2-Aminothiophene-3-carboxylates and carboxamides as adenosine A1 receptor allosteric enhancers

Article abstract of DOI:10.1016/j.bmc.2005.11.018

Three series of 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene and 2-amino-5,6,7,8-tetrahydrocyclohepta[b]thiophenes with 3-carboxylates and carboxamides have been prepared using the Gewald synthesis and evaluated as A₁AR allosteric enhancers. The

Full text of DOI:10.1016/j.bmc.2005.11.018

Bioorganic & Medicinal Chemistry 14 (2006) 2358–2365
2-Aminothiophene-3-carboxylates and carboxamides as
adenosine A1 receptor allosteric enhancers
George Nikolakopoulos,a Heidi Figler,b Joel Lindenb and Peter J. Scammellsa,*
aDepartment of Medicinal Chemistry, Victorian College of Pharmacy, Monash University,
381 Royal Parade, Parkville, Vic. 3052, Australia
bDepartments of Medicine (Cardiology) and Pharmacology, University of Virginia, Charlottesville, VA 22908, USA
Received 13 September 2005; revised 8 November 2005; accepted 8 November 2005
Available online 28 November 2005
Abstract—Three series of 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene and 2-amino-5,6,7,8-tetrahydrocyclohepta[b]thiophenes
with 3-carboxylates and carboxamides have been prepared using the Gewald synthesis and evaluated as A1AR allosteric enhancers.
The structure–activity relationships of these classes of compound are described. A number of compounds, notably 7b, are more
potent and efficacious than PD81,723 (1).
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
group maintained allosteric enhancer activity, but in-
creased competitive antagonist activity of the resulting
compounds.8 Bulky (or hydrophobic) substituents at the
meta- and para-positions of the 3-benzoyl group and also
3-naphthoyl groups greatly enhanced enhancer activity.
Thus, the A1AR is thought to contain an allosteric bind-
ing site able to accommodate 3-aroyl substituents that are
bulky and/or hydrophobic but not necessarily planar. A
second region in the allosteric binding site interacts
constructively with alkyl substituents at thiophene
C-4 and/or C-5.6
The first allosteric enhancers (AEs) acting at the adeno-
sine A1 receptor (A1AR) were reported by Bruns et al.
in 1990.1,2 These compounds were primarily 2-amino-3-
benzoylthiophenes and 2-amino-3-benzoyl-4,5,6,7-tetra-
hydrothieno[2,3-c]pyridines and were found to decrease
the rate of dissociation of agonist, but not antagonist
radioligand from the orthosteric binding site. In addition
to allosteric activity, some of these compounds also have
weak activity as competitive antagonists of the A1AR.
PD81,723 (1) was one of the more potent and effective
of the initial series of enhancers and has subsequently
been commonly used for benchmarking new AEs. Since
this initial discovery, other researchers have directed
significant effort to refining the structure–activity
relationships of the 3-, 4- and 5-positions of the 2-amino-
thiophene core.3–7 The 2-amino and 3-keto groups were
found to be crucial for activity, while replacement of the
thiophene by benzene resulted in a marked reduction of
activity. Substitution at the 4-position increased activity
by a factor of 3 (phenyl > methyl > H), while substitution
at the 5-position had little effect on activity. The benzoyl
group was reported to be important for activity and sub-
stituents were identified which further improved activity
(e.g., trifluoromethyl, chloro and methoxy). The replace-
ment of the phenyl at the 3-position with a 2- or 3-thienyl
H3C
H3C
S
S
NH2
O
NH2
O
H3C
O
CF3
1
2
CF3
Relatively few 2-aminothiophenes with esters or amides
in the 3-position have been tested as allosteric
enhancers. The initial study by Bruns and co-workers
included two 2-amino-3-ethoxycarbonyl-4,5,6,7-tetrahy-
drothieno[2,3-c]pyridines.1,2 Whilst these compounds
maintained some enhancing ability, they proved to be
Keywords: Adenosine; Allosteric enhancer; Adenosine A1 receptor.
*
Corresponding author. Tel.: +61 3 9903 9542; fax: +61 3 9903
9582; e-mail: peter.scammells@vcp.monash.edu.au
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.11.018

G. Nikolakopoulos et al. / Bioorg. Med. Chem. 14 (2006) 2358–2365
2359
Table 1. 3-Ethoxycarbonyl and 3-benzoyl substituted allosteric
enhancers reported by Bruns et al.1,2
ing CPX and GTPcS). Three of the five alkoxycarbonyl
compounds that were tested at 50 lM proved to be
clearly more effective than PD81,723 (1). 3-Trifluorom-
ethylbenzyl 2-amino-4-(4-methylphenyl)-5-phenylthio-
phene-3-carboxylate (2) proved to be the most effective
compound in this group with an AE score of 63% (com-
pared with 19% for PD81,723). The sole amide tested
recorded a slightly lower AE score than PD81,723.
Although the test set was too small to draw many firm
conclusions about structure–activity relationships, this
investigation demonstrates that 2-aminothiophenes with
esters and amides in the 3-position can exhibit substan-
tial AE activity. This current study further explores the
structure–activity relationships of this class of com-
pounds as A1AR allosteric enhancers.
R
S
X
N
NH2
O
R
X
Enhancement (%)a
Me
Me
OEt
Ph
6
14
CH2Ph
CH2Ph
OEt
Ph
66
102
a Allosteric enhancement was evaluated using an assay that measured
the % increase over control in specific [3H]N6-cyclohexyladenosine
binding to rat brain membranes after addition of 100 lM R-PIA for
2 h.
2. Results and discussion
2-Aminothiophene targets were prepared using the Ge-
wald synthesis.10,11 This synthesis initially involves the
Knoevenagel condensation of an aldehyde or ketone with
an activated nitrile (such as an a-cyanoketone, cyanoace-
tate or cyanoacetamide), followed by a sulfur mediated
cyclization to afford the desired 2-aminothiophene. There
are two common variations on this synthesis: namely (i) a
one-pot procedure in which the ketone and activated ni-
trile react in the presence of sulfur and base, and (ii) a
two-step procedure in which the alkene produced by the
Knoevenagel condensation is isolated prior to cyclization
with sulfur and base. The former is more convenient and
has been used for the synthesis of numerous 2-aminothi-
ophenes, while the latter is generally higher yielding.11
In this study, the one-pot procedure was used to prepare
the 2-aminothiophene-3-carboxylates (Series 1, Scheme
1), but gave poor yields of the corresponding phenyl
less potent than the corresponding 3-benzoyl substituted
(see
2-amino-4,5,6,7-tetrahydrothieno[2,3-c]pyridines
Table 1).
We recently synthesised a series of substituted 2-amino-
3-benzoyl-4,5-diphenylthiophene allosteric enhancers.
En route to these target compounds we also prepared
some novel 2-aminothiophene-3-carboxylates and carb-
oxamides to explore the effect of these substituents on
AE activity. Candidate allosteric enhancers were evalu-
ated using a rapid in vitro assay which measures their
ability to stabilize the agonist–A1AR–G protein ternary
complex.9 More specifically, the A1AR agonist [125I]4-
amino-3-iodo-N6-benzyladenosine is incubated with
membranes from CHO-K1 cells stably expressing the
hA1AR and the ability of the candidate AE to stabilize
the agonist–A1AR–G protein ternary complex is scored
from 0 to 100 as the percentage of the ternary complex
remaining after 10 min of dissociation (initiated by add-
2-aminothiophene carboxamides. As
a result, the
two-step variant was employed for the preparation of
these compounds (Series 2A, Scheme 1). It was possible
Series 1
Series 2B
S
(iv)
S
(v)
S
( )n
( )n
( )n
CN
NH2
O
NH2
O
NH2
O
4a
[R = Et]
O
RO
5
7
8
RO
HO
HN
(i)
( )n
O
(ii)
Series 2A
3
X
CN
CN
(iii)
S
( )n
( )n
NH2
O
O
O
ArN
H
4b
HN
6
HN
X
X
Scheme 1. Reagents: (n = 1, 2). (i) Sulfur, NEt3, DMF; (ii) NH4OAc, AcOH, C6H6; (iii) sulfur, NHEt2, EtOH; (iv) KOH, EtOH [R = Et]; (v) BnNH2,
EDC Æ HCl, DMAP, HOBt, CH2Cl2.

2360
G. Nikolakopoulos et al. / Bioorg. Med. Chem. 14 (2006) 2358–2365
to prepare the benzyl 2-aminothiophene-3-carboxamides
Series 2B, Scheme 1) in a more convergent fashion from
the appropriate ethyl 2-aminothiophene-3-carboxylate
in two steps (saponification followed by carbodiimide
coupling).
oxamides (5a, 5i, 8a and 8k) were amongst the most effec-
tive compounds tested. Thus, in a number of cases
further substitution of the benzyl group may lead to
unfavourable interaction with the AE binding site.
The relationship between the size of the C4–C5
polymethylene bridge and enhancing activity was less
clear cut for the 2-aminothiophene-3-carboxamides
(8a–j vs 8k–t) than was previously observed for
The activity of the new compounds as allosteric enhanc-
ers of the human adenosine A1 receptors was evaluated
based on their ꢀscoreꢁ to inhibit the kinetics of agonist
radioligand dissociation upon the addition of 25 lM
GTPcS and 100 lM CPX. As shown in Figure 1,
dose–response curves relating score to enhancer concen-
tration (0.1–50 lM) were well fit by hyperbolic equa-
tions from which the maximal score could be
accurately calculated, provided the ED50 was <20 lM.
The calculated maximal scores are recorded in Table
2. For compounds with ED50 values > 20 lM maximal
scores could not be accurately calculated, and the score
at 50 lM is recorded as an approximate maximum.
Potent compounds (low ED50) were not always highly
efficacious. For example, compound 6g and PD81,723
are potent but have limited efficacy (Fig. 1). In general,
the benzyl 2-aminothiophene-3-carboxamides proved to
be more efficacious than the corresponding benzyl
2-aminothiophene-3-carboxylates (compare compounds
8a–g with 5a–g). The benzyl 2-aminothiophene-3-carb-
oxamides were also more active than the corresponding
phenyl 2-aminothiophene-3-carboxylates (compare 8b–g
with 6b–g). Two 2-aminothiophene-3-carboxylic acids
(7a and 7b) were also tested. Interestingly, high efficacy
and unusually high potency were observed for 2-amino-
5,6,7,8-tetrahydrocyclohepta[b]thiophene-3-carboxylic
acid (7b, Fig. 1).
2-amino-3-aroylthiophenes.
2-Amino-4,5,6,7-tetra-
hydrobenzo[b]thiophene-3-carboxamides were more
effective than the corresponding 2-amino-5,6,7,8-tetra-
hydrocyclohepta[b]thiophene-3-carboxamides in five
cases (3-trifluoromethylbenzyl, 3-methoxybenzyl, 4-
chlorobenzyl, 4-bromobenzyl and 4-iodobenzyl). How-
ever, the larger polymethylene bridge [–(CH2)5–] was
more effective for the benzyl, 3-chlorobenzyl, 3-nitro-
benyl and 4-nitrobenzylcarboxamides.
Since some aminothiophenes have been found to have
activity as competitive antagonists of the A1AR, the
antagonist activity of all AEs with ED50 values less
than 20 lM was measured in a [3H]CPX competitive
binding assay. All of the new AEs showed lower
antagonist activity than PD81,723 (Table 2). In the
case of compound 7b the % inhibition of [3H]CPX
binding was approximately half that of PD81,723
(9% inhibition compared with 19% exerted by
PD81,723).
3. Experimental
Melting points were determined with an Electrothermal
melting point apparatus and are uncorrected. All NMR
spectra were recorded on a Bruker Avance DPX 300
The effect of further substitution of the phenyl and
benzyl of the 2-aminothiophene-3-carboxylates and
3-carboxamides was also investigated. A panel of substit-
uents that had previously been reported to confer high
AE activity in a related series of 2-amino-3-aroylthioph-
enes (primarily halo, methoxy, trifluoromethyl and phen-
yl) was chosen for this purpose. In this case, no clear
correlation between the steric or electronic properties
of the substituent and enhancing activity was observed.
The benzyl 2-aminothiophene-3-carboxylates and carb-
1
spectrometer. H and 13C NMR spectra were recorded
at 300.13 and 75.4 MHz, respectively, and, unless stated
otherwise, samples were dissolved in CDCl3. Thin-layer
chromatography was conducted on 0.2 mm plates using
Merck silica gel 60 F254. Column chromatography was
achieved using Merck silica gel 60 (particle size 0.063–
0.200 lm, 70–230 mesh).
3.1. General procedure for 2-aminothiophene-3-carbox-
ylate formation (5a–k)
100
The benzyl cyanoacetate (1.0 molar equiv), sulfur (1.1
equiv), ketone (cyclohexanone or cycloheptanone, 1.0
equiv) and triethylamine (2 mL) were dissolved in
DMF (2 mL) and stirred at room temperature for
16 h. The reaction mixture was diluted with CHCl3,
washed with brine (4 · 25 mL), dried over anhydrous
MgSO4 and filtered. The crude product was purified
by chromatography using a deactivated SiO2 column
eluted with hexane/ethyl acetate (5:1).
7b
80
60
40
6g
20
PD81,723
0
3.1.1. Benzyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thio-
phene-3-carboxylate (5a). Yield 69%. H NMR d 1.74–
1.80 (m, 4H, 2· CH2), 2.50–2.51 (m, 2H, CH2), 2.74–
2.77 (m, 2H, CH2), 5.30 (s, 2H, CH2Ph), 6.08 (br s,
2H, NH2), 7.31–7.45 (m, 5H, ArH). HR-ES MS: m/z,
288.10595 [M+H].
0
10
20
30
40
50
60
1
[AE] (µM)
Figure 1. Dose–response curves relating AE score to concentration.
Data were fit to rectangular hyperbolae to calculate maximal scores
and ED50 values listed in Table 2.

G. Nikolakopoulos et al. / Bioorg. Med. Chem. 14 (2006) 2358–2365
Table 2. AE activity of 2-aminothiophene-3-carboxylates and carboxamides
2361
R5
R4
S
NH2
X
R
O
Compound
X
R4/R5
R
ScoreMaxa
ED50 (lM)
% Inhibitionb
5a
5b
5c
5d
5e
5f
O
O
O
O
O
O
O
O
O
O
O
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)5–
–(CH2)5–
–(CH2)5–
PhCH2–
72.4 1.2%
31 6.5%
10.4 1.2
>20
>20
15.2 1.89
3-CF3PhCH2–
3-OCH3PhCH2–
4-ClPhCH2–
4-BrPhCH2–
4-IPhCH2–
1.5 1.5%
12.0 1.0%
19.5 3.5%
6.5 0.5%
21.0 1.0%
25.5 1.5%
88.0 1%
>20
>20
>20
>20
5g
5h
5i
4-CF3PhCH2–
4-PhPhCH2–
PhCH2–
>20
>20
5j
5k
3-CF3PhCH2–
PhCH2CH2–
59.0 6.0%
48 3.0%
>20
>20
6b
6c
6d
6e
6f
NH
NH
NH
NH
NH
NH
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
3-CF3Ph–
3-OCH3Ph–
4-ClPh–
32.5 3.5%
19.0 1.0%
39.5 1.5%
24.5 2.5%
50.0 2.0%
38.5 1.5%
>20
>20
>20
>20
4-BrPh–
4-IPh–
4-CF3Ph–
>20
14.2 2.0
6g
7.46 0.56
9.23 1.20
7a
7b
O
O
–(CH2)4–
–(CH2)5–
H
H
49.5 3.0%
79.5 1.5%
>20
1.35 0.17
8a
8b
8c
8d
8e
8f
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
—
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)4–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–(CH2)5–
–CH3/–CH3
PhCH2–
79.5 1.5%
55.5 3.5%
42.5 3.5%
39.5 1.5%
50.5 1.5%
70.0 4.0%
70.0 1.0%
79.0 3.0%
1.8 1.8%
58.5 0.5%
68.5 7.5%
17.0 3.0%
12.5 1.5%
24.0 3.0%
22.0 1.0%
23.0 3.0%
15.0 2.0%
88.0 4.0%
85.5 1.5%
87.0 1.0%
28 1.1
10.4 0.9
3.0 0.7
>20
6.53 1.10
9.47 1.13
3-CF3PhCH2–
3-OCH3PhCH2–
4-ClPhCH2–
4-BrPhCH2–
4-IPhCH2–
>20
>20
>20
8g
8h
8i
4-CF3PhCH2–
3-ClPhCH2–
3-NO2PhCH2–
4-NO2PhCH2–
PhCH2–
16.5 1.5
13.0 1.8
>20
10.8 0.73
3.67 0.52
8j
15.0 0.75
>20
>20
8.10 1.18
8k
8l
3-CF3PhCH2–
3-OCH3PhCH2–
4-ClPhCH2–
4-BrPhCH2–
4-IPhCH2–
8m
8n
8o
8p
8q
8r
8s
8t
>20
>20
>20
>20
4-CF3PhCH2–
3-ClPhCH2–
3-NO2PhCH2–
4-NO2PhCH2–
3-CF3Ph–
>20
6.8 0.4
6.0 0.22
10.3 1.4
13.6 2.1
4.51 1.75
7.03 1.06
4.67 0.89
18.8 2.46
PD
a For compounds with an ED50 < 20 lM this value is based on curve fitting relating AE score to AE concentration using the equation Score = Score-
Max [AE]/(ED50 + [AE]). Data points are means SEM, N = 2–3. For compounds with ED50 values > 20 lM the score at 50 lM is recorded.
*
b % inhibition of specific [3H]CPX binding by 10 lM allosteric enhancer, N = 3.
3.1.2. 3-Trifluoromethylbenzyl 2-amino-4,5,6,7-tetra-
hydrobenzo[b]thiophene-3-carboxylate (5b). Yield 34%.
1H NMR d 1.71–1.75 (m, 4H, 2· CH2), 2.48–2.52 (m,
2H, CH2), 2.69–2.73 (m, 2H, CH2), 5.31 (s, 2H, CH2Ph),
6.02 (br s, 2H, NH2), 7.46–7.68 (m, 4H, ArH). HR-ES
MS calcd for C17H17F3NO2S+ (M+1) 356.0927, found
356.0921.
CH2Ph), 5.99 (br s, 2H, NH2), 6.86 (d, J = 8.1 Hz, 1H,
ArH), 6.98 (d, J = 8.7 Hz, 2H, ArH), 7.30 (d,
J = 7.5 Hz, 1H, ArH). HR-ES MS calcd for
C17H20NO3S+ (M+1) 318.1158, found 318.1148.
3.1.4. 4-Chlorobenzyl 2-amino-4,5,6,7-tetrahydrobenzo[b]-
thiophene-3-carboxylate (5d). Yield 46%. 1H NMR d
1.70–1.81 (m, 4H, 2· CH2), 2.48–2.51 (m, 2H, CH2),
2.67–2.71 (m, 2H, CH2), 5.23 (s, 2H, CH2Ph), 5.77
(br s, 2H, NH2), 7.30–7.37 (m, 4H, ArH). HR-ES
MS calcd for C16H17ClNO2S+ (M+1) 322.0663, found
322.0661.
3.1.3. 3-Methoxybenzyl 2-amino-4,5,6,7-tetrahydro-
benzo[b]thiophene-3-carboxylate (5c). Yield 29%. 1H
NMR d 1.75 (m, 4H, 2· CH2), 2.49 (m, 2H, CH2),
2.74 (m, 2H, CH2), 3.82 (s, 3H, OCH3), 5.26 (s, 2H,

2362
G. Nikolakopoulos et al. / Bioorg. Med. Chem. 14 (2006) 2358–2365
3.1.5.
4-Bromobenzyl
2-amino-4,5,6,7-tetrahydro-
ammonium acetate (1.3 equiv) and glacial acetic acid
benzo[b]thiophene-3-carboxylate (5e). Yield 54%. 1H
NMR d 1.70–1.81 (m, 4H, 2· CH2), 2.49 (m, 2H,
CH2), 2.69 (m, 2H, CH2), 5.21 (s, 2H, CH2Ph),
5.81 (br s, 2H, NH2), 7.27 (d, J = 8.3 Hz, 2H,
ArH), 7.49 (d, J = 8.3 Hz, 2H, ArH). HR-ES MS
calcd for C16H17BrNO2S+ (M+1) 366.0158, found
366.0148.
(3.5 equiv) in benzene (50 mL) were refluxed for 16 h
in a Dean–Stark apparatus. The reaction mixture
was cooled and then diluted with CHCl3. The mixture
was washed with H2O (100 mL), 10% Na2CO3
(100 mL) then H2O (100 mL) and the organic phase
was dried over anhydrous MgSO4 and filtered. Evap-
oration of the solvent afforded the crude Knoevenagel
product, which was used without further purification.
This material was dissolved in dry EtOH (11 mL)
and prior to the addition of sulfur (4.0 equiv) and
N,N-diethylamine (4.0 equiv). The reaction mixture
was stirred for 1.5 h at 40–50 ꢁC and then cooled to
0 ꢁC. After filtration and evaporation of the solvent,
the target compound was purified by chromatography
on a SiO2 column eluting with CH2Cl2/MeOH/NH3
(99%:0.5%:0.5%).
3.1.6.
4-Iodobenzyl
2-amino-4,5,6,7-tetrahydro-
benzo[b]thiophene-3-carboxylate (5f). Yield 42%. 1H
NMR d 1.68–1.81 (m, 4H, 2· CH2), 2.49 (m, 2H,
CH2), 2.68 (m, 2H, CH2), 5.20 (s, 2H, CH2Ph), 5.70
(br s, 2H, NH2), 7.14 (d, J = 8.3 Hz, 2H, ArH), 7.69
(d, J = 8.3 Hz, 2H, ArH). HR-ES MS calcd for C16H17I-
NO2S+ (M+1) 414.0019, found 414.0010.
3.1.7. 4-Trifluoromethylbenzyl 2-amino-4,5,6,7-tetra-
hydrobenzo[b]thiophene-3-carboxylate (5g). Yield 53%.
1H NMR d 1.70–1.82 (m, 4H, 2· CH2), 2.51 (m, 2H,
CH2), 2.71 (m, 2H, CH2), 5.33 (s, 2H, CH2Ph), 5.57
(br s, 2H, NH2), 7.51 (d, J = 8.1 Hz, 2H, ArH), 7.63
(d, J = 8.1 Hz, 2H, ArH). HR-ES MS calcd for
C17H17F3NO2S+ (M+1) 356.0927, found 356.0928.
3.2.1. 3-Trifluoromethylphenyl 2-amino-4,5,6,7-tetra-
hydrobenzo[b]thiophene-3-carboxamide (6b). Yield 20%.
1H NMR d 1.86 (m, 4H, 2· CH2), 2.57 (m, 2H, CH2),
2.78 (m, 2H, CH2), 5.97 (br s, 2H, NH2), 7.34 (d, 1H,
ArH), 7.43 (t, 1H, ArH), 7.71 (t, 2H, ArH), 7.84 (br s,
1H, NH). HR-ES MS calcd for C16H16F3N2OS+
(M+1) 341.0930, found 341.0948.
3.1.8.
4-Phenylbenzyl
2-amino-4,5,6,7-tetrahydro-
benzo[b]thiophene-3-carboxylate (5h). Yield 15%. 1H
NMR d 1.70–1.82 (m, 4H, 2· CH2), 2.51 (m, 2H,
CH2), 2.74 (m, 2H, CH2), 5.32 (s, 2H, CH2Ph), 5.97
(br s, 2H, NH2), 7.33–7.61 (m, 9H, ArH). HR-ES MS
calcd for C22H22NO2S+ (M+1) 364.1366, found
364.1366.
3.2.2. 3-Methoxyphenyl 2-amino-4,5,6,7-tetrahydro-
benzo[b]thiophene-3-carboxamide (6c). Yield 28%. 1H
NMR d 1.84–1.85 (m, 2H, 2· CH2), 2.57 (m, 2H,
CH2), 2.76 (m, 2H, CH2), 3.82 (s, 3H, OCH3), 5.95 (br
s, 2H, NH2), 6.65 (d, 1H, ArH), 6.99 (d, 1H, ArH),
7.18–7.31 (m, 2H, ArH), 7.51 (br s, 1H, NH). HR-ES
MS calcd for C16H19N2O2S+ (M+1) 303.1162, found
303.1150.
3.1.9. Benzyl 2-amino-5,6,7,8-tetrahydrocyclohepta[b]thi-
1
ophene-3-carboxylate (5i). Yield 49%. H NMR d 1.60–
1.68 (m, 4H, 2· CH2), 1.77–1.80 (m, 2H, CH2), 2.54–
2.58 (m, 2H, CH2), 2.98–3.01 (m, 2H, CH2), 5.30 (s,
2H, CH2Ph), 5.41 (br s, 2H, NH2), 7.30–7.41 (m, 5H,
ArH). HR-ES MS calcd for C17H20NO2S+ (M+1)
302.12093, found 302.11954.
3.2.3.
4-Chlorophenyl
2-amino-4,5,6,7-tetrahydro-
benzo[b]thiophene-3-carboxamide (6d). Yield 29%. 1H
NMR d 1.82 (m, 4H, 2· CH2), 2.55 (m, 2H, CH2),
2.73 (s, 2H, CH2), 5.94 (br s, 2H, NH2), 7.25 (d,
J = 8.7 Hz, 2H, ArH), 7.46 (d, J = 8.7 Hz, 2H, ArH),
7.57 (br s, 1H, NH). HR-ES MS calcd for
C15H16ClN2OS+ (M+1) 307.0666, found 307.0663.
3.1.10. 3-Trifluoromethylbenzyl 2-amino-5,6,7,8-tetrahy-
drocyclohepta[b]thiophene-3-carboxylate (5j). Yield 23%.
1H NMR d 1.57–1.68 (m, 4H, 2· CH2), 1.77–1.81 (m,
2H, CH2), 2.56–2.59 (m, 2H, CH2), 2.96–2.99 (m, 2H,
CH2), 5.33 (s, 2H, CH2Ph), 5.86 (br s, 2H, NH2),
7.46–7.68 (m, 5H, ArH). HR-ES MS calcd for
C18H19F3NO2S+ (M+1) 370.1083, found 370.1094.
3.2.4.
4-Bromophenyl
2-amino-4,5,6,7-tetrahydro-
benzo[b]thiophene-3-carboxamide (6e). Yield 88%. 1H
NMR d 1.82 (m, 4H, 2· CH2), 2.54 (m, 2H, CH2),
2.71 (m, 2H, CH2), 5.93 (br s, 2H, NH2), 7.37–7.43
(m, 4H, ArH), 7.58 (br s, 1H, NH). HR-ES MS
calcd for C15H16BrN2OS+ (M+1) 351.0161, found
351.0157.
3.1.11. Phenethyl 2-amino-5,6,7,8-tetrahydrocyclohep-
ta[b]thiophene-3-carboxylate (5k). Yield 71%. H NMR
1
d 1.53–1.71 (m, 4H, 2· CH2), 1.78–1.86 (m, 2H, CH2),
2.56–2.59 (m, 2H, CH2), 2.89–2.93 (m, 2H, CH2), 3.05
(t, J = 6.8 Hz, 2H, CH2), 4.51 (t, J = 6.8 Hz, 2H,
CH2Ph), 5.55 (br s, 1H, NH2), 7.22–7.36 (m, 4H,
ArH). HR-ES MS calcd for C18H22NO2S+ (M+1)
316.1366, found 316.1377.
3.2.5.
4-Iodophenyl
2-amino-4,5,6,7-tetrahydro-
benzo[b]thiophene-3-carboxamide (6f). Yield 24%. 1H
NMR d 1.82 (m, 4H, 2· CH2), 2.55 (m, 2H, CH2),
2.72 (m, 2H, CH2), 5.91 (br s, 2H, NH2), 7.30 (d,
J = 8.7 Hz, 2H, ArH), 7.58 (d, J = 8.7 Hz, 2H, ArH).
HR-ES MS calcd for C15H16IN2OS+ (M+1) 399.0023,
found 399.0021.
3.2. General procedure for N-phenyl 2-aminothiophene-3-
carboxamide formation (6b–g)
3.2.6. 4-Trifluoromethylphenyl 2-amino-4,5,6,7-tetra-
hydrobenzo[b]thiophene-3-carboxamide (6g). Yield 29%.
1H NMR d 1.82 (m, 4H, 2· CH2), 2.54 (m, 2H, CH2),
2.74 (m, 2H, CH2), 5.70 (br s, 2H, NH2), 7.53 (d,
The appropriate N-phenyl cyanoacetamide (1.0 molar
equiv), cyclohexanone or cycloheptanone (4.0 equiv),

G. Nikolakopoulos et al. / Bioorg. Med. Chem. 14 (2006) 2358–2365
2363
J = 8.7 Hz, 2H, ArH), 7.64 (d, J = 8.7 Hz, 2H, ArH),
7.79 (br s, 1H, NH). HR-ES MS calcd for
C16H16F3N2OS+ (M+1) 341.0930, found 341.0947.
3.4.3. 3-Methoxybenzyl 2-amino-4,5,6,7-tetrahydro-
benzo[b]thiophene-3-carboxamide (8c). Yield 30%. 1H
NMR d 1.78 (m, 4H, 2· CH2), 2.53–2.59 (m, 4H, 2·
CH2), 3.80 (s, 3H, OCH3), 4.55 (d, 2H, CH2Ph), 5.99
(br s, 2H, NH2), 6.80–6.94 (m, 3H, ArH), 7.25 (m, 1H,
ArH). HR-ES MS calcd for C17H21N2O2S+ (M+1)
317.1318, found 317.1314.
3.3. General procedure for 2-aminothiophene-3-carboxylic
acid formation (7a,b)
Ethyl 2-aminothiophene-3-carboxylate (1.0 equiv)
was dissolved in EtOH (10 mL) and KOH (4.0 mo-
lar equiv) in H2O (10 mL) was added and the
reaction was refluxed for ꢀ5 h. After cooling, the
reaction mixture was diluted with H2O and washed
with diethyl ether. The aqueous phase was then
adjusted to pH 6 with 1 M HCl and a pure solid
precipitated, which was collected by suction
filtration.
3.4.4.
4-Chlorobenzyl
2-amino-4,5,6,7-tetrahydro-
benzo[b]thiophene-3-carboxamide (8d). Yield 16%. 1H
NMR d 1.78 (m, 4H, 2· CH2), 2.53–2.58 (m, 2H, 2·
CH2), 4.53 (d, 2H, CH2Ph), 6.01 (br s, 3H, NH2 and
NH), 7.24 (d, J = 8.4 Hz, 2H, ArH), 7.29 (d,
J = 8.4 Hz, 2H, ArH). HR-ES MS calcd for
C16H18ClN2OS+ (M+1) 321.0823, found 321.0839.
3.4.5.
4-Bromobenzyl
2-amino-4,5,6,7-tetrahydro-
3.3.1.
2-Amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-
benzo[b]thiophene-3-carboxamide (8e). Yield 16%. 1H
NMR d 1.77 (m, 4H, 2· CH2), 2.53–2.58 (m, 4H, 2·
CH2), 4.50 (d, 2H, CH2Ph), 6.01 (m, 2H, NH2 and
NH), 7.18 (d, J = 8.3 Hz, 2H, ArH), 7.44 (d,
J = 8.3 Hz, 2H, ArH). HR-ES MS calcd for
C16H18BrN2OS+ (M+1) 365.0317, found 365.0332.
1
carboxylic acid (7a). Yield 62%. Mp = 116–118 ꢁC. H
NMR (DMSO-d6) d 1.65 (m, 4H, 2· CH2), 2.39 (m,
2H, CH2), 2.56 (m, 2H, CH2), 7.15 (br s, 2H, NH2).
LR-ES MS calcd for C9H10NO2Sꢁ (Mꢁ1) 196.2, found
196.1.
3.3.2. 2-Amino-5,6,7,8-tetrahydrocyclohepta[b]thiophene-
3-carboxylic acid (7b). Yield 77%. Mp = 100–101 ꢁC. 1H
NMR (DMSO-d6) d 1.50–1.52 (m, 4H, 2· CH2), 1.70–
1.77 (m, 2H, CH2), 2.49 (m, 2H, CH2), 2.88–2.92 (m,
2H, CH2), 7.03 (br s, 2H, NH2). LR-ES MS calcd for
C10H12NO2Sꢁ (Mꢁ1) 210.1, found 210.1.
3.4.6.
4-Iodobenzyl
2-amino-4,5,6,7-tetrahydro-
benzo[b]thiophene-3-carboxamide (8f). Yield 33%. Mp
1
152–154 ꢁC. H NMR d 1.78 (m, 4H, 2· CH2), 2.53–
2.58 (m, 2H, 2· CH2), 4.50 (d, 2H, CH2Ph), 6.01 (br s,
3H, NH2), 7.06 (d, J = 8.4 Hz, 2H, ArH), 7.64 (d,
J = 8.4 Hz, 2H, ArH). HR-ES MS calcd for C16H18I-
N2OS+ (M+1) 413.0179, found 413.0158.
3.4. General procedure for N-benzyl 2-aminothiophene-3-
carboxamide formation (8a–t)
3.4.7. 4-Trifluoromethylbenzyl 2-amino-4,5,6,7-tetra-
hydrobenzo[b]thiophene-3-carboxamide (8g). Yield 34%.
1H NMR d 1.79 (m, 4H, 2· CH2), 2.53–2.61 (m, 4H,
2· CH2), 4.61 (d, 2H, CH2Ph rotamers), 6.02 (br s,
2H, NH2), 6.12 (br s, 1H, NH), 7.42 (d, J = 8.1 Hz,
ArH), 7.58 (d, J = 8.1 Hz, 2H, ArH). HR-ES MS calcd
for C17H18F3N2OS+ (M+1) 355.1086, found 355.1083.
A solution of the 2-aminothiophene-3-carboxylic acid 7
(1.0 molar equiv) in CH2Cl2 (3 mL) was cooled to 0 ꢁC.
The appropriate benzyl amine (1.0 equiv) and a solu-
tion of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (1.0 equiv), 1-hydroxybenzotriazole
hydrate (1.0 equiv) and N,N-(dimethylamino)pyridine
(1.0 equiv) in CH2Cl2 (3 mL) were added. The mixture
was stirred for 16 h at room temperature before being
diluted with CHCl3 (10 mL) and washed with brine
(3 · 30 mL). After drying (anhydrous MgSO4) and fil-
tration, the solvent evaporated and the crude product
was purified by chromatography on a deactivated
SiO2 column eluting with hexane/ethyl acetate (2:1).
3.4.8.
3-Chlorobenzyl
2-amino-4,5,6,7-tetrahydro-
benzo[b]thiophene-3-carboxamide (8h). Yield 22%. 1H
NMR d 1.79 (m, 4H, 2· CH2), 2.53–2.61 (m, 4H, 2·
CH2), 4.55 (d, 2H, CH2Ph rotamers), 5.89 (br s, 1H,
NH), 6.05 (br s, 2H, NH2), 7.19–7.30 (m, 4H, ArH).
HR-ES MS calcd for C16H18ClN2OS+ (M+1)
321.0823, found 321.0829.
3.4.1. Benzyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thio-
phene-3-carboxamide (8a). Yield 13%. 1H NMR d
1.77 (m, 4H, 2· CH2), 2.53–2.59 (m, 4H, 2· CH2),
4.58 (d, 2H, CH2Ph), 5.57 (br s, 2H, NH2), 6.02 (br
s, 1H, NH), 7.24–7.39 (m, 5H, ArH). HR-ES MS
calcd for C16H19N2OS+ (M+1) 287.1213, found
287.1215.
3.4.9. 3-Nitrobenzyl 2-amino-5,6,7,8-tetrahydrocyclohep-
ta[b]thiophene-3-carboxamide (8i). Yield 6%. 1H NMR d
1.78 (m, 4H, 2· CH2), 2.52 (m, 2H, CH2), 2.63 (m, 2H,
CH2), 4.65 (d, 2H, CH2Ph), 6.02 (br s, 2H, NH2), 6.23
(br s, 1H, NH), 7.48 (t, J = 7.8 Hz, 1H, ArH), 7.66 (d,
J = 7.5 Hz, 1H, ArH), 8.07 (d, J = 7.5 Hz, 1H, ArH),
8.14 (s, 1H, ArH). HR-ES MS calcd for C16H18N3O3S+
(M+1) 332.1063, found 332.1064.
3.4.2. 3-Trifluoromethylbenzyl 2-amino-4,5,6,7-tetra-
hydrobenzo[b]thiophene-3-carboxamide (8b). Yield 36%.
1H NMR d 1.79 (m, 4H, 2· CH2), 2.54–2.61 (m, 4H,
2· CH2), 4.62 (d, 2H, CH2Ph), 6.02 (br s, 1H, NH),
6.10 (br s, 2H, NH2), 7.36–7.56 (m, 4H, ArH). HR-ES
MS calcd for C17H18F3N2OS+ (M+1) 355.1086, found
355.1082.
3.4.10. 4-Nitrobenzyl 2-amino-5,6,7,8-tetrahydrocyclo-
hepta[b]thiophene-3-carboxamide (8j). Yield 11%. 1H
NMR d 1.81 (m, 4H, 2· CH2), 2.55 (m, 2H, CH2),
2.62 (m, 2H, CH2), 4.67 (d, 2H, CH2Ph), 6.03 (br s,
2H, NH2), 6.17 (br s, 1H, NH), 7.47 (d, J = 8.7 Hz,
2H, ArH), 8.18 (d, J = 8.7 Hz, 2H, ArH). HR-ES MS

2364
G. Nikolakopoulos et al. / Bioorg. Med. Chem. 14 (2006) 2358–2365
calcd for C16H18N3O3S+ (M+1) 332.1063, found
332.1062.
3.4.18. 3-Chlorobenzyl 2-amino-5,6,7,8-tetrahydrocyclo-
hepta[b]thiophene-3-carboxamide (8r). Yield 23%. Mp
121–122 ꢁC. H NMR d 1.65–1.66 (m, 4H, 2· CH2),
1.79–1.81 (m, 2H, CH2), 2.58–2.61 (m, 2H, CH2),
2.73–2.76 (m, 2H, CH2), 4.55 (d, 2H, CH2Ph), 5.15 (br
s, 2H, NH2), 5.93 (br s, 1H, NH), 7.21–7.31 (m, 4H,
ArH). HR-ES MS calcd for C17H20ClN2OS+ (M+1)
335.0979, found 335.0986.
1
3.4.11.
Benzyl
2-amino-5,6,7,8-tetrahydrocyclohep-
ta[b]thiophene-3-carboxamide (8k). Yield 25%. 1H
NMR d 1.59–1.69 (m, 4H, 2· CH2), 1.76–1.84 (m, 2H,
CH2), 2.57–2.61 (m, 2H, CH2), 2.72–2.76 (m, 2H, CH2),
4.57 (d, 2H, CH2Ph), 5.12 (br s, 2H, NH2), 5.88 (br s,
1H, NH), 7.25–7.37 (m, 5H, ArH). HR-ES MS calcd for
C17H21N2OS+ (M+1) 301.1369, found 301.1368.
3.4.19. 3-Nitrobenzyl 2-amino-5,6,7,8-tetrahydrocyclo-
hepta[b]thiophene-3-carboxamide (8s). Yield 26%. Mp
141–143 ꢁC. 1H NMR d 1.64 (m, 4H, 2· CH2), 1.78–1.80
(m, 2H, CH2), 2.56–2.59 (m, 2H, CH2), 2.75–2.78 (m,
2H, CH2), 4.64 (d, 2H, CH2Ph), 4.88 (br s, 2H, NH2),
6.24 (br s, 1H, NH), 7.48 (t, J = 8.0 Hz, 1H, ArH), 7.66
(d, J = 7.5 Hz, 1H, ArH), 8.08 (d, J = 8.1 Hz, 1H, ArH),
8.15 (s, 1H, ArH). HR-ES MS calcd for C17H20N3O3S+
(M+1) 346.1220, found 346.1233.
3.4.12. 3-Trifluoromethylbenzyl 2-amino-5,6,7,8-tetrahy-
drocyclohepta[b]thiophene-3-carboxamide (8l). Yield
1
29%. Mp 118–120 ꢁC. H NMR d 1.65–1.68 (m, 4H, 2·
CH2), 1.78–1.82 (m, 2H, CH2), 2.59–2.62 (m, 2H, CH2),
2.74–2.78 (m, 2H, CH2), 4.64 (d, 2H, CH2Ph), 5.99 (br
s, 2H, NH2), 7.43–7.58 (m, 4H, ArH). HR-ES MS calcd
for C18H20F3N2OS+ (M+1) 369.1243, found 369.1241.
3.4.13. 3-Methoxybenzyl 2-amino-5,6,7,8-tetrahydrocy-
clohepta[b]thiophene-3-carboxamide (8m). Yield 29%.
Mp 103–105 ꢁC. 1H NMR d 1.63–1.66 (m, 4H, 2·
CH2), 1.78–1.80 (m, 2H, CH2), 2.57–2.60 (m, 2H,
CH2), 2.73–2.76 (m, 2H, CH2), 3.79 (s, 3H, OCH3),
4.54 (d, 2H, CH2Ph), 4.61 (br s, 1H, NH), 5.90 (br s,
2H, NH2), 6.80–6.91 (m, 3H, ArH), 7.22–7.27 (m, 1H,
ArH). HR-ES MS calcd for C18H23N2O2S+ (M+1)
331.1475, found 331.1471.
3.4.20. 4-Nitrobenzyl 2-amino-5,6,7,8-tetrahydrocyclo-
hepta[b]thiophene-3-carboxamide (8t). Yield 36%. Mp
155–157 ꢁC. H NMR d 1.67 (m, 4H, 2· CH2), 1.81–
1.82 (m, 2H, CH2), 2.60–2.63 (m, 2H, CH2), 2.76–2.78
(m, 2H, CH2), 4.67 (d, 2H, CH2Ph), 6.15 (br s, 1H,
NH), 7.49 (d, J = 8.1 Hz, 2H, ArH), 8.19 (d,
J = 8.1 Hz, 2H, ArH). HR-ES MS calcd for
C17H20N3O3S+ (M+1) 346.1220, found 346.1228.
1
3.4.14. 4-Chlorobenzyl 2-amino-5,6,7,8-tetrahydrocyclo-
hepta[b]thiophene-3-carboxamide (8n). Yield 26%. Mp
100–102 ꢁC. 1H NMR d 1.63–178 (m, 6H, 3· CH2),
2.57 (m, 2H, CH2), 2.71 (m, 2H, CH2), 4.49 (br s, 2H,
CH2Ph), 5.05 (br s, 2H, NH2), 6.05 (br s, 1H, NH),
7.25 (m, 4H, ArH). HR-ES MS calcd for
C17H20ClN2OS+ (M+1) 335.0979, found 335.0981.
3.5. Assay of AE activity6,7
The assay of AE activity consisted of three phases: (1)
binding to equilibrium of the agonist, 125I-ABA to the
A1AR–G protein ternary complex; (2) stabilization of
that complex by adding vehicle or AE for 5 min, and (3)
dissociation of the complex by adding a combination of
an A1AR antagonist, 100 lM BW-1433 and 25 lM
GTPcS for 10 min. Compounds were scored between
0% (no different than AE vehicle) and 100% (complete
abolition of 125I-ABA dissociation). The assay employed
membranes from CHO-K1 cells stably expressing the
hA1AR. For agonist binding to equilibrium (phase 1)
the buffer consisted of 10 mM HEPES, pH 7.2, containing
0.5 mM MgCl2, 1 U/mL adenosine deaminase, 0.5 nM
125I-ABA and 10 lg membrane protein in a final volume
of 100 lL applied to 96-well Millipore GF/C glass fibre
filter plates. After 90 min at room temperature, the addi-
tion 50 lL of AE (0.1–50 lM, final) initiated stabilization
of the ternary complex (phase 2). Five minutes later 50 lL
solution containing BW-1433 and GTPcS was added to
initiate the dissociation of the ternary complex. Ten min-
utes later membranes were filtered, washed, dried and
counted for residual 125I-ABA. The percentage of specif-
ically bound agonist remaining after 10 min of dissocia-
tion served as an index of AE activity:
3.4.15. 4-Bromobenzyl 2-amino-5,6,7,8-tetrahydrocyclo-
hepta[b]thiophene-3-carboxamide (8o). Yield 19%. Mp
1
145–146 ꢁC. H NMR d 1.60–1.69 (m, 4H, 2· CH2),
1.77–1.84 (m, 2H, CH2), 2.58–2.61 (m, 2H, CH2), 2.71–
2.75 (m, 2H, CH2), 4.51 (d, 2H, CH2Ph), 4.69 (br s, 2H,
NH2), 5.90 (br s, 1H, NH), 7.20 (d, J = 8.3 Hz, 2H,
ArH), 7.45 (d, J = 8.3 Hz, 2H, ArH). HR-ES MS calcd
for C17H20BrN2OS+ (M+1) 379.0474, found 379.0470.
3.4.16. 4-Iodobenzyl 2-amino-5,6,7,8-tetrahydrocyclohep-
ta[b]thiophene-3-carboxamide (8p). Yield 13%. Mp 162–
1
163 ꢁC. H NMR d 1.64–1.66 (m, 4H, 2· CH2), 1.77–
1.81 (m, 2H, CH2), 2.58 (m, 2H, CH2), 2.72 (m, 2H,
CH2), 4.51 (d, 2H, CH2Ph), 5.18 (br s, 1H, NH), 5.87
(br s, 2H, NH2), 7.08 (d, J = 8.1 Hz, 2H, ArH), 7.66
(d, J = 8.1 Hz, 2H, ArH). HR-ES MS calcd for C17H20I-
N2OS+ (M+1) 427.0336, found 427.0342.
3.4.17. 4-Trifluoromethylbenzyl 2-amino-5,6,7,8-tetrahy-
drocyclohepta[b]thiophene-3-carboxamide (8q). Yield
22%. Mp 130–132 ꢁC. H NMR d 1.63–1.68 (m, 4H,
%AE score ¼ 100 ꢂ ðB ꢁ BoÞ=ðBeq ꢁ BoÞ;
1
2· CH2), 1.76–1.83 (m, 2H, CH2), 2.56–2.60 (m, 2H,
CH2), 2.72–2.76 (m, 2H, CH2), 4.60 (d, 2H, CH2Ph),
6.10 (br s, 2H, NH2), 7.41 (d, J = 8.1 Hz, 2H, ArH),
7.57 (d, J = 8.1 Hz, 2H, ArH). HR-ES MS calcd for
C18H20F3N2OS+ (M+1) 369.1243, 369.1253.
where B is the residual binding (cpm) bound at the end of
10 min of dissociation in the presence of an AE, Bo is the
residual binding (cpm) at the end of 10 min of dissociation
in the absence of an AE and Beq is the cpm bound at the
end of 90 min of equilibrium binding.

G. Nikolakopoulos et al. / Bioorg. Med. Chem. 14 (2006) 2358–2365
2365
The percentage of specific binding remaining after
References and notes
10 min of dissociation constitutes an index of AE activ-
ity for ranking candidate compounds. A score of 100%
means no dissociation and a score of zero means com-
plete dissociation.
1. Bruns, R. F.; Fergus, J. H. Mol. Pharmacol. 1990, 38, 939–
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2. Bruns, R. F.; Fergus, J. H.; Coughenour, L. L.; Court-
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Pharmacol. 1990, 38, 950–958.
3. van der Klein, P. A. M.; Kourounakis, A. P.; IJzermann,
A. P. J. Med. Chem. 1999, 42, 3629–3635.
4. Kourounakis, A. P.; van der Klein, P. A. M.; IJzerman, A.
P. Drug Dev. Res. 2000, 49, 227–237.
5. Baraldi, P. G.; Zaid, A. N.; Lampronti, I.; Fruttarolo, F.;
Pavani, M.; Tabrizi, M. A.; Shryock, J. C.; Leung, E.;
Romagnoli, R. Bioorg. Med. Chem. Lett. 2000, 10, 1953–
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6. Tranberg, C. E.; Zickgraf, A.; Giunta, B. N.; Luetjens, H.;
Figler, H.; Murphree, L. J.; Falke, R.; Fleischer, H.;
Linden, J.; Scammells, P. J.; Olsson, R. A. J. Med. Chem.
2002, 45, 382–389.
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A.; Scammells, P. J. J. Med. Chem. 2003, 46, 1870–
1877.
3.6. Assay of A1AR antagonist activity
CHO-K1 membranes expressing human A1 adenosine
receptors were resuspended at 400 lg/mL in HE buffer
containing 1 U/mL adenosine deaminase. Fifty microli-
tres of membrane solution was added to 50 lL HE buffer
containing [3H]CPX (2 nM). Hundred microlitres of HE
buffer with either vehicle, enhancer or NECA (to define
non-specific) was added. The final drug concentrations
were 10 lM for enhancer and 100 lM for NECA. Sam-
ples were incubated for 90 min at room temperature, fil-
tered and counted on a liquid scintillation counter.
Binding was performed in triplicate and expressed as %
inhibition as compared to control binding.
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Acknowledgment
The authors thank the Victorian College of Pharmacy,
Monash University, for providing a postgraduate schol-
arship for G.N. The authors also wish to acknowledge
financial support from NIH Grant R01 HL56111.
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