Allosteric modulation of the adenosine A1 receptor. Synthesis and biological evaluation of novel 2-amino-3-benzoylthiophenes as allosteric enhancers of agonist binding

Article abstract of DOI:10.1021/jm991051d

Novel allosteric enhancers of agonist binding to the rat adenosine A₁ receptor are described. The lead compound for the new series was PD 81,723 ((2-amino-4,5-dimethyl-3-thienyl)[3-(trifluoromethyl)phenyl]methanone), a compound previously reported by Bruns and co-workers (Mol. Pharmacol. 1990, 38, 950-958). The 4,5-dimethyl group and the benzoyl moiety were targets for further modifications, leading to series of 4,5-dialkyl (12a-g), of tetrahydrobenzo (12h-u), and of tetrahydropyridine (13a-g) derivatives. A number of compounds, in particular 12b, 12e, 12j, 12n, and 12u, proved superior to PD 81,723. Their EC50 values for enhancing the binding of the adenosine A₁ receptor agonist N⁶-cyclopentyladenosine to the receptor were lower, and/or their antagonistic activity on the adenosine A₁ receptor was shown to be diminished.

Full text of DOI:10.1021/jm991051d

J . Med. Chem. 1999, 42, 3629-3635
3629
Alloster ic Mod u la tion of th e Ad en osin e A1 Recep tor . Syn th esis a n d Biologica l
Eva lu a tion of Novel 2-Am in o-3-ben zoylth iop h en es a s Alloster ic En h a n cer s of
Agon ist Bin d in g†
Pieter A. M. van der Klein,* Angeliki P. Kourounakis, and Ad P. IJ zerman
Division of Medicinal Chemistry, Leiden/ Amsterdam Center for Drug Research, P.O. Box 9502,
2300 RA Leiden, The Netherlands
Received March 24, 1999
Novel allosteric enhancers of agonist binding to the rat adenosine A1 receptor are described.
The lead compound for the new series was PD 81,723 ((2-amino-4,5-dimethyl-3-thienyl)[3-
(trifluoromethyl)phenyl]methanone), a compound previously reported by Bruns and co-workers
(Mol. Pharmacol. 1990, 38, 950-958). The 4,5-dimethyl group and the benzoyl moiety were
targets for further modifications, leading to series of 4,5-dialkyl (12a -g), of tetrahydrobenzo
(12h -u ), and of tetrahydropyridine (13a -g) derivatives. A number of compounds, in particular
12b, 12e, 12j, 12n , and 12u , proved superior to PD 81,723. Their EC50 values for enhancing
the binding of the adenosine A1 receptor agonist N6-cyclopentyladenosine to the receptor were
lower, and/or their antagonistic activity on the adenosine A1 receptor was shown to be
diminished.
In tr od u ction
concept. Such compounds inhibit the intracellular up-
take of extracellular adenosine, thereby effectively
Extracellular adenosine is regarded as a local hor-
mone with profound and ubiquitous physiological ef-
fects. This nucleoside is currently known to interact with
increasing its concentration outside the cell.11
We chose to focus on the second category, i.e., com-
pounds that enhance agonist action. In the early 1990s
Bruns and co-workers reported on various allosteric
modulators, 2-amino-3-benzoylthiophene derivatives ca-
pable of enhancing the binding and activity of reference
A1 receptor agonists, such as N6-cyclopentyladenosine
(CPA).12,13 One of these “allosteric modulators”, PD
81,723, has been examined pharmacologically in greater
detail by various independent research groups.14,15 It
was convincingly demonstrated that PD 81,723 also
enhances the action of endogenous adenosine, which
corroborates the concept mentioned above.16,17 Also,
long-term exposure of cells expressing the human ad-
enosine A1 receptor to PD 81,723 caused only little
desensitization and down-regulation, which is consid-
ered to be encouraging in terms of therapeutic poten-
tial.18
four different subtypes of adenosine receptors (A1, A2A
,
A2B, and A3), all members of the large superfamily of G
protein-coupled receptors.1-3 The wide distribution of
adenosine receptors offers both opportunities and draw-
backs for therapeutic intervention.4,5 As an example, A1
adenosine receptors are found in the CNS, in heart and
adipose tissue. Hence, agonists are capable of reducing
free-fatty-acid levels in the blood through their interac-
tion with adenosine A1 receptors on fat cells. This is a
useful feature in noninsulin-dependent diabetes melli-
tus (type II diabetes), since insulin’s action is then
sensitized.6 However, the concomitant bradycardia and
drop in mean arterial pressure due to interference with
cardiovascular adenosine receptors are to be considered
as serious side effects.7
Here we report our progress on a fundamentally
different approach. The formation of extracellular ad-
enosine as a breakdown product of ATP is a local
phenomenon, induced by a tissue at risk, e.g., under
hypoxic or anoxic conditions (heart failure, stroke,
epilepsy). This adenosine production is considered to be
tissue-protective.8-10 As a consequence, compounds that
would either augment the concentration of adenosine
or enhance its action locally might have a better
therapeutic profile than the agonists described above.
Marketed nucleoside transport blockers such as dipy-
ridamole and dilazep have already proven the first
In this study we describe the synthesis and biological
evaluation of mostly novel PD 81,723 analogues. We
have identified a number of compounds that appear
superior to PD 81,723 in their enhancing activity.
Ch em istr y
The synthesis of the 2-amino-3-benzoylthiophene
derivatives (12a -u , 13a -g, 14) was accomplished via
the routes illustrated in Schemes 1-3. The methods
used in these schemes were described previously.19,20
The appropriate carbonyl compounds (6a -c, 7, 9-11)
were reacted with benzoylacetonitrile derivatives (4a -
m ) and sulfur in ethanol in the presence of diethyl-
amine to provide the products in moderate to low
yields (Scheme 3). These benzoylacetonitrile derivatives
(Scheme 1), when not commercially available, were
synthesized by condensation of the acetonitrile anion
with the appropriate substituted methyl benzoates (1a -
† Abbreviations: [3H]DPCPX, [3H]-1,3-dipropyl-8-cyclopentylxan-
thine; [3H]CCPA, [3H]-2-chloro-N6-cyclopentyladenosine; CH3CN, ac-
etonitrile; CPA, N6-cyclopentyladenosine; HOAc, acetic acid; KOtBu,
potassium tert-butoxide; PD 81,723, (2-amino-4,5-dimethyl-3-thienyl)-
[3-(trifluoromethyl)phenyl]methanone; THF, tetrahydrofuran; TLC,
thin-layer chromatography.
* Corresponding author. Tel: 31-71-5274607. Fax: 31-71-5274565.
E-mail: klein_p@chem.leidenuniv.nl.
10.1021/jm991051d CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/19/1999

3630 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Sch em e 1. Synthesis of Benzoylacetonitrilesa
van der Klein et al.
ence compound PD 81,723 and many of the analogues
in the present study were capable of shifting the CPA
displacement curve to the left with an approximately
2-fold affinity shift. In this experimental setup it ap-
peared that many of the compounds at low CPA
concentrations displaced some of the [3H]DPCPX bind-
ing themselves. Therefore we decided to analyze the
enhancing activity more directly through its influence
on the dissociation rate of a radiolabeled agonist for the
adenosine A1 receptor.13 We used [3H]CCPA (2-chloro-
N6-cyclopentyladenosine) for that purpose. After 2.5 h
of incubation of receptor preparation with radioligand,
dissociation was induced by addition of 100 µM of CPA
in the absence or presence of 10 µM of putative
enhancer. After 60 min the reaction was stopped and
the difference in [3H]CCPA binding between the two
conditions was taken as a measure for enhancing
activity. In all experiments PD 81,723 was incorporated
as a reference substance. Its enhancing activity served
as a control and was set to 100%. Some of the more
potent compounds were evaluated at different concen-
trations which allowed the construction of concentra-
tion-effect curves and the determination of EC50 values.
The antagonistic effects of the compounds were assessed
by their inhibition of [3H]DPCPX binding at one con-
centration of 10 µM.
a
Reagents and conditions: (i) KOtBu, CH3CN, 50 °C; (ii) THF,
n-BuLi, -70 °C; (iii) 20% HCl.
Sch em e 2. Synthesis of Substituted Piperidonesa
Resu lts a n d Discu ssion
In Table 3 the biological effects of all compounds are
summarized. We first examined close analogues of PD
81,723, i.e., 4,5-dialkyl substituted 2-amino-3-benzoylth-
iophenes. 4,5-Dimethyl substitution as in PD 81,723 and
4-ethyl,5-methyl substitution led to compounds 12a -
g. Of those compounds, none was a more potent en-
hancer than PD 81,723, although chloro substitution in
either the meta or para position on the benzoyl ring led
to activities comparable with the meta trifluoromethyl
substituent of PD 81,723. In general, however, the
compounds were somewhat less active as antagonists
(Table 3), improving the window between enhancing and
antagonistic activity. This was particularly true for 12e
and 12g. Compound 12e has been reported before as
RS-74513-000. This PD 81,723 analogue displayed
enhancing activity on a field-stimulated isolated guinea
pig ileum preparation.23 An obvious extension to these
series were the “cyclized” PD 81,723 analogues 12h -u .
In this series we applied a “Topliss” approach24 by
varying substituents on the benzoyl ring system. Some
of the compounds (12a , 12d , 12h -j, 12k (PD 71,605),
12n , 13a , and 13e (PD 117,975)) were also reported on
by Bruns and co-workers.12,13 On the para position
halogen substitution was preferred over nitro (12q),
carboxylate (12t), and carboxylic ester (12s) groups.
Also, methyl and trifluoromethyl substitution on this
position yielded compounds (12r , 12m ) with higher
enhancing activity than PD 81,723. For those com-
pounds with the same substituent on either the meta
or the para position, it appeared that the para substitu-
tion was better (12p vs 12l and 12n vs 12k ). Interest-
ingly, combined meta/ para substitution as in the dichlo-
ro compound 12u seemed to have an additive effect,
since this derivative showed enhancing activity more
pronouncedly than either the meta or the para substi-
tuted chloro derivative. In a third series of 6-benzyl
substituted 2-amino-3-benzoyl-4,5,6,7-tetrahydrothieno-
a
Reagents and conditions: (i) benzyl chloride derivatives (8a -
c), (CH2Cl)2, Et3N, ∆T; (ii) BzCl, CH2Cl2, Et3N.
c) in moderate to low yields.21 Unfortunately, this
procedure failed for 4-nitro- and 4-iodobenzoylacetoni-
trile. However, the latter two benzoylacetonitriles (4d ,
4e) could be obtained in reasonable yield by condensa-
tion of 4-nitro- and 4-iodobenzoyl chloride (3a ,b) with
the dilithium salt of cyanoacetic acid (2).22 The ap-
propriate carbonyl compounds were commercially avail-
able except for the acylated and alkylated piperidones
(see Scheme 2). Alkylation was effected by reaction of
the hydrochloric salt of 4-piperidone (5) with substituted
benzyl chloride (8a -c) in dichloroethane in the presence
of triethylamine under reflux conditions for 16 h. After
workup and distillation, products 6a -c were obtained
in high yields. Acylation of the hydrochloric salt of
4-piperidone (5) with benzoyl chloride was easily ac-
complished in dichloromethane, using triethylamine as
a base, to afford 1-benzoyl-4-piperidone (7) in good yield.
All synthetic and physicochemical characteristics of
the compounds are listed in Tables 1 and 2.
Biology
All compounds were tested in radioligand binding
assays on adenosine A1 receptors, both for their enhanc-
ing and antagonistic activity. The enhancement of
agonist binding was demonstrated in two ways. First,
displacement studies with [3H]DPCPX as the radiola-
beled antagonist and CPA as the displacing agonist were
recorded in the absence and presence of 10 µM of a
putative allosteric enhancer. In such studies the refer-

Synthesis of Novel 2-Amino-3-benzoylthiophenes
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3631
Sch em e 3. Synthesis of 2-Amino-3-benzoylthiophenes
Ta ble 1. Physical and Synthetic Data for 2-Amino-3-benzoylthiophenes 12a -u
compd
R0
R4
CH3
CH3
CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
R5
mp, °C
cryst solventa
yield,b %
formulac
EI-MS m/z [M+]
12a 13
12b
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
133-134
114-116
123-125
129-131
100-101
99-102
108-110
152-153
145-147
122-123
114-115
160-162
145-147
140-142
152-153
150-152
202-204
155-156
159-160
232-233
152-154
a
a
a
a
a
a
a
b
c
c
a
a
a
a
a
c
a
a
a
d
a
12
15
10
17
12
16
21
52
10
37
34
20
30
46
35
36
15
25
15
93d
40
C13H13NOS
232
266
266
246
314
280
280
258
292
326
292
384
326
292
336
384
303
272
316
302
326
3-Cl
4-Cl
C13H12ClNOS
C13H12ClNOS
C14H15NOS
C15H14F3NOS
C14H14ClNOS
C14H14ClNOS
C15H15NOS
C15H14ClNOS
C16H14F3NOS
C15H14ClNOS
C15H14INOS
C16H14F3NOS
C15H14ClNOS
C15H14BrNOS
C15H14INOS
C15H14N2O3S
C16H17NOS
12c
12d 13
12e23
12f
H
3-CF3
3-Cl
4-Cl
12g
12h 19
12i20
12j13
12k 13,20
12l
H
-(CH2)4-
2-Cl
3-CF3
3-Cl
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
-(CH2)4-
3-I
12m
12n 13
12o
12p
12q
12r
12s
12t
12u
4-CF3
4-Cl
4-Br
4-I
4-NO2
4-CH3
4-CO2CH3
4-CO2H
3,4-Cl
C17H17NO3S
C16H15NO3S
C15H13Cl2NOS
a
Code: a, dichloromethane/n-hexane; b, toluene/n-hexane; c, ethanol; d, H2O. b Yields were not optimized. c All compounds were analyzed
d
for C, H, N; analytical results were within 0.4% of theoretical values. Prepared from 12s.
[2,3-c]pyridines (see also Table 2), other potent enhanc-
ers were identified. The two phenyl rings in this class
of compounds allow combined structural variations of
which we explored only chloro substitution in this study.
Again, on the benzoyl moiety, 3,4-dichloro substitution
was most favored (13g vs 13a and 13e). The benzyl
moiety was best substituted with a meta chloro sub-
stituent (13b vs 13a , 13c and 13d ). A benzoyl rather
than a benzyl moiety as in 14 provided only little
enhancing activity.
EC50 values were determined for the more potent
compounds. In our hands PD 81,723 had an EC50 value
of 15 µM, quite comparable to the value of 10 µM
reported before.13 It should be mentioned, however, that
its limited solubility precluded the recording of a full
concentration-effect curve (see also Figure 1). This was
easily achieved for the more potent compounds, of which
12j, 12m , 12p , and 12u proved to be 3-fold more potent
than PD 81,723 (Table 3 and Figure 1). These materials
proved to have the same efficacy, i.e., they achieved the
same maximal effect.
Subsequently, we analyzed the antagonistic behavior
of the compounds in a simple binding assay with [3H]-
DPCPX as the radioligand and the individual com-
pounds at one single concentration of 10 µM (Table 3).
PD 81,723 displaced approximately 40% of the radioli-
gand, which on further analysis of more concentrations
yielded a Ki value of 11 µM. All other compounds in the
present study showed some antagonistic behavior too,
although to varying extents. Apparently, it is possible
to achieve some separation between enhancing and
antagonistic activity. In this respect, a few compounds
warrant further discussion. The close PD 81,723 ana-
logues 12b and 12e are equipotent to PD 81,723 in their

3632 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
van der Klein et al.
Ta ble 2. Physical and Synthetic Data for 2-Amino-3-benzoyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridines 13a -g and 14
compd
R0
R1
mp, °C
cryst solventa
yield,b %
formulac
EI-MS m/z [M+]
13a 13
13b
13c
13d
13e13
13f
H
H
H
H
4-Cl
4-Cl
H
178-180
152-153
148-149
186-188
144-146
100-102
85-87
a
a
a
a
b
c
a
a
50
30
18
44
38
45
50
40
C21H20N2OS
349
383
383
417
383
451
417
363
3-Cl
4-Cl
3,4-Cl
H
3,4-Cl
H
C
C
C
21H19ClN2OS
21H19ClN2OS
21H18Cl2N2OS
C21H19ClN2OS
C
C
C
21H17Cl3N2OS
21H18Cl2N2OS
21H18N2O2S
13g
14
3,4-Cl
233-235
a
b
Code: a, ethanol; b, ether/n-hexane; c, 2-propanol. Yields were not optimized. c All compounds were analyzed for C, H, N; analytical
results were within 0.4% of theoretical values.
Ta ble 3. Enhancing and Antagonistic Activity of Compounds
12a -u , 13a -g, and 14
compd
% enhancementa
EC50 (µM)b % antagonismc
PD 81,723
12a
12b
12c
12d
12e
12f
12g
12h
12i
12j
12k
12l
12m
12n
12o
12p
12q
12r
12s
12t
12u
13a
13b
13c
100.0
8.0 ((5.5)
80.3 ((19.0)
93.5 ((31.6)
31.4 ((4.4)
111.9 ((10.5)
29.9 ((7.5)
97.2 ((24.9)
47.2 ((4.6)
72.5 ((19.3)
122.0 ((19.3)
92.7 ((6.4)
14.7 ((1.4)
39.5 ((4.3)
13.9 ((3.2)
18.7 ((4.1)
41.0 ((6.1)
12.6 ((3.0)
5.4 ((10.9)
22.3 ((2.0)
19.8 ((12.2)
34.7 ((5.8)
35.1 ((3.4)
32.2 ((8.4)
50.6 ((4.9)
65.8 ((0.9)
56.7 ((3.9)
39.9 ((5.5)
41.6 ((3.7)
64.4 ((8.1)
19.5 ((2.5)
30.3 ((2.9)
28.9 (n ) 1)
nd
34.7 ((4.5)
67.1 ((5.5)
80.1 ((1.2)
52.2 ((2.5)
4.0 ((2.0)
60.9 ((0.1)
45.7 ((2.1)
51.0 ((0.0)
71.8 ((2.1)
56.2 ((5.1)
6.0 ((1.0)
10.5 ((0.9)
113.4 ((18.4)
131.2 ((11.0)
122.8 ((14.6)
128.2 ((18.3)
154.7 ((21.0)
33.9 ((21.7)
137.2 ((20.9)
43.9 ((8.9)
F igu r e 1. Concentration-effect curves for derivative 12u and
PD 81,723 from one representative experiment. Enhancement
of 100% is equivalent to the maximum decrease in [3H]CCPA
dissociation by the highest concentration of 12u .
4.7 ((0.9)
6.8 ((0.2)
16.4 ((0.3)
4.5 ((0.2)
A quantitative structure-activity analysis (QSAR)
was not feasible due to solubility problems that pre-
vented the determination of some EC50 values. However,
a more qualitative approach suggests that lipophilicity
is of prime importance on the benzoyl ring system of
series 12 and 13. Lipophilic meta and/or para substit-
uents such as halogen are preferred for enhancing
activity, whereas more hydrophilic groups such as nitro
(12q) and carboxylate (12t) are not favorable. Electronic
influences are probably small, since substituents dif-
ferent in this respect (e.g., methyl and chloro) have
similar enhancing effects (cf. 12n and 12r ).
34.9 ((1.4)
28.6 ((3.3)
151.0 ((24.2)
52.8 ((36.6)
105.7 ((27.3)
69.0 ((23.5)
57.0 ((35.8)
132.4 ((21.0)
106.5 ((30.6)
173.6 ((37.5)
13.8 ((27.3)
14.9 ((7.5)
6.2 ((0.9)
15.1 (n ) 1)
13d
13e
13f
13g
14
theophylline
11.3 ((1.1)
32.0 ((1.1)
9.2 ((1.4)
a
Enhancing activity by 10 µM of test compound is expressed
as percent decrease ((SEM) in [3H]CCPA dissociation over control
b
In conclusion, we have identified novel allosteric
enhancers of agonist binding to adenosine A1 receptors.
Some of the compounds proved superior to the reference
material PD 81,723. It is hoped that these derivatives
may be of help in better understanding adenosine
receptor function. It is tempting to speculate that
allosteric modulation of G protein-coupled receptors is
a more generally valid concept. This prospect seems
realistic, since examples for other receptors such as
muscarinic,25 R2-adrenergic,26 and serotonin27 are al-
ready at hand. In that case, the development of new
chemical entities with a mechanism of action funda-
mentally different from “classical” agonists and antago-
nists is anticipated.
(0%) and that of 10 µM PD 81,723 (100.0%, n ) 3). EC50 for
enhancing activity ((SEM, n ) 3). c Antagonistic activity is
expressed as percent displacement ((SEM, n ) 3-5) of 0.4 nM of
[3H]DPCPX by 10 µM of test compound. nd: not determined.
enhancing activity, but have less antagonistic activity
(Table 3). Compounds 12j, 12n , and 12u are more
potent enhancers than PD 81,723, but have comparable
antagonistic activity (Table 3). For reasons of compari-
son, data for theophylline, a classical competitive ad-
enosine receptor antagonist, were also incorporated in
Table 3. It had negligible enhancing activity while
showing modest antagonistic activity at 10 µM, compa-
rable to PD 81,723 and derivatives.

Synthesis of Novel 2-Amino-3-benzoylthiophenes
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3633
for 16 h, CH2Cl2 (50 mL) was added. The mixture was washed
with water (20 mL), dried (MgSO4), and evaporated. The
residual oil was distilled (oil pump, 0.05-0.1 mmHg) to give
pure product.
1-(3-Ch lor oben zyl)-4-p ip er id on e (6a ) was prepared as
described above in a yield of 85% (oil pump, 0.05-0.1 mmHg,
145 °C): 1H NMR (CDCl3) δ 2.42-2.48 (m, 4H, 2 × CH2), 2.71-
2.77 (m, 4H, 2 × CH2), 3.60 (bs, 2H, CH2Ph), 7.25-7.38 (m,
4H, Harom).
1-(4-Ch lor oben zyl)-4-p ip er id on e (6b) was prepared as
described above in a yield of 80% (oil pump, 0.05-0.1 mmHg,
140-150 °C): 1H NMR (CDCl3) δ 2.42-2.45 (m, 4H, 2 × CH2),
2.68-2.71 (m, 4H, 2 × CH2), 3.56 (bs, 2H, CH2Ph), 7.29-7.32
(m, 4H, Harom).
1-(3,4-Dich lor oben zyl)-4-p ip er id on e (6c) was prepared
as described above in a yield of 75% (oil pump, 0.05-0.1
mmHg, 175 °C): 1H NMR (CDCl3) δ 2.43-2.48 (m, 4H, 2 ×
CH2), 2.71-2.74 (m, 4H, 2 × CH2), 3.57 (bs, 2H, CH2Ph), 7.23-
7.49 (m, 3H, Harom).
1-Ben zoyl-4-p ip er id on e (7). To a mixture of 4-piperidone
mono hydrate mono hydrochloric acid (5, 3.9 g, 25 mmol) and
benzoyl chloride (2.95 mL, 25 mmol) in CH2Cl2 (50 mL) was
added Et3N (10 mL). After the mixture was stirred for 16 h,
CH2Cl2 (50 mL) was added. The mixture was washed with
water (20 mL), dried (MgSO4), and evaporated. The residue
was purified by column chromatography to afford 7 in 80%:
MS m/z 204 [MH]+.
Exp er im en ta l Section
Ch em ica ls a n d Solven ts. All chemicals and solvents used
were commercially available unless stated otherwise. [3H]-
DPCPX ([3H]-8-cyclopentyl-1,3-dipropyl-xanthine, 120 Ci/
mmol, antagonist) and [3H]CCPA (2-chloro-N6-[3H]-cyclopen-
tyladenosine, 30 Ci/mmol, agonist) were purchased from NEN,
and N6-cyclopentyladenosine (CPA, agonist) was purchased
from RBI.
Ch r om a togr a p h y. Thin-layer chromatography (TLC) was
carried out using aluminum sheets (20 cm × 20 cm) with silica
gel F254 from Merck. Spots were visualized under UV light (254
nm). Column chromatography was performed on columns of
silica gel 60 (Merck 230-400 mesh).
In str u m en ts a n d An a lyses. Elemental analyses were
performed for C, H, and N (Microanalytical Laboratory,
Department of Chemistry, University College, Dublin, Ire-
land). 1H NMR spectra were measured at 200 MHz with a
J EOL J NM-FX 200 spectrometer equipped with a PG 200
computer operating in the Fourier transform mode. Chemical
shifts are given in ppm (δ) relative to tetramethylsilane (TMS)
as internal standard.
All high-resolution mass spectra were measured on
a
Finnigan MAT900 mass spectrometer equipped with a direct
insertion probe for EI experiments (70 eV with resolution
1000).
Melting points were determined in a Bu¨chi capillary melting
point apparatus and are uncorrected.
Gen er a l P r oced u r e for th e Syn th esis of 2-Am in o-3-
ben zoylth iop h en es 12a-u , 13a -g, a n d 14.19,20 To a suspen-
sion of 5 mmol of carbonyl compound, 5 mmol of benzoylace-
tonitrile derivative, and 5.05 mmol of sulfur in 1.5 mL of
ethanol was added 1 mL of Et2NH. The mixture was stirred
for 2 h at 50 °C. The mixture was cooled to room temperature.
If the product crystallized from the crude reaction mixture,
the precipitate was collected and recrystallized from the
appropriate solvent. When no crystallization occurred, the
mixture was evaporated and crystallized from the appropriate
solvent, after column chromatography purification.
(2-Am in o-4,5-d im et h yl-3-t h ien yl)(p h en yl)m et h a n on e
(12a )13 was prepared as described above, starting from methyl
ethyl ketone and benzoylacetonitrile: 1H NMR (CDCl3) δ 1.54
(s, 3H, CH3), 2.13 (s, 3H, CH3), 6.40 (bs, 2H, NH2), 7.39-7.54
(m, 5H, Harom).
(2-Am in o-4,5-dim eth yl-3-th ien yl)(3-ch lor oph en yl)m eth -
a n on e (12b) was prepared as described above, starting from
methyl ethyl ketone and 3-chlorobenzoylacetonitrile: 1H NMR
(CDCl3) δ 1.52 (s, 3H, CH3), 2.10 (s, 3H, CH3), 6.75 (bs, 2H,
NH2), 7.30-7.49 (m, 4H, Harom).
(2-Am in o-4,5-dim eth yl-3-th ien yl)(4-ch lor oph en yl)m eth -
a n on e (12c) was prepared as described above, starting from
methyl ethyl ketone and 4-chlorobenzoylacetonitrile: 1H NMR
(CDCl3) δ 1.55 (s, 3H, CH3), 2.13 (s, 3H, CH3), 7.35-7.49 (m,
6H, NH2, Harom).
Gen er a l P r oced u r e for th e Syn th esis of Ben zoyla c-
eton itr ile Der iva tives.21,22 Meth od A. To a mixture of 50
mmol of methyl benzoate derivative 1a -c and 62 mmol of
acetonitrile was added 45 mmol of KOtBu. The suspension was
heated at 50 °C for 4 h. Water (25 mL) was added, and this
solution was washed with CH2Cl2. The aqueous phase was
acidified with concentrated HCl and extracted with CH2Cl2
(3×). The combined organic layers were washed with 10%
NaHCO3, dried (MgSO4), and evaporated to give a solid.
Meth od B. A solution of cyanoacetic acid (2, 2 equiv) was
dissolved in THF and cooled to -70 °C. To the mixture was
added n-BuLi (4 equiv), and the reaction temperature was then
allowed to rise to 0 °C. The mixture was stirred for 0.5 h at 0
°C and then recooled to -70 °C, and a solution of acid chloride
3a ,b (1 equiv) in THF was added. After being stirred for 1 h,
the mixture was allowed to gradually come to room temper-
ature over a period of 1 h. Then aqueous HCl (20%) was added,
and the solution was extracted with CH2Cl2 (2×). The com-
bined organic layers were washed with 10% NaHCO3, dried
(MgSO4), and evaporated to give a solid. After purification by
column chromatography the products were used in the next
step.
4-Tr iflu or om et h ylben zoyla cet on it r ile (4a ) was pre-
pared according to method A, starting from methyl 4-trifluo-
romethylbenzoate in a yield of 15%: 1H NMR (CDCl3) δ 3.25
(bs, 2H, CH2), 7.21-8.09 (m, 4H, Harom).
(2-Am in o-4-et h yl-5-m et h yl-3-t h ien yl)(p h en yl)m et h a -
n on e (12d )13 was prepared as described above, starting from
3-pentanone and benzoylacetonitrile: 1H NMR (CDCl3) δ 0.71
(t, 3H, CH3), 2.09 (q, 2H, CH2), 2.16 (s, 3H, CH3), 6.10 (bs, 2H,
NH2), 7.30-7.65 (m, 5H, Harom).
(2-Am in o-4-eth yl-5-m eth yl-3-th ien yl)[3-(tr iflu or om eth -
yl)p h en yl]m eth a n on e (12e)23 was prepared as described
above, starting from 3-pentanone and 3-trifluoromethylben-
zoylacetonitrile: 1H NMR (CDCl3) δ 0.68 (t, 3H, CH3), 2.00
(q, 2H, CH2), 2.16 (s, 3H, CH3), 6.55 (bs, 2H, NH2), 7.54-7.80
(m, 4H, Harom).
(2-Am in o-4-eth yl-5-m eth yl-3-th ien yl)(3-ch lor op h en yl)-
m eth a n on e (12f) was prepared as described above, starting
from 3-pentanone and 3-chlorobenzoylacetonitrile: 1H NMR
(CDCl3) δ 0.71 (t, 3H, CH3), 2.08 (q, 2H, CH2), 2.13 (s, 3H,
CH3), 6.40 (bs, 2H, NH2), 7.30-7.60 (m, 4H, Harom).
(2-Am in o-4-eth yl-5-m eth yl-3-th ien yl)(4-ch lor op h en yl)-
m eth a n on e (12g) was prepared as described above, starting
from 3-pentanone and 4-chlorobenzoylacetonitrile: 1H NMR
(CDCl3) δ 0.71 (t, 3H, CH3), 2.09 (q, 2H, CH2), 2.16 (s, 3H,
CH3), 6.00 (bs, 2H, NH2), 7.36-7.55 (m, 4H, Harom).
4-Br om oben zoyla ceton itr ile (4b) was prepared according
to method A, starting from methyl 4-bromobenzoate in a yield
of 27%: 1H NMR (CDCl3) δ 2.91 (s, 2H, CH2), 7.85-8.25 (m,
4H, Harom).
4-Meth ylben zoyla ceton itr ile (4c) was prepared according
to method A, starting from methyl 4-methylbenzoate in a yield
of 30%: 1H NMR (CDCl3) δ 1.61 (s, 3H, CH3), 4.05 (s, 2H, CH2),
7.29-7.83 (m, 4H, Harom).
4-Nitr oben zoyla ceton itr ile (4d ) was prepared according
to method B, starting from 4-nitrobenzoyl chloride in a yield
of 51%: 1H NMR (CDCl3) δ 4.01 (bs, 2H, CH2), 7.69-7.94 (m,
4H, Harom).
4-Iod oben zoyla ceton itr ile (4e) was prepared according
to method B, starting from 4-iodobenzoyl chloride in a yield
of 20%: 1H NMR (CDCl3) δ 4.85 (bs, 2H, CH2), 7.54-7.90 (m,
4H, Harom).
Gen er a l P r oced u r e for th e Syn th esis of Su bstitu ted
1-Ben zyl-4-p ip er id on es 6a -c. To a mixture of 50 mmol of
4-piperidone monohydrate mono hydrochloric acid (5) and 50
mmol of benzyl chloride derivative 8a -c in dichloroethane (50
mL) was added Et3N (14 mL). After the mixture was refluxed

3634 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
van der Klein et al.
(2-Am in o-4,5,6,7-tetr ah ydr oben zo[b]th ioph en -3-yl)(ph e-
n yl)m eth a n on e (12h )19 was prepared as described above,
starting from cyclohexanone and benzoylacetonitrile: mp 155-
KOH (1 M, 5 mL). The mixture was stirred for 0.5 h at 50 °C.
Then MeOH was evaporated, and aqueous HCl (1 M) was
added until the product precipitated. Now the mixture was
stirred at 0 °C for 0.5 h and filtered. The resulting solid was
dried to afford 12t (140 mg, 93%): 1H NMR (CD3OD) δ 1.40-
1.49 (m, 2H, CH2), 1.64-1.70 (m, 4H, 2 × CH2), 2.42-2.48 (m,
2H, CH2), 7.43-8.09 (m, 4H, Harom).
(2-Am in o-4,5,6,7-tetr a h ydr oben zo[b]th iop h en -3-yl)(3,4-
d ich lor op h en yl)m eth a n on e (12u ) was prepared as de-
scribed above, starting from cyclohexanone and 3,4-dichlo-
robenzoylacetonitrile: 1H NMR (CDCl3) δ 1.46-1.54 (m, 2H,
CH2), 1.67-1.83 (m, 4H, 2 × CH2), 2.45-2.51 (m, 2H, CH2),
7.11 (bs, 2H, NH2), 7.27-7.7.56 (m, 3H, Harom).
(2-Am in o-6-ben zyl-4,5,6,7-tetr a h yd r oth ien o[2,3-c]p yr i-
d in -3-yl)(p h en yl)m eth a n on e (13a )13 was prepared as de-
scribed above, starting from 1-benzyl-4-piperidone and ben-
zoylacetonitrile: 1H NMR (DMSO-d6) δ 1.71 (m, 2H, CH2CH2N),
2.34 (m, 2H, CH2CH2N), 3.27 (m, 2H, NCH2), 3.53 (m, 2H, CH2-
Ph), 7.27-7.44 (m, 10H, Harom), 8.20 (bs, 2H, NH2).
(2-Am in o-6-(3-ch lor oben zyl)-4,5,6,7-tetr a h yd r oth ien o-
[2,3-c]p yr id in -3-yl)(p h en yl)m eth a n on e (13b) was prepared
as described above, starting from 1-(3-chlorobenzyl)-4-piperi-
done and benzoylacetonitrile: 1H NMR (CDCl3) δ 1.90-1.96
(m, 2H, CH2CH2N), 2.43-2.49 (m, 2H, CH2CH2N), 3.42 (bs,
2H, NCH2), 3.59 (bs, 2H, CH2Ph), 6.80 (bs, 2H, NH2), 7.19-
7.51 (m, 9H, Harom).
1
153 °C (lit.19 155 °C); H NMR (CDCl3) δ 1.40-1.62 (m, 2H,
CH2), 1.68-1.80 (m, 4H, 2 × CH2), 2.48-2.55 (m, 2H, CH2),
6.65 (bs, 2H, NH2), 7.38-7.60 (m, 5H, Harom).
(2-Am in o-4,5,6,7-tetr a h yd r oben zo[b]th iop h en -3-yl)(2-
ch lor op h en yl)m eth a n on e (12i) was prepared as described
above, starting from cyclohexanone and 2-chlorobenzoylaceto-
nitrile: mp 145-147 °C (lit.20 155-157 °C); 1H NMR (CDCl3)
δ 1.44-1.71 (m, 6H, 3 × CH2), 2.44-2.49 (m, 2H, CH2), 7.21-
7.37 (m, 4H, Harom).
(2-Am in o-4,5,6,7-tetr a h yd r oben zo[b]th iop h en -3-yl)[3-
(tr iflu or om eth yl)p h en yl]m eth a n on e (12j) was prepared as
described above, starting from cyclohexanone and 3-trifluo-
romethylbenzoylacetonitrile: 1H NMR (CDCl3) δ 1.41-1.63 (m,
2H, CH2), 1.68-1.79 (m, 4H, 2 × CH2), 2.47-2.54 (m, 2H, CH2),
7.00 (bs, 2H, NH2), 7.48-7.72 (m, 4H, Harom).
(2-Am in o-4,5,6,7-tetr a h yd r oben zo[b]th iop h en -3-yl)(3-
ch lor op h en yl)m eth a n on e (12k )13,20 was prepared as de-
scribed above, starting from cyclohexanone and 3-chloroben-
zoylacetonitrile: mp 114-115 °C (lit.20 108-110 °C); 1H NMR
(CDCl3) δ 1.42-1.58 (m, 2H, CH2), 1.70-1.87 (m, 4H, 2 × CH2),
2.47-2.55 (m, 2H, CH2), 6.88 (bs, 2H, NH2), 7.31-7.44 (m, 4H,
H
arom).
(2-Am in o-4,5,6,7-tetr a h yd r oben zo[b]th iop h en -3-yl)(3-
iod op h en yl)m eth a n on e (12l) was prepared as described
above, starting from cyclohexanone and 3-iodobenzoylaceto-
nitrile: 1H NMR (CDCl3) δ 1.47-1.82 (m, 6H, 3 × CH2), 2.49-
2.55 (m, 2H, CH2), 6.80 (bs, 2H, NH2), 7.14-7.79 (m, 4H, Harom).
(2-Am in o-4,5,6,7-tetr a h yd r oben zo[b]th iop h en -3-yl)[4-
(tr iflu or om eth yl)p h en yl]m eth a n on e (12m ) was prepared
as described above, starting from cyclohexanone and 4-tri-
fluoromethylbenzoylacetonitrile: 1H NMR (CDCl3) δ 1.48-1.50
(m, 2H, CH2), 1.69-1.76 (m, 4H, 2 × CH2), 2.48 (m, 2H, CH2),
6.91 (bs, 2H, NH2), 7.54-7.69 (m, 4H, Harom).
(2-Am in o-4,5,6,7-tetr a h yd r oben zo[b]th iop h en -3-yl)(4-
ch lor op h en yl)m eth a n on e (12n )13 was prepared as described
above, starting from cyclohexanone and 4-chlorobenzoylaceto-
nitrile: 1H NMR (CDCl3) δ 1.47-1.52 (m, 2H, CH2), 1.71-1.81
(m, 4H, 2 × CH2), 2.47-2.54 (m, 2H, CH2), 6.71 (bs, 2H, NH2),
7.34-7.45 (m, 4H, Harom).
(2-Am in o-6-(4-ch lor oben zyl)-4,5,6,7-tetr a h yd r oth ien o-
[2,3-c]p yr id in -3-yl)(p h en yl)m eth a n on e (13c) was prepared
as described above, starting from 1-(4-chlorobenzyl)-4-piperi-
done and benzoylacetonitrile: 1H NMR (CDCl3) δ 1.89-1.91
(m, 2H, CH2CH2N), 2.42-2.47 (m, 2H, CH2CH2N), 3.40 (bs,
2H, NCH2), 3.57 (bs, 2H, CH2Ph), 6.80 (bs, 2H, NH2), 7.24-
7.47 (m, 9H, Harom).
(2-Am in o-6-(3,4-d ich lor ob e n zyl)-4,5,6,7-t e t r a h yd r o-
th ien o[2,3-c]p yr id in -3-yl)(p h en yl)m eth a n on e (13d ) was
prepared as described above, starting from 1-(3,4-dichloroben-
zyl)-4-piperidone and benzoylacetonitrile: 1H NMR (CDCl3) δ
1.92-1.95 (m, 2H, CH2CH2N), 2.43-2.48 (m, 2H, CH2CH2N),
3.40 (bs, 2H, NCH2), 3.55 (bs, 2H, CH2Ph), 6.79 (bs, 2H, NH2),
7.19-7.47 (m, 8H, Harom).
(2-Am in o-6-ben zyl-4,5,6,7-tetr a h yd r oth ien o[2,3-c]p yr i-
d in -3-yl)(4-ch lor op h en yl)m eth a n on e (13e)13 was prepared
as described above, starting from 1-benzyl-4-piperidone and
4-chlorobenzoylacetonitrile: 1H NMR (CDCl3) δ 1.90-1.96 (m,
2H, CH2CH2N), 2.46-2.51 (m, 2H, CH2CH2N), 3.41 (bs, 2H,
NCH2), 3.61 (bs, 2H, CH2Ph), 6.79 (bs, 2H, NH2), 7.27-7.45
(m, 9H, Harom).
(2-Am in o-4,5,6,7-tetr a h yd r oben zo[b]th iop h en -3-yl)(4-
br om op h en yl)m eth a n on e (12o) was prepared as described
above, starting from cyclohexanone and 4-bromobenzoylaceto-
nitrile: 1H NMR (CDCl3) δ 1.46-1.54 (m, 2H, CH2), 1.67-1.83
(m, 4H, 2 × CH2), 2.46-2.51 (m, 2H, CH2), 6.80 (bs, 2H, NH2),
7.33-7.55 (m, 4H, Harom).
(2-Am in o-6-(3,4-d ich lor ob e n zyl)-4,5,6,7-t e t r a h yd r o-
t h ien o[2,3-c]p yr id in -3-yl)(4- ch lor op h en yl)m et h a n on e
(13f) was prepared as described above, starting from 1-(3,4-
dichlorobenzyl)-4-piperidone and 4-chlorobenzoylacetonitrile:
1H NMR (CDCl3) δ 1.93-1.97 (m, 2H, CH2CH2N), 2.45-2.48
(m, 2H, CH2CH2N), 3.38 (bs, 2H, NCH2), 3.56 (bs, 2H, CH2-
Ph), 6.86 (bs, 2H, NH2), 7.15-7.48 (m, 7H, Harom).
(2-Am in o-4,5,6,7-tetr a h yd r oben zo[b]th iop h en -3-yl)(4-
iod op h en yl)m eth a n on e (12p ) was prepared as described
above, starting from cyclohexanone and 4-iodobenzoylaceto-
nitrile: 1H NMR (CDCl3) δ 1.41-1.87 (m, 6H, 3 × CH2), 2.43-
2.52 (m, 2H, CH2), 6.82 (b, 2H, NH2), 7.17-7.75 (m, 4H, Harom).
(2-Am in o-4,5,6,7-tetr a h yd r oben zo[b]th iop h en -3-yl)(4-
m eth ylp h en yl)m eth a n on e (12q) was prepared as described
above, starting from cyclohexanone and 4-methylbenzoylac-
etonitrile: 1H NMR (CDCl3) δ 1.45-1.50 (m, 2H, CH2), 1.70-
1.91 (m, 4H, 2 × CH2), 2.38 (s, 3H, CH3), 2.47-2.54 (m, 2H,
CH2), 6.05-6.60 (b, 2H, NH2), 7.16-7.42 (m, 4H, Harom).
(2-Am in o-4,5,6,7-tetr a h yd r oben zo[b]th iop h en -3-yl)(4-
n itr op h en yl)m eth a n on e (12r ) was prepared as described
above, starting from cyclohexanone and 4-nitrobenzoylaceto-
nitrile: 1H NMR (CDCl3) δ 0.85-1.16 (m, 6H, 3 × CH2), 1.88-
2.00 (m, 2H, CH2), 6.43 (bs, 2H, NH2), 7.01-7.69 (m, 4H, Harom).
Meth yl 4[(2-Am in o-4,5,6,7-tetr ah ydr oben zo[b]th ioph en -
3-yl)ca r bon yl]ben zoa te (12s) was prepared as described
above, starting from cyclohexanone and methyl 4-(2-cyanoacetyl-
)benzoate: 1H NMR (CDCl3) δ 1.41-1.47 (m, 2H, CH2), 1.68-
1.73 (m, 4H, 2 × CH2), 2.45-2.51 (m, 2H, CH2), 3.94 (s, 3H,
OCH3), 7.12 (bs, 2H, NH2), 7.49-8.10 (m, 4H, Harom).
4-[(2-Am in o-4,5,6,7-tetr a h yd r oben zo[b]th iop h en -3-yl)-
ca r bon yl]ben zoic Acid (12t). To a solution of methyl 4-[(2-
amino-4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl)carbonyl]-ben-
zoate (12s, 160 mg, 0.5 mmol) in MeOH (2 mL) was added
(2-Am in o-6-ben zyl-4,5,6,7-tetr a h yd r oth ien o[2,3-c]p yr i-
d in -3-yl)(3,4-d ich lor op h en yl)m eth a n on e (13g) was pre-
pared as described above, starting from 1-benzyl-4-piperidone
and 3,4-dichlorobenzoylacetonitrile: 1H NMR (CDCl3) δ 1.92-
1.96 (m, 2H, CH2CH2N), 2.44-2.47 (m, 2H, CH2CH2N), 3.39
(bs, 2H, NCH2), 3.61 (bs, 2H, CH2Ph), 7.16-7.55 (m, 10H, NH2,
H
arom).
(2-Am in o-6-ben zoyl-4,5,6,7-tetr a h yd r oth ien o[2,3-c]p y-
r id in -3-yl)(p h en yl)m eth a n on e (14) was prepared as de-
scribed above, starting from 1-benzoyl-4-piperidone and ben-
zoylacetonitrile: 1H NMR (DMSO-d6) δ 1.79-1.82 (m, 2H,
CH2CH2N), 3.12-3.57 (m, 2H, CH2CH2N), 4.31-473 (m, 2H,
NCH2), 7.18-7.42 (m, 10H, Harom), 8.22 (bs, 2H, NH2).
Bin d in g Stu d ies. The adenosine A1 binding assays were
carried out on membranes of rat brain cortex. Membranes were
prepared according to a method described previously,28 except
that the membranes were incubated with 2 U/mL ADA at 37
°C before storage.29 Protein concentrations were measured
with the BCA method.30

Synthesis of Novel 2-Amino-3-benzoylthiophenes
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3635
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1. Disp la cem en t Assa y. The assays were performed as
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)
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2. Dissocia tion Assa y.13 Rat brain cortex membranes were
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program grant in the area of Cell Communication in
Neurosciences (#BIO4-CT97-5138).
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