[3H]OSIP339391, a selective, novel, and high affinity antagonist radioligand for adenosine A2B receptors

Article abstract of DOI:10.1016/j.bcp.2004.03.026

Until recently, the characterization of adenosine A_2B receptors has been hampered by the lack of high affinity radioligands. This study describes the synthesis and in vitro characterization of the radiolabeled derivative of OSIP339391, a novel,

Full text of DOI:10.1016/j.bcp.2004.03.026

Biochemical Pharmacology 68 (2004) 305–312
[3H]OSIP339391, a selective, novel, and high affinity antagonist
radioligand for adenosine A2B receptors
Mike Stewart, Arno G. Steinig, Chienling Ma, Jian-Ping Song1, Bryan McKibben2,
Arlindo L. Castelhano*, Stephen J. MacLennan3
OSI Pharmaceuticals Inc., Bioscience Park Drive, Farmingdale, NY 11735, USA
Received 17 December 2003; accepted 23 March 2004
Abstract
Until recently, the characterization of adenosine A2B receptors has been hampered by the lack of high affinity radioligands. This study
describes the synthesis and in vitro characterization of the radiolabeled derivative of OSIP339391, a novel, potent, and selective
pyrrolopyrimidine A2B antagonist. OSIP339391 had a selectivity of greater than 70-fold for A2B receptors over other human adenosine
receptor subtypes. The radiolabel was introduced by hydrogenation of the acetylenic precursor with tritium gas resulting in the
incorporation (on average) of three tritium atoms in the molecule, yielding a ligand with specific activity of 87 Ci/mmol (3.2 TBq/mmol).
Using membranes from HEK-293 cells expressing the human recombinant A2B receptor, [3H]OSIP339391 was characterized in kinetic,
saturation, and competition binding experiments. From the association and dissociation rate studies, the affinity was 0.41 nM and in close
agreement with that found in saturation binding experiments (0.17 nM). In competition, binding studies using 0.5 nM [3H]OSIP339391,
the affinity of a range of agonists and antagonists was consistent with previously reported data. Thus, [3H]OSIP339391 is a novel,
selective, and high affinity radioligand that can be a useful tool in the further exploration and characterization of recombinant and
endogenous adenosine A2B receptors.
# 2004 Published by Elsevier Inc.
Keywords: Adenosine receptors; Radioligand binding; Pharmacology; Radiolabel synthesis; Non-xanthine A2B receptor antagonist; Receptor
characterization
1. Introduction
receptor. The filtration and washing procedures routinely
employed to terminate radioligand binding assays reduce
the free radioligand concentration in the assay medium,
thus disturbing the equilibrium and driving dissociation of
the receptor–radioligand complex. Hence, binding assays
utilizing low affinity radioligands such as [3H]-NECA will
suffer a marked loss in specific binding during the filtration
and washing process. This in turn may compromise assay
reproductibility and hinder the ability to detect low recep-
tor expression levels. The binding of high affinity radi-
oligands (i.e. slowly dissociating) is not significantly
affected by the termination process and hence tends to
be more robust and reproducible [1,11]. More recently, a
high affinity radioligand for A2B receptors ([3H]MRS
1754) has been described [12] but, this compound was
not commercially available at the time of this study. In the
course of our studies we have identified a series of novel
pyrrolopyrimidine compounds with high affinity for the
human A2B receptor. One such compound is OSIP339391,
Characterization of adenosine A2B receptors using radi-
oligand binding techniques has, until recently, necessitated
the use of relatively low affinity ligands, for example [3H]-
DPCPX [19], which actually has greater affinity for A1
receptors, and [3H]-NECA, a non-selective A2 receptor
agonist [3]. By definition, the inherent low affinity (KD
values >20 nM) of these ligands for the human adenosine
A
2B receptor translates to a rapid dissociation rate from the
Abbreviations: TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl-
uronium tetrafluoroborate; DMF, dimethyl formamide; DEPT, distortion-
less enhancement by polarization transfer
* Corresponding author.
E-mail address: arlindo.castelhano@osip.com (A.L. Castelhano).
1 Present address: Athersys Inc., Cleveland, OH 44114, USA.
2 Present address: Boehringer-Ingelheim Pharmaceuticals, Ridgefield,
CT 06877, USA.
3 Present address: King Pharmaceuticals R&D, Cary, NC 27513, USA.
0006-2952/$ – see front matter # 2004 Published by Elsevier Inc.
doi:10.1016/j.bcp.2004.03.026

306
M. Stewart et al. / Biochemical Pharmacology 68 (2004) 305–312
(N-(2-{2-phenyl-6-[4-(3-phenylpropyl)-piperazine-1-
carbonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino}-ethyl)-
acetamide). The present study describes the development
of a receptor binding assay for human adenosine A2B
receptors expressed in HEK-293 cells utilizing
[3H]OSIP339391 as a high affinity radioligand.
2850, 1654, 1589, 1573, 1531, 1455, 1429, 1386, 1367,
1327, 1298, 1270, 1171, 1135, 1027, 999, 970, 803, 776,
755, 704, 691. 1H NMR (CDCl3, 200 MHz): d ¼ 1:80
(s, 3H), 2.73 (mc, 4H), 3.50–3.61 (m, 4H), 3.80–4.00
(m, 6H), 5.93 (brs, 1H), 6.68 (s, 1H), 6.82 (brs, 1H),
7.25–7.35 (m, 3H), 7.35–7.55 (m, 5H), 8.37–8.47 (m,
2H), 9.63 (brs, 1H). 13C NMR (CDCl3, 50.3 MHz, addi-
tional DEPT135): d ¼ 23:03 (þ), 29.66 (À), 40.60 (À),
41.70 (À), 47.67 (À), 51.93 (À), 83.63 (Cquart), 85.81
(Cquart), 101.97 (þ), 101.20 (Cquart), 122.70 (Cquart),
126.40 (Cquart), 128.04 (þ), 128.28 (þ), 129.81 (þ),
131.69 (þ), 138.70 (Cquart), 151.13 (Cquart), 157.67 (Cquart),
159.89 (Cquart), 161.60 (Cquart), 171.17 (Cquart). tR
ðHPLCÞ ¼ 7:5 min. C30H31N7O2Á0.5H2O (530.64): Calcd.
C 67.91, H 6.08, N 18.48; found C 67.88, H 6.09, N 18.46.
2. Methods
2.1. Synthesis
[3H]OSIP339391 ([3H]-4) was synthesized as shown
below (Scheme 1).
2.1.1. N-(2-{2-Phenyl-6-[4-(3-phenylprop-2-ynyl)-
piperazine-1-carbonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-
ylamino}-ethyl)-acetamide (3)
2.1.2. N-(2-{2-Phenyl-6-[4-(2,2,3,3-tetratritio-3-
phenylpropyl)-piperazine-1-carbonyl]-7H-pyrrolo[2,3-
d]pyrimidin-4-ylamino}-ethyl)-acetamide ([3H]-4)
TBTU (37 mg, 0.12 mmol) is added to a solution of acid
1 (30 mg, 0.088 mmol) [4,5] in dimethyl formamide
(DMF) (1.3 ml), cooled by ice/water. After 30 min, amine
2 (19 mg, 0.095 mmol) [21] and triethylamine (20 ml,
0.14 mmol) are added, the cooling bath is removed,
and the reaction mixture is stirred at ambient temperature
for 3 h. Water (10 ml) is then added, and the mixture
is extracted with EtOAc (4 ꢀ 15 ml). The combined
organic layers are washed with 2N NaOH, water (3ꢀ),
and brine, dried over MgSO4, filtered and concentrated.
The crude material is purified by chromatography on silica
gel eluting with CH2Cl2 ! 5% MeOH in CH2Cl2 ! 7.5%
MeOH in CH2Cl2 followed by trituration with Et2O,
yielding 21 mg (0.040 mmol, 46% yield) of 3 as an off-
white solid, mp 218–219 8C. MS (ES): m/z 522 (100)
[MHþ], 322 (100) [MHþ—(3-phenylprop-2-ynyl)pipera-
zine]. IR (film): n ¼ 3302 cmÀ1, 3061, 3022, 2924,
A solution of the alkyne 3 (2.6 mg, 4.95 mmol) in
methanol (2 ml) was reacted for 5 h at room temperature
in an atmosphere of tritium gas using 10% Pd/BaSO4
(18 mg). The catalyst was removed by filtration, and labile
tritium was removed by repeated evaporations to dryness
from methanol. The crude product was purified using
an ultrasphere ODS column (10 mm ꢀ 250 mm) and a
MeOH:H2O:NEt3 gradient system (solvent A: water with
0.1% NEt3, solvent B: methanol with 0.1% NEt3; elution:
0–18 min, 60% B isocratic; 18–37 min, 65% B isocratic;
37 min, 70% B isocratic). The purified material (72%
yield) had a radiochemical purity of 98% and co-chroma-
tographed with the non-labeled reference. The triple-bond
reduction gives a mixture of species with zero to four
tritium ions incorporated, presumably, as a consequence
of scrambling. Mass spectrometric analysis showed an
Scheme 1.

M. Stewart et al. / Biochemical Pharmacology 68 (2004) 305–312
307
average of 3.02 tritiums, MW ¼ 531:7 with a specific
Commercially available anhydrous solvents and HPLC-
grade solvents were used without further purification.
activity of 87 Ci/mmol (3.2 TBq/mmol).
2.1.3. N-(2-{2-Phenyl-6-[4-(3-phenylpropyl)-piperazine-
1-carbonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino}-
ethyl)-acetamide (4)
2.3. Pharmacological methods
The human A2B receptor was cloned and expressed
in Human Embryonic Kidney cells (HEK-293) in our
laboratory using standard techniques [17]. Cells stably
expressing the human A2B receptor were grown to
confluency in DMEM containing 10% fetal bovine
serum (FBS), penicillin/neomycin/streptomycin antibio-
tic (PNS) and 0.1 mg/ml G418. Cells were washed once
with phosphate-buffered saline (PBS) and harvested by
scraping and centrifugation for 10 min at 1000 ꢀ g. Cell
pellets were resuspended in lysis buffer (5 mM Tris
containing 5 mM EDTA and 5 mM EGTA, pH 7.4 at
4 8C), homogenized and centrifuged for 30 min at
30,000 ꢀ g. This process was repeated with the final
pellet being resuspended in ice-cold 10 mM HEPES,
pH 7.4 at 4 8C and frozen at À80 8C until use. Protein
concentration was determined using a standardized Coo-
massie Blue protocol (Pierce Life Sciences).
The methods used for radioligand binding were mod-
ified from those described by Stehle et al. [19]. All assays
were performed in 10 mM HEPES containing 1 mM
EDTA, 2 U/ml adenosine deaminase (ADA), 0.2 mM
benzamidine, and 5 mg/ml fatty acid free BSA (pH 7.4
at 22 8C). A total assay volume of 100 ml was employed
in which final protein levels varied between 1 and 5 mg
per well depending upon protocol. The time to equili-
brium and the association rate constant at 22 8C were
determined by incubating 25 ml of 1 nM [3H]OSIP339391
with 50 ml of adenosine A2B receptor membrane homo-
genate for various durations. The dissociation rate con-
stant was determined by adding 10 mM DPCPX to the
assay mixture following an initial 60 min incubation
period at 22 8C. This excess concentration of unlabeled
competitor acts to initiate radioligand dissociation and
effectively displaces all radioligand from the receptor
over time. Total and non-specific binding was determined
in quadruplicate in both association and dissociation
experiments at time points ranging from 1 to 180 min.
Saturation assays were performed by measuring specific
binding at concentrations ranging from 0.02 to 12 nM
with total and non-specific binding being determined in
duplicate. NSB was defined using an excess of cold
OSIP339391 (10 mM). Competition assays were per-
formed with 0.5 nM [3H]OSIP339391 using 10 concen-
trations of test compounds. All binding assays were
initiated by addition of membrane homogenate and were
terminated by rapid vacuum filtration using a Packard
UNIFILTER-96 cell harvester through GF/C grade filters
presoaked in ice-cold 0.5% PEI. Trapped membrane
was then given 6 ml ꢀ 300 ml washes with ice-cold
10 mM HEPES (pH 7.4 at 4 8C). Filters were dried
and receptor bound radioligand was quantified using
White solid, mp 198–200 8C. MS (ES): m/z 526 (51)
[MHþ]. IR (film): n ¼ 3295 cmÀ1, 3062, 3024, 2929,
2857, 1654, 1589, 1573, 1530, 1454, 1432, 1387, 1328,
1
1297, 1170, 1132, 1027, 1001, 776, 749, 703. H NMR
(CDCl3, 400 MHz): d ¼ 1:79 (s, 3H), 1.83 (quint,
J ¼ 7:4 Hz, 2H), 2.39 (t, J ¼ 7:6 Hz, 2H), 2.47 (mc,
4H), 2.65 (t, J ¼ 7:6 Hz, 2H), 3.52–3.56 (m, 2H), 3.80–
3.90 (m, 6H), 6.19 (brs, 1H), 6.67 (s, 1H), 7.01 (brs, 1H),
7.16–7.42 (m, 8H), 8.37–8.40 (m, 2H), 10.12 (brs, 1H). 13
C
NMR (CDCl3/CD3OD, 50.3 MHz, additional DEPT135):
d ¼ 22:97 (þ), 28.31 (À), 33.50 (À), 40.85 (À), 41.19 (À),
41.35 (À), 53.01 (À), 57.66 (À), 101.95 (2C, þ, Cquart),
125.85 (þ), 126.35 (Cquart), 127.96 (þ), 128.33 (þ),
129.91 (þ), 138.64 (Cquart), 141.84 (Cquart), 150.95 (Cquart),
157.59 (Cquart), 160.07 (Cquart), 161.49 (Cquart), 171.42
(Cquart). tR ðHPLCÞ ¼ 4:4 min. C30H35N7O2 (525.66):
Calcd. C 68.55, H 6.71, N 18.65; found C 68.93, H
6.78, N 18.26.
2.2. Analytical methods
LC–MS analysis was performed using a Gilson 215
autosampler and Gilson 819 autoinjector, attached to a
Hewlett Packard HP110. Mass spectra were obtained on a
Micromass Platform II mass spectrometer, using positive
electrospray ionization. LC analysis was undertaken at
254 nm using a UV detector. Samples were eluted on a
Phenomenex Luna C18(2) column (5 mm, 4:6 mm ꢀ
150 mm) using a linear gradient of 15–99% solvent A
in solvent B over 10 min. The solvent A was 100%
acetonitrile, solvent B was 0.01% formic acid, which
was observed to have no noticeable effect on sample
retention time, in water.
IR spectra were recorded on a Perkin-Elmer Spectrum
1000 FT-IR spectrometer as thin films using diffuse reflec-
tance.
1H NMR and 13C NMR spectra were recorded with
Varian instruments (400 or 200 MHz for 1H, 100.6 or
50.3 MHz for 13C) at ambient temperature with TMS
1
(for H) or the residual solvent peak (for 13C) as internal
standards. The line positions or multiplets are given in ppm
(d) and the coupling constants (J) are given as absolute
values in Hertz, while the signal multiplicities are abbre-
viated as follows: s (singlet), d (doublet), t (triplet), q
(quartet), quint (quintet), m (multiplet), mc (centered mul-
tiplet), br (broadened).
All melting points were determined with a Mel-Temp II
apparatus and are uncorrected.
Elemental analyses were carried out at Atlantic Micro-
lab Inc., Norcross, GA.

308
M. Stewart et al. / Biochemical Pharmacology 68 (2004) 305–312
liquid scintillation counting on a Packard TopCount NXT.
Radioligand concentrations were determined by adding
an aliquot of the working solution to 5 ml of Ecoscint,
capped, and counted using a tritium dpm program on a
Beckman LS6500 Liquid Scintillation Counter.
minÀ1) and the dissociation rate constant (koff in units of
minÀ1) such that
kobs À koff
kon
¼
½Lꢂ
The KD can then be derived from koff/kon. The koff and
kobs are derived directly from the association and dissocia-
tion curves generated in PRISM.
2.4. Materials
All standard compounds were purchased from Sigma
(St. Louis, MO, USA). All compounds were initially
prepared as 10 mM stocks in DMSO with subsequent
dilutions performed in assay buffer containing 4% DMSO,
hence 1% final DMSO concentration.
3. Results
Using membranes from parental (non-transfected)
HEK-293 cells, we failed to detect any specific binding
using 10 nM [3H]OSIP339391. Hence, the presence of
very low levels of the endogenous A2B receptor [7] would
not prove an interfering factor in assay conditions (0.5 nM
[3H]OSIP339391) routinely employed to screen com-
pounds in competition assays.
Initial experiments revealed a discrepancy between the
concentration of [3H]OSIP339391 achieved and that
expected. A common cause of such a difference is the
adsorption of the radioligand into plastic labware, such as
the 96-well plates or pipette tips, and can often be mini-
mized by the addition of an anti-adsorbant such as bovine
serum albumin (BSA). Table 1 shows the extent and the
time dependent nature of the adsorption together with the
protective effects of increasing BSA concentrations. BSA
(0.5%), was therefore, routinely included in the assay
buffer for all subsequent experiments.
Specific binding of [3H]OSIP339391 to the human
A2B receptor was time dependent with equilibrium estab-
lished after 30 min at 22 8C with a half-life of approxi-
mately 4.5 min and an association rate constant (kon) of
0.095 minÀ1 nMÀ1 (Fig. 1A). The standard time of incu-
bation for subsequent experiments was 60 min. At equili-
brium, non-specific binding did not exceed 20% of the
total [3H]OSIP339391 bound. Specific binding remained
stable for up to 180 min (the longest time point tested) and
2.5. Data analysis
Data are reported as mean Æ S:E: mean of at least three
experiments. Equilibrium and kinetic constants were
obtained by fitting to the appropriate nonlinear regression
functions within PRISM (GraphPad Software, San Diego,
CA, USA). The equilibrium dissociation constant (KD)
and the binding site density (Bmax) were derived from
radioligand saturation curves using the Langmuir equa-
tion RL ¼ RtL/(KD þ L) where L is the free ligand con-
centration, RL is the concentration of receptor-bound
ligand at equilibrium, and Rt is the total receptor con-
centration. Since these studies revealed that the cell line
employed had a very high level of receptor expression
(ꢁ21 pmol/mg protein, see Section 3) the methods
employed here resulted in a modest extent of ligand
depletion. Hence, we used the free concentration of
radioligand to analyse saturation data which is one
approach to minimize the effects of depletion in such
analyses [11]. Saturation curves were routinely analyzed
by fitting to one- and two-site isotherms but the data were
never adequately described by the latter model. As with
the analyses of saturation curve data, competition curve
data were routinely fitted to one- and two-site models;
invariably the data for all compounds were described
better by a one-site model and hence the data reported
are from such analyses. IC50 (the concentration of com-
pound producing 50% inhibition of specific binding) and
Hill Coefficients were derived from fitting to a four-
parameter logistic equation. Curve fitting was not con-
strained when data fully reached maximum and minimum
plateaus but was constrained where compounds had low
affinity and did not fully inhibit binding. Apparent Ki
values were derived using the equation of Cheng and
Prusoff [6]: Ki ¼ IC50/(1 þ ½Lꢂ/KD) where [L] is the free
concentration of radioligand in each assay and KD is the
appropriate equilibrium dissociation constant derived
from the saturation analysis. In kinetic experiments,
Table 1
Influence of BSA on the recovery of [3H]OSIP339391 from the assay
medium
Assay
Assay
Assay
buffer À BSA
buffer þ 0.2%
BSA
buffer þ 0.5%
BSA
Expected dpm
38,500
38,500
38,500
Obtained dpm at time
0 min
8 min
19,641 Æ 300
25,578 Æ 369
25,695 Æ 504
25,837 Æ 594
26,346 Æ 286
33,447 Æ 1831
38,432 Æ 146
38,175 Æ 714
38,291 Æ 597
12,825 Æ 285
8,996 Æ 70
7,545 Æ 91
16 min
24 min
Aliquots (25 ml) of 8 nM [3H]OSIP339391 were removed and counted at
various time points after preparation (see Section 2 for details). The data
are expressed as mean Æ S:E: mean of four individual determinations. The
expected dpm was calculated using the specific activity (87 Ci mmolÀ1) of
the radioligand.
the association rate constant (kon in units of minÀ1 nMÀ1
)
was derived by the pseudo first-order method [20] which
takes into account the concentration of radioligand used,
the observed association rate constant (kobs in units of

M. Stewart et al. / Biochemical Pharmacology 68 (2004) 305–312
309
(A)
24000
20000
16000
12000
8000
100
80
60
40
20
0
3.0
2.5
2.0
1.5
1.0
0.5
0.0
KD 0.17 nM + 0.05
Bmax 21102 + 1503 fmol/mg
4000
0
5
10
15
20
time (mins)
0
0.0
2.5
5.0
7.5
10.0
12.5
0
20 40 60 80 100 120 140 160 180
Free [3H-OSI339391] nM
Incubation time (mins)
Rosenthal plot
3.0
2.5
2.0
1.5
1.0
0.5
0.0
(B)
100
80
60
40
20
0
0.00
-0.25
-0.50
-0.75
-1.00
-1.25
-1.50
-1.75
0
10 20 30 40
time (mins)
0
5000
10000
15000
20000
0
20 40 60 80 100 120 140 160 180
B (fmol/mg)
Incubation time (mins)
Fig. 2. Mean saturation isotherm of [3H]OSIP339391 binding to HEK-293
cell membranes expressing the human A2B receptor. The data are
expressed as mean Æ S:E: mean of three individual experiments performed
in triplicate.
Fig. 1. Binding kinetics of [3H]OSIP339391 to HEK-293 cell membranes
expressing the human recombinant A2B receptor. (A) Association of 1 nM
[3H]OSIP339391 with 1 mg receptor protein per well. (B) Dissociation of
[3H]OSIP339391 initiated by 10 mM DPCPX. Insets show the logarithmic
transformations of the kinetic data where ln is natural logarithm, Be is
specific binding at equilibrium, BT is specific binding at each time point
and B0 is specific binding at time 0. Linear relationships are generally
indicative of a non-complex binding interaction. The data are expressed as
mean Æ S:E: mean of three individual experiments.
good internal consistency within the assay. In general, the
affinity of the ligands employed in this study is consistent
with previously reported data (see Section 4).
OSIP339391 is selective for the human adenosine
A2B over A1, A2A or A3 receptors; in radioligand binding
assays for these receptors employing [3H]-DPCPX,
[3H]ZM241385 and [125I]AB-MECA, respectively, the
was fully reversible as measured by addition of 10 mM
DPCPX to the assay mixture once equilibrium had been
achieved. The dissociation half life was approximately
19 min and the slow dissociation rate constant of
0.039 minÀ1 confirmed the suitability of termination of
the binding assay by vacuum filtration without significant
loss of specific binding (Fig. 1B). From kon and koff values
a KD of 0:41 Æ 0:06 nM was derived.
Table 2
Apparent equilibrium inhibition constants (Ki) and slope factors for
compounds at the human A2B receptor using [3H]OSIP339391
Compound
Ki (nM)
nH
Saturation assays confirmed the high affinity nature of
the interaction between [3H]OSIP339391 and the human
A2B receptors yielding a KD value of 0:17 Æ 0:05 nM, a
value less than three fold different from that derived from
the kinetic constants (Fig. 2). High receptor expression was
reflected in a Bmax value of over 20 pmol/mg of protein.
Table 2 and Fig. 3 shows a range of adenosine receptor
ligands tested for their ability to inhibit 0.5 nM specific
[3H]OSIP339391 binding to the human A2B receptor.
The most potent inhibitor of [3H]OSIP339391 was
OSIP339391 itself with a Ki value of 0.5 nM consistent
with the KD values for the radiolabeled version derived
from the saturation and kinetic studies. This is indicative of
OSIP339391
XAC
0.5 Æ 0.1
1.17
0.82
0.85
0.83
1.09
0.68
0.81
–
1.6 Æ 0.4
ZM 241385
CGS15943
DPCPX
5.8 Æ 1.3
7.6 Æ 1.5
51.1 Æ 11.5
MRS-1220
NECA
332 Æ 29
489 Æ 109
Theophylline
Enprofylline
CPA
17 Æ 1% inhibition @ 10 mM
18 Æ 2% inhibition @ 10 mM
No inhibition at 5 mM
No inhibition at 5 mM
No inhibition at 10 mM
–
–
CHA
–
CGS-21680
–
The data are expressed as mean Æ S:E: mean of at least three individual
experiments.

310
M. Stewart et al. / Biochemical Pharmacology 68 (2004) 305–312
this receptor subtype or evaluating potential novel ligands.
OSIP339391
CGS15943
DPCPX
NECA
CGS21680
100
80
60
40
20
0
Specifically, its high affinity (slow dissociation rate) makes
it suitable for a filtration based binding assay and is likely
to produce more robust, reliable, and reproducible Ki
values than the lower affinity [3H]-DPCPX or [3H]-NECA,
both of which suffered marked dissociation during the
assay termination process. This characteristic could also
make it the radioligand of choice in systems where low
expression of A2B receptors is suspected. Furthermore, the
selectivity of OSIP339391 among adenosine receptor sub-
types (greater than 70-fold) further points to a utility in
tissues or cell lines with multiple receptor subtypes. This
receptor selectivity is not shared by DPCPX, NECA or
ZM241385, whose radiolabeled derivatives have been used
before now for adenosine A2B receptor studies. The
recently described radiolabeled derivative of MRS1754
[12] appears to have similar attributes with respect to high
affinity and selectivity. Although saturation data fitted well
to the one-site model, and did not fit adequately to the two-
site model, a slight curvilinearity was observed in the
derived Rosenthal plot. This analysis might suggest bind-
ing to the endogenous A2B receptors in HEK-293 cells at
very high radioligand concentrations. However, initial
experiments using non-transfected HEK-293 cells does
not support this idea. Further, the presence of a second
binding component is difficult to reconcile given that the
association and dissociation kinetics were apparently
monophasic. However, it is possible that significant recep-
tor occupancy of a lower affinity component, which would
have allowed detection, may not have been achieved, given
that the kinetic assays were performed using a single, low
concentration of [3H]OSIP339391. Hence, the curvilinear-
ity may simply reflect that lower radioligand concentra-
tions had not achieved equilibrium during the incubation
period employed [15].
-11
-10
-10
-10
-9
-9
-9
-8
-7
-6
-5
-5
-5
-4
-4
-4
Log [Compound]
100
80
60
40
20
0
CPA
CHA
ZM241385
XAC
-11
-8
-7
-6
Log [Compound]
100
80
60
40
20
0
MRS-1220
Theophylline
Enprofylline
-11
-8
-7
-6
Log [Compound]
We found that the cell line used in these studies had a
high level of receptor expression (Bmax ꢁ 21 pmol/mg
protein) which resulted in a small degree of ligand deple-
tion under the experimental conditions employed here
(receptor concentration, Rt ¼ 210 pmol). We do not
believe this extent of depletion resulted in a meaningful
(greater than two-fold) underestimation of KD as it has
been shown that Rt must at least exceed KD for this to occur
[14]. KD from kinetic studies was 410 pmol, and hence
Rt ¼ 0:5KD. Indeed, KD from saturation analyses (170 pM)
was actually lower then that derived from kinetic studies.
With regard to the influence of ligand depletion on
Rosenthal plots, it has been shown that this results in a
convex relationship [14], whereas we found a concave
relationship. Ligand depletion in competition binding stu-
dies also leads to underestimation of IC50, but it has been
calculated this difference is only two-fold when depletion
is 50% [9]. Under the experimental conditions employed
here the depletion is ꢁ20%, hence the error in IC50
estimation is very minimal, indeed smaller than the stan-
dard deviation between experiments.
Fig. 3. Inhibition of [3H]OSIP339391 binding to HEK-293 cell mem-
branes expressing the human A2B receptor by various adenosine receptor
ligands. The data are expressed as mean Æ S:E: mean of at least three
individual experiments performed in triplicate. Bt is total bound
radioactivity.
affinity for these human subtypes is 37 Æ 7, 328 Æ 75, and
450 Æ 55 nM (authors’ unpublished data). This suggests
that OSIP339391 is at least 70-fold selective for the A2B
receptor over other adenosine receptor subtypes.
4. Discussion
We have shown that [3H]OSIP339391 binds with high
affinity (KD ¼ 410 pM in kinetic studies) to a homogenous
population of human recombinant A2B receptors stably
expressed in membranes prepared from HEK-293 cells.
This high affinity makes it a more attractive radioligand
than [3H]-NECA or [3H]-DPCPX, when characterizing

M. Stewart et al. / Biochemical Pharmacology 68 (2004) 305–312
311
OSIP339391 is one compound of a series of pyrrolpyr-
imidines that were synthesized in a program to identify
selective A2B receptor antagonists. Many of the com-
pounds in this series, but not OSIP339391, were tested
in functional assays to help further characterize their
properties. One such assay was A2B receptor-mediated
accumulation of cAMP in recombinant cell lines. These
compounds neither augmented nor reduced basal levels of
cAMP, suggesting this series of compounds behave as
neutral antagonists, and blocked NECA-induced cAMP
responses with affinities consistent with radioligand bind-
ing-derived estimates. Clearly, the characterization of
OSIP339391 will be more complete, and its possible utility
enhanced, by further characterization in functional assays
utilizing endogenous and recombinant receptor systems.
In competition binding experiments, the affinity of test
compounds was in good agreement with the previously
reported values from studies that have employed different
radioligands including [3H]-DPCPX, [3H]MRS1754 and
responses in cells due to the activation of endogenous
receptors which cannot be detected using radioligands is
not without precedent, a similar observation having been
made of 5-HT1B receptors in CHO cells [8].
In conclusion, [3H]OSIP339391 represents a novel,
selective and high affinity radioligand which will be useful
in the further exploration and characterization of recom-
binant and endogenous adenosine A2B receptors.
Acknowledgments
We wish to acknowledge Peter Iafrate (Amersham, Buck-
inghamshire, UK) for the synthesis of [3H]OSIP339391
from the alkyne precursor.
References
[1] Bennett JP. Receptor binding methods. In: Yamamura HI, Enna SJ,
Kuhar MJ, editors. Neurotransmitter receptor binding. New York:
Raven Press; 1978. p. 57–90.
[
125I]-ABOPX [2,12,16]. There are some data which do not
agree so well and worthy of comment. The high affinity
displayed by the antagonist XAC for the human A2B
receptor (1.6 nM) is somewhat more potent than that
previously reported (16 nM) [12]. The triazolotriazine
ZM 241385 had a Ki of 5.8 nM in line with findings
employing [3H]ZM241385 as the radioligand [13] but
contrasting with the lower affinity obtained against the
recently tritiated xanthine derivative [3H]MRS1754
(145 nM) [12]. Theophylline and enprofylline, both A2B
antagonists used as antiasthmatic therapies, inhibited
[3H]OSIP339391 binding only at 10 mM and not to an
extent to permit accurate determination of affinity due to
the low solubility of these compounds. Nevertheless, the
affinities of these compounds are less than 10 mM, con-
sistent with published data [12] The adenosine A1 receptor
agonists CPA and CHA showed no significant inhibition
of specific binding even at 5 mM. Similarly, the selective
A2A agonist, CGS 21680 did not inhibit specific
[3H]OSIP339391 binding to the human A2B receptor even
at a concentration of 10 mM. This observation is consistent
with functional studies indicating this agonist to be inactive
at A2B receptors and selective for the A2A subtype [10].The
choice of HEK-293 cells for transfection with the human
A2B gene may be considered somewhat controversial,
given that this cell line expresses an endogenous A2B
receptor which is coupled to cAMP generation [7]. We
attempted to transfect other cell lines such as CHO, which
do not appear to express adenosine A2B receptors [18]
but without success. Nevertheless, using parental HEK-293
cells we were not able to detect a significant level
of specific binding at high concentrations of either
[3H]OSIP339391 or [3H]-DPCPX (results not shown).
Thus, although there appears to be only a small number
of endogenous A2B receptors in HEK-293 cells, these must
be efficiently coupled to adenylyl cyclase in order to
measure a functional response. Measurement of functional
[2] Beukers MW, Den Dulk H, Van Tilburg EW, Brouwer J, Ijzerman AP.
Why are A2B receptors low-affinity receptors? Mutation of Asn273 to
TYR increases affinity of human A2B receptor for 2-(1-hexynyl)ade-
nosine. Mol Pharmacol 2000;58:1349–56.
[3] Bruns RF, LuPugsley GH, Pugsley TA. Characterisation of the A2
adenosine receptor labeled by [3H]-NECA in rat striatal membranes.
Mol Pharmacol 1986;29:331–46.
[4] Campbell RM, Cartwright C, Chen W, Chen Y, Duzic E, Fu JM, et al.
Selective A1-adenosine receptor antagonists identified using Sacchar-
omyces cerevisiae functional assays. Bioorg Med Chem Lett 1999;9:
2413–8.
[5] Castelhano AL, McKibben B, Steinig AG. Preparation of pyrrolopyr-
imidine A2b selective antagonist compounds, method of synthesis and
therapeutic use. PCT Int Appl 2003;WO2003053361.
[6] Cheng YC, Prusoff WH. Relationship between the inhibition constant
(Ki)andtheconcentrationofinhibitorwhichcauses50percentinhibition
(I50) of an enzymatic reaction. Biochem Pharmacol 1973;22:3099–108.
[7] Cooper JH, Hill SJ, Alexander SPH. An endogenous A2B receptor
coupled to cyclic AMP generation in human embryonic kidney
(HEK 293) cells. Br J Pharmacol 1997;122(3):546–50.
[8] Giles H, Lansdell SJ, Bolofo ML, Wilson HL, Martin GR. Character-
ization of a 5-HT1B receptor on CHO cells: functional responses in the
absence of radioligand binding. Br J Pharmacol 1996;117:1119–26.
[9] Goldstein A, Barrett RW. Ligand dissociation constants from compe-
tition binding assays: errors associated with ligand depletion. Mol
Pharmacol 1987;31(6):603–9.
[10] Hide I, Padgett WL, Jacobson KA, Daly JW. A2A-adenosine receptors
from rat striatum and rat pheochromocytoma PC12 cells: character-
isation with radioligand binding and by activation of adenylate
cyclase. Mol Pharmacol 1992;41:352–9.
[11] Hulme EC, Birdsall NJM. Strategy and tactics in receptor binding
studies. In: Hulme EC, editor. Receptor–ligand interactions: a prac-
tical approach. Oxford: IRL Press; 1992. p. 63–176.
[12] Ji XD, Kim YC, Ahern DG, Linden J, Jacobsen KA. [3H]MRS 1754, a
selective antagonist radioligand for A2B adenosine receptors. Biochem
Pharmacol 2001;61:657–63.
[13] Ji XD, Jacobsen KA. Use of the triazolotriazine [3H]ZM241385 as a
radioligand at recombinant human A2B adenosine receptors. Drug Des
Discovery 1999;16:217–26.
[14] Kenakin T. Radioligand binding experiments. In: Pharmacologic
analyses of drug–receptor interactions, 2nd ed. New York: Raven
Press; 1993. p. 385–410.

312
M. Stewart et al. / Biochemical Pharmacology 68 (2004) 305–312
[15] Limbird LE. Identification of receptors using direct radioligand
techniques. In: Limbird LE, editor. Cell surface receptors: a short
course on theory and methods. Boston: Kluwer Academic Publishers;
1987. p. 51–96.
[18] Schonbrunn A. 1999, http://www.tumor-gene.org/GPCR/gpcr.html.
[19] Stehle JH, Rivkees SA, Lee JJ, Weaver DR, Deeds JD, Reppert SM.
Molecular cloning and expression of the cDNA for a novel A2-
adenosine receptor subtype. Mol Endocrinol 1992;6:384–93.
[20] Weiland GA, Molinoff PB. Quantitative analysis of drug-receptor
interactions: I. Determination of kinetic and equilibrium properties.
Life Sci 1981;29:313–30.
[16] Linden J, Thai T, Figler H, Jin X, Robeva AS. Characterization of
human A2B adenosine receptors: radioligand binding, western blot-
ting, and coupling to Gq in Human Embryonic Kidney 293 cells and
HMC-1 mast cells. Mol Pharmacol 1999;56:705–13.
[21] Wieringa J, Van Den Meerendonk A, Steenvoorden J, Heeres G, Van
Bakel F, Roeters T, et al. Plate R monoamine re-uptake inhibiting 1-[2-
[(phenoxyphenyl)methoxy]ethyl]piperazines as potential antidepres-
sants. Recl Trav Chim Pays-Bas 1993;112:143–50.
[17] Pierce KD, Furlong TJ, Selbie LA, Shine J. Molecular cloning and
expression of an adenosine A2b receptor from human brain. Biochem
Biophys Res Commun 1992;187:86–93.