Dopamine D2, D3, and D4 selective phenylpiperazines as molecular probes to explore the origins of subtype specific receptor binding

Article abstract of DOI:10.1021/jm900690y

Assembling phenylpiperazines with 7a-azaindole via different spacer elements, we developed subtype selective dopamine receptor ligands of types 1a,c, 2a, and 3a preferentially interacting with D₄, D₂, and D₃, respectively.

Full text of DOI:10.1021/jm900690y

J. Med. Chem. 2009, 52, 4923–4935 4923
DOI: 10.1021/jm900690y
Dopamine D2, D3, and D4 Selective Phenylpiperazines as Molecular Probes To Explore the
Origins of Subtype Specific Receptor Binding
Katharina Ehrlich,† Angela Gotz,†,‡ Stefan Bollinger,† Nuska Tschammer,† Laura Bettinetti,† Steffen Harterich,†
Harald Hubner,† Harald Lanig,‡ and Peter Gmeiner*,†
†Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich Alexander University, Schuhstrasse 19, 91052 Erlangen,
Germany, and ‡Department of Chemistry and Pharmacy, Computer Chemistry Center, Friedrich-Alexander-Universitat,
Nagelsbachstrasse, 25, 91052 Erlangen, Germany
Received May 22, 2009
Assembling phenylpiperazines with 7a-azaindole via different spacer elements, we developed subtype
selective dopamine receptor ligands of types 1a,c, 2a, and 3a preferentially interacting with D4, D2, and
D3, respectively. To complete this set, the methylthio analogues 2b and 3b exceeding the affinity of 2a
and 3a by one order of magnitude and the structural intermediate 1b were synthesized. These chemically
similar but biologically divergent target compounds served as molecular probes for radioligand
displacement experiments, mutagenesis, and docking studies on homology models based on the recent
crystal structure of the β2-adrenergic receptor. Specific interactions with the highly conserved amino
acids Asp3.32 and His6.55 and less conserved residues at positions 2.61, 2.64, 3.28, and 3.29 were
identified. Inclusion of a carefully modeled extracellular loop 2 displayed two nonconserved residues in
EL2 that differently contribute to ligand binding. Obviously, subtype selectivity is caused by non-
conserved but frequently mediated by conserved amino acids.
Introduction
TM3, and TM7 to be liable for subtype selectivity at the D2-
like dopamine receptors.9-11
The D2-like dopamine receptors including the subtypes D2,
D3, and D4 are members of the large family of G-protein-
coupled receptors (GPCRsa), also referred to as seven trans-
membrane receptors. Many central nervous system diseases
like schizophrenia, Parkinson’s disease, drug addiction, and
erectile dysfunction are associated with D2-like dopamine
receptors.1
Highly selective ligands are needed to specifically target
receptor subtypes involved in these diseases. For example,
D3-selective drug candidates are discovered for the treatment
of schizophrenia and drug addiction.1-4 On the other hand,
D4-selective agonists and partial agonists facilitate a promis-
ing approach in the therapy of erectile dysfunction.5-7 There-
fore, knowledge about selectivity generating properties of the
ligands and their receptors is important for drug design.
On the basis of rhodopsin, comparative molecular model-
ing of the D2-like dopamine receptors and ligand docking
revealed differences in the size and shape of a common ligand
binding site when we and others suggested that particular
microdomains in transmembrane helix 2 (TM2), TM3, and
TM7 might be relevant for ligand selectivity.1,8 These results
are in accordance with mutagenesis studies, which also pro-
posed changes in the hydrophobic interface between TM2,
By application of CoMFA (comparative molecular field
analysis) and CoMSIA (comparative molecular similarities
indices analysis), 3D-QSAR studies guided us to a rational
development of D3-selective phenylpiperazines as radioli-
gands for PET (positron emission tomography).12-19
Among aminergic GPCR ligands, phenylpiperazines are
known as privileged structural moieties simulating native
biogenic amines, like dopamine, serotonin, and (nor)epin-
ephrine (Chart 1).20 Containing an aromatic ring system and a
basic nitrogen, the phenylpiperazine scaffold can be regarded
as the primary recognition element targeting the neurotrans-
mitter binding site of aminergic GPCRs.21-24 Carbocyclic or
heterocyclic appendages have been linked via a spacer element
to enhance both affinity and subtype selectivity. We identified
the 7a-azaindolesystem as an excellent heterocyclic moietyfor
D2, D3, and D4 selective phenylpiperazines when subtype
specificity could be controlled by the nature of the spacer
element. Thus, use of an N-butylcarboxamide chain linking
postion 2 of the azaindole ring with the phenylpiperazine
moiety led to the D3-selective partial agonist 3a (Ki=4.3 nM,
D3/D2 = 72, D3/D4 = 30). Interestingly, our SAR studies
displayed that shortening of the spacer by one carbon and
change of the substitution at the azaindole ring resulted
in preferential D2 binding when compared to D3. Thus, the
N-propylcarboxamide derivative 2a revealed a Ki of 11 nM
compared to 150 nM of D3 and 14 nM for D4. On the other
hand, we discovered highly selective D4 receptor partial
agonists and antagonists of type 1a (FAUC 213, Ki=2.2 nM,
D4/D2=1500; D4/D3=2400), 1c and 1d (FAUC 113) when
using a methylene unit to link the primary recognition element
*To whom correspondence should be addressed. Phone: þ49 9131 85-
29383. Fax: þ49 9131 85-22585. E-mail: peter.gmeiner@medchem.
uni-erlangen.de.
a Abbreviations: GPCR, G-protein-coupled receptor; EL, extracel-
lular loop; TM, transmembrane helix; CHO, Chinese hamster ovary;
HEK, human embryonic kidney; HR-EIMS, high resolution electron
ionization mass spectrometry; EI-MS, electron ionization mass spectro-
metry.
r
2009 American Chemical Society
Published on Web 07/16/2009
pubs.acs.org/jmc

4924 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Ehrlich et al.
Chart 1
Chart 2
with the heterocyclic appendage (Chart 2).5,25-28 Taking
advantage of the finding that these target compounds show
both high chemical similarity and significantly different bio-
logical binding patterns, we chose them as molecular probes to
better understand the molecular origins of subtype selectivity.
The identification of conserved and nonconserved residues
that determine subtype selectivity of the binding pocket was
based on previous SAR, mutagenesis, and docking studies.
Whereas the phenylpiperazine substructure obviously simu-
lates the biogenic amine, the data clearly indicate that the
relative topicity of the heterocyclic appendage is responsible
for subtype specific recognition.
Recent mutagenesis and preliminary docking studies sug-
gest that two adjacent amino acid side chains in TM3 (3.28
and 3.29), two critical residues in TM2 (2.61 and 2.64), and
two positions within EL2 are in proximity to the spacer
elements and the heterocyclic appendages and, thus, deter-
mine subtype selectivity. For the primary recognition element,
our preliminary molecular modeling results identified His6.55
as a key residue for the interaction with the common phenyl-
piperazine unit. Finally, Asp3.32 should be modified because
its carboxylate function has been proved to be responsible for
a reinforced H-bond provided by the basic nitrogen of the
ligands.
Results and Discussion
Compound Selection. To be able to confirm that mutages-
esis experiments are, in fact, diagnostic for a family of
compounds, at least two representatives for each subtype
should be investigated. Taking advantage of our in-house
compound library, we identified an ortho-positioned
methylthio group to be advantageous for dopamine receptor
affinity. Because high binding affinity was expected to be
beneficial for mutagenesis experiments, the test compounds
2b and 3b should be synthesized as structural analogues of
methoxyphenylpiperazines 2a and 3a. As a structural inter-
mediate between the methylene linked probe 1a and the D2
and D3 selective N-alkylcarboxamides 2a and 3a (FAUC
329), respectively,26 the pentylene bridged azaindole 1b
should be prepared. In analogy to the D2 selective congeners
2a,b, compound 1b displays a five-atom spacer. However, a
carboxamide function is replaced by an ethylene unit.
Synthesis. The azaindole derivative 2b was synthesized as
shown in Scheme 1. Nucleophilic substitution of 3-bromo-
propionitrile with 1-(2-methylsulfanylphenyl)piperazine (4)
resulted in formation of the tertiary amine 5. Reduction of
the nitrile function with lithium aluminum hydride followed
by TBTU mediated coupling of the resulting aminopropyl-
piperazine 6 with pyrazolo[1,5-a]pyridine-3-carboxylic acid
gave the target compound 2b (Scheme 1).30,31
Extending the above-mentioned dopamine receptor ligands
by three further molecular probes to be synthesized, we herein
describe mutagenesis studies and homology modeling of D2,
D3, and D4 based on the recent crystal structure of the
β2-adrenergic receptor.29
To approach to the azaindole derivative 1b, the building
block 7 was reacted with 1-aminopyridinium iodide to give
the heteroarene 8.32-35 Intermediate 8 was subjected to

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4925
hydrolysis and decarboxylation with 40% H2SO4 to afford 9.
Subsequent HOAt mediated DCC coupling with 1-(4-chlor-
ophenyl)piperazine furnished the carboxamide 10, which
was reduced by LiAlH4 to get the final product 1b
(Scheme 2). Test compounds 1a, 1c, 2a, and 3a were prepared
according to our previously described protocols.5,25,26,36
Ligand Binding. Radioligand displacement assays were
performed to determine binding affinities and the selectivity
profiles of the new dopamine receptor ligands 1b, 2b, and 3b
in comparison to the described phenylpiperazines 1a,c, 2a,
and 3a. The binding data were generated by measuring the
ligands’ ability to compete with [3H]spiperone for the cloned
human dopamine receptor subtypes stably expressed in
CHO (Chinese hamster ovary) cells (Table 1). Formal ex-
change of the methoxy group of the preferential D2 agonist
2a by a methylthio group to give the methylthiophenylpiper-
azine 2b resulted in an 8-fold increase in binding affinity
and substantially improved selectivity over the D4 subtype.
An analogous exchange for the D3 ligand 3a, giving the thio
analogue 3b, resulted in a 12-fold increase in binding affinity
at D3. Elongation of the spacer of the small, highly D4
selective ligand 1a gave the structural intermediate 1b which
displayed a large decrease in affinity for the D4 receptor,
whereas affinity for the D2 and the D3 receptor was increased
when compared to 1a.
Scheme 1a
Modeling of the Dopamine D2, D3, and D4 Receptors.
Receptor models of the dopamine D2, D3, and D4 receptor
were constructed using the crystal structure of the human
β2-adrenergic receptor (PDB entry 2RH1) as a template.37
The artificially engineered intracellular loop 3 present in the
crystal structure was omitted in receptor modeling, since it
was found to have no direct effect on ligand binding.38
On the basis of agreement with experimental data, one
receptor model was selected for each of the D2 and the D3
receptor, whereas for the D4 receptor, two diverse models
existed that showed different χ1 rotamers atPhe2.61. These two
diverse models were chosen by docking experiments with the
D4-selective ligand 1a and its 3-substituted regioisomer 1d.28
In rhodopsin and in the β2-adrenergic receptor, the part of
the EL2 that is C-terminal to the conserved disulfide bond
connecting EL2 and TM3 is involved in receptor-ligand
interaction.37,39 SCAM (substituted cysteine accessibility
method) studies at the D2 receptor also identified Ile184 to
be involved in ligand binding.40 Therefore, we decided to
complete our D2, D3, and D4 receptor structures by the
inclusion of a carefully modeled EL2. The C-terminal region
of the EL2, which is supposed to take part in ligand binding,
was modeled according to the β2-adrenergic crystal struc-
ture. The N-terminal region was selected from the loop
database of the Swiss-PdbViewer.41 The resulting structure
was equilibrated by means of molecular dynamics simulation
and energy minimization at a D4 ligand-receptor complex.
This loop was then reinserted into the original D4 receptor
model and also provided the basis for modeling of the EL2 at
the D2 and the D3 receptor models. This procedure resulted
in conformations of the EL2 that agreed with experimental
data; the C-terminal end of EL2 is close to the binding site
crevice; Ile184 in D2, Ile183 in D3, and Leu187 in D4 point
into the binding pocket, thereby enabling interactions with
the ligand.
a Reaction conditions: (i) 3-bromopropionitrile, K2CO3, NaI,
MeCN, reflux 24 h (67%); (ii) Et2O, LiAlH4, 0 °C to room temp, 1 h
(18%); (iii) pyrazolo[1,5-a]pyridine-3-carboxylic acid, CH2Cl2, DIPEA,
TBTU, 0 °C to room temp, 2 h (87%).
Scheme 2a
a Reaction conditions: (i) 1-aminopyridinium iodide, K2CO3, DMF,
50 °C, 2 h (13%); (ii) 40% H2SO4, 110 °C 3 h (93%); (iii) 1-(4-
chlorophenyl)piperazine, DCC, HOAt, room temp, 15 h (81%); (iv)
LiAlH4, Et2O, 0 °C, 1 h, to room temp, 5 h (59%).
Ligand Docking and Analysis. The final receptor models
were used for docking experiments by means of AUTO-
DOCK4. The high affinity ligands 2b, 3a, and 1a were
docked in the D2, D3, and D4 receptor model, respectively.
Table 1. Receptor Binding and Selectivity Ratios for 1a-3b
Ki a[3H]spiperone
ratio of Ki values
compd
X
position
D2L
D3
D4
D2L/D3
D2/D4
D3/D4
2400
8.0
1000
11
2.0
1ab
1b
2
2
3
3
3
2
2
3400 ( 450
160 ( 90
280 ( 130
11 ( 1.4
1.4 ( 0.26
310 ( 34
16 ( 2.2
5300 ( 720
370 ( 40
1200 ( 240
150 ( 17
18 ( 2.5
4.3 ( 0.29
0.35 ( 0.036
2.2 ( 0.23
46 ( 5.2
1.2 ( 0.2
14 ( 2.5
8.8 ( 1.4
130 ( 16
65 ( 11
0.64
0.43
0.23
0.07
0.08
72
1500
3.5
1cc
2ad
2b
230
0.79
0.16
2.4
O
S
3ad
3b
O
S
0.033
0.005
46
0.25
a Ki ( SEM, in nM, are based on the mean values of 2-9 experiments each done in triplicate. b Reference 25. c Reference 5. d Reference 26.

4926 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Ehrlich et al.
Figure 1. Phenylpiperazines of types 1a, 2b, and 3a docked into the respective D2, D3, and D4 receptor models. Residues involved in ligand
binding and the ligands are represented as sticks. Receptor backbone is represented as gray ribbon, except for TM3 which is colored blue, for
clarity. D2 and D3 ligand-receptor complexes as well as four different D4 ligand-receptor complexes (two receptor models with two binding
modes, each) are displayed.
Only ligand-receptor complexes in which the protonated
nitrogen of the phenylpiperazine scaffold established an
essential charge reinforced H-bond with Asp3.32 were con-
sidered. The resulting receptor-ligand complexes were clus-
is in proximity to His6.55 and Ile184 originating from EL2. The
amide group interacts with Thr7.39 via the carbonyl oxygen. The
azaindole moiety points toward TM2 and interacts with a
lipophilic microdomain formed by Ile183 from EL2, Val2.61
,
tered and scored by Drugscore
.
online 42 After a final selection
Leu2.64, and Trp7.40
.
according to experimental data, the best receptor-ligand
complexes were subjected to energy minimization. The mini-
mized complexes are shown in Figure 1.
The D3 selective ligand 3a was docked into the D3 receptor
model. Analogous to the orientation of 2b in the D2 receptor
model, the methoxyphenyl moiety of 3a interacts with
critical residues in TM5, TM6, Ile183 in EL2, and Val3.33
in TM3. It is worthy of note that the methoxy group was
positioned in proximity to His6.55. The heterocyclic moiety
points toward TM2, interacting with a hydrophobic cluster
In addition to the polar interaction with Asp3.32, all
ligands interacted with two receptor microdomains. The first
microdomain comprises the less conserved residue His6.55 in
TM6 and the highly conserved residues Trp6.48, Phe6.51
,
Phe6.52, and Val3.33 in TM6 and TM3, respectively. The
formed by Val180 from EL2, and Leu2.64
.
second microdomain consists of residues in TM7 (Thr7.39
,
Ligands of types 2 and 3 interact in a very similar binding
mode with the D2 and D3 binding site, respectively, with the
phenylpiperazine moiety pointing toward TM5 and the
methoxy or methylthio substituent pointing toward an
isoleucine originating from the EL2 (Figure 2). The hetero-
cyclic arene moiety, however, interacts differently at the
D2 and the D3 ligand-receptor complexes. The azaindole
Trp7.40, and Tyr7.43) and at positions 2.61, 2.64, 3.28, and
3.29 at TM2 and TM3, respectively.
Docking of the preferential D2 ligand 2b into the D2 receptor
model yielded a receptor-ligand complex in which the methyl-
thiophenyl moiety of 2b lies in a lipophilic pocket formed by
TM3, TM5, TM6 and capped by EL2. The methylthio group

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4927
In summary, docking of the phenylpiperazines 2b and 3a
into our D2 and D3 models, respectively, yielded one ligand-
receptor complex each. The substituted phenyl moiety dis-
plays favorable interactions with a lipophilic cluster in TM6,
whereas the azaindole moiety points at TM2. Docking of the
methylene-bridged test compound 1a into D4 resulted in two
alternative orientations. In mode-1, the heteroarene moiety
interacts with the aromatic residues in TM6 and the chlor-
ophenyl moiety is involved in interactions with TM2 and
TM3. In mode-2, the chlorophenyl moiety lies within the
hydrophobic pocket formed by TM6 and TM3 whereas the
pyrazolopyridine moiety interacts with a hydrophobic clus-
ter at the TM2-TM3 interface. Although scoring values of
D4-A2, showing an orientation of the ligand analogous to the
D2 and D3 ligand-receptor complexes, were much better
than those of D4-A1, previous experimental data could not
yet prove in which orientation type 1 ligands bind to the
dopamine D4 receptor. Because of the symmetry of the short
methylene-bridged ligand, both binding modes are concei-
vable. Experiments published by Schetz et al. revealed earlier
that small changes at the ligand can cause a switch from
mode-1 in which the azaindole moiety is oriented toward
TM5 (models D4-A1 and D4-B1) to mode-2 in which the
phenylpiperazine points toward TM5 (models D4-A2 and
D4-B2).10 According to our studies, the observed heteroge-
neity in the binding mode and the 4-chloro substitution of the
phenylpiperazine moiety led to a deviation in the position of
the basic amine and its interaction with Asp3.32 compared to
the D2 and D3 ligand-receptor complexes. We suggest that
the binding mode of the elongated structural analogue 1b
resembles mode-2. Because of its longer spacer arm, the
basic amine of the phenylpiperazine scaffold is no longer
at the center of the molecule but further away from the
azaindole. In mode-1, the longer distance would impede the
simultaneous interaction with Asp3.32 and the aromatic/
hydrophobic pocket formed at TM3, TM5, and TM6.
Receptor Mutagenesis. Site directed mutagenesis was ap-
plied in the D2 and D3 dopamine receptor background to
determine the influence of selected residues on ligand binding
and subtype selectivity. Thus, diagnostic amino acids of D2
and D3 were mutated and mutant receptors transiently ex-
pressed in HEK 293 cells. To characterize the binding proper-
ties, saturation experiments were conducted to determine
Bmax and KD values. Subsequently, the impact on the recogni-
tion properties of our test compounds 1a-c, 2a,b, and 3a,b
was investigated when comparing binding affinities with those
of transiently expressed wildtype receptor protein. The bind-
ing site crevice of D2-like receptors is expected to be lined by
Figure 2. Ligand binding at the EL2. Representation of the D2
(green) and the D3 (orange) ligand- receptor complexes. Hydro-
phobic residues from EL2 and the hydrophobic cluster at TM2 and
TM3 (Val2.61, Leu2.64, Trp100/Trp96 from EL1, Phe3.28, Val3.29) are
shown as sticks. Important residues and sequence differences at the
EL2 are highlighted by residue numbers.
of the preferential D2 ligand 2b interacts with Val2.61, Leu2.64
,
Trp7.40, and Ile183 from the EL2. The elongated heterocyclic
appendage of the D3 selective ligand 3a stretches further
toward the extracellular side, interacting primarily with
Leu2.64, and Val180 from the EL2. The position of Ile183 at
the D2 receptor is occupied by Ser182 at the D3 receptor,
whereas Val180 of the D3 receptor corresponds to Glu181 at
the D2 receptor (Figure 2). Both residues are located next to
the conserved cysteine, forming a disulfide bond with Cys3.25
from TM3. These differences within the ligand binding pocket
are specifically addressed by the heterocyclic appendage of the
respective D2 and D3 selective receptor ligands. We therefore
presume that these nonconserved residues are directly in-
volved in D2/D3 subtype selectivity and that the heterocyclic
appendage greatly contributes to subtype selectivity.
The D4 selective phenylpiperazine 1a was docked into two
relevant D4 receptor models (A and B). In model A, Phe2.61 is
oriented toward TM7, thereby facilitating interactions bet-
ween the ligand and residues originating from TM3. In model
B, Phe2.61 points toward TM3 partly obstructing interactions
between the ligand and TM3. Docking resulted in two alter-
native orientations of 1a in both models A and B. In mode-1,
the azaindole moiety interacts with the lipophilic pocket
formed by aromatic residues in TM6, Leu187 in EL2, and
Val3.33 in TM3. The chlorophenyl moiety, however, interacts
differently in both models. In model A, the aromatic moiety is
situated between Phe2.61 and TM3 interacting with the hydro-
several highly conserved amino acids, among which Asp3.32
,
Ser5.42, Ser5.43, Ser5.46, Phe6.51, Phe6.52, and Tyr7.43 seem to be
of crucial importance.1,43-46 These results are now corrobo-
rated by the recent GPCR crystal structures.29,47,48 However,
nonconserved residues within or next to the binding pocket
have the highest probability to be involved in subtype selec-
tivity. On the basis of our computational predictions, we
selected four residues, differentiating the D2 and D3 from
the D4 receptor (positions 2.61, 2.64, 3.28, and 3.29), and two
residues from EL2, differentiating the D2 from the D3 subtype
(Glu181/Val180and Ile183/Ser182). Butwealsoexamined the
key contacts of the conserved amino acids Asp3.32 and His6.55
that were expected to interact with the cationic ammonium
center and the catechol bioisostere, respectively. Asp3.32 is
regarded to be essential for ligand binding at aminergic
GPCRs but might differ in its contribution to ligand binding
phobic microdomain formed by Phe2.61, Leu3.28, and Met3.29
whereas in model B the conformation of Phe2.61 hinders the
interaction between the ligand and Leu3.28
,
.
Mode-2 is similar to the orientation of the long chain
analogues 2b and 3a in the D2 and D3 receptor model,
respectively. The chlorophenyl moiety interacts with the
hydrophobic microdomain that spans the TM6 and TM3
interface, and the chloro substituent points at TM5. In model
A, the azaindole moiety lies between Phe2.61 and TM3,
interacting with the hydrophobic cluster at the TM2-TM3
interface. In model B, the heteroarene points at TM7 lacking
the interaction with Val3.29. In a comparison of mode-1 and
mode-2, the position of the charged nitrogen of 1a varies by
˚
2-3 A.

4928 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Table 2. Kd and Bmax Values at Wild Type and Mutant Receptorsa
Ehrlich et al.
D2L receptor
D3 receptor
b
c
b
c
mutation
wild type
D3.32E
Kd
Bmax
Kd
Bmax
0.44 ( 0.06
0.34 ( 0.09
0.75 ( 0.16
0.53 ( 0.15
1.3 ( 0.24
0.67 ( 0.19
1.1 ( 0.24
1600 ( 180
1300 ( 230
1100 ( 200
2500 ( 760
2200 ( 440
2300 ( 360
1900 ( 340
0.54 ( 0.07
2.0 ( 0.20
0.55 ( 0.08
0.54 ( 0.12
0.81 ( 0.17
0.84 ( 0.09
1600 ( 180
390 ( 89
1600 ( 200
330 ( 53
12009 ( 160
2200 ( 270
V2.61F
L2.64F
FV3.28,3.29LM
V2.61FþFV3.28,3.29LM
D2E181 VþI183S
D3V180EþS182I
H6.55A
0,70 ( 0.04
1.0 ( 0.13
NBDd
290 ( 100
1200 ( 170
NBDd
0.41 ( 0.09
0.86 ( 0.13
1600 ( 310
1400 ( 500
H6.55F
a Values were determined by saturation binding experiments with [3H]spiperone at receptors transiently expressed in HEK 293 cells. b Kd ( SEM
values, in nM, are based on the mean of 2-43 independent experiments each done in triplicate. c Bmax in fmol/mg protein. d NBD = no binding detected.
Table 3. Binding Data of Compounds 1a-c, 2a,b, and 3a,b at Wild Type and Mutant Receptors (Ki values ( SEM in nM)a
receptor
1a
1b
1c
2a
2b
3a
3b
D2wt
2300 ( 360
220 ( 46
350 ( 83
13 ( 2.8
2.2 ( 0.4
150 ( 35
37 ( 7
D2D3.32E
D2V2.61F
D2L2.64F
5100 ( 360 (2.2)
1200 ( 220 (0.53)
2000 ( 200 (0.89)
1500 ( 130 (4.4)
94 ( 32 (0.27)
610 ( 100 (1.8)
720 ( 140 (55)
450 ( 75 (34)
390 ( 70 (30)
110 ( 11 (8.5)
83 ( 26 (6)
160 ( 29 (74)
100 ( 25 (45)
660 ( 92 (303)
35 ( 7 (16)
3500 ( 240 (23) 1300 ( 75 (34)
740 ( 140 (3.3)
920 ( 230 (6.0)
940 ( 38 (6)
220 ( 53 (1.4)
130 ( 6.0 (3.4)
120 ( 17 (3)
65 ( 15 (1.8)
D2FV3.28,3.29LM 300 ( 61 (0.13)
1100 ( 250 (4.9) 46 ( 3.5 (0.13)
D2V2.61Fþ
FV3.28,3.29LM
D2E181 VþI183S
D2H6.55A
D2H6.55F
D3wt
66 ( 14 (0.03)
470 ( 90 (2.1)
54 ( 8 (0.15)
20 ( 8 (9)
120 ( 7.5 (0.81) 26 ( 1.0 (0.70)
47000 ( 19000 (19)
320 ( 100 (0.14)
1600 ( 550 (0.7)
8300 ( 1200
3600 ( 460 (10.5) 81 ( 6.6 (6.2)
26 ( 3.8 (12)
1000 ( 250 (6.8) 130 ( 23 (3.4)
170 ( 29 (0.74)
220 ( 52
63 ( 5 (0.18)
590 ( 100 (1.2)
770 ( 38
1.6 ( 0.40 (0.12) 0.1 ( 0.014 (0.05) 21 ( 1.1 (0.14)
69 ( 15 (5.3)
320 ( 61
11 ( 0.6 (0.3)
160 ( 43 (4.3)
0.5 ( 0.08
17 ( 4 (7.7)
26 ( 4
340 ( 69 (2.2)
7.4 ( 1.1
D3D3.32E
D3V2.61F
D3L2.64F
12000 ( 1900 (1.4)
9200 ( 1700 (1.1)
1600 ( 170 (0.19)
680 ( 100 (0.9)
1900 ( 460 (8.8) 2300 ( 110 (3.0)
720 ( 140 (0.9)
44 ( 3 (0.06)
1100 ( 260 (5.1) 51 ( 11 (0.07)
390 ( 61 (1.2)
1030 ( 170 (3.3) 740 ( 100 (29)
630 ( 120 (2)
640 ( 140 (2.0)
34 ( 7 (0.11)
650 ( 67 (25)
93 ( 9.0 (13)
500 ( 81 (68)
290 ( 47 (40)
97 ( 4 (13)
230 ( 21 (466)
58 ( 12 (116)
33 ( 8.5 (66)
18 ( 3 (36)
430 ( 110 (17)
320 ( 34 (12)
8 ( 0.9 (0.31)
D3FV3.28,3.29LM 730 ( 85 (0.09)
530 ( 120 (2.4)
D3V2.61Fþ
FV3.28,3.29LM
D3V180EþS182I
D3H6.55A
76 ( 10 (0.009)
130 ( 19 (18)
40 ( 12 (80)
2500 ( 1100 (0.31)
46 ( 3.3 (0.005)
6.8 ( 0.7
340 ( 59 (0.44)
61 ( 1.0 (0.08)
2.3 ( 0.3
16 ( 4 (0.05)
56 ( 11 (0.18)
52 ( 8
3.4 ( 0.99 (0.13)
12 ( 3 (0.5)
26 ( 3.4
7.6 ( 1.5 (1.0)
2 ( 0.21 (0.27)
400 ( 38
0.76 ( 0.11 (1.5)
0.63 ( 0.15 (1.3)
310 ( 43
D4wt
46 ( 5b
a Binding data of compounds 1a-c, 2a,b, and 3a,b were determined in competition binding experiments with [3H]spiperone at wild type and mutant
receptors transiently expressed in HEK 293 cells. Values given are mean Ki values ( SEM, in nM, of 2-18 independent experiments each done in
triplicate. Fold changes in affinity compared to those of wild type are indicated in parentheses. b Binding data determined in competition binding
experiments with [3H]spiperone at wild type stably expressed in CHO cells.
at the different receptor subtypes.49 His6.55 is conserved
among the D2-like dopamine receptors and involved in bind-
ing of dopamine, whereas no effect on binding of butyrophe-
none ligands could be detected upon mutation to cysteine or
leucine.50-52
receptor. While elongation of the amino acid side chain by
one CH2 group was tolerated, the acid functionality itself
seems to be essential for the binding of the radioligand.
Compared to the D2 wild type receptor, the exchange of
aspartate by glutamate resulted in a 55-fold and a 74-fold
decrease in affinity for the preferential D2 ligands 2a and 2b,
respectively (Table 3). At the D3 receptor, 2a was unaffected
by D3D3.32E, while 2b exhibited a 25-fold decrease in
affinity. The D3 selective phenylpiperazine 3a displayed a
23-fold and 13-fold drop in affinity and its thio analogue 3b a
34-fold and 466-fold drop in affinity at the respective
D2D3.32E and D3D3.32E receptor mutants. The D4 selec-
tive compounds 1a and 1c showed a significant change in
binding affinity at neither the D2D3.32E nor the D3D3.32E
receptor mutant.
Except for D2D3.32N and D3H6.55F, our mutations had
no noteworthy effect on binding of [3H]spiperone, a ligand
that does not show subtype selectivity. As apparent from the
Bmax values, expression levels remained unchanged and
showed only minor fluctuations. Altogether, binding of
[3H]spiperone was hardly influenced at our D2 and D3
receptor mutants, thereby distinguishing itself as the ideal
radioligand for competition binding studies (Table 2).
To probe specific receptor ligand interactions of the
ligands’ cationic ammonium center, the highly conserved
Asp3.32 was mutated to glutamate at D2 and D3 receptors
(D2D3.32E, D3D3.32E), which was least likely to disturb the
protein structure either locally or in its overall folding path-
way according to the guidelines for “safe amino acid sub-
stitution”.43,53,54 In fact, the Bmax value of D2D3.32E was
comparable to that of wild type (Table 2). On the other hand,
mutation of Asp3.32 to asparagine resulted in a complete loss
of [3H]spiperone binding at the D2 receptor, thereby exclud-
ing further experiments with test compounds at this mutant
Asp3.32 is one of the most conserved amino acids among
aminergic GPCRs and is supposed to form a reinforced
hydrogen bond with the basic amine of the ligand.49 Inter-
estingly, the D4 selective compounds 1a and 1c were not
affected by the D3.32E mutations at the D2 and D3 receptors.
Ligands showing a low Ki value, however, seem to be more
affected by the D3.32E mutant receptors. It may be assumed
that selectivity involves tight binding at a specific receptor
subtype. This strict binding mode is very susceptible to small

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4929
changes, whereas at subtypes bound with lower affinity the
substitution of Asp3.32 by glutamate is easily tolerated.
Since preliminary molecular modeling studies indicated
spatial proximity to the arene part of the phenylpiperazine
unit, we decided to change His6.55 into alanine at the D2 and
the D3 receptor, thereby eliminating both its aromatic and
basic side chain properties (D2H6.55A, D3H6.55A). Inter-
estingly, our test compounds exhibited a gain in affinity at
both the D2H6.55A and the D3H6.55A receptor mutants.
The most prominent result was a 180-fold increase in affinity
for the phenylpiperazine 1a at D3H6.55A. The mutation into
phenylalanine (H6.55F) led to a complete loss of radioligand
binding at the D3 receptor, thereby excluding further experi-
ments with this mutant. H6.55F mutation in the D2 receptor
background led to a decrease in affinity for the D2 selective
ligands 2a,b, whereas the affinity of 1a and 3a,b was hardly
affected.
His6.55 is positioned one helix turn above Phe6.52 and
Phe6.51, two highly conserved residues taking part in ligand
binding. We suppose that the additional space created by the
H6.55A mutation leads to an increase in conformational
freedom of these two phenylalanine residues, thereby en-
abling a better accommodation of the phenylpiperzine
moiety to the aromatic microdomain of TM6. The observed
loss of binding at the D2H6.55F mutant receptor further
supports this hypothesis as exchange of His6.55 by a sterically
more demanding phenylalanine leads to less conformational
freedom of Phe6.52 or Phe6.51, thereby hindering favorable
receptor-ligand interactions.
The described D2 model clearly showed an aberration
from the D3 and D4 receptor models at the helix turn
containing His6.55. Phe7.38, which is represented by a threo-
nine at the D3 and D4 subtypes, sterically interacts with
Thr6.54, resulting in a sideward shift of the helix turn contain-
ing Thr6.54 and His6.55. At the D2 model, the latter points
downward in the direction of the ligand binding site, whereas
at the D3 receptor model, His6.55 stretches upward in the
direction of the EL2. This upward position enables the sp2
nitrogen of the imidazole ring to accept an H-bond from the
side chain of Tyr7.35 (Figure 3). Although the residues
involved in ligand binding are conserved, nonconserved
residues in EL2 and at the TM6/TM7 helical interface lead
to a subtype specific difference in the spatial arrangement
next to the binding site. This explains why the H6.55F
mutation was tolerated at the D2 receptor, while at the D3
receptor, it led to a complete loss of radioligand binding.
A similar effect was also observed in the direct comparison
of the β1 and β2 adrenergic receptor crystal structures.
All residues involved in ligand binding are conserved, but
differences at more distant receptor sites are supposed
to provide subtype selectivity, be it by influencing the con-
served residues’ rotameric states, binding kinetics, or recep-
tor dynamics.29,47 Extensive molecular modeling studies of
the D2-like dopamine receptors also indicated a different
behavior and differing proline kink angles within the trans-
membrane helices, influencing the geometrical properties of
the ligand binding site.1 Therefore, nonconserved residues
though distant from the ligand binding site can have a
substantial influence on selectivity by changes in local or
even overall geometry.
Figure 3. Differences between the D2 and D3 receptor models at
His6.55. Representation of the D2 (green) and D3 (orange) receptor
models including the docked ligands. The EL2 was omitted
for clarity. Important residues from TM6 and TM7 and Asp3.32
are shown as sticks. The images display the influence of the
interaction between positions 7.38 and 6.54 on the position and
orientation of His6.55
.
which are conserved among the D2 and D3 receptors, into the
corresponding amino acids present at the D4 receptor.
Starting from both D2 and D3 wildtype, we constructed
the mutants V2.61F and FV3.28,3.29LM and the triple
mutant V2.61FþFV3.28,3.29LM, which were reported to
be involved in D2/D4 subtype selectivity.11
To gain further insight into the determinants of D2/D4 and
D3/D4 ligand selectivity and to probe the molecular interactions
of the heterocyclic appendages including parts of the spacer
element, we decided to change Val2.61, Phe3.28, and Val3.29
,

4930 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Ehrlich et al.
Figure 4. Radioligand displacement curves of 1a at the D3 and D4
wild type and the D3V2.61F, D3FV3.28,3.29LM, and D3V2.61Fþ
FV3.28,3.29LM mutant receptors. Data shown are normalized results
of competitive binding assays, showing the shift in affinity from D3
toward D4 receptor upon mutation at Val2.61, Phe3.28, andVal3.29 of the
D3 receptor.
Figure 5. Receptor mutants at TM2 and TM3. Representation of
the different spatial arrangement within the ligand binding site at
positions 2.61, 3.28, and 3.29. The backbone of the D4 receptor
model A is shown as gray ribbon. Important residues (green, D2;
dark-red, D4 model A; pink, D4 model B) and compound 2b docked
into the D2 receptor (olive green) are shown as sticks. This figure
highlights the steric influence of the amino acid in position 3.28 on
the conformation of Phe2.61 and its interaction with the docked
ligand.
In accordance with previously recorded mutagenesis
data,11 the single mutant V2.61F had hardly any effect on
binding of 1a and the double mutant FV3.28,3.29LM ex-
hibited an 8-fold and 11-fold increase in affinity in the D2 and
D3 background, respectively. Because of the D4 like recogni-
tion properties, the affinity of the D4 ligand 1a was improved
35-fold and 109-fold at D2V2.61FþFV3.28,3.29LM and
D3V2.61FþFV3.28,3.29LM, respectively (Figure 4). The
D4 selective methoxyphenylpiperazine derivative 1c also
improved in affinity at the double and the triple mutants.
On the other hand, the structural homologue 1b incorporat-
ing an elongated spacer arm exhibited a 2- to 9-fold loss of
binding affinity at the mutant receptors.
While the mutation V2.61F had no noteworthy effect on
the phenylpiperazine 1a and 1c, the long chain analogues 2a,b
and 3a,b displayed a substantial loss of binding affinity.
The susceptibility of FV3.28,3.29LM on ligand binding of
2a,b and 3a,b was very similar but less pronounced. For both
cases, the impact on ligands with high affinity was signifi-
cantly higher than on poor binders.
As expected, a negative effect on ligand binding was
observed for the preferential D2 ligands 2a,b at the
D2V2.61FþFV3.28,3.29LM and the D3 selective phenylpi-
perazines 3a,b at the D3V2.61FþFV3.28,3.29LM receptor
mutant. Because of the low affinity at the wild types,
significant loss of binding could not be detected for 2a,b in
the D3 and for 3a,b in the D2 background.
For a further validation of our binding hypothesis, Leu2.64
was also mutated into the sterically more demanding amino
acid phenylalanine. The mutation exhibited a distinct de-
crease in affinity for the preferential D2 ligands 2a,b at the
D2L2.64F and the D3 selective phenylpiperazines 3a,b at the
D3L2.64F receptor mutants. Affinity of the shorter D4
ligands 1a,c was hardly affected.
is restricted by steric interactions with Phe3.28, which is still
present at the D2 and D3 receptor mutant V2.61F. We
suggest that the resulting conformation of Phe2.61 is similar
to the one observed in our D4 receptor model A (D4-A),
which would lead to disadvantageous steric interactions with
2b as docked into the D2 receptor model (Figure 5). Addi-
tional mutation of Phe3.28 and Val3.29 to the sterically less
demanding amino acids leucine and methionine, respec-
tively, allows Phe2.61 to move out of the ligand’s way,
adopting a conformation similar to the one observed in the
D4 receptor model B (D4-B). This hypothesis is further
supported by our experimental data indicating a negative
effect of the mutation V2.61F on ligand binding compared to
wild type but no effect or even an improvement in binding
affinity when comparing FV3.28,3.29LM with V2.61Fþ
FV3.28,3.29LM mutant receptors. Additionally, our D4
receptor modeling indicates the propensity of Phe2.61 to exist
in two different rotameric states (D4-A and D4-B). Thus,
binding affinity seems to be specifically generated by the
heterocyclic appendage and the linker system and is sub-
stantially perturbed by the mutation at position 2.61.
The shorter D4 ligands do not stretch that far into the
hydrophobic pocket at TM2/TM3 but interact directly with
Phe2.61, Leu3.28, and Met3.29. Interestingly, the effect of the
mutation at Val2.61 on the binding affinity of D4 selective
compounds (1a,c) is similar when comparing wild type to
V2.61F and FV3.28,3.29LM to V2.61FþFV3.28,3.29LM.
This leads to the assumption that the aforementioned con-
formational changes at Phe2.61, depending on the amino acid
at position 3.28, do not occur for the binding of D4 ligands,
thus indicating model D4-A. These conclusions are corrobo-
rated by the fact that the scoring values of model D4-A were
significantly better than those of D4-B (see Supporting
Information).
Our molecular docking experiments showed that the
phenylpiperazine moiety of the longer D2 and D3 ligands
binds to a primary recognition site provided by crucial
microdomains of TMs 3, 5, and 6 forming the described
favorable contacts with Asp3.32, Val3.33, Trp6.48, Phe6.51
,
Phe6.52, and His6.55. The heterocyclic arene moiety resides
within a hydrophobic region at the TM2/TM3 interface
which cannot be reached by the shorter D4 selective com-
pounds. This hydrophobic interface mainly consists of posi-
tions 3.28, 3.29, 2.64, 2.61 and a conserved tryptophane from
the EL1. Mutation of Leu2.64 or Val2.61 to phenylalanine
partly blocks this hydrophobic pocket which explains the
observed loss of affinity. Conformational freedom of Phe2.61
According to our newly established homology models,
two amino acids preceding and following the disulfide
forming cysteine residue of EL2 were expected to be involved
in ligand binding. To investigate the influence of these

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4931
residues in D2/D3 selectivity, we decided to create both the
D2E181VþI183S and the D3V180EþS182I double mutants.
In fact, mutation of Val180 and Ser182 of the D3 receptor
into the corresponding amino acids present at the D2 recep-
tor (Glu and Ile, respectively) resulted in a 20-fold increase in
binding affinity for the preferential D2 ligand 2a at the
mutant D3 receptor. Accordingly, the thio analogue 2b
displayed an 8-fold enhancement of affinity. The resulting
Ki values were comparable to those measured for both test
compounds at the D2 wild type. Interestingly, this mutation
did not affect the affinity of the ligands 3a,b. The reciprocal
double mutation D2E181VþI183S resulted in a 3-fold to 12-
fold decrease in affinity of both the preferential D2 (2a,b) and
D3 (3a,b) ligands.
Although the longer D3 selective phenylpiperazines 3a,b
could not profit from the mutations at the D2 receptor, these
results confirm that Glu181/Ile183 at the D2 and Val180/
Ser182 at the D3 receptor are involved in D2/D3 subtype
selectivity. In agreement with our ligand-receptor models
(Figure 2), we assume that the mutation of Ser182 of the D3
receptor into an isoleucine introduces a D2-like hydrophobic
binding region next to the conserved cysteine in EL2. This
additional hydrophobicity is beneficial for high affinity bind-
ing of the azaindole moiety in the preferantial D2 ligands 2a,b.
solvents indicated (J values in Hz). IR spectra were performed
on a Jasco FT/IR 410 spectrometer using a film of substance on
a NaCl pill. Purification by flash chromatography was per-
formed using silica gel 60 if not stated otherwise. TLC analyses
were performed using Merck 60 F254 aluminum sheets and
analyzed by UV light (254 nm) or by spraying with ninhydrin
reagent. Analytical HPLC/MS was performed on Agilent 1100
HPLC systems employing a VWL detector connected to a
Bruker Esquire 2000. The purity of all SAR compounds was
determined to be >95% by reverse phase HPLC. As column, a
Zorbax SB-C18 (4.6 mm i.d. ꢀ 150 mm, 5 μm) with a flow rate of
0.5 mL/min was used (eluent, methanol/water/0.1 N formic
acid, 10% methanol for 3 min to 100% methanol in 15 min,
100% for 6 min to 10% in 3 min, 10% for 3 min). Compound 1b
was further checked for its purity via combustion analysis
on a EA 1110 CHNS (CE) instrument performed at the
Chair of Organic Chemistry, Friedrich-Alexander-Universitat,
Erlangen-Nurnberg.
3-[4-(2-Methylsulfanylphenyl)piperazin-1-yl]propionitrile (5).
1-(2-Methylsulfanylphenyl)piperazine (744 mg, 3.15 mmol),
K2CO3 (1000 mg, 7.26 mmol), and NaI (88.9 mg, 0.59 mmol)
were dissolved in CH3CN (20.0 mL). 3-Bromopropionitrile (559
mg, 4.17 mmol) was added, and the mixture was heated to reflux
for 24 h. The solvent was removed under reduced pressure and
the resulting residue was purified using flash chromatography
(hexane/EtOAc 1:1) to give 5 (554 mg, 67% related to 1-(2-
methylsulfanylphenyl)piperazine as white crystalline solid. Mp:
88 °C. IR ν 3054, 2942, 2919, 2878, 2819, 2683, 2247, 1579, 1472,
1440 cm-1. 1H NMR (CDCl3, 600 MHz) δ 2.41 (s, 3H), 2.56 (t,
J=7.2, 2H), 2.65-2.74 (m, 4H), 2.78 (t, J=7.0, 2H), 2.99-3.07
(m, 4H), 7.03-7.07 (m, 1H), 7.09-7.14 (m, 3H). 13C NMR
(CDCl3, 90 MHz) δ 14.3, 15.9, 51.4, 53.0, 53.4, 118.8,
119.6, 124.2, 124.6, 124.9, 135.0, 149.2. HPLC/MS (254 nm)
purity >99% (tR = 14.0 min). APCI-MS, [M þ H]þ: calcd,
261.1; found, 262.0. EI-MS: m/z 261 (Mþ). HR-EIMS: calcd,
261.1300; found, 261.1300.
Conclusions
Chemical synthesis of specific molecular probes and SAR
studies combined with computational predictions and site
directed mutagenesis proved to be a powerful tool facilitating
the discovery of the structural origins of subtype selectivity.
Homology modeling and ligand docking have significantly
profited from the recent β2-adrenergic receptor crystal struc-
ture and the inclusion of a carefully modeled EL2. We were
able to further examine amino acids present at position 2.61 in
TM2 and positions 3.28 and 3.29 in TM3 and their impor-
tance in D4/D2 and D3/D4 receptor subtype selectivity.
Mutagenesis of Asp3.32 and His6.55 revealed that, in spite of
their conservation, the residues contribute differently to
ligand binding at the subtypes D2 and D3. Thus, differences
in interhelical interfaces can lead to distortions in the spatial
arrangement of the ligand binding pocket mediated by con-
served residues like His6.55. Careful modeling of the extra-
cellular loop 2 revealed Glu181/Val180 and Ile183/Ser182 to
be involved in ligand binding and selectivity of the D2 and D3
receptor, together with the critical residues 2.61, 2.64, 3.28,
and 3.29. These results could be corroborated by site directed
mutagenesis, thereby validating our ligand-receptor com-
plexes and showing the importance of these residues in D2/D3
selectivity. Interactive processes between computational pre-
dictions and experimental validation will guide new experi-
ments that further elucidate the molecular origins of subtype
specificity and a general rational design of subtype selective
GPCR ligands.
3-[4-(2-Methylsulfanylphenyl)piperazin-1-yl]propylamine (6).30,31
3-[4-(2-Methylsulfanylphenyl)piperazin-1-yl]propionitrile (100 mg,
0.35 mmol) was dissolved in dry Et2O (4.0 mL) and cooled to -5 to
0 °C. LiAlH4 (1 M in Et2O, 0.83 mL, 0.83 mmol) was added, and
the solution was stirred at room temperature for 1 h. After being
cooled to 0 °C, the solution was quenched with a saturated solution
of NaHCO3. The solution was stirred with MgSO4 and filtered
through a short column of Celite and MgSO4. The filtrate was
evaporated under reduced pressure to give 6 (18.2 mg, 18%) as
light-brown oil. IR ν 3353, 3281, 3055, 2941, 2876, 2816, 2680, 2184,
1579, 1472, 1440 cm-1. 1H NMR (CDCl3, 360 MHz) δ 1.69 (tt, J=
7.0, 7.1, 2H), 2.41 (s, 3H, CH3), 2.49 (t, J=7.4, 2H), 2.49-2.72 (m,
4H), 2.78 (t, J=6.8, 2H), 2.99-3.11 (m, 4H), 7.02-7.16 (m, 4H).
13C NMR (CDCl3, 90 MHz) δ 14.4, 30.7, 40.9, 46.5, 51.7 (2C), 53.8
(2C), 56.6, 119.6, 124.2, 124.4, 124.9, 135.0, 149.5. HR-EIMS:
calcd, 265.1613; found, 265.1613.
N-[3-[4-(2-Methylsulfanylphenyl)piperazin-1-yl]propyl]pyrazolo-
[1,5-a]pyridine-3-carboxamide (2b). Pyrazolo[1,5-a]pyridine-3-car-
boxylic acid (11.0 mg, 0.068 mmol) was dissolved in dry CH2Cl2
(2.2 mL) under nitrogen, and DIPEA (0.04 mL, 0.02 mmol) was
added. The mixture was cooled to 0 °C while stirring. A solution
of TBTU (21.4 mg, 0.067 mmol) in DMF (0.2 mL) was then
added dropwise to the cooled reaction mixture. The mixture was
allowed to reach room temperature. 3-[4-(2-Methylsulfanylphe-
nyl)piperazin-1-yl]propylamine (6) (18.0 mg, 0.068 mmol) dis-
solved in CH2Cl2 was added dropwise at room temperature, and
stirring was continued for 2 h. The mixture was then washed with a
saturated aqueous solution of NaHCO3. The layers were sepa-
rated, and the aqueous layer was extracted with CH2Cl2. The
combined organic layers were then washed with brine, dried over
Na2SO4, filtered, and evaporated. Purification using flash chro-
matography with CH2Cl2/MeOH, 95:5, revealed 2b (24 mg, 87%
related to 6) as a light-brown foam. Mp: 75-80 °C. IR ν 3314,
Experimental Section
Chemistry. All reactions were carried out under nitrogen
atmosphere. Dry solvents and reagents were commercial quality
and used as purchased. MS were run on a Finnigan MAT TSQ
700 spectrometer by EI (70 eV) with solid inlet or with an ion-
trap mass with APCI ionization. HR-EIMS were run on a
Finnigan MAT 8200 using peak Peak-Matching (M/ΔM =
10 000). NMR spectra were obtained on a Bruker Avance 360
or a Bruker Avance 600 spectrometer relative to TMS in the
2922, 2816, 2233, 1638, 1552, 1530 cm-1 1H NMR (CDCl3,
.

4932 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Ehrlich et al.
360 MHz) δ 1.89 (tt, J=6.0, 6.0, 2H), 2.41 (s, 3H), 2.70 (t, J=6.0,
2H), 2.62-2.86 (m, 4H), 3.04-3.16 (m, 4H), 3.62 (dt, J=5.7, 5.8,
2H), 6.90 (ddd, J=6.8, 7.5, 1.1, 1H), 7.05-7.17 (m, 4H), 7.33 (ddd,
J=8.9, 6.8, 1.1, 1H), 7.66 (br s, 1H), 8.29 (s, 1H), 8.34 (ddd, J=8.9,
1.1, 1.1, 1H), 8.48 (ddd, J=6.9, 1.0, 0.9, 1H). 13C NMR (CDCl3,
90 MHz) δ 13.3, 23.8, 38.5, 50.4, 52.8, 57.0, 106.2, 112.4, 118.7,
118.7, 123.3, 123.7, 124.0, 125.1, 127.8, 133.9, 139.5, 139.6, 147.9,
162.4. HPLC/MS (254 nm) purity 99% (tR=15.8 min). APCI-MS,
[M þ H]þ: calcd, 409.6; found:, 410.2. EI-MS: m/z 409 (Mþ). HR-
EIMS: calcd, 409.1937; found, 409.1936.
IR ν 2936, 2860, 2816, 1634, 1496, 1235 cm-1. 1H NMR (CDCl3,
360 MHz) δ 1.40-1.49 (m, 2H), 1.55-1.63 (m, 2H), 1.75-1.84
(m, 2H), 2.37-2.41 (m, 2H), 2.56-2.59 (m, 4H), 2.81-2.85 (m,
2H), 3.14-3.17 (m, 4H), 6.28 (s, 1H), 6.65 (brdd, J = 7.1, J =
6.7, 1H), 6.81-6.85 (m, 2H), 7.04 (brdd, J = 8.9, 6.7, 1H), 7.17-
7.21 (m, 2H), 7.41 (brd, J = 8.9, 1H), 8.37 (brd, J = 7.1, 1H).
13C NMR (CDCl3, 90 MHz) δ 26.7, 27.4, 28.5, 29.7, 49.1, 53.1,
58.6, 95.1, 110.8, 117.1, 117.3, 123.1, 124.3, 128.2, 128.9, 141.0,
150.0, 156.2. EI-MS: m/z 382 (Mþ), 384 (Mþ þ 2). Anal. Calcd
for C22H27N4Cl 0.6H2O: C, 67.11; H, 7.22; N, 14.23. Found: C,
3
Methyl 2-(4-Ethoxycarbonylbutyl)pyrazolo[1,5-a]pyridine-3-
carboxylate (8). The reaction was carried out using 1-aminopyr-
idinium iodide (3.0 g, 13.5 mmol), K2CO3 (1.5 g, 11 mmol),
and 3-oxooctanedioic acid 8-ethyl ester 1-methyl ester (7) (3.3 g,
14.3 mmol) in DMF (10 mL) and stirred at 50 °C for 2 h.32-35
After addition of saturated NaHCO3 solution, the mixture was
extracted with Et2O and washed with water. The organic solvent
was dried over MgSO4 and evaporated. The crude product was
purified by flash chromatography (hexane/EtOAc, 8:2) yielding
8 as a solid (0.5 g, 13%). Mp: 46 °C. IR ν 2980, 2950, 2872, 1732,
67.62; H, 7.22; N, 13.72.
Residue Numbering Scheme. For reasons of clarity, amino
acid positions are numbered according to Ballesteros and
Weinstein. Residues are numbered consecutively as [TMx].n
relative to the most conserved residue within each TM, which is
designated as [TMx].50.55
Dopamine Receptor Binding Studies. Receptor binding studies
were carried out as described.56 In brief, competition experi-
ments with human D2L, D3, and D4 receptors were run with
preparations of membranes from CHO cells stably expressing
the corresponding receptor and [3H]spiperone at a final con-
centration of 0.1-0.3 nM according to the individual Kd values.
The assays were carried out with a protein concentration of
5-20 μg/assay tube and Kd values of 0.06-0.14, 0.08-0.35, and
0.15-0.40 nM for the D2L, D3, and D4 receptors, respectively.
The corresponding Bmax values were in the range of 750-
1400 fmol/mg for D2L, 1500-4300 fmol/mg for D3, and 700-
1800 fmol/mg for D4, respectively. Protein concentration
was established by the method of Lowry using bovine serum
albumin as standard.57
Data Analysis. The resulting competition curves of the re-
ceptor binding experiments were analyzed by nonlinear regres-
sion using the algorithms in PRISM 3.0 (GraphPad Software,
San Diego, CA). The data were initially fit using a sigmoid
model to provide a slope coefficient (nH) and an IC50 value,
representing the concentration corresponding to 50% of max-
imal displacement of the radioligand. The IC50 values were
transformed to Ki values according to the equation of Cheng
and Prusoff.58
Site Directed Mutagenesis. The hDRD2 cDNA, subcloned
into a pcDNA3.1(þ) eukaryotic expression vector, was pur-
chased from UMR cDNA Resource Center. The pcDNA3.1(þ)
of hDRD3 receptor and of the hDRD4.4 were used as described
previously.53 Oligonucleotidic primers were purchased from
Biomers.net or MWG Biotech AG. Site directed mutagenesis
was performed by polymerase chain reaction (PCR) using
oligonucleotides bearing the desired mutation.59 Fidelity of
PCR amplification and introduction of mutations in the recep-
tor cDNA was confirmed by sequencing with the ABI sequencer
system (ABI Systems, Weiterstadt, Germany) at the laboratory
of C.-M. Becker (Department of Biochemistry, FAU Erlangen,
Germany) using oligonucleotidic primers.
1703, 1636, 1519, 1095 cm-1 1H NMR (CDCl3, 360 MHz)
.
δ 1.24 (t, J=7.1, 3H), 1.71-1.89 (m, 4H), 2.35-2.39 (m, 2H),
3.10-3.14 (m, 2H), 3.92 (s, 3H), 4.12 (q, J=7.1, 2H), 6.89 (ddd,
J=7.1, 6.7, 1.4, 1H), 7.36 (ddd, J=8.9, 6.7, 1.1, 1H), 8.09 (br d,
J = 8.9, 1H), 8.43 (dd, J = 7.1, 1.1, 1H). 13C NMR (CDCl3,
90 MHz) δ 14.2, 24.9, 27.8, 28.5, 34.2, 50.9, 60.2, 100.5, 113.2,
119.1, 127.1, 128.7, 142.2, 159.2, 173.7, 164.3. EI-MS: m/z 304
(Mþ). Anal. Calcd for C16H20N2O4: C, 63.14; H, 6.62; N, 9.20.
Found: C, 63.24; H, 6.75; N, 8.83.
5-(Pyrazolo[1,5-a]pyridine-2-yl)pentanoic Acid (9). A suspen-
sion of 8 (400 mg, 1.31 mmol) in 40% H2SO4 solution (13 mL)
was stirred at 110 °C for 3 h. After cooling to room temperature,
the solution was neutralized with NaOH (5 M), and HCl (2 M)
was added to get pH 3. Extraction with CHCl3 gave a white solid
of compound 9 (255 mg, 93%). Mp: 121 °C. IR ν 3522, 2944,
2869, 1709, 1635 cm-1. 1H NMR (DMSO, 360 MHz) δ 1.54-
1.60 (m, 2H), 1.65-1.72 (m, 2H), 2.25 (t, J = 7.1, 2H), 2.73 (t,
J = 7.1, 2H), 6.38 (s, 1H), 6.77 (ddd, J = 7.1, 6.7, 1.4, 1H), 7.13
(ddd, J = 8.9, 6.7, 1.1, 1H), 7.56 (br d, J = 8.9, 1H), 8.55 (dd,
J = 7.1, 1.1, 1H), 11.98 (s, 1H). 13C NMR (DMSO, 90 MHz)
δ 24.2, 27.6, 28.5, 33.4, 94.0, 111.0, 117.2, 123.3, 128.3, 140.3,
155.0, 174.4. EI-MS: m/z 218 (Mþ).
1-[4-(4-Chlorophenyl)piperazin-1-yl]-5-[pyrazolo[1,5-a]pyridin-2-
yl]pentan-1-one (10). The reaction was carried out using 9 (218 mg,
1.0 mmol), HOAt (150 mg, 1.1 mmol), DCC (227 mg, 1.1 mmol),
and 4-chlorophenylpiperazine (236 mg, 1.2 mmol) in CH2Cl2
(10 mL) under nitrogen atmosphere. The mixture was stirred at
room temperature for 15 h and filtered, and the solvent was
evaporated. The crude product was purified by flash chromatog-
raphy (CH2Cl2/MeOH, 98:2), obtaining a white solid of 10 (322
mg, 81%). Mp: 117 °C. IR ν 2930, 2857, 2826, 1644, 1635, 1231,
1
1029 cm-1. H NMR (CDCl3, 360 MHz) δ 1.70-1.86 (m, 4H),
2.39-2.43 (m, 2H), 2.84-2.89 (m, 2H), 3.05-3.10 (m, 4H), 3.56-
3.59 (m, 2H), 3.73-3.77 (m, 2H), 6.30 (s, 1H), 6.65 (br dd, J = 7.1,
6.7, 1H), 6.79-6.83 (m, 2H), 7.03 (brdd, J = 8.9, 6.7, 1H), 7.19-
7.24 (m, 2H), 7.40 (br d, J = 8.9, 1H), 8.34 (br d, J = 7.1, 1H).
13C NMR: (CDCl3, 90 MHz) δ 24.9, 28.1, 29.3, 33.0, 41.3, 45.4,
49.4, 49.7, 95.2, 110.8, 117.4, 117.8, 123.1, 125.4, 128.1, 129.1,
141.0, 149.6, 155.8, 171.5. EI-MS: m/z 396 (Mþ). Anal. Calcd for
C22H25N4OCl: C, 66.57; H, 6.35; N, 14.12. Found: C, 66.52; H,
6.49; N, 13.93.
2-[5-[4-(4-Chlorophenyl)piperazin-1-yl]pentyl]pyrazolo[1,5-a]-
pyridine (1b). A suspension of 10 in dry, absolute Et2O was
treated with 1.0 M LiAlH4 solution in Et2O (2 equiv) and stirred
under nitrogen atmosphere at 0 °C for 1 h and subsequently at
room temperature for 5 h. Saturated NaHCO3 solution was
added dropwise to quench the remaining LiAlH4. The mixture
was filtered on Celite and washed with MeOH. The crude
product was purified by flash chromatography (CH2Cl2/MeOH
98:2) to obtain a white solid of 1b (114 mg, 59%). Mp: 72 °C.
Mutant Receptor Preparation. HEK-293 cells, transiently
transfected with the wildtype and mutant receptor cDNA by
CaHPO4 method or using TransIT-293 transfection reagent
(Mirus Bio Corporation), were cultured in 150 mm Petri
plates containing 20 mL of MEM R medium supplemented
with 10% (v/v) fetal bovine serum, 100 U/mL penicillin G,
100 μg/mL streptomycin, and 2 mM L-glutamine at 37 °C and
5% CO2.
HEK-293 cells were harvested 48 h after transfection. Cells
were harvested by removal of the medium, followed by a wash
with phosphate buffered saline, which was discarded, resuspen-
sion in 10 mL of harvest puffer (10 mM Tris-HCl, 0.5 mM
EDTA, 5.4 mM KCl, and 140 mM NaCl, pH 7.4), scraping of
the cells with a rubber spatula into a centrifuge tube, and
collection of the cells by centrifugation at 220g for 8 min. The
cellular pellet was resuspended in 5 mL of homogenate buffer for
D2 and D4 receptor (50 mM Tris-HCl, 5 mM EDTA, 1,5 mM
CaCl2, 5 mM MgCl2, 5 mM KCl, and 120 mM NaCl, pH 7,4)

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4933
and for D3 receptor (10 mM Tris-HCl and 5 mM MgSO4,
pH 7,4). Cells were used as they were or stored at -80 °C.
After thawing or directly, the cells were diluted in homoge-
nate buffer, homogenized using a Polytron (20 000 rpm, 5 ꢀ 5 s
each in an ice bath), and spun at 50000g for 18 min. The mem-
brane pellet was always resuspended in homogenate puffer for
D2 and D4 receptor, homogenized with a Potter-Elvehjem
homogenizer, and stored in small aliquots at -80 °C. Protein
concentration was estimated by the method of Lowry et al.,
using bovine serum albumin as a standard.57
500 cycles of steepest descent minimization, followed by 4500
cycles of conjugate gradient minimization. All calculations were
carried out in a water box with periodic boundary conditions
˚
and a nonbonded cutoff of 8.0 A. The all atom force field ff99SB
was applied.65 Parameterization of 1d was accomplished with
antechamber. The charges for the ligand atoms were calculated
using Gaussian 98, with a 6-31** basis set, and then the RESP
procedure was applied as described in the literature.66,67
A formal charge of þ1 was attributed to the ligand because
the piperazine nitrogen connected to the aliphatic chain is
assumed to be protonated at physiological pH.
Radioligand Binding Studies. The radioligand [3H]spiperone
was purchased from Amersham/GE Healthcare UK Limited
and obtained with a specific activity of 84-114 Ci/mmol.
KD and Bmax values for wild type and mutants were received
from saturation experiments, and the Ki values for the com-
pounds were obtained by competition experiments.
In the next step the minimized structure was submitted to MD
simulations. By application of Shake bond-length restraints to
bonds involving hydrogens, it was possible to set the time step to
2 fs. Nonbonded interactions were updated every 25 fs. The
system was gradually heated to 300 K and equilibrated for 10 ns
-2
˚
A
The competition binding assay followed the previously
published protocol.56 In brief, binding assays with D2, D3, and
D4.4 wild type and mutant receptors were performed with
[3H]spiperone at the final concentrations of 0.1-0.7 nM. The
assays were carried out in the 96-well plates at the protein
concentration of 20-200 μg/mL in a final volume of 200 μL.
Ki values were derived from the corresponding EC50 data.58
Dopamine Receptor Modeling. The crystal structure of the
human β2-adrenergic receptor (PDB entry 2RH1) was used as a
template to construct the dopaminergic receptors.37 The amino
acid sequences of the human dopamine D2, D3, and D4 receptors
and the β2-adrenergic receptor were retrieved from the SWISS-
PROT database.60 Sequence alignment was performed by
means of ClustalX by employing the Gonnet series matrix with
a gap open penalty of 10 and a gap extension penalty of 0.2.61 To
define highly conserved residues, 10 additional sequences of
members of the family A GPCRs were included in the alignment
that was manually refined to ensure a perfect alignment of the
highly conserved residues of the GPCR superfamily according
to Baldwin et al.62
with a harmonic force constant of 1.0 kcal mol-1
on the
main chain atoms of the protein except EL2. Then 20 ns of MD
simulations without restraints followed. The resulting final
structure of EL2 was inserted into the original D4 receptor
model using Swiss-PdbViewer.
Because of the same sequence length, part of this EL2,
ranging from the conserved cysteine to the extracellular end of
TM5 (position 5.36), was also used to construct the EL2 of the
D2 and the D3 receptors. Nonconserved amino acids were
mutated using Swiss-PdbViewer. Because of big differences in
sequence length between the D4 and the D2 and D3 receptors, the
part of the EL2 ranging from the extracellular end of TM4
(position 4.61) to the conserved cysteine was modeled using the
loop database of the Swiss-PdbViewer.
Docking Procedure. 1a, 2b, and 3a were geometry optimized
by means of Gaussian 98 at the 6-31** level, attributing a formal
charge of þ1 to all ligands. The ligands were then docked using
the program AUTODOCK4.68-70 A grid of 70 points in each
of the x, y, and z directions was used to completely enclose the
binding pocket surrounded by the extracellular parts of TM2,
˚
TM3, TM5, TM6, and TM7. A grid spacing of 0.375 A was used,
On the basis of the alignment and the crystal structure of the
β2-adrenergic receptor, 100 models of each receptor (D2, D3,
and D4) were constructed using the MODELLER program.63
The extracellular loop 2 (EL2) was omitted because of the
difference in sequence length between the dopaminergic recep-
tors and the β2-adrenergic receptor. The intracellular loop 3 is
already absent in the crystal structure and omitted in receptor
modeling.
and all ligands were subjected to 50 runs of the AUTODOCK
Lamarckian genetic algorithm with a randomly selected starting
position. The docking procedure was free of constraints, thereby
exploring all the available grid space. The 50 docking conforma-
tions of each compound were clustered manually and scored by
DrugScoreONLINE (www.agklebe.de), applying the knowledge-
based DrugScoreCSD scoring function.42 On the basis of this
scoring, the best conformation of each cluster was selected.
After a final selection according to experimental data, the
receptor-ligand complexes were subjected to energy minimiza-
tion. Parametrization of the ligands, calculation of charges, and
energy minimization were accomplished as described above.
One model was selected for each of the D2 and D3 receptor.
For the D4 receptor, two diverse models showing different χ1
rotamers at Phe2.61 were selected by docking the D4 ligand 1d
into each of the initial 100 model structures and subsequent
scoring with DrugScoreONLINE 28,42
Further validation was
.
achieved by additionally docking 1a into these two D4 receptor
models.
Acknowledgment. The authors thank Dr. H. H. M.
Van Tol (Clarke Institute of Psychiatry, Toronto, Canada),
Dr. J.-C. Schwartz and Dr. P. Sokoloff (INSERM, Paris), and
Dr. J. Shine (The Garvan Institute of Medical Research,
Sydney) for providing dopamine D4, D3, and D2 receptor
expressing cell lines, respectively, and Dr. R. Maggio
(University of L’Aquila) for providing the cDNA of a human
D3 receptor chimera.
Modeling of the Extracellular Loop 2. A prototype of the
extracellular loop 2 (EL2) was modeled into the D4 receptor
model using the crystal structure of the β2-adrenergic receptor,
the loop database of the Swiss-PdbViewer, and molecular
dynamics simulations by means of the SANDER program of
the AMBER10 suite.41,64
Because of the similarity in sequence length, the crystal
structure of the β2-adrenergic receptor was used to model the
EL2 ranging from the conserved cysteine, which forms a disul-
fide bond with Cys3.25 in TM3, to the extracellular end of TM5
(position 5.36). The relevant amino acids were mutated using
Swiss-PdbViewer. Because of a big difference in sequence length
between the β2-adrenergic and the dopamine receptors, the part
of the EL2 ranging from the extracellular end of TM4 (position
4.61) to the conserved cysteine was modeled using the loop
database of the Swiss-PdbViewer.
Supporting Information Available: NMR spectra, LC/MS
purity data including MS spectra, used oligonucleotide primer
sequences, amino acid sequence alignment, and scoring results.
This material is available free of charge via the Internet at
http://pubs.acs.org. Described ligand-receptor complexes are
available from the corresponding author upon personal request.
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