1-Alkyl-8-(piperazine-1-sulfonyl)phenylxanthines: Development and characterization of adenosine A2B receptor antagonists and a new radioligand with subnanomolar affinity and subtype specificity

Article abstract of DOI:10.1021/jm900413e

A new series of 1-alkyl-8-(piperazine-1-sulfonyl)phenylxanthines was designed, synthesized, and characterized in radioligand binding and functional assays at A_2B adenosine receptors. A_2B antagonists with subnanomolar affinity and hig

Full text of DOI:10.1021/jm900413e

3994
J. Med. Chem. 2009, 52, 3994–4006
1-Alkyl-8-(piperazine-1-sulfonyl)phenylxanthines: Development and Characterization of
Adenosine A2B Receptor Antagonists and a New Radioligand with Subnanomolar Affinity and
Subtype Specificity
Thomas Borrmann, Sonja Hinz, Daniela C. G. Bertarelli, Wenjin Li, Nicole C. Florin, Anja B. Scheiff, and Christa E. Mu¨ller*
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, An der Immenburg 4, D-53121 Bonn, Germany
ReceiVed March 31, 2009
A new series of 1-alkyl-8-(piperazine-1-sulfonyl)phenylxanthines was designed, synthesized, and
characterized in radioligand binding and functional assays at A2B adenosine receptors. A2B antagonists
with subnanomolar affinity and high selectivity were discovered. The most potent compounds were
1-ethyl-8-(4-(4-(4-trifluoromethylbenzyl)piperazine-1-sulfonyl)phenyl)xanthine (24, PSB-09120, Ki
(human A2B) ) 0.157 nM) and 8-(4-(4-(4-chlorobenzyl)piperazine-1-sulfonyl)phenyl)-1-propylxanthine
(17, PSB-0788, Ki (human A2B) ) 0.393 nM). Moreover, 8-(4-(4-(4-chlorophenyl)piperazine-1-
sulfonyl)phenyl)-1-propylxanthine (35, PSB-603) was developed as an A2B-specific antagonist exhibiting
a Ki value of 0.553 nM at the human A2B receptor and virtually no affinity for the human and rat A1
and A2A and the human A3 receptors up to a concentration of 10 µM. A tritiated form of the compound
was prepared as a new radioligand and characterized in kinetic, saturation, and competition studies. It
was shown to be a useful pharmacological tool for the selective labeling of human as well as rodent
A2B receptors (KD human A2B 0.403 nM, mouse A2B 0.351 nM).
Introduction
the inflammatory cytokines interleukin-4 (IL-4), IL-8, and
IL-13.10,12,13 A series of experiments described the A2B receptor-
mediated release of proinflammatory cytokines from a number
of cells and tissues, including bronchial smooth muscle and
epithelial cells, lung fibroblasts, and intestinal epithelial cells.14-17
It was demonstrated that activation of the A2BAdoR promotes
the differentiation of human fibroblasts into myofibroblasts via
IL-6 release,16 resulting in airway remodeling, which is known
Adenosine is an endogenous purine nucleoside activating four
subtypes of G protein-coupled receptors (adenosine receptors,
AdoR) that have been cloned and termed A1, A2A, A2B, and
A3.1 Adenosine induces bronchoconstriction in asthmatics,2 and
the exhaled breath condensate3 and bronchoalveolar lavage fluid4
of asthmatic patients contains increased concentrations of
adenosine. Studies with adenosine deaminase (ADAa)-deficient
mice revealed a causal connection between increased lung
adenosine levels and a phenotype of pulmonary inflammation.5,6
These findings strongly suggested that adenosine plays an
important role in the pathophysiology of chronic inflammatory
lung diseases.
to play an important role in the pathophysiology of inflammatory
18
airway diseases.
A antagonists proved to attenuate the release
2B
of IL-19 from human bronchial epithelial cells15 and to abolish
the secretion of IL-13 from mouse bone marrow-derived mast
cells.19
In another set of experiments, it was observed that 5′-N-
ethylcarboxamidoadenosine (NECA), a nonselective AdoR
agonist, inhibited tumor necrosis factor alpha (TNFR) release
in mouse peritoneal macrophages of A2AAdoR knockout mice,
an effect that could be blocked by the A2BAdoR antagonist
MRS-1754 (8).20 Yang et al. reported that A2BAdoR knockout
mice developed low-grade inflammation due to increases in IL-6
and TNFR.21 Both studies suggested that, in addition to the
predominant A2A-mediated anti-inflammatory effect of adenos-
ine, activation of A2BAdoR might also lead to a reduction of
inflammation.
The A2BAdoR is a low affinity receptor that is thought to
remain silent under physiological conditions and to be activated
in consequence of increased extracellular adenosine levels.7,8
It is mainly expressed in the gastrointestinal tract, bladder, lung,
and on mast cells.1,9 Activation of A2BAdoR can stimulate
adenylate cyclase and phospholipase C through activation of
Gs and Gq proteins, respectively. Coupling to mitogen-activated
protein kinases has also been described.10
There is ample evidence suggesting that the A2BAdoR
mediates proinflammatory effects in the lung.11 Stimulation of
human mast cells, which is pivotal to the pathophysiology of
asthma, is potentiated by the activation of A2BAdoR, releasing
However, follow-up experiments with A2BAdoR knockout and
wild-type mice and with mouse peritoneal macrophages, isolated
from A2BAdoR knockout and wild-type mice, demonstrated that
NECA induced a release of the proinflammatory cytokine IL-6
in wild-type mice, which was abrogated by A2BAdoR antagonists
or by genetic ablation of A2BAdoR. In this study, it could also
be shown that the NECA-induced suppression of TNFR in mouse
peritoneal macrophages observed by Kreckler et al.20 was neither
affected by A2B antagonists nor by genetic ablation of
A2BAdoR.22 These results indicated that activation of A2BAdoR
by adenosine might not contribute significantly to the primarily
A2A-mediated suppression of TNFR release.23,24
* To whom correspondence should be addressed: Phone: +49-228-73-
2301. Fax: +49-228-73-2567. E-mail: christa.mueller@uni-bonn.de.
a Abbreviations ADA, adenosine deaminase; AdoR, adenosine receptor;
CADO, 2-chloroadenosine; [3H]CCPA, [3H]2-chloro-N6-cyclopentyladenos-
ine; CHO cells, Chinese hamster ovary cells; DPCPX, 8-cyclopentyl-1,3-
dipropylxanthine; DSS, dextran sodium sulfate; EDC, N-(3-(dimethylami-
no)propyl)-N′-ethylcarbodiimide; HMDS, 1,1,1,3,3,3-hexamethyldisilazane;
[
125I]I-ABOPX, [125I]3-(4-amino-3-iodobenzyl)-8-phenyl-(4-oxyacetic acid)-
1-propylxanthine; IL, interleukin; NECA, 5′-N-ethylcarboxamidoadenosine;
PPSE, polyphosphoric acid trimethylsilyl ester; PSA, polar surface area;
PSB, Pharmaceutical Sciences Bonn; TNBS, 2,4,6-trinitrobenzene sulfonic
acid; TNFR, tumor necrosis factor alpha; VEGF, vascular endothelial growth
factor.
10.1021/jm900413e CCC: $40.75  2009 American Chemical Society
Published on Web 05/27/2009

1-Alkyl-8-(piperazine-1-sulfonyl)phenylxanthines
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3995
Chart 1. A2B Antagonists and their Ki Values at AdoR Subtypes (h ) Human, r ) Rat)9,27,40-47
In vivo experiments showed that the A2B antagonist CVT-
6883 (5) reduced pulmonary inflammation and fibrosis in ADA-
deficient mice as well as in mice with lung injury induced by
bleomycin pretreatment.25 It was also found that A2B antagonist
treatment reduced airway reactivity to NECA, AMP (which is
converted to adenosine in vivo), or allergen challenge in an
allergic mouse model of asthma.26 Compound 5 is currently
evaluated for cardiopulmonary disease in an ascending-dose
phase I clinical study by CV Therapeutics, representing the first
A2B antagonist in clinical trials.27 Thus, the asthmatic response
to adenosine is likely to be mediated via A2BAdoR, providing
a firm basis for the development of A2B antagonists as new
antiasthmatic agents.28
The A2BAdoR antagonists 8-(p-bromophenyl)-1-propargyl-
xanthine (PSB-50), 4-(1-butylxanthin-8-yl)benzoic acid (PSB-
53), 8-(4-(2-(4-benzylpiperazin-1-yl)-2-oxoethoxy)phenyl)-1-
butylxanthine (PSB-55), and PSB-1115 (3) were tested among
other subtype-selective AdoR antagonists for their analgesic
potency in mice using the hot plate test. Unlike the tested A1-,
A2A-, and A3-selective antagonists, all four A2BAdoR antagonists
exhibited dose-dependent antinociceptive effects.29 Moreover,
3 in combination with morphine showed a synergistic effect in
the hot plate test and potently diminished inflammatory pain in
mice during the second inflammatory phase after formalin
injection.29,30 These results suggest that selective A2BAdoR
antagonists could serve as drugs for the treatment of inflam-
matory pain, as a monotherapy or in combination with other
analgesic drugs.
Adenosine was shown to stimulate IL-6 secretion to the
luminal compartment in the human intestinal epithelial cell line
T84 via cyclic AMP (cAMP) formation by activation of
A2BAdoR.31,32 Upregulation of A2BAdoR in mice with dextran
sodium sulfate (DSS)-induced colitis and in intestinal epithelial
mucosa of patients with colitis has been observed as well as
TNFR-induced upregulation of A2BAdoR.33 The potential proin-
flammatory role of the A2BAdoR in colon was further investi-
gated with A2BAdoR knockout mice, which were found to
develop an attenuated grade of colonic inflammation in com-
parison to the wild-type mice after treatment with DSS, 2,4,6-
trinitrobenzene sulfonic acid (TNBS), or Salmonella typhimu-
rium, respectively.34 Moreover, it has been shown that the
A2BAdoR antagonist N-(5-(1-cyclopropyl-2,6-dioxo-3-propyl-
2,3,6,7-tetrahydro-1H-purin-8-yl)pyridin-2-yl)-N-ethylnicotina-
mide (ATL-801) significantly suppressed proinflammatory
cytokines and reduced the symptoms of colitis in DSS-treated
and piroxicam-treated IL-10-/- mice, indicating that A2BAdoR
antagonists might serve as anti-inflammatory drugs for the
treatment of colonic inflammation.35,36
Another potential field of application for A2B antagonists
might be the attenuation of neurodegenerative disorders such
as Alzheimer′s disease. A2BAdoR have been shown to mediate
IL-6 release in human astrocytoma cells37 and to enhance
astrogliosis in the presence of TNFR in human astrocytoma
cells,38 both effects being important for the development of
neurodegeneration.

3996 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Borrmann et al.
Chart 2. Selective A2B Antagonist Radioligands with KD Values and Specific Activities for the A2BAdoR and AdoR Affinities of Their
Unlabeled Analogues41,43,44,46,47,52-55
A recent study showed that A2BAdoR knockout mice exhibit
attenuated tumor growth and decreased levels of vascular endot-
helial growth factor (VEGF) in the tumors after inoculation with
Lewis lung carcinoma when compared to wild-type mice, pointing
at the involvement of A2BAdoR activation in the neovascularization
of tumors. The release of VEGF was increased by the nonselective
agonist NECA but not by the A2A-selective AdoR agonist 2-p-(2-
carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine
(CGS-21680), whereas a combination of NECA and the selective
A2BAdoR antagonist 5 did not show this effect, indicating that A2B
antagonists might have the potential to inhibit tumor vasculariza-
tion.39
In Chart 1, selected A2B antagonists are shown. Theophylline
(1) and enprofylline (2)40 are currently used for the treatment
of asthma. Their antiasthmatic effects have recently been
attributed to the antagonism of the A2BAdoR; however both
compounds are weak A2B antagonists and not or only moderately
selective.12 Intensified research led to the development of more
selective high-affinity A2B antagonists with a xanthine scaffold,
namely 3,41 PSB-601 (4),42 5,27 PSB-298 (7),41 8,43 and MRE-
2029F20 (9),44 the deazaxanthine 6,45 and the deazaadenine
derivative OSIP-339391 (10).46 However, the majority of the
nonxanthine A2B antagonists developed so far is characterized
by only moderate selectivity toward other AdoR subtypes. The
xanthine-based A2B antagonists currently show the most promis-
ing combination of high affinity and selectivity, but they often
suffer from poor water solubility and thus low oral bioavail-
ability.47-49
affinities to the rat than to the human A2BAdoR.51 On the other
hand, Kim et al.43 as well as Yan et al.42 observed that the
xanthine derivatives tested in their studies were more potent at
rat than at human A1AdoR. This could mean that A2B antago-
nists, which are very selective in humans may be less selective
or even nonselective in rodents. For example, 8 is selective in
humans but nonselective vs A1 in rodents (see Chart 2).
High affinity, selectivity, and sufficient water solubility are
pivotal properties for the development of new A2B radioligands.
The recently developed antagonist radioligands [3H]7,52 [3H]8,53
[3H]9,54 and [3H]1055 show KD values in the low nanomolar
range for the human A2BAdoR. However, 7, 9, and 10 show
selectivities of less than 100-fold vs human hA1AdoR, and
further improvement in selectivity is desirable. Compound 8
was recently tested for its affinity at A2B receptors of different
species, revealing a Ki value of 12.8 nM for the rA2BAdoR,
which means that the compound is nonselective in rat because
it is similarly potent at rat A1AdoR (Ki ) 16.8 nM).50 Its 205-
fold selectivity in humans (A2B vs A1) further supports the
hypothesis mentioned above that A2B selectivity in rodents seems
to be more difficult to achieve than in humans. To our
knowledge, the recently developed high-affinity A2B antagonists
were exclusively tested at the human and not at rodent A2B
receptors. So far, only antagonist radioligands have been
developed for the A2BAdoR and the use of nonselective A2B
radioligands is still frequently reported.
On the basis of the most potent A2B antagonists, a pharma-
cophore model has recently been developed by our group (Chart
3).42 This model is characterized by a flat central core, which
is a deazapurine or frequently a xanthine scaffold for most of
the selective A2B antagonists. The core is characterized by a
hydrogen bond acceptor (e.g., C6-oxygen in xanthines) and a
hydrogen bond donor (e.g., N7-hydrogen in xanthines). The
lipophilic domains L1 and L2 allow for an introduction of alkyl
The A2BAdoR has been cloned from rats, mice, and humans,
showing high structural homologies between these species (87%
human vs rat or mouse; 95% rat vs mouse).50 Nevertheless, it
has been reported that AdoR affinities of A2B antagonists can
vary between different species. Kim et al. found that the xanthine
derivatives that they investigated bound with 2-9-fold lower

1-Alkyl-8-(piperazine-1-sulfonyl)phenylxanthines
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3997
Chart 3. Pharmacophore Model for A2B Antagonists and Structure of 442
Scheme 1. Preparation of Sulfonamides 15-43a,b
a Reagents and conditions: (a) EDC, MeOH, rt, 4 h. (b) For 14a and 14b: PPSE; (1) 120 °C, 10 min; (2) 170 °C, 2 h, MeOH. (c) For 14c: HMDS,
(NH4)2SO4, reflux, 16 h, MeOH. (d) (1) DMF, rt, 2 d; (2) reflux, 1 h (method A). (e) (1) DMF, rt, 2 d; (2) reflux, 1 h; (3) aq NH4OH (12.5%), 60 °C, 20
b
min (method B). (f) (1) DMF, rt, 2 d; (2) aq NH4OH (12.5%), 60 °C, 20 min (method C). For R1, R2, and R3, see Table 1.
residues at positions 1 and 3. A small alkyl substituent (propyl,
ethyl, methyl) at position 1 proved to increase A2BAdoR affinity,
whereas additional alkylation at position 3 led to a decrease in
A2BAdoR affinity and an increase in A1 and A2AAdoR affinity.41
Position 8 is substituted with a spacer featuring hydrogen bond
donor and acceptor groups, which is further connected to an
aromatic residue. Taking 4 as a lead structure, we varied the
substituent L1, the spacer, and the aromatic residue, whereas
residue L2 was retained as a hydrogen atom, which was optimal
for high affinity as well as selectivity.41
The aim of this study was to develop high-affinity A2B
antagonists exhibiting high selectivity in humans as well as
rodents and to provide a new, improved A2B antagonist
radioligand on the basis of the obtained structure-activity
relationships.
of 1-ethyl and 1-propyl substitution (14a, 14b).59 It had been
reported that the use of PPSE in the case of 1-propargyl
substitution leads to the formation of tricyclic oxazolo[3,2-a]-
purinones.60 Therefore, heating of 13c with 1,1,1,3,3,3-hexa-
methyldisilazane (HMDS) in the presence of ammonium sul-
fate61 was applied instead to afford compound 14c. Sulfona-
mides 15-43 were obtained by aminolysis of p-nitrophenyl-
sulfonates42 and were subsequently purified by reverse-phase
high performance liquid chromatography (RP-HPLC) (method
A). However, in the cases of phenylpiperazinyl-substituted
derivatives 34-38, the sulfonamides could not be separated from
unreacted starting compounds (esters 14a-c) by chromato-
graphic methods. Therefore, the product mixtures were subjected
to alkaline hydrolysis in ammonia (pH 11; 60 °C; 20 min),
resulting in hydrolysis of the esters 14a-c, which were present
as impurities. After evaporation of the ammonia and purification
by RP-HPLC (methods B and C), the pure sulfonamides 34-38
were obtained. The synthesized xanthine-8-yl-benzenesulfona-
mides are collected in Table 1. The structures of all final
Results and Discussion
The target compounds were synthesized as depicted in
Scheme 1. 3-Substituted 5,6-diaminouracils 11a-c56-58 and
4-(p-nitrophenoxysulfonyl)benzoic acid 1259 were obtained
according to previously described procedures. Amide coupling
of 11a-c with 12 was performed with N-(3-(dimethylamino)-
propyl)-N′-ethylcarbodiimide (EDC) as condensing agent to
yield amides 13a-c.59 Ring closure of 13a-c to the corre-
sponding xanthine derivatives 14a-c was achieved by heating
with polyphosphoric acid trimethylsilyl ester (PPSE) in cases
1
products were confirmed by H NMR and 13C NMR spectros-
copy and by mass spectrometry employing electron spray
ionization (ESI). Purity was determined by elemental analysis
and by RP-HPLC analysis coupled to UV detection at 254 nm
and found to be g95% in all cases.
Biological Evaluation. The affinities of the xanthine-8-yl-
benzenesulfonamide derivatives were determined in radioligand

3998 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Borrmann et al.
Table 1. Methods, Isolated Yields, and Melting Points of Synthesized Xanthine-8-yl-benzenesulfonamide Derivatives
compd
R1
R2
precursor
methoda
yield, %
melting point, °C (dec.)
Benzylpiperazine Derivatives
15
16
ethyl
ethyl
propyl
ethyl
propyl
ethyl
propyl
ethyl
propyl
ethyl
propyl
ethyl
propyl
ethyl
benzyl
14a
14a
14b
14a
14b
14a
14b
14a
14b
14a
14b
14a
14b
14a
14bv
14a
14b
14a
14b
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
77
63
64
66
62
46
49
63
80
50
68
56
65
38
75
61
69
57
59
>308
>289
>292
>315
>293
>289
>286
>310
>295
>310
>290
>288
>294
>283
>283
>278
>278
>288
>276
4-chlorobenzyl
4-chlorobenzyl
3-chlorobenzyl
3-chlorobenzyl
4-fluorobenzyl
4-fluorobenzyl
3-fluorobenzyl
3-fluorobenzyl
4-(trifluoromethyl)benzyl
4-(trifluoromethyl)benzyl
3-(trifluoromethyl)benzyl
3-(trifluoromethyl)benzyl
4-methylbenzyl
17 (PSB-0788)
18
19
20
21
22
23
24 (PSB-09120)
25
26
27
28
29
30
31
32
33
propyl
ethyl
propyl
ethyl
4-methylbenzyl
4-methoxybenzyl
4-methoxybenzyl
3,4-(methylenedioxy)benzyl
3,4-(methylenedioxy)benzyl
propyl
Phenylpiperazine Derivatives
34
ethyl
4-chlorophenyl
4-chlorophenyl
4-chlorophenyl
4-methoxyphenyl
4-methoxyphenyl
14a
14b
14c
14a
14b
B
B
C
B
B
49
62
38
39
47
>308
>298
>274
>265
>270
35 (PSB-603)
propyl
propargyl
ethyl
36
37
38
propyl
Phenethylpiperazine Derivatives
39
40
ethyl
propyl
phenethyl
phenethyl
14a
14b
A
A
71
69
>280
>288
Piperidine Derivatives
41
42
ethyl
propyl
see structure above
see structure above
14a
14b
A
A
71
78
>343
>332
Benzylpiperidinylamine Derivative
see structure above 14b
a For methods A, B, and C, see general procedures in Experimental Section.
43
propyl
A
38
>271
binding studies at A1, A2A, A2B, and A3AdoR using [3H]2-chloro-
N6-cyclopentyladenosine ([3H]CCPA), [3H](E)-3-(3-hydroxypro-
pyl)-8-(2-(3-methoxyphenyl)vinyl)-7-methyl-1-prop-2-ynyl-3,7-
dihydropurine-2,6-dione ([3H]MSX-2), [3H]8-(4-(4-(4-chloro-
phenyl)piperazine-1-sulfonyl)phenyl)-1-propyl-3,7-dihydropu-
rine-2,6-dione ([3H]35), and [3H]2-phenyl-8-ethyl-4-methyl-
(8R)-4,5,7,8-tetrahydro-1H-imidazo[2.1-i]purin-5-one ([3H]PSB-
11) as A1, A2A, A2B, and A3 radioligands, respectively.62-65 Rat
brain cortical membranes were used as a source for the rat
A1AdoR, rat brain striatal membranes for rat A2AAdoR. Assays
at the A2B and A3AdoR were performed with human receptors
recombinantly expressed in Chinese hamster ovary (CHO)
cells.66 Selected compounds were additionally tested at the
human A1 and A2AAdoR, and the mouse A2BAdoR recombi-
nantly expressed in CHO cells.66 Functional calcium assays of
selected compounds were performed at Jurkat T cells, a human
lymphoma cell line.67
the human A2BAdoR by measuring the inhibition of the NECA-
induced A2B-mediated increase in the intracellular calcium
concentration ([Ca2+]i) in Jurkat T cells.67 A2B receptors were
stimulated by NECA (10 µM),67 resulting in an increase in
calcium concentration (Figure 1). This effect appeared to be
due to A2B as well as A2AAdoR activation. The A2A component
was blocked by a high concentration of the A2A-selective
antagonist MSX-268 (200 nM) (Figure 1). Therefore, the
functional experiments were performed in the presence of 200
nM MSX-2 to exclude potential A2A-mediated effects of the
tested compounds.68
Selectivities over the other AdoR were determined by
radioligand binding studies at the rat A1 and A2AAdoR and
at the human recombinant A1, A2A, and A3AdoR. The best
compound was further tested at recombinant mouse A2B
receptors in order to investigate potential species differences.
After development of the new potent and selective A2B
radioligand 35, all compounds were investigated in radioli-
gand binding assays versus [3H]35 at human A2B receptors
Structure-Activity Relationships. The first 13 compounds
synthesized in this series were tested in a functional assay at

1-Alkyl-8-(piperazine-1-sulfonyl)phenylxanthines
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3999
Figure 1. NECA (10 µM) induced calcium signal in the presence and in the absence of the A2AAdoR antagonist MSX-2 (200 nM). A representative
curve out of three independent experiments is shown.
Limited. The identity of [3H]35 was confirmed by mass
spectrometric analysis, and [3H]35 displayed a specific activity
of 2.70 TBq/mmol (73 Ci/mmol) and a radiochemical purity of
98.9% as determined by HPLC/UV (254 nm).
[3H]35 was found to bind with high affinity to human
A2BAdoR stably expressed in CHO cells. Membrane prepara-
tions thereof containing 30 µg protein/vial were used to
characterize the new radioligand in kinetic, saturation, and
competition binding experiments. Kinetic as well as competition
experiments were performed with a radioligand concentration
of 0.3 nM in the presence of 2 U/mL adenosine deaminase with
a dimethylsulfoxide (DMSO) concentration not exceeding 2.5%
in 50 mM Tris buffer pH 7.4. The specific binding amounted
Figure 2. Concentration-dependent inhibition of A2B-mediated calcium
to ca. 70% of total binding. Thus, the nonspecific binding of
[3H]35 was comparable to the degree of nonspecific binding
reported for other selective A2B antagonist radioligands.52,53,55,69
A direct comparison with literature data is not possible because
the relationship of specific to nonspecific binding is highly
dependent on the receptor expression level. Incubation was
terminated by rapid filtration through GF/B glass-fiber filters
and subsequent washing with 4 × 3 mL of ice-cold Tris-buffer
containing 0.1% bovine serum albumin (BSA). For kinetic
studies, 30 µg of protein was incubated with the radioligand
for 180 min (association kinetics). Dissociation was initiated
by addition of 10 µM 8-cyclopentyl-1,3-dipropylxanthine
(DPCPX) after 95 min of preincubation. Equilibrium binding
was reached after less than 60 min and was stable for at least
3 h at room temperature (Figure 3A). Radioligand binding was
reversible after addition of DPCPX. A kinetic KD value of 0.652
( 0.269 nM was calculated from the association and dissociation
constants (Figure 3).
response in Jurkat T cells by 35. Cells were stimulated with 10 µM
NECA in the presence of 200 nM MSX-2 to block A2A receptors.
Oregon Green was used as a fluorescent indicator of the intracellular
calcium concentration. Data points are means ( SEM of three
experiments.
expressed in CHO cells. All tested compounds showed high
potency at the A2BAdoR with IC50 values in the nanomolar
range (Table 2).
All of the initial series of compounds tested were potent A2B
antagonists at Jurkat T cells exhibiting IC50 values between 1.13
nM (35) and 16.2 nM (31). Propyl and ethyl substitution at N1
were similarly well tolerated (compare 26 and 27). The terminal
benzyl residues at the piperazine ring could be substituted in
the meta- or para-position (compare 17/19 and 25/27) without
much change in potency. However, 3,4-methylenedioxybenzyl
substituents were somewhat less well tolerated (33) when
combined with a 1-propyl residue. Phenethyl substitution as in
compound 40 was also slightly disadvantageous when compared
to the benzyl derivatives. The most potent and selective A2B
antagonist in this series was compound 35, exhibiting an IC50
value of 1.13 nM and virtually no affinity for all other AdoR
subtypes up to a concentration of 10 µM. The concentration-
inhibition curve for compound 35 at the human A2B receptor in
Jurkat T cells is shown in Figure 2.
The saturation experiments were performed with 30 µg of
protein (CHO cell membranes expressing the human A2BAdoR)
and [3H]35 in a concentration range of 0.05 nM to 1 nM. A KD
value of 0.403 ( 0.188 nM was obtained and a Bmax value of
502 ( 57 fmol/mg protein was calculated. This KD value was
in close agreement with the KD value obtained from the kinetic
experiments (Figure 4).
Competition binding experiments with standard AdoR ago-
nists and antagonists were performed using a radioligand
concentration of 0.3 nM, an incubation time of 75 min in 50
mM Tris buffer pH 7.4, and a protein amount of 30 µg per
tube (Figure 5). The Ki values determined with the new
radioligand binding assay were in accordance with previously
published data (Table 3). The rank order of potency was
consistent with specific labeling of an A2BAdoR, demonstrating
Thus, we selected this A2B-specific antagonist for radiola-
beling in order to obtain an A2B radioligand for further studying
the structure-activity relationships of this class of compounds.
This was achieved by catalytic hydrogenation of the 1-propargyl-
substituted precursor 36 with tritium gas in the presence of Pd/C
(10%) in ethanol/dimethylformamide (DMF) affording the new
radioligand [3H]35 after purification with RP-HPLC (Scheme
2). The labeling reaction was performed by GE Healthcare UK

4000 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Borrmann et al.
Table 2. AdoR Affinities of Xanthine-8-yl-benzenesulfonamide Derivatives
Ki ( SEM [nM] (or % inhibition of radioligand binding at indicated concentration ( SEM)
IC50 ( SEM [nM]
A1 rat brain
A1 human
A2A rat brain
A2A human
A2B human
A3 human
A2B Jurkat
cortical membranesa
recombinanta
striatal membranesb
recombinantb
recombinantc
recombinantd
T cells (human)e
Benzylpiperazine Derivatives
4
260 ( 042
250 ( 49
2730 ( 1430
386 ( 98
96.7 ( 7.8
178 ( 38
296 ( 43
2067 ( 26142
1840 ( 530
>10000 (18 ( 9)h
2240 ( 959
nd
1090 ( 250
1820 ( 660
nd
2070 ( 1120
2300 ( 430
>10000 (25 ( 3)h
5630 ( 490
2300 ( 410
2610 ( 222
nd
93.7 ( 32.142
484 ( 11542
152 ( 66
6.49 ( 2.19
333 ( 84
nd
161 ( 42
92.0 ( 41.3
nd
144 ( 62
278 ( 121
22.7 ( 8.5
145 ( 14
69.4 ( 57.2
306 ( 140
nd
3.6 ( 0.442f >100042 (29)g
10.1 ( 1.6
4.49 ( 2.88
nd
3.64 ( 0.98
nd
1.91 ( 0.08
nd
nd
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
335 ( 52
1.15 ( 0.07
>10000 (27 ( 10)h
445 ( 141
1730 ( 790
263 ( 93
799 ( 358
40.2 ( 14.2
194 ( 55
388 ( 149
782 ( 236
122 ( 36
1540 ( 271
635 ( 212
1700 ( 217
341 ( 76
0.215 ( 0.067 >10000 (10 ( 18)h
0.393 ( 0.085 >1000 (18 ( 1)g
0.610 ( 0.239 557 ( 217
0.782 ( 0.195 24200 ( 9700
1.27 ( 0.60
>1000 (21 ( 2)g
33.8 ( 2.0
359 ( 86
0.595 ( 0.101 758 ( 229
0.695 ( 0.199 5780 ( 960
nd
111 ( 43
0.446 ( 0.129 >1000 (32 ( 7)g
0.157 ( 0.062 >10000 (13 ( 5)h
0.303 ( 0.153 >10000 (44 ( 7)h
0.281 ( 0.016 >10000 (38 ( 4)h
0.775 ( 0.214 30000 ( 22000
3.23 ( 0.19
nd
1.62 ( 0.69
2.29 ( 0.58
3.56 ( 0.20
nd
>1000 (14 ( 2)g
463 ( 122
564 ( 180
273 ( 47
39.0 ( 9.0
46.7 ( 12.4
354 ( 134
83.8 ( 18.9
272 ( 108
79.4 ( 4.7
1.58 ( 0.35
>1000 (22 ( 10)g
nd
1750 ( 480
914 ( 343
1020 ( 561
436 ( 48
nd
0.858 ( 0.151 156 ( 35
nd
3800 ( 820
>10000 (38 ( 6)h
>10000 (28 ( 9)h
443 ( 113
236 ( 40
328 ( 59
102 ( 33i
112 ( 3
1.19 ( 0.37
>1000 (29 ( 15)g
>10000 (23 ( 10)h
5.26 ( 1.00
16.2 ( 6.3
5.34 ( 2.06
23.4 ( 10.3
0.944 ( 0268
0.875 ( 0.224 >10000 (32 ( 7)h
590 ( 29
1.06 ( 0.37
17600 ( 1400
Phenylpiperazine Derivatives
34
35
36
37
38
>10000 (48 ( 3)h
>10000 (18 ( 1)h
1440 ( 50
>1000 (-7 ( 4)g
119 ( 83
48.4 ( 10.9
0.406 ( 0.142 434 ( 143
nd
>10000 (10 ( 3)h
>10000 (19 ( 5)h
198 ( 88
>10000 (7 ( 14)h 0.553 ( 0.103 >10000 (10 ( 15)h
1.13 ( 0.39
nd
nd
nd
nd
0.473 ( 0.067 >1000 (35 ( 2)g
nd
nd
nd
>1000 (30 ( 6)g
>1000 (27 ( 4)g
367 ( 44
41.5 ( 15.3
11.7 ( 1.8
1.99 ( 0.37
>1000 (-19 ( 20)g
>1000 (1 ( 7)g
>10000(-17 ( 14)h
3720 ( 1060
Phenethylpiperazine Derivatives
39
40
86.8 ( 46.1
nd
ca. 1000(56 ( 7)g
681 ( 337
nd
7.49 ( 1.20
7.51 ( 1.84
>1000 (-17 ( 12)g
>1000 (30 ( 16)g
nd
>10000 (42 ( 8)h
>10000 (39 ( 4)h
271 ( 127i
10.9 ( 3.3
Piperidine Derivatives
41
42
77.1 ( 22.9
91.1 ( 25.0
nd
nd
251 ( 87
714 ( 122
nd
nd
5.43 ( 0.61
19.7 ( 4.7
209 ( 57
140 ( 20
nd
nd
Benzylpiperidinylamine Derivative
43
38.5 ( 4.5
1170 ( 64
136 ( 16 94.5 ( 30.8
3.03 ( 0.55
2080 ( 410
nd
b
c
d
a Versus [3H]CCPA (n ) 3). Versus [3H]MSX-2 (n ) 3, unless otherwise noted). Versus [3H]35 (n ) 3). Versus [3H]PSB-11 (n ) 3). e Calcium
assay (n ) 3). Versus [3H]7.42 g Inhibition of radioligand binding at 1 µM ( SEM. h Inhibition of radioligand binding at 10 µM ( SEM. i n ) 4.
f
Scheme 2. Preparation of the New A2B-Specific Radioligand
[3H]35 by Catalytic Hydrogenation of its Propargyl Precursor
mouse A2B receptor (see Figure 6). Applying the same conditions
as for the human A2B receptors, we observed a similarly high
affinity for the mouse A2B receptor as well. The calculated KD
value was 0.351 nM at the mouse A2BAdoR and thus almost
identical to that determined at the human receptor (0.403 nM).
These results show that [3H]35 can be used for the labeling of
human as well as rodent A2B receptors; it does not only show
high affinity but also very high selectivity in humans as well as
rodents (see Table 2).
All synthesized compounds of the xanthine-8-yl-benzene-
sulfonamide series were then tested in competition binding
experiments at human A2BAdoR versus the new radioligand
[3H]35. The obtained Ki values correlated quite well with the
IC50 values obtained in the functional calcium assays at Jurkat
T cells, confirming the suitability of the newly established assays
(Table 2). The A2B Ki values of 12 compounds of the present
series were in the nanomolar range, and 17 compounds exhibited
affinities even in the subnanomolar range. This fact demonstrated
clearly that the synthesized xanthine-8-yl-benzenesulfonamides
represent a class of very potent A2B antagonists. As expected,
most of the tested compounds showed virtually no affinity for
the human A3AdoR and a good selectivity versus A1 and
A2AAdoR, confirming the previously reported finding that
1-monosubstitution leads to higher A2B affinity and enhanced
selectivity when compared to 1,3-disubstitution.41,42,59 Interest-
that [3H]35 will be a useful tool for the identification of new
A2BAdoR ligands.
To investigate whether the new radioligand was also suitable
for the labeling of rodent A2BAdoR, we performed a homologous
competition experiment at CHO cells that stably express the

1-Alkyl-8-(piperazine-1-sulfonyl)phenylxanthines
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 4001
Figure 3. Kinetics of [3H]35 binding to the human A2BAdoR recombinantly expressed in CHO cells. (A) Association curve, (B) dissociation curve.
Dissociation was initiated by the addition of 10 µM DPCPX after 95 min of preincubation. Data points are means of two experiments performed
in duplicates.
in affinity) when compared to the corresponding benzylpiper-
azinyl- or phenylpiperazinyl-substituted derivatives, which
mostly exhibited Ki values in the subnanomolar range. The most
potent A2B antagonist in this series was the 4-trifluoromethyl-
benzylpiperazine derivative 24 with a Ki value of 0.157 nM
showing no detectable affinity for the A1 and A3AdoR. However,
as many benzylpiperazine and phenylpiperazine derivatives, it
exhibited some affinity for the A2A receptor (Ki ) 22.7 nM).
Nevertheless, 24 was still >100-fold selective. Compound 17
showed the best profile of affinity and selectivity in the
benzylpiperazine series (Ki ) 0.393 nM; 5700-fold (vs hA1),
870-fold (vs hA2A), >2544-fold (vs hA3)). The most selective
A2B antagonist was the corresponding 4-chlorophenyl-piperazi-
nyl derivative 35 exhibiting a Ki value of 0.553 nM for the
A2BAdoR and no detectable affinity for the A1, A2A, and
A3AdoR, respectively, and therefore being, to our knowledge,
the first A2B-specific high-affinity antagonist. Because 35 is the
corresponding cold ligand of the new radioligand described in
this study, it can be stated that [3H]35 is the first A2B-specific
high-affinity radioligand.
As mentioned above, a major problem of many xanthine-
based A2B antagonists is their low water solubility and thus
limited peroral bioavailability. To improve these properties, we
had introduced basic nitrogen atoms as a part of the spacer in
the substituted piperazine and 4-aminopiperidine derivatives
offering the potential to form salts with increased solubility and
bioavailability. To assess the physicochemical properties of this
new series of A2B antagonists, we calculated their pKA values,
polar surface areas (PSA), and log P values, which are important
predictive parameters, employing the software MarvinSketch
(pKA and log P values) and Molinspiration (PSA values). The
obtained results are listed in Table 4.
The benzyl- and phenethyl-piperazine derivatives 15-33, and
39-40 showed calculated pKA values in the range of 6.96-7.50,
indicating that these compounds will be partly protonated under
physiological conditions, which increases water solubility.
Compounds 34-38 exhibited pKA values in the range of 1-3
and will therefore be not protonated under physiological
conditions, unless perhaps in the acidic medium of the stomach.
Most of the compounds showed polar surface area (PSA) values
of <140 Å2, indicating that they may be intestinally absorbable.71
The calculated log P values of all compounds were lower than
5, which is, according to Lipinski’s rules,72 the upper limit for
drugs exhibiting suitable pharmacokinetic properties.
Figure 4. Saturation binding of [3H]35 to the human A2BAdoR
recombinantly expressed in CHO cells. Data points represent means
from a typical experiment performed in duplicates.
ingly, the tested A2B antagonists were about 10-fold more potent
at the rat A1AdoR than at the human A1AdoR, whereas the Ki
values at the rat A2AAdoR were in most cases 3-10-fold higher
than those determined at the human A2AAdoR. This observation
is illustrated by a correlation of the pKi values of the synthesized
A2B antagonists for the rat and the human A1 and A2AAdoR
subtypes (Figure 7). For the A1AdoR, all data points are above
the line meaning higher affinity for the rat than for the human
A1AdoR, whereas for the A2AAdoR, the correlation is much
better. These findings are in accordance with previously
observed species differences,42,43,51 suggesting that the depen-
dency of AdoR affinities on the species represents an important
issue that should be addressed in further studies evaluating AdoR
ligand affinities and selectivities.50
As already observed in the initial functional experiments,
1-ethyl- and 1-propyl-substituted xanthines showed very similar
affinities for the A2BAdoR (compare 16/17, 18/19, 24/25, 30/
31, 39/40). Likewise, 1-propargyl substitution in case of
compound 36 resulted in an A2BAdoR affinity (Ki ) 0.473 nM),
which was very similar to those of the corresponding ethyl (34,
Ki ) 0.406 nM) and propyl (35, Ki ) 0.553 nM) analogues.
On the other hand, most 1-ethyl derivatives showed higher
affinity than the corresponding 1-propyl derivatives for the rat
A2A receptor, indicating that 1-propyl substitution was favorable
in terms of improved A2B/A2A selectivity.
The variation of the spacer and the aromatic residue at
position 8 of the xanthine core led to the main finding that
benzylpiperazinyl and phenylpiperazinyl substitution appeared
to be the best tolerated modification for the A2BAdoR. Piperidine
analogues 41 and 42 exhibited lower affinities than nearly all
of the corresponding piperazine derivatives (Ki values of 5.43
nM and 19.7 nM, respectively). Also, phenethylpiperazine and
benzylpiperidinylamino substituents (39, 40, and 43) were less
tolerated by the A2BAdoR (about 1 order of magnitude decrease
Conclusions
A series of xanthine-8-yl-benzenesulfonamide derivatives was
synthesized as new selective high-affinity A2B antagonists, tested
in radioligand binding studies, and functionally characterized.
On the basis of the obtained structure-activity relationships,

4002 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Borrmann et al.
Figure 5. Inhibition of 0.3 nM [3H]35 binding to the human A2BAdoR recombinantly expressed in CHO cells, (A) by the agonists NECA, CGS-
21680, and 2-chloroadenosine (CADO), and (B) by the antagonists 9-chloro-2-(2-furanyl)-1,2,4-triazolo[1.5-c]quinazolin-5-amine (CGS-15943), 3,
caffeine, and unlabeled 35 (homologous competition). Data points are means ( SEM of three experiments performed in duplicates.
Table 3. A2BAdoR Affinities of Selected Standard Agonists and Antagonists
Ki ( SEM [µM] (or % inhibition of radioligand binding at indicated concentration)
human A2BAdoR vs [3H]35
human A2BAdoR vs other radioligands
Agonists
,
,
NECA
CADO
CGS-21680
1.89 ( 0.24
0.570 ( 0.17053 a 0.262 ( 0.030,69 b
25.5 ( 6.240 c
,
21.4 ( 5.7
,
(12 ( 1% at 10 µM)
(<10% at 100 µM)70 d
Antagonists
,
,
,
CGS-15943
3
caffeine
4
0.0300 ( 0.0039
0.237 ( 0.050
33.8 ( 1.2
0.0342 ( 0.001053 a 0.0164 ( 0.003670 d 0.0098 ( 0.000969 b
0.0534 ( 0.018241 d
,
10.4 ( 1.851 c
,
,
0.00133 ( 0.00050
d
0.0036 ( 0.000442 e
b
c
e
a Versus [3H]8. Versus [3H]9. Versus [125I]I-ABOPX. Versus [3H]ZM-241385. Versus [3H]7.
layer chromatography (TLC) using aluminum sheets coated with
silica gel 60 F254 (Merck). Compounds were visualized under UV
light (254 nm). Preparative HPLC was performed on a Knauer
HPLC system using a Wellchrome K-1800 pump, a WellChrome
K-2600 spectrophotometer, and a Eurospher 100 C18 column (250
mm × 20 mm, particle size 10 µm). A gradient of methanol in
water was used as indicated below with a flow rate of 20 mL/min.
The purity of all tested compounds was determined by elemental
analysis and by HPLC-UV obtained on an LC-MS instrument
(Applied Biosystems API 2000 LC-MS/MS, HPLC Agilent 1100)
using the following procedure: Compounds were dissolved at a
concentration of 0.5 mg/mL in methanol. Then, 10 µL of the sample
were injected into a Phenomenex Luna C18 HPLC column (50 mm
× 2.00 mm, particle size 3 µm) and were chromatographed using
a gradient of water: methanol (containing 2 mM ammonium acetate)
from 90:10 to 0:100 for 30 min at a flow rate of 250 µL/min, starting
the gradient after 10 min. UV absorption was detected from 200 to
950 nm using a diode array detector. Purity of the compounds was
determined at 254 nm and proved to be g95%. Elemental
microanalyses (C, H, N) were performed on a VarioEL apparatus
and determined values are generally within (0.4% of calculated
values. Mass spectra were recorded on an API 2000 mass
spectrometer (electron spray ion source, Applied Biosystems,
Darmstadt, Germany) coupled with an Agilent 1100 HPLC system.
1H and 13C NMR spectra were recorded on a Bruker Avance 500
MHz NMR spectrometer. DMSO-d6 was used as solvent. NMR
spectra were recorded at rt. Shifts are given in parts per million
(ppm) relative to the remaining protons of the deuterated solvent
used as internal standard. Coupling constants J are given in Hertz,
and spin multiplicities are given as s (singlet), d (doublet), t (triplet),
q (quartet), m (multiplet), br (broad). Melting points were deter-
mined on a Bu¨chi B-545 melting point apparatus and were
uncorrected. Lyophilization was performed with a CHRIST ALPHA
1-4 LSC freeze-dryer.
Figure 6. Homologous competition experiment: concentration-de-
pendent inhibition of radioligand binding (0.3 nM [3H]35) by compound
35 (unlabeled 35) at membrane preparations of CHO cells recombi-
nantly expressing the mouse A2B receptor. The calculated KD value
was 0.351 ( 0.126 nM (n ) 3). A Bmax value of 645 ( 51 fmol/mg
protein was determined.
the new A2B-specific antagonist radioligand [3H]35 was devel-
oped and utilized in radioligand binding assays. In this study
we developed, to our knowledge, the most potent A2B antago-
nists known to date, 24 (Ki ) 0.157 nM, 150-fold selective vs
hA2A, >60000-fold selective vs hA1 and hA3) and 17 (Ki ) 0.393
nM, 850-fold selective vs hA2A, >2500-fold selective vs hA1
and hA3). Both compounds feature a basic nitrogen atom that
will increase water solubility and may improve bioavailability.
The unlabeled analogue of the radioligand [3H]35 (Ki ) 0.553
nM) represents, to our knowledge, the first high-affinity A2B-
specific antagonist for humans and rodents exhibiting no
measurable affinity to any other AdoR subtype.
Experimental Section
All reagents were obtained from various producers (Acros,
Aldrich, Fluka, Merck, and Sigma) and used without further
purification. Solvents were used without additional purification or
drying unless otherwise noted. Reactions were monitored by thin-
General Procedure for the Amide Coupling of 11a-c with
12. 5,6-Diaminouracil 11a-c (8.30 mmol) was dissolved in 40 mL
methanol. 4-(4-Nitrophenoxysulfonyl)benzoic acid 12 (8.30 mmol)
and EDC (9.10 mmol) were added, and the mixture was stirred for

1-Alkyl-8-(piperazine-1-sulfonyl)phenylxanthines
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 4003
Figure 7. Comparison of the pKi values of synthesized A2B antagonists for the human and rat AdoR ((A) A1AdoR; (B) A2AAdoR).
Table 4. Calculated Physicochemical Properties of New A2B
Antagonists
Compound 14c. A mixture of carboxamido derivative 13c (0.50
mmol), 0.1 g (NH4)2SO4, and 10 mL HMDS was heated under
reflux for 16 h. After cooling to rt, 20 mL of methanol were added
and the resulting suspension was filtered off and washed with
methanol, yielding the product in 70% yield.
General Procedures for the Aminolysis of p-Nitrophenylsul-
fonates. Method A. p-Nitrophenylsulfonate 14a-c (0.22 mmol)
was dissolved in 5 mL DMF, and the appropriate amine was added
(2.2 mmol). The solution was stirred for 48 h at rt and subsequently
refluxed for 1 h. The solvent was removed under reduced pressure,
and the residue was dissolved in dichloromethane:methanol
(20:1). Diethyl ether was added to precipitate the product. The
crystals were collected by filtration and excess amine was removed
by washing with 20 mL of diethyl ether. The residue was purified
by RP-HPLC using a gradient from water:methanol:triethylamine
(90:10:0.5) to methanol:triethylamine (100:0.5). Yields ranged from
38% to 80%.
Method B. The reaction, precipitation, and washing was
performed as described for method A. To hydrolyze unreacted
p-nitrophenylsulfonate, the crystals were stirred in 12.5% aq
NH4OH solution (30 mL) at 60 °C for 20 min. NH3 was partially
removed under reduced pressure until pH 9 was reached. The
precipitate was then filtered off and washed with water. The residue
was purified by RP-HPLC using a gradient from water:methanol:
triethylamine (90:10:0.5) to methanol:triethylamine (100:0.5). Yields
ranged from 39% to 62%.
calcd calcd
pKA
calcd
a
compd
R1
R2
Benzylpiperazine Derivatives
1-benzyl 7.26 124.168
4-chlorobenzyl
4-chlorobenzyl
3-chlorobenzyl
3-chlorobenzyl
4-fluorobenzyl
4-fluorobenzyl
PSAb log Pa
15
16
17
18
19
20
21
22
23
24
25
26
27
29
30
31
32
33
ethyl
ethyl
propyl
ethyl
propyl
ethyl
propyl
ethyl
propyl
ethyl
propyl
ethyl
propyl
propyl
ethyl
2.84
3.36
3.83
3.36
3.83
2.98
3.45
2.98
3.45
3.72
4.19
3.72
4.19
4.19
2.59
3.06
2.52
2.99
7.11 127.09
7.11 124.168
7.07 127.09
7.07 124.168
7.04 127.09
7.04 127.09
6.96 127.09
6.96 124.168
7.11 127.09
7.11 124.168
7.07 124.168
7.07 124.168
7.11 127.09
7.16 133.402
7.16 133.402
3-fluorobenzyl
3-fluorobenzyl
4-(trifluoromethyl)benzyl
4-(trifluoromethyl)benzyl
3-(trifluoromethyl)benzyl
3-(trifluoromethyl)benzyl
4-methylbenzyl
4-methoxybenzyl
4-methoxybenzyl
propyl
ethyl
propyl
3,4-(methylenedioxy)benzyl 6.96 142.436
3,4-(methylenedioxy)benzyl 6.96 142.436
Method C. This method corresponds to method B, but no heating
under reflux was applied. The product was isolated in 38% yield.
8-(4-(4-(4-Chlorobenzyl)piperazine-1-sulfonyl)phenyl)-1-pro-
pyl-3,7-dihydropurine-2,6-dione (17). 1H NMR (500 MHz, DMSO-
d6) δ 0.88 (t, 3H, J ) 7.25 Hz, CH3), 1.58 (m, 2H, CH2-CH3), 2.42
(m (br), 4H, CH2(piperazinyl)), 2.94 (m (br), 4H, CH2(piperazinyl)),
Phenylpiperazine Derivatives
34
35
36
37
38
ethyl
propyl
4-chlorophenyl
4-chlorophenyl
1.80 127.09
1.80 124.168
1.78 127.09
3.02 133.402
3.02 133.402
3.74
4.20
3.83
2.97
3.43
propargyl 4-chlorophenyl
ethyl
propyl
4-methoxyphenyl
4-methoxyphenyl
3.44 (s, 2H, CH2(benzyl)), 3.82 (t, 2H,
J ) 7.25 Hz,
N-CH2(propyl)), 7.23 (d, 2H, J ) 8.20 Hz, CH(benzyl)), 7.30 (d,
2H, J ) 8.20 Hz, CH(benzyl)), 7.83 (d, 2H, J ) 8.15 Hz,
CH(phenyl)), 8.32 (d, 2H, J ) 8.20 Hz, CH(phenyl)), 11.93 (s (br),
1H, NH), 13.99 (s (br), 1H, NH). 13C NMR (125 MHz, DMSO-d6)
δ 11.32 (CH3), 21.00 (CH2-CH3), 41.62 (N-CH2(propyl)), 46.10,
51.48 (CH2(piperazinyl)), 60.48 (CH2(benzyl)), 108.87 (C-5),
127.07, 128.26, 128.38, 130.60 (CH(phenyl)), 131.65, 133.30,
135.74, 136.94 (C(phenyl)), 147.71 (C-4), 148.17 (C-8), 151.09
(C-2), 155.13 (C-6). LC/ESI-MS: negative mode m/z ) 541 ([M
- H]-), positive mode m/z ) 543 ([M + H]+). Purity (HPLC-UV
254 nm) 99.5%. Anal. (C25H27ClN6O4S·H2O) C, H, N.
Phenethylpiperazine Derivatives
39
40
ethyl
propyl
phenethyl
phenethyl
7.50 127.09
7.50 124.168
3.09
3.56
a log P values and pKA values for the benzyl-, phenyl-, or phenethyl-
substituted nitrogen atom were calculated with the software MarvinSketch.
b PSA values were calculated using the software Molinspiration.
5 h at rt. The formed precipitate was filtered off and washed with
water. A sample was recrystallized from ethanol for analytical
characterization. Yields ranged from 65% to 77%.
1-Ethyl-8-(4-(4-(4-trifluoromethylbenzyl)piperazine-1-sulfo-
nyl)phenyl)-3,7-dihydropurine-2,6-dione (24). 1H NMR (500
MHz, DMSO-d6) δ 1.14 (t, 3H, J ) 6.95 Hz, CH3), 2.45 (m (br),
4H, CH2(piperazinyl)), 2.95 (m (br), 4H, CH2(piperazinyl)), 3.55
(s, 2H, CH2(benzyl)), 3.92 (q, 2H, J ) 6.95 Hz, CH2(ethyl)), 7.45
(d, 2H, J ) 7.85 Hz, CH(benzyl)), 7.61 (d, 2H, J ) 8.20 Hz,
CH(benzyl)), 7.84 (d, 2H, J ) 8.50 Hz, CH(phenyl)), 8.33 (d, 2H,
General Procedures for the Cyclization of the Xanthine
Heterocycles. Compounds 14a-b. A mixture of carboxamido
derivative 13a-b (2.00 mmol) and 3.0 g of PPSE was heated for
10 min at 120 °C and then for 2 h at 170 °C. After cooling to rt,
20 mL of methanol were added and the resulting suspension was
filtered and washed with methanol. Yields ranged from 80% to 86%.

4004 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Borrmann et al.
J ) 8.50 Hz, CH(phenyl)), 11.94 (s (br), 1H, NH), 14.01 (s (br),
1H, NH). 13C NMR (125 MHz, DMSO-d6) δ 13.35 (CH3), 35.19
(CH2-CH3), 46.11, 51.58 (CH2(piperazinyl)), 60.66 (CH2(benzyl)),
108.83 (C-5), 124.42 (q, J ) 270.13 Hz, CF3), 125.16 (q, J ) 3.73
Hz, CH(benzyl)), 127.08 (CH(phenyl)), 127.81 (q, J ) 31.48 Hz,
C-CF3), 128.40 (CH(phenyl)), 129.41 (CH(benzyl)), 133.23, 135.74
(C(phenyl)), 143.00 (C(benzyl)), 147.72 (C-4), 148.09 (C-8), 150.89
(C-2), 154.89 (C-6). LC/ESI-MS: negative mode m/z ) 561 ([M
- H]-), positive mode m/z ) 563 ([M + H]+). Purity (HPLC-UV
254 nm) 96.8%. Anal. (C25H25F3N6O4S·0.25H2O) C, H, N.
8-(4-(4-(4-Chlorophenyl)piperazine-1-sulfonyl)phenyl)-1-pro-
pyl-3,7-dihydropurine-2,6-dione (35). 1H NMR (500 MHz, DMSO-
d6) δ 0.87 (t, 3H, J ) 7.25 Hz, CH3), 1.57 (m, 2H, CH2-CH3),
3.06 (m (br), 4H, CH2(piperazinyl)), 3.20 (m (br), 4H,
CH2(piperazinyl)), 3.82 (t, 2H, J ) 7.25 Hz, N-CH2(propyl)), 6.90
(d, 2H, J ) 8.85 Hz, CH(phenyl)), 7.20 (d, 2H, J ) 8.85 Hz,
CH(phenyl)), 7.88 (d, 2H, J ) 8.50 Hz, CH(phenyl)), 8.34 (d, 2H,
J ) 8.20 Hz, CH(phenyl)), 11.93 (s (br), 1H, NH), 14.00 (s (br),
1H, NH). 13C NMR (125 MHz, DMSO-d6) δ 11.31 (CH3), 20.98
(CH2-CH3), 41.62 (N-CH2(propyl)), 45.81, 47.91 (CH2(pipera-
zinyl)), 108.71 (C-5), 117.76 (CH(phenyl)), 123.37 (C(phenyl)),
127.14, 128.44, 128.80 (CH(phenyl)), 133.29, 135.69 (C(phenyl)),
147.73 (C-4), 148.07 (C-8), 149.29 (C(phenyl)), 151.07 (C-2),
155.08 (C-6). LC/ESI-MS: negative mode m/z ) 527 ([M - H]-),
positive mode m/z ) 529 ([M + H]+). Purity (HPLC-UV 254 nm)
99.8%. Anal. (C24H25ClN6O4S·0.25H2O) C, H, N.
described.59,66,73-75 [3H]2-chloro-N6-cyclopentyladenosine ([3H]C-
CPA, 42.6 Ci/mmol, 1 nM), [3H](E)-3-(3-hydroxypropyl)-8-(2-(3-
methoxyphenyl)vinyl)-7-methyl-1-prop-2-ynyl-3,7-dihydropurine-
2,6-dione ([3H]MSX-2, 84 Ci/mmol, 1 nM), and [3H]2-phenyl-8-
ethyl-4-methyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2.1-i]purin-5-
one ([3H]PSB-11, 53 Ci/mmol, 1 nM) were used as A1, A2A, and
A3 radioligands, respectively.62-65 The radioligands were custom-
labeled by GE Healthcare from suitable precursors as described.62-65
Membrane Preparations for A2B Receptor Assays. Membranes
of CHO cells expressing the human A2B receptor were prepared by
scratching the cells off the previously frozen cell culture dishes in ice-
cold hypotonic buffer (5 mM Tris-HCl, 2 mM EDTA, pH 7.4). The
cell suspension was homogenized on ice for 20 s with an Ultra-Turrax
and spun down for 10 min (4 °C) at 1,000 g. The supernatant was
subsequently centrifuged for 60 min at 48000g. The obtained mem-
brane pellets were resuspended in 10 mL of 50 mM Tris-HCl buffer,
pH 7.4, and again centrifuged under the same conditions. Then the
membrane pellets were resuspended and homogenized in the required
amount of 50 mM Tris-HCl puffer, pH 7.4, to obtain a protein
concentration of 1-3 mg/mL. Aliquots of the membrane preparation
(1 mL each) were stored at -80 C° until used.
A2B Radioligand Binding Assays. Competition experiments with
[3H]35 were performed in a final volume of 500 µL containing 25
µL of test compound dissolved in 50% DMSO/50% Tris-HCl buffer
(50 mM, pH 7,4), 275 µL buffer (50 mM Tris-HCl, pH 7,4), 100
µL of radioligand solution in the same buffer (final concentration
0.3 nM), and 100 µL of membrane preparation (30 µg protein per
tube in buffer containing adenosine deaminase (2 U/mL, 20 min
incubation). Nonspecific binding was determined in the presence
of 10 µM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). After an
incubation time of 75 min at rt, the assay mixture was filtered
through GF/B glass fiber filters using a Brandel harvester (Brandel,
Gaithersburg, MD). Filters were washed four times (3-4 mL each)
with ice-cold 50 mM Tris-HCl buffer, pH 7.4, containing 0.1%
bovine serum albumin (BSA). Then filters were transferred to vials,
incubated for 9 h with 2.5 mL of scintillation cocktail (Beckmann
Coulter), and counted in a liquid scintillation counter (Tricarb
2700TR) with a counting efficiency of ∼54%. Three separate
experiments were performed in duplicates.
Association studies were performed over a time range of 180
min with 12 different points. Dissociation was initiated by the
addition of 10 µM 8-cyclopentyl-1,3-dipropylxanthine after 95
min of preincubation. Two separate experiments were performed
in duplicates. The other conditions were as described for competition
experiments. Saturation studies were performed over a concentration
range of 0.05-1 nM using nine different concentrations of [3H]35.
All other conditions were as described for competition experiments.
Three separate experiments were performed in duplicates. All data
were analyzed with GraphPad Prism, Version 4.1 (GraphPad Inc.,
La Jolla, CA).
Preparation of [3H]35. The labeling reaction was performed
by GE Healthcare UK Limited by catalytic hydrogenation of the
1-propargyl-substituted precursor 36 with tritium gas in the presence
of Pd/C (10%) using a mixture of ethanol and DMF as solvent.
The radioligand [3H]35 was purified by RP-HPLC. The identity
was confirmed by mass spectrometric analysis (see Supporting
Information). [3H]35 displayed a specific activity of 2.70 TBq/mmol
(73 Ci/mmol) and a radiochemical purity of 98.9% as determined
by HPLC/UV (254 nm).
Fluorimetric Measurement of Intracellular Calcium Con-
centrations in Jurkat T Cells. Fluorimetric measurements were
performed with a FLUOStar Galaxy (BMG Labtech, Offenburg,
Germany) at 30 °C. Jurkat T cells were grown in RPMI 1640 medium
supplemented with 10% fetal calf serum, 2 mM GlutaMAX (Invitro-
gen), and 100 IU penicillin and 100 µg/mL streptomycin. The cells
were transferred to Falcon tubes, aliquots of cells were counted using
a hemocytometer, and the cells were then spun down at 200g and 4
°C for 5 min. After removal of the supernatant, the cells were incubated
with Oregon Green BAPTA-1 AM (Invitrogen, 3 µL of a 1 mM
solution in DMSO) and with 3 µL of a 25% aq solution of Pluronic
F-127) in a final volume of 1 mL of Krebs-Ringer-Hepes (KRH)
buffer consisting of NaCl (118.6 mM), KCl (4.7 mM), KH2PO4 (1.2
mM), NaHCO3 (4.2 mM), D-glucose (11.7 mM), HEPES (10 mM),
CaCl2 (1.3 mM), MgSO4 (1.2 mM), pH 7.4 (with NaOH) in the dark
with shaking (600 rpm) at 37 °C for 60 min. Then the cells were
washed twice with 1 mL KRH buffer each by centrifugation at 7000
rpm and 4 °C for a few seconds. Then the cells were suspended in
fresh KRH buffer and pipetted into 96-well plates at a density of ca.
200000 cells in a volume of 160 µL per well. Before addition of the
cells, 20 µL of a solution containing test compound (1-alkyl-8-
(piperazine-1-sulfonyl)phenylxanthine derivative) and 3-(3-hydroxy-
propyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine (final con-
centration: 200 nM) to block A2AAdoR had been added. After 10 min
of preincubation and measurement of basic fluorescence levels, 20 µL
of a solution containing NECA (10 µM final concentration) was added
automatically to each well and changes in fluorescence levels were
recorded. The DMSO concentration did not exceed 2.5%. A2B
antagonists were tested in 8-10 concentrations spanning at least 3-4
orders of magnitude in order to calculate IC50 values. At least three
separate experiments were performed each in triplicates or quadruplicates.
Radioligand Binding Assays at A1, A2A and A3 Receptors.
Membrane preparations and radioligand binding assays at rat A1
(rat brain cortex), human A1 (expressed in CHO cells), rat A2A (rat
brain striatum), human A2A (expressed in CHO cells), and human
A3 receptors (expressed in CHO cells) were performed as previously
Acknowledgment. T.B. was supported by a stipend provided
by the Bischo¨fliche Studienfo¨rderung Cusanuswerk. D.C.G.B.
was supported by a scholarship of the Deutsche Forschungs-
gemeinschaft (DFG) within the Graduiertenkolleg GRK 677.
W.L. is grateful for a STIBET scholarship by the Deutscher
Akademischer Austauschdienst (DAAD).
Supporting Information Available: Elemental analyses of
synthesized A2B antagonists; mass spectrum of [3H]PSB-603;
experimental data of all synthesized new compounds except 17,
24, and 35. This material is available free of charge via the Internet
at http://pubs.acs.org.
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