Synthesis and biological investigations of dopaminergic partial agonists preferentially recognizing the D4 receptor subtype

Article abstract of DOI:10.1016/j.bmcl.2006.02.075

Aminomethyl-substituted biaryls bearing a pyrazole or triazole moiety were synthesized and investigated for dopamine and serotonin receptor binding. The N-arylpyrazoles 3b,f,g and 4 revealed K_i values in the subnanomolar range (0.28-0.70 nM) fo

Full text of DOI:10.1016/j.bmcl.2006.02.075

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2955–2959
Synthesis and biological investigations of dopaminergic
partial agonists preferentially recognizing the D4 receptor subtype
Stefan Lo¨ber, Harald Hubner and Peter Gmeiner*
¨
Department of Medicinal Chemistry, Emil Fischer Center, Friedrich-Alexander University,
Schuhstraße 19, D-91052 Erlangen, Germany
Received 16 January 2006; revised 24 February 2006; accepted 25 February 2006
Available online 24 March 2006
Abstract—Aminomethyl-substituted biaryls bearing a pyrazole or triazole moiety were synthesized and investigated for dopamine
and serotonin receptor binding. The N-arylpyrazoles 3b,f,g and 4 revealed Ki values in the subnanomolar range (0.28–0.70 nM) for
the dopamine D4 receptor subtype. Employing both mitogenesis and GTPcS assays, ligand efficacy was evaluated indicating partial
agonist properties. Interestingly, the tetrahydropyrimidine 4 (FAUC 2020) displayed significant intrinsic selectivity for D2long over
D2short
.
Ó 2006 Elsevier Ltd. All rights reserved.
N
The dopamine D4 receptor subtype received much
attention as a pharmacological target for the treatment
of schizophrenia, Parkinson’s disease, depression, and
attention deficit hyperactivity disorder (ADHD).1 As a
consequence, ongoing efforts have been made to find
selective ligands revealing high affinity for the D4 recep-
tor. Thus, an N-arylpiperazine framework proved to be
a privileged structural unit,2 which can be labeled as an
integral part of a majority of potent D4 receptor ligands.
The second nitrogen atom of the piperazine moiety is
preferably attached to a benzylic CH2 position of a
fused heteroaryl or a biaryl substructure (Chart 1).
Starting from the highly selective D4 ligand L-745,870
displaying weak partial agonist properties,3,4 structural
variations proved to be highly beneficial when ligand
efficacy could be tuned. Thus, FAUC 213 turned out
to be a complete antagonist exhibiting atypical antipsy-
chotic properties in behavioral and neurochemical
models of schizophrenia.5,6 Very recently, the D4
agonist ABT-724 was discovered as a drug candidate
for the treatment of erectile dysfunction (ED).7
N
N
N
N
Cl
N
N
N
N
H
N
L-745,870
N
NGD 94-1
H
N
N
N
N
N
NH
NH
ABT-724
A
N
N
N
N
Cl
R
N
Y
N
N
N
FAUC 213
B
X
Chart 1. Structures of D4 selective lead and target compounds.
The phenylimidazole NGD 94-1 is a prominent member
of the biaryl type class of D4 selective ligands being
reported to act as a full antagonist.8 Following studies
indicated partial agonist effects.9 Recently, we reported
on the synthesis and receptor binding of dehydroimidaz-
ole and pyrrole analogs of type A,10,11 demonstrating
that the phenyl nucleus can be displaced by a 1,1-dicy-
anovinyl substituent.12 Having in mind that a negative
molecular electrostatic potential (MEP) exerted by the
lone pair of an endocyclic sp2 nitrogen proved to be
beneficial for preferential D4 binding of the fused
Keywords: Dopamine; D4; Partial agonist; Regioselective synthesis;
Subtype selectivity.
*
Corresponding author. Tel.: +49 9131 85 29383; fax: +49 9131 85
22585; e-mail: gmeiner@pharmazie.uni-erlangen.de
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.075

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S. Lo¨ber et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2955–2959
heteroarene derivatives,13 we envisioned to prepare het-
erocyclic biaryl analogs of type B revealing a pyrazole or
triazole nucleus in combination with a benzene or pyri-
dine ring.14 Further structural manipulations were
envisaged including the exchange of the positions of
the five- and the six-membered heteroarene units and
the introduction of a tetrahydropyridine moiety replac-
ing the piperazine ring.15
CN
CHO
CN
a
b
N
N
N
N
F
5
6
7
N
N
c
N
N
8
Starting from N-phenylpyrazoles 1a,b16 and the pyridine
analog 1c,17 we employed a Vilsmeier formylation reac-
tion to obtain the respective pyrazole carbaldehydes
2a–c, which could be transformed into the piper-
azinylmethylpyrazoles 3a–i by reductive amination in
78–88% yield (Scheme 1). Regiodirected lithiation of
the azole moiety of 3c followed by alkylation with
MeI gave access to the 5-methyl derivative 3j. Treatment
of 2a with 4-phenyl-1,2,5,6-tetrahydropyridine in the
presence of Na(OAc)3BH resulted in formation of the
aminomethylpyrazole 4.
Scheme 2. Reagents and conditions: (a) pyrazole, K2CO3, DMSO,
120 °C, 16 h (84%); (b) DIBAL-H, toluene, ꢀ60 °C (55%); (c)
phenylpiperazine, Na(OAc)3BH, CH2Cl2, rt, 16 h (80%).
N
N
Ar
N
N Ar
HN
N Ar
a
b
N
N
N
9a: Ar= Ph
9b: Ar= 2-MeO-Ph
10a: Ar= Ph
10b: Ar= 2-MeO-Ph
11a: Ar= Ph
11b: Ar= 2-MeO-Ph
N
Ph
N
Ph
HN
Ph
To prove our hypothesis that the relative orientation of
the negative MEP is crucial for preferential D4 binding,
we synthesized the regioisomer of the phenylpyrazole 3c
featuring an exchange of the rings within the biaryl
moiety. Thus, 4-fluorobenzonitrile (5) was reacted with
pyrazole to give the N-arylazole 6.18 DIBAL-H reduc-
tion furnished the respective carbaldehyde 7, which
was converted into the desired phenylpiperazine 8 by
reductive amination (Scheme 2).
a
b
N
N
N
12
13
14
Scheme 3. Reagents and conditions: (a) Ref. 20; (b) phenylazide,
CuCl2, Na-ascorbate, i-PrOH, rt, 24 h (62–68%).
cycloaddition with phenyl azide in the presence of Cu(II)
and Na-ascorbate (Scheme 3).
As an extension, we synthesized triazole analogs of the
above-mentioned pyrazoles 3c,g and 4. Thus, alkylation
of the secondary amines 9a,b and 12 with propargyl bro-
mide gave the 3-aminopropynes 10a,b and 13, respec-
tively. Employing a slightly modified ‘click chemistry’
protocol,19 we transformed the respective intermediates
into the triazoles 11a,b and 14 employing 1,3-dipolar
The final products 3a–j, 4, 8, 11a,b, and 1420 and the
phenylpyrrole A as a reference compound were evaluat-
ed in vitro for their abilities to displace [3H]spiperone
from the cloned human dopamine receptors D2long
21
,
D2short
,
D3,22 and D4.423 being stably expressed in
CHO cells.24 The D1 affinities were determined by
employing porcine striatal membrane preparations and
the D1 selective antagonist [3H]SCH 23390.
Ar
N
N
R
Receptor binding studies clearly indicated that the target
compounds reveal only poor affinity to the D1 subtype.
Depending on the substitution pattern of the aryl piper-
azine group, Ki values ranged from 11 to 6000 nM, 8 to
14,000 nM, and 33 to 4900 nM for D2long, D2short, and
D3, respectively (Table 1). Interestingly, incorporation
of a halogen atom in para-position (3d,e) strongly
decreases D2 and D3 binding whereas ortho-substituents
bearing a negative electrostatic potential caused an
increase of affinity (3b,f,g and 11b). In contrast, the
2-pyrimidyl-substituted NGD 94-1 analog 3i displayed
poor receptor binding for all GPCRs investigated. With
the exception of the pyrimidine 3i and the reversed
regioisomer 8, all target compounds displayed extra-
ordinarily high D4 affinity with Ki values in the sub-
nanomolar and single digit nanomolar range. Thus,
the arylpyrazole and aryltriazole scaffold causes a sub-
stantial increase of affinity when compared to the phe-
nylpyrrole substructure of the reference D4 ligand of
type A. SAR observations indicate that the introduction
of a further sp2 nitrogen into the six- or five-membered
arenes resulting in formation of the pyridylpyrazoles
CHO
R
R
N
N
N
a
b
N
N
N
X
X
X
3a: X= N, R= H, Ar= Ph
2a: X= CH, R= H
2b: X= CH, R= Me
2c: X= N, R= H
1a: X= CH, R= H
1b: X= CH, R= Me
1c: X= N, R= H
3b: X= N, R= H, Ar= 2-MeO-Ph
3c: X= CH, R= H, Ar= Ph
3d: X= CH, R= H, Ar= 4-Cl-Ph
3e: X= CH, R= H, Ar= 4-F-Ph
3f: X= CH, R= H, Ar= 2-F-Ph
3g: X= CH, R= H, Ar= 2-MeO-Ph
3h: X= CH, R= Me, Ar= Ph
Ph
N
3i: X= CH, R= H, Ar= 2-pyrimidyl
N
b
N
2a
4
Ph
N
N
Me
c
N
N
3c
3j
Scheme 1. Reagents and conditions: (a) Ref. 18; (b) arylpiperazine,
Na(OAc)3BH, CH2Cl2, rt, 16 h (78–88%); (c) 1—n-BuLi, THF, ꢀ78 to
ꢀ30 °C, 2 h; 2—MeI, ꢀ78 to 0 °C, 1.5 h (62%).

S. Lo¨ber et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2955–2959
2957
Table 1. Receptor binding data (Ki valuesa of the arylazoles 3a–j, 4, 8, 11a,b, and 14, and the reference agent A based on means of 3–14 experiments
each performed in triplicate) (nM)
N
N
R'
R
N
N
X
Compound
X
R
R0
H
D1
1600
D2l
D2s
190
D3
160
D4
2.6
3a
3b
3c
3d
3e
3f
N
H
340b
40
N
H
2-MeO
H
1900
1600
7500
1900
680
31
75
72
140
1900
540
67
0.44
1.0
CH
CH
CH
CH
CH
CH
H
140
2900
450
63
H
4-Cl
4-F
1900
400
37
2.9
2.9
H
H
2-F
2-MeO
H
0.70
0.28b
1.0
310b
3g
3h
3i
H
3-CH3
1000
3900
7100
560
11
71
8.0
35
43
14,000
250
360b
2900b
320
6000
630
3j
CH
5-CH3
H
5.3
z
Y
N
N
N
X
R'
Compound
X
Y
Z
R0
D1
D2l
D2s
D3
D4
11a
11b
4
N
N
N
C
CH2
CH2
CH
H
21,000
27,000
3000
1500
120
860
65
1200
340
33
9.9
2.7
N
CH
OMe
H
38
28
0.59
8
7600
10,000
2100
3000
220
230b
2400
110
170
2100
94
20
3.2
12
14
A
N
C
CH
H
514
a All standard deviations ( SEM) are within <25% of the appropriate Ki values.
b Standard deviation in the range of 25–35%.
3a,b and the phenyltriazoles 11a,b, respectively, leads to
a significant reduction of dopamine receptor binding.
C-Methylation in position 3 or 5 of the pyrazole ring
resulting in the test compounds 3h and j was not crucial
for D4 affinity and selectivity. It is interesting to note
that the replacement of the piperazine ring by a tetrahy-
dropyridine unit proved to be possible without signifi-
cant reduction of receptor binding for the bioisosteres
4 and 14.
tetrahydropyridine 4 (FAUC 2020). In detail, ligand
efficacy of 54% relative to the dopaminergic full agonist
quinpirole significantly exceeded a 25% relative efficacy
upon the D2short mediated transduction system. To
confirm these results, a GTPcS assay was performed
when FAUC 2020 (4) led to 45% intrinsic activity via
D2long and only 17% via D2short corroborating the re-
sults of the mitogenesis assay. To our knowledge,
FAUC 2020 is the first example of a dopamine recep-
tor ligand revealing nearly strong partial agonist prop-
erties at the D2long splice variant in combination with
an only weak agonist effect at the D2short isoform
and, thus might be of great interest as a pharmacolog-
ical tool.
To investigate the intrinsic effect of the most active D4
ligands of the piperazine and tetrahydropyridine class
of compounds, respectively, in vitro functional assays
with 3g and 4 were envisaged measuring the [3H]thymi-
dine uptake in growing CHO cells stably expressing the
dopamine D2long, D2short, D3, and D4.225 receptor,
respectively, and GTPcS binding for D2 and D4.26
Due to the structural similarity of the test compounds to
the atypical neuroleptic agent bifeprunox27 displaying a
combination of dopaminergic properties and single digit
nanomolar 5-HT1A receptor affinity, porcine striatal
membranes were employed for serotonin receptor bind-
ing studies. In fact, the phenylpyrazoles 3g and 4 have
been capable to efficiently displace the radioligand
[3H]WAY100635, whereas 5-HT2 binding was only
weak.
Employing the mitogenesis assay, the aminomethylpy-
razoles 3g and 4 proved to be partial agonists for
D2long, D2short, D3, and D4.2 displaying intrinsic activ-
ities between 30% and 66% (Table 2). The partial ago-
nist effect of 4 at the D4 subtype was further confirmed
when antagonizing the effect of 100 nM quinpirole in a
dose-dependent manner (Fig. 1). Interestingly, the D4
preference demonstrated for the binding experiments
could not be observed in the functional assay. Having
in mind that ligand binding and functional properties
of the two isoforms D2long and D2short have been de-
scribed to be more or less identical, it was very surpris-
ing that the data indicated an intrinsic selectivity of the
In conclusion, SAR experiments on dopaminergic
arylazole derivatives lead to highly active D4 partial
agonist showing substantial 5-HT1A binding, as well.
The tetrahydropyridine analog FAUC 2020 (4) indicat-
ed significant intrinsic selectivity for D2long over the
isoform D2short
.

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S. Lo¨ber et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2955–2959
Table 2. Agonist effects of the arylpyrazoles 3g, 4 and quinpirole at the D2long, D2short, D3, and D4.2 receptor investigated by measuring the
stimulation of mitogenesis and GTPcS binding as well as serotonin receptor binding
Test compounds
3g
4
Quinpirole
D2l
D2s
Efficacy mitogenesisa (%)
56
54
3.0
45
25
100
7.4
b
EC50 (nM)
0.28
nd
Efficacy GTPcS (%)a
EC50 (nM)
100
330
b
—
Efficacy mitogenesisa (%)
66
30
1.7
17
32
100
10
b
EC50 (nM)
0.22
nd
Efficacy GTPcS (%)a
EC50 (nM)
100
230
b
—
D3
D4
Efficacy mitogenesisa (%)
62
0.21
44
3.3
100
3.4
b
EC50 (nM)
Efficacy mitogenesisa (%)
43
39
100
11
b
EC50 (nM)
0.60
nd
5.6
38
Efficacy GTPcS (%)a
EC50 (nM)
100
23
b
—
9.5
5-HT1A
5-HT2
Kic (nM)
Kic (nM)
6.8
1300
37
2100
nd
nd
a Rate of incorporation of [3H]thymidine (in %) relative to the maximal effect of the full agonist quinpirole (100%); the results are means of
quadruplicate from 2 to 14 experiments.
b EC50 values derived from the mean curves of 2–14 experiments; nd, not determined.
c Ki values for 5-HT1A and 5-HT2 binding were derived from two experiments each done in triplicate.
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75
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4 + 100 nM quinpirole
4
-11
-10
-9
-8
-7
-6
concentration of test compound [logM]
Figure 1. Partial agonism of the tetrahydropyridine 4 at the D4
receptor indicated by stimulation of [3H]thymidine incorporation and
confirmed by partially antagonizing the effect of quinpirole.
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Acknowledgments
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We thank Dr. J. -C. Schwartz and Dr. P. Sokoloff (IN-
SERM, 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
D3, D4.4, and D2 receptor expressing cell lines.
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14. For previously described aminomethyl-substituted
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