Discovery of dopamine D4 receptor antagonists with planar chirality

Article abstract of DOI:10.1016/j.bmc.2013.01.065

Employing the D₄ selective phenylpiperazine 2 as a lead compound, planar chiral analogs with paracyclophane substructure were synthesized and evaluated for their ability to bind and activate dopamine receptors. The study revealed that the introduction of a [2.2]paracyclophane moiety is tolerated by dopamine receptors of the D₂ family. Subtype selectivity for D₄ and ligand efficacy depend on the absolute configuration of the test compounds. Whereas the achiral single-layered lead 2 and the double-layered paracyclophane (R)-3 showed partial agonist properties, the enantiomer (S)-3 behaved as a neutral antagonist.

Full text of DOI:10.1016/j.bmc.2013.01.065

Bioorganic & Medicinal Chemistry 21 (2013) 1680–1684
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of dopamine D4 receptor antagonists with planar chirality
Fabrizio Sanna, Birgit Ortner, Harald Hübner, Stefan Löber, Nuska Tschammer,
⇑
Peter Gmeiner
Department of Medicinal Chemistry, Emil Fischer Center, Friedrich Alexander University, Schuhstrasse 19, D-91052 Erlangen, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Employing the D4 selective phenylpiperazine 2 as a lead compound, planar chiral analogs with paracyclo-
phane substructure were synthesized and evaluated for their ability to bind and activate dopamine
receptors. The study revealed that the introduction of a [2.2]paracyclophane moiety is tolerated by dopa-
mine receptors of the D2 family. Subtype selectivity for D4 and ligand efficacy depend on the absolute
configuration of the test compounds. Whereas the achiral single-layered lead 2 and the double-layered
paracyclophane (R)-3 showed partial agonist properties, the enantiomer (S)-3 behaved as a neutral
antagonist.
Received 3 December 2012
Revised 18 January 2013
Accepted 24 January 2013
Available online 5 February 2013
Keywords:
GPCR
Dopamine
Ó 2013 Elsevier Ltd. All rights reserved.
Subtype selectivity
Ligand efficacy
Paracyclophane
Planar chirality
1. Introduction
subtype selectivity and intrinsic activity.8 Thus, we could demon-
strate that the attachment position of heteroarenes that served
The dopamine D4 receptor is a subtype of the D2-like family
belonging to the rhodopsin like G-protein coupled receptors
(GPCRs).1 D4 receptor can be found in distinct areas of the central
nervous system including the cerebral cortex, striatum, hippocam-
pus, and amygdala.2 Genetic studies have demonstrated that a var-
iable number of tandem repeat polymorphisms, which has been
detected for the D4 receptor, is associated with an increased risk
of developing attention deficit hyperactivity disorder (ADHD).3
Moreover, an association between this genetic vulnerability for
pathologic gambling and the presence of the seven-repeat allele
of the D4 receptor was shown.4 D4 antagonists might be also very
useful for the treatment of psychotic diseases, although the theory
that the D4 receptor might play a crucial part in the etiology of
schizophrenia remains controversial.5 Because D4 receptor activa-
tion causes penile erection in rodents, a promising approach to a
therapeutic use of D4 selective drug candidates results from the
impact of the dopaminergic system on sexual stimulation.6
as lipophilic appendages strongly influenced the intrinsic activity
of the test compounds at the D4 receptor.9 As an extension of our
recent studies on [2.2]paracyclophanes as bioisosteres for arene
derivatives including D4 receptor ligands of type 1 (Fig. 1),10 we
envisaged to investigate if binding affinity and intrinsic activity
of the promising lead compounds of type 27d,11 could be varied
by the introduction of a paracyclophane moiety. Because both par-
tial agonists and neutral antagonists are of interest for D4-related
dysfunctions and paracyclophanes are useful molecular probes to
investigate the size of binding sites, our results will be valuable
for further work in GPCR based drug discovery. We were also intri-
gued by the question how ligand efficacy depends on the stereo-
chemistry of the planar chiral unit. Thus, the target compound 3
should be synthesized in enantiomerically pure form. To learn
more about the relationships between D4 receptor binding and
the position of the double-layered unit within the 1,4-DAP phar-
macophore, we intended to prepare and biologically study also
the cyclophanylpiperazine 4 as a regiosiomer of 3.
We have previously developed a number of 1,4-disubstituted
arylpiperidines and piperazines (1,4-DAPs), displaying subtype
selectivity for the dopamine receptor subtypes D2, D3 or D4.7
Employing a toolbox of head groups, amine moieties, linker ele-
ments and lipophilic appendages, the 1,4-DAP privileged structure
proved to be a valuable starting point to fine-tune both GPCR
2. Results and discussion
2.1. Synthesis
The synthesis of the enantiomerically pure cyclophanylpyraz-
oles 3 started from the racemic alkohol ( )-5, which was obtained
from bromo substituted [2.2]paracyclophane by metallation and
subsequent boronation (Scheme 1).12 According to our previously
⇑
Corresponding author. Tel.: +49 9131 852 9383; fax: +49 9131 852 2585.
E-mail addresses: peter.gmeiner@medchem.uni-erlangen.de, peter.gmeiner@
fau.de (P. Gmeiner).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.065

F. Sanna et al. / Bioorg. Med. Chem. 21 (2013) 1680–1684
1681
Cl
ref. 9c
Br
N
N
N
N
HN
N
9
10
N
N
a
N
H
(R)-1
(S)-1
2
N
N
N
N
N
N
N
N
4
(+)-
-
N
N
N
N
Scheme 2. Reagents and conditions: (a) phenylpyrazole-4-carbaldehyde,
Na(OAc)3BH, CH2Cl2, rt, 24 h.
(R)-3
(S)-3
4
15
16
17
receptor subtypes D2long, D2short
,
D3 and D4.4 each stably ex-
Figure 1. Dopamine D4 receptor ligands.
pressed in Chinese hamster ovary (CHO) cells and the radioligand
[3H]spiperone.18 Binding affinities to the dopamine D1 receptor
were determined when using membranes from bovine striatum
and the radioligand [3H]SCH23390.18 As displayed in Table 1, all
compounds show moderate binding to the D1 subtype in the
micromolar range. Interestingly, the planar chiral test compounds
(R)-3 and (S)-3 incorporating a double-layered lipophilic append-
enzym.
kinetic
resolution
+
OH
ref. 9b
NH
N
HN
N
Ph
Ph
age displayed substantial binding affinity to the GPCRs D2long
,
(rac)-5
D2short, D3 and D4.4 with significant preference to the D4 receptor.
(R)-6
6
(S)-
Ph
Ph
In detail, Ki values in the single digit nanomolar range (Ki = 4.5 nM
and 2.5 nM for (R)-3 and (S)-3, respectively) were slightly higher
than the data of our lead compound 2 (Ki = 1.0 nM) and an enantio-
specific difference in affinity could be observed. On the one hand,
(S)-3 showed a higher selectivity over D3 than its (R)-enantiomer.
On the other hand, the data for (R)-3 clearly indicated stronger
D4 preference over D2long and D2short binding (53- and 78-fold).
Compared to the endogenous ligand dopamine, the planar chiral
test compounds (R)-3 and (S)-3 displayed superior D4 binding
affinity and subtype selectivity. Further SAR studies led us to the
1,4-DAP 4, which represents a regioisomer of the test compound
3 that includes a paracyclophane unit within the head group. In
fact, this structural modification induced a substantial reduction
of D4 affinity (Ki = 45 nM) while the binding properties for D2 and
D3 were similar to (S)-3.
a
R
N
c
N
N
N
N
N
(R)-3
(S)-3
(R)-7: R= H
(S)-7: R= H
b
8
(R)- : R= CHO
(S)-8: R= CHO
Scheme 1. Reagents and conditions: (a) (1) TosOH, HCl in EtOH, ethylene glycol,
80 °C, 18 h; (2) 1,1,3,3-tetramethoxypropane, HCl in EtOH, 80 °C, 1.5 h; (b) POCl3,
DMF, 1,2-dichloroethane, 0 °C to reflux, 2.5 h; (c) 1-phenylpiperazine, Na(OAc)3BH,
CH2Cl2, rt, 5 h.
2.3. Functional experiments
published synthetic route,10b ( )-5 was converted into the hydra-
zones (R)-6 and (S)-6, whereas an enzymatic kinetic resolution of
the respective 4-acetyloxy-[2.2]paracyclophane by Candida cylindr-
acea lipase was the key step.13 The pyrazoles (R)-7 and (S)-7 were
obtained by treatment of (R)-6 and (S)-6 with HCl in ethanol in the
presence of ethylene glycol and subsequent cyclocondensation
with tetramethoxypropane. Finally, Vilsmeier–Haack formylation
led to the pyrazole carbaldehydes (R)-8 and (S)-8, which were con-
verted into the desired target compounds (R)-3 and (S)-3 by reduc-
tive amination with 1-phenylpiperazine and Na(OAc)3BH. Analysis
by chiral HPLC gave ee values >98% for both enantiomers.
The activation of D4 receptors can be measured as a concentra-
tion-dependent inhibition of the forskolin-stimulated cyclic AMP
Table 1
Receptor binding data for the test compounds (R)-3, (S)-3 and 4 at the bovine
dopamine D1 receptor and the human subtypes D2long, D2short, D3 and D4.4 in
comparison to the reference agents dopamine and 2
Compound Ki valuea (nM)
D1
D2long
D2short
D3
D4.4
For the preparation of the reference ligand 4 we took advantage
of our established synthesis of the piperazine derivative 10,10c
including a palladium free aryl-amine coupling reaction (Scheme
2).14 Reductive alkylation with 1-phenylpyrazole-4-carbalde-
hyde7d gave the corresponding racemic tertiary amine 4.
[3H]SCH23390
[3H]spiperone
(R)-3
(S)-3
4
Dopamine
2
1700 63
1500 75
2600 920b
440 72
350 52
55 15
39 13
190 28
140 7.8
240 40
33 7.3
28 9.3
110 49
23 5.2 4.5 0.19
20 3.0 2.5 0.29
18 1.8
21 6.7 8.9 2.2
1.0 0.12
45 10
1600 570b
75 8.1 140 13
2.2. Receptor-ligand binding experiments
a
Ki values are the means of 3–8 individual experiments SEM each done in
triplicate.
b
Ki values are the means of two individual experiments SD each done in
triplicate.
Receptor binding data were evaluated in radioligand displace-
ment experiments using preparations of the human dopamine

1682
F. Sanna et al. / Bioorg. Med. Chem. 21 (2013) 1680–1684
was performed with silica gel plates on aluminium (Silica Gel 60
F254 from Merck). The ee values were determined by HPLC-analy-
sis using the following HPLC equipment: Gilson 305 HPLC pump
with pulsation suppressor (type 805); UV detector from Knauer;
chiral CHIRALCEL OD column from Diacel (length 20 cm and diam-
eter 0.64 cm); solvent system: petroleum ether/i-PrOH 6:4.
4.2. (R)-1-[2.2]Paracyclophane-4-yl-1H-pyrazole ((R)-7)
A solution of (R)-N-benzhydrylidene-N’-[2.2]paracyclophane-4-
yl-hydrazine (50 mg, 0.13 mmol), toluene sulfonic acid hydrate
(118 mg, 0.62 mmol) and ethylene glycol (0.1 mL) in ethanolic
HCl (1 mL) are stirred in a pressure vial under nitrogen at 80 °C.
After 18 h,
a
solution of tetramethoxypropane (20.4 mg,
0.13 mmol) in ethanol (0.1 mL) saturated with HCl was added.
After stirring at 80 °C for 90 min, the solution was allowed to cool
to room temperature, evaporated and the residue was added to an
aqueous solution of saturated aqueous NaHCO3. The mixture was
extracted with CH2Cl2. The organic layer was dried (Na2SO4) and
evaporated and the residue was purified by flash chromatography
(petroleum ether/ethyl acetate 95:5) to give pure (R)-7 (26.1 mg,
77%) as a colorless solid (mp 114 °C). EI-MS: m/z 274 (M+); 1H
NMR: (CDCl3, 360 MHz) d (ppm): 2.55–2.63 (m, 1H), 2.83–3.22
(m, 6H), 3.45–3.53 (m, 1H), 6.48 (dd, J = 2.1 Hz, 2.1 Hz, 1H), 6.55
(dd, J = 8.0 Hz, 1.9 Hz, 1H), 6.56 (dd, J = 8.0 Hz, 1.9 Hz, 1H), 6.60
(d, J = 7.5 Hz, 1H), 6.61 (dd, J = 8.0 Hz, 1.9 Hz, 1H), 6.62 (dd,
J = 8.0 Hz, 1.9 Hz, 1H), 6.66 (d, J = 2.0 Hz, 1H), 6.67 (dd, J = 7.5 Hz,
2.0 Hz, 1H), 7.71 (d, J = 2.1 Hz, 1H), 7.79 (d, J = 2.1 Hz, 1H); IR:
Figure 2. Adenylyl cyclase assay indicating the ability of the test compounds 2, (R)-
3 and (S)-3 to inhibit forskolin stimulated cAMP accumulation. The D4.4 mediated
inhibition of adenylyl cyclase was measured in the CHO cells expressing D4.4. The
compounds were tested in the presence of 10
reference full agonist.
lM forskolin. Quipirole served as a
(cAMP) levels.17 To evaluate the efficacy of the D4 ligands (R)-3 and
(S)-3 and the lead compound 2, intrinsic activity of the test com-
pounds was determined in a luminescence based adenylyl cyclase
activity assay (Fig. 2). The assay was performed in the presence of
10 lM forskolin. Quinpriole was used as a reference compound
(KBr)
m
(cmꢀ1): 1596. ½a 2D0
¼ ꢀ98:8 (c 1, CHCl3). Anal. Calcd for
ꢁ
yielding full agonist response. Whereas a 40% ligand efficacy
clearly indicated partial agonist properties for the lead compound
2, intrinsic activity was strongly reduced for the planar chiral para-
cyclophanes (R)-3 and (S)-3. The (R)-enantiomer displayed 19% li-
gand efficacy and (S)-3 proved to be a neutral antagonist. Thus,
intrinsic activity depends on the chirality of the double-layered
lipophilic appendage.
C19H18N2: C, 83.18; H, 6.61; N, 10.21. Found: C, 82.98; H, 6.52; N,
10.44. (S)-7 was prepared under identical conditions.
¼ þ100:2 (c 1, CHCl3).
½ ꢁ
a 2D0
4.3. (R)-1-[2.2]Paracyclophane-4-yl-1H-pyrazole-4-carbaldehyde
((R)-8)
To
0.27 mmol) in 1,2-dichloroethane (dry, 0.5 mL) was added a solu-
tion of dimethylformamide (21 L, 0.27 mmol) in 1,2-dichloroeth-
a pre-cooled solution of phosphoryl chloride (26 lL,
3. Conclusion
l
The intrinsic activity of a GPCR ligand determines whether it
may be used to treat disorders that require stimulation or attenu-
ation of specific signaling events, respectively. Planar chiral ana-
logs of type 3 and 4 were synthesized and evaluated for their
ability to bind and activate dopamine receptors. The study revealed
that the introduction of a paracyclophane moiety is tolerated by
dopamine receptors of the D2 family. D4 subtype selectivity and li-
gand efficacy depend on the absolute stereochemistry of the test
compounds. Whereas the achiral single-layered lead 2 and the
double-layered paracyclophane (R)-3 showed partial agonist prop-
erties, the enantiomer (S)-3 behaved as a neutral antagonist.
ane and the solution was stirred at 0 °C for 0.5 h. Then, a solution of
(R)-7 (50 mg, 0.18 mmol) in dry 1,2-dichloroethane (dry, 0.5 mL)
was added and the mixture was refluxed under N2 for 2 h. After
addition of saturated aqueous sodium acetate at room tempera-
ture, stirring was continued for 0.5 h. After neutralization by addi-
tion of NaHCO3, the mixture was extracted with dichloromethane.
The organic layer was dried (Na2SO4) and evaporated and the res-
idue was purified by flash chromatography (petroleum ether/ethyl
acetate 85:15) to give pure (R)-8 (41 mg, 74%) as a colorless solid
(mp 139 °C). EI-MS: m/z 302 (M+); 1H NMR: (CDCl3, 360 MHz) d
(ppm): 2.61–2.70 (m, 1H), 2.86–3.23 (m, 6H), 3.35–3.54 (m, 1H),
6.55–6.70 (m, 7H), 8.20 (s, 1H), 8.24 (s, 1H), 10.01 (s, 1H); IR:
(cmꢀ1): 1681. ½a 2D0
¼ ꢀ77:9 (c 0.31, CHCl3). Anal. Calcd
ꢁ
4. Experimental section
4.1. General
(KBr)
m
for C20H18N2O: C, 79.44; H, 6.00; N, 9.26. Found: C, 79.62; H,
5.80; N, 8.98. (S)-8 was prepared under identical conditions.
½
a 2D0
ꢁ
¼ þ78:8 (c 0.75, CHCl3).
Melting points were determined on a Büchi 530 apparatus and
are uncorrected. All 1H NMR and 13C NMR spectra were recorded in
CDCl3 solution with Bruker AC spectrometers at 360 MHz as well
as 90 and 63 MHz. IR spectra were recorded with a Jasco FT/IR
410 spectrometer. Elemental analyses were performed by the
Department of Organic Chemistry, University of Erlangen-Nürnberg.
Optical rotations were measured with
spectropolarimeter. All reagents were of commercial quality and
used as purchased. Flash chromatography was carried out with sil-
4.4. (R)-1-(1-[2.2]Paracyclophane-4-yl-1H-pyrazol-4-ylmethyl)-
4-phenyl-piperazine ((R)-3)
A suspension of (R)-8 (21 mg, 0.07 mmol), sodium triacetoxy-
borohydride (18.6 mg, 0.10 mmol) and 1-phenylpiperazine
(11.5 mg, 0.07 mmol) in dichloromethane (1.5 mL) was stirred for
5 h at room temperature. The mixture was added to a saturated
aqueous solution of NaHCO3 and was extracted with dichloro-
methane. The organic layer was dried (Na2SO4) and evaporated
and the residue was purified by flash chromatography (ethyl ace-
a Perkin–Elmer 241
ica gel 60 (4.0–6.3
stated v/v proportions. Analytical thin-layer chromatography (TLC)
lm) eluting with the appropriate solution in the

F. Sanna et al. / Bioorg. Med. Chem. 21 (2013) 1680–1684
1683
tate/petroleum ether 7:3) to give pure (R)-3 (25 mg, 80%) as a col-
orless solid (mp 131 °C). EI-MS: m/z 448 (M+); 1H NMR: (CDCl3,
360 MHz) d (ppm): 2.54–2.64 (m, 1H), 2.65–2.75 (m, 4H), 2.78–
3.21 (m, 6H), 3.22–3.30 (m, 4H), 3.52 (ddd, J = 13.0 Hz, 9.9 Hz,
2.7 Hz, 1H), 3.60 (d, J = 13.7 Hz, 1H), 3.64 (d, J = 13.7 Hz, 1H), 6.55
(dd, J = 7.8 Hz, 1.8 Hz, 2H), 6.59 (d, J = 7.8 Hz, 1H), 6.62 (dd,
J = 7.8 Hz, 1.8 Hz, 1H) 6.63 (dd, J = 7.8 Hz, 1.8 Hz, 1H), 6.66 (d,
J = 1.8 Hz, 1H), 6.69 (dd, J = 7.8 Hz, 1.8 Hz, 1H), 6.69–6.89 (m, 1H),
6.91–6.98 (m, 2H), 7.23–7.30 (m, 2H), 7.67 (d, J = 2.1 Hz, 1H),
in PRISM 5.0 (GraphPad Software, San Diego, CA). Competition curves
were fitted to a sigmoid curve by non-linear regression analysis in
which the logEC50 value and the Hill coefficient were free param-
eters. EC50 values were transformed to Ki values according to the
equation of Cheng and Prusoff.20
4.8. Functional assays on the Inhibition of cAMP Accumulation
Bioluminescence based cAMP-Glo Promega assay was per-
formed according to the manufacturer instructions (Promega
Corporation, Madison, WI, USA). In this assay, the activation of
cAMP-dependent protein kinase (PKA) is monitored by measuring
ATP utilization in a kinase reaction by using a luciferase/luciferin
luminescent reaction.21 The D4.4 receptor expressing CHO cells17
were seeded into a white 96 well plate at the density of 10,000
cells/well 24 h before the assay. The cells were first washed
once with Krebs Ringer Buffer, pH 7.4, and then incubated with
different concentrations of the compounds under study in pres-
7.73 (d, J = 2.1 Hz, 1H); IR: (KBr)
0.5, CHCl3). Anal. Calcd for C30H32N4: C, 80.32; H, 7.19; N, 12.49.
m
(cmꢀ1): 1598. ½a 2D0
¼ ꢀ75:8 (c
ꢁ
Found: C, 79.99; H, 7.08; N, 12.35. [ee] >98%. (S)-3 was prepared
under identical conditions. ½a D20
¼ þ74:8 (c 1, CHCl3). [ee] >98%.
ꢁ
4.5. 1-(1-Phenyl-1H-pyrazol-4-ylmethyl)-4-[2.2]paracyclophan-
4-yl-piperazine (4)
A
mixture of 1-phenylpyrazole-4-carbaldehyde (18.5 mg,
ence of 10
l
M
forskolin in Krebs Ringer Buffer containing
M 4-(3-
0.11 mmol), paracyclophanyl piperazine (31.4 mg, 0.11 mmol)
and sodium triacetoxyborohydride (27.5 mg, 0.13 mmol) in dichlo-
romethane (1.5 mL) was stirred at room temperature for 24 h.
Then, the mixture was added to a saturated aqueous solution of
NaHCO3 and extracted with dichloromethane. The organic layer
was dried (Na2SO4) and evaporated and the residue was purified
by flash chromatography (petroleum ether/ethyl acetate 4:6) to
give pure 4 (35.2 mg, 72%) as a colorless solid (mp 78 °C). EI-MS:
m/z 448 (M+); 1H NMR: (CDCl3, 360 MHz) d (ppm): 2.60–2.81 (m,
5H), 2.85–3.12 (m, 9H), 3.25 (ddd, J = 12.3 Hz, 9.0 Hz, 6.0 Hz, 1H),
3.37 (ddd, J = 12.7 Hz, 9.7 Hz, 2.4 Hz, 1H), 3.60 (s, 2H), 5.71 (d,
J = 1.8 Hz, 1H), 6.27 (dd, J = 7.8 Hz, 1.8 Hz, 1H), 6.35 (dd,
J = 7.9 Hz, 1.9 Hz, 1H), 6.40 (d, J = 7.8 Hz, 1H), 6.44 (dd, J = 7.9 Hz,
1.9 Hz, 1H), 6.52 (dd, J = 7.9 Hz, 1.9 Hz, 1H), 6.69 (dd, J = 7.9 Hz,
1.9 Hz, 1H), 7.24–7.31 (m, 1H), 7.41–7.48 (m, 2H), 7.67–7.73 (m,
100 M 3-isobutyl-1-methylxanthine (IBMX) and 100
l
l
butoxy-4-methoxybenzyl)imidazoline (Ro-20-1724), pH 7.4. After
incubation for 20 min at 25 °C, cells were treated as previously
described.22 Luminescence was measured on a microplate reader
Victor 3V (Perkin Elmer, Waltham, MA, USA).
The EC50 and Emax values were calculated as means SEM of
data from at least three experiments with each concentration in
triplicate.
Acknowledgments
F.S. is recipient of a Master and Back grant (Code No. PRR-MAB-
A2011–19138) from ARS (Autonomous Region of Sardinia).
m
(cmꢀ1): 1596. Anal. Calcd for
References and notes
3H), 7.92 (s, 1H); IR: (KBr)
C30H32N4: C, 80.32; H, 7.19; N, 12.49. Found: C, 80.63; H, 7.13; N,
12.32.
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6. (a) Brioni, J. D.; Moreland, R. B.; Cowart, M.; Hsieh, G. C.; Stewart, A. O.;
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15
16
17
D
2long, D2short
,
D3 and D4.4 receptors were run on membrane
preparations from CHO cells stably expressing the corresponding
receptor. Assays were run with membranes at protein concentra-
tions per well of 14–20 lg/mL, 8 lg/mL, 6–20 lg/mL and 20 lg/
mL for D2long, D2short, D3 and D4.4, respectively and with the radio-
ligand [3H]spiperone (specific activity = 84 Ci/mmol, PerkinElmer,
Rodgau, Germany) at a final concentration of 0.5 nM in binding
7. (a) Ehrlich, K.; Gotz, A.; Bollinger, S.; Tschammer, N.; Bettinetti, L.; Harterich, S.;
Hubner, H.; Lanig, H.; Gmeiner, P. J. Med. Chem. 2009, 52, 4923; (b) Lober, S.;
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buffer (50 mM Tris, 1.0 mM EDTA, 5.0 mM MgCl2, 100
lg/ml baci-
tracin and 5 g/ml soybean trypsin inhibitor at pH 7.4). The KD val-
l
ues of the membrane preparations used were 0.10–0.12 nM,
0.10 nM, 0.10–0.40 nM and 0.13 nM for D2long, D2short, D3 and
D
4.4, respectively at a receptor density expressed as Bmax in the
range of 220–1100 fmol/mg. For the determination of D1 affinity
homogenate from bovine striatum was incubated with the radioli-
gand [3H]SCH 23390 (specific activity = 75 Ci/mmol, Biotrend, Co-
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4.7. Data analysis
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