Cyclic AMP-dependent inhibition of human neutrophil oxidative activity by substituted 2-propynylcyclohexyl adenosine A2A receptor agonists

Article abstract of DOI:10.1038/sj.bjp.0703893

1. Novel 2-propynylcyclohexyl-5′-N-ehtylcarboxamidoadenosines, trans-substituted in the 4-position of the cyclohexyl ring, were evaluated in binding assays to the four subtypes of adenosine receptors (ARs). Two esters, 4-{(3-[6-amino-9-(5-ethylcarbamoyl-3

Full text of DOI:10.1038/sj.bjp.0703893

British Journal of Pharmacology (2001) 132, 1017 ± 1026
ã
2001 Nature Publishing Group All rights reserved 0007 ± 1188/01 $15.00
www.nature.com/bjp
Cyclic AMP-dependent inhibition of human neutrophil oxidative
activity by substituted 2-propynylcyclohexyl adenosine A2A
receptor agonists
2
1
2
1Gail W. Sullivan, Jayson M. Rieger, W. Michael Scheld, Timothy L. Macdonald &
*,1Joel Linden
2
1Department of Medicine, University of Virginia, Charlottesville, Virginia, U.S.A. and Department of Chemistry, University of
Virginia, Charlottesville, Virginia, U.S.A.
1
Novel 2-propynylcyclohexyl-5'-N-ehtylcarboxamidoadenosines, trans-substituted in the 4-position
of the cyclohexyl ring, were evaluated in binding assays to the four subtypes of adenosine receptors
(ARs). Two esters, 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-pur-
in-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester (ATL146e) and acetic acid 4-{3-[6-
amino-9-(5-ethylcarbamoyl-3, 4-dihydroxy-tetrahydro-furan -2-yl)-9H-purin-2-yl] -prop-2-ynyl}-cyclo-
hexylmethyl ester (ATL193) were 4506more potent than 2-[4-(2-carboxyethyl)phenethylamino]-5'-
N-ethylcarboxamidoadenosine (CGS21680) for human A2A AR binding. Human A2A AR anity for
substituted cyclohexyl-propynyladenosine analogues was inversely correlated with the polarity of the
cyclohexyl side chain. There was a comparable order of potency for A2A AR agonist stimulation of
human neutrophil [cyclic AMP]i, and inhibition of the neutrophil oxidative burst. ATL146e and
CGS21680 were *equipotent agonists of human A3 ARs.
2
We measured the e€ects of selective AR antagonists on agonist stimulated neutrophil [cyclic
AMP]i and the e€ect of PKA inhibition on A2A AR agonist activity. ATL193-stimulated neutrophil
[cyclic AMP]i was blocked by antagonists with the potency order: ZM241385 (A2A
-
selective)4MRS1220 (A3-selective)44N-(4-Cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-
hexahydro-1H-purin-8-yl)-phenoxy]-acetamide (MRS1754; A2B-selective) & 8-(N-methylisopropyl)-
amino-N6-(5'-endohydroxy-endonorbornyl)-9-methyladenine (WRC0571; A1-selective). The type IV
phosphodiesterase inhibitor, rolipram (100 nM) potentiated ATL193 inhibition of the oxidative
burst, and inhibition by ATL193 was counteracted by the PKA inhibitor H-89.
3 The data indicate that activation of A2AARs inhibits neutrophil oxidative activity by activating
[cyclic AMP]i/PKA.
British Journal of Pharmacology (2001) 132, 1017 ± 1026
Keywords: A2A adenosine receptors; neutrophil; oxidative burst; cyclic AMP-dependent protein kinase
Abbreviations: ATL146a, 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-
cyclohexanecarboxylic acid; ATL146e, 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-
yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester; ATL193, acetic acid 4-{3-[6-amino-
9- (5-ethylcarbamoyl-3, 4-dihydroxy-tetrahydro-furan-2-yl) -9H-purin-2-yl] -prop-2-ynyl} -cyclohexylmethyl ester;
ATL2037, 5-{6-amino-2-[3-(4-hydroxymethyl-cyclohexyl)-prop-1-ynyl]-purin-9-yl}-3,4-dihydroxy-tetrahydro-fur-
an-2-carboxylic acid ethylamide; BW-1433, 8-(4-carboxyethenylphenyl)-1,3-dipropylxanthine; CGS21680, 2-[4-
(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine; HE-NECA, 5-(6-amino-2-hex-1-ynyl-purin-
9-yl)-3,4-dihydroxy-tetrahydro-furan-2-carboxylic acid ethylamide; MRE0470, 2-[6-amino-2-(N'-cyclohexyl-
methylene-hydrazino)-purin-9-yl]-5-hydroxymethyl-tetrahydro-furan-3,4-diol; MRS1220, N-(9-chloro-2-furan-
2-yl-[1,2,4]triazolo[1,5-c]quinazolin-5-yl)-2-phenyl-acetamide; MRS1754, N-(4-cyano-phenyl)-2-[4-(2,6-dioxo-
1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide; NECA, 5'-N-ethylcarboxamidoadeno-
sine; rolipram, 4-(3'-cyclopentyloxy-4'-methoxyphenyl)-2-pyrrolidone; WRC0571, 8-(N-methylisopropyl)amino-
N6-(5'-endohydroxy-endonorbornyl)-9-methyladenine; XAC, 8-(4-((2-aminoethyl)aminocarbonylmethyloxy)
phenyl)-1,3-dipropylxanthine; ZM241385, 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]
ethyl)-phenol
Introduction
There are four subtypes of G protein coupled adenosine
receptors (ARs), A1, A2A, A2B and A3. A2A ARs have been
extensively characterized in striatum (Rosin et al., 1998),
coronary arteries (Belardinelli et al., 1998) and platelets
(Dionisotti et al., 1996). A2AAR agonists have been reported
to produce anti-in¯ammatory responses mediated by recep-
tors on neutrophils (Cronstein et al., 1983; Sullivan et al.,
1999; reviewed by Gessi et al., 2000) and other types of
in¯ammatory cells (reviewed by Sullivan & Linden, 1998).
Among the most potent and selective agonists of A2AARs are
the 2-substituted amine, 2-[4-(2-carboxyethyl)phenethylami-
no]-5'-N-ethylcarboxamidoadenosine (CGS21680) (Hutchison
*Author for correspondence at: University of Virginia, Department
of Medicine, MR4 Box 801395, Charlottesville, Virginia, VA 22908,
U.S.A.; E-mail: jlinden@virginia.edu

1018
G.W. Sullivan et al
Anti-inflammatory 2-propynylcyclohexyl-adenosines
et al., 1989) the hydrazines, (Niiya et al., 1992) and the
2-alkynyls, e.g. 5-(6-amino-2-hex-1-ynyl-purin-9-yl)-3,4-di-hy-
droxy-tetrahydro-furan-2-carboxylic acid ethylamide (HE-
NECA) (Cristalli et al., 1992) (Figure 1). In this study we
describe the properties of new compounds designed to
combine the cyclohexyl element of a very active hydrazine,
MRE0470, and the alkynyl element of HE-NECA to produce
the ®rst 2- (4-substituted-cyclohexyl)propynyl NECAs, 4-
{ 3- [ 6-amino-9- ( 5-ethylcarbamoyl-3, 4-dihydroxy-tetrahydro-
furan-2-yl)- 9H- purin-2-yl] -prop-2-ynyl} - cyclohexanecarbox-
ylic acid methyl ester (ATL146e) and acetic acid 4-{3-[6-
amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-
yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexylmethyl ester (ATL
193); Figure 1). We observed that the nature of the 4-
substitution markedly a€ects the potency and selectivity of
these compounds.
(Bouma et al., 1997; Bowlin et al., 1997; Cronstein et al.,
1990; Sullivan et al., 1999) and in vivo (Bowlin et al., 1997;
Hasko et al., 1998; Hasko et al., 1996; Salvatore et al., 2000;
Sullivan et al., 1999; Szabo et al., 1998). Because there is
potential cross-reactivity between binding of adenosine
agonists to A2A ARs and A3 ARs, we have used the new
A2A AR agonists and selective receptor antagonists to
con®rm the neutrophil AR subtype responsible for inhibiting
neutriophil oxidative activity. Since there is disagreement in
the literature concerning the role of [cyclic AMP]i and cyclic
AMP-dependent protein kinase (PKA) in adenosine inhibi-
tion of the neutrophil oxidative burst (Cronstein et al., 1988;
Fredholm et al., 1996; Sullivan et al., 1995; 1999), we used
the phosphodiesterase inhibitor, rolipram, and the PKA
inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline
sulfonamide (H-89), to further investigate the role of cyclic
AMP as a mediator of the A2AAR response in neutrophils.
The data indicate that ATL146e and ATL193 are very potent
inhibitors of neutrophil oxidative activity and that they signal
predominantly through A2AARS, cyclic AMP and PKA.
We and others have observed that both A2A AR and A3
AR agonists have anti-in¯ammatory activities in vitro
Methods
Materials
4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-
amino]ethyl)-phenol (ZM241385) (Poucher et al., 1995) was a
gift from Simon Poucher, Astra-Zeneca Pharmaceuticals,
Cheshire, U.K. Stock solutions (1 mM and 10 mM in
dimethyl sulphoxide [DMSO]) were stored at 7208C.
Recombinant human tumor necrosis factor-a (TNF) was a
gift from Dainippon Pharmaceutical Co. Ltd Osaka, Japan
(speci®c activity=600 pg u71). Stock solutions were made in
Hanks balanced salt solution (HBSS)-0.1% human serum
albumin (HAS) at 26105 u ml71, aliquoted into single day
samples and frozen at 7708C. Rolipram (4-[3'-cyclopenty-
loxy-4'-methoxyphenyl]-2-pyrrolidone) was a gift from Berlex
Laboratories (Cedar Knolls, NJ, U.S.A.). Stock solutions
(10 mM) were made in DMSO and stored at 7708C, 8-(4-
carboxyethenylphenyl)-1,3-dipropylxanthine (BW1433) was a
gift from Susan Daluge (Glaxo-Wellcome, Research Triangle
Park, NC, U.S.A.). N-(4-cyano-phenyl)-2-[4-(2,6-dioxo-1,3-
dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acet-
amide (MRS1754) (Kim et al., 2000) and, N-(9-chloro-2-
furan-2-yl-[1,2,4]triazolo[1,5-c]quinazolin-5-yl)-2-p henyl-acet-
amide (MRS1220) (Jacobson, 1998) were gifts from
Kenneth Jacobson (National Institutes of Health; Bethesda,
MD, U.S.A.). 8-(N-methylisopropyl)amino-N6-(5'-endohy-
droxy-endonorbornyl)-9-methyladenine (WRC0571) (Martin
et al., 1996) and, 2-[6-amino-2-(N'-cyclohexylmethylene-
hydrazino)-purin-9-yl]-5-hydroxymethyl-tetrahydro-furan-3,4-
dio (MRE0470) (Martin et al., 1997) were gifts from Pauline
Martin (Discovery Therapeutics; Richmond, VA, U.S.A.).
CGS21680 was purchased from Research Biochemicals
Incorporated (Natick, MA, U.S.A.). Neutrophil isolation
medium (Ficoll-Hypaque) was purchased from ICN Biome-
dicals (Aurora, OH), Accurate Chemicals and Scienti®c
(Westbury, NY, U.S.A.), and Cardinal Associates (Santa
Fe, NM, U.S.A.). The following reagents were purchased
from Sigma Chemical (St. Louis, MO, U.S.A.), luminol,
8-(4-((2-a minoethyl) aminocarbony lmethyloxy)phenyl)-1,3-di-
Figure 1 Molecular structures of some A2A AR agonists discussed
in the study.
British Journal of Pharmacology vol 132 (5)

G.W. Sullivan et al
Anti-inflammatory 2-propynylcyclohexyl-adenosines
1019
propylxanthine (XAC) and formyl-methionyl-leucyl-phenyla-
lanine (fMLP). Adenosine deaminase was purchased from
Boehinger Mannheim (Indianapolis, IN, U.S.A.), and Sigma
Chemical. N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoli-
nesulfonamide (H-89), was purchased from Calbiochemical
(La Jolla, CA, U.S.A.).
vacuo to yield a syrup that was puri®ed via silica gel column
chromatography using 6% methanol/chlorform to yield pure
products. These products were characterized via 1H NMR,
13C NMR and MS. Purity was con®rmed via LC/MS and was
greater than 95% for all compounds tested.
Human neutrophil preparation
Synthesis of novel 2-cyclohexyl-propynyl-NECAs
A one-step Ficoll-Hypaque separation procedure (Ferrante &
Thong, 1980) was used to purify human neutrophils from
The synthesis of ATL146e involved the coupling of 2-iodo-5'-
(N-ethylcarboxamido) adenosine with the acetylenic methyl(-
cyclohexylcarboxylate). This synthetic strategy has been
described previously (Cristalli et al., 1992) and (Matsuda et
al., 1992) for the making 2-alkynyl A2A AR agonists and has
the value of enabling considerable synthetic manipulation of
the adenosine nucleus. The principal focus of our search for
novel A2A agonists was modifying the adenosine C2-position
to a€ect selectivity and potency. The conversion of the
adenosine 5'-position into an ethyl carboxamide was retained
because it improves A2A selectivity and precludes the possible
formation of phosphorylated metabolites from the action of
adenosine kinase at this position. Studies of A2A structure-
normal heparinized (10 u ml71
) venous blood yielding
approximately 98% neutrophils; 495% viable as determined
with trypan blue containing 550 pg ml71 of endotoxin.
Following separation, the neutrophils were washed with
Hank's balanced salt solution three times.
Radioligand binding assays
Our binding methodology has been described previously
(Sullivan et al., 1999). In brief, all four subtypes of
recombinant human ARs were stably expressed in HEK-
293 cells (A3, A2A, and A2B receptors) or CHO-K1 cells (A1
receptors). Crude membrane preparations from these
transfected cells were diluted in HE bu€er (50 mM HEPES;
1 mM EDTA pH 7.4) at concentrations between 2 ± 35 mg
tube71 in 50 ml. Dilutions of agonist and antagonist
compounds were prepared in HE plus ADA (2 u ml71),
MgCl2 (10 mM), and an appropriate radioligand (125I-ABA
for A1 and A3 receptors, 125I-ZM241385 for A2A receptors
and 125I-ABOPX for A2B receptors). The ®nal concentrations
in binding assays are ADA (1 u ml71) and MgCl2 (5 mM).
Diluted agonists or antagonists (50 ml) were added to each
membrane sample (50 ml), and the tubes incubated for 2 h at
room temperature and then ®ltered through glass ®bre ®lters
and counted. Samples were assayed in triplicate. Non-
speci®c binding was measured in the presence of XAC
(50 mM) (A1, A2A, and A2B receptors) or BW1433 (10 mM)
(A3 receptors).
activity relationships suggest that these sites exhibit
a
substantial level of independence ± that is, each site may be
modi®ed independently with an additive e€ect on activity.
Other analogues we have synthesized include the de-esteri®ed
hydrolysis product of ATL146e, 4-{3-[6-amino-9-(5-ethyl-
carbamoyl-3, 4-dihydroxy-tetrahydro-furan-2-yl) -9H-purin-2-
yl]-prop-2-ynyl}-cyclohexanecarboxylic acid (ATL146a), and
the inverse ester of ATL146e (ATL193) and its hydrolysis
product, 5-{6-amino-2-[3-(4-hydroxymethyl-cyclohexyl)-prop-
1-ynyl] - purin-9-yl} -3, 4- dihydroxy - tetrahydro - furan- 2 - car-
boxylic acid ethylamide (ATL2037).
The novel 2-alkynyladenosines (2-AAs) designed and
evaluated in this report required the coupling of a series of
terminal alkynes to 2-iodo 5'-N-ethylcarboxamidoadenosine
(NECA) (Cristalli et al., 1992) utilizing trietylamine (TEA)
and copper (I) iodide (CuI) in dimethylformamide (DMF)/
Acetonitrile with catalytic Pd(triphenylphosphine[PPh3])4 in a
classical Sonogashira cross-coupling fashion. These novel
alkynes were realized from modi®cation of commercially
available trans-1,4-cyclohexanedimethanol. This diol was ®rst
mono-protected with tertbutyldimethylsilyl chloride (TBSCl)
under classical conditions using imidazole and DMF as
solvent. The remaining free hydroxyl group was then
activated by formation of the tosylate using p-toluenesulpho-
nyl (TosCl) and pyridine in chloroform. This tosylate was
then displaced with the acetylene group through the use of
lithium acetylide. After deprotection with tetrabutylammo-
nium ¯uoride (TBAF), the alkyne precursor to ATL2037 was
complete. This compound was then either acetalated with
acetic anhydride to form the precursor to ATL193, or
oxidized via Jones oxidation to form the acid precursor
ATL146a. Finally, the ATL146e precursor was formed
through esteri®cation of ATL146a with trimethylsilyl (TMS)
diazomethane.
Neutrophil intracellular cyclic AMP concentrations
[cyclic AMP]i
Puri®ed human neutrophils were incubated in HBSS
containing 0.1% HSA (26106 PMN 250 ml71) and ADA
(1 u ml71),+ATL146e, ATL146a, ATL193, or CGS21680,
plus rolipram (1 mM), and+ZM241385 (100 nM) for 15 min
at 378C in a shaking water bath. The reaction was stopped
with 0.5 ml of 0.15 N HCl. The cells were pelleted by
centrifugation and 0.5 ml samples were frozen for cyclic
AMP analysis in the University of Virginia Radioimmuno-
assay Core Laboratory.
Neutrophil oxidative activity (luminol-enhanced
chemiluminescence)
Activated neutrophils emit light from unstable high-energy
oxygen species produced by the plasma membrane associated
NADPH oxidase and metabolized by cytoplasmic and
granule enzymes. The light signal from activated neutrophils
can be enhanced by the addition of luminol to the samples.
Luminol-enhanced emission of light is stimulated by singlet
oxygen, a reactive oxygen species, dependent on both the
To a solution of 2-Iodo NECA (0.025 mmol) and alkyne
(0.045 mmol) in DMF/acetonitrile (0.5 ml ml71) was added
TEA
(0.76 mmol),
Pd(PPh3)4
(0.0038 mmol),
CuI
(0.0046 mmol), and PPh3 (0.0038 mmol). The clear yellow
solution was heated to 608C for 12 h under nitrogen to yield
a brown/black solution. The solvent was then removed en
British Journal of Pharmacology vol 132 (5)

1020
G.W. Sullivan et al
Anti-inflammatory 2-propynylcyclohexyl-adenosines
production of superoxide and mobilization of myeloperox-
idase from primary granules (DeChatelet et al., 1982).
Puri®ed neutrophils (16106 ml71) were incubated in HBSS
containing 0.1% HSA (1 ml), plus adenosine deaminase
(1 u ml71),+ATL193, ATL2037, CGS21680, ATL146e or
ATL146a,+ZM241385,+H-89,+rolipram (100 nM), and
with TNFa (10 u ml71) for 30 min at 378C in a shaking
water bath. Then luminol (16104 M) enhanced f-met-leu-phe
Comparison of binding and functional activities of A2A
agonists
The e€ects of cyclohexyl substitutions had not been explored
previously. We found that these compounds, particularly
ATL146e and ATL193, were among the most potent and
selective A2A agonists yet synthesized. The polarity of the 4-
cyclohexyl substituent has a large e€ect on anity (see Figure
2 and Table 1). The ester, ATL146e, was 440 times more
(1 mM)-stimulated chemiluminescence was read with
a
Chronolog Photometer (Chronolog Corp., Havertown, PA,
U.S.A.) at 378C for 8 min. Chemiluminescence is reported as
relative peak light emitted (=height of the curve) compared
to TNFa-primed fMLP-stimulated control samples.
potent at human A2AARs than its de-esteri®ed acid
metabolite, ATL146a, or CGS21680. Compared to the
ATL146e, the inverse ester, ATL193, had similar anity,
but the de-esteri®ed metabolites di€er in anity; the
moderately polar alcohol, ATL2037, was more potent that
the more polar acid, ATL146a.
A2AAR agonist bioactivity in human plasma
Although the novel A2AAR agonist, ATL146e bound to
transfected human A2AARs with 4.8, 13 and 46 fold greater
anity than NECA, MRE0470 and CGS21680, respectively
(Figure 2 and Table 1), its absolute potency to uncoupled
recombinant human A2AARs was moderate; the Ki was
83 nM. CGS21680 and MRE0470 are widely recognized as
A2AAR selective compounds based on their relative binding
anities for A1 and A2A receptors in rat brain and based on
Heparinized (10 u ml71) platelet free human plasma was
prepared from normal blood by di€erential centrifugation
and then frozen (7808C). ATL193 and ATL146e (15 ml)
were added to 1.5 ml aliquots of plasma from 1 mM stock
solutions in DMSO and incubated at 378C in a 5% CO2
environment. Aliquots (200 ml) were sampled at 0, 15, 30,
60, 120, and 240 min and immediately added to 1.8 ml of
cold acetone to arrest compound metabolism and to
precipitate plasma proteins. Following centrifugation, the
supernatants were stored at 7208C. The acetone was dried
in a Speed Vac (Savant, Farmingdale, NY, U.S.A.) and
residual compound reconstituted in PBS and assayed by
competition for 125I-ZM241385 binding to recombinant
human A2AARs (see radioligand binding assays above).
The concentrations of ATL193 and ATL146e present were
calculated from an appropriate standard curve run con-
currently with the samples. Control serum samples contain-
ing vehicle did not compete for 125I-ZM241385 binding to
A
2AARs.
Statistical analysis Binding and functional data values
(mean, SEM, EC50 and KI) were determined using GraphPad
PRISM (San Diego, CA, U.S.A.). Schild analysis (Schild,
1957) was used to calculate pA2 values of inhibitors.
Results
Design of novel A2AAR agonists
Selectivity of adenosine analogues has been conferred by
modifying the 5'-hydroxyl of adenosine to make 5'-N-
ethylcarboxamine and by adding various 2-substitutions.
CGS21680 (Figure 1) is an adenosine analogue that is
substituted on the 5' ribose with N-ethylcarboxamide and
on the 2 position with amino-ethyl-phenyl-propionic acid.
This compound has emerged as the most widely used A2A
-
selective agonist (Jarvis et al., 1989). Among a series of 2-
hydrazino adenosine analogues, a very potent compound was
a 2-cyclohexyl derivative, MRE0470 (Niiya et al., 1992). Of
the various 2-substituted compounds that have been
synthesized, 2-propynyl compounds such as HE-NECA
appear to have high potency and selectivity (Cristalli et al.,
1992). These considerations led us to synthesize 2-propynyl-
cyclohexyl NECAs with various substitutions on the 4
position of the cyclohexyl ring.
Figure 2 Competition by A2A AR agonists for binding of 125I-
ZM241385 to recombinant human A2A ARs. (A) ATL146e,
ATL146a and CGS21680. (B) ATL193 and ATL2037. Membranes
(2 mg protein tube71) prepared from HEK-293 cells stably transfected
with human A2A ARs were incubated with 125I-ZM241385 (0.5 nM)
and various concentrations of competing compounds. Speci®c
binding was 480% of total binding. Each point is the mean+
s.e.mean of triplicate determinations. Similar results were obtained
in two or more additional experiments. Ki values are reported in
Table 1.
British Journal of Pharmacology vol 132 (5)

G.W. Sullivan et al
Anti-inflammatory 2-propynylcyclohexyl-adenosines
1021
Table 1 Binding to membranes derived from cells stably transfected with recombinant human ARs. Each value is the mean+s.e.mean
of 3 ± 10 replicate experiments
Receptor
A2A
A3
A2B
A1
Radioligand
(affinity)*
125I-ZM241385
(low)
(
125I-ABA)
(high)
(
125I-ABOPX)
(low)
(
125I-ABA)
(high)
Agonists
ATL146e
ATL146a
ATL193
ATL2037
NECA
KI (nM) [fold less affinity than ATL146e]
83+20 [1]
45+15 [1]
29+10 [0.6]
231+91 [5.1]
76+6 [1.7]
32+9 [0.7]
41+8 [0.9]
82+18 [1.8]
410,000
410,000
410,000
410,000
2200+532
410,000
410,000
77+12 [1]
3820+1249 [46]
187+25 [2.3]
694+69 [8.4]
401+106 [4.8]
1090+252 [13]
3820+733 [46]
189+19 [2.4]
71+14 [0.9]
19+1 [0.2]
2.0+1.4 [0.02]
113+21 [1.7]
316+59 [4.1]
MRE0470
CGS21680
Antagonists
WRC0571
MRS1220
ZM241385
MRS1754
KI (nM)
4110+2530
25+2
1.9+0.5{
508+62
410,000
1.3+0.1
774+124
570+151
410,000
355+60
33+10{
2.2+0.3
5.6+0.2
231+23
250+89{
403+16
*Agonists are known to bind to two anity states of G protein coupled receptors. Low anity binding re¯ects receptors uncoupled
from G proteins. High anity refers to receptor-G protein complexes that are selectively detected with agonist radioligands. {(Sullivan
et al., 1999).
functional assays, such as coronary dilator potency (Shryock
et al., 1998). We evaluated for the ®rst time the anity of
various ligands to all four subtypes of recombinant human
ARs in competition radioligand binding assays. None of the
agonists appear to be highly selective for human A2AARs
over A1ARs or A3ARs (Table 1). However, these binding
parameters are not a direct measure of functional potency,
since agonist anity is in¯uenced by G protein coupling and
spare receptors in tissues. The relative potency at A2AARs is
probably underestimated (see Discussion). Functionally, the
potency of ATL146e to inhibit oxidative burst in human
neutrophils (EC50=0.32 nM, Figure 3) was 260 fold greater
than the potency for binding to human A2AARs (KI=83 nM;
Table 1). The potency orders in assays of binding to
proteins. Hence, it would be predicted that A2A and A2B AR
antagonists will decrease cyclic AMP accumulation stimu-
lated by their respective receptor agonists and that A1 and A3
AR antagonism would result in increased [cyclic AMP]i in
response to A1 and A3 AR agonist stimulation, respectively.
The accumulation of cyclic AMP stimulated by ATL193 was
potently blocked by ZM241385 (100 nM) and not by 1 mM
WRC0571 nor by MRS1754 (Figure 4). It is signi®cant that
the A3 AR-selective antagonist, MRS1220, has only moderate
selectivity (19 fold) for A3 over A2A ARs. If used at
concentrations in the range of 100 nM ± 1 mM, MRS1220 will
not distinguish well between responses mediated by human
A2A and A3 receptors and consequently (as would be
predicted of an A2A AR antagonist but not an A3 AR
antagonist) decreased ATL193-stimulated cyclic AMP accu-
mulation. In contrast, ZM241385 is 407 fold selective for
human A2A over A3 ARs, and produces selective A2A
blockade of [cyclic AMP]i stimulated by ATL 193 in the
range of 100 nM ± 1 mM. ZM241385 also antagonized ATL193
inhibition of neutrophil oxidative activity (Figure 5). Schild
analysis of the data indicated a pA2=8.86 (KD=1.4 nM;
Figure 5, inset) which agrees closely with the KI of
ZM241385 for binding to A2AARs (1.9 nM).
human
and inhibition of oxidative burst in all cases were
the same: ATL146e4ATL1934NECA4ATL20374
A2AARs, neutrophil cyclic AMP accumulation
MRE04704CGS21680 & ATL146a (Figures 2 and 3, Table
1). In contrast, ATL146e and ATL193 had comparable
binding anity compared to CGS21680 and ATL146a for
binding to human A3ARs (Table 1). ATL146e and ATL193
had * 30 fold lower anity for human A1ARs compared to
NECA, and all of the agonists tested had relatively low
anity for human
A2BARs. These results suggest that
inhibition of the oxidative burst in neutrophils by ATL146e
and ATL193 are mediated by A2A ARs.
The results of cyclic AMP assays with selective
antagonists are consistent with the conclusion that
The role of cyclic AMP and cyclic AMP-dependent
kinase (PKA) in mediating the functional effects of A2A
AR agonists
the agonists are acting through
order of antagonists for human
A
2AARs. The anity
2AARs was
Phosphodiesterase (PDE) type IV is the most abundant
isozyme found in in¯ammatory cells. Selective PDE IV
inhibitors such as rolipram (Schneider et al., 1986) increase
cellular [cyclic AMP]i by blocking the breakdown of cyclic
AMP to 5' AMP. Although we have observed that higher
concentrations of rolipram decrease neutrophil oxidative
activity (Sullivan et al., 1995), rolipram (100 nM) alone had
little e€ect on TNFa-primed fMLP-stimulated human
neutrophil oxidative activity, but synergistically decreased
the oxidative burst of neutrophils when added with sub-
A
ZM2413854MRS122044MRS175444WRC0571 (Table
1). ZM241385 (100 nM) selectively blocks A2A and not
A3ARs; WRC0571 selectively blocks A1 and not A2A
receptors; and MRS1754 is a highly selective antagonist of
human A2BARs (Kim et al., 2000). Agonist activity at A2A
ARs and A2B ARs stimulate adenylyl cyclase through
coupling to Gs proteins. In contrast, agonist activity at A1
and A3 ARs inhibit adenylyl cyclase through coupling to Gi
British Journal of Pharmacology vol 132 (5)

1022
G.W. Sullivan et al
Anti-inflammatory 2-propynylcyclohexyl-adenosines
Figure 4 Competitive antagonism of ATL193-stimulated neutrophil
cyclic AMP accumulation. (A) ZM241385 (100 nM). (B) ATL193
(1 mM), WRC0571, MR1754, MRS1220, and ZM241385 (10 ±
Figure 3 Inhibition of the neutrophil oxidative burst and stimula-
tion of neutrophil [cyclic AMP]i by agonists of A2A ARs. (A)
ATL146e, ATL146a, and CGS21680. (B) ATL193 and ATL2037.
Luminol-enhanced chemiluminescence was assayed in human neu-
trophils (16106 ml71) primed with TNFa (10 u ml71; 30 min; 378C)
and then stimulated with fMLP (1 mM; 10 min; 378C)+A2A AR
agonists+ADA (1 u ml71). The results are reported as peak
chemiluminescence as per cent of a TNF-primed control. Each point
1000 nM). Human neutrophils (86106 ml71
)
were incubated
(15 min; 378C) with 1 mM rolipram and 1 u ml71 ADA)+ATL193
(0.01 ± 100 nM) and+adenosine receptor antagonists (WRC0571,
MRS1754; MRS1220 or ZM241385; 10 ± 1000 nM). Neutrophil [cyclic
AMP]i was measured by radioimmunoassay. Each point (cyclic AMP
pmol ml71) is the mean+s.e.mean (n=6).
is the mean+s.e.mean, n=476 experiments. The EC50s for
decreasing oxidative activity are: ATL146e=0.3 nM; ATL193=
0.8 nM; ATL146a=16.7 nM; ATL2037=4.4 nM; and CGS21680=
9.2 nM. Inset: Human neutrophils (86106 ml71
) were incubated
(15 min; 378C; with 1 mM rolipram and 1 u ml71 ADA)+A2A AR
agonists. Neutrophil [cyclic AMP]i was measured by radio-
immunoassay. Each point (cyclic AMP pmol ml71) is the mean+
s.e.mean (n=6). The EC50 values for increased neutrophil [cyclic
AMP]i are ATL146e=7.1 nM; ATL193=5.6 nM; ATL146a=
19.7 nM; ATL2037=8.6 nM and CGS21680=17.1 nM.
optimal concentrations of ATL193. The EC50 was shifted 20
fold from 0.81 nM to 0.04 nM upon the addition of 100 nM
rolipram (Figure 6). The PKA inhibitor H-89 (Chijiwa et al.,
1990) did not a€ect TNFa-primed fMLP-stimulated human
neutrophil oxidative activity (Figure 7), did not prime
neutrophils in the absence of TNFa (data not shown), did
not bind to human A2A receptors (data not shown), but did
completely counteract the inhibitory e€ect of ATL193
(10 nM) on the neutrophil oxidative burst (Figure 7). The
EC50 of H-89 for reversing ATL193 (10 nM)-inhibited
neutrophil oxidative burst was 2.2 mM.
Figure 5 E€ect of ZM241385 on ATL193-inhibited neutrophil
oxidative activity. Luminol-enhanced chemiluminescence was assayed
in neutrophils (16106 ml71) primed with TNFa (10 u ml71; 30 min;
378C) and then stimulated with fMLP (1 mM; 10 min;
378C)+ATL193 (0.01 nM ± 100 nM)+ZM241385+ADA (1 u ml71).
The results are reported as percent TNF-primed control. Each point
is the mean+s.e.mean (n=6). Inset: Schild analysis, pA2=8.86.
A2AAR agonist bioactivity in human plasma
ATL146e bioactivity was lost after 240 min, ATL193 activity
reached a plateau at about one-third of the initial bioactivity
(Figure 8). These results are consistent with the possibility
that ATL146e and ATL193 are de-esteri®ed to the acid,
ATL146a and the alcohol, ATL2037, respectively.
We observed that both ATL146e and ATL193 lost bioactivity
when incubated in human plasma with a half-life of 55 and
41 min, respectively (Figure 8). While almost all of the
British Journal of Pharmacology vol 132 (5)

G.W. Sullivan et al
Anti-inflammatory 2-propynylcyclohexyl-adenosines
1023
Figure 6 Synergistic e€ects of rolipram and ATL193 on the
oxidative burst in human neutrophils. Luminol-enhanced chemilumi-
nescence was measured in human neutrophils (16106 ml71) primed
with TNFa (10 u ml71; 30 min; 378C) and then stimulated with
fMLP (1 mM 10 min; 378C)+ATL193 (0.01 nM ± 100 nM)+rolipram
(100 nM)+ADA (1 u ml71). The results are reported as per cent of
TNF-primed control. Each point is the mean+s.e.mean (n=4 ± 5).
Figure 8 Kinetics of declines in the bioactivity of ATL146e and
ATL193 in human plasma. (A) ATL146e. (B) ATL193. ATL193 and
ATL146e (15 ml of 1 mM) were added to 1.5 ml aliquots of human
plasma and incubated (378C; 5% CO2) for 0 ± 240 min. Aliquots
(200 ml) were transferred to 1.8 ml of cold acetone at each time point.
Following removal of precipitated proteins and rapid evaporation
bioactivity was assayed by competitive radioligand binding to A2A
ARs. Each point is the mean+s.e.mean (n=3 ± 4).
Figure 7 H-89 counteracts ATL193-inhibited neutrophil oxidative
activity. Luminol-enhanced chemiluminescence was measured in
human neutrophils (16106 ml71) primed with TNFa (10 u ml71
;
the fact that agonists bind to two anity states of G protein
coupled receptors. Low anity binding sites re¯ect agonist
binding to receptors uncoupled from G proteins, and high
anity sites re¯ect binding to receptor-G protein complexes
that are selectively detected with agonist radioligands.
A2AARs are poorly coupled to Gs in striatum and
particularly to overexpressed recombinant receptors in
transfected cells (Luthin et al., 1995). These uncoupled
receptors are best detected with antagonist radioligands.
Binding of agonists to uncoupled receptors was measured in
competition with antagonist radioligands, 125I-ZM241385 for
A2A ARs and 125I-ABOPX for A2B ARs. This and possible
spare receptors on neutrophils may account for the large
discrepancy (4250 fold) between A2A agonist ligand Ki
values and EC50 values to inhibit neutrophil oxidative
activity. In contrast, agonist binding to the Gi/o coupled
A1 and A3ARs detects primarily GTP-sensitive high anity
binding sites of these well-coupled receptor-G protein
complexes.
30 min; 378C) and then stimulated with fMLP (1 mM;
10 min; 378C)+ATL193 (10 nM)+H-89 (100 nM ± 10 mM)+ADA
(1 u ml71). The results are reported as percent of a TNF-primed
control. Each point is the mean+s.e.mean (n=6).
Discussion
We have investigated the properties of novel 2-propylylcy-
clohexyl NECAs as potent and selective agonists of human
A
2AARs. These compounds combine the best features of a
series of 2-substituted hydrazine and 2-substituted propynyl
compounds to make 2-propynyl-cyclohexyl derivatives. Of
interest was the new ®nding that the substituent on the 4-
position of the cyclohexyl ring had a profound e€ect on
binding anity to human A2AARs. In particular, the ester,
ATL146e, had 440 times higher anity than the acids,
ATL146a and CGS21680. Our hypothesis for the present
studies was that the novel A2A-selective agonists would
inhibit pro-in¯ammatory neutrophil activity by binding A2A
receptors via a cyclic AMP-dependent mechanism.
The potency order of both agonists (ATL146e4ATL1934
NECA4ATL20374MRE04704CGS21680
and antagonists (ZM2413854MRS122044MRS1754
&
ATL146a)
&
Based on radioligand binding assays, none of the
compounds we evaluated, including ATL146e or CGS21680,
were highly selective for human A2A receptors over human
A1 and A3 receptors. This incongruity can be explained by
WRC057) in neutrophil functional assays (i.e. oxidative
activity and [cyclic AMP]i) correlate well with the potency
order of these compounds in binding assays. These data
support the conclusion that the agonists act through A2AARs
British Journal of Pharmacology vol 132 (5)

1024
G.W. Sullivan et al
Anti-inflammatory 2-propynylcyclohexyl-adenosines
to increase neutrophil [cyclic AMP]i and decrease the
oxidative burst, rather than through A3ARs (where ATL146e
consistently lower than the EC50 for increasing neutrophil
[cyclic AMP]i. This suggests the possibility that e€ector
function stimulated by A2A AR agonists may also act
through cyclic AMP/PKA independent pathway(s). It has
been recently reported that stimulation of b-adrenergic
receptors on thymocytes in addition to activating PKA, can
stimulate through Gs and the Src family tyrosine kinase Lck
to induce apoptosis independent of cyclic AMP/PKA (Gu et
al., 2000). It is not known if neutrophils have analogous Src
family tyrosine kinase dependent cyclic AMP independent
pathway(s) stimulated by A2A AR agonists.
The e€ects of ATL193 on neutrophil oxidative activity
were markedly potentiated by the type IV PDE inhibitor
rolipram (Figure 6), and ATL193 suppression of the
oxidative burst was completely counteracted by the PKA
inhibitor H-89 (Figure 7). These data taken together strongly
indicate that the substituted 2-propynylcyclohexyl adenosine
agonists are anti-in¯ammatory predominantly by increasing
neutrophil [cyclic AMP]i. Possible e€ects of PKA activation
in neutrophils include phosphorylation of Rap1A, preventing
incorporation of cytochrome b558 into the NADPH oxidase
complex (Bokoch et al., 1991), and phosphorylation of RhoA
(Laudanna et al., 1997) resulting in diminished phospholipase
D activity (Thibault et al., 2000) and reduced oxidase activity
(Frank et al., 1994). In addition, PKA activation causes
reduced fMLP-stimulated phosphorylation of the NADPH
oxidase component p47 phox upstream of protein kinase C
(Bengis-Garber & Gruener, 1996).
We observed that the binding anities and functional
activities of the relatively nonpolar compounds, ATL146e
and ATL193, were much greater at human A2A ARs than
their more polar de-esteri®ed products, ATL146a and
ATL2037 or the polar agonist CGS21680 (Figure 1). The
rapid decay of bioactivity of ATL146e and ATL193 in
human plasma are consistent with the possibility that serum
esterase(s) convert the esters to the corresponding acid,
ATL146a, and alcohol, ATL2037, respectively. ATL146a had
little detectable residual bioactivity in plasma since ATL146a
had 46 times less binding anity than ATL146e for A2AARs.
The residual activity of ATL2037 in plasma could be because
it bound to A2AARs with only 3.6 fold lower anity than
ATL193.
&
CGS21680 in binding anity) or A1ARs (where
NECA4ATL146e and ATL20374ATL193 in binding
anity). Although the A2A AR agonists are potent inhibitors
of the neutrophil oxidative burst, they do not completely
inhibit the oxidative burst. This suggests that some of the
oxidative burst is resistant to inhibition by A2A AR agonist
binding. This is consistent with our previous observations
(Sullivan, et al., 1995) that A2A AR agonists do not decrease
unstimulated neutrophil oxidative activity nor oxidative
activity induced by some stimuli (e.g. phorbol myristate
acetate). These data indicate that the neutrophil oxidase is
not directly a€ected by A2A AR binding, but rather upstream
process(es) such as coupling of the f-met-leu-phe receptor to
its signalling pathway (Cronstein et al., 1992a) may be
a€ected.
In addition to
A2AARs, neutrophils express A1ARs
(Cronstein et al., 1992b), and possibly A3 receptors that
may control human neutrophil degranulation (Bouma et al.,
1997). Unlike binding to A2AARs, when adenosine binds to
A1ARs the e€ects are pro-in¯ammatory, resulting in
increased adhesion of neutrophils to endothelial cells
(Cronstein et al., 1992b), augmented phagocytosis (Salmon
& Cronstein, 1990), and enhanced stimulation of oxidative
burst by immune complexes (Salmon & Cronstein, 1990). In
contrast, 2-propynylcyclohexyl adenosine receptor agonists
displayed only anti-in¯ammatory activity, substantiating their
selectivity for A2AARs.
Whereas agonist binding to either A2A or A2B ARs
stimulates adenylyl cyclase via Gs coupling, agonist binding
to A1 and A3 ARs signal via Gi coupling to decrease adenylyl
cyclase activity (Ongini & Fredholm, 1996). 2-Propynylcyclo-
hexyl adenosine receptor agonists only stimulated neutrophil
[cyclic AMP]i, and this e€ect was selectively blocked by the
A2A AR antagonist, ZM241385. Consistent with the expecta-
tion of little binding by the new compounds to human A2B
ARs, the selective A2BAR antagonist MRS1754 had little
e€ect on agonist-stimulated [cyclic AMP]i.
Like
A2AAR agonists, other agents that stimulate
neutrophil cyclic AMP accumulation also decrease neutrophil
oxidative activity (Bengis-Garber & Gruener, 1996). How-
ever, the roles of cyclic AMP and cyclic AMP-dependent
PKA in the A2A receptor-mediated anti-in¯ammatory e€ects
of adenosine on neutrophil function are controversial. There
are con¯icting reports regarding the correlation between
adenosine-stimulated [cyclic AMP]i and e€ects on neutrophil
function (Cronstein et al., 1988). Supporting a role for cyclic
AMP is the ®nding of synergistic e€ects of A2AAR agonists
and type IV phosphodiesterase inhibitors (Sullivan et al.,
1995). However, another study reported additive activity
(Cronstein et al., 1988). In addition, some PKA inhibitors
only partially counteract inhibition of neutrophil stimulated
oxidative activity (Mueller et al., 1992). Hence, the role of
cyclic AMP in the A2A adenosine receptor anti-in¯ammatory
response in neutrophils is unresolved.
Several anti-in¯ammatory responses occur when A2AARs
are activated on neutrophils. Adenosine and A2AAR agonists
decrease expression of adhesion molecules (Wollner et al.,
1993), which results in decreased neutrophil adherence to
matrix proteins (Sullivan et al., 1999) and to vascular
endothelium (Cronstein et al., 1992b).
A2AAR agonists
reduce the oxidative activity of both suspended and adherent
neutrophils (Cronstein et al., 1983; De La Harpe & Nathan,
1989; Sullivan et al., 1999), and inhibit neutrophil degranula-
tion (Richter, 1992).
There is also evidence in vivo that adenosine is anti-
in¯ammatory by binding to A2AARs. For example, metho-
trexate appears to act by increasing adenosine concentrations
within in¯amed tissues. This endogenous adenosine binds to
neutrophil A2AARs and attenuates the in¯ammatory re-
sponse (Cronstein et al., 1993). Moreover, we have observed
that the A2AAR agonist, MRE0470, inhibits endotoxin-
induced pleocytosis and blood brain barrier permeability in
a rat model of bacterial meningitis (Sullivan et al., 1999). In
recent studies we have shown that ATL146e can protect rat
In the present study, we observed a correlation between
stimulation of neutrophil [cyclic AMP]i and inhibition of the
oxidative burst by the substituted 2-propynylcyclohexyl
adenosine agonists. Although there is a correlation between
neutrophil [cyclic AMP]i and inhibition of the oxidative
burst, the EC50 for decreasing the oxidative burst is
British Journal of Pharmacology vol 132 (5)

G.W. Sullivan et al
Anti-inflammatory 2-propynylcyclohexyl-adenosines
1025
The authors wish to thank Dr Simon Poucher of Astra-Zeneca
(Cheshire, U.K.) for his gift of ZM241385, Dr Susan Daluge of
Glaxo-Wellcome (Research Triangle Park, NC, U.S.A.) for
BW1433 and Dr Pauline Martin of Discovery Therapeutics
(Richmond, VA, U.S.A.) for WRC0571 and MRE0470. We thank
Melissa Marshall for expert technical assistance with the ligand
binding assays. Supported in part by RO1HL37942 (Joel Linden)
from the NIH.
kidneys from ischaemia/reperfusion induced damage (Okusa
et al., 1999), and lung reperfusion injury in
transplantation model (Ross et al., 1999).
We conclude that substituted 2-propynylcyclohexyl adeno-
sine agonists, ATL146e and ATL193, inhibit the neutrophil
oxidative burst by binding predominantly to A2A ARs and
act principally by a mechanism mediated by cyclic AMP-
dependent PKA. Hence, compounds in this class may have
therapeutic potential for reducing in¯ammation due to
infection or ischaemia.
a rabbit
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(Received September 29, 2000
Revised November 23, 2000
Accepted December 8, 2000)
British Journal of Pharmacology vol 132 (5)