Synthesis and biological evaluation of 2-aminothiazoles and their amide derivatives on human adenosine receptors. Lack of effect of 2-aminothiazoles as allosteric enhancers

Article abstract of DOI:10.1016/j.bmc.2005.01.006

A number of 2-aminothiazoles (2a-e) and their amide derivatives (4-10) were prepared. The 2-aminothiazoles themselves were tested as allosteric enhancers of agonist binding to human adenosine A₁ receptors. In a variety of experimental set-ups the compounds did not show any such effect, in contrast to earlier findings by another research group. Subsequently the 2-aminothiazoles were used as intermediates in the synthesis of a number of amide derivatives of either aromatic (4-6) or aliphatic nature (7-10). Some of the compounds emerged as moderately active antagonists on human adenosine A₁ and/or A _2A receptors with lower or negligible potency at adenosine A ₃ receptors.

Full text of DOI:10.1016/j.bmc.2005.01.006

Bioorganic & Medicinal Chemistry 13 (2005) 2079–2087
Synthesis and biological evaluation of 2-aminothiazoles and
their amide derivatives on human adenosine receptors.
Lack of effect of 2-aminothiazoles as allosteric enhancers
´
Aniko Go¨blyo¨s, Sabrina Neves Santiago, Daniele Pietra, Thea Mulder-Krieger,
Jacobien von Frijtag Drabbe Kunzel, Johannes Brussee and Adriaan P. IJzerman*
¨
Leiden/Amsterdam Center for Drug Research, Division of Medicinal Chemistry, PO Box 9502, 2300 RA Leiden, The Netherlands
Received 21 September 2004; revised 22 December 2004; accepted 7 January 2005
Available online 26 January 2005
Abstract—A number of 2-aminothiazoles (2a–e) and their amide derivatives (4–10) were prepared. The 2-aminothiazoles themselves
were tested as allosteric enhancers of agonist binding to human adenosine A1 receptors. In a variety of experimental set-ups the
compounds did not show any such effect, in contrast to earlier findings by another research group. Subsequently the 2-aminothiaz-
oles were used as intermediates in the synthesis of a number of amide derivatives of either aromatic (4–6) or aliphatic nature (7–10).
Some of the compounds emerged as moderately active antagonists on human adenosine A1 and/or A2A receptors with lower or neg-
ligible potency at adenosine A3 receptors.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
S
NH
2
S
NH
2
Adenosine receptors are membrane-bound proteins
belonging to the large family of G protein-coupled
receptors. The four subtypes of adenosine receptors
are the target for the endogenous ligand adenosine and
as such they play an important role in intra- and inter-
cellular communication. Their important role in physio-
logy (e.g., in cardiac and metabolic homeostasis, in
inflammation, and sleep/wake control mechanisms) has
evoked the interest of medicinal chemists who, over
the years, have generated a vast library of synthetic
ligands interacting with these adenosine receptors.1
O
N
O
S
N
O
Cl
O
HN
CF3
1 (PD 81,723)
2
3
Figure 1. Chemical structures of putative allosteric enhancers (1, 2)
and antagonist (3) for adenosine A1 receptors.
structures with similar characteristics.3–6 Although the
receptor binding site for these allosteric modulators
has not been elucidated yet, it is believed that such com-
pounds indeed bind to another site on the receptor than
the primary ligand binding site for adenosine and syn-
thetic derivatives thereof.
Among these ligands an interesting class of the so-called
ꢀallosteric enhancersꢁ of adenosine binding and function
at the adenosine A1 receptor was discovered next to
ꢀclassicꢁ agonists and antagonists. The reference com-
pound is PD 81,723,2 a 2-amino-3-aroylthiophene (1,
Fig. 1), which served as a template for a range of related
Interestingly, in a recent report a novel class of allosteric
enhancers was identified. Certain 2-aminothiazoles were
described to be potent enhancers of agonist binding sur-
passing the potency of PD 81,723 with 2 being the most
active (Fig. 1).7 Our attention was drawn by these find-
ings, also because we had previously reported on a series
Keywords: 2-Aminothiazoles; Adenosine receptors; Allosteric modula-
tion; Antagonism.
*
Corresponding author. Tel.: +31 071 527 4651; fax: +31 071 527
4565; e-mail: ijzerman@lacdr.leidenuniv.nl
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.01.006

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A. Go¨blyo¨s et al. / Bioorg. Med. Chem. 13 (2005) 2079–2087
of functionalized 2-aminothiazoles as adenosine recep-
tor antagonists (e.g., 3 in Fig. 1).8
centration of 1 lM on human adenosine A1, A2A, and
A3 receptors (Table 1). For A1 adenosine receptors both
a radiolabeled agonist ([3H]CCPA) and a radiolabeled
antagonist ([3H]DPCPX) were used. Compounds 5c,
4d, 9b, and 10b significantly inhibited [3H]DPCPX bind-
ing, that is, they displaced the radioligand for more than
50% (Table 1). We determined the apparent affinity of
these compounds (Table 2). Of the four compounds 9b
proved to be the most potent with a Ki value of
144 30 nM. Interestingly none of the compounds ap-
peared to enhance [3H]CCPA binding.
We therefore decided to synthesize and study a number
of known as well as new 2-aminothiazoles with respect
to their activity on adenosine receptors, including hybrid
structures of compounds 2 and 3. We were not able to
confirm the allosteric enhancing capacity of the 2-
aminothiazoles. Some of the compounds, however,
proved to be antagonists for the adenosine receptors
and showed affinity in the higher nanomolar range.
Subsequently, all compounds were studied on adenosine
A2A receptors with [3H]ZM241385 as the radiolabeled
antagonist. Compounds 4d, 6d, 8b–10b significantly
(i.e., >50%) inhibited [3H]ZM241385 binding at the sin-
gle concentration of 1 lM (Table 1). Of these eight com-
pounds 8b proved to be the most active with a Ki value
of 262 48 nM (Table 2). The compounds were also
studied on adenosine A3 receptors with [125I]AB-MECA
as the radiolabeled agonist. None of the compounds dis-
placed the radioligand by more than 50% (Table 1), and
hence we refrained from determining their Ki values.
2. Results
2.1. Chemistry
To prepare the compounds we applied a known syn-
thetic route with minor modifications.7,9 2-Amino-
thiazolium salts were prepared by a one-step synthesis
from the appropriate ketones, thiourea, and iodine. 2-
Aminothiazoles (2a–e) were obtained from the above
HI salts after alkaline treatment. The syntheses of amide
derivatives of 2-aminothiazoles (4a–e–6a–e; 7b–10b)
were achieved by condensation reactions of 2-amino-
thiazoles (2a–e) with the appropriate acylchlorides either
under classic8 or microwave conditions, which allowed
us to prepare the compounds in approximately 20 min
instead of 2–5 h. Optimized yields were not aimed for,
and were in the range of 13–99% (Schemes 1 and 2).
Selected 2-aminothiazoles 2b and 2c (at concentrations
of 10 lM) were used for further characterization as
putative allosteric enhancers by studying their influence
on association and dissociation kinetics of the adenosine
A1 receptor agonist radioligand [3H]CCPA. The associ-
ation kinetics of [3H]CCPA were slightly affected by the
two compounds with negligible effects on its dissociation
characteristics. Compounds 2b and 2c appeared to in-
crease the extent of association somewhat. For com-
pound 2b the highest value was 121%, for 2c it was
110%, compared to the control of 93%. The rate con-
stants for the two processes did not differ significantly
between the treatment protocols (Fig. 2, Table 3).
2.2. Biology
All compounds were initially characterized in equili-
brium radioligand displacement studies at a final con-
NH2
O
N
R1
R2
R1
R2
Finally the most active compounds in the adenosine A1
receptor binding studies were also analyzed in a func-
tional assay, that is, their influence on the cAMP pro-
duction in cells expressing the human adenosine A1
receptor. Compounds 9b and 10b were capable of block-
ing the effect induced by the reference agonist N6-cyclo-
i
S
( )
( )
ii
n
n
R3
R3
2a-e
pentyladenosine (CPA),
antagonistic activity (Fig. 3).
demonstrating
their
a: n = 1, R1 = R2 = R3 = H; b: n = 2, R1 = R2 = R3 = H;
c: n = 3, R1 = R2 = R3 = H; d: n = 2, R1 = R2 = H, R3 = OMe;
e: n = 2, R1 = R2 = OMe, R3 = H
3. Discussion
Scheme 1. Reagents and conditions: (i) iodine, thiourea, heating at
100 ꢁC, (ii) NaOH.
Recently 2-aminothiazoles were introduced as a new
class of agonist allosteric enhancers of adenosine A1
receptors.7 We first synthesized and analyzed such 2-
aminothiazoles, both known (2a–c) and new derivatives
(2d, 2e). At a concentration of 1 lM none of these five
compounds appeared to affect significantly either radio-
labeled antagonist ([3H]DPCPX) or radiolabeled agonist
([3H]CCPA) binding to the adenosine A1 receptor. Par-
ticularly the lack of effect on [3H]CCPA binding sur-
prised us, since an increase in its binding had been
anticipated due to the presence of a putative allosteric
enhancer. We then decided to study the influence of
two compounds also reported by Chordia et al.7 (2a
H
NH2
O
N
N
R1
R2
N
R1
R4
iii
S
S
( )
( )
n
n
R2
R3
R3
2a-e
4a-e – 6a-e; 7b-10b
4a-e: R4 = Ph; 5a-e: R4 = pNO2-Ph; 6a-e: R4 = pCl-Ph; 7b: R4 = Me;
8b: R4 = Et; 9b: R4 = tBu; 10b: R4 = cPe
Scheme 2. Reagents and conditions: (iii) R4–COCl, EtN3, 1,4-dioxane.

A. Go¨blyo¨s et al. / Bioorg. Med. Chem. 13 (2005) 2079–2087
2081
Table 1. Binding affinity of compounds 2a–e, 4a-e–6a–e, and 7b–10b at human A1, A2A, and A3 adenosine receptor subtypes (concentration of
compounds was 10ꢀ6 M)
H
NH2
O
N
N
R1
R2
N
R1
R4
S
S
( )
(
)
n
n
R2
R3
R3
2a-e
4a-e – 6a-e; 7b-10b
% Specific binding of radioligand remaining
Compd.
n
R1
R2
R3
R4
A1
A2A
A3
[
125I]-AB-MECA
[3H]DPCPX
[3H]CCPA
[3H]ZM241385
2a
2b
2c
2d
2e
4a
5a
6a
4b
5b
6b
4c
5c
6c
4d
5d
6d
4e
5e
6e
7b
8b
9b
10b
1
2
3
2
2
1
1
1
2
2
2
3
3
3
2
2
2
2
2
2
2
2
2
2
H
H
H
—
101
104
96
96
95
100
101
90
87
96
59
61
82
66
54
69
50
52
51
43
86
44
95
100
93
54
43
34
40
105
103
97
H
H
H
—
—
—
—
H
H
H
103
97
H
H
OMe
H
93
105
105
68
OMe
H
OMe
H
100
51
98
81
H
Ph
H
H
H
p-NO2–Ph
p-Cl–Ph
Ph
58
88
85
93
92
86
H
H
H
H
H
H
74
70
87
81
66
80
H
H
H
p-NO2–Ph
p-Cl–Ph
Ph
H
H
H
81
55
86
87
71
67
H
H
H
H
H
H
p-NO2–Ph
p-Cl–Ph
Ph
48
60
90
94
67
65
H
H
H
H
H
OMe
OMe
OMe
H
39
75
92
58
80
H
H
p-NO2–Ph
p-Cl–Ph
Ph
100
65
H
H
79
57
73
OMe
OMe
OMe
H
OMe
OMe
OMe
H
101
97
100
109
109
89
H
p-NO2–Ph
p-Cl–Ph
Me
90
H
97
84
102
65
H
H
H
H
Et
t-Bu
70
18
82
47
54
69
H
H
H
H
H
H
c-Pe
21
54
88
Data are expressed as means from two independent experiments performed in duplicate; individual values varied less than 10% for [3H]CCPA
binding, less than 15% for [3H]DPCPX binding, and less than 20% for [3H]ZM241385 and [125 I]-AB-MECA binding. The results are given as
percentage specific binding of radioligand remaining, where total control binding is 100% and nonspecific binding is 0%.
Table 2. Effects of various compounds as displacing agents of
[3H]DPCPX and [3H]ZM241385 binding to human adenosine A1 and
A2A receptors
ation of the dissociation kinetics with EC50 values of 38
and 17 lM for 2a and 2b, respectively. The reason for
this discrepancy is unclear to us, although slight differ-
Compound
A1
Ki (nM)
A2A
Ki (nM)
ences in experimental conditions may be a trivial expla-
nation (e.g., the use of a different radioligand, [125I]-N6-
aminobenzyladenosine rather than [3H]CCPA). The
association kinetics of [3H]CCPA were somewhat af-
fected by the two compounds. The extent of association
was increased by approximately 20% without an effect
on kon values (Fig. 2A, Table 3). The association kinet-
ics had not been studied by Chordia et al.7
4c
5c
—
1040 411
—
801 73
374 73
—
4d
6d
8b
9b
10b
369 67
410 60
262 48
343 96
274 17
—
144 30
171 15
We then focused our attention to the resemblance be-
tween these aminothiazole structures and a 2-amino-4-
phenylthiazole template that we had used before to ob-
tain adenosine receptor antagonists (e.g., compound 3
in Fig. 1). From research in our group it had been estab-
lished that 4-phenyl-2-(phenylcarboxamido)-1,3-thiazole
derivatives are indeed antagonists for the adenosine A1
receptor with little effect on adenosine A2A and A3 recep-
tors.8 Later Jung et al. identified a series of related 4-(4-
methoxyphenyl)-2-aminothiazole derivatives as potent
and 2b) on the association and dissociation kinetics of
[3H]CCPA. Alteration of the dissociation kinetics in
particular is regarded as experimental evidence for allo-
steric modulation.10 However, at a concentration of
10 lM we did not observe any effect on the dissociation
kinetics with identical t1/2 and koff values under all con-
ditions (Fig. 2B, Table 3). This finding is in contrast with
data by Chordia et al.,7 who reported a significant retard-

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A. Go¨blyo¨s et al. / Bioorg. Med. Chem. 13 (2005) 2079–2087
100
130
120
110
100
90
80
70
60
50
40
30
A
B
90
80
70
60
50
40
30
20
10
0
20
10
0
0
5
10 15 20 25 30 35 40 45 50 55 60
0
5
10 15 20 25 30 35 40 45 50 55 60
time (min)
time (min)
Figure 2. Association (A) and dissociation kinetics (B) of [3H]CCPA binding to and from human adenosine A1 receptors. Data are from a
representative experiment performed in duplicate. Radioligand binding is expressed as percentage of specific binding. Concentration of compounds
2b (.) and 2c (d) was 10 lM. Control (j) was without allosteric modulators.
Table 3. Association and dissociation kinetic parameters of [3H]CCPA
binding to and from human adenosine A1 receptors in the presence or
that these compounds were modestly active on either the
adenosine A1 or A2A receptor or on both. From the
three compounds that were tested on both receptors
absence of 2b and 2c (10 lM)
Association
Dissociation
(4d, 9b, and 10b) we learned that selectivity for either
one of the receptors was negligible. It is interesting to
compare compound 3, synthesized and tested before
with a Ki value of 18 nM on (rat) adenosine A1 recep-
tors,8 with compounds 6a–c that show only little affinity
for the (human) adenosine A1 receptor. Apparently,
bridging the thiazole and phenyl rings with 1–3 methyl-
ene moieties and thus rigidifying the structures is not
favorable for affinity toward adenosine A1 receptors.
The most potent compounds (9b and 10b) contained ali-
phatic rather than (substituted) benzoyl amides. These
two compounds were also tested in a cAMP assay dem-
onstrating their antagonistic characteristics. In view of
their structure, that is without a ribose group, this was
as expected, although we have recently reported on
ribose lacking compounds that behave as agonists
for adenosine receptors.13
t1/2 (min) kon (nMꢀ1 minꢀ1
)
t1/2 (min) koff (minꢀ1
)
Control 1.9 0.2 0.4 0.03
11.8 3.2 0.06 0.02
11.1 1.2 0.06 0.01
10.7 0.4 0.07 0.01
2b
2c
2.4 0.3 0.3 0.05
2.7 0.2 0.3 0.03
7
6
5
4
3
2
1
0
4. Conclusion
In our experiments 2-aminothiazoles (2a–e) did not be-
have as allosteric enhancers of agonist binding to adeno-
sine A1 receptors as suggested by Chordia et al.7 We
therefore suggest to examine and reconsider the alloste-
ric activity of this type of compounds. Amides derived
from the 2-aminothiazoles (4–10) were also tested in
radioligand binding studies and some of them proved
to have modest affinity for adenosine A1 and/or adeno-
sine A2A receptors. They were classified as antagonists
based on the results obtained in a functional assay of
cAMP production.
Figure 3. Modulation of forskolin-induced cAMP production in CHO
cells expressing the human adenosine A1 receptor. CPA (100 nM)
reduced the forskolin-induced cAMP production by approximately
60%. This reduction was reversed by the addition of either 9b
(partially) or 10b (fully), both at 10 lM.
and selective human adenosine A3 receptor antago-
nists.11 A further extension of the chemical structure with
a 5-pyridin-4-yl substituent also provided a number of
potent adenosine A3 receptor antagonists.12
With this in mind we prepared a number of amides (4–
10) from the above ꢀbridgedꢁ 2-aminothiazoles (2a–e).
5. Experimental
5.1. Materials and methods
The amide derivatives were also tested on all three adeno-
sine receptor subtypes at a concentration of 1 lM. It
appeared that in contrast to the aminothiazoles 2a–e
some of the amides showed significant affinity for aden-
osine A1 and/or A2A receptors (Table 1). We determined
Ki values if the compounds in the initial screen displaced
more than 50% of the radioligand. The affinities were all
in the range from 100 nM to 1 lM (Table 2), suggesting
5.1.1. Chemicals. [3H]DPCPX (128 Ci/mmol) and
[
chased from Amersham Pharmacia Biosciences (The
Netherlands). [3H]CCPA (54.9 Ci/mmol) was obtained
from NEN (The Netherlands). DPCPX and CPA were
from Sigma-RBI (The Netherlands). [3H]ZM 241385
was obtained from Tocris Cookson (United Kingdom),
125I]AB-MECA (approx. 2000 Ci/mmol) were pur-

A. Go¨blyo¨s et al. / Bioorg. Med. Chem. 13 (2005) 2079–2087
2083
[3H]cAMP was obtained from Perkin Elmer Life Sci-
ences (The Netherlands). HEK 293 cells stably express-
ing the human adenosine A3 receptor were a gift from
5.2.3.
5,6-Dihydro-4H-benzo(6,7)cyclohepta(1,2-d)thi-
azol-2-ylamine (2c).7 Starting material was 1-benzosub-
eron (6.3 mmol, 1.01 g). The eluent for column
chromatography was diisopropylether. The product
was recrystallized from CH2Cl2 to give the product as
off-white crystals. Yield: 0.72 g, 53%. Mp: 129–131 ꢁC.
1H NMR (CDCl3): d 2.07–2.20 (m, 2H, CH2), 2.80–2.78
(m, 4H, CH2), 4.89 (s, 2H, NH2), 7.13–7.31 (m, 4H,
Ar), 7.91 (d, 1H, J = 7.3 Hz, Ar). 13C NMR (MeOD):
26.6, 30.9, 34.9, 123.0, 127.1, 129.3, 130.0, 135.5, 141.8,
145.5, 167.5. Anal. Calcd for C12H12N2S: C, 66.63; H,
5.59; N, 12.95. Found: C, 66.46; H, 5.80; N, 13.11.
Dr. K.-N. Klotz (University of Wurzburg, Germany).
¨
All compounds made were tested in radioligand binding
assays to determine their affinities at the human adeno-
sine A1, A2A, and the A3 receptors. All other chemicals,
including starting materials, were from standard sources
and of the highest purity commercially available.
1
5.1.2. Instruments and analysis. H NMR spectra were
measured at 200 MHz with a Bruker AC 200 or Bruker
DMX 600 spectrometer. 13C NMR spectra were mea-
5.2.4. 6-Methoxy-4,5-dihydro-naphto(1,2-d)thiazol-2-yl-
amine (2d). Starting material was 5-methoxy-1-tetralone
(11.3 mmol, 2.00 g). The eluent for column chromato-
graphy was 5% methanol in CH2Cl2. The product was
recrystallized from chloroform–methanol = 80:20, to
give the product as white crystals. Yield: 1.40 g, 53%.
sured at 50 or 150 MHz. Chemical shifts for 1H and 13
C
are given in ppm (d) relative to tetramethylsilane (TMS)
as internal standard, coupling constants are given in Hz.
Melting points were determined with a Buchi capillary
¨
melting point apparatus and are uncorrected. Combus-
tion analyses of new target compounds were performed
by the analytical department of the Gorlaeus Laborato-
ries, Leiden University (The Netherlands) and are within
0.4% of theoretical values unless otherwise specified.
1
Mp: 196–198 ꢁC. H NMR (CDCl3): d 2.79–2.87 (m,
2H, CH2), 2.99–3.08 (m, 2H, CH2), 3.85 (s, 3H,
OCH3), 4.94 (s, 2H, NH2), 6.79 (d, 1H, J = 8.0 Hz,
Ar), 7.17–7.26 (m, 1H, Ar), 7.36 (d, 1H, J = 7.3 Hz,
Ar). 13C NMR (CDCl3): 19.7, 20.7, 55.1, 109.2, 115.0,
119.3, 121.9, 126.9, 131.9, 156.0, 166.8. Anal. Calcd
for C12H12N2OS: C, 62.04; H, 5.21; N, 12.06. Found:
C, 61.88; H, 5.00; N, 12.16.
5.2. General procedure for the preparation of 2-amino-
thiazoles7,9
Thiourea (3.0 equiv) and iodine (1.1 equiv) were added
to a solution of the appropriate ketone in absolute eth-
anol (1.0 mmol/2 mL). The mixture was heated for 2–3 h
in an open vessel at 100 ꢁC, which yielded 2-aminothiaz-
ole hydroiodide salts. 2-Aminothiazoles were obtained
from the above hydroiodides by alkaline treatment
(5% NaOH), extraction (CH2Cl2), and evaporation un-
der reduced pressure. The crude products were purified
by column chromatography on silica gel and recrystal-
lized afterwards.
5.2.5. 7,8-Dimethoxy-4,5-dihydro-naphto(1,2-d)thiazol-2-
ylamine (2e). Starting material was 6,7-dimethoxy-1-
tetralone (8.2 mmol, 1.69 g). The eluent for column
chromatography was 5% methanol in CH2Cl2. The
product was recrystallized from chloroform–metha-
nol = 90:10, to give the product as white crystals. Yield:
0.69 g, 32%. Mp: 236–227 ꢁC. Lit.14 Mp: 225–228 ꢁC. 1H
NMR (CDCl3): d 2.78–2.86 (m, 2H, CH2), 2.97–3.01 (m,
2H, CH2), 3.91 (d, 6H, J = 8.78 Hz, 2CH3), 4.89 (s, 2H,
NH2), 6.72 (s, 1H, Ar), 7.29 (s, 1H, Ar). 13C NMR
(CDCl3): 21.5, 28.4, 55.6, 55.7, 106.3, 111.5, 116.8,
124.2, 126.8, 144.1, 147.2, 147.4, 167.0. Anal. Calcd
for C13H14N2O2S: C, 59.52; H, 5.38; N, 10.68. Found:
C, 59.25; H, 5.10; N, 10.74.
5.2.1. 8H-Indeno(1,2-d)thiazol-2-ylamine (2a). Starting
material was 1-indanone (4.5 mmol, 0.59 g). The eluent
for column chromatography was 1% methanol in
CH2Cl2. The product was recrystallized from CH2Cl2,
to give off-white crystals. Yield: 0.72 g, 85%. Mp: 202–
207 ꢁC. Lit.9 Mp: 213–214 ꢁC. MS (ESI) m/z: 189.1
5.3. General procedure for the syntheses of the amides
1
[M+H]+. H NMR (CDCl3): d 3.70 (s, 2H, CH2), 5.16
(s, 2H, NH2), 7.15–7.37 (m, 2H, Ar), 7.45 (d, 1H,
J = 7.3 Hz, Ar), 7.57 (d, 1H, J = 7.3 Hz, Ar). 13C
NMR (MeOD): d 32.9, 118.6, 124.6, 125.3, 127.4,
138.4, 146.6, 156.4, 175.7. Anal. Calcd for C10H8N2S:
C, 63.80; H, 4.28; N, 14.88. Found: C, 63.78; H, 4.10;
N, 15.07.
Method A: Triethylamine (1.1 equiv) and the appropri-
ate acid chloride (1.5 equiv) were added to a solution
of 2-aminothiazole in 1,4-dioxane (1 mmol/3–4 mL).
The mixture was refluxed for 2–5 h. After the reaction
was completed the mixture was extracted with CH2Cl2
and washed with water. The water layer was extracted
with CH2Cl2. The organic layers were collected and
dried on MgSO4. The products were purified by column
chromatography and recrystallized.8
5.2.2. 4,5-Dihydro-naphto(1,2-d)thiazol-2-ylamine (2b).
Starting material was a-tetralone (4.5 mmol, 0.66 g).
The product was recrystallized from diisopropylether,
to give white crystals. Yield: 0.72 g, 79%. Mp: 129–
131 ꢁC. Lit.9 Mp: 133–134 ꢁC. 1H NMR (CDCl3): d
2.85–2.90 (m, 2H, CH2), 3.00–3.08 (m, 2H, CH2), 5.04
(s, 2H, NH2), 7.10–7.29 (m, 3H, Ar), 7.68 (d, 1H,
J = 7.3 Hz, Ar). 13C NMR (DMSO): 21.1, 28.5, 117.6,
122.2, 126.3, 126.7, 127.7, 131.7, 134.3, 144.4, 166.7.
Anal. Calcd for C11H10N2S: C, 65.32; H, 4.98; N,
13.85. Found: C, 65.00; H, 4.56; N, 14.00.
Method B (microwave-assisted chemistry): Triethyl-
amine (1.1 equiv) and the appropriate acid chloride
(1.5 equiv) were added to a solution of 2-aminothiazole
in 1,4-dioxane (1 mmol/2.5–3 mL). Conditions: prestir-
ring 120 s, temperature 140 ꢁC, time 1200 s, normal sam-
ple absorption, no fixed hold time. Work up and
purification of the products were the same as described
in Method A.

2084
A. Go¨blyo¨s et al. / Bioorg. Med. Chem. 13 (2005) 2079–2087
5.3.1. N-(8H-Indeno[1,2-d]thiazol-2-yl)-benzamide (4a).
Scale: 1.0 mmol. Eluent for column chromatography
was CH2Cl2. The product was recrystallized from ethyl-
acetate–ethanol = 95:5, to give white crystals. Yield:
0.22 g, 75%. Mp: 221–223 ꢁC. 1H NMR (CDCl3): d
3.86 (s, 2H, CH2), 7.16–47.26 (m, 2H, Ar), 7.33–7.55
(m, 5H, Ar), 7.98 (d, 2H, J = 9.5 Hz, Ar), 10.86 (br s,
1H, NH). 13C NMR (CDCl3): d 32.3, 118.2, 124.5,
124.8, 126.5, 128.6, 130.7, 131.8, 132.5, 136.3, 145.3,
155.2, 162.7, 165.0. Anal. Calcd for C17H12N2OSÆ0.1-
H2O: C, 69.41; H, 4.18; N, 9.52. Found: C, 69.26; H,
3.91; N, 9.67.
156.2, 163.7. Anal. Calcd for C18H13N3O3S: C, 61.53;
H, 3.73; N, 11.96. Found: C, 61.43; H, 3.53; N, 12.09.
5.3.6. N-(4,5-Dihydro-naphto[1,2-d]thiazol-2-yl)-4-chloro-
benzamide (6b). Scale: 1.0 mmol. Eluent for column
chromatography was CH2Cl2. The product was recrys-
tallized from ethanol, to give off-white crystals. Yield:
0.28 g, 82%. Mp: 204–205 ꢁC. 1H NMR (CDCl3): d
2.95–3.11 (m, 4H, 2CH2), 7.01–7.26 (m, 5H, Ar), 7.41–
7.50 (m, 1H, Ar), 7.65–7.72 (m, 2H, Ar), 11.32 (s, 1H,
NH). 13C NMR (DMSO-d6): d 20.8, 28.3, 122.1, 124.3,
127.0, 128.1, 128.7, 130.1, 130.9, 131.0, 134.8, 137.5,
143.6, 156.5, 164.0. Anal. Calcd for C18H13ClN2OS: C,
63.43; H, 3.84; N, 8.22. Found: C, 63.53; H, 3.65; N,
8.58.
5.3.2. N-(8H-Indeno[1,2-d]thiazol-2-yl)-4-nitro-benzam-
ide (5a). Scale: 1.0 mmol. Eluent for column chromato-
graphy was CH2Cl2. The product was recrystallized
from N,N-dimethyl-formamide and after recrystalliza-
tion the crystals were washed three times with a solution
of petroleum ether–methanol = 95:5, to give orange
crystals. Yield: 0.29 g, 86%. Mp: 270–275 ꢁC. 1H
NMR (DMSO-d6): d 3.95 (s, 2H, CH2), 7.25–7.44 (m,
4H, Ar), 7.61 (d, 4H, J = 8.0 Hz, Ar), 11.89 (br s, 1H,
NH). 13C NMR (DMSO-d6): d 32.5, 123.8, 125.3,
127.0, 128.3, 128.6, 129.8, 131.2, 132.0, 135.6, 146.1,
149.7, 166.8, 167.1. Anal. Calcd for C17H11N3O3SÆ1.0-
H2O: C, 57.46; H, 3.69; N, 11.82. Found: C, 57.42; H,
4.00; N, 12.19.
5.3.7. N-(4,5-Dihydro-naphto[1,2-d]thiazol-2-yl)acetam-
ide (7b). Scale: 1.0 mmol. Eluent for column chromato-
graphy was CH2Cl2. The product was recrystallized
from methanol, to give white crystals. Yield: 0.22 g,
90%. Mp: 214–216 ꢁC. Lit.9 Mp: 233–234 ꢁC. 1H
NMR (CDCl3): d 2.12 (s, 3H, CH3), 2.93–3.12 (m, 4H,
2CH2), 7.15–7.42 (m, 3H, Ar), 7.67–7.80 (m, 1H, Ar),
10.20 (br s, 1H, NH). 13C NMR (CDCl3): d 21.5, 22.9,
28.8, 122.1, 124.3, 126.8, 127.1, 127.9, 130.6, 134.7,
143.2, 157.5, 168.3. Anal. Calcd for C13H12N2OS: C,
63.91; H, 4.95; N, 11.47. Found: C, 63.62; H, 5.19; N,
11.07.
5.3.3. N-(8H-Indeno[1,2-d]thiazol-2-yl)-4-chloro-benzam-
ide (6a). Scale 0.50 mmol. Eluent for column chroma-
tography was CH2Cl2. The product was recrystallized
from chloroform–ethanol = 75:25, to give white crystals.
Yield: 0.16 g, 98%. Mp: 232–235 ꢁC. 1H NMR (CDCl3):
d 3.87 (s, 2H, CH2), 7.18–7.34 (m, 3H, Ar), 7.38–7.45
(m, 3H, Ar), 7.50–7.53 (m, 2H, Ar), 10.43 (br s, 1H,
NH). 13C NMR (DMSO-d6): d 30.0 (CH2), 118.0,
125.1, 125.2, 126.9, 128.3, 128.7, 130.1, 130.9, 137.0,
137.6, 146.0, 162.3, 164.0, 175.5. Anal. Calcd for
C17H11ClN2OSÆ0.5H2O: C, 60.80; H, 3.60; N, 8.34.
Found: C, 60.74; H, 3.20; N, 8.55.
5.3.8.
N-(4,5-Dihydro-naphto[1,2-d]thiazol-2-yl)-prop-
ionamide (8b). Scale: 1.0 mmol. Eluent for column chro-
matography was petroleum ether–ethylacetate = 80:20.
The product was recrystallized from ethanol, to give
off-white crystals. Yield: 0.23 g, 89%. Mp: 147–148 ꢁC.
1H NMR (CDCl3): d 2.20–2.30 (m, 3H, CH3), 2.72–
2.78 (m, 4H, 2CH2), 2.91 (t, 2H, J = 7.3, 6.6 Hz, CH2),
7.16–7.29 (m, 2H, Ar), 7.35–7.63 (m, 1H, Ar), 7.93 (d,
1H, J = 7.3 Hz, Ar). 13C NMR (CDCl3): d 8.8, 21.3,
28.9, 29.0, 122.1, 124.2, 126.8, 127.1, 127.8, 130.6,
134.6, 143.2, 157.5, 172.0. Anal. Calcd for C14H14N2OS:
C, 65.09; H, 5.46; N, 10.84. Found: C, 65.33; H, 5.26; N,
10.96.
5.3.4. N-(4,5-Dihydro-naphto[1,2-d]thiazol-2-yl)-benzam-
ide (4b). Scale 1.0 mmol. Eluent for column chromato-
graphy was CH2Cl2. The product was recrystallized
from CH2Cl2, to give off-white crystals. Yield: 0.30 g,
5.3.9.
N-(4,5-Dihydro-naphto[1,2-d]thiazol-2-yl)-2,2-
dimethyl-propionamide (9b). Scale: 1.0 mmol. Eluent
for column chromatography was 12.5% ethylacetate in
petroleum ether. The product was recrystallized from
1% n-hexane in methanol, to give off-white crystals.
Yield: 0.25 g, 87%. Mp: 133–135 ꢁC. 1H NMR (CDCl3):
d 1.34 (s, 9H, 3CH3), 2.89–3.08 (m, 4H, 2CH2), 7.13–
7.31 (m, 3H, Ar), 7.70 (d, 1H, J = 7.3 Hz, Ar), 9.91 (br
s, 1H, NH). 13C NMR (CDCl3): d 21.2, 26.9, 28.7,
38.9, 122.2, 124.0, 126.8, 127.0, 127.7, 130.6, 134.5,
143.0, 156.9, 176.3. Anal. Calcd for C16H18N2OS: C,
67.10; H, 6.34; N, 9.78. Found: C, 66.79; H, 5.94; N,
9.85.
1
98%. Mp: 119–123 ꢁC. H NMR (CDCl3): d 2.95–3.13
(m, 4H, CH2), 7.13–7.28 (m, 3H, Ar), 7.35–7.63 (m,
4H, Ar), 7.95–8.00 (m, 2H, Ar). 13C NMR (CDCl3):
21.3, 28.8, 122.2, 124.4, 126.7, 127.6, 128.2, 128.4,
130.4, 131.8, 132.4, 134.5, 157.3, 165.0, 188.1. Anal.
Calcd for C18H14N2OSÆ1.0H2O: C, 66.64; H, 4.97; N,
8.64. Found: C, 66.35; H, 5.15; N, 8.84.
5.3.5. N-(4,5-Dihydro-naphto[1,2-d]thiazol-2-yl)-4-nitro-
benzamide (5b). Scale: 1.0 mmol. Eluent for column
chromatography was CH2Cl2. The product was recrys-
tallized from ethylacetate, to give yellow crystals. Yield:
0.33 g, 94%. Mp: 246–248 ꢁC. 1H NMR (CDCl3): d 1.58
(s, 2H, CH2), 3.04 (s, 2H, CH2), 7.06–7.13 (m, 2H, Ar),
7.42–7.55 (m, 2H, Ar), 7.94 (d, 2H, J = 8.8 Hz, Ar), 8.14
(d, 2H, J = 8.8 Hz), 11.21 (br s, 1H, NH). 13C NMR
(DMSO-d6): d 20.8, 28.2, 122.1, 123.6, 124.6, 127.0,
127.1, 128.2, 129.8, 130.9, 134.9, 137.8, 143.8, 149.7,
5.3.10. N-(4,5-Dihydro-naphto[1,2-d]thiazol-2-yl)-cyclo-
pentanecarboxamide (10b). Scale: 1.0 mmol. Eluent for
column chromatography was tert-butyl methyl ether–
petroleum ether = 1:1. The product was recrystallized
from methanol, to give off-white crystals. Yield: 0.21 g,
1
70%. Mp: 148–150 ꢁC. H NMR (CDCl3): d 1.25–1.42

A. Go¨blyo¨s et al. / Bioorg. Med. Chem. 13 (2005) 2079–2087
2085
(m, 4H, 2CH2), 1.68–1.97 (m, 4H, 2CH2), 2.54–
2.69 (m, 1H, CH), 2.79–3.09 (m, 4H, 2CH2), 7.14–7.32
(m, 3H, Ar), 7.73 (d, 1H, J = 7.3 Hz, Ar), 11.37
(br s, 1H, NH). 13C NMR (CDCl3): d 21.2, 25.6, 28.9,
30.1, 45.0, 122.2, 124.0, 127.1, 127.7, 130.5, 134.6,
143.0, 157.9, 174.6, 182.3. Anal. Calcd for C17H18N2OS:
C, 68.41; H, 6.08; N, 9.39. Found: C, 68.15; H, 5.85; N,
9.51.
132.3, 143.8, 156.2, 157.1, 165.2. Anal. Calcd for
C19H16N2O2SÆ0.2H2O: C, 67.12; H, 4.86; N, 8.24.
Found: C, 67.09; H, 4.91; N, 8.51.
5.3.15. N-(6-Methoxy-4,5-dihydro-naphto[1,2-d]thiazol-
2-yl)-4-nitro-benzamide (5d). Scale: 1.0 mmol. Eluent
for column chromatography was petroleum ether–ethyl-
acetate = 70:30. The product was recrystallized from
CH2Cl2–methanol = 90:10, to give yellow crystals.
Yield: 0.05 g, 13%. Mp: 267–268 ꢁC. 1H NMR (CDCl3):
d 2.94–3.08 (m, 4H, 2CH2), 3.84 (s, 3H, OCH3), 6.67–
6.72 (m, 1H, Ar), 6.97–7.10 (m, 2H, Ar), 7.86–7.92 (m,
2H, Ar), 8.08 (d, 2H, J = 8.8 Hz, Ar), 11.37 (br s, 1H,
NH). 13C NMR (DMSO-d6): d 20.2, 20.6, 55.5, 110.1,
115.0, 122.1, 123.6, 124.5, 127.5, 129.8, 130.0, 131.7,
137.8, 149.6, 149.8, 155.4, 156.3, 163.5. Anal. Calcd
for C19H15N3O4SÆ0.3H2O: C, 59.00; H, 4.06; N, 10.86.
Found: C, 58.86; H, 3.81; N, 11.19.
5.3.11. N-(5,6-Dihydro-4H-benzo[6,7]cyclohepta[1,2-d]-
thiazol-2-yl)-benzamide (4c). Scale: 1.0 mmol. Eluent
for column chromatography was CH2Cl2. The product
was recrystallized from 1,4-dioxane–n-hexane = 80:20,
to give white crystals. Yield: 0.24 g, 74%. Mp: 60–
1
62 ꢁC. H NMR (CDCl3): d 2.16–2.29 (m, 2H, CH2),
2.71–2.77 (m, 2H, CH2), 2.94 (t, 2H, J = 7.3 Hz, CH2),
7.10–7.21 (m, 3H, Ar), 7.27–7.34 (m, 1H, Ar), 7.41–
7.50 (m, 1H, Ar), 7.73–7.81 (m, 3H, Ar). 13C NMR
(CDCl3): d 25.1, 29.9, 33.6, 126.1, 127.2, 127.4, 128.0,
128.9, 129.9, 130.3, 131.9, 132.0, 133.0, 133.6, 140.4,
143.9, 156.8, 165.5, 171.4. Anal. Calcd for C19H16-
N2OSÆ1.0 C4O2: C, 67.45; H, 5.98; N, 6.91. Found:
C, 67.54; H, 5.94; N, 6.81.
5.3.16. N-(6-Methoxy-4,5-dihydro-naphto[1,2-d]thiazol-
2-yl)-4-chloro-benzamide (6d). Scale: 1.0 mmol. Eluent
for column chromatography was 1% methanol in
CH2Cl2. The product was recrystallized from ethanol,
to give light brown crystals. Yield: 0.06 g, 16%. Mp:
216–218 ꢁC. 1H NMR (CDCl3): d 2.93–3.12 (m, 4H,
2CH2), 3.86 (s, 3H, OCH3), 6.71–6.75 (m, 1H, Ar),
7.02 (t, 1H, J = 7.3 Hz, Ar), 7.13–7.26 (m, 3H, Ar),
7.66–7.71 (m, 2H, Ar), 11.26 (br s, 1H, NH). 13C
NMR (CDCl3): d 20.8, 21.0, 55.4, 109.6, 114.9, 122.4,
124.8, 127.0, 128.3, 128.8, 130.7, 131.3, 138.3, 143.8,
156.3, 157.5, 164.9. Anal. Calcd for C19H15ClN2O2S:
C, 61.54; H, 4.08; N, 7.55. Found: C, 61.46; H, 4.19;
N, 7.95.
5.3.12. N-(5,6-Dihydro-4H-benzo[6,7]cyclohepta[1,2-d]-
thiazol-2-yl)-4-nitro-benzamide (5c). Scale: 0.90 mmol.
Eluent for column chromatography was CH2Cl2. The
product was recrystallized from ethylacetate–metha-
nol = 1:1, to give dark yellow crystals. Yield: 0.22 g,
1
67%. Mp: 163–164 ꢁC. H NMR (CDCl3): d 2.18–2.24
(m, 2H, CH2), 2.60–2.66 (m, 2H, CH2), 2.93 (t, 2H,
J = 7.3 Hz, CH2), 7.00–7.05 (m, 4H, Ar), 7.73 (d, 2H,
J = 8.8 Hz, Ar), 7.97 (d, 2H, J = 8.8 Hz, Ar). 13C
NMR (CDCl3): d 24.6, 30.2, 33.0, 122.8, 126.1, 127.3,
127.6, 127.9, 128.3, 128.7, 133.3, 137.7, 140.3, 143.9,
149.2, 156.9, 164.4. Anal. Calcd for C19H15N3O3SÆ
0.8H2O: C, 60.08; H, 4.41, N, 11.06. Found: C, 60.06;
H, 4.79; N, 11.09.
5.3.17. N-(7,8-Dimethoxy-4,5-dihydro-naphto[1,2-d]thi-
azol-2-yl)-benzamide (4e). Scale: 1.0 mmol. Eluent for
column chromatography was 0.5% methanol in CH2Cl2.
The product was recrystallized from ethanol, to give off-
1
white crystals. Yield: 0.18 g, 49%. Mp: 180–182 ꢁC. H
5.3.13. N-(5,6-Dihydro-4H-benzo[6,7]cyclohepta[1,2-d]-
thiazol-2-yl)-4-chloro-benzamide (5d). Scale: 0.80 mmol.
Eluent for column chromatography was CH2Cl2. The
product was recrystallized from CH2Cl2, to give color-
NMR (CDCl3): d 2.99 (s, 4H, 2CH2), 3.88 (d, 6H,
J = 4.38 Hz, 2OCH3), 6.74 (s, 1H, Ar), 7.25 (d, 1H,
J = 4.38 Hz, Ar), 7.40–7.89 (m, 3H, Ar), 7.91 (d, 2H,
J = 6.58 Hz, Ar), 10.28 (br s, 1H, NH). 13C NMR
(CDCl3): d 21.5, 28.4, 55.6, 55.8, 105.9, 111.3, 119.3,
122.4, 123.6, 127.0, 127.2, 128.5, 131.8, 132.4, 143.9,
147.4, 147.7, 156.5, 164.6. Anal. Calcd for
C20H18N2O3S: C, 65.56; H, 4.95; N, 7.64. Found: C,
65.38; H, 4.96; N, 7.96.
1
less crystals. Yield: 0.28 g, 99%. Mp: 180–182 ꢁC. H
NMR (CDCl3): d 2.19–2.29 (m, 2H, CH2), 2.71–2.77
(m, 2H, CH2), 2.95 (t, 2H, J = 7.8 Hz, CH2), 7.05–7.21
(m, 6H, Ar), 7.51–7.70 (m, 2H, Ar), 11.66 (br s, 1H,
NH). 13C NMR (CDCl3): d 24.9, 30.0, 33.4, 126.0,
127.4, 127.5, 128.0, 128.6, 130.6, 133.5, 138.0, 140.3,
144.0, 156.9, 165.1. Anal. Calcd for C19H15ClN2OS: C,
64.31; H, 4.26; N, 7.89. Found: C, 64.10; H, 4.13; N,
8.04.
5.3.18. N-(7,8-Dimethoxy-4,5-dihydro-naphto[1,2-d]thi-
azol-2-yl)-4-nitro-benzamide (5e). Scale: 1.0 mmol.
Eluent for column chromatography was ethylacetate–
petroleum ether = 1:1. The product was recrystallized
from 2% methanol in CH2Cl2, to give dark red crystals.
Yield: 0.08 g, 20%. Mp: 269–273 ꢁC. 1H NMR (CDCl3):
d 2.95 (s, 4H, 2CH2), 3.79 (s, 6H, 2OCH3), 6.94 (s, 1H,
Ar), 7.32 (s, 1H, Ar), 8.28–8.39 (m, 4H, Ar), 12.11 (br s,
1H, NH). 13C NMR 600 MHz (DMSO-d6): d 20.9,
27.7, 55.5, 55.6, 106.2, 112.4, 123.5, 126.5, 127.2,
127.3, 129.7, 129.9, 130.2, 134.3, 147.5, 147.8, 149.0,
149.5, 165.8, 175.6. Anal. Calcd for C20H17N3O5SÆ1.8-
H2O: C, 54.12; H, 4.68; N, 9.47. Found: C, 53.86; H,
4.65; N, 9.85.
5.3.14. N-(6-Methoxy-4,5-dihydro-naphto[1,2-d]thiazol-
2-yl)-benzamide (4d). Scale: 1.0 mmol. Eluent for col-
umn chromatography was 1% methanol in CH2Cl2.
The product was recrystallized from diisopropylether,
to give light yellow crystals. Yield: 0.07 g, 21%. Mp:
177–179 ꢁC. 1H NMR (CDCl3): d 2.93–3.13 (m, 4H,
2CH2), 3.84 (s, 3H, OCH3), 6.75–6.80 (m, 1H, Ar),
7.13–7.63 (m, 5H, Ar), 7.87–9.91 (m, 2H, Ar), 10.57
(br s, 1H, NH). 13C NMR (CDCl3): d 20.9, 55.4,
109.6, 115.2, 122.4, 124.8, 127.1, 128.4, 131.6, 132.0,

2086
A. Go¨blyo¨s et al. / Bioorg. Med. Chem. 13 (2005) 2079–2087
5.3.19. N-(7,8-Dimethoxy-4,5-dihydro-naphto[1,2-d]thi-
azol-2-yl)-4-chloro-benzamide (6e). Scale: 1.0 mmol.
Eluent for column chromatography was 2% methanol
in CH2Cl2. The product was recrystallized from 2% eth-
anol in CH2Cl2, to give light brown crystals. Yield:
0.29 g, 72%. Mp: 190–192 ꢁC. 1H NMR (CDCl3): d
2.99 (s, 4H, 2CH2), 3.93 (d, 6H, J = 4.38 Hz, 2OCH3),
6.78 (s, 1H, Ar), 7.28 (d, 1H, J = 8.04 Hz, Ar), 7.44–
7.50 (m, 2H, Ar), 7.98–8.08 (m, 2H, Ar). 13C NMR
(CDCl3): d 21.4, 28.4, 55.6, 55.7, 105.9, 111.2, 122.2,
122.7, 127.3, 128.6, 129.0, 130.1, 130.9, 138.8, 143.0,
147.8, 157.9, 164.1, 170.5. Anal. Calcd for
C20H17ClN2O3SÆ1.3H2O: C, 56.61; H, 4.66; N, 6.60.
Found: C, 56.60; H, 4.88; N, 6.67.
binding was measured in the presence of 100 lM R-
PIA. Binding reactions were terminated by dilution with
ice-cold buffer. Samples were then filtered through
Whatman GF/B glass-fiber filters using a Brandell cell
harvester. Filters were washed three times with 2–3 mL
of the same buffer. Bound radioactivity was measured
in a Minaxi c Auto-gammaꢂ 5000 series gamma
counter.
Compounds 9b and 10b were tested in functional as-
says for their ability to influence the levels of cAMP
in the test system. CHO cells expressing the human
adenosine A1 receptor were grown overnight as a
monolayer in 24-well tissue culture plates (400 lL/well;
2 · 105 cells/well). cAMP generation was performed
in Dulbeccoꢁs Modified Eagles Medium (DMEM)/
5.4. Radioligand binding assays
N-2-hydroxyethylpiperazin-N0-2-ethansulfonic
acid
For displacement experiments, membranes of CHO
cells expressing recombinant human adenosine A1
receptors (20 lg of protein for [3H]CCPA and 10 lg
of protein for [3H]DPCPX), were incubated at 25
ꢁC for 60 min with ꢁ1.0 nM of [3H]CCPA, or
ꢁ1.6 nM of [3H]DPCPX, and a fixed concentration or
increasing concentrations of the compounds, in a final
volume of 0.4 mL Tris–HCl buffer. Nonspecific binding
was measured in the presence of 10 lM DPCPX
([3H]CCPA) or 10 lM CPA ([3H]DPCPX). Binding
reactions were terminated by dilution with ice-cold
50 mM Tris–HCl buffer. Samples were then filtered
through Whatman GF/B glass-fiber filters using a
Brandell cell harvester or a Millipore manifold. Filters
were washed three times with 2–3 mL of the same buffer.
Bound radioactivity was measured in a liquid scintilla-
tion counter (LKB Wallac) after the addition
of 3.5 mL of scintillation liquid (Emulsifier-Safe,
Packard).
(HEPES) buffer (0.60 g HEPES/50 mL DMEM
pH 7.4). Each well was washed twice with HEPES/
DMEM buffer (250 lL), and the following added,
adenosine deaminase (0.8 IU/mL), rolipram (50 lM),
cilostamide (50 lM). This was then incubated for
30 min at 37 ꢁC followed by the introduction of the
compound of interest. After a further 10 min of incu-
bation, forskolin was added (10 lM). After a subse-
quent 15 min, incubation was stopped by aspirating
the assay medium and by adding 200 lL of ice-cold
0.1 M HCl. The amount of cAMP was determined by
competition with [3H]cAMP for protein kinase A
(PKA). Briefly, the sample, approximately 1.8 nM
[3H]cAMP, and 100 lL PKA solution were incubated
on ice for 2.5 h. The incubations were stopped by rapid
dilution with 2 mL of ice-cold Tris–HCl buffer (pH 7.4)
and bound radioactive material was then recovered by
filtration through Whatman GF/C filters. Filters were
additionally rinsed with 2 · 2 mL Tris–HCl buffer and
then the radioactivity counted in Packard Emulsifier
Safe scintillation fluid (3.5 mL). All data reflect three
independent experiments performed in duplicate.
In kinetic studies, the association of the radiolabeled
agonist [3H]CCPA (1 nM) was started by addition of
the membranes (30 lg) to the radioligand, in the pres-
ence or absence of either 2b or 2c. To study dissociation
of [3H]CCPA, membranes were preincubated with
[3H]CCPA (1 nM) at 25 ꢁC for 60 min. Dissociation of
[3H]CCPA was then initiated by the addition of
100 lM 8-cyclopentyltheophylline (CPT) in the presence
or absence of either 2b or 2c. Samples were handled as
mentioned before.
5.5. Statistical analysis
Binding parameters were estimated using a nonlinear
regression curve-fitting program (GraphPad Prism 4,
GraphPAD Software Inc. San Diego, CA, USA). KD
values of the radioligands were 1.6, 1.0, 1.0, and
5.0 nM for [3H]DPCPX, [3H]CCPA, [3H]ZM 241385,
and [125I]AB-MECA, respectively. Data are expressed
as mean SEM for the number of experiments
indicated.
The conditions for adenosine A2A receptor binding were
slightly different. In competition experiments, mem-
branes of HEK293 cells expressing recombinant human
A2A receptors (22.5 lg of protein) were incubated with
ꢁ1.7 nM [3H]ZM241385 for 120 min at 25 ꢁC. Nonspe-
cific binding was measured in the presence of 100 lM
CPA.
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