Synthesis of 2-amino-3-heteroaroylthiophenes and evaluation of their activity as potential allosteric enhancers at the human A1 receptor

Article abstract of DOI:10.1016/j.ejmech.2004.06.009

2-Amino-3-benzoylthiophenes are allosteric enhancers of agonist binding to the adenosine A₁ receptor. New compounds bearing an heteroaroyl instead of the benzoyl moiety at the 3-position of the thiophene were synthesized. The phenyl ring was re

Full text of DOI:10.1016/j.ejmech.2004.06.009

European Journal of Medicinal Chemistry 39 (2004) 855–865
www.elsevier.com/locate/ejmech
Original article
Synthesis of 2-amino-3-heteroaroylthiophenes and evaluation of their
activity as potential allosteric enhancers at the human A1 receptor
Pier Giovanni Baraldi a,*, Maria Giovanna Pavani a, John C. Shryock b, Allan R. Moorman c,
Valeria Iannotta d, Pier Andrea Borea d, Romeo Romagnoli a
a Dipartimento di Scienze Farmaceutiche, Università di Ferrara, 44100 Ferrara, Italy
b Department of Medicine, University of Florida, Gainesville, Florida, USA
c King Pharmaceuticals Research and Development Inc., Cary, NC 27513, USA
d Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Università di Ferrara, 44100 Ferrara, Italy
Received 26 March 2004; accepted 24 June 2004
Available online 12 August 2004
Abstract
2-Amino-3-benzoylthiophenes are allosteric enhancers of agonist binding to the adenosine A1 receptor. New compounds bearing an
heteroaroyl instead of the benzoyl moiety at the 3-position of the thiophene were synthesized. The phenyl ring was replaced with heterocycles
that possess heteroatoms able to form hydrogen bonds (2-furanyl, 2-benzofuranyl, 2-pyridinyl in compounds 2–13) or with a thienyl moiety as
isoster of the phenyl ring (2-thienyl, 3-thienyl and 5-halo-2-thienyl in compounds 14–29). The effect of several alkyl substituents at positions
4 and 5 of the thiophene ring to increase enhancer activity was determined. The ability of the new molecules to reduce the cAMP content in
CHO cells expressing the human adenosine A1 receptor was evaluated. Compounds 2–13 with hydrogen bond-forming heteroatoms did not
show significant activity as allosteric enhancers. On the other hand, compounds 15–16 and 19–20 with an unsubstituted thienyl moiety as
replacement for the phenyl ring were nearly as efficacious as PD 81,723, the prototypicalA1 allosteric enhancer.Alkyl substituents at positions
4 and 5 of the thiophene ring were tolerated while a substituted piperidine ring was not tolerated. We conclude that hydrogen bonds could not
be formed in the domain of the receptor that accommodates the phenyl ring of 2-amino-3-benzoylthiophene derivatives, indicating that this
domain is hydrophobic.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Allosteric enhancer; Adenosine A1 receptor; 2-Amino-3-heteroaroylthiophenes; Cyclic AMP
1. Introduction
would be limited to times and locations at which significant
release of adenosine occurs [2].
Adenosine is cardioprotective during periods of ischemia
and a similar effect has been demonstrated in brain. These
and other important physiological effects of adenosine on the
cardiovascular and central nervous systems are mediated
through the interaction with the A1 receptor subtype [1]. The
therapeutic potential of A1 adenosine receptor agonists is
undermined by a poor side effect profile due to the wides-
pread distribution of adenosine receptors throughout the
body. One way to avoid these side effects is offered by
positive A1 allosteric adenosine modulators whose action
A series of 2-amino-3-benzoylthiophene derivatives has
been shown to enhance the apparent binding of the radioli-
gand agonist [3H]CHA to A1 adenosine receptors and to
decrease the rate of dissociation of [3H]CHA from the recep-
tor. It has been suggested that these compounds act on an
allosteric site, distinct from the orthosteric adenosine binding
site, to stabilize the high affinity state of the receptor for
agonist binding [3].
Among these compounds, PD 81,723 (1) has been pro-
posed as a lead compound and chosen for pharmacological
investigations. It increased agonist potencies 4- to 10-fold
both in membrane and whole-cell function assays. As a
drawback, this compound was also able to inhibit the binding
* Corresponding author. Tel.: +39-0-532-291293;
fax: +39-0-532-291296.
E-mail address: baraldi@unife.it (P.G. Baraldi).
0223-5234/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.06.009

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P.G. Baraldi et al. / European Journal of Medicinal Chemistry 39 (2004) 855–865
of [3H]DPCPX, implying a collateral antagonistic activity
[4–8].
2. Chemistry
Compounds 2–29 were obtained by a method analogous
to that described by Gewald et al. [14–16] (Scheme 1). The
appropriate b-ketonitriles (30–36) were reacted with the ap-
propriate commercially available ketones (butanone, cyclo-
pentanone, cyclohexanone and 4-benzylpiperidone) in the
presence of a base (morpholine) to give the Knoevenagel
intermediate. This intermediate, in turn, was reacted with
elemental sulfur to provide the desired thiophene ring forma-
tion.
Ketonitriles 30–35 were easily synthesized following pro-
cedures reported in the literature [17]. Compound 36 was
obtained from the corresponding bromo derivative 37 [18]
through a reaction of change bromine-cyanide.
Analysis of the structure–activity effects of the first series
of 2-amino-3-benzoylthiophene derivatives, reported by
Bruns in 1990, indicated that the 2-amino and 3-keto carbo-
nyl groups were both essential for activity. Further, it was
suggested that alkyl substitutions at position 4 of the
thiophene ring as well as various substitutions on the phenyl
ring increased activity. Interestingly, the evaluation of the
substitution of the benzoyl group at position 3 of the
thiophene with carbamoyl, acetyl and cyclohexyl carbonyl
moieties suggested that the phenyl ring could not be replaced
[9].
In the series of 2-amino-3-benzoylthiophenes that fol-
lowed the original paper of Bruns et al., a number of com-
pounds proved to be superior to PD 81,723 (1) in enhancing
the agonist binding to the A1 receptor and their collateral
antagonistic activity was low [10–13]. These studies gave
further information about the structural requirements for
enhancement. In particular it was found that a fused ring
between positions 4 and 5 of the thiophene led to good
activity. Optimally, this fused ring was composed of three or
four methylene units or formed N-benzyltetrahydropyridine
ring.
3. Pharmacology
The new compounds were evaluated in a functional assay
to test their potential A1 enhancer activity and the putative
enhancers identified by this test were then evaluated at the
receptor level in equilibrium binding assays.
Allosteric enhancers of the action of adenosine are be-
lieved to stabilize a conformation of the A1 adenosine recep-
tor that has a high affinity for agonists. This effect is mani-
fested as a slowing of the rate of dissociation of an agonist
from the receptor [3]. In addition, an allosteric enhancer
appears to stabilize an active conformation of the receptor
even in the absence of an agonist. Thus, in cells with A1
adenosine receptors that are active spontaneously in the ab-
sence of an agonist (such as the CHO cells used in our study),
an allosteric enhancer may increase the number of receptors
that are active at any given time, and thus cause a change in
cell function. Compounds with the potential to be allosteric
enhancers of activation of human A1 adenosine receptors are
expected to decrease the content of cAMP in CHO cells
expressing humanA1 adenosine receptor. Not all compounds
that decrease cAMP content of CHO cells are allosteric
enhancers, however. Compounds that are rapidly toxic to
cells, compounds that directly inhibit adenylyl cyclase, and
agonists of A1 adenosine receptors may cause reductions of
cAMP content similar to those caused by allosteric enhancers
in our assay. Therefore, the results of the experiments re-
ported here can identify compounds as putative allosteric
enhancers, but not definitively demonstrate that allosteric
enhancement is the mechanism of action.
The cyclic AMP assay was carried out to determine the
ability of compounds 2–29 to activate the human A1 adenos-
ine receptor [19–21]. The experiments were performed using
CHO cells expressing recombinant human A1 receptors. Al-
losteric enhancement was measured as the action of a test
compound at different concentrations (0.01, 0.1, 1 and
10 µM) to reduce the cAMP content in the presence of a
sub-optimal (0.01 nM) concentration of an agonist (CPA).
The reference compound for comparison was PD 81, 723 (1)
(Table 1).
The aim of the present research was to more thoroughly
investigate the nature of the receptor domain that accommo-
dates the phenyl ring of 2-amino-3-benzoylthiophene deriva-
tives. Realizing that the number of compounds considered by
Bruns was not sufficient to state that the phenyl ring was the
only moiety accepted by the receptor within this domain, we
prepared a series of 2-amino-3-heteroaroylthiophenes where
we maintained the best substitutions for enhancement at
positions 4 and 5 of the thiophene and we replaced the phenyl
ring with different heterocycles.
We first replaced the phenyl ring with 2-furanyl,
2-benzofuranyl and 2-pyridyl moieties to test the possibility
of hydrogen bond formation within the domain. In addition,
especially with pyridine, we could also increase the hydro-
philic properties of the molecules, since low water-solubility
is one of the major limitations of the 2-amino-3-
benzoylthiophene derivatives [3].
In case the domain to which the phenyl ring binds was
merely hydrophobic, as Bruns suggested, we thought that
replacement of the phenyl ring by an isosteric 2- or 3-thienyl
moiety might lead to improved activity. To further increase
the lipophilicity of these moieties and further probe the size
of this hydrophobic domain, we evaluated the introduction of
an halogen (chlorine and bromine) at the 5-position of the
thiophene-2-carbonyl moiety.
Our research would confirm or refute the structure–activ-
ity relationships initially proposed by Bruns for enhancement
of adenosine A1 receptors and further complete our under-
standing of these relationships. (Fig. 1)

P.G. Baraldi et al. / European Journal of Medicinal Chemistry 39 (2004) 855–865
857
Fig. 1. PD81,723 and structures of compounds 2–29.
4. Results and discussion
important, because 2-thienyl derivatives 18–21 possessed the
same activity as the 3-thienyl counterparts 14–17. Thus,
results of functional assay of both the hydrophilic com-
pounds 10–13 and hydrophobic derivatives 14–21, seem to
confirm that the aroyl receptor domain is principally hydro-
phobic in nature.
Replacement of the phenyl ring in position 3 of the
2-aminothiophene with heterocycles such as furan (2–5),
benzofuran (6–9)) and pyridine (10–13), that potentially
could form an hydrogen bond with the allosteric site of theA1
adenosine receptor, led to a significant loss of activity. The
most hydrophilic compounds, the pyridine derivatives
10–13, were inactive as allosteric enhancers at any concen-
tration. Among the furan (2–5) and benzofuran (6–9) com-
pounds, some showed slight activity (3–5, 7, 8) with com-
pound 3 being the most efficacious at concentrations of 0.1,
1 and 10 µM. Furan compounds were more active than
benzofuran derivatives bearing the same substitution pattern
at the 4 and 5 positions of the 2-aminothiophene ring. This
finding suggests that limited steric bulk is preferred at that
position.
Replacement of the phenyl ring with a thiophene (14–21),
which is electronically similar, was well tolerated. When
there was a methylene chain of three or four carbon atoms
between positions 4 and 5 of the 2-aminothiophene (com-
pounds 15,16 and 19,20), enhancer activity was only slightly
reduced relative to that of PD 81,723 at 10 µM and was better
than PD 81,723 at lower concentrations. The position at
which thiophene is connected to carbonyl does not seem
To investigate the size of the hydrophobic domain in the
receptor to which the phenyl ring at position 3 of the
2-aminothiophene binds, the phenyl group was replaced with
5-chloro- and 5-bromo-2-thienyl moieties (22–29). Com-
pounds 22–29 all proved to be inactive as enhancers, with no
significant difference between chloro and bromo derivatives.
This suggests that an halogen is not tolerated in that position.
We also evaluated the effects of different substitutions at
positions 4 and 5 of 2-aminothiophene. With the exception of
compounds 23–24 and 27–28, enhancement was better when
there was a chain of three or four carbons linking the two
positions. Compounds bearing a methyl group at positions
4 and 5 (2, 6, 10, 14, 18, 22, 26) were weak enhancers and
only at some concentrations. Derivatives 5, 9, 13, 17, 21, 25
and 29, where there was an N-benzyl-tetrahydropyridine
ring, were the worst enhancers. They mainly showed a re-
verse value of the cAMP cyclic level (positive change of
cAMP content in Table 1), appearing they stabilized the
receptor in a conformation that reduced receptor activation.

858
P.G. Baraldi et al. / European Journal of Medicinal Chemistry 39 (2004) 855–865
Scheme 1
.
Reagents (a) butanone (compounds with R1=R2=CH3), cyclopentanone [compounds with R1,R2=-(CH2)3-], cyclohexanone [compounds with
R1,R2=-CH2)4-], S8, morpholine, EtOH, 70 °C for 1 h then 18 h at rt; (b) KCN, EtOH/water, 2 h, rt; (c) N-benzylpiperidone, s8, morpholine, EtOH, 70 °C for
1 h then 18 h at rt.
Because the known allosteric enhancers are also A1-
adenosine receptor antagonists at some (usually high)
concentration, the fact that compounds 15–16 and 19–20
did not show a greater efficacy than did 10 µM PD 81,
723 could be explained by a possible antagonist property of
these compounds. Saturation and inhibition assays were car-
ried out to evaluate the presence of a competitive antagonist
effect.
Compounds 16 and 20 were selected for saturation bind-
ing experiments that were performed using membranes pre-
pared from CHO:hA1, rat cortex, and human brain (Table 2).
In the current study PD 81, 723 did not affect the KD value
of the agonist [3H]CCPA to recombinant human A1 adenos-
ine receptors in CHO cells membranes and to A1 receptors in
human brain and rat cortex membranes but it increased the
BMAX value. These findings are in accordance with previous
reports [22,23].
The KD value of [3H]CCPA was slightly increased by the
tested compounds. This data indicated that the selected com-
pounds 16 and 20 possessed an adenosine A1 antagonist
activity that partly reversed their allosteric property.
Compounds 15, 16 and 20 were tested at a concentration
of 10 µM for their ability to displace the binding of [3H]D-
PCPX, [3H]ZM 241385, and [3H]MRE 3008F20 to the
ligand binding site of CHO:hA1, CHO:hA2A, and CHO:hA3
adenosine receptors, respectively (Table 3).

P.G. Baraldi et al. / European Journal of Medicinal Chemistry 39 (2004) 855–865
859
Table 1
Percentage change in CHO cell cAMP content in presence of compounds 2–29
Compound
Change in cAMP content from control (mean SEM) concentration of test compounds
0.01 µM
0.1 µM
1 µM
–13
10 µM
PD 81, 723 (1)
2
3
4
5
6
7
8
–1
–4
3
2
5
–7
11
–14
2
2
5
1
–50
5
1
–7
5
10
4
5
3
4
–0.8
–3
3
–23
–10
2
7
–10
–27
6
2
3
6
6
5
3
0.6
–7
–13
–3
14
–8
0.8
–8
7
4
–15
16
3
–8 11
4
7
6
–5
–13
4
3
3
3
5
–4
–5
11
14
2
6
4
4
4
5
4
4
3
–4
–10
4
9
2
5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
0.7
–3
–2
2
3
4
4
4
7
–1
–11
6
7
3
5
8
4
5
–0.1
–19
–15
–16
14
5
31
3
3
–16
–12
–19
–8
15
–3
–11
–1
–1
5
4
4
5
–20
–5
–15
–10
22
–4
–11
3
4
3
5
5
3
4
–38
–43
47
3
7
6
4
3
5
7
4
9
5
4
6
6
26
46 10
–6
–33
–40
75
3
2
3
5
–16
23
3
5
6
4
4
4
5
6
4
–7
–15
2
5
3
9
–10
11
6
5
4
4
2
5
6
5
6
–14
8
5
–2
–10
13
10
15
35
16
0.1
–21
1
4
6
20
–13
13
14
6
4
8
8
9
6
4
0.3
–5
4
7
6
6
7
12
11
4
7
28
5
92 15
The prototype enhancer PD 81, 723 did not inhibit the
binding of a radiolabelled antagonist toA1 andA2A receptors,
but it reduced by 21% the binding of [3H]MRE 3008F20 to
the A3 receptor [21].
None of the selected compounds inhibited binding at the
hA2AAR, but they inhibited binding to the hA1AR to some
extent (18–35%) and to the hA3AR to similar degree (15–
24%).
Table 2
Saturation parameters of [3H]CCPA in the absence (control) and presence of tested compounds (10 µM) on different systems
Compound
KD control
2.9 0.6
0.7 0.6
1.7 0.2
1.9 0.1
0.5 0.1
1.3 0.1
1.4 0.1
0.6 0.1
1.4 0.1
KD + compound
1.9 0.4
0.7 0.1
1.4 0.1
2.1 0.2
0.8 0.1
2.0 0.2
1.7 0.2
0.9 0.1
2.5 0.2
BMAX control
111 39
BMAX + compound
179 50
PD 81, 723
h-CHO-A1
521 52
604 90
RAT CORTEX
HUMAN BRAIN
h-CHO-A1
736 58
957 65
16
20
1970 69
642 73
1872 77
685 59
RAT CORTEX
HUMAN BRAIN
h-CHO-A1
950 107
2436 23
679 29
1007
4
2099 87
623 30
RAT CORTEX
HUMAN BRAIN
1087 16
1007 105
Table 3
Inhibition activity of compounds 15, 16 and 20
Compound (10 µM)
% A1 antagonism
% A2A antagonism
0.0 0.0
% A3 antagonism
PD 81, 723
0.0 0.0
0.0 0.0
15
16
20
18.0 3.2
23.5 4.6
35.0 5.5
0.0 0.0
15.0 3.0
24.0 5.1
23.5 4.6
0.0 0.0
0.0 0.0
Inhibition was expressed as percent displacement value ( STD, n = 3) of 1 nM of [3H]DPCPX, of 2 nM of [3H]ZM 241385 and of 2 nM [3H]MRE 3008F20 by
10 µM of tested compound.

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P.G. Baraldi et al. / European Journal of Medicinal Chemistry 39 (2004) 855–865
5. Conclusion
gel column using ethyl acetate–petroleum ether 2:8 as eluant.
Final products were recrystallized from petroleum ether.
To conclude, among the new compounds reported in the
present study, the functional cAMP assay indicated deriva-
tives 15–16 and 19–20 as potential allosteric enhancers at the
human A1 receptor. The replacement of the phenyl at the
3 position of thiophene ring with a 2- or 3-thienyl moiety
seemed to maintain the enhancing activity of the reference
compound PD 81, 723. However, the substitution added a
collateral antagonist A1 activity that was detected both in
saturation and inhibition experiments. This activity possibly
reduced the enhancing potential of the compounds.
6.1.3.
methanone (2)
(2-Amino-4,5-dimethyl-thiophen-3-yl)-furan-2-yl-
2-(2-Furanoyl)acetonitrile 30, butanone, morpholine and
sulfur were reacted according to the general procedure to
afford
methanone 2.Yield: 41%; yellow solid; mp 95–97 °C (petro-
leum ether); IR (KBr) cm–1: 3265, 1573, 1426, 1293, 1021,
750; 1H-NMR (CDCl3) d: 1.24 (s, 3H), 2.05 (s, 3H), 6.52 (bs,
2H), 7.00 (m, 1H), 7.31 (d, J = 3.4 Hz, 1H), 7.54 (d,
J = 3.4 Hz, 1H); C11H11NO2S.
(2-amino-4,5-dimethyl-thiophen-3-yl)-furan-2-yl-
6. Experimental protocols
6.1.4. (2-Amino-5,6-dihydro-4H-cyclopenta[b]thiophen-3-
yl)-furan-2-yl-methanone (3)
6.1. Chemistry
2-(2-Furanoyl)acetonitrile 30, cyclopentanone, morpho-
line and sulfur were reacted according to the general proce-
dure to afford 2-amino-5,6-dihydro-4H-cyclopenta[b]
thiophen-3-yl)-furan-2-yl-methanone 3. Yield: 59%; yellow
solid; m.p. 149–151 °C (petroleum ether); IR (KBr) cm–1:
Reactions were monitored by thin-layer chromatography
on silica gel (precoated F254 Merck plates); the spots were
examined with UV light and visualized with aqueous
KMnO4. Flash chromatography was performed using Merck
1
3115, 1560, 1476, 1288, 1021, 746; H-NMR (CDCl3) d:
2.30 (m, 2H), 2.73 (m, 4H), 6.53 (m, 1H), 6.81 (bs, 2H), 7.05
1
silica gel (230–240 mesh). H-NMR spectra were recorded
on a Bruker AC 200 MHz spectrometer using TMS as inter-
nal standard. IR spectra were recorded on a Perkin-Elmer
FT-IR Paragon 500 spectrometer. Melting points were deter-
mined on a Buchi-Tottoli apparatus and are uncorrected.
Elemental analyses were performed by the microanalytical
laboratory of Dipartimento di Chimica, University of Fer-
rara.
(d, J = 3.4 Hz, 1H), 7.55 (d, J = 3.4 Hz, 1H); C12H11NO2S.
6.1.5. (2-Amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-yl)-
furan-2-yl-methanone (4)
2-(2-Furanoyl)acetonitrile 30, cyclohexanone, morpho-
line and sulfur were reacted according to the general proce-
dure to afford (2-amino-4,5,6,7-tetrahydro-benzo[b]
thiophen-3-yl)-furan-2-yl-methanone 4. Yield: 40%; yellow
solid; m.p. 122 °C (petroleum ether); IR (KBr) cm–1: 3320,
1577, 1433, 1260, 1080, 726; 1H-NMR (CDCl3) d: 1.63 (m,
2H), 1.82 (m, 2H), 2.28 (m, J = 5.8 Hz, 2H), 2.57 (t,
J = 5.8 Hz, 2H), 6.20 (bs, 2H), 6.53 (m, 1H), 6.99 (d,
J = 3.4 Hz, 1H), 7.55 (d, J = 3.4 Hz, 1H); C13H13NO2S.
6.1.1. Synthesis of 3-(5-bromo-thiophen-2-yl)-3-oxo-
propionitrile (36)
A solution of potassium cyanide (15 mmol, 977 mg, 3 eq.)
in water (5 ml) was added in one portion at 0 °C to a solution of
2-bromo-1-(5-bromo-thiophen-2-yl)-ethanone 37 (5 mmol,
1.42 g.) in 95% EtOH (15 ml). The mixture was stirred at
room temperature overnight and then poured onto a mixture
of crushed ice and water and acidified with glacial acetic acid
(pH 5–6) to precipitate the nitrile. The pale brown solid was
filtered, washed with water and dried, affording 3-(5-bromo-
thiophen-2-yl)-3-oxo-propionitrile 36 (yield: 66%) as pale
6.1.6. (2-Amino-6-benzyl-4,5,6,7-tetrahydro-thieno[2,3-c]
pyridin-3-yl)-furan-2-yl-methanone (5)
2-(2-Furanoyl)acetonitrile 30, 1-benzyl-4-piperidone,
morpholine and sulfur were reacted according to the general
procedure to afford (2-amino-6-benzyl-4,5,6,7-tetrahydro-
thieno[2,3-c]pyridin-3-yl)-furan-2-yl-methanone 5. Yield:
62%; yellow solid; m.p. 115–117 °C (petroleum ether); IR
1
brown solid; mp 159–160 °C (petroleum ether); H-NMR
(CDCl3) d: 4.08 (s, 2H), 7.59 (d, J = 2.8 Hz, 1H), 8.16 (d,
J = 2.8 Hz, 1H); C7H4BrNOS.
1
(KBr) cm–1: 3337, 1579, 1485, 1433, 1287, 734; H-NMR
(CDCl3) d: 2.47 (t, J = 5.6 Hz, 2H), 2.61 (t, J = 5.6 Hz, 2H),
3.47 (s, 2H), 3.69 (s, 2H), 6.36 (bs, 2H), 6.52 (m, 1H), 7.01
(d, J = 3.4 Hz, 1H), 7.31 (m, 5H), 7.55 (d, J = 3.4 Hz, 1H);
C19H18N2O2S.
6.1.2. General procedure for the synthesis
of 2-amino-3-heteroaroylthiophenes 2–29
A mixture of the appropriate b-ketonitrile (5 mmol), the
appropriate ketone (5 mmol), morpholine (0.44 ml, 5 mmol),
and sulfur (164 mg, 5 mmol) was heated at 70 °C for 1 h, and
then stirred at room temperature for 20 h. The solvent was
evaporated under reduced pressure and the residue was di-
luted with ethyl acetate (30 ml), washed with water (10 ml),
brine (10 ml), dried over Na2SO4 and concentrated in vacuo.
The crude residue was purified by chromatography on a silica
6.1.7. (2-Amino-4,5-dimethyl-thiophen-3-yl)-benzofuran-2-
yl-methanone (6)
2-(Benzofuran-2-carbonyl)acetonitrile 31, butanone,
morpholine and sulfur were reacted according to the general
procedure to afford 2-amino-4,5-dimethyl-thiophen-3-yl)-
benzofuran-2-yl-methanone 6. Yield: 44%; yellow solid;

P.G. Baraldi et al. / European Journal of Medicinal Chemistry 39 (2004) 855–865
861
m.p. 113–115 °C (petroleum ether); IR (KBr) cm–1: 3305,
1574, 1434, 1257, 750; H-NMR (CDCl3) d: 1.87 (s, 3H),
6.1.12. (2-Amino-5,6-dihydro-4H-cyclopenta[b]thiophen-
3-yl)-pyridin-2-yl-methanone (11)
1
2-(Pyridin-2-carbonyl)acetonitrile 32, cyclopentanone,
morpholine and sulfur were reacted according to the general
procedure to afford (2-amino-5,6-dihydro-4H-cyclo-
penta[b]thiophen-3-yl)-pyridin-2-yl-methanone 11. Yield:
52%; yellow solid; m.p. 145–146 °C (petroleum ether); IR
(KBr) cm–1: 3338, 2853, 1560, 1146, 673; 1H-NMR (CDCl3)
d: 2.14 (m, 4H), 2.64 (m, 2H), 7.09 (bs, 2H), 7.36 (m,1H),
7.58 (d, J = 7.6 Hz, 1H), 7.80 (m, 1H), 8.62 (d, J = 4.6 Hz,
1H); C13H12N2OS.
2.19 (s, 3H), 6.31 (bs, 2H), 7.32 (m, 2H), 7.38 (t, J = 7.8 Hz,
1H), 7.57 (d, J = 7.8 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H); anal.
calcd for C15H13NO2S: C, 66.40; H, 4.83; N, 5.16; found: C,
66.43; H, 4.85; N, 5.14.
6.1.8. (2-Amino-5,6-dihydro-4H-cyclopenta[b]thiophen-3-
yl)-benzofuran-2-yl-methanone (7)
2-(Benzofuran-2-carbonyl)acetonitrile 31, cyclopen-
tanone, morpholine and sulfur were reacted according to the
general procedure to afford (2-amino-5,6-dihydro-4H-
cyclopenta[b]thiophen-3-yl)-benzofuran-2-yl-methanone 7.
Yield: 67%; yellow solid; m.p. 128–129 °C (petroleum
ether); IR (KBr) cm–1: 3298, 1570, 1422, 1288, 1034, 751;
1H-NMR (CDCl3) d: 2.33 (m, 2H), 2.74 (m, 4H), 7.03 (bs,
2H), 7.31 (m, 2H), 7.42 (t, J = 7.8 Hz, 1H), 7.57 (d,
J = 7.8 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H); C16H13NO2S.
6.1.13. (2-Amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-
yl)-pyridin-2-yl-methanone (12)
2-(Pyridin-2-carbonyl)acetonitrile 32, cyclohexanone,
morpholine and sulfur were reacted according to the general
procedure to afford (2-amino-4,5,6,7-tetrahydro-benzo
[b]thiophen-3-yl)-pyridin-2-yl-methanone 12. Yield: 58%;
yellow solid; m.p. 191-193 °C (petroleum ether); IR (KBr)
cm–1: 3249, 2949, 1573, 1450, 1286, 676; 1H-NMR (CDCl3)
d: 1.46 (m, 2H), 1.71 (m, 4H), 2.50 (t, J = 6.2 Hz, 2H), 6.99
(bs, 2H), 7.37 (m, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.81 (m, 1H),
8.62 (d, J = 4.6 Hz, 1H); C14H14N2OS.
6.1.9.
(2-Amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-
yl)-benzofuran-2-yl-methanone (8)
2-(Benzofuran-2-carbonyl)acetonitrile 31, cyclohex-
anone, morpholine and sulfur were reacted according to the
general procedure to afford (2-amino-4,5,6,7-tetrahydro-
6.1.14. (2-Amino-6-benzyl-4,5,6,7-tetrahydro-thieno[2,3-
c]pyridin-3-yl)-pyridin-2-yl-methanone (13)
benzo[b]thiophen-3-yl)-benzofuran-2-yl-methanone
8.
Yield: 64%; yellow solid; m.p. 88–89 °C (petroleum ether);
IR (KBr) cm–1: 3430, 1575, 1430, 752; 1H-NMR (CDCl3) d:
1.56 (m, 2H), 1.81 (m, 2H), 2.29 (t, J = 5.9 Hz, 2H), 2.59 (t,
J = 5.9 Hz, 2H), 6.55 (bs, 2H), 7.32 (m, 2H), 7.42 (t,
J = 7.8 Hz, 1H), 7.58 (d, J = 7.8 Hz, 1H), 7.69 (d, J = 7.8 Hz,
1H); C17H15NO2S.
2-(Pyridin-2-carbonyl)acetonitrile 32, 1-benzyl-4-
piperidone, morpholine and sulfur were reacted accord-
ing to the general procedure to afford (2-amino-6-benzyl-
4,5,6,7-tetrahydro-thieno[2,3-c]pyridin-3-yl)-pyridin-2-
yl-methanone 13. Yield: 60%; yellow solid; m.p. 69–
71 °C (petroleum ether); IR (KBr) cm–1: 3368, 1578,
1442, 1130, 671; 1H-NMR (CDCl3) d: 1.89 (t, J = 5.8 Hz,
2H), 2.51 (t, J = 5.8 Hz, 2H), 3.41 (s, 2H), 3.62 (s, 2H), 7.07
(bs, 2H), 7.32 (m, 6H), 7.54 (d, J = 7.8 Hz, 1H), 7.80 (m, 1H),
8.63 (d, J = 4.8 Hz, 1H); C20H19N3OS.
6.1.10. (2-Amino-6-benzyl-4,5,6,7-tetrahydro-thieno[2,3-c]
pyridin-3-yl)-benzofuran-2-yl-methanone (9)
2-(Benzofuran-2-carbonyl)acetonitrile 31, 1-benzyl-4-
piperidone, morpholine and sulfur were reacted according to
the general procedure to afford (2-amino-6-benzyl-4,5,6,7-
tetrahydro-thieno[2,3-c]pyridin-3-yl)-benzofuran-2-yl-me-
thanone 9. Yield: 59%; yellow solid; m.p. 85–87 °C (petro-
leum ether); IR (KBr) cm–1: 3435, 1580, 1551, 1441, 749;
1H-NMR (CDCl3) d: 2.45 (t, J = 5.2 Hz, 2H), 2.59 (t,
J = 5.2 Hz, 2H), 3.48 (s, 2H), 3.69 (s, 2H), 6.64 (bs, 2H), 7.33
(m, 8H), 7.58 (d, J = 7.8 Hz, 1H), 7.66 (d, J = 7.8 Hz, 1H);
C23H20N2O2S.
6.1.15. (2-Amino-4,5-dimethyl-thiophen-3-yl)-thiophen-3-
yl-methanone (14)
2-(Thiophene-3-carbonyl)acetonitrile 33, butanone, mor-
pholine and sulfur were reacted according to the general
procedure to afford (2-amino-4,5-dimethyl-thiophen-3-yl)-
thiophen-3-yl-methanone 14. Yield: 38%; yellow solid; m.p.
122–123 °C (petroleum ether); IR (KBr) cm–1: 3337, 1576,
1427, 1265, 719; 1H-NMR (CDCl3) d: 1.71 (s, 3H), 2.15 (s,
3H), 6.17 (bs, 2H), 7.30 (dd, J = 5.0 Hz, J′ = 2.7, 1H), 7.36
(dd, J = 5.0 Hz, J′ = 0.9 Hz, 1H), 7.63 (t, J = 2.7 Hz,
J′ = 0.9 Hz, 1H); C11H11NOS2.
6.1.11. (2-Amino-4,5-dimethyl-thiophen-3-yl)-pyridin-2-yl-
methanone (10)
2-(Pyridin-2-carbonyl)acetonitrile 32, butanone, morpho-
line and sulfur were reacted according to the general proce-
dure to afford (2-amino-4,5-dimethyl-thiophen-3-yl)-
pyridin-2-yl-methanone 10. Yield: 39%; yellow solid; m.p.
125–127 °C (petroleum ether); IR (KBr) cm–1: 3233, 1565,
1436, 1279, 750; 1H-NMR (CDCl3) d: 1.46 (s, 3H), 2.12 (s,
3H), 6.76 (bs, 2H), 7.37 (m, 1H), 7.62 (m, 1H), 7.81 (d,
J = 7.5 Hz, 1H), 8.63 (d, J = 4.6 Hz, 1H); C12H12N2OS.
6.1.16. (2-Amino-5,6-dihydro-4H-cyclopenta[b]thiophen-3-
yl)-thiophen-3-yl-methanone (15)
2-(Thiophene-3-carbonyl)acetonitrile 33, cyclopen-
tanone, morpholine and sulfur were reacted according to the
general procedure to afford (2-amino-5,6-dihydro-4H-
cyclopenta[b]thiophen-3-yl-thiophen-3-yl)-methanone 15.
Yield: 71%; yellow solid; m.p. 125–127 °C (petroleum

862
P.G. Baraldi et al. / European Journal of Medicinal Chemistry 39 (2004) 855–865
ether); IR (KBr) cm–1: 3330, 3117, 1569, 1435, 715; H-
NMR (CDCl3) d: 2.24 (m, 4H), 2.71 (m, 2H), 6.83 (bs, 2H),
7.24 (dd, J = 5.0 Hz, J′ = 2.7, 1H), 7.30 (dd, J = 5.0 Hz,
J′ = 0.9 Hz, 1H), 7.56 (t, J = 2.7 Hz, J′ = 0.9 Hz, 1H);
C12H11NOS2.
6.1.21. (2-Amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-
yl)-thiophen-2-yl-methanone (20)
1
2-(Thiophene-2-carbonyl)acetonitrile 34, cyclohexanone,
morpholine and sulfur were reacted according to the general
procedure
to
afford
(2-amino-4,5,6,7-tetrahydro-
benzo[b]thiophen-3-yl)-thiophen-2-yl-methanone 20. Yield:
56%; orange solid; m.p. 117–118 °C (petroleum ether); IR
(KBr) cm–1: 3384, 1576, 1436, 1056, 765; 1HNMR: (CDCl3)
d: 1.59 (m, 2H), 1.81 (m, 2H), 2.20 (m, 2H), 2.54 (m, 2H),
6.10 (bs, 1H), 7.06 (dd, J = 4.5 Hz, J′ = 3.6 Hz, 1H), 7.38 (dd,
J = 3.6 Hz, J′ = 0.8 Hz, 1H), 7.60 (dd, J = 4.5 Hz, J′ = 0.8 Hz,
1H); C13H13NOS2.
6.1.17. (2-Amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-
yl)-thiophen-3-yl-methanone (16)
2-(Thiophene-3-carbonyl)acetonitrile 33, cyclohexanone,
morpholine and sulfur were reacted according to the general
procedure
to
afford
(2-amino-4,5,6,7-tetrahydro-
benzo[b]thiophen-3-yl)-thiophen-3-yl-methanone 16. Yield:
67%; yellow solid; m.p. 133–135 °C (petroleum ether); IR
(KBr) cm–1: 3323, 1576, 1433, 1080, 726; 1H-NMR (CDCl3)
d: 1.55 (m, 2H), 1.77 (m, 2H), 2.01 (t, J = 6.2 Hz, 2H), 2.54 (t,
J = 6.2 Hz, 2H), 6.46 (bs, 2H), 7.24 (dd, J = 5.0 Hz, J′ = 2.5,
1H), 7.30 (dd, J = 5.0 Hz, J′ = 0.9 Hz, 1H), 7.58 (t, J = 2.5 Hz,
J′ = 0.9 Hz, 1H); C13H13NOS2.
6.1.22. (2-Amino-6-benzyl-4,5,6,7-tetrahydro-thieno[2,3-c]
pyridin-3-yl)-thiophen-2-yl-methanone (21)
2-(Thiophene-2-carbonyl)acetonitrile 34, 1-benzyl-4-
piperidone, morpholine and sulfur were reacted according to
the general procedure to afford (2-amino-6-benzyl-4,5,6,7-
tetrahydro-thieno[2,3-c]pyridin-3-yl)-thiophen-2-yl-metha-
none 21. Yield: 78%; yellow solid; m.p. 122–123 °C (petro-
leum ether); IR (KBr) cm–1: 3412, 2928, 1570, 1485, 1355,
6.1.18. (2-Amino-6-benzyl-4,5,6,7-tetrahydro-thieno[2,3-
c]pyridin-3-yl)-thiophen-3-yl-methanone (17)
1
700; H-NMR (CDCl3) d: 2.35 (t, J = 5.4 Hz, 2H), 2.59 (t,
2-(Thiophene-3-carbonyl)acetonitrile 33, 1-benzyl-4-
piperidone, morpholine and sulfur were reacted according to
the general procedure to afford (2-amino-6-benzyl-4,5,6,7-
tetrahydro-thieno[2,3-c]pyridin-3-yl)-thiophen-3-yl-metha-
none 17. Yield: 68%; yellow solid; m.p. 132–133 °C (petro-
leum ether); IR (KBr) cm–1: 3350, 2805, 1578, 1429, 722;
1H-NMR (CDCl3) d: 2.14 (t, J = 5.6 Hz, 2H), 2.54 (t,
J = 5.6 Hz, 2H), 3.43 (s, 2H), 3.65 (s, 2H), 6.61 (bs, 2H), 7.24
(dd, J = 5.0 Hz, J′ = 2.5, 1H), 7.33 (dd, J = 5.0 Hz, J′ = 1.0 Hz,
1H), 7.58 (t, J = 2.5 Hz, J′ = 1.0 Hz, 1H); C19H18N2OS2.
J = 5.4 Hz, 2H), 3.47 (s, 2H), 3.67 (s, 2H), 6.26 (bs, 2H), 7.05
(dd, J = 4.0 Hz, J′ = 3.4 Hz, 1H), 7.30 (dd, J = 3.4 Hz,
J′ = 0.9 Hz, 1H), 7.35 (m, 5H), 7.53 (dd, J = 4.0 Hz,
J′ = 0.9 1H); C19H18N2OS2.
6.1.23.
(2-Amino-4,5-dimethyl-thiophen-3-yl)-(5-chloro-
thiophen-2-yl)-methanone (22)
2-(5-Chlorothiophene-2-carbonyl)acetonitrile 35, bu-
tanone, morpholine and sulfur were reacted according to the
general procedure to afford 2-amino-4,5-dimethyl-thiophen-
3-yl)-(5-chloro-thiophen-2-yl)-methanone 22. Yield: 46%;
red solid; m.p. 125–127 °C (petroleum ether); IR (KBr)
6.1.19. (2-Amino-4,5-dimethyl-thiophen-3-yl)-thiophen-2-
yl-methanone (18)
1
cm–1: 3377, 1551, 1424, 1003, 764; H-NMR (CDCl3) d:
2-(Thiophene-2-carbonyl)acetonitrile 34, butanone, mor-
pholine and sulfur were reacted according to the general
procedure to afford (2-amino-4,5-dimethyl-thiophen-3-yl)-
thiophen-2-yl-methanone 18. Yield: 44%; orange solid; m.p.
191–193 °C (petroleum ether); IR (KBr) cm–1: 3390, 1552,
1429, 1272, 772; 1H-NMR (CDCl3) d: 1.86 (s, 3H), 2.17 (s,
3H), 5.78 (bs, 2H), 7.07 (dd, J = 4.5 Hz, J′ = 3.5 Hz, 1H), 7.56
(d, J = 3.5 Hz, J′ = 0.7 Hz, 1H), 7.58 (dd, J = 4.5 Hz,
J′ = 0.7 Hz, 1H); C11H11NOS2.
1.90 (s, 3H), 2.17 (s, 3H), 5.77 (bs, 2H), 6.89 (d, J = 4.0 Hz,
1H), 7.16 (d, J = 4.0 Hz, 1H); C11H10ClNOS2.
6.1.24. (2-Amino-5,6-dihydro-4H-cyclopenta[b]thiophen-3-
yl)-(5-chloro-thiophen-2-yl)-methanone (23)
2-(5-Chlorothiophene-2-carbonyl)acetonitrile 35, cyclo-
pentanone, morpholine and sulfur were reacted according to
the general procedure to afford (2-amino-5,6-dihydro-4H-
cyclopenta[b]thiophen-3-yl)-(5-chloro-thiophen-2-yl)-me-
thanone 23. Yield: 69%; red solid; m.p. 170-171 °C (petro-
leum ether); IR (KBr) cm–1: 3351, 2851, 1550, 1270, 1002,
698; 1H-NMR (CDCl3) d: 2.28 (m, 2H), 2.52 (t, J = 7.2 Hz,
2H), 2.70 (t, J = 7.2 Hz, 2H), 6.62 (bs, 2H), 6.88 (d,
J = 4.0 Hz, 1H), 7.16 (d, J = 4.0 Hz, 1H); C12H10ClNOS2.
6.1.20. (2-Amino-5,6-dihydro-4H-cyclopenta[b]thiophen-
3-yl)-thiophen-2-yl-methanone (19)
2-(Thiophene-2-carbonyl)acetonitrile 34, cyclopen-
tanone, morpholine and sulfur were reacted according to the
general procedure to afford (2-amino-5,6-dihydro-4H-
cyclopenta[b]thiophen-3-yl)-thiophen-2-yl-methanone 19.
Yield: 67%; yellow solid; m.p. 133–134 °C (petroleum
6.1.25. (2-Amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-
yl)-(5-chloro-thiophen-2-yl)-methanone(24)
1
ether); IR (KBr) cm–1: 3344, 1566, 1435, 1031, 747; H-
NMR (CDCl3) d: 2.27 (m, 2H), 2.47 (t, J = 7.2 Hz, 2H), 2.70
(t, J = 7.2 Hz, 2H), 6.55 (bs, 2H), 7.06 (dd, J = 4.6 Hz,
J′ = 3.5 Hz, 1H), 7.51 (dd, J = 3.5 Hz, J′ = 1.1 Hz, 1H), 7.53
(dd, J = 4.6 Hz, J′ = 1.1 Hz, 1H); C12H11NOS2.
2-(5-Chlorothiophene-2-carbonyl)acetonitrile 35, cyclo-
hexanone, morpholine and sulfur were reacted according to
the general procedure to afford (2-amino-4,5,6,7-tetrahydro-
benzo[b]thiophen-3-yl)-(5-chloro-thiophen-2-yl)-

P.G. Baraldi et al. / European Journal of Medicinal Chemistry 39 (2004) 855–865
863
methanone 24. Yield: 76%; yellow solid; m.p. 173–175 °C
(petroleum ether); IR (KBr) cm–1: 3349, 2911, 1577, 1430,
765; 1H-NMR (CDCl3) d: 1.59 (m, 2H), 1.65 (m, 2H), 2.35 (t,
J = 5.8 Hz, 2H), 2.55 (t, J = 5.8 Hz, 2H), 6.07 (bs, 2H), 6.88
(d, J = 4.0 Hz, 1H), 7.16 (d, J = 4.0 Hz, 1H); C13H12ClNOS2.
according to the general procedure to afford (2-amino-6-
benzyl-4,5,6,7-tetrahydro-thieno[2,3-c]pyridin-3-yl)-(5-bro-
mo-thiophen-2-yl)-methanone 29. Yield: 72%; orange solid;
m.p. 87–89 °C (petroleum ether); IR (KBr) cm–1: 3411,
1
1570, 1411, 978; H-NMR (CDCl3) d: 2.38 (t, J = 5.4 Hz,
2H), 2.59 (t, J = 5.4 Hz, 2H), 3.48 (s, 2H), 3.67 (s, 2H), 6.28
(bs, 2H), 7.01 (d, J = 4.0 Hz, 1H), 7.15 (d, J = 4.0 Hz, 1H),
7.32 (m, 5H); C19H17BrNOS2.
6.1.26. (2-Amino-6-benzyl-4,5,6,7-tetrahydro-thieno[2,3-
c]pyridin-3-yl)-(5-chloro-thiophen-2-yl)-methanone (25)
2-(5-Chlorothiophene-2-carbonyl)acetonitrile
35,
1-benzyl-4-piperidone, morpholine and sulfur were reacted
according to the general procedure to afford (2-amino-6-
benzyl-4,5,6,7-tetrahydro-thieno[2,3-c]pyridin-3-yl)-(5-
chloro-thiophen-2-yl)-methanone 25. Yield: 63%; yellow
solid; m.p. 81–83 °C (petroleum ether); IR (KBr) cm–1:
7. Biological evaluation
7.1. Biological materials
1
3306, 2807, 1574, 1428, 699; H-NMR (CDCl3) d: 2.39 (t,
[3H]DPCPX (specific activity, 112 Ci/mmol) and [3H]C-
CPA (specific activity, 55 Ci/mmol) were obtained from
NEN Research Products (Boston, MA); [3H]ZM241385
(specific activity, 17 Ci/mmol) was obtained from Tocris
Cookson (Bristol, UK); [3H]MRE 3008F20 (specific activity
67 Ci/mmol) was obtained from Amersham International
(Buckinghamshire, UK). CHO cells transfected with the hu-
man recombinant A1 adenosine receptor (hCHO-A1) were
obtained by Prof. K.N. Klotz, University of Wurzburg. Hu-
man cerebral cortex was obtained from the University of
Ferrara, Medicina Legale Section, with approval of human
tissue use protocol. Rat cerebral cortex was harvested from
adult male Wistar rats. All tissue culture reagents were ob-
tained from Sigma.
J = 5.2 Hz, 2H), 2.59 (t, J = 5.2 Hz, 2H), 3.45 (s, 2H), 3.67 (s,
2H), 6.26 (bs, 2H), 6.87 (d, J = 3.9 Hz, 1H), 7.19 (d,
J = 3.9 Hz, 1H), 7.35 (m, 5H); C19H17ClN2OS2.
6.1.27. (2-Amino-4,5-dimethyl-thiophen-3-yl)-(5-bromo-
thiophen-2-yl)methanone (26)
2-(5-Bromothiophene-2-carbonyl)acetonitrile 36, bu-
tanone, morpholine and sulfur were reacted according to the
general procedure to afford (2-Amino-4,5-dimethyl-
thiophen-3-yl)-(5-bromo-thiophen-2-yl)methanone
26.
Yield: 51%; orange solid; m.p. 128–130 °C (petroleum
1
ether); IR (KBr) cm–1: 3348, 1559, 1448, 1263, 769; H-
NMR (CDCl3) d: 2.16 (s, 6H), 5.90 (bs, 2H), 7.02 (d,
J = 4.0 Hz, 1H), 7.11 (d, J = 4.0 Hz, 1H); C11H10BrNOS2.
6.1.28. (2-Amino-5,6-dihydro-4H-cyclopenta[b]thiophen-
3-yl)-(5-bromo-thiophen-2-yl)-methanone (27)
7.2. CHO: huA1 assay
2-(5-Bromothiophene-2-carbonyl)acetonitrile 36, cyclo-
pentanone, morpholine and sulfur were reacted according to
the general procedure to afford (2-amino-5,6-dihydro-4H-
cyclopenta[b]thiophen-3-yl)-(5-bromo-thiophen-2-yl)-
methanone 27. Yield: 73%; red solid; m.p. 154–156 °C (pe-
troleum ether); IR (KBr) cm–1: 3349, 1560, 1431, 1269, 975;
1H-NMR (CDCl3) d: 2.28 (m, 2H), 2.51 (t, J = 7.4 Hz, 2H),
2.70 (t, J = 7.4 Hz, 2H), 6.64 (bs, 2H), 7.02 (d, J = 4.0 Hz,
1H), 7.13 (d, J = 4.0 Hz, 1H); C12H12BrNOS2.
The preparation of Chinese hamster ovary cells express-
ing human recombinant A1 adenosine receptors was done as
previously described [19]. For experiments, aliquots of cells
were placed into 24-well plates with culture medium, serum,
and antibiotic for 48 h, by which time the cells had grown to
a confluent monolayer. Allosteric enhancement was mea-
sured as the action of a test compound at different concentra-
tions (0.01, 0.1, 1 and 10 µM) to reduce the cAMP content of
CHO: huA1 cells in the presence of the adenosine A1 agonist
N6-cyclopentyladenosine (CPA). To begin an experiment,
growth medium was removed from the plates and cells were
washed once with warm Hanks’ buffered saline solution. The
wash solution was then removed and replaced with fresh
Hanks’ solution containing forskolin (1 µM), rolipram
(20 µM), (CPA, 0.01 nM), adenosine deaminase (2 U/ml),
and the allosteric enhancer to be tested. Forskolin was used to
stimulate the activity of adenylyl cyclase, rolipram to inhibit
cAMP phosphodiesterase, adenosine deaminase to degrade
endogenous adenosine and CPA to cause a small increase of
the number of activated adenosine receptors. After 6 min of
incubation at 36 °C in the presence of drugs, the incubation
solution was removed and hydrochloric acid (final concen-
tration 50 mM) was added to cells to terminate drug action.
The content of cAMP in acidified extracts of cells was deter-
mined by radioimmunoassay [19]. Because the magnitude of
6.1.29. (2-Amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-
yl)-(5-bromothiophen-2-yl)-methanone (28)
2-(5-Bromothiophene-2-carbonyl)acetonitrile 36, cyclo-
hexanone, morpholine and sulfur were reacted according to
the general procedure to afford (2-Amino-4,5,6,7-
tetrahydro-benzo[b]thiophen-3-yl)-(5-bromothiophen-2-yl)-
methanone 28. Yield: 66%; orange solid; m.p. 161–163 °C
(petroleum ether); IR (KBr) cm–1: 3379, 2918, 1577, 1432,
761; 1H-NMR (CDCl3) d: 1.63 (m, 2H), 1.79 (m, 2H), 2.24 (t,
J = 5.8 Hz, 2H), 2.55 (t, J = 5.8 Hz, 2H), 6.11 (bs, 2H), 7.02
(d, J = 4.0 Hz, 1H), 7.13(d, J = 4.0 Hz, 1H); C13H12BrNOS2.
6.1.30. (2-Amino-6-benzyl-4,5,6,7-tetrahydro-thieno[2,3-c]
pyridin-3-yl)-(5-bromo-thiophen-2-yl)-methanone (29)
2-(5-Bromothiophene-2-carbonyl)acetonitrile
36,
1-ben-zyl-4-piperidone, morpholine and sulfur were reacted

864
P.G. Baraldi et al. / European Journal of Medicinal Chemistry 39 (2004) 855–865
the effects of allosteric enhancers on CHO:huA1 cells
changed subtly with passage number and differed slightly
among different aliquots of cells, the actions of the test
compounds and the action of the reference compound PD 81,
723 were assayed in each experiment.
Non-specific binding was defined as binding in the presence
of 1 µM R-PIA.
7.6. Saturation
Saturation binding experiments of [3H]CCPA (0.05–
10 nM) to A1 receptors expressed in CHO:hA1, rat, and
human brain membranes were performed in triplicate at
25 °C for 90 min in 50 mM Tris–HCl, pH 7.4, in the absence
and presence of test compounds. Non-specific binding was
defined as binding in the presence of 1 µM R-PIA.
7.3. Membrane preparation from CHO-A1 cells
For membrane preparation the culture medium was re-
moved. The cells were washed with PBS and scraped off T75
flasks in ice-cold hypotonic buffer (5 mM Tris–HCl, 2 mM
EDTA, pH 7.4). The cell suspension was homogenized using
a Polytron, and the homogenate was spun for 10 min at
1000g. The supernatant was then centrifuged for 30 min at
100 000g. The membrane pellet was resuspended in 50 mM
Tris–HCl buffer pH 7.4 and incubated with 2 U/ml of ADA
for 30 min at 37 °C. Then the suspension was stored at
–80 °C. The protein concentration was determined according
to a Bio-Rad method with bovine albumin as a standard
reference.
7.7. Competition assay
Competition experiments of 1 nM [3H]-DPCPX to
CHO:hA1 membranes were performed incubating mem-
branes (100 µg of protein/assay) at 25 °C for 150 min.
Competition experiments were performed in duplicate in a
final volume of 250 µl in test tubes containing 50 µM Tris–
HCl buffer, pH 7.4 and 100 µl of membranes and at least six
to eight different concentrations of the tested compounds.
Non-specific binding was defined as the binding in the pres-
ence of 1 µM DPCPX and was about 25% of total binding.
Competition experiments of 2 nM [3H]ZM241385 to
CHO:hA2A membranes were performed incubating mem-
branes (100 µg of protein/assay) at 4 °C for 60 min. Compe-
tition experiments were performed in duplicate in a final
volume of 250 µl in test tubes containing 50 µM Tris–HCl
buffer, 10 µM MgCl2, pH 7.4 and 100 µl of membranes and at
least six to eight different concentrations of the tested com-
pounds. Non-specific binding was defined as the binding in
the presence of 1 µM ZM241385 and was about 30% of total
binding. Competition experiments of 2 nM [3H]MRE
3008F20 to CHO:hA3 membranes were performed incubat-
ing membranes (100 µg of protein/assay) at 4 °C for 150 min.
Competition experiments were performed in duplicate in a
final volume of 250 µl in test tubes containing 50 µM Tris–
HCl buffer, 10 µM MgCl2, 1 mM EDTA, pH 7.4 and 100 µl of
membranes and at least six to eight different concentrations
of the tested compounds. Non-specific binding was defined
as the binding in the presence of 1 µM MRE 3008F20 and
was about 30% of total binding.
7.4. Membrane preparation from rat cortex and human
brain
Cerebral cortical tissue from each species was homog-
enized using a Polytron (setting 6, 20 s) in 20 volumes of ice
cold 50 mM Tris–HCl, pH 7.4. This crude membrane homo-
genate was then centrifuged at 48 000g for 15 min at 4 °C.
The resulting pellet was resuspended in buffer containing
2 IU/ml ADA to 20 mg/ml original tissue weight and incu-
bated at 37 °C for 30 min to remove endogenous adenosine.
This membrane homogenate was recentrifuged at 48 000g
for 15 min at 4 °C. The resulting membrane pellet was
resuspended and recentrifuged at 48 000g for 15 min at 4 °C.
The final membrane pellets were stored at –80 °C until the
time of assay.
7.5. Adenosine receptor binding
To determine the effect of the new series of derivatives of
PD 81, 723 on the binding of ligands to A1, A2A, and A3
receptors, membranes from CHO: hA1, hA2A, hA3, rat cor-
tex, and human brain were incubated in a buffer solution in
the absence and presence of test compounds. Test agents
were dissolved in DMSO and added to the assay from a
100-fold concentrated solution in DMSO. Control incuba-
tions also contained 1% DMSO. Bound and free radioactivi-
ties were separated by filtering the assay mixture through
Whatman GF/B glass-fiber filters using a Micro-Mate
196 cell harvester (Packard Instrument Company). The filter-
bound radioactivity was counted on Top Count Microplate
Scintillation Counter (efficiency 57%) with Micro-Scint 20.
Binding of 1 nM [3H]CCPA toA1 receptors in CHO:hA1, rat,
and human brain membranes in the absence and presence of
increasing concentrations of test compounds was carried out
in triplicate at 25 °C for 90 min in 50 mM Tris–HCl, pH 7.4.
7.8. Data analysis
All values are expressed as mean (SEM of three indepen-
dent experiments. A weighted non-linear least-squares curve
fitting program LIGAND was used for computer analysis of
saturation and competition experiments. For experiments
with two comparison groups, statistical analysis was per-
formed with a two-tailed t test. Differences between group
mean values were considered significant at P e 0.05.
Acknowledgments
We thank King Pharmaceuticals and MIUR (60% and
40%) for financial support of this research.

P.G. Baraldi et al. / European Journal of Medicinal Chemistry 39 (2004) 855–865
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