 at the human A2B
AR. The modeling results suggest that bulky substituents at the
A successful statistical CoMFA/CoMSIA model was derived
for the NECA-related potency of the studied A2B receptor

2096 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
IVanoV et al.
Scheme 2. Synthetic Routes to the Novel 6-Arylaminopurine-9-riboside Derivativesa
a Reagents and conditions: (i) EtOH, TEA, 110°, 18 h; (ii) KOH; (iii) aniline, PyBop, DIPEA, DMF.
various publications, the RP of compounds was calculated as RP
) pEC50(comp)i – pEC50(NECA)i, where i represents the same
reference publication. The entire DS was randomly divided into
the test set of 54 compounds and training set of 18 compounds
(25% of the total number of compounds). All types of compounds
included in the entire DS were also included in both training and
test sets of compounds.
agonists based on their receptor-docked conformation. The
distribution of steric, electrostatic, H-bond donor and acceptor,
and hydrophobic contours around the ligands was analyzed. The
best statistical parameters were obtained with a joint CoMFA
and CoMSIA model, which displayed an R2 of 0.960. On the
basis of the modeling results, four novel adenosine analogues
having elongated substitutions at the N6 and/or 2 positions were
synthesized and evaluated biologically. All of proposed com-
pounds were shown to be moderately potent, full agonists of
the A2B AR in adenylate cyclase studies. Thus, as predicted by
the QSAR modeling in conjunction with receptor docking,
combinations of elongated and bulky substituents in adenosine
analogues are tolerated for recognition by the A2B AR. These
substituents bind to distinct pockets in the putative binding site
for nucleosides in the TM region of the receptor. The QSAR
models were consistent with the orientation of the N6-amino
group toward TM V and the substituent at the adenine 2 position
toward EL2.
The entire DS of compounds with their RP values are shown in
the Supporting Information (Table S1).
Molecular Docking. Throughout this paper we used the
Ballesteros-Weinstein GPCR residue indexing system.49
The molecular model of the A2B AR15,16 was used in the ligand
docking studies. All 72 compounds included in the DS, as well as
compounds 7–10 synthesized in the present study, were docked to
the A2B AR and subjected to the MCMM conformational search
analysis as recently described.16 MacroModel software was used
in the automated docking studies.33 The initial position of the
receptor was the same for each compound.
CoMFA and CoMSIA Models. The CoMFA and CoMSIA
models were built using the Sybyl 7.3 software.43 The Gasteiger-
Hückel charges were assigned to each compound. In addition, the
AM1, PM3, and MNDO partial atomic charges were calculated
for each compound in the DS using MOE software.52 The
conformations of the A2B AR agonists obtained after molecular
docking were used. Since during the molecular docking studies the
receptor position was the same for each compound, additional
alignment of the structures was not performed. The individual
Experimental Section
Data Set of Compounds. The structures and experimental values
of EC50 of 72 known agonists of the adenosine A2B AR were
collected from the literature.16,25,26,46–48 The 2- and N6-substituted
derivatives of adenosine and NECA 1 were included in the DS.
Since the EC50 values were measured with different protocols in

Distal Regions of A2B Adenosine Receptor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2097
CoMFA and CoMSIA models, as well as combined CoMFA/
CoMSIA models, were built. The both steric and electrostatic
contours were calculated for CoMFA and the steric, electrostatic,
H-bond donor and acceptor, and hydrophobic contours for CoMSIA
were used. The four types of atomic charges mentioned above and
the lattice space of 2 or 1 Å were examined in each model. All other
parameters were set to their default values. The final model 3 was
derived with the attenuation factor of R ) 0.5 instead of its default
value of R ) 0.3. The Use SAMPLS option was deselected, and the
value of the column filtering option was set to 2.0. The automatically
determined optimal number of PLS components was used.
mg, 0.030 mmol), was dissolved in 1.0 mL of anhydrous ethanol.
2-(4-Aminophenoxy)-N-phenylacetamide 78 (8.0 mg, 0.033 mmol)
and 5.0 µL of triethylamine (TEA) were added. The flask was then
sealed and heated to 110 °C for 18 h. The solvent was evaporated
under a vacuum, and the crude residue was subjected to preparative
TLC developed with a mixture of toluene and acetone (1:1) to give
79(16 mg, 50% yield). 1H NMR (CDCl3) δ 7.81 (1H, s), 7.72 (2H,
d, J ) 8.2 Hz), 7.69(2H, d, J ) 8.2 Hz), 7.60 (2H, d, J ) 9.0 Hz),
7.45 (2H, d, J ) 9.0 Hz), 7.35 (1H, t, 8.2 Hz), 7.12 (1H, s), 7.08
(1H, dd, J ) 1.5 and 8.5 Hz), 7.02(d, 2H, J ) 9.0 Hz), 6.11 (1H,
d, J ) 5.7 Hz), 5.96 (1H, t, J ) 5.0 Hz), 5.52 (1H, t, J ) 7.0 Hz),
4.62, (2H, S), 4.33 (1H, dd, J ) 3.1 Hz and 5.2 Hz), 3.20 (2H, t,
J ) 7.0 Hz), 2.31 (3H, s), 2.13 (3H, s), 2.08 (3H, s), 2.06 (3H, s).
HRMS (ESI MS m/z) calcd for C47H44BrN7O12S (M + H)+
1010.8609, found 1010.8622.
Chemical Synthesis. Materials and Instrumentation. Anhy-
drous hydrazine, 2-furoic acid, benzotriazol-1-yloxytripyrrolidino-
phosphonium hexafluorophosphate (PyBop), diisopropylethylamine
(DIPEA), and other reagents and solvents were purchased from
Sigma-Aldrich (St. Louis, MO). 2-(4-Aminophenoxy)-N-phenyl-
acetamide 78 was prepared as reported.26
2-(3′′-(6′′-Bromo-1′′-N-indolyl)ethyloxy)-6-(4-carboxylmethoxy)
phenylamino)-3′,4′,5′-triacetyladenosine (80). Compound 79 (16
mg, 0.015 mmol) was stirred with 1.0 mL of 0.25 M KOH in
MeOH. The reaction mixture was stirred under N2 at room
temperature for 18 h. The solvent was evaporated under a vacuum,
and the crude residue was subjected to preparative TLC developed
with a mixture of chloroform and methanol (4:1) to give 80 (7 mg,
1H NMR spectra were obtained with a Varian Gemini 300
spectrometer using CDCl3 and CD3OD as solvents. Chemical shifts
are expressed in δ values (ppm) with tetramethylsilane (δ0.00) for
CDCl3 and water (δ3.30) for CD3OD.
TLC analysis was carried out on aluminum sheets precoated with
silica gel F254 (0.2 mm) from Aldrich. Low-resolution mass
spectrometry was performed with a JEOL SX102 spectrometer with
6 kV Xe atoms following desorption from a glycerol matrix or on
an Agilent LC/MS 1100 MSD, with a Waters (Milford, MA)
Atlantis C18 column. High-resolution mass spectroscopic (HRMS)
measurements were performed on a proteomics optimized Q-TOF-2
(Micromass-Waters) using external calibration with polyalanine.
Observed mass accuracies are those expected on the basis of the
known performance of the instrument as well as trends in masses
of standard compounds observed at intervals during the series of
measurements. Reported masses are observed masses uncorrected
for this time-dependent drift in mass accuracy.
2-(3′′-(6′′-Bromoindolyl)ethyloxy)-6-hydrazinoadenosine (7).
A solution of 2-(3′′-(5′′-(bromo-1′′-(p-toluenesulfonyl)indolyl)ethy-
loxy)-6-chloro-3′,4′,5′-triacetyladenosine, 77 (24.0 mg, 0.030 mmol)16
was stirred with 0.50 mL of anhydrous hydrazine in a sealed flask.
The reaction mixture was stirred at room temperature for 48 h. The
solvent was evaporated under a vacuum, and the crude residue was
subjected to preparative TLC developed with a mixture of chloroform
and methanol (8:2) to give 7(7 mg, 43% yield). 1H NMR (CD3OD) δ
8.15 (1H, s), 7.50 (1H, d, J ) 8.5 Hz), 7.46 (1H, d, J ) 1.5 Hz), 7.18
(1H, S), 7.14 (1H, dd, J ) 1.5 and 8.5 Hz), 5.90 (1H, d, J ) 5.7 Hz),
4.70 (1H, t, J ) 5.0 Hz), 4.65 (2H, t, J ) 7.0 Hz), 4.35 (1H, dd, J )
3.1 Hz and 5.2 Hz), 4.11 (1H, q, J ) 3.0), 3.85 (1H, dd, J ) 3.1 Hz
and 12.5 Hz), 3.75 (1H, dd, J ) 3.1 Hz and 12.5 Hz). 3.21 (2H, t, J
) 7.0 Hz); HRMS (ESI MS m/z) calcd for C20H21BrN7O5 (M - H)-
518.0788, found 518.0794.
1
51% yield). H NMR (CD3OD) δ 7.78 (2H, d, J ) 8.2 Hz), 7.72
(2H, d, J ) 8.2 Hz), 7.68 (2H, d, J ) 9.0 Hz), 7.62 (2H, d, J ) 9.0
Hz), 7.35 (1H, t, 8.2 Hz), 7.12 (1H, s), 7.08 (1H, dd, J ) 1.5 and
8.5 Hz), 6.95(d, 2H, J ) 9.0 Hz), 5.90 (1H, d, J ) 5.7 Hz), 4.74
(1H, t, J ) 5.0 Hz), 4.65, (1H, S), 4.60 (2H, t, J ) 7.0 Hz), 4.33
(1H, dd, J ) 3.1 and 5.2 Hz), 4.15 (1H, q, J ) 3.0), 3.85 (1H, dd,
J ) 3.1 and 12.5 Hz), 3.75 (1H, dd, J ) 3.1 and 12.5 Hz), 3.65
(1H, s), 3.20 (2H, t, J ) 7.0 Hz); LRMS (ESI MS m/z) calcd for
C28H26BrN6O8 (M - H)- 653.1, found 653.1.
2-(3′′-(6′′-Bromo-1′′-N-indolyl)ethyloxy)-6-(4-((phenylcarbam-
oyl)methoxy)phenylamino)adenosine (9) and 2-(3′′-(6′′-Bromo-
1′′-N-indolyl)ethyloxy)-6-(4-((N-pyrrolidinecarbamoyl)methoxy)
phenylamino)adenosine (10). A solution of 80 (5.0 mg, 0.007
mmol), PyBop (8.0 mg, 0.015 mmol), and DIPEA (5.4 µL, 0.030
mmol) in 0.500 mL of anhydrous DMF was stirred at room
temperature under N2 for 30 min. Aniline (5.0 µL, 0.050 mmol)
was added to the reaction mixture, and stirring continued at room
temperature for 18 h. The solvent was evaporated under a vacuum,
and the crude residue was subjected to preparative TLC developed
with a mixture of chloroform and methanol (9:1) to give 9 (2 mg,
1
40% yield). H NMR (CD3OD) δ 8.16 (1H, s), 7.82 (2H, d, J )
8.2 Hz), 7.72(2H, d, J ) 8.2 Hz), 7.68 (2H, d, J ) 9.0 Hz), 7.62
(2H, d, J ) 9.0 Hz), 7.35 (1H, t, 8.2 Hz), 7.12 (1H, s), 7.08 (1H,
dd, J ) 1.5 and 8.5 Hz), 6.95(d, 2H, J ) 9.0 Hz), 5.90 (1H, d, J
) 5.7 Hz), 4.74 (1H, t, J ) 5.0 Hz), 4.65, (1H, S), 4.60 (2H, t, J
) 7.0 Hz), 4.33 (1H, dd, J ) 3.1 and 5.2 Hz), 4.15 (1H, q, J )
3.0), 3.85 (1H, dd, J ) 3.1 and 12.5 Hz), 3.75 (1H, dd, J ) 3.1
and 12.5 Hz), 3.65 (1H, s), 3.20 (2H, t, J ) 7.0 Hz); HRMS (ESI
MS m/z) calcd for C34H32BrN7O7 (M + H)+ 730.1625, found
730.1663.
2-(3′′-(6′′-Bromoindolyl)ethyloxy)-6-(N-furan-2-carbonyl)hy-
drazinoadenosine (8). A solution of 2-furoic acid (11.2 mg, 0.10
mmol), PyBop (104 mg, 0.20 mmol), DIPEA (70.0 µL, 0.4 mmol)
and 2.50 mL of anhydrous DMF was stirred at room temperature
under N2 for 30 min. Then 250 µL of the mixture was added to a
stirring solution of 7 (3.0 mg, 0.005 mmol) and 250 µL of
anhydrous DMF. The reaction mixture was stirred at room
temperature for 18 h. The solvent was evaporated under vacuum,
and the crude residue was subjected to preparative TLC developed
with a mixture of chloroform and methanol (8:2) to give 8 (2 mg,
Compound 10 was also isolated from the preparative TLC, (1.0
mg, 18% yield). 1H NMR (CD3OD) δ 8.16 (1H, s), 7.82 (2H, d, J
) 8.2 Hz), 7.72(2H, d, J ) 8.2 Hz), 7.68 (2H, d, J ) 9.0 Hz), 7.62
(2H, d, J ) 9.0 Hz), 7.35 (1H, t, 8.2 Hz), 7.12 (1H, s), 7.08 (1H,
dd, J ) 1.5 and 8.5 Hz), 6.95(d, 2H, J ) 9.0 Hz), 5.90 (1H, d, J
) 5.7 Hz), 4.74 (1H, t, J ) 5.0 Hz), 4.65, (1H, S), 4.60 (2H, t, J
) 7.0 Hz), 4.33 (1H, dd, J ) 3.1 and 5.2 Hz), 4.15 (1H, q, J )
3.0), 3.85 (1H, dd, J ) 3.1 and 12.5 Hz), 3.75 (1H, dd, J ) 3.1
and 12.5 Hz), 3.65 (1H, s), 3.52 (2H, m), 3.20 (2H, t, J ) 7.0 Hz),
2.02 (1H, m), 1.90 (1H, m); HRMS (ESI MS m/z) calcd for
C32H35BrN7O7 (M + H)+ 708.1781, found 708.1775.
HPLC Analysis of Compounds 7–10. The purity of the
nucleoside products was demonstrated by HPLC as in our previous
study,16 using an Agilent 1100 series HPLC (Santa Clara, CA)
equipped with a Phenomenex Luna 5 µm C18(2) 100A analytical
column (250 mm × 10 mm; Torrance, CA). All compounds were
tested with a flow rate of 2 mL/min and the following acetonitrile/
water linear gradient: 0 min, 20% acetonitrile; 20 min, 80%
acetonitrile. Peaks were detected by UV absorption using a diode
1
60% yield). H NMR (CD3OD) δ 8.20 (1H, s), 7.47 (1H, d, J )
1.5 Hz), 7.42 (1H, d, J ) 8.5 Hz), 7.18 (1H, S), 7.06 (1H, dd, J )
1.5 and 8.5 Hz), 7.01 (1H, m), 6.54 (1H, m), 6.32 (1H. m), 5.90
(1H, d, J ) 5.7 Hz), 4.70 (1H, t, J ) 5.0 Hz), 4.65 (2H, t, J ) 7.0
Hz), 4.35 (1H, dd, J ) 3.1 Hz and 5.2 Hz), 4.11 (1H, q, J ) 3.0),
3.85 (1H, dd, J ) 3.1 Hz and 12.5 Hz), 3.75 (1H, dd, J ) 3.1 Hz
and 12.5 Hz). 3.21 (2H, t, J ) 7.0 Hz); HRMS (ESI MS m/z) calcd
for C25H25BrN7O7 (M+H)+ 614.0999, found 614.0981.
2-(3′′-(6′′-Bromo-1′′-(p-toluenesulfonyl)indolyl)ethyloxy)-6-(4-
((phenylcarbamoyl)methoxy)phenylamino)-3′,4′,5′-triacetylad-
enosine (79). A solution of 2-(3′′-(5′′-(bromo-1′′-(p-toluenesulfo-
nyl)indolyl)ethyloxy)-6-chloro-3′, 4′, 5′-triacetyladenosine, 77 (24.0

2098 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
IVanoV et al.
(10) Ivanov, A. A.; Baskin, I. I.; Palyulin, V. A.; Piccagli, L.; Baraldi,
P. G.; Zefirov, N. S. Molecular modeling and molecular dynamics
simulation of the human A2B adenosine receptor. The study of the
possible binding modes of the A2B receptor antagonists. J. Med. Chem.
2005, 48, 6813–6820.
(11) Ferretti, V.; Bertoni, E. A preliminary docking study of a potent and
selective xanthinic antagonist to the adenosinic A2B receptor. MinerVa
Biotecnol. 2005, 16, 79–83.
(12) Kim, S. K.; Gao, Z.-G.; Jeong, L. S.; Jacobson, K. A. Docking studies
of agonists and antagonists suggest an activation pathway of the A3
adenosine receptor. J. Mol. Graphics Modell. 2006, 25, 562–577.
(13) 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 selectiv human A3 adenosine
receptor antagonists. Synthesis, pharmacological evaluation, and
ligand-receptor modeling studies. J. Med. Chem. 2007, 50, 4061–
4074.
(14) Palaniappan, K. K.; Gao, Z.-G.; Ivanov, A. A.; Greaves, R.; Adachi,
H.; Besada, P.; Kim, H. O.; Kim, A. Y.; Choe, A. A.; Jeong, L. S.;
Jacobson, K. A. Probing the binding site of the A1adenosine receptor
reengineered for orthogonal recognition by tailored nucleosides.
Biochemistry 2007, 46, 7437–7448.
(15) Ivanov, A. A.; Palyulin, V. A.; Zefirov, N. S. Computer aided
comparative analysis of the binding modes of the adenosine receptor
agonists for all known subtypes of adenosine receptors. J. Mol.
Graphics Modell. 2007, 25, 740–754.
(16) Adachi, H.; Palaniappan, K. K.; Ivanov, A. A.; Bergman, N.; Gao,
Z.-G.; Jacobson, K. A. Structure-activity relationships of 2, N6, 5′-
substituted adenosine derivatives with potent activity at the A2B
adenosine receptor. J. Med. Chem. 2007, 50, 1810–1827.
(17) Loging, W.; Harland, L.; Williams-Jones, B. High-throughput elec-
tronic biology: mining information for drug discovery. Nature ReV.
Drug DiscoVery 2007, 6, 220–230.
(18) Muegge, I.; Oloff, S. Advances in virtual screening. Drug DiscoVery
Today 2006, 3, 405–411.
(19) Akamatsu, M. Current state and perspectives of 3D-QSAR. Curr. Top.
Med. Chem. 2002, 2, 1381–1394.
(20) Tchilibon, S.; Kim, S. K.; Gao, Z. G.; Harris, B. A.; Blaustein, J.;
Gross, A. S.; Melman, N.; Jacobson, K. A. Exploring distal regions
of the A3 adenosine receptor binding site: sterically-constrained N6-
(2-phenylethyl)adenosine derivatives as potent ligands. Bioorg. Med.
Chem. 2004, 12, 2021–2034.
(21) Li, A.-H.; Moro, S.; Melman, N.; Ji, X.-d.; Jacobson, K. A. Structure
activity relationships and molecular modeling of 3,5-diacyl-2,4-
dialkylpyridine derivatives as selective A3 adenosine receptor antago-
nists. J. Med. Chem. 1998, 41, 3186–3201.
(22) Moro, S.; Braiuca, P.; Deflorian, F.; Ferrari, C.; Pastorin, G.; Cacciari,
B.; Baraldi, P. G.; Varani, K.; Borea, P. A.; Spalluto, G. Combined
target-based and ligand-based drug design approach as a tool to define
a novel 3D-pharmacophore model of human A3 adenosine receptor
antagonists: pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives
as a key study. J. Med. Chem. 2005, 48, 152–162.
array detector. All compounds were found to be >98% pure by
HPLC analysis. The compounds eluted at the following retention
times: 7, 9.9 min; 8, 8.1 min; 9, 10.0 min; 10, 9.9 min.
Pharmacological Methods. Binding Assay. Competitive ra-
dioligand binding assays were performed as described previously.50
[3H]CCPA (2-chloro-N6-cyclopentyladenosine, 42.6 Ci/mmol) and
[3H]CGS21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-eth-
ylcarboxamidoadenosine, 40.5 Ci/mmol) were purchased from
Perkin-Elmer (Waltham, MA). Human A1 ARs were stably
expressed in Chinese hamster ovary (CHO) cells, while the human
A2A AR was stably expressed in HEK293 cells. Cell membrane
suspensions were prepared as described.51 For the binding deter-
mination, each tube contained 50 µL of the appropriate radioligand
(A1, [3H]CCPA, final concentration of 1.0 nM; A2A, [3H]CGS21680,
final concentration of 10 nM), 50 µL of increasing concentrations
of the compounds, and 100 µL of cell membrane suspension
expressing the appropriate receptor diluted with Tris-HCl buffer
(50 mM, pH 7.4) containing 10 mM MgCl2. Nonspecific binding
was determined using 10 µM NECA. The tubes were incubated at
25 °C for 1 h and then filtered through Whatman GF/B filters under
reduced pressure using an MT-24 cell harvester (Brandell; Gaith-
ersburg, MD). Filters were washed rapidly three times with ice-
cold buffer. After the filters were washed, they were placed in
scintillation vials containing 5 mL of Hydrofluor scintillation buffer
and counted using a Perkin-Elmer liquid scintillation analyzer. The
Ki values were determined using Prism (GraphPad, San Diego, CA)
for all assays.
cAMP Assay. cAMP was measured in CHO cells stably
expressing the human A2B AR.51 cAMP accumulation was measured
with cAMP enzyme immunoassay kit according to the instructions
in the assay manual (Sigma; St. Louis, MO).
Acknowledgment. We thank Dr. John Lloyd (NIDDK) for
mass spectral determinations. This research was supported by
the Intramural Research Program of the NIH, National Institute
of Diabetes and Digestive and Kidney Diseases. We thank Dr.
Stefano Costanzi (NIDDK) for helpful discussions.
Supporting Information Available: Table of A2B AR ligands
in the training and test sets, adenylate cyclase concentration–re-
sponse curves, and HPLC analysis results of the newly synthesized
nucleosides 7-10. This material is available free of charge via the
Internet at http://pubs.acs.org.
References
(23) Rieger, J. M.; Brown, M. L.; Sullivan, G. W.; Linden, J.; Macdonald,
T. L. Design, synthesis, and evaluation of novel A2A adenosine receptor
agonists. J. Med. Chem. 2001, 44, 531–539.
(24) Fossa, P.; Pestarino, M.; Menozzi, G.; Mosti, L.; Schenone, S.; Ranise,
A.; Bondavalli, F.; Trincavelli, M. L.; Lucacchini, A.; Martini, C. New
pyrazolo[3,4-b]pyridones as selective A1 adenosine receptor antago-
nists: synthesis, biological evaluation and molecular modelling studies.
Org. Biomol. Chem. 2005, 3, 2262–2270.
(25) Baraldi, P. G.; Preti, D.; Tabrizi, M. A.; Fruttarolo, F.; Romagnoli,
R.; Carrion, M. D.; Cara, L. C. L.; Moorman, A. R.; Varani, K.; Borea,
P. A. Synthesis and biological evaluation of novel 1-deoxy-1-[6-
[((hetero)arylcarbonyl)hydrazine]-9H-purin-9-yl]-N-ethyl-ꢀ-D-ribofura-
nuronamide derivatives as useful templates for the development of
A2B adenosine receptor agonists. J. Med. Chem. 2007, 50, 374–380.
(26) Baraldi, P. G.; Preti, D.; Tabrizi, M. A.; Fruttarolo, F.; Saponaro, G.;
Baraldi, S.; Romagnoli, R.; Moorman, A. R.; Gessi, S.; Varani, K.; Borea,
P. A. N6-[(Hetero)aryl/(cyclo)alkyl-carbamoyl-methoxy-phenyl]-(2-chloro)-
5′-N-ethylcarboxamido-adenosines: the first example of adenosine-related
structures with potent agonist activity at the human A2B adenosine receptor.
Bioorg. Med. Chem. 2007, 15, 2514–2527.
(27) Dooley, M. J.; Quinn, R. J. The three binding domain model of
adenosine receptors: molecular modeling aspects. J. Med. Chem. 1992,
35, 211–216.
(28) Elzein, E.; Palle, V.; Wu, Y.; Maa, T.; Zeng, D.; Zablocki, J.
2-Pyrazolyl-N6-substituted adenosine derivatives as high affinity and
selective adenosine A3 receptor agonists. J. Med. Chem. 2004, 47,
4766–4773.
(29) Okada, T.; Sugihara, M.; Bondar, A.-N.; Elstner, M.; Entel, P.; Buss,
V. The retinal conformation and its environment in rhodopsin in light
of new 2.2 Å crystal structure. J. Mol. Biol. 2004, 342, 571–583.
(1) Jacobson, K. A.; Gao, Z.-G. Adenosine receptors as therapeutic targets.
Nat. ReV. Drug. DiscoVery 2006, 5, 247–264.
(2) Burnstock, G. Purine and pyrimidine receptors. Cell. Mol. Life Sci.
2007, 64, 1471–1483.
(3) Gao, Z.-G.; Jacobson, K. A. Emerging adenosine receptor agonists.
Expert Opin. Emerging Drugs 2007, 12, 479–492.
(4) Eckle, T.; Krahn, T.; Grenz, A.; Köhler, D.; Mittelbronn, M.; Ledent,
C.; Jacobson, M. A.; Osswald, H.; Thompson, L. F.; Unertl, K.;
Eltzschig, H. K. Cardioprotection by ecto-5′-nucleotidase (CD73) and
A2B adenosine receptors. Circulation 2007, 115, 1581–1590.
(5) Németh, Z. H.; Bleich, D.; Csóka, B.; Pacher, P.; Mabley, J. G.; Himer,
L.; Vizi, E. S.; Deitch, E. A.; Szabó, C.; Cronstein, B. N.; Haskó, G.
Adenosine receptor activation ameliorates type 1 diabetes. FASEB J.
2007, 10, 2379–2388.
(6) Sum, C. S.; Tikhonova, I. G.; Neumann, S.; Engel, S.; Raaka, B. M.;
Costanzi, S.; Gershengorn, M. C. Identification of residues important
for agonist recognition and activation in GPR40. J. Biol. Chem. 2007,
282, 29248–29255.
(7) Ivanov, A. A.; Voronkov, A. E.; Baskin, I. I.; Palyulin, V. A.; Zefirov,
N. S. The study of the mechanism of binding of human ML1A
melatonin receptor ligands using molecular modeling. Dokl. Biochem.
Biophys. 2004, 394, 49–52.
(8) Li, J. H.; Han, S.-J.; Hamdan, F. F.; Kim, S.-K.; Jacobson, K. A.;
Bloodworth, L. M.; Zhang, X.; Wess, J. Distinct structural changes
in a G protein-coupled receptor caused by different classes of agonist
ligands. J. Biol. Chem. 2007, 282, 26284–26293.
(9) Rodina, E. V.; Vorobyeva, N. N.; Kurilova, S. A.; Belenikin, M. S.;
Fedorova, N. V.; Nazarova, T. I. ATP as effector of inorganic
pyrophosphatase of Escherichia coli. Identification of the binding site
for ATP. Biochemistry (Moscow) 2007, 72, 93–99.

Distal Regions of A2B Adenosine Receptor
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2099
(30) Lodowski, D. T.; Salom, D.; Le Trong, I.; Teller, D. C.; Ballesteros,
J. A.; Palczewski, K.; Stenkamp, R. E. Crystal packing analysis of
rhodopsin crystals. J. Struct. Biol. 2007, 158, 455–462.
(31) Schueler-Furman, O.; Wang, C.; Bradley, P.; Misura, K.; Baker, D.
Progress in modeling of protein structures and interactions. Science
2005, 310, 638–642.
(32) Fanelli, F.; De Benedetti, P. G. Computational modeling approaches
to structure-function analysis of G protein-coupled receptors. Chem.
ReV. 2005, 105, 3297–3351.
(33) Mohamadi, F. N.; Richards, G. J.; Guida, W. C.; Liskamp, R.; Lipton,
M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. Macro-
Models an integrated software system for modeling organic and
bioorganic molecules using molecular mechanics. J. Comput. Chem.
1990, 11, 440–467.
(34) Costanzi, S.; Ivanov, A. A.; Tikhonova, I. G.; Jacobson, K. A. Structure
and function of G protein-coupled receptors studied using sequence
analysis molecular modeling and receptor engineering: adenosine
receptors. Front. Drug Des. DiscoVery 2007, 3, 63–79.
(35) Ivanova, A. A.; Ivanov, A. A.; Oliferenko, A. A.; Palyulin, V. A.;
Zefirov, N. S. Highly diverse, massive organic data as explored by a
composite QSPR strategy: an advanced study of boiling point. SAR
QSAR EnViron. Res. 2005, 16, 231–246.
Kononowicz, K. Phenylethyl-substituted pyrimido[2,1-f]purinediones
and related compounds: structure-activity relationships as adenosine
A1 and A2A receptor ligands. Bioorg. Med. Chem. 2007, 15, 6956–
6974.
(42) Tikhonova, I. G.; Baskin, I. I.; Palyulin, V. A.; Zefirov, N. S. CoMFA
and homology-based models of the glycine binding site of N-methyl-
D-aspartate receptor. J. Med. Chem. 2003, 46, 1609–1616.
(43) SYBYL, version 7.3; Tripos Inc. (1699 South Hanley Road, St. Louis,
MO 63144).
(44) Klebe, G.; Abraham, U.; Mietzner, T. Molecular similarity indices in
a comparative analysis (CoMSIA) of drug molecules to correlate and
predict their biological activity. J. Med. Chem. 1994, 37, 4130–4146.
(45) San Juan, A. A. Structural investigation of PAP derivatives by CoMFA
and CoMSIA reveals novel insight towards inhibition of Bcr-Abl
oncoprotein. J. Mol. Graphics Modell. 2007, 26, 482–493.
(46) de Zwart, M.; Link, R.; von Frijtag Drabbe Künzel, J. K.; Cristalli,
G.; Jacobson, K. A.; Townsend-Nicholson, A.; IJzerman, A. P.
Nucleosides Nucleotides 1998, 17, 969–985.
(47) Vittori, S.; Costanzi, S.; Lambertucci, C.; Portino, F. R.; Taffi, S.;
Volpini, R.; Klotz, K.-N.; Cristalli, G. A2B adenosine receptor agonists:
synthesis and biological evaluation of 2-phenylhydroxypropynyl
adenosine and NECA derivatives. Nucleosides, Nucleotides Nucleic
Acids 2004, 23, 471–481.
(36) Kim, S. K.; Jacobson, K. A. Three-dimensional quantitative structure-
activity relationship of nucleosides acting at the A3 adenosine receptor:
analysis of binding and relative efficacy. J. Chem. Inf. Model. 2007,
47, 1225–1233.
(37) Moro, S.; Bacilieri, M.; Ferrari, C.; Spalluto, G. Autocorrelation of
molecular electrostatic potential surface properties combined with
partial least squares analysis as alternative attractive tool to generate
ligand-based 3D QSARs. Curr. Drug DiscoVery Technol. 2005, 2,
13–21.
(38) Li, A.-H.; Moro, S.; Forsyth, N.; Melman, N.; Ji, X.; Jacobson, K. A.
Synthesis, CoMFA analysis, and receptor docking of 3,5-diacyl-2,4-
dialkylpyridine derivatives as selective A3 adenosine receptor antago-
nists. J. Med. Chem. 1999, 42, 706–721.
(39) Palyulin, V. A.; Radchenko, V. A.; Zefirov, N. S. Molecular field
topology analysis method in QSAR studies of organic compounds.
J. Chem. Inf. Comput. Sci. 2000, 40, 659–667.
(40) Costanzi, S.; Tikhonova, I. G.; Ohno, M.; Joshi, B. V.; Colson, A. O.;
Houston, D.; Maddileti, S.; Harden, T. K.; Jacobson, K. A. P2Y1
antagonists: combining receptor-based modeling and QSAR for a
quantitative prediction of the biological activity based on consensus
scoring. J. Med. Chem. 2007, 50, 3229–3241.
(41) Drabczyn´ska, A.; Müller, C. E.; Schiedel, A.; Schumacher, B.; Karolak-
Wojciechowska, J.; Fruzin´ski, A.; Zobnina, W.; Yuzlenko, O.; Kiec´-
(48) Volpini, R.; Costanzi, S.; Lambertucci, C.; Taffi, S.; Vittori, S.; Klotz,
K.-N.; Cristalli, G. N6-Alkyl-2-alkynyl derivatives of adenosine as
potent and selective agonists at the human adenosine A3 receptor and
a starting point for searching A2B ligands. J. Med. Chem. 2002, 45,
3271–3279.
(49) Ballesteros, J.; Weinstein, H. Integrated methods for the construction
of three-dimensional models of structure-function relations in G
protein-coupled receptors. Methods Neurosci. 1995, 25, 366–428.
(50) Perreira, M.; Jiang, J. K.; Klutz, A. M.; Gao, Z. G.; Shainberg, A.;
Lu, C.; Thomas, C. J.; Jacobson, K. A. “Reversine” and its 2-substi-
tuted adenine derivatives as potent and selective A3 adenosine receptor
antagonists. J. Med. Chem. 2005, 48, 4910–4918.
(51) Gao, Z. G.; Mamedova, L. M.; Chen, P.; Jacobson, K. A. 2-Substituted
adenosine derivatives: affinity and efficacy at four subtypes of human
adenosine receptors. Biochem. Pharmacol. 2004, 68, 1985–1993.
(52) Molecular Operating EnVironment, version 2008.05; Chemical Com-
puting Group: Montreal, Canada.
(53) Rosenbaum, D. M.; Cherezov, V.; Hanson, M. A.; Rasmussen, S. G. F.;
Thian, F. S.; Kobilka, T. S.; Choi, H. J.; Yao, X. J.; Weis, W. I.;
Stevens, R. C.; Kobilka, B. K. GPCR engineering yields high-
resolution structural insights into ꢀ2 adrenergic receptor function.
Science 2007, 318, 1266–1273.
JM701442D