Interactive SAR studies: Rational discovery of super-potent and highly selective dopamine D3 receptor antagonists and partial agonists

Article abstract of DOI:10.1021/jm025558r

Starting from dopamine receptor ligand BP897, an interactive drug discovery process leading to heterocyclic bioisosteres is demonstrated. The four step strategy involved a careful optimization of geometric and electronic properties by systematic modificat

Full text of DOI:10.1021/jm025558r

4594
J . Med. Chem. 2002, 45, 4594-4597
Ch a r t 1
In ter a ctive SAR Stu d ies: Ra tion a l
Discover y of Su p er -P oten t a n d High ly
Selective Dop a m in e D3 Recep tor
An ta gon ists a n d P a r tia l Agon ists
Laura Bettinetti, Karin Schlotter,
Harald Hu¨bner, and Peter Gmeiner*
Department of Medicinal Chemistry, Emil Fischer Center,
Friedrich-Alexander University, Schuhstrasse 19,
D-91052 Erlangen, Germany
Received J une 27, 2002
Abstr a ct: Starting from dopamine receptor ligand BP897, an
interactive drug discovery process leading to heterocyclic
bioisosteres is demonstrated. The four step strategy involved
a careful optimization of geometric and electronic properties
by systematic modification of the attachment points and
heteroatoms, respectively. Efficacy tuning by modification of
the phenyl substituents led to both D3 partial agonists and
full antagonists. The benzothiophenes 3c (FAUC346) and 3d
(FAUC365) revealed outstanding D3 affinity and subtype
selectivity.
Sch em e 1a
In tr od u ction . The dopamine D3 receptor identified
in 1990 by Sokoloff, Schwartz, and co-workers is pref-
erentially expressed in the nucleus accumbens, where
dopamine is released by neurons originating from the
ventral tegmental area.1 Convincing pharmacological
studies implicate D3-mediated neurotransmission in the
reinforcing effects of cocaine.2 Very recently, the pref-
erential dopamine D3 receptor partial agonist BP 897
(Chart 1) was designed and investigated inhibiting
cocaine-seeking behavior without revealing any intrin-
sic, primary rewarding effects.3
a
Reagents and conditions: (a) Refs 8 and 9; (b) (1) (COCl)2,
toluene, 40 °C until gas development, and then 1 h at room
temperature, and then 4 h at 60 °C; (2) 6a -d , CH2Cl2, -60 °C to
room temperature, 30 min (from 5a : 1a , n ) 1, 97%; 1b, n ) 2,
99%; 1c, n ) 3, 93%; 1d , n ) 4, 84%; from 5b: 1e, n ) 1, 78%; 1f,
n ) 2, 88%; 1g, n ) 3, 76%; 1h , n ) 4, 74%).
On the basis of the lead structure of BP 897 and
related dopaminergic benzamides,4,5 we started to de-
velop more selective D3 partial agonists. Our plan of
investigation involved the incorporation of the pyrazolo-
[1,5-a]pyridine unit as a heterocyclic bioisostere that
proved to be excellent for a fine-tuning of selectivity and
ligand efficacy within our recent structure-activity
relationship (SAR) studies.6 To modify the spatial orien-
tation of the ring system, all of the possible attachment
points of the five- and six-membered partial structures
should be exploited leading to regioisomers of types 1
and 2, respectively. Furthermore, the length of the chain
connecting the arylamide function with the methox-
yphenylpiperazine unit should be optimized. Within
subsequent interactive SAR studies, further suitable
heteroarenes and phenyl substitutents, which are taken
into account within formula 3, should be evaluated.
Resu lts a n d Discu ssion . For the synthesis of the
test compounds of type 1, we started from N-aminopy-
ridinium iodide 47 when 1,3-dipolar cycloaddition with
ethyl propiolate under oxidative conditions and subse-
quent saponification afforded the pyrazolo[1,5-a]pyri-
dine derivative 5b (Scheme 1).8 Employing dimethyl
acetylenedicarboxylate as a dipolarophile, the regioiso-
mer 5a could be isolated after hydrolysis and site
selective decarboxylation.9
For our initial investigations, we chose the amino-
ethyl-, aminopropyl-, aminobutyl-, and aminopentyl-
substituted arylpiperazines 6a -d as suitable building
blocks, which were readily prepared by N-alkylation of
o-methoxyphenylpiperazine with phthaloyl-protected
alkyl bromides and subsequent hydrazinolysis, following
previously described protocols.10 Amide bond formation
was performed by oxalyl chloride-induced activation of
the heterocyclic carboxylic acids 5a ,b and addition of
the primary amines 6a -d to the crude acid chloride
when the final products 1a -h were formed in 74-99%
yield. As central intermediates for the synthesis of the
target compounds of type 2, we needed to prepare the
respective pyrazolo[1,5-a]pyridine carbaldehydes re-
giospecifically. In the case of the 5-substituted isomer
8c, we started from 4-hydroxymethylpyridine, which
could be N-aminated by hydroxylamine-O-mesitylene-
sulfonate to give the 1,3-dipolar intermediate 7 (Scheme
2). Subsequent cycloaddition with methyl propiolate
furnished the 7a-azaindole 8a that could be transformed
into the degradation product 8b under acidic conditions.6a
Oxidation of the primary alcohol function succeeded by
MnO2 in dichloromethane gave access to the carbalde-
hyde 8c.
Employing identical reaction conditions, the regio-
isomers 9 and 10 were obtained from their hydroxy-
methyl-substituted precursors6a whereas the 7-carbal-
dehyde 11 was prepared by ortho-directed metalation
* To whom correspondence should be addressed. Tel: +49(9131)-
8529383. Fax: +49(9131)8522585. E-mail: gmeiner@pharmazie.uni-
erlangen.de.
10.1021/jm025558r CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/06/2002

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4595
Sch em e 2a
tion that the cocaine-seeking behavior inhibiting lead
compound BP 897 reveals serotoninergic and adrenergic
activity as well,3 selected test compounds were inves-
tigated for their potency to displace [3H]8-OH-DPAT and
[3H]ketanserin when employing porcine 5-HT1A, 5-HT-
2, and R1 receptors (Table 2).
The first steps of our interactive SAR studies should
be directed to the optimization of the chain length
controlling the spatial relationship of the heteroaryl-
carboxamide and the methoxypiperazine moiety when
the investigations involved the pyrazolo[1,5-a]pyridines
of type 1. With respect to the D3 affinities, both the 2-
and 3-substituted regioisomers showed only moderate
receptor recognition when n ) 1, 2, and 4. On the other
hand, substantial D3 binding was observed for the
aminobutyl derivatives 1c,g (n ) 3) resulting in Ki
values of 4.3 and 18 nM, respectively. It was interesting
to see that the attachment position 2 of the 7a-azaindole
system proved to be advantageous as compared to
position 3 causing a more bent orientation of the
π-system. Actually, the carboxamide 1c (D1/D3 ) 350,
D2long/D3 ) 72, D2short/D3 ) 72, and D4/D3 ) 30)
showed a binding profile that was comparable to the
naphththalene-2-carboxamide BP 897 (Ki ) 1.4 nM, D1/
D3 ) 540, D2long/D3 ) 150, D2short/D3 ) 150, and D4/
D3 ) 28). Substantial 5-HT1A and R1 binding was
detected for both regioisomers. On the other hand,
5-HT2 receptor recognition was only weak. It is inter-
esting to note that the 3-substituted test compounds 1e
(n ) 1) and 1f (n ) 2) displayed strong and selective
D4 (Ki ) 0.67 nM) and D2 binding (Ki ) 11 nM for D2long
and 3.9 nM for D2short), respectively. To further evaluate
the relationship between the regiochemistry of the
heteroaromatic unit and D3 binding, we pharmacologi-
cally characterized the target compounds of type 2 (for
n ) 3). For the 5- and 6-substituted regioisomers, the
data indicated receptor binding profiles that were
similar to 1c. By contrast, the attachment positions 4
and 7 exerted reduced affinity and selectivity. On the
basis of these observations, we concluded that the bent
geometry that is a common structural feature of the 3-,
4-, and 7-substituted derivatives disfavors D3 binding
whereas a more linear shape that is associated with the
attachment positions 2, 5, and 6 is favorable. A sche-
matic representation of both structural families is
depicted in Figure 1.
a
Reagents and conditions: (a) Methyl propiolate, K2CO3, air-
O2, DMF, room temperature, 2 h (21%). (b) H2SO4 (v/v 40%), 110
°C, 3 h (90%). (c) MnO2, CH2Cl2, room temperature, 19 h (91%).
Sch em e 3a
a
Reagents and conditions: (a) (1) MnO2, NaCN, AcOH, MeOH,
room temperature, 3 h; (2) NaOH (v/v 50%), reflux, 1 h (12a , 90%;
12b, 72%; 12c, 89%; 12d , 33%). (b) Compound 6c, HOAt, DCC,
CH2Cl2, room temperature, 23 h (2a , 64%); 6c, HOBt/H2O, DCC,
CH2Cl2-DMF (9:1), room temperature, 23 h (2b, 90%; 2c, 86%;
2d , 88%).
Sch em e 4a
a
Reagents and conditions: SOCl2, DMF, CHCl3-toluene (1:1),
90 °C, 30 min. Compound 6c or 14, Et3N, CHCl3, 0 °C, 30 min
(3a : X ) O, Ar ) 2-MeO-Ph, 68%; 3b: X ) O, Ar ) 2,3-di-Cl-
Ph, 57%; 3c: X ) S, Ar ) 2-MeO-Ph, 50%; 3d : X ) S, Ar ) 2,3-
di-Cl-Ph, 68%). Compound 6c, HATU-HOAt (1:1), DIPEA, DMF,
0 °C, 4 h (3e: X ) Te, Ar ) 2-MeO-Ph, 58%); 14, HATU, DIPEA,
DMF, 0 °C, 2 h (3f: X ) Te, Ar ) 2,3-di-Cl-Ph, 45%).
and formylation following a recently described protocol
(Scheme 3).11
Smooth transformation into the carboxylic acids
12a -d was accomplished by utilizing a reaction mixture
composed of MnO2, sodium cyanide, acetic acid, and
methanol12 furnishing the respective methyl carboxy-
lates, which were saponified by NaOH to give the
building blocks 12a -d . Finally, DCC coupling assisted
by HOBt or HOAt13 resulted in formation of the test
compounds 2a -d when utilizing the diaminobutane
derivative 6c. Benzofuran-2-carboxylic acid (13a ) as well
as the thia- and tellura-analogues 13b,c, respectively,
were activated by thionyl chloride or HATU13 and
subsequently reacted with 6c and the 2,3-dichlorophenyl
analogue 14 to give the potential dopamine receptor
ligands 3a -f (Scheme 4).
As a measure of functional activity, ligand efficacy of
1c was confirmed by a mitogenesis assay measuring the
rate of [3H]thymidine incorporation into growing CHO
dhfr- cells stably expressing the human D3 receptor.18,19
The data listed in Table 3 clearly show substantial
ligand efficacy of 1c (52%, 1.4 nM) that proved to be
similar to those we obtained for the partial agonist BP
897 with respect to both intrinsic activity and EC50
values. Starting from the pyrazolopyridine-2-carboxa-
mide 1c (FAUC 329) that combines substantial D3
affinity and subtype selectivity and intrinsic activity,
we tried to further improve the pharmacological profiles
by modifying the electronic properties of the heteroaro-
matic unit. Within the pyrazolo[1,5-a]pyridine nucleus,
the nitrogen atom in position 8 is part of the aromatic
10π-system whereas the lone pair of the nitrogen in
position 1 induces a negative electrostatic potential that
might influence the receptor recognition process. We
Radioligand binding assays and mitogenesis experi-
ments were employed to analyze affinity, selectivity
profiles, and ligand efficacy of the target compounds.
The binding data were generated by measuring their
ability to compete with [3H]spiperone for the cloned
14
human dopamine receptor subtypes D2long, D2short
,
D3,15 and D4.416 stably expressed in Chinese hamster
ovary cells (CHO).17 D1 receptor affinities were deter-
mined utilizing porcine striatal membranes and the D1
selective radioligand [3H]SCH 23390.17 The resulting Ki
values are listed in Table 1 as compared to the dopam-
inergic lead compound BP 897. Because of the observa-

4596 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
Letters
Ta ble 1. Receptor Binding and Selectivity Ratios for 1a -3f and BP 897 Employing Porcine D1 as Well as Human D2long, D2short, D3,
and D4.4 Receptorsa
Ki values (nM)
compd
pos
[3H]SCH 23390
pD1
[3H]spiperone
ratio of Ki values
D2long/3 D2short/3
no.
1a
1b
1c
1d
1e
1f
1g
1h
2a
2b
2c
2d
3a
3b
3c
X
n
R
hD2long
hD2short
hD3
hD4
D4/3
2
2
2
2
3
3
3
3
4
5
6
7
2
2
2
2
2
2
1
2
3
4
1
2
3
4
3
3
3
3
3
3
3
3
3
3
1200 ( 100
1200 ( 0
1500 ( 50
1400 ( 200
1200 ( 0
2200 ( 200
1500 ( 100
1100 ( 160
2300 ( 150
1500 ( 50
2000 ( 100
1200 ( 0
1100 ( 50
2900 ( 150
670 ( 15
520 ( 110
830 ( 120
310 ( 34
71 ( 0.50
290 ( 60
11 ( 1.4
110 ( 0
480 ( 40
970 ( 5.0
310 ( 30
76 ( 3.0
140 ( 8.2
250 ( 45
650 ( 180
4.3 ( 0.29
51 ( 1. 0
360 ( 9.6
30 ( 5.0
41 ( 10
130 ( 16
78 ( 11
0.67 ( 0.19
14 ( 2.5
44 ( 7.4
68 ( 5.5
87 ( 10
67 ( 8.7
40 ( 4.1
8.9 ( 1.5
30 ( 8.0
93 ( 18
2.1
1.3
1.9
1.5
0.12
0.063
72
72
30
1.4
1.5
1.5
0.81
0.07
6.1
0.39
0.026
3.6
0.0019
0.093
2.4
1.1
4.4
3.9 ( 0.41 150 ( 17
65 ( 4.2
25 ( 2.5
180 ( 14
120 ( 12
84 ( 9.7
98 ( 6.3
84 ( 6.0
80 ( 31
18 ( 1.3
64 ( 6.0
20 ( 1.6
2.8 ( 0.48
4.3 ( 0.68
29 ( 4.7
1.1 ( 0.048
1.5 ( 0.22
0.23 ( 0.016
30 ( 1.2
270 ( 16
190 ( 12
140 ( 14
150 ( 18
110 ( 0
0.47
14
0.39
9
68
33
5.2
43
20
3.4
24
9.3
0.31
O
O
S
S
Te
Te
2-MeO
2,3-di-Cl
2-MeO
2,3-di-Cl
2-MeO
2,3-di-Cl
100
210
380
7200
93
76
53
230
5200
57
27
62
65
680
51
320 ( 10
87 ( 0.50
52 ( 1.0
15 ( 1.5
340 ( 10
35 ( 8.5
150 ( 0
3d
3e
3f
8800 ( 1300
380 ( 15
1400 ( 720
760 ( 60
3600 ( 950 2600 ( 730 0.50 ( 0.12
63 ( 7.5
91 ( 20
210 ( 18
39 ( 6.0
48 ( 5.5
210 ( 28
0.68 ( 0.061
0.55 ( 0.10
1.4 ( 0.075
170
150
87
150
270
28
BP897
39 ( 4.1
a
Ki values in nM ( SEM are based on the means of 2-9 experiments each done in triplicate.
Ta ble 2. Receptor Binding Data for Selected Test Compounds
in Comparison to BP 897 Employing Porcine 5-HT1A, 5-HT-2,
and R1 Receptorsa
Ta ble 3. Intrinsic D3 Activities of 1c, 3a -f, and BP 897
Derived from the Stimulating Effect on Mitogenesis of D3
Receptor Expressing CHO Cells
Ki values (nM ( SEM)
agonist
effecta
EC50
agonist
effecta
EC50
test compd
(nM)b
test compd
(nM)b
[3H]8-OH-DPAT
[3H]ketanserin
1c
3a
3b
3c
3d
52%
53%
no effect
49%
no effect
1.4
1.5
3e
3f
no effect
no effect
56%
compd
5-HT1A
5-HT2
R1c
1c
1g
2a
2b
2c
2d
3a
3b
3c
3d
3e
3f
24 ( 3.4
14 ( 0.50
11 ( 1.3
15 ( 1.0
19 ( 0
1200 ( 50b
1300 ( 50b
1600 ( 300b
740 ( 70b
26 ( 9.7
48 ( 9.0
28 ( 8.3
32 ( 10
19 ( 5.8
18 ( 4.0
8.6 ( 1.7
BP 897
quinpirole 100%
1.8
3.1
0.36
Rate of [3H]thymidine uptake related to the full agonist
quinpirole (100%); quadruplicates from 4 to 12 experiments. b EC50
values derived from mean curve of all experiments.
a
950 ( 55b
22 ( 7.0
17 ( 1.0
480 ( 5.0
41 ( 4.2
360 ( 10
69 ( 11
2000 ( 200b
660 ( 10b
11 000 ( 500
>10 000
15 ( 6.0
>2000
15 ( 5.0
>1000
25 ( 6.9
observed for the nitrogen- and oxygen-containing het-
erocycles. For the less electronegative sulfur and tel-
lurium analogues, positive ESP charges were calculated
(for 3D representations including the resulting MEP
maps, see the Supporting Information). To find out
whether the resulting electrostatic properties might
control the receptor activity profiles, the methoxyphen-
ylpiperazinylbutylamides 3a ,c,e were included in our
study. Interestingly, D3 binding significantly increased,
especially for the thia-derivative 3c that showed a Ki of
0.23 nM and a 380, 230, and 65-fold selectivity over
D2long, D2short, and D4, respectively. Unless organo
tellurium derivatives are scarcely described as phama-
cologically active compounds,20 the tellurophene car-
boxamide 3e displayed superior D3 affinity (Ki ) 0.68
nM). Finally, exchange of the 2-methoxyphenylpipera-
zine substructure by a 2,3-dichlorophenylpiperazine
moiety, which showed to be a highly suitable framework
according to recent findings in the field of D3 antagon-
ists,4a was performed. As a matter of fact, the dichloro
derivatives 3b,d ,f revealed D3 affinities that are com-
parable to the methoxy-substituted analogues; however,
the selectivities over 5HT-1A, 5-HT2, and R1 were
substantially higher. Interestingly, extraordinary se-
lectivity ratios of 17 600, 7200, 5200, and 680 over D1,
D2long, D2short, and D4, respectively, were determined
for thebenzothiophene-2-carboxamide 3d (Ki ) 0.50 nM).
To the best of our knowledge, the family of compounds
presented herein shows by far the highest D3 selectiv-
350 ( 70b
2000 ( 600
500 ( 110b
730 ( 190
840 ( 120b
125 ( 5.0
81 ( 8.0
BP897
a
Ki values as means of 2-5 experiments each done in duplicate
or triplicate. Determined in the presence of 10 µM prazosine.c Ki
b
values derived from the high affinity binding site of a biphasic
curve when labeled with [3H]ketanserin.
F igu r e 1. Schematic presentation of the different structural
features of 2-,5-,6- and 3-,4-,7-substituted heteroarene car-
boxamides showing linear (left) or bent geometry (right),
respectively.
were intrigued by the question whether oxa-, thia-, or
even tellura-analogues might serve as bioisosteres.
According to ab initio molecular orbital calculations that
we performed on the 3-21G level of theory for the
2-aminocarbonyl substituted pyrazolo[1,5-a]pyridine,
benzo[b]furan, benzo[b]thiophene, and benzo[b]tel-
lurophene ring systems, negative partial charges were

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ites reported yet. The intrinsic activities determined
within the mitogenesis assays were also highly structure-
dependent. The benzothiophene 3c (FAUC 346) and its
oxa analogue 3a proved partial agonist character with
EC50 values at 0.36 and 1.5 nM, respectively. On the
other hand, exchange of the methoxyphenyl group by a
dichlorophenyl moiety or introduction of the tellurium
into the heteroarene led to a complete loss of ligand
efficacy.
In conclusion, highly selective dopamine D3 partial
agonists and also complete antagonists including the
pyrazolo[1,5-a]pyridine FAUC 329 (1c) and the ben-
zothiophenes FAUC 346 (3c) and FAUC 365 (3d ) were
discovered by a rational and interactive SAR sequence.
Whereas the antagonists are of potential interest for the
treatment of schizophrenia, the partial agonists could
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Ack n ow led gm en t. We thank Dr. J .-C. Schwartz
and Dr. P. Sokoloff (INSERM, Paris), Dr. H. H. M. Van
Tol (Clarke Institute of Psychiatry, Toronto), and Dr.
J . Shine (The Garvan Institute of Medical Research,
Sydney) for providing dopamine D3, D4, and D2 recep-
tor-expressing cell lines, respectively. Thanks are also
due to Dr. W. Utz for helpful discussions. This work was
supported by the BMBF and the Fonds der Chemischen
Industrie.
Su p p or tin g In for m a tion Ava ila ble: Complete Experi-
mental Section including details on synthesis, analytical
characterization, and biological studies, as well as a graphical
representation of molecular electrostatic isopotential maps.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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