Discovery of LAS101057: A potent, selective, and orally efficacious A 2B Adenosine Receptor Antagonist

Article abstract of DOI:10.1021/ml100249e

The structure-activity relationships for a series of pyrazine-based A _2B adenosine receptor antagonists are described. From this work, LAS101057 (17), a potent, selective, and orally efficacious A_2B receptor antagonist, was identifie

Full text of DOI:10.1021/ml100249e

LETTER
pubs.acs.org/acsmedchemlett
Discovery of LAS101057: A Potent, Selective, and Orally Efficacious A2B
Adenosine Receptor Antagonist
Paul Eastwood,† Cristina Esteve,† Jacob Gonzꢀalez,† Silvia Fonquerna,† Josep Aiguadꢀe,† Inꢀes Carranco,†
Teresa Domꢀenech,† Mꢁonica Aparici,† Montserrat Miralpeix,† Joan Albertí,† Mꢀonica Cꢀordoba,†
Raquel Fernꢀandez,†,§ Mercꢁe Pont,† Nꢀuria Godessart,† Neus Prats,† María Isabel Loza,‡ María Isabel Cadavid,‡
Arsenio Nueda,† and Bernat Vidal*,†
†Almirall, R&D Centre, Ctra. Laureꢁa Mirꢀo 408, 08980-Sant Feliu de Llobregat, Barcelona, Spain
‡Discovery group “BioFarma”, Faculty of Pharmacy, University of Santiago de Compostela, 15782-Santiago de Compostela, Spain
S Supporting Information
b
ABSTRACT: The structure-activity relationships for a series of pyrazine-based A2B adenosine receptor
antagonists are described. From this work, LAS101057 (17), a potent, selective, and orally efficacious
A2B receptor antagonist, was identified as a clinical development candidate. LAS101057 inhibits
agonist-induced IL-6 production in human fibroblasts and is active in an ovalbumin (OVA)-sensitized
mouse model after oral administration, reducing airway hyperresponsiveness to methacholine, Th2
cytokine production, and OVA-specific IgE levels.
KEYWORDS: A2B adenosine receptor antagonist, ovalbumin mouse model, clinical candidate
he purinergic, G protein-coupled receptor A2B has a low
Herein are detailed further optimization efforts in a structu-
rally related series which ultimately led to the discovery of a
clinical candidate LAS101057 (17) intended for use in oral asthma
therapy.
Studies conducted in parallel to those described above revealed
that replacement of the central pyridine scaffold of compounds
from the amide-based series by a pyrazine moiety gave compounds
with good activity/selectivity profiles (Table 1).
The pharmacokinetic profiles of the pyrazines 1 and 2 in the
rat were determined, and the results are given in Table 4. Despite
showing excellent bioavailability and acceptable plasma levels,
the acetamide analogue 2 suffered from a very short intravenous
half-life (t1/2 = 0.4 h). The corresponding cyclopropyl analogue
1, in addition to showing complete oral bioavailability, demon-
strated an improvement in the intravenous half-life (t1/2 = 0.7 h)
when compared to the acetamide derivative.
Encouraged by these preliminary findings, further exploration
of the pyrazine series was undertaken. A range of amide deri-
vatives were prepared (Table 1), and although several derivatives
showed reasonable potency and selectivity, no compound showed
an improvement upon the overall profile of the cyclopropyl
derivative 1.
T
affinity for the endogenous mediator adenosine.1-3 Evidence
published over the past decade has shown that the A2B receptor
mediates the release of inflammatory mediators from several cell
types, including many which are relevant for the pathophysiology
of asthma, such as mast cells, smooth muscle cells, airways
epithelial cells, and fibroblasts.4,5
The A2B receptor has a pro-inflammatory role in the gastro-
intestinal tract,6 and it has been shown to contribute to the deve-
lopment of colitis in a murine A2B receptor knockout model.7
Some of the initial evidence obtained using A2B receptor knock-
out mice suggested an anti-inflammatory role for this receptor.8,9
However, subsequent studies do not entirely support this
10-12
concept.
A
2B
activates cystic fibrosis transmembrane regu-
lator chloride channel-mediated liquid release in the lungs,13 and
it could play a role in severe watery infectious diarrhea through
activation of a chloride secretory response.14 A2B receptor knock-
out mice have also shown that A2B receptor prevents endothelial
damage during inflammation and stimulates endothelial cell
proliferation and capillary tube formation, mediating the release
of the pro-angiogenic factors VEGF, bFGF, and IGF-1,15 suggest-
ing a possible role in tumorigenesis.16 Expression of A2B is up-
regulated in several types of cancer cells.17,18
Replacement of the two aromatic rings attached to the pyrazine
central core was then investigated, keeping the cyclopropyl motif
constant. Of particular interest was the replacement of the furan
moiety, which is a potential metabolic liability and toxicophore.22
Findings from parallel studies20,21 led us to focus on specific
replacements of these two rings which were most likely to deliver
A2B adenosine antagonists that have been or are in clinical
trials are the dual A2B/A3 antagonist QAF 805 and the selective
A
2B antagonist CVT-6883.19
In previous disclosures,20,21 it was revealed that appropriately
substituted N-(5,6-diarylpyridin-2-yl)amides are potent and se-
lective A2B adenosine receptor antagonists. Although particular
compounds from these series demonstrated good oral bioavail-
ability in rat pharmacokinetic studies, in general all compounds
tested presented rapid systemic clearance.
Received: October 14, 2010
Accepted: December 14, 2010
Published: December 20, 2010
r
2010 American Chemical Society
213
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Table 1. SAR of Amide Substituent
Ki (nM) or % inhibition of radioligand binding at indicated compound concentration (μM)
compd
R
hA2B
hA2A
hA1
hA3
1
2
3
4
5
6
7
8
cyclopropyl
Me
2.2 ( 0.2
6 ( 1
44 ( 11
55 ( 17
454 ( 134
42 ( 7
398 ( 63
727 ( 143
55 ( 21
1563 ( 543
373 ( 118
1920 ( 631
4405 ( 7
Et
3.5 ( 2
10 ( 4
3 ( 1
i-Pr
197 ( 62
140 ( 96
145 ( 23
24 ( 5% (1)
105 ( 5
18 ( 7
cyclobutyl
cyclopentyl
4-F-phenyl
cyclopentylmethyl
17 ( 6
13 ( 2
63 ( 2
12 ( 4
27 ( 5
4076 ( 538
17 ( 16% (1)
ND
1040 ( 454
98 ( 6
a set of potent and selective A2B receptor antagonists. Com-
pounds synthesized and biological results are given in Table 2,
and a representative synthesis, as exemplified by the preparation
of 17 (LAS101057), is shown in Scheme 1.
Regioselective iodination of chloropyrazine (a) followed by a
metal-catalyzed Stille coupling reaction with 3-fluoro-4-(tributyl-
stannyl)pyridine23 furnished the flexible intermediate (c). Amide
formation followed by Suzuki coupling furnished compound 17
(LAS101057) in four steps in an overall yield of 21%.
risk of interaction in a clinical setting with drugs which are
predominantly metabolized by CYP2C19.
The pharmacokinetic profiles of selected analogues were
determined in the rat (Table 4). The compounds demonstrated
moderate to excellent oral bioavailability in rat, and the combina-
tion of moderate clearance values and moderate volumes of
distribution resulted in half-lives ranging from 0.8 to 1.6 h.
Based on the preliminary pharmacological and pharmacokinetic
profile in the rat, compound 17 (LAS101057) was selected for further
characterization. Additional pharmacokinetic studies (Table 5)
showed that 17 had low plasma clearance in both dog and monkey
with moderate to long half-lives: excellent bioavailability was
observed in both species. Plasma protein binding (determined
by ultrafiltration) was moderate in all species, accounting for 82%,
82%, 79%, and 89% in mouse, rat, dog, and human, respectively.
Since cAMP mediated signaling is one of the two main path-
ways elicited through activation of the A2B receptor,24 the antago-
nist activity of 17 was confirmed using a cell based functional assay
measuringA2B receptor-dependent intracellularcAMPlevelsusing
5-(N-ethylcarboxamido)adenosine (NECA) as agonist.
In order to better interpret in vivo efficacy results obtained in
the mouse, both human and mouse receptors were used for these
assays. The results (IC50 (nM): human A2B = 120 ( 21; mouse
A2B = 512 ( 67) indicated that 17 was active in inhibiting A2B
receptor-dependent cAMP signaling in both the human and
mouse forms of the receptor and that the compound was
approximately 4-fold more active in inhibiting signaling of the
human receptor than that of the mouse receptor.
Given that the A2B receptor is known to mediate the release
of the pro-inflammatory cytokine IL-6 through a mechanism
dependent on the cAMP/cAMP response element binding
(CREB) signaling pathway,24 it was decided to test the effect
of 17 on NECA-induced IL-6 release in human primary dermal
fibroblasts as an additional means of demonstrating blockade of
A2B receptor function. In this assay, 17 effected a concentration-
dependent down-regulation of IL-6 production with a potency
(67% ( 2 at 100 nM) that was in a similar range to that seen in
both the A2B receptor radioligand binding and cAMP assays.
The general selectivity of 17 was found to be excellent. When
tested at a concentration of 10 μM in a diverse panel of more than
340 enzymes, receptors, channels, and transporters, 17 was found
to be >400-fold selective.
As can be seen from the table, the majority of analogues
prepared were found to potently displace radioligand binding to
the A2B receptor. Compounds with receptor selectivity profiles
comparable to that of 1 could be obtained by replacement of the
furan ring at C-6 with other aromatic nuclei, such as pyridyl,
2-fluorophenyl, and oxazolyl (compounds 10-15 and 17-23),
albeit at the expense of potency in the majority of cases. Nevertheless,
desired levels of potency could be attained by the introduction of
halogen atoms, particularly fluorine, into the pyridyl ring system at
C-5 of the pyrazine. When combined, these particular changes at C-5
and C-6 led to a number of nonfuran containing compounds with
excellent activity/selectivity profiles (compounds 17-23).
The in vitro metabolism, permeability, and cytochrome P450
(CYP450) inhibition profiles of selected derivatives are given in
Table 3. All compounds tested presented excellent Caco-2 per-
meabilities (Papp >30 ꢀ 10-6 cm/s) and were not found to be
substrates of the P-glycoprotein efflux transporter (P-gp). Incu-
bation of compounds with liver microsomes revealed a range of
metabolic stabilities, although, in general, turnover was found to
be low in both rat and human. N- and C-oxidation of the pyridine
rings were found to be the major in vitro metabolic routes. In
addition, minor amounts of metabolites derived from the hydrolysis
of the amide bond were found in the liver microsome studies,
although such compounds were not detected in plasma, blood, and
liver subcellular fractions from different animal species and humans.
The majority of compounds showed no relevant inhibition of
human CYP450 cytochromes CYP1A2, 2C9, and 2D6 (IC50
g
25 μM). However, in certain cases, a moderate inhibition of
CYP3A4 (IC50: 1-25 μM) and CYP2C19 (IC50: 1-5 μM) was
observed. The potential for drug-drug interactions involving
CYP2C19 was assessed by determination of the inhibition con-
stant (Ki) for the most promising candidates (compounds 17,
Ki = 2.6 μM; 20, Ki = 0.7 μM). These results indicated a potential
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Table 2. SAR of Aromatic Substituents
The efficacy of 17 was then determined in an ovalbumin
(OVA)-sensitized mouse model: an in vivo functional model
that recapitulates some of the pathological features of human
asthma.25 Relevant parameters that were determined from these
studies included the airway hyperresponsiveness (AHR) to
methacholine, numbers of inflammatory cells and levels of IL-
4, IL-13, and γIFN present in bronchial alveolar lavage fluid
(BAL), the extent of mucus accumulation, and plasma levels of
OVA-specific IgE, total IgE, and IgG.
As shown in Figure 1, 17 reduced the increase of lung
resistance induced by methacholine. LAS101057 (17) was active
in preventing methacholine-induced AHR at 3 mg/kg, and at 10
mg/kg it inhibited AHR to methacholine to a level virtually equal
to that seen with dexamethasone at 1 mg/kg.
Additionally, at 10 mg/kg, 17 reduced the levels of Th2
cytokines IL-4 (42 ( 12% inhibition) and IL-13 (44 ( 14%
inhibition) in BAL but did not show any effect on the Th1
cytokine INFγ levels. 17 produced a slight but nonstatistically
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Table 3. Profiles of Selected Compoundsa
human CYP450 inhibition (IC50, μM)d
compd
% turnoverb rat/human
Papp (ꢀ10-6 cm/s)c A-B/B-A
1A2
2C9
2C19
<5
2D6
3A4
1
46/18
50/22
20/11
20/14
15/12
13/5
36/26
-
5
5-25
-
5-25
-
≈25
-
9
-
-
10
11
12
17
18
19
20
21
22
23
35/20
38/21
34/22
37/23
-
-
-
-
-
-
≈25
>25
>25
-
≈25
>25
>25
-
-
>25
5-25
>25
-
5-25
1-5
5-25
-
-
2.6 (Ki)
-
17/3
19/10
4/2
-
-
>25
>25
-
≈1
0.7 (Ki)
-
>25
>25
-
>25
1-5
-
33/22
-
>25
-
75/27
33/9
-
>25
≈25
-
≈1
>25
-
>25
5-25
6/6
-
≈25
>25
a -: Not tested. b % turnover after a 30 min incubation period at 37 °C of a 5 μM solution of test compound with hepatic microsomes (1 mg/mL).
c Passive permeability through a CACO-2 monolayer determined using a 12.5 μM solution of test compound. d IC50 values were estimated by incubation
of test compounds (1, 5, and 25 μM) with human liver microsomes using as selective CYP substrates 0.5 μM 7-ethoxyresorufin (CYP1A2), 10 μM
diclofenac (CYP2C9), 50 μM S-mephenytoin (CYP2C19), 5 μM dextromethorphan (CYP2D6), and 50 μM testosterone (CYP3A4). Inhibition
constants (Ki) for CYP2C19 were determined using different concentrations of S-mephenytoin (25-100 μM) and test substances (0.5-10 μM).
Inhibition profiles obtained were consistent with a pure competitive model.
Table 4. Pharmacokinetic Properties of Selected Derivatives in Wistar Ratsa
i.v. (1 mg/kg)
Clc (mL/(min kg))
p.o. (10 mg/kg)
t1/2 (h)
AUCb (μg h/mL)
Vssd (L/kg)
Cmax (μg/mL)
tmax (h)
Fe (%)
3
1
0.7
0.4
1.0
0.9
0.8
1.6
1.2
933
751
18.3
21.7
12.8
17.3
18.3
15.0
24.5
1.1
0.8
2140
2294
0.87
3.75
7.11
2.88
1.46
0.4
1.1
2.5
0.6
0.25
0.6
2.5
100
93
2
10
11
17
19
20
1.31
0.98
0.92
1.11
0.68
0.74
0.93
1.2
100
100
100
68
1.4
1.3
53
a Mean values (n = 2). b Area under the curve. c Total plasma clearance. d Volume of distribution at steady state. e Bioavailability.
Scheme 1. Synthesis of 17 (LAS101057)a
Table 5. Pharmacokinetic Properties of 17 (LAS101057) in
Preclinical Speciesa
i.v.b
oralc
Cl
(mL/
(μg h/ (min Vss (L/ Vβ (L/ (μg/ tmax
AUC
Cmax
t1/2
F
3
species
(h)
mL)
kg))
kg)
kg) mL) (h) (%)
mouse
monkey
dog
-
-
3.4
-
-
-
17.4 0.25
-
3.8
3.4
0.4
0.8
5.9 3.0 100
8.6
13.6
1.7
0.8
0.9 0.82 2.0 98
a -: Not tested. Mean values (n = 2). b iv administration: 1 mg/kg (male
Beagle dogs) and 0.5 mg/kg (male Cynomolgus monkeys). c Oral
administration: 10 mg/kg (female BALB/C mouse) and 1 mg/kg
(male Beagle dogs and male Cynomolgus monkeys).
a Reagents and conditions: (i) N-iodosuccinimide, DMSO/H2O, 70%; (ii)
3-fluoro-4-(tributylstannyl)pyridine, PdCl2(Ph3P)2, CuI, DMF, 150 °C,
57%; (iii) cyclopropanecarbonyl chloride, pyridine, 70 °C, 78%; (iv)
pyridin-3-yl boronic acid, 2 M Cs2CO3, 1,10-bis(diphenylphosphino)-
ferrocene-palladium(II) dichloride (PdCl2dppf), 1,4-dioxane, 90 °C, 70%.
As shown in Table 6, 17 inhibited OVA-specific IgE produc-
tion in a dose-dependent fashion, but neither the total IgE nor
total IgG were affected.
significant reduction in mucus accumulation in the airway
epithelia and demonstrated no effect in inhibiting lung tissue
cell infiltrates (see Supporting Information).
Overall, the activity and efficacy data obtained with
LAS101057 (17) indicates that this compound is active through
selective inhibition of the A2B receptor, adding to other pre-
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Table 6. Effect of 17 on Immunoglobulin Levels 24 h after the Last OVA Challengea
IgE OVA
total IgE
total IgG
plasma levels (μg/mL)
plasma levels (μg/mL)
inhibition (%)
plasma levels (μg/mL)
inhibition (%)
inhibition (%)
PBS
2.2 ( 0 02b
330 ( 104
165 ( 52
63 ( 31b
68 ( 21b
0.7 ( 0.04b
13 ( 0.8
11 ( 0.8
11 ( 1
275 ( 13b
613 ( 22
571 ( 29
632 ( 29
590 ( 18
OVA
LAS101057 3 mg/kg
LAS101057 10 mg/kg
dexamethasone 1 mg/kg
50 ( 16
81 ( 9
80 ( 6
12 ( 8
7 ( 5
12 ( 8
0 ( 9
7 ( 5
8 ( 0.8b
41 ( 7
a Data represent mean ( SEM of 6-8 animals per group. b p < 0.05 Comparison with t-student test versus OVA group.
Present Addresses
§Laboratorios Esteve, Barcelona, Spain.
Author Contributions
Bernat Vidal was the chemistry project leader, the main inventor
of the chemical series, the person directly responsible for the
design and synthesis of LAS101057, and the key decision maker
in the selection of LAS101057. Arsenio Nueda was the biology
project leader, the designer and coordinator of the experiments
performed, and the key decision maker in the selection of
LAS101057. Teresa Domenech was the person responsible for
performing radioligand binding assays. María Isabel Loza and
María Isabel Cadavid set up and provided data on the cAMP
in vitro assays and performed some of the radioligand binding
assays. Neus Prats was responsible for the histology studies.
Nꢀuria Godessart and Mercꢁe Pont designed the experiments and
performed experimental work on the human fibroblasts studies.
Joan Albertí was mainly responsible for the selection, prioritiza-
tion, and interpretation of in vitro and in vitro ADME studies and
for performing dog and monkey studies. Mꢀonica Cꢀordoba per-
formed in vitro metabolism, permeability, and CYP studies. Raquel
Fernꢀandez performed pharmacokinetic studies in rats. Montserrat
Miralpeix and Mꢁonica Aparici designed experiments and performed
experimental work on OVA efficacy studies. Paul Eastwood,
Cristina Esteve, Jacob Gonzꢀalez, Silvia Fonquerna, Josep Aiguadꢀe,
and Inꢀes Carranco explored the structure-activity relationships,
optimization of substituents, optimization of synthetic routes and
yields, and identification and characterization of compounds.
Figure 1. Effect of 17 on airway hyperresponsiveness to methacholine
24 h after the last OVA challenge. Data represent mean; numbers
represent the inhibition percentages compared to the OVA group. *p <
0.05, one way ANOVA with Dunnett's post-test.
clinical studies which have shown that the anti-inflammatory
effects mediated by selective blockade of the A2B receptor may be
beneficial in a range of disease states.2,3
In conclusion, herein is reported the discovery of 17, a novel
potent, selective, and orally efficacious A2B antagonist. LAS101057
(17) displays excellent potency in both mechanistic and cell based
functional assays and exhibits good in vivo activity in the OVA-
sensitized mouse model of asthma. Antagonism of the A2B receptor
is expected to have a beneficial effect on asthma therapy, and as such,
LAS101057, after successfully undergoing further preclinical safety
and toxicology characterization, was subsequently advanced into a
phase I, open-label, dose-escalation study in healthy subjects.
’ ACKNOWLEDGMENT
We thank Josep Maria Huerta for spectroscopic data; Josꢀe
Prieto for synthesis of key intermediates; Ester Tor for samples
bioanalysis; Francisco Jimꢀenez for animal dosing; Mꢀonica Bravo
for expert technical assistance (OVA experiments); Rosa Lꢀopez
and Marta Calbet for providing biological material used for
radioligand binding and cAMP assays; Jorge de Alba for guidance
on statistical data analysis; Estrella Lozoya for molecular model-
ing; and Victor Matassa for helpful discussions.
’ ASSOCIATED CONTENT
S
Supporting Information. Detailed experimental procedures
b
for the synthesis of compounds 1-23, in vitro assay protocols,
procedures for the pharmacokinetic studies, protocol for the
ovalbumin (OVA)-sensitized mouse model, and full selectivity
profile. This material is available free of charge via the Internet at
http://pubs.acs.org.
’ REFERENCES
(1) Polosa, R. Adenosine-receptor subtypes: their relevance to
adenosine-mediated responses in asthma and chronic obstructive pul-
monary disease. Eur. Respir. J. 2002, 20, 488–496.
(2) Fredholm, B. B.; Irenius, E.; Kull, B.; Schulte, G. Comparison of
the potency of adenosine as an agonist at human adenosine receptors expres-
sed in Chinese hamster ovary cells. Biochem. Pharmacol. 2001, 61, 443–448.
(3) Reviewed in: Haskꢀo, G.; Linden, J.; Cronstein, B.; Pacher, P.
Adenosine receptors: therapeutic aspects for inflammatory and immune
diseases. Nat. Rev. Drug Discov. 2008, 7, 759–770.
’ AUTHOR INFORMATION
Corresponding Author
*Telephone: þ34 93-291-3996. Fax: þ34 93-312-8635. E-mail:
bernat.vidal@almirall.com.
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LETTER
(4) Holgate, S. T. The Quintiles Prize Lecture 2004. The identifica-
tion of the adenosine A2B receptor as a novel therapeutic target in
asthma. Br. J. Pharmacol. 2005, 145, 1009–1015.
(5) Polosa, R.; Blackburn, M. Adenosine receptors as targets for
therapeutic intervention in asthma and chronic obstructive pulmonary
disease. Trends Pharmacol. Sci. 2009, 30, 528–535.
(6) Sitaraman, S. V.; Merlin, D.; Wang, L.; Wong, M.; Gewirtz, A. T.;
Si-Tahar, M.; Madara, J. L. Neutrophil-epithelial crosstalk at the
intestinal lumenal surface mediated by reciprocal secretion of adenosine
and IL-6. J. Clin. Invest. 2001, 107, 861–869.
(7) Kolachala, V. L.; Vijay-Kumar, M.; Dalmasso, G.; Yang, D.;
Linden, J.; Wang, L.; Gewirtz, A.; Ravid, K.; Merlin, D.; Sitaraman, S. V.
A2B adenosine receptor gene deletion attenuates murine colitis. Gastro-
enterology 2008, 135 (3), 861–870.
(22) Kalgutkar, A. S.; Gardner, I.; Obach, R. S.; Shaffer, C. L.;
Christopher, L.; Callegari, E.; Henne, K. R.; Mutlib, A. E.; Dalvie, D. K.;
Lee, J. S.; Nakai, Y.; O’Donnell, J. P.; Boer, J.; Harriman, S. P. A
Comprehensive Listing of Bioactivation Pathways of Organic Functional
Groups. Curr. Drug Metab. 2005, 6, 161–225.
(23) Prepared according to EP1104754 (Reference Example 197).
(24) Murakami, S.; Terakura, M.; Kamatani, T.; Hashikawa, T.;
Saho, T.; Shimabukuro, Y.; Okada, H. Adenosine regulates the produc-
tion of interleukin-6 by human gingival fibroblasts via cyclic AMP/
protein kinase A pathway. J. Periodontal Res. 2000, 35, 93–101.
(25) Lloyd, C. M.; Gonzalo, J. A.; Nguyen, T.; Delaney, T.; Tian, J.;
Oettgen, H.; Coyle, A. J.; Gutierrez-Ramos, J. C. Resolution of bronchial
hyperresponsiveness and pulmonary inflammation is associated with
IL-3 and tissue leukocyte apoptosis. J. Immunol. 2001, 166, 2033–2040.
(8) Yang, D.; Zhang, Y.; Nguyen, H. G.; Koupenova, M.; Chauhan,
A. K.; Makitalo, M.; Jones, M. R.; St Hilaire, C.; Seldin, D. C.; Toselli, P.;
Lamperti, E.; Schreiber, B. M.; Gavras, H.; Wagner, D. D.; Ravid, K. The
A2B adenosine receptor protects against inflammation and excessive
vascular adhesion. J. Clin. Invest. 2006, 116 (7), 1913–1923.
(9) Hua, X.; Kovarova, M.; Chason, K. D.; Nguyen, M.; Koller, B. H.;
Tilley, S. L. Enhanced mast cell activation in mice deficient in the A2B
adenosine receptor. J. Exp. Med. 2007, 204 (1), 117–128.
(10) Ryzhov, S.; Zaynagetdinov, R.; Goldstein, A. E.; Novitskiy,
S. V.; Dikov, M. M.; Blackburn, M. R.; Biaggioni, I.; Feoktistov, I. Effect
of A2B adenosine receptor gene ablation on proinflammatory adenosine
signaling in mast cells. J. Immunol. 2008, 180, 7211–7220.
(11) Ryzhov, S.; Zaynagetdinov, R.; Goldstein, A. E.; Novitskiy, S.;
Blackburn, M. R.; Biaggioni, I.; Feoktistov, I. Effect of A2B adenosine
receptor gene ablation on adenosine-dependent regulation of proin-
flammtory cytokines. J. Pharmacol. Exp. Ther. 2008, 324, 694–700.
(12) Zaynagetdinov, R.; Ryzhov, S.; Goldstein, A. E.; Huiyong, Y.;
Novitskiy, S. V.; Goleniewska, K.; Polosukhin, V. V.; Newcomb, D. C.;
Mitchell, D.; Morschl, E.; Zhou, Y.; Blackburn, M. R.; Peebles, R. S.;
Biaggioni, I.; Feoktistov, I. Attenuation of chronic pulmonary inflamma-
tion in A2B adenosine receptor knockout mice. Am. J. Respir. Cell Mol.
Biol. 2010, 42, 564–571.
(13) Lazarowski, E. R.; Boucher, R. C. Purinergic receptors in airway
epithelia. Curr. Opin. Pharmacol. 2009, 3, 262–267.
(14) Crane, J. K.; Olson, R. A.; Jones, H. M.; Duffey, M. E. Release of
ATP during host cell killing by enteropathogenic E. coli and its role as a
secretory mediator. Am. J. Physiol. Gastrointest. Liver Physiol. 2002, 283,
G74–G86.
(15) Haskꢀo, G.; Csꢀoka, B.; Nꢀemeth, Z. H.; Vizi, E. S.; Pacher, P. A2B
adenosine receptors in immunity and inflammation. Trends Immunol.
2009, 30, 263–70.
(16) Ryzhov, S.; Novitskiy, S. V.; Zaynagetdinov, R.; Goldstein,
A. E.; Carbone, D. P.; Biaggioni, I.; Dikov, M. M.; Feoktistov, I. Host A2B
adenosine receptors promote carcinoma growth. Neoplasia 2008, 10,
987–995.
(17) Li, S.; Huang, S.; Peng, S. B. Overexpression of G protein-
coupled receptors in cancer cells: involvement in tumor progression. Int.
J. Oncol. 2005, 27, 1329–1339.
(18) Ma, D. F.; Kondo, T.; Nakazawa, T.; Niu, D. F.; Mochizuki, K.;
Kawasaki, T.; Yamane, T.; Katoh, R. Hypoxia-inducible adenosine A2B
receptor modulates proliferation of colon carcinoma cells. Hum. Pathol.
2010, PMID: 20619442.
(19) Polosa, R.; Blackburn, M. R. Adenosine receptors as targets for
therapeutic intervention in asthma and chronic obstructive pulmonary
disease. Trends Pharmacol. Sci. 2009, 30 (10), 528–535.
(20) Eastwood, P.; Gonzalez, J.; Paredes, S.; Fonquerna, S.; Cardꢀus,
A.; Alonso, J. A.; Nueda, A.; Domenech, T.; Reinoso, R. F.; Vidal, B.
Discovery of potent and selective bicyclic A2B adenosine receptor
antagonists via bioisosteric amide replacement. Bioorg. Med. Chem. Lett.
2010, 20, 1634–1637.
(21) Eastwood, P.; Gonzalez, J.; Paredes, S.; Nueda, A.; Domenech,
T.; Alberti, J.; Vidal, B. Discovery of N-(5,6-diarylpyridin-2-yl)amide
derivatives as potent and selective A2B adenosine receptor antagonists.
Bioorg. Med. Chem. Lett. 2010, 20, 1697–1700.
218
dx.doi.org/10.1021/ml100249e |ACS Med. Chem. Lett. 2011, 2, 213–218