Novel 1,3-dipropyl-8-(3-benzimidazol-2-yl-methoxy-1-methylpyrazol-5-yl) xanthines as potent and selective A2B adenosine receptor antagonists

Article abstract of DOI:10.1021/jm201292w

Molecular modeling studies, including the comparative molecular field analysis (CoMFA) method, on 52 antagonists of the A_2B adenosine receptor with known biological activity were performed to identify the three-dimensional features responsible

Full text of DOI:10.1021/jm201292w

Article
pubs.acs.org/jmc
Novel 1,3-Dipropyl-8-(3-benzimidazol-2-yl-methoxy-1-
methylpyrazol-5-yl)xanthines as Potent and Selective A2B Adenosine
Receptor Antagonists
Pier Giovanni Baraldi,*,† Stefania Baraldi,† Giulia Saponaro,† Delia Preti,† Romeo Romagnoli,†
Laura Piccagli,§ Andrea Cavalli,‡ Maurizio Recanatini,‡ Allan R. Moorman,∥ Abdel Naser Zaid,⊥
Katia Varani,‡ Pier Andrea Borea,‡ and Mojgan Aghazadeh Tabrizi*,†
‡
†Department of Pharmaceutical Sciences, Department of Clinical and Experimental Medicine, University of Ferrara, Via Fossato di
Mortara 17−19, 44100 Ferrara, Italy
§Department of Biochemistry and Molecular Biology, University of Ferrara, 44100 Ferrara, Italy
∥King Pharmaceuticals Inc., Research & Development, 4000 CentreGreen Way, Suite 300, Cary, North Carolina 27513, United States
⊥College of Pharmacy, An-Najah National University, Nablus, Palestine
S
* Supporting Information
ABSTRACT: Molecular modeling studies, including the comparative molecular field analysis (CoMFA) method, on 52
antagonists of the A2B adenosine receptor with known biological activity were performed to identify the three-dimensional
features responsible for A2B adenosine receptor antagonist activity. On the basis of these and previous results on the potent
antagonist effect of 8-pyrazolyl-xanthines at human A2BAR, a new series of compounds was synthesized and evaluated in binding
studies against the human A1, A2A, A3, and A2BARs. A remarkable improvement in selectivity with respect to the previous series,
maintaining the potency at human A2B receptor, was achieved, as exemplified by the 8-[3-(4-chloro-6-trifluoromethyl-1H-
benzoimidazol-2-yl-methoxy)-1-methyl-1H-pyrazol-5-yl]-1,3-dipropyl-3,7-dihydro-purine-2,6-dione derivative 66: Ki A2B = 9.4
nM, IC50 hA2B = 26 nM hA1/hA2B = 269, hA2A/hA2B > 106, hA3/hA2B >106. This study also led to the identification of a series of
pyrazole-xanthine compounds with a simplified structure, exemplified by 8-(3-hydroxy-1-methyl-1H-pyrazol-5-yl)-xanthine 80
displaying very high affinity at A2BAR with good selectivity over AR subtypes (Ki = 4.0 nM, IC50 hA2B = 20 nM hA1/hA2B = 183,
hA2A,hA3/hA2B > 250).
production.4 Recent findings support the involvement of A2BAR
INTRODUCTION
Adenosine mediates its effects through activation of a family of
■
subtype in adenosine-mediated coronary vasodilation.5,6
Activation of A2BAR may prevent cardiac remodelling after
four G-protein coupled adenosine receptors (ARs). These
myocardial infarction.7 A protective effect from infarction has
receptors differ in their affinity for adenosine, in the type of G
been also attributed to A2BAR in ischemic postconditioning
through a pathway involving protein kinase C and phospha-
tidylinositol-3-kinase.8
According to mRNA analysis, revealing high levels of A2BAR
message in the cecum and large intestine, it has been reported
that A2BARs in intestinal epithelial cells trigger an increase in
cAMP levels that is responsible for Cl− secretion. This Cl−
proteins that they recruit, and finally in the downstream
signaling pathways that are activated in the target cells.1,2
The A2BAR has long been known to couple to adenylyl
cyclase activation through Gs proteins. However, other
intracellular signaling pathways have been demonstrated to be
associated with A2BAR, including Ca2+ mobilization through Gq
proteins and mitogen-activated protein kinase (MAPK)
activation.3 A2BAR-induced stimulation of phospholipase C
results in mobilization of intracellular calcium in human mast
cell line (HMC-1) and promotion of interleukin-8 (IL-8)
Received: September 28, 2011
Published: December 12, 2011
© 2011 American Chemical Society
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Chart 1. Chemical Structures and Biological Data for A2BAR Antagonists
secretion pathway results in movement of isotonic fluid into the
lumen, a process that naturally serves to hydrate the mucosal
A2BAR function in alveolar macrophages as a novel treatment
for COPD.17
surface but, in extreme, produces secretory diarrhea.9−11
Adenosine constricts airways of asthmatic patients through
the release of histamine and leukotrienes from sensitized mast
cells.12 The receptor involved seems to be the A2BAR in
humans or the A3AR in rats. Recently, A2BARs have been
reported to mediate several proinflammatory effects of
adenosine in inflammatory cells of the lung. In addition to
mast cells, functional A2BARs have been found in bronchial
smooth muscle cells and lung fibroblasts. In these cells,
adenosine increases the release of various inflammatory
cytokines through stimulation of the A2B subtype, supporting
the evidence that the A2BAR plays a key role in the
inflammatory response associated with asthma.13−15
It has been also reported that adenosine deaminase-deficient
mice treated with the selective A2BAR antagonist 1a (CVT-
6883,18 Chart 1) showed reduced elevations in proinflamma-
tory cytokines and chemokines as well as mediators of fibrosis
and airway destruction.
Because of the biological effects described above, selective
ligands of the A2BAR could be useful as antiasthmatic and
antiallergic, antidiabetic, antidiarrheal, antiatherosclerotic, and
oncolytic drugs, as well as for the treatment of cardiovascular
and ocular disorders.
Many selective and high affinity antagonists have been
developed for adenosine A2B receptors.19−22 The major
representative A2BAR antagonists are shown in Chart 1. On
the basis of early A2BAR selective ligands such as 1b (MRS
1754)23 (Ki hA2B = 2 nM, Ki hA1 = 403 nM, Ki hA2A= 503, and
Ki hA3 = 570 nM), several compounds have been discovered as
A2BAR antagonists. Zablocki and co-workers have described the
preparation and affinity of 3-phenyl-1,2,4-oxadiazoles and 5-
phenyl-1,2,4-oxadiazoles as amide surrogates for 1b. In this
study, they found that the metabolic stability of MRS analogues
was enhanced by replacing the anilide moiety with 1,2,4-
oxadiazole nucleus. Within this series, 1c (CVT-5440)24 had
the highest selectivity and an affinity of 50 nM for the A2BAR.
The 8-(4-pyrazolyl)-xanthine derivative 1a18,25 is another
selective, potent, and orally available A2BAR antagonist (Ki hA2B
= 22 nM, Ki hA1 = 1940 nM, Ki hA2A = 3280 nM, and Ki hA3 =
1070 nM) which had been in early clinical development for the
treatment of asthma and for the treatment of cardiovascular
disease. Preclinical data have also suggested its potential utility
The first evidence for the involvement of A2BAR in asthma
derived from studies concerning the selectivity of enprofylline, a
methylxanthine structurally related to theophylline.16 Further
support for the role of A2BAR in asthma comes from studies
demonstrating their presence on different type of cells
important for the cytokine release in asthmatic disease such
as smooth muscle cells, lung fibroblasts, endothelial cells,
bronchial epithelium, and mast cells. Low expression of A2BARs
was also found in lung parenchyma of patients affected by
chronic obstructive pulmonary disease (COPD).13 In bron-
choalveolar lavage macrophages from COPD patients, A2BARs
were downregulated compared to smokers with normal lung
function. In addition, A2BAR mRNA and protein expression was
selectively decreased by oxidative/nitrosative stress but not by
inflammatory mediators in a human leukemic monocyte-like
cell line (U937 cells) supporting the potential for modulating
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Chart 2. Experimental and Predicted Potencies of the Novel Antagonists at hA2BAR and Experimental Binding Affinities
a
Measured at Other AR
a
The predictions (pKi calcd) were obtained by using the model A (Supporting Information Table S3). (a) Data are expressed as the geometric mean
with 95% confidence limits in parentheses and derived from inhibition binding experiments and cAMP assays as described in the Experimental
Section. (b) Displacement of [3H]MRE2029-F20 binding to hA2BARs (n = 3−6). (c) Displacement of specific [3H]DPCPX binding to hA1ARs (n =
3−6). (d) Displacement of specific [3H]ZM241385 binding to hA2AARs (n = 3−6). (e) Displacement of specific [3H]MRE3008F20 binding to
hA3ARs (n = 3−6). Data are expressed as geometric means with 95% confidence limits.
in chronic obstructive pulmonary disease and pulmonary
fibrosis. It is being developed by Gilead Sciences.
The pyrazine-based A2BAR antagonists discovered by the
Almirall group include 1d (101057),26 a potent, selective, and
orally efficacious A2BR antagonists that was identified as a
clinical development candidate.
Chart 3. Structural Modifications Considered on the 8-[3-
(1H-Benzimidazol-2-ylmethoxy)-1-methyl-1H-pyrazol-5-yl]-
a
1,3-dipropyl-3,7-dihydro-1H-purine-2,6-dione Template
A novel, potent, and selective pyrrolopyrimidine A2B
antagonist 1e (OSIP339391)27 was discovered by the OSI
group that had a selectivity of greater than 70-fold for A2B
receptors over other adenosine receptor subtypes (Ki hA2B
=
0.5 nM, Ki hA1 = 37 nM, Ki hA2A = 328 nM, and Ki hA3 = 450
nM) .
Our group has discovered a series of 8-(5-pyrazolyl)-xanthine
analogues as selective, high affinity A2BAR antagonists.28 Within
this series, 30 (MRE2029-F20,29 Chart 1, Supporting
Information Table S1) (Ki hA2B = 5.5 nM, Ki hA1 = 200 nM,
Ki hA2A, A3 > 1000) has been radiolabeled for use in
pharmacological studies.
In the present paper, we performed a CoMFA study on the
previous series of A2BAR antagonists.28 In this respect, the
series of antagonists is indeed uniform in terms of biological
assays. The 3D QSAR model that we obtained then led to the
design of two new derivatives, including a bioisosteric
replacement of the anilide moiety of 30 with benzimidazole
or quinazoline rings (compounds 53 and 54 respectively, Chart
2).
a
The template compound 54 is shown.
The amide bond was also replaced with the 5-phenyl-1,2,4-
oxadiazole nucleus on the basis of other adenosine
pharmacophores such as those reported previously by Zablocki
et al.24 In this context, considering the good antagonistic
potencies of deazaxanthines at human adenosine receptors,30,31
a comparison of the affinity and selectivity profiles of 9-
deazaxanthines with the corresponding xanthines was afforded
by the preparation of four 9-deaza direct analogues. The new
compounds were tested in competitive binding assays toward
four hARs expressed in CHO cells. cAMP assays were
performed to determine their functionality.
We then undertook the synthesis and biological evaluation of
a new series of 1,3-dipropyl-xanthines that carry a N-
methylpyrazolo or isoxazolo group at position 8. The selection
of the benzimidazol-2-yl-methoxy group at the 3-position of the
pyrazole/isoxazole was based on the greater potency of the
benzimidazole derivative 54 over that of quinazoline 53. In
addition, the commercial availability of substituted-phenylendi-
amines permitted the easier optimization of the benzimidazole
series. As shown in Chart 3, various structural modifications
were realized using the novel A2B antagonist 54 as the template.
CHEMISTRY
■
To explore the present class of A2B antagonists, the new
compounds reported in Tables 1−3 were synthesized as
outlined in Schemes 1−4. The carboxylic acid derivatives I or
II,28 in anhydrous DMF, were condensed with phenylendi-
amines (as expect for compounds 53 and 78, 2-amino-
benzamide and pyrimidine-4,5-diamine were used, respectively)
using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydro-
chloride (EDC) to furnish the amides that were subsequently
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a
Scheme 1. Preparation of Xanthine Derivatives 53−78
a
Reagents and conditions: (i) (1) appropiate diamine, EDC, DMF, rt, (2) CH3COOH, 60−70 °C; (ii) (1) 2-amino-benzamide, EDC, DMF, rt, (2)
NaOH 2.5 N, 80 °C.
cyclized in the presence of acetic acid at 80−100 °C to provide
the 8-heteroaryl derivatives 53−78 (Scheme 1).
the pyrazolo carboxylic acid XV28 to afford the intermediate
pyrrolo[3,2-d]pyrimidin-6-yl)-pyrazol-3-yloxy]-acetic acid XIX
(Scheme 4). Target compound 82 was obtained by the same
procedure used for analogues 54−69, while direct condensation
of carboxylic acid XIX and the suitable anilines in DMF in the
presence of EDC as coupling reagent yielded 83 and 84.
The target xanthine derivatives 79 and 80 were prepared as
shown in Scheme 2. 3-(Benzyloxy)-1-methyl-1H-pyrazole-5-
carboxylic acid V was prepared by direct alkylation of methyl 3-
hydroxy-1-methyl-1H-pyrazole-5-carboxylate28 III with benzyl
bromide in acetone using K2CO3 as a base, followed by ester
hydrolysis. Coupling of V with 1,3-dipropyl-5,6-diaminouracil32
using EDC afforded the amide intermediate, which was cyclized
in NaOH to afford compound VI. Debenzylation with
ammonium formate and palladium-on-carbon yielded the target
compound 80. Treatment of VI with 2-(trimethylsilyl)-
ethoxymethyl chloride (SEM-Cl) and K2CO3 introduced the
SEM protection group at N-7. Debenzylation yielded our key
intermediate VIII. This compound was then alkylated with 3-
chloromethyl-5-(3-methoxy-phenyl)-[1,2,4]oxadiazole33 (IX)
to yield X. SEM deprotection with 3 N HCl in ethanol at 80
°C afforded the target compound 79.
RESULTS AND DISCUSSION
■
Three-Dimensional QSAR Models. The 3D QSAR model
was developed using 52 xanthine compounds as a training set
(Supporting Information Table S1). The most active
compound of the series (30, Supporting Information Table
S1, Table 3) was selected as the template for the alignment in
the CoMFA analyses. A frequently used approach in
pharmacophore modeling is to identify common motifs
contributing to the interaction with the receptor. In the
present study, the xanthine core was assumed as the common
anchoring moiety, and the flexible chains linked at the 8-
position were considered as biological modulators. The
investigation of conformational data for the template showed
the existence of several low energy structures (within an energy
window of 1.0 kcal/mol). As a consequence, the selection of
the candidate conformations (i.e., the putative bioactive
conformations) for the eventual alignment was not trivial. To
this aim, we first selected representative (i.e., the most potent)
molecules (22, 23, 30, and 51, Supporting Information Table
S1) of four different subclasses of the series of compounds
under investigation. Subsequently, we aligned all the low energy
conformations of the four representative antagonists onto the
common xanthine moiety. Considering that all ligands bind at
the same site of the receptor, they should adopt a common
three-dimensional geometry that is responsible for their affinity
and functional activity. Thus, all the low energy conformations
were clustered based on geometrical root-mean-square
deviation (rmsd). On the basis of the result of the rmsd
clustering procedure, two low energy conformations of
compound 30 were used as templates in the following
The synthesis of 9-deaza-xanthines 81−84 analogues was
performed as described in published procedures31,34 with some
modifications to reflect the change from benzaldehyde to the
pyrazolo carboxaldehyde (Schemes 3 and 4).
3-(Benzyloxy)-1-methyl-1H-pyrazole-5-carboxaldehyde XI
was prepared by conversion of V into a mixed anhydride
with ethyl chloroformate to yield the corresponding alcohol,
which was reacted with pyridinium chlorochromate (PCC)
(Scheme 3). Condensation of the 1,3-dipropyl-6-methyl-5-
nitrouracil (XII)35 with 3-benzyloxy-1-methyl-1H-pyrazole-5-
carboxaldehyde XI in dry toluene and catalytic morpholine
allowed us to obtain the corresponding (E)-6-styryl derivative
XIII in satisfactory yields. The ring closure of XIII to
intermediate XIV was performed by reductive cyclization
using iron in acetic acid and ethanol under reflux.
Debenzylation of XIV in a manner similar to that for the
synthesis of 80 yielded the deaza derivative 81 (Scheme 3).
The target 9-deaza derivatives 82−84 were prepared by the
same synthetic route reported for compound 81 starting from
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a
Scheme 2. Preparation of 8-Pyrazolylxanthines 79 and 80
a
Reagents and conditions: (i) benzyl bromide, K2CO3, acetone, rt; (ii) KOH, dioxane, rt; (iii) (1) 5,6-diamino-1,3-dipropyl-1H-pyrimidine-2,4-
dione, EDC, methanol, rt, (2) NaOH, 80 °C; (iv) Pd/C, HCOONH4, methanol, reflux; (v) SEM-Cl, K2CO3, DMF, 70 °C; (vi) K2CO3, acetone;
(vii) HCl, ethanol, 80 °C.
a
Scheme 3. Preparation of Deaza-xanthine 81
a
Reagents: (i) (1) ethyl chloroformate, TEA, NaBH4, THF, −10 °C, (2) PCC, CH2Cl2, rt; (ii) morpholine, pTsOH, toluene, reflux; (iii) Fe,
CH3COOH, ethanol, reflux; (iv) HCOONH4, Pd/C, methanol, reflux.
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a
Scheme 4. Preparation of Deaza-xanthines 82−84
a
Reagents and conditions: (i) (1) ethyl chloroformate, TEA, NaBH4, THF, −10 °C, (2) PCC, CH2Cl2, rt; (ii) morpholine, pTsOH, toluene, reflux;
(iii) Fe, CH3COOH, ethanol, reflux; (iv) NaOH 10%, methanol; (v) (1) 1,2-phenylendiamine, EDC, DMF, rt, (2) CH3COOH, 60 °C; (vi) aniline,
EDC, DMF, reflux.
CoMFA studies. Namely, the chosen putative bioactive
conformations of 30 were those placed in the two clusters
with the greatest number of low energy conformations of 22,
23, and 51. The statistical results of the PLS analysis were
satisfactory; the best model was obtained with five latent
variables (components) showing q2 = 0.655, scross = 0.290, r2 =
0.901, and s = 0.155 (see Supporting Information for further
details).
Steric (green and yellow) and electrostatic (blue and red)
CoMFA contours are shown in Figures 1 and 2. The regions of
the space around the molecule describe where the steric and
the electrostatic fields have the greatest effect on binding
affinity as predicted by the CoMFA model. The green and
yellow zones represent regions where an increase of steric bulk
increases or decreases the biological activity, respectively. The
red area represents a region, where negatively charged groups
favor the activity, while the blue one indicates a favorable
contribution from positive groups.
The green CoMFA contour (Figure 1) located around the
phenyl ring may be attributed mainly to the N1-methyl-pyrazole
oxyacetamide derivatives (28−40, Supporting Information
Table S1). One of the three sterically unfavorable regions
(yellow in Figure 2a, left) is close to the phenyl rings of
structurally different compounds: pyrazole acetamide (7−15,
19−21), urea (25−27), and pyridine oxyacetamide (5)
derivatives (Supporting Information Table S1). In particular,
the phenyl moiety of 5 (relatively a low potency compound,
orange in Figure 2a, left), protrudes completely into this
sterically negative contour map. Moreover, negative steric
contours are located around the N1-methyl-pyrazole oxy-
acetamide derivatives, which bear bulky groups in the
paraposition of the phenyl ring (32, 34, 35) (Figure 2a,
right). Electrostatic negative regions are close to both the
negatively charged oxygen atom of the carbonyl group and to
the phenyl ring of N1-methyl-pyrazole oxyacetamide xanthine
derivatives (28−40) (Figure 2b). This means that in such a
region, where relatively more potent compounds containing
electron-releasing functions orient their aromatic ring, an
increase of negative electrostatic potential should provide
more potent A2B antagonists. Concerning the positive electro-
static CoMFA contours, the main region is located around the
amide hydrogen atoms of N1-methyl-pyrazole oxyacetamide
derivatives (28−40) (Figure 2b).
The present CoMFA results are in a good agreement with
the experimental data reported in the literature.28 For instance,
the good biological activity of the pyrazole oxyacetamide
derivative 30 may be well explained on the basis of the present
3D QSAR model, considering that the negatively charged
oxygen atom of the carbonyl group point toward the negative
electrostatic regions, while the N-benzodioxol-phenyl structure
is placed in a sterically and electrostatically favorable region
(Figure 1a). In addition, the lower biological activity of 43 and
44 as compared to 28 and 29 might be due to the presence of
the methyl substitutent on the N2-position of the pyrazole
structure, which forces the flexible chains of 43 (Figure 1b) and
44 far from the electrostatic and steric positive regions.
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Structure−Activity Relationships. Binding affinities of
the synthesized compounds for A1, A2A, A3, and A2BARs are
presented in Tables 1−3. A general comparison of similarly
substituted phenyl analogues between the pyrazole and
isoxazole series (e.g., 56 vs 71, 59 vs 73, etc.) favors the
selectivity of the pyrazole series for A2BARs versus A1ARs.
As shown in Table 1, the parent unsubstituted phenyl
analogue 54 was highly potent at the human A2BAR, with a Ki
value of 18 nM, although not selective for A2BAR vs A1AR
(hA1/hA2B = 12). The 5-substituted-phenyl analogues 55−60
and 62−64 showed high affinity at the A2BAR, and the most
potent compound was the 5-Cl derivative 56 (Ki A2B = 5 nM).
Introducing a stronger electron-withdrawing group (Br or CF3)
in place of the 5-Cl group, as in compounds 58 and 59, resulted
in a 2−3-fold loss in binding affinity (58, Ki = 15.9 nM; 59, Ki =
8.1 nM). However, compound 59 showed improved selectivity
for the A2BAR versus the A1AR. The 5-F derivative 55 exhibited
lower affinity and selectivity in comparison to 5-Cl 56. The 5-
nitro analogue 62 showed 3-fold lower affinity although
comparable selectivity for the A2BAR relative to the 5-Cl
analogue 56.
Introduction of substituents containing a carbonyl function,
such as the 5-carboxylic acid ethyl ester 63, produced good
affinity and selectivity for A2BAR (Ki = 12 nM A1/A2B = 42).
The carboxylic acid derivative 64 exhibited less affinity at both
A1 and A2BAR (hA2B Ki = 88 nM, A1/A2B = 52).
The 5-methoxy-phenyl analogue 57 and the 5-methyl
analogue 60 had favorable binding affinities at the A2BAR
(hA2B Ki = 14.2 nM, hA2B Ki = 10.2 nM, respectively). They
also showed greater selectivity for the A2BAR versus the A1AR,
relative to 56. Shifting the methyl group from the 5- to the 4-
position in analogue 65 had no significant effect on affinity or
selectivity for the A2BAR versus A1AR. The 5,6-dimethyl
analogue 69 was slightly less potent at A2BAR (Ki = 22 nM),
with lower selectivity versus A1AR, compared with 5-methyl
analogue 60.
Figure 1. CoMFA stdev*coeff contour plots (model A). Green
contours (0.0162 level): regions where bulky groups increase activity.
Yellow contours (−0.0100 level): regions where bulky groups decrease
activity. Red contours (0.0150 level): regions where negative groups
increase activity. Blue contours (−0.0150 level): regions where
positive charge increases activity. (a) The molecule shown is the
most potent compound (30). (b) The molecules shown are 28 and 43
(carbon atoms are orange).
On the basis of the CoMFA model, the new xanthine
derivatives 53 and 54 (Chart 2) were designed and synthesized,
and a fairly good agreement was found between the calculated
and observed pKi values (7.31 vs 7.77 and 7.74 vs 7.71,
respectively; Chart 2).
Interestingly, replacement of the phenyl ring with the
naphthyl nucleus produced compound 61, which had similar
Figure 2. CoMFA stdev*coeff contour plots (model A). Some aligned molecules are displayed. (a) Left: compounds 5 (carbon and hydrogen atoms
are in orange), 7−15, 19−21, 25−27. Right: compounds 32,34, and 35. (b) Compounds 28−40.
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Table 1. Chemical Structures and Biological Data of Pyrazolo Xanthine Derivatives 54−69
a
The data are expressed as the geometric mean with 95% confidence limits in parentheses and derived from inhibition binding experiments and
b
c
cAMP assays as described in the Experimental Section. Displacement of specific [3H]DPCPX binding to hA1ARs (n = 3−6). Displacement of
d
specific [3H]ZM241385,binding to hA2AARs (n = 3−6). Displacement of specific [3H]MRE2029-F20 binding at hA2BARs (n = 3−6).
e
Displacement of specific [3H]MRE3008-F20 binding to hA3ARs (n = 3−6). Data are expressed as geometric means with 95% confidence limits.
binding affinity (Ki = 20 nM) as the unsubstituted phenyl
analogue 54, while it showed nearly 10-fold more selectivity for
the A2BAR versus A1AR, suggesting the presence of an
increased binding domain in the A2BAR versus the A1AR.
The 4,6-disubstituted analogue 66 showed high affinity and
selectivity at A2BAR. In fact, compound 66 (4-Cl, 6-CF3) is the
most selective analogue among the two classes of synthesized
compounds (Ki = 9.4 nM A1/A2B = 269). Introducing a
stronger electron-withdrawing group (CF3) in place of the 4-Cl
group, such as in compound 67 (4,6-CF3), resulted in a 3-fold
loss in binding affinity (Ki =25 nM). The 5-Cl, 6-F analogue 68
displayed similar affinity as the 5,6-dimethyl-substituted
analogue 69, even though the 5,6-dimethyl substitution
produced a 3-fold increase in selectivity versus the A1AR.
In general, the benzimidazole-isoxazole series was less
selective for the A2BAR versus the A1AR when compared to
the benzimidazole-pyrazole class of compounds. These data are
in contrast with the pyrazole-oxyacetamide derivatives synthe-
sized in our previous work28 that were more selective when
compared to the pyrazole parent compounds. A similar trend
was observed when comparing the isoxazole analogue 70 to the
pyrazole analogue 54. The 5-halogen analogues 71−73
displayed similar affinities at the A2BAR compared to that of
the 5-halogen-substituted pyrazole analogues (55, 56, 58) but
were slightly less selective versus the A1AR. A noticeable
exception was the 5-bromo analogue 72 that was 4-fold more
active at A2BAR in comparison to analogue 58. Further
substitution of the 5-position of the phenyl ring by a methoxy
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Table 2. Chemical Structures and Biological Data of 8-Heterocyclyl Xanthine Derivatives 70−77
a
Ki (nM)
b
c
d
e
a
compd
R
hA1AR
hA2AAR
hA2BAR
hA3AR
hA1/A2B
cAMP hA2BAR IC50 (nM)
70
71
72
73
74
75
76
77
H
277 (227−338)
66 (57−76)
89 (77−104)
175 (134−229)
304 (257−361)
65 (55−78)
>1,000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
23 (17−30)
3.5 (2.9−4.2)
4.1 (2.9−5.7)
10.0 (7.5−13.1)
35 (27−47)
5.5 (4.8−6.3)
22 (16-31)
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
12
19
74 (65−84)
6.4 (4.9−8.3)
11 (7−17)
51 (38−68)
114 (86−151)
8.0 (7.2−8.9)
64 (50−81)
197 (136−287)
5-Cl
5-Br
22
5-CF3
18
5-OCH3
5-Cl, 6-F
4-Cl, 6-CF3
4,6 CF3
9
12
>50
>13
>1,000
76 (65−87)
a
The data are expressed as the geometric mean with 95% confidence limits in parentheses and derived from inhibition binding experiments and
b
c
cAMP assays as described in the Experimental Section. Displacement of specific [3H]DPCPX binding to hA1ARs (n = 3−6). Displacement of
d
specific [3H]ZM241385 binding to hA2AARs (n = 3−6). Displacement of specific [3H]MRE2029-F20 binding at hA2BARs (n = 3−6).
e
Displacement of specific [3H]MRE3008-F20 binding to hA3ARs (n = 3−6). Data are expressed as geometric means with 95% confidence limits.
group produced compound 74 that was both less potent (hA2B
Ki =35 nM) than 57 (hA2B Ki =14.2 nM) and less selective
versus the A1AR.
The deaza analogue 81, compared to 80, displayed 2-fold less
affinity at A2BAR and 10-fold less selectivity versus A1AR. The
deaza analogue 82 showed an affinity comparable to the
xanthine derivative 54, although it was 4-fold less selective
versus A1AR. The deaza oxyacetamides 83 and 84 showed less
affinity and selectivity at A2BAR relative to the corresponding
xanthine analogues 28 and 30.
The compounds were also tested in cAMP assays, evaluating
their capability to inhibit NECA-stimulated cAMP production
(Tables 1−3). The majority of these ligands showed IC50 values
in the nanomolar range, suggesting good potency in cAMP
assays. Figure 3B shows concentration−response curves of
some of the most interesting compounds.
The introduction of a fluorine in the ortho-position of the 5-
chloro analogue 71 produced nonsignificant effects on affinity
and selectivity at A2BAR, as shown by analogue 75. In the
isoxazole series, as with the pyrazole analogues, the most
selective compound was the 4-Cl,6-CF3-disubstituted phenyl
analogue 76 (hA2B Ki = 22 nM, hA1/hA2B > 50). Introduction
of a trifluoromethyl group at the 4- and 6-positions produced a
decrease of affinity and selectivity, such as in compound 77 in
comparison with analogue 67.
The enlargement of the benzimidazole nucleus, suggested
from modeling studies, did not improve affinity at the A2BAR,
as demonstrated by 4-oxo-3,4-dihydro-quinazoline 53 (Table
1) but did increase the selectivity 3-fold versus A1AR.
Replacement of the phenyl ring with a pyrimidine, such as in
compound 78, afforded a complete loss of affinity at the A2BAR.
The benzimidazole nucleus was also replaced by 5-m-
methoxyphenyl-1,2,4-oxadiazole to afford analogue 79 (Ki =
61 nM). This analogue displayed 5-fold lower affinity compared
to the 5-(methoxy)benzimidazole analogue 57 (Tables 1 and2;
Figure 3A).
All ligands tested in functional assays in vitro showed very
interesting antagonist activities to hA2BAR, in good agreement
with their affinities measured in the binding assays. (Figure 3C)
CONCLUSION
■
In this paper, we constructed a 3D QSAR model for xanthine
antagonists at the A2BAR. Such a model was then validated by
synthesizing two new xanthine compounds which displayed
good agreement between the observed and calculated pKi
values.
The 8-(3-hydroxy-1-methyl-1H-pyrazol-5-yl)-xanthine 80
was synthesized as a simplified chemical structure of the
pyrazole-xanthine series. This compound displayed very high
affinity at A2BAR with good selectivity over A1AR. (Table 3, Ki
= 4.0 nM, hA1/hA2B = 183; Figure 3A).
The 9-deaza-xanthines 81−84, differing by the absence of a
nitrogen atom at the 9-position of the heterocyclic xanthine
system, were synthesized based on their noted antagonistic
activity at the A2B receptor.30,34 The AR binding affinities of the
deaza analogues, in comparison with the corresponding
xanthines, are reported in Table 3. The analogues 83 and 85
were prepared comparing to those recently published 8-
pyrazolyl-oxyacetamide derivatives24,28 28 and 30.
To further explore the potential of the 8-pyrazolyl-xanthine
chemotype, we synthesized a series of novel potent A2BAR
antagonists by varying the pyrazole moiety. Compound 66 (8-
[3-(4-chloro-6-trifluoromethyl-1H-benzoimidazol-2-yl-me-
thoxy)-1-methyl-1H-pyrazol-5-yl]-1,3-dipropyl-xanthine) dis-
played high affinity (Ki = 9.4 nM) and selectivity for the
A2BAR relative to the A1, A2A, and A3ARs. In addition, this study
led to the identification of compound 80 (8-(3-hydroxy-1-
methyl-1H-pyrazol-5-yl)-1,3-dipropylxanthine; Ki = 4 nM). To
the best of our knowledge, this compound has the highest
reported affinity and selectivity described, thus far. These target
compounds show high potency in inhibiting the accumulation
of NECA-induced cAMP in HEK-A2BAR, with IC50 values of 26
and 20 nM, respectively. These selective A2BAR antagonists can
be useful in understanding the physiological role of the A2BAR
In general, the new deaza analogues showed less selectivity
over A1AR, even though they displayed high affinity at A2BAR.
805
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Article
compounds revealed a purity of not less than 95%. The mass spectra
were obtained on a ESI Micromass ZMD 2000 mass spectrometer.
8-{1-Methyl-3-[(4-oxo-1,4-dihydroquinazolin-2-yl)methoxy]-
1H-pyrazol-5-yl}-1,3-dipropyl-3,7-dihydro-1H-purine-2,6-
dione (53). To a solution of I (100 mg, 0.25 mmol) and EDC (60
mg, 0.30 mmol) in DMF (5 mL) was added 2-aminobenzamide (35
mg, 0.25 mmol). The reaction mixture was stirred at room
temperature for 2 h. The solvent was evaporated in vacuo and the
residue poured into water. The amide intermediate was filtered and
reacted with NaOH (2.5 N, 4 mL) at 80 °C for 2 h. After cooling to 0
°C, the reaction was acidified to pH 2−3, the resultant solid was
filtered and washed with water. The residue was purified by column
chromatography (ethyl acetate/methanol 9:1). Yield, 60%; mp >300
°C.
1H NMR (200 MHz, DMSO-d6): δ 0.82−0.91 (m, 6H), 1.56 (q, J =
7.4 Hz, 2H), 1.72 (q, J = 7.0 Hz, 2H), 3.85 (t, J = 7.2 Hz, 2H), 4.03 (t,
J = 6.6 Hz, 2H), 4.07 (s, 3H), 5.08 (s, 2H), 6.50 (s, 1H), 7.52−8.17.82
(m, 3H), 8.10−8.14 (m, 1H), 12.55 (bs, 1H), 13.88 (bs, 1H). MS m/z
491 (M H+). Anal. (C24H26N8O4) C, H, N.
8-[3-(1H-Benzoimidazol-2-ylmethoxy)-1-methyl-1H-pyrazol-
5-yl]-1,3-dipropyl-3,7-dihydro-purine-2,6-dione 54−69 and 8-
[3-(1H-Benzoimidazol-2-ylmethoxy)-isoxazol-5-yl]-1,3-diprop-
yl-3,7-dihydro-purine-2,6-dione (70−77). General Procedure.
(Except Compound 64 That Was Prepared from 63). To a mixture
of the carboxylic acid (I or II, see Scheme 1) (0.25 mmol), EDC (0.25
mmol), and anhydrous DMF (5 mL) was added the appropriate
phenylenediamine (0.25 mmol), and the mixture was stirred at room
temperature for 20 h under argon atmosphere. The resulting solution
was evaporated under reduced pressure, and the residue was dissolved
in acetic acid (5 mL) and heated under stirring at 60−70 °C for 1 h.
The resulting solution was cooled to room temperature and poured
into water. The precipitate was collected by filtration and washed with
water . The resulting crude was purified by flash column
chromatography on silica gel or crystallized when necessary.
8-[3-(1H-Benzoimidazol-2-ylmethoxy)-1-methyl-1H-pyrazol-
5-yl]-1,3-dipropyl-3,7-dihydro-purine-2,6-dione (54). Purifica-
tion by crystallization from dioxane. Yield 96%; mp 290 °C. 1H
NMR (200 MHz, DMSO-d6): δ 0.83−0.90 (m, 6H), 1.53 (q, J = 7.2
Hz, 2H), 1.70 (q, J = 7.2 Hz, 2H), 3.84 (t, J = 7.8 Hz, 2H), 3.98 (t, J =
7.0 Hz, 2H), 4.08 (s, 3H), 5.35 (s, 2H), 6.53 (s, 1H), 7.16−7.75 (m,
4H), 12.63 (bs, 1H), 14.00 (bs, 1H). MS m/z 443 (M H+). Anal.
(C23H26N8O3) C, H, N.
Figure 3. Competition binding and functional assays. (A) Affinity (Ki)
of selected novel adenosine compounds. (B) Potency (IC50) of
selected novel adenosine compounds. (C) Correlation between
binding and functional parameters of the novel adenosine compounds.
8-[3-(5-Fluoro-1H-benzoimidazol-2-ylmethoxy)-1-methyl-
1H-pyrazol-5-yl]-1,3-dipropyl-3,7-dihydro-purine-2,6-dione
(55). Purification by using column chromatography (ethyl acetate).
Yield 48%; mp 280 °C.
1H NMR (200 MHz, DMSO-d6): δ 0.83−0.92 (m, 6H), 1.57 (q, J =
7.0 Hz, 2H), 1.72 (q, J = 7.2 Hz, 2H), 3.86 (t, 2H), 4.01 (t, 2H), 4.15
(s, 3H), 5.36 (s, 2H), 6.54 (s, 1H), 7.00−7.68 (m, 3H), 12.77 (bs,
1H), 13.97 (bs, 1H). MS m/z 481 (M H+). Anal. (C23H25FN8O3) C,
H, N.
and may serve as leads toward the discovery of therapeutically
useful agents for the treatment of asthma.
EXPERIMENTAL SECTION
■
8-{3-[(5-Chloro-1H-benzimidazol-2-yl)methoxy]-1-methyl-
1H-pyrazol-5-yl}-1,3-dipropyl-3,7-dihydro-1H-purine-2,6-
dione (56). Purification by crystallization from dioxane/Et2O. Yield
52%; mp 285−286 °C.
Reagent grade solvents were dried according to standard techniques.
Sodium sulfate was used as a drying agent for water containing organic
phases. All reported yields are of isolated products and are not
optimized. Reactions were routinely monitored by thin-layer
chromatography (TLC) on silica gel (F245 Merck plates). Chromato-
graphic spots were visualized by UV light. Purification of crude
compounds and separation of reaction mixtures were carried out by
column chromatography on silica gel 60 (230−400 mesh from
Merck). Melting points (uncorrected) were determined in a 240
Buchi-Tottoli melting point apparatus. Chemical shifts (δ) are
reported in parts per million (ppm) relative to the solvent central
1H NMR (200 MHz, DMSO-d6): δ 0.83−0.92 (m, 6H), 1.57 (q, J =
7.2 Hz, 2H), 1.73 (q, J = 7.6 Hz, 2H), 3.86 (t, J = 7.2 Hz, 2H), 4.01 (t,
J = 7.2 Hz, 2H), 4.12 (s, 3H), 5.38 (s, 2H), 6.53 (s, 1H), 7.21−7.67
(m, 3H), 12.92 (bs, 1H), 13.97 (bs, 1H). MS m/z 497 (M H+). Anal.
(C23H25ClN8O3) C, H, N.
8-[3-(5-Methoxy-1H-benzoimidazol-2-ylmethoxy)-1-methyl-
1H-pyrazol-5-yl]-1,3-dipropyl-3,7-dihydro-purine-2,6-dione
(57). Purification by crystallization from dioxane/water. Yield 55%;
mp 271 °C.
1
peak. H NMR spectra were recorded at 200 MHz on a Bruker AC
1H NMR (200 MHz, DMSO-d6): δ 0.83−0.92 (m, 6H), 1.57 (q, J =
7.2 Hz, 2H), 1.73 (q, J = 7.2 Hz, 2H), 3.77 (s, 3H), 3.86 (t, J = 7.2 Hz,
2H), 4.00 (t, J = 7.0 Hz, 2H), 4.10 (s, 3H), 5.31(s, 2H), 6.53 (s, 1H),
6.79−7.58 (m, 3H), 12.48 (bs, 1H), 13.97 (bs, 1H). MS m/z 493 (M
H+). Anal. (C24H28N8O4) C, H, N.
200 spectrometer. Electron spray ionization mass spectrometry (ESI/
MS) was performed with an Agilent 1100 series LC/MSD model in
positive scan mode. The molecular weights from the MS spectra were
in full agreement with the proposed chemical structures of target
compounds. Elemental analyses were performed by the microanalytical
laboratory of Dipartimento di Chimica, University of Ferrara, and were
within 0.4% of the theoretical values for C, H, and N. All final
8-[3-(5-Bromo-1H-benzoimidazol-2-ylmethoxy)-1-methyl-
1H-pyrazol-5-yl]-1,3-dipropyl-3,7-dihydro-purine-2,6-dione
806
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Table 3. Chemical Structures and Binding Affinities (Ki) at AR of Various Sets of Xanthines and Corresponding 9-Deaza
f
Derivatives
a
The data are expressed as the geometric mean with 95% confidence limits in parentheses and derived from inhibition binding experiments and
b
c
cAMP assays as described in the Experimental Section. Displacement of specific [3H]DPCPX binding to hA1ARs (n = 3−6). Displacement of
d
e
specific [3H]ZM241385 binding to hA2AARs (n = 3−6). Displacement of [3H]MRE2029-F20 binding at hA2BARs (n = 3−6). Displacement of
f
specific [3H]MRE3008 F20 binding to hA3ARs (n = 3−6). Data are expressed as geometric means with 95% confidence limits. *Numbered in the
Supporting Information.
(58). Purification by crystallization from dioxane/water. Yield 52%;
mp 299 °C.
J = 6.6 Hz, 2H), 4.09 (s, 3H), 5.44 (s, 2H), 6.55 (s, 1H), 7.50−8.00
(m, 3H), 13.00 (bs, 1H), 13.99 (bs, 1H). MS m/z 531 (M H+). Anal.
(C24H25F3N8O3) C, H, N.
1H NMR (200 MHz, DMSO-d6): δ 0.85−0.92 (m, 6H), 1.58 (q, J =
7.2 Hz, 2H), 1.75 (q, J = 7.2 Hz, 2H), 3.85 (t, J = 7.0 Hz, 2H), 4.00 (t,
J = 7.2 Hz, 2H), 4.09 (s, 3H), 5.37 (s, 2H), 6.66 (s, 1H), 7.22−7.80
(m, 3H), 12.95 (bs, 1H), 12.99 (bs, 1H). MS m/z 541 (M H+). Anal.
(C23H25BrN8O3) C, H, N.
8-{1-Methyl-3-[(5-methyl-1H-benzimidazol-2-yl)methoxy]-
1H-pyrazol-5-yl}-1,3-dipropyl-3,7-dihydro-1H-purine-2,6-
dione (60). Purification by using column chromatography (ethyl
acetate). Yield 86%; mp 276 °C.
8-(1-Methyl-3-{[5-(trifluoromethyl)-1H-benzimidazol-2-yl]-
methoxy}-1H-pyrazol-5-yl)-1,3-dipropyl-3,7-dihydro-1H-pu-
rine-2,6-dione (59). Purification by using column chromatography
(ethyl acetate). Yield 50%; mp 299 °C.
1H NMR (200 MHz, DMSO-d6): δ 0.830.92 (m, 6H), 1.57 (q, J =
7.2 Hz, 2H), 1.73 (q, J = 7.2 Hz, 2H), 2.4 (s, 3H), 3.86 (t, J = 7.2 Hz,
2H), 4.00 (t, J = 7.2 Hz, 2H), 4.10 (s, 3H), 5.33 (s, 2H), 6.48 (s, 1H),
6.93−7.00 (m, 1H), 7.27−7.49 (m, 2H), 12.5 (bs, 1H), 13.96 (bs,
1H). MS m/z 477 (M H+). Anal. (C24H28N8O3) C, H, N.
1H NMR (200 MHz, DMSO-d6): δ 0.83−0.92 (m, 6H), 1.57 (q, J =
7.0 Hz, 2H), 1.71 (q, J = 6.8 Hz, 2H), 3.82 (t, J = 7.2 Hz, 2H), 3.89 (t,
807
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dione (68). Purification by crystallization from dioxane. Yield 55%;
mp 288 °C.
8-[1-Methyl-3-(1H-naphtho[2,3-d]imidazol-2-ylmethoxy)-
1H-pyrazol-5-yl]-1,3-dipropyl-3,7-dihydro-1H-purine-2,6-
dione (61). Purification by crystallization from DMF. Yield 85%; mp
297 °C. 1H NMR (200 MHz, DMSO-d6): δ 0.83−0.92 (m, 6H), 1.55
(q, J = 7.2 Hz, 2H), 1.71 (q, J = 7.0 Hz, 2H), 3.86 (t, J = 7.0 Hz, 2H),
4.00 (t, J = 7.0 Hz, 2H), 4.10 (s, 3H), 5.47 (s, 2H), 6.58 (s, 1H), 7.35−
7.39 (m, 2H), 7.97−8.16 (m, 4H), 12.73 (bs, 1H), 13.98 (bs, 1H)).
MS m/z 513 (M H+). Anal. (C27H28N8O3) C, H, N.
1H NMR (200 MHz, DMSO-d6): δ 0.83−0.92 (m, 6H), 1.55 (q, J =
7.4 Hz, 2H), 1.78 (q, J = 7.2 Hz, 2H), 3.84 (t, J = 6.6 Hz, 2H), 3.99 (t,
J = 7.0 Hz, 2H), 4.07 (s, 3H), 5.35 (s, 2H), 6.50 (s, 1H), 7.88 (s, 2H),
13.00 (bs, 1H), 14.00 (bs, 1H)). MS m/z 515 (M H+). Anal.
(C23H24ClFN8O3) C, H, N.
8-{3-[(5,6-Dimethyl-1H-benzimidazol-2-yl)methoxy]-1-
methyl-1H-pyrazol-5-yl}-1,3-dipropyl-3,7-dihydro-1H-purine-
2,6-dione (69). Purification by crystallization from methanol. Yield
75%; mp 281 °C.
8-{1-Methyl-3-[(6-nitro-1H-benzimidazol-2-yl)methoxy]-1H-
pyrazol-5-yl}-1,3-dipropyl-3,7-dihydro-1H-purine-2,6-dione
(62). Purification by crystallization from dioxane. Yield 58%; mp 263
°C.
1H NMR (200 MHz, DMSO-d6): δ 0.83−0.92 (m, 6H), 1.58 (q, J =
7.2 Hz, 2H), 1.70 (q, J = 7.4 Hz, 2H), 2.29 (s, 6H), 3.84 (t, J = 6.8 Hz,
2H), 4.00 (t, J = 7.0 Hz, 2H), 4.10 (s, 3H), 5.31 (s, 2H), 6.53 (s, 1H),
7.30 (s, 2H), 12.45 (bs, 1H), 14.00 (bs, 1H). MS m/z 491 (M H+).
Anal. (C25H30N8O3) C, H, N.
1H NMR (400 MHz, DMSO-d6): δ 0.85−0.90 (m, 6H), 1.57 (q, J =
7.6 Hz, 2H), 1.72 (q, J = 7.2 Hz, 2H), 3.86 (t, J = 7.0 Hz, 2H), 3.99 (t,
J = 7.8 Hz, 2H), 4.01 (s, 3H), 5.49 (s, 2H), 6.57 (s, 1H), 7.75 (d, J =
8.8 Hz, 1H), 8.13 (dd, J1 = 8.8, J2 = 2.4, Hz, 1H), 8.48 (d, J = 2.0 Hz,
1H), 12.95 (bs, 1H), 14.00 (bs, 1H). MS m/z 508 (M H+). Anal.
(C23H25N9O5) C, H, N.
8-[3-(1H-Benzimidazol-2-ylmethoxy)isoxazol-5-yl]-1,3-di-
propyl-3,7-dihydro-1H-purine-2,6-dione (70). Purification by
crystallization from methanol. Yield 70%; mp 282 °C.
1H NMR (200 MHz, DMSO-d6): δ 0.85−0.92 (m, 6H), 1.58 (q, J =
7.2 Hz, 2H), 1.80 (q, J = 7.6 Hz, 2H), 3.98 (t, J = 7.2 Hz, 2H), 4.10 (t,
J = 7.8 Hz, 2H), 5.53 (s, 2H), 6.97 (s, 1H), 7.20−7.59 (m, 4H), 12.76
(bs, 1H), 14.59 (bs, 1H). MS m/z 450 (M H+). Anal. (C22H23N7O4)
C, H, N.
8-{3-[(5-Chloro-1H-benzimidazol-2-yl)methoxy]isoxazol-5-
yl}-1,3-dipropyl-3,7-dihydro-1H-purine-2,6-dione (71). Purifica-
tion by crystallization from methanol. Yield 68%; mp 260 °C.
1H NMR (200 MHz, DMSO-d6): δ 0.83−0.92 (m, 6H), 1.55 (q, J =
7.6 Hz, 2H), 1.70 (q, J = 7.2 Hz, 2H), 3.86 (t, J = 7.4 Hz, 2H), 3.94 (t,
J = 7.2 Hz, 2H), 5.54 (s, 2H), 6.99 (s, 1H), 7.20−7.77 (m, 3H), 13.00
(bs, 1H), 14.55 (bs, 1H). MS m/z 484 (M H+). Anal.
(C22H22ClN7O4) C, H, N.
8-{3-[(5-Bromo-1H-benzimidazol-2-yl)methoxy]isoxazol-5-
yl}-1,3-dipropyl-3,7-dihydro-1H-purine-2,6-dione (72). Purifica-
tion by crystallization from methanol. Yield 70%; mp 230 °C.
1H NMR (200 MHz, DMSO-d6): δ 0.82−0.92 (m, 6H), 1.58 (q, J =
7.6 Hz, 2H), 1.72(q, J = 7.2 Hz, 2H), 3.89 (t, J = 7.2 Hz, 2H), 3.98 (t, J
= 7.4 Hz, 2H), 5.54 (s, 2H), 6.98 (s, 1H), 7.36−7.80 (m, 3H), 13.00
(bs, 1H), 14.60 (bs, 1H). MS m/z 529 (M H+). Anal.
(C22H22BrN7O4) C, H, N.
2-({[5-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-
8-yl)-1-methyl-1H-pyrazol-3-yl]oxy}methyl)-1H-benzimida-
zole-5-carboxylic Acid Ethyl Ester (63). Purification by crystal-
lization from methanol. Yield 63%; mp >300 °C.
1H NMR (200 MHz, DMSO-d6): δ 0.83−0.92 (m, 6H), 1.32 (t, J =
7.0 Hz, 3H), 1.55 (q, J = 7.4 Hz, 2H), 1.72 (q, J = 7.2 Hz, 2H), 3.86 (t,
J = 7.4 Hz, 2H), 3.97 (t, J = 7.6 Hz, 2H), 4.10 (s, 3H), 4.34 (q, J = 7.2
Hz, 2H), 5.42 (s, 2H), 6.53 (s, 1H), 7.76−8.22 (m, 3H), 13.00 (bs,
1H), 13.99 (bs, 1H). MS m/z 535 (M H+). Anal. (C26H30N8O5) C, H,
N.
2-({[5-(2,6-Dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-
8-yl)-1-methyl-1H-pyrazol-3-yl]oxy}methyl)-1H-benzimida-
zole-5-carboxylic Acid (64). The ester 63 (0.15 mmol) was
dissolved in a mixture of 2:1 MeOH/10% NaOH. The solution was
stirred at 50 °C for 3 h. The MeOH was evaporated, and the aqueous
solution was acidified to pH 4−5 by using 10% HCl. The precipitate
formed was collected by filtration, washed with water, and air-dried
under vacuum to yield the corresponding carboxylic acid 64, which
was purified by crystallization (dioxane). Yield, 68%; mp >300 °C.
1H NMR (200 MHz, DMSO-d6): δ 0.83−0.92 (m, 6H), 1.33 (q, J =
7.0 Hz, 2H), 1.77 (q, J = 7.2 Hz, 2H), 4.01 (t, J = 7.0 Hz, 2H), 4.05 (t,
J = 7.2 Hz, 2H), 4.10 (s, 3H), 5.41 (s, 2H), 6.66 (s, 1H), 7.82−8.20
(m, 3H), 12.99 (s, 1H), 13.00 (bs, 1H), 13.99 (bs, 1H). MS m/z 507
(M H+). Anal. (C24H26N8O5) C, H, N.
1,3-Dipropyl-8-(3-{[5-(trifluoromethyl)-1H-benzimidazol-2-
yl]methoxy}isoxazol-5-yl)-3,7-dihydro-1H-purine-2,6-dione
(73). Purification by crystallization from methanol. Yield 65%; mp 268
°C.
8-{1-Methyl-3-[(4-methyl-1H-benzimidazol-2-yl)methoxy]-
1H-pyrazol-5-yl}-1,3-dipropyl-3,7-dihydro-1H-purine-2,6-
dione(65). Purification by using column chromatography (ethyl
acetate−methanol 9.5:0.5). Yield 86%; mp 276−277 °C.
1H NMR (200 MHz, DMSO-d6): δ 0.83−0.92 (m, 6H), 1.55 (q, J =
7.6 Hz, 2H), 1.69 (q, J = 7.2 Hz, 2H), 3.86 (t, J = 7.6 Hz, 2H), 3.98 (t,
J = 7.4 Hz, 2H), 5.61 (s, 2H), 7.00 (s, 1H), 7.66−8.00 (m, 3H), 13.20
(bs, 1H), 14.70 (bs, 1H). MS m/z 518 (M H+). Anal. (C23H22F3N7O4)
C, H, N.
1H NMR (200 MHz, DMSO-d6): δ 0.83−0.92 (m, 6H), 1.57 (q, J =
7.2 Hz, 2H), 1.73 (q, J = 7.4 Hz, 2H), 2.41 (s, 3H), 3.86 (t, J = 6.6 Hz,
2H), 3.98 (t, J = 7.4 Hz, 2H), 4.12 (s, 3H), 5.33 (s, 2H), 6.54 (s, 1H),
6.99−7.32 (m, 3H), 12.58 (bs, 1H), 13.97 (bs, 1H). MS m/z 477 (M
H+). Anal. (C24H28N8O3) C, H, N.
8-{3-[(5-Methoxy-1H-benzimidazol-2-yl)methoxy]isoxazol-
5-yl}-1,3-dipropyl-3,7-dihydro-1H-purine-2,6-dione (74). Purifi-
cation by crystallization from methanol. Yield 75%; mp 199 °C.
1H NMR (200 MHz, DMSO-d6): δ 0.83−0.92 (m, 6H), 1.59 (q, J =
7.6 Hz, 2H), 1.69 (q, J = 7.4 Hz, 2H), 3.78 (s, 3H), 3.86 (t, J = 7.8 Hz,
2H), 3.98 (t, J = 7.6 Hz, 2H), 5.48 (s, 2H), 6.86 (dd, J1 = 8.8 Hz, J2 =
2.2 Hz, 1H), 6.97 (s, 1H), 7.46 (d, J = 8.8 Hz, 1H), 13.01 (bs, 1H),
14.30 (bs, 1H). MS m/z 480 (M H+). Anal. (C23H25N7O5) C, H, N.
8-{3-[(5-Chloro-6-fluoro-1H-benzimidazol-2-yl)methoxy]-
isoxazol-5-yl}-1,3-dipropyl-3,7-dihydro-1H-purine-2,6-dione
(75). Purification by crystallization from methanol. Yield 55%; mp 249
°C.
8-(1-Methyl-3-{[4-chloro-6-(trifluoromethyl)-1H-benzimida-
zol-2-yl]methoxy}-1H-pyrazol-5-yl)-1,3-dipropyl-3,7-dihydro-
1H-purine-2,6-dione (66). Purification by crystallization from
methanol. Yield 48%; mp 267 °C.
1H NMR (200 MHz, DMSO-d6): δ 0.81−0.91 (m, 6H), 1.55 (q, J =
7.4 Hz, 2H), 1.72 (q, J = 7.2 Hz, 2H), 3.83 (t, J = 7.2 Hz, 2H), 3.96 (t,
J = 7.4 Hz, 2H), 4.09 (s, 3H), 5.48 (s, 2H), 6.55 (s, 1H), 7.63 (s, 1H),
7.86 (s, 1H), 13.50 (bs, 1H), 14.00 (bs, 1H). MS m/z 565 (M H+).
Anal. (C24H24ClF3N8O3) C, H, N.
8-(3-{[4,6-Bis(trifluoromethyl)-1H-benzimidazol-2-yl]-
methoxy}-1-methyl-1H-pyrazol-5-yl)-1,3-dipropyl-3,7-dihydro-
1H-purine-2,6-dione (67). Purification by crystallization from
methanol. Yield 55%; mp 281 °C.
1H NMR (200 MHz, DMSO-d6): δ 0.86−0.92 (m, 6H), 1.55 (q, J =
7.2 Hz, 2H), 1.69 (q, J = 7.4 Hz, 2H), 3.88 (t, J = 7.6 Hz, 2H), 3.97 (t,
J = 7.8 Hz, 2H), 5.54 (s, 2H), 6.98 (s, 1H), 7.65−7.80 (m, 2H), 13.06
(bs, 1H), 14.61(bs, 1H). MS m/z 502 (M H+). Anal.
(C22H21ClFN7O4) C, H, N.
8-(3-{[4-Chloro-6-(trifluoromethyl)-1H-benzimidazol-2-yl]-
methoxy}isoxazol-5-yl)-1,3-dipropyl-3,7-dihydro-1H-purine-
2,6-dione (76). Purification by crystallization from methanol. Yield
50%; mp 251 °C.
1H NMR (200 MHz, DMSO-d6): δ 0.83−0.92 (m, 6H), 1.59 (q, J =
7.2 Hz, 2H), 1.78 (q, J = 7.0 Hz, 2H), 3.93 (t, J = 7.4 Hz, 2H), 4.09 (t,
J = 7.0 Hz, 2H), 4.10 (s, 3H), 5.51 (s, 2H), 6.51 (s, 1H), 7.90 (s, 1H),
8.20 (s, 1H), 13.55 (bs, 1H), 14.00 (bs, 1H). MS m/z 599 (M H+).
Anal. (C25H24F6N8O3) C, H, N.
8-{3-[(5,6-Dichloro-1H-benzimidazol-2-yl)methoxy]-1-meth-
yl-1H-pyrazol-5-yl}-1,3-dipropyl-3,7-dihydro-1H-purine-2,6-
808
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1H NMR (200 MHz, DMSO-d6): δ 0.86−0.92 (m, 6H), 1.59 (q, J =
7.4 Hz, 2H), 1.69 (q, J = 7.2 Hz, 2H), 3.86 (t, J = 7.0 Hz, 2H), 3.97 (t,
J = 7.2 Hz, 2H), 5.96 (s, 2H), 6.95 (s, 1H), 7.48 (s, 1H), 7.98 (s, 1H),
10.00 (bs, 1H), 14.20 (bs, 1H). MS m/z 552 (M H+). Anal.
(C23H21ClF3N7O4) C, H, N.
with water. The resulting crude was purified by column chromatog-
raphy (ethyl acetate−methanol 9:1).
1
83. Yield 65%; mp 215 °C. H NMR (200 MHz, DMSO-d6): δ
0.82−0.93 (m, 6H), 1.52−1.65 (m, 4H), 3.82 (s, 3H), 3.86−3.99 (m,
4H), 4.73 (s, 2H), 6.24 (s, 1H), 6.57 (s, 1H), 7.15−7.20 (m, 2H),
7.61−7.66 (m, 2H), 10.15 (bs, 1H), 12.40 (bs, 1H). MS m/z 483 (M
H+). Anal. (C24H27FN6O4) C, H, N.
8-(3-{[4,6-Bis(trifluoromethyl)-1H-benzimidazol-2-yl]-
methoxy}isoxazol-5-yl)-1,3-dipropyl-3,7-dihydro-1H-purine-
2,6-dione (77). Purification by crystallization from methanol. Yield
60%; mp 233 °C.
1
84. Yield 60%; mp: 226 °C. H NMR (200 MHz, DMSO-d6): δ
0.86 (m, 6H), 1.62 (m, 4H), 3.82 (s, 3H), 3.86 (m, 4H), 4.70 (s, 2H),
5.98 (s, 2H), 6.23 (s, 1H), 6.56 (s, 1H), 6.85 (d, J = 8.4 Hz 1H), 7.00
(d, J = 8.4 Hz 1H), 7.32 (s, 1H), 9.99 (s, 1H), 12.44 (s, 1H). MS m/z
509 (M H+). Anal. (C25H28N6O6) C, H, N.
1H NMR (200 MHz, DMSO-d6): δ 0.81−0.91 (m, 6H), 1.52 (q, J =
7.4 Hz, 2H), 1.67 (q, J = 8.0. Hz, 2H), 3.86 (t, J = 7.0 Hz, 2H), 3.94 (t,
J = 6.8 Hz, 2H), 5.64 (s, 2H), 6.68 (s, 1H), 7.84 (s, 1H), 8.26 (s, 1H),
13.06 (bs, 1H), 14.00 (bs, 1H). MS m/z 586 (M H+). Anal.
(C24H21F6N7O4) C, H, N.
Determination of Affinity (Ki) and Potency (IC50) Values of
the Novel Adenosine Compounds. Cell membranes preparation.
The human ARs have been transfected in CHO or HEK293 cells
according with the method previously described.36,37 Briefly, the cells
were grown adherently and maintained in Dulbecco’s Modified Eagles
Medium (DMEM) with nutrient mixture F12 (DMEM/F12) without
nucleosides, containing 10% fetal calf serum, penicillin (100 U/mL),
streptomycin (100 μg/mL), L-glutamine (2 mM), and Geneticin
(G418, 0.2 mg/mL) at 37 °C in 5% CO2/95% air. For membrane
preparation the culture medium was removed and the cells were
washed with phosphate-buffered saline and scraped off T75 flasks in
ice-cold hypo tonic buffer (5 mM Tris HCl, 1 mM EDTA, pH 7.4).
The cell suspension was homogenized with Polytron, the homogenate
was spun for 10 min at 1000g, and the supernatant was then
centrifuged for 30 min at 100000g. The membrane pellet was
suspended in 50 mM Tris HCl buffer (pH 7.4) for A1ARs, in 50 mM
Tris HCl, 10 mM MgCl2 (pH 7.4) for A2AARs, in 50 mM Tris HCl, 10
mM MgCl2, 1 mM EDTA (pH 7.4) for A2B and A3ARs.
Competition Binding Experiments to Human A1, A2A, A2B,
and A3ARs. All new synthesized compounds have been tested to
evaluate their affinity to human A1, A2A, A2B, and A3ARs. Displacement
experiments of [3H]-DPCPX (1,3-[3H]-dipropyl-8-cyclopentylxan-
thine) to CHO cells transfected with the human recombinant
A1ARs were carried out for 120 min at 25 °C incubating diluted
membranes (50 μg of protein/assay) and at least 6−8 different
concentrations of examined antagonists.38 Nonspecific binding was
determined in the presence of DPCPX 10 μM, and this was always
≤10% of the total binding. Binding of [3H]-ZM 241385 (4-(2-[7-
amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]-
ethyl)phenol) to CHO cells transfected with the human recombinant
A2AARs was performed by using a suspension of membranes (50 μg of
protein/assay) and at least 6−8 different concentrations of studied
antagonists for an incubation time of 60 min at 4 °C.39 Nonspecific
binding was determined in the presence of 1 μM ZM 241385 and was
about 20% of total binding. Competition binding experiments of
tritiated-30 to HEK293 cells transfected with the human recombinant
A2BARs were carried out incubating for 120 min at 4 °C diluted
membranes (50 μg of protein/assay) and at least 6−8 different
concentrations of examined compounds.40 Nonspecific binding was
defined as binding in the presence of 1 μM MRE 2029F20 and was
about 25% of total binding. Inhibition binding assays of [3H]-MRE
3008F20 (5-N-(4-methoxyphenylcarbamoyl)amino-8-propyl-2-(2-
furyl)pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) in CHO cells
transfected with the human recombinant A3ARs were performed
incubating for 120 min at 4 °C diluted membranes (50 μg of protein/
assay) and at least 6−8 different concentrations of examined ligands.41
Bound and free radioactivity was separated by filtering the assay
mixture through Whatman GF/B glass fiber filters using a Brandel cell
8-[1-Methyl-3-(7H-purin-8-ylmethoxy)-1H-pyrazol-5-yl]-1,3-
dipropyl-3,7-dihydro-1H-purine-2,6-dione (78). The same pro-
cedure as reported for the preparation of compounds 54−69, except a
temperature of 100 °C was necessary for the ring closure of the amide
intermediate.
Purification by crystallization from DMF/water. Yield 60%; mp
293−294 °C.
1H NMR (200 MHz, DMSO-d6): δ 0.80−0.89 (m, 6H), 1.54 (q, J =
7.6 Hz, 2H), 1.70 (q, J = 7.2 Hz, 2H), 3.83 (t, J = 7.2 Hz, 2H), 3.97 (t,
J = 7.2 Hz, 2H), 4.05 (s, 3H), 5.44 (s, 2H), 6.50 (s, 1H), 8.87 (s, 1H),
9.03 (s, 1H), 13.77 (bs, 2H). MS m/z 465 (M H+). Anal.
(C21H24N10O3) C, H, N.
8-(3-{[5-(3-Methoxyphenyl)-1,2,4-oxadiazol-3-yl]methoxy}-
1-methyl-1H-pyrazol-5-yl)-1,3-dipropyl-3,7-dihydro-1H-pu-
rine-2,6-dione (79). The SEM protected X (50 mg, 0.07 mmol) was
dissolved in ethanol (2 mL) and treated with HCl 1N (0.5 mL) for 4 h
at 80 °C. After cooling to room temperature, the precipitate formed
was filtered and washed with ethanol to afford pure 79. Yield 85%; mp
242 °C.
1H NMR (200 MHz, DMSO-d6): δ 0.83−0.92 (m, 6H), 1.55 (q, J =
7.8 Hz, 2H), 1.73 (q, J = 7.8 Hz, 2H), 3.87 (s, 3H), 3.89 (t, J = 7.2 Hz,
2H), 4.01 (t, J = 7.6 Hz, 2H), 4.09 (s, 3H), 5.40 (s, 2H), 6.53 (s, 1H),
7.31−7.74 (m, 4H), 13.98 (bs, 1H). MS m/z 521 (M H+). Anal.
(C25H28N8O5) C, H, N.
8-(3-Hydroxy-1-methyl-1H-pyrazol-5-yl)-1,3-dipropyl-3,7-di-
hydro-1H-purine-2,6-dione (80). The same procedure as reported
for the preparation of compound VIII, except used VI. Yield 80%; mp
>300 °C.
1H NMR (200 MHz, DMSO-d6): δ 0.83−0.92 (m, 6H), 1.58 (q, J =
7.2 Hz, 2H), 1.75 (q, J = 7.8 Hz, 2H), 3.86 (t, J = 7.6 Hz, 2H), 3.89 (t,
J = 8.0 Hz, 2H), 4.04 (s, 3H), 6.25 (s, 1H), 9.95 (s, 1H), 13.80 (bs,
1H). MS m/z 333 (M H+). Anal. (C15H20N6O3) C, H, N.
6-(3-Hydroxy-1-methyl-1H-pyrazol-5-yl)-1,3-dipropyl-1H-
pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-dione (81). Same procedure
as for compound VIII except used XIV. Yield 80%; mp 282 °C. 1H NMR
(DMSO-d6): δ − 0.87 (m, 6H), 1.62 (m, 2H), 3.76 (s, 3H), 3.85 (m,
4H), 5.96 (s, 2H), 6.51 (s, 1H), 9.77 (bs, 1H), 12.3 (bs, 1H). MS m/z
332 (M H+). Anal. (C16H21N5O3) C, H, N.
6-[3-(1H-Benzimidazol-2-ylmethoxy)-1-methyl-1H-pyrazol-
5-yl]-1,3-dipropyl-1H-pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-
dione (82). The same procedure as for compound 54 except used XIX.
Yield 85%; mp 224 °C. 1H NMR (200 MHz, DMSO-d6): δ 0.82−0.93
(m, 6H), 1.60−1.75 (m, 4H), 3.86 (s, 3H), 3.89−3.99 (m, 4H), 5.34
(s, 2H), 6.26 (s, 1H), 6.55 (s, 1H), 7.20−7.62 (m, 4H), 12.50 (bs,
1H), 12.65 (bs, 1H). MS m/z 462 (M H+). Anal. (C24H27N7O3) C, H,
N.
Preparation of 2-{[5-(2,4-Dioxo-1,3-dipropyl-2,3,4,5-tetrahy-
dro-1H-pyrrolo[3,2-d]pyrimidin-6-yl)-1-methyl-1H-pyrazol-3-
yl]oxy}-N-(4-fluorophenyl)acetamide (83) and N-1,3-Benzo-
dioxol-5-yl-2-{[5-(2,4-dioxo-1,3-dipropyl-2,3,4,5-tetrahydro-
1H-pyrrolo[3,2-d]pyrimidin-6-yl)-1-methyl-1H-pyrazol-3-yl]-
oxy}acetamide (84). To a mixture of the carboxylic acid XIX (0.15
mmol), EDC (0.15 mmol), and anhydrous DMF (5 mL) was added
the appropriate aniline (0.15 mmol), and the mixture was stirred at
room temperature for 5 h under argon atmosphere. The resulting
solution was evaporated under reduced pressure, and treated with
water. The precipitate formed was collected by filtration and washed
harvester (Brandel Instruments, Unterfohring, Germany). The filter-
̈
bound radioactivity was counted by a Packard Tri Carb 2810 TR
(Perkin-Elmer) scintillation counter.
Measurement of Cyclic AMP Levels in CHO Cells Trans-
fected with Human A2BARs. CHO cells transfected with human
A2BARs were washed with phosphate-buffered saline, diluted trypsine,
and centrifuged for 10 min at 200g. The pellet resulting was suspended
in 0.5 mL of incubation mixture: NaCl 150 mM, KCl 2.7 mM,
NaH2PO4 0.37 mM, MgSO4 1 mM, CaCl2 1 mM, Hepes 5 mM,
MgCl2 10 mM, glucose 5 mM, pH 7.4 at 37 °C. Then 2.0 IU/mL
809
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adenosine deaminase and 0.5 mM 4-(3-butoxy-4-methoxybenzyl)-2-
imidazolidinone (Ro 20−1724) as phosphodiesterase inhibitor were
added and preincubated for 10 min in a shaking bath at 37 °C.41 The
potencies of antagonists studied were determined by antagonism of
NECA (100 nM)-induced stimulation of cyclic AMP levels. The
reaction was terminated by the addition of cold 6% thrichloroacetic
acid (TCA). The TCA suspension was centrifuged at 2000g for 10 min
at 4 °C, and the supernatant was extracted four times with water
saturated diethyl ether. The final aqueous solution was tested for cyclic
AMP levels by a competition protein binding assay. Samples of cyclic
AMP standard (0−10 pmoles) were added to each test tube
containing the incubation buffer (trizma base 0.1 M, aminophylline
8.0 mM, 2 mercaptoethanol 6.0 mM, pH 7.4) and [3H] cyclic AMP in
a total volume of 0.5 mL. The binding protein previously prepared
from beef adrenals, was added to the samples previously incubated at 4
°C for 150 min, and after the addition of charcoal were centrifuged at
2000g for 10 min. The clear supernatant was counted with 4 mL of
Atomlight liquid scintillator and counted in a Tri Carb 2810 TR
(Perkin-Elmer) scintillation counter .
Data Analysis. The protein concentration was determined
according to a Bio-Rad method42 with bovine albumin as a standard
reference. Inhibitory binding constant, Ki, values were calculated from
those of IC50 according to Cheng and Prusoff equation Ki = IC50/(1 +
[C*]/KD*), where [C*] is the concentration of the radioligand and
KD* its dissociation constant.43 In addition, a weighted nonlinear least-
squares curve fitting program LIGAND was used for computer analysis
of the inhibition binding experiments.44 The IC50 values obtained in
cyclic AMP assays were calculated by nonlinear regression analysis
using the equation for a sigmoidal concentration response curves
(Graph Pad Prism, San Diego, CA). All experimental data are
expressed as the geometric mean with 95% confidence limits in
parentheses of three or four independent experiments performed in
duplicate.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Additional synthetic, spectroscopic, analytical, and molecular
modeling data. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*For P.G.B.: phone, +39-0532-455921; fax, +39-0532-455921;
E-mail, baraldi@dns.unife.it. For M.A.T.: phone, +39-0532-
455926; fax, +39-0532-455921; E-mail, tbj@dns.unife.it.
(12) Bjorck, T.; Gustafsson, L. E.; Dahlen, S. E. Isolated bronchi from
asthmatics are hyperresponsive to adenosine, which apparently acts
indirectly by liberation of leukotrienes and histamine. Am. Rev. Respir.
Dis. 1992, 145, 1087−1091.
ACKNOWLEDGMENTS
■
(13) Varani, K.; Caramori, G.; Vincenzi, F.; Adcock, I.; Casolari, P.;
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We thank King Pharmaceuticals, Inc., Research and Develop-
ment, 4000 CentreGreen Way, Suite 300, Cary, North Carolina
27513, for financial support.
ABBREVIATIONS USED
■
AR, adenosine receptor; cAMP, cyclic adenosine mono-
phosphate; ADA, adenosine deaminase; CHO, Chinese
hamster ovary; CoMFA, comparative molecular field analysis;
COPD, chronic obstructive pulmonary disease; DMF,
dimethylformamide; DMSO, dimethylsulfoxide; DMEM, Dul-
becco’s Modified Eagle Medium; EDC, N-(3-(dimethylamino)-
propyl)-N′-ethylcarbodiimide; HMC-1, human mast cell line;
HEK, human embryonic kidney; h, human; MAPK, mitogen-
activated protein kinase; IL, interleukin; mitogen-activated
protein kinase; NECA, 5′-N-ethylcarboxamidoadenosine; PCC,
pyridinium chlorocromate; QSAR, quantitative structure−
activity relationship; rmsd, root-mean-square deviation;
mRNA, messenger ribonucleic acid; SEM-Cl, 2-(trimethylsilyl)-
ethoxymethyl chloride
(17) Varani, K.; Caramori, G.; Vincenzi, F.; Tosi, A.; Barczyk, A.;
Contoli, M.; Casolari, P.; Triggiani, M.; Hansel, T.; Leung, E.;
Maclennan, S.; Barnes, P. J.; Chung, K. F.; Adcock, I.; Papi, A.; Borea,
P. A. Oxidative/nitrosative stress selectively altered A2B adenosine
receptors in chronic obstructive pulmonary disease. FASEB J. 2010, 24,
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Molina, J. G.; Belardinelli, L.; Zeng, D.; Blackburn, M. R. Role of A2B
810
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