Rationally based efficacy tuning of selective dopamine D4 receptor ligands leading to the complete antagonist 2-[4-(4-Chlorophenyl)piperazin-1-ylmethyl]pyrazolo[1,5-α]pyridine (FAUC 213)

Full text of DOI:10.1021/jm015522j

J . Med. Chem. 2001, 44, 2691-2694
2691
Ch a r t 1
Ra tion a lly Ba sed Effica cy Tu n in g of
Selective Dop a m in e D4 Recep tor Liga n d s
Lea d in g to th e Com p lete An ta gon ist
2-[4-(4-Ch lor op h en yl)p ip er a zin -1-
ylm eth yl]p yr a zolo[1,5-a ]p yr id in e
(F AUC 213)
Stefan Lo¨ber, Harald Hu¨bner, Wolfgang Utz, and
Peter Gmeiner*
Department of Medicinal Chemistry, Emil Fischer Center,
Friedrich-Alexander University, Schuhstrasse 19, D-91052
Erlangen, Germany
Received April 6, 2001
Abstr a ct: Structure dependent efficacy studies in the field
of selective D4 ligands led to the 2-aminomethyl substituted
azaindole 2 (FAUC 213) that displayed strong D4 binding, high
subtype selectivity, and complete antagonist properties in
ligand-induced mitogenesis experiments. According to our
schematic molecular model, the intrinsic activity of the regio-
isomers investigated is controlled by the ability of the hetero-
cyclic unit to interact with both elements of the D4 binding-
site crevice, the aromatic microdomain in TM6, and a serine
residue in TM5.
Sch em e 1a
In tr od u ction . Dopamine D4 receptors have been
attributed to involvement in the pathogenesis of neu-
ropathological diseases, dependencies, and normal per-
sonality trait.1 Due to previous studies, indicating an
increase in the density of D4 receptors of schizophrenia
brains2 and high D4 affinity for the atypical anti-
psychotic clozapine,3 selective dopamine D4 receptor
antagonists have been pursued as potential anti-
psychotic agents. The azaindole L-745,870 (3) and
further drug candidates were entered into clinical trials
but failing to demonstrate clinical efficacy.4,5 However,
the ineffectiveness can be explained by the partial
agonist properties that were found very recently.6 To
find a complete antagonist, structure dependent efficacy
studies in the field of selective D4 ligands are of
particular relevance.
When rationally approaching this goal we studied
previous work on the molecular mechanisms of agonist-
receptor interaction clearly indicating the involvement
of a cluster of conserved serine residues in the fifth
transmembrane domain cooperating with aromatic side
chains in TM6.7-9 On the basis of site-directed mu-
tagenesis data and docking studies with the D4 ligand
3, the Ar subunit of the lead structure A simulating the
catechole fragment of dopamine interacts with the TM5-
TM6 microdomain of the D4 receptor controlling recep-
tor activation.10,11 On the other hand, the (chloro)phenyl
moiety interacts with a divergent microdomain in TM2-
TM3-TM7 which is responsible for D4/D2 selectivity. As
a consequence, we envisioned manipulating ligand
efficacy by subtle structural modification of Ar when
employing the D4 selective partial agonists L-745,870
(3), FAUC 113 (4),12 and FAUC 299 (5)13 as lead
a
Reagents and conditions: (a) K2CO3, air-O2, DMF, rt, 2 h
(45%); (b) (1) H2SO4 (v/v 50%), 80 °C, 3 h, (2) EtOH, H2SO4, reflux,
16 h (72%); (c) 1-(4-chlorophenyl)piperazine, LiAlH4, THF, rt to
60 °C, 45 min (35%); (d) K2CO3, air-O2, DMF, rt, 1.5 h (72%); (e)
H2SO4 (v/v 40%), reflux, 3 h (86%); (f) n-BuLi, HCOOEt, THF, -78
°C, 0.5 h (82%); (g) 1-(4-chlorophenyl)piperazine, Na(OAc)3BH,
CH2Cl2, rt, 16 h (87%).
compounds (Chart 1). In this paper, we describe the
synthesis, receptor binding, and ligand efficacy of the
regioisomeric azaindole derivatives 1,2, 6, and 7, gaining
insights to the question whether the relative orientation
of Ar can determine the amount of receptor activation.
R esu lt s a n d Discu ssion . The synthesis of the
2-aminomethyl pyrazolo[1,5-a]pyridine derivative 2 and
its 7-substituted regioisomer 7 started from N-amino-
pyridinium iodide 814 when 1,3-dipolar cycloaddition
with dimethyl acetylenedicarboxylate afforded the pyr-
azolo[1,5-a]pyridine-2,3-dicarboxylate 9 (Scheme 1).15
Hydrolysis and regioselective decarboxylation with sul-
furic acid (50%) followed by esterification gave the ethyl
azaindole-2-carboxylate 10 in 72% yield. Employing a
reductive amination protocol reported by Wright,16 this
* To whom correspondence should be addressed. Tel:
+49(9131)8529383. Fax: +49(9131)8522585. E-mail: gmeiner@
pharmazie.uni-erlangen.de.
10.1021/jm015522j CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/24/2001

2692 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Letters
Ta ble 1. Receptor Binding Data of the Pyrazolo[1,5-a]pyridines 1, 2, 4, 6, 7, 17, and 18 to Human and Bovine Dopamine Receptorsa
Ki (nM) ( SEMb
[3H]spiperone
positions
CH2N<
[3H]SCH 23390
bovine D1
compd
R
human D2long
human D2short
human D3
human D4.4
1
2
4
6
7
H
H
H
H
H
4
2
3
6
7
4
4
32000 ( 9500
5500 ( 1300
12000 ( 500
7500 ( 950
18000 ( 5000
19000 ( 2000
70000 ( 14000
420 ( 50
16000 ( 2000
3400 ( 450
21000 ( 1500
6300 ( 1900
4300 ( 650
15000 ( 2500
67000 ( 33000
45000 ( 1500
53000 ( 1500
28 ( 0.50
12000 ( 3900
5300 ( 720
5000 ( 650
4900 ( 1100
10000 ( 2100
12000 ( 500
23000 ( 4500
960 ( 45
64 ( 7.0 (n ) -1.0)
2.2 ( 0.23 (n ) -1.1)
3.6 ( 0.87 (n ) -0.9)
3.1 ( 0.12 (n ) -0.9)
1300 ( 150 (n ) -0.9)
6800 ( 3200 (n ) -1.0)
3600 ( 550 (n ) -0.9)
16 ( 0.50 (n ) -0.9)
3200 ( 400
13000 ( 500
31000 ( 8000
37000 ( 9000
39000 ( 11000
41 ( 1.5
17
18
clozapine
3-CO2Et
3-CH2OH
a
Ki values are the means of two to four independent experiments ( SEM using eight different concentrations each as triplicates.
b
Steepness of the competition curves and the derived hill slopes indicate a one-site binding model typical for antagonists; the results of
the functional test characterizing 4 and 6 as partial agonists led to the conclusion that the binding data at least for the D4.4 receptor
represent a two-state model and should be expressed as K0.5 values.
Sch em e 2a
ester could be transformed into the piperazinylmethyl
derivative 2 within a one-step process using LiAlH4 in
THF.
Formation of the unsubstituted pyrazolo[1,5-a]pyri-
dine (12)17 was achieved by cycloaddition of the amino-
pyridinium salt 8 with ethyl propiolate in the presence
of O218 followed by hydrolysis and decarboxylation of the
ethylester 11 in 62% overall yield. Regioselective lithia-
tion of the azaindole 12 by BuLi at dry ice temperature
followed by treatment with ethyl formiate and acid
workup to avoid Cannizzaro reaction resulted in forma-
tion of the aldehyde 13.19 Finally, reductive amination
with 1-(4-chlorophenyl)piperazine gave the 7-amino-
methyl substituted final product 7 in 87% yield.
The target compounds 1 and 6 were synthesized
starting from N-amino-3-(hydroxymethyl)pyridinium
mesityl sulfonate 14 which was converted into the
regioisomeric (hydroxymethyl)pyrazolo[1,5-a]pyridine
carboxylates 15 and 16.20 Subsequent hydrolysis and
decarboxylation under strong acidic conditions furnished
a
Reagents and conditions: (a) K2CO3, air-O2, DMF, rt, 1.5 h
the (hydroxymethyl)pyrazolo[1,5-a]pyridines 19 and 20,
respectively (Scheme 2). Activation of these benzylic
alcohols by MsCl followed by nucleophilic substitution
with 1-(4-chlorophenyl)piperazine gave the (azaindolyl-
methyl)piperazines 1 and 6 in 63% and 61% yield,
respectively. To synthesize 3-substituted analogues of
1, the alcohol functionality of 15 was activated and
transformed into the tertiary amine without former
removing of the ethoxycarbonyl side chain. Subsequent
reduction of the ester derivative 17 gave the corre-
sponding 3-(hydroxymethyl)pyrazolo[1,5-a]pyridine 18.
Radioligand binding assays were employed to char-
acterize the dopamine receptor affinity profiles of the
pyrazolo[1,5-a]pyridines 1, 2, 6, and 7 representing
regioisomers of the recently described D4 selective
ligand 4 (FAUC 113). Comparative data were collected
for the 3,4-disubstituted derivatives 17 and 18 and for
the atypical neuroleptic agent clozapine. The selected
compounds were investigated for their ability to displace
(25% for 15; 17% for 16); (b) (1) MsCl, Et3N, THF, rt, 2 h; (2) 1-(4-
chlorophenyl)piperazine, DMF, K2CO3, rt, 16 h (63% for 1; 61%
for 6; 61% for 17); (c) H2SO4 (v/v 40%), 100 °C, 1.5 h (83%); (d)
H2SO4 (v/v 40%), 100 °C, 3 h (78%); (e) LiAlH4, THF, 0 °C to rt, 5
h (86%).
D1 selective radioligand [3H]SCH 23390.24 The Ki values
of the 2- and 6-aminomethyl derivatives 2 and 6
depicted in Table 1 indicate strong D4 binding (Ki ) 2.2,
3.1 nM) and substantial selectivity over D1, D2 long, D2
short, and D3 (>1000). Significant loss of D4 receptor
recognition was observed for the 4-aminomethyl pyr-
azolo[1,5-a]pyridine 1 and for the 3,4-disubstituted
derivatives 17 and 18. Migration of the aminomethyl
substituent into position 7 resulted in strong reduction
of D4 binding. The steepness of the competition curves
and the derived Hill slopes gave one-site binding models.
Agonist activation of dopamine receptors is known to
increase mitogenesis in heterologously transfected cell
lines that can be determined by observing the rate of
[3H]thymidine incorporation into growing cells.25 The
functional experiment can be quantified by determina-
tion of the effective test compound concentration (EC50)
and by comparing the stimulating effect to the [3H]thy-
midine incorporation that is caused by a full agonist.
[3H]spiperone from the cloned human dopamine recep-
21
tor subtypes D2 long, D2
,
D3,22 and D4.423 stably
short
expressed in Chinese hamster ovary cells (CHO).24 D1
affinities were assessed via competition experiments
using bovine striatal membrane preparations and the

Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17 2693
F igu r e 1. Stimulation of mitogenesis as a functional assay
to assess the agonist effects of 2, 3, 4, and 6 at the human
dopamine D4.2 receptor in relation to the full agonist quin-
pirole and the antagonist clozapine, derived from 16, 7, 11, 8,
10, and 6 experiments, respectively.
Ta ble 2. Intrinsic Activity of the Azaindoles 2, 3, 4, and 6 in
Relation to the Reference Compounds Quinpirole and Clozapine
at the Dopamine D4 Receptor Established by Measuring the
Stimulation of Mitogenesis
test compounds
2
3
4
6
quinpirole clozapine
agonistic effecta <3% 23% 26% 17%
100%
10
<3%
nd
EC50 (nM)
nd
31
12
24
a
Rate of incorporation of [3H]thymidine (percentage) as evi-
dence for mitogenetic activity related to the full agonistic effect of
quinpirole (100%) as the means of quadruplicates from 6 to 16
experiments. EC50 values are derived from the mean curves of the
experiments. nd ) not determined.
To investigate whether the relative orientation of the
azaindole substructure can determine the amount of
receptor activation, the intrinsic effects of the pyrazolo-
[1,5-a]pyridines 2, 4, and 6, which emphasized the most
promising D4 ligands in the series of tested regio-
isomers, were determined. CHO10001 cells stably ex-
pressing the human D4.2 receptor were established for
the mitogenesis assay.13,26 Comparative experiments
were performed with the full agonist quinpirole, the
antagonist clozapine, and the 7-azaindole 3 (L-745,870)
that is known as a partial agonist.6 Figure 1 shows dose
response curves clearly indicating partial agonist effects
for the 3-substituted azaindoles 3 (L-745,870) and 4
(FAUC 113) with an efficacy of approximately one-
fourth (23% and 26%) of the effect of quinpirole. The
ligand efficacy of the 6-aminomethyl derivative 6 was
slightly reduced (17%). The results including the EC50
values for 3, 4, and 6 (31, 12, and 24 nM) are shown in
Table 2. As outlined in Figure 1, the most interesting
biological properties were observed for the 2-amino-
methyl substituted regioisomer. As a matter of fact, the
highly selective D4 ligand 2 (FAUC 213) proved com-
plete antagonist properties, comparable to those of the
atypical neuroleptic agent clozapine. Thus, ligand ef-
ficacy of the investigated family of drug candidates
seems really to be a function of the orientation of the
heteroarene subunit.
F igu r e 2. Schematic molecular model illustrating the poten-
tial D4 receptor binding of the partial agonist 4 (top) and the
antagonist 2 (bottom). In contrast to the binding situation of
4 (FAUC 113) involving an H-bond (approximate distance )
2.3 Å) between the lone pair in position 1 and the HO function
of Ser-196 (red line), analogous docking of 2 (FAUC 213)
results in a significantly higher N-HO distance (approxi-
mately 4.1 Å).
directed mutagenesis studies and docking experiments,
the protonated aliphatic amine functionality of the D4
ligands described herein is expected to interact with the
conserved Asp115 (3.32) in the third membrane-span-
ning segment (TM3) whereas the chlorophenyl moiety
is supposed to contribute to D4 affinity and selectivity
by recognizing a phenylalanine at position 89 (2.61) in
the second transmembrane-spanning domain.10,11 As a
consequence, the azaindole subunit is located next to
an aromatic cluster in TM6 that is in proximity to the
conserved serine residues in TM5 being known as the
molecular determinants for agonist-induced signaling.7,8
We propose that the intrinsic activity of the D4 ligands
investigated is controlled by the ability of the hetero-
cyclic unit to interact with both elements: the aromatic
microdomain in TM6 and a serine residue in TM5. A
schematic molecular model illustrating the potential
binding situation of the partial agonist 4 and the
antagonist 2 at the human D4 receptor indicates
On the basis of a pharmacophore model derived from
a CoMFA study of a series of D4 receptor ligands, a
specific spatial orientation of two π-systems and a
cationic nitrogen seems to be necessary for receptor
binding.27 Taking into account recently reported site-

2694 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 17
Letters
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Ack n ow led gm en t. The authors thank Dr. H. H. M.
Van Tol (Clarke Institute of Psychiatry, Toronto), Dr.
J .-C. Schwartz, and Dr. P. Sokoloff (INSERM, Paris) as
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Huff (Pharmacia & Upjohn, Inc., Kalamazoo, MI) is
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