Synthesis and characterization of selective dopamine D2 receptor ligands using aripiprazole as the lead compound

Article abstract of DOI:10.1016/j.bmc.2011.04.021

A series of compounds structurally related to aripiprazole (1), an atypical antipsychotic and antidepressant used clinically for the treatment of schizophrenia, bipolar disorder, and depression, have been prepared and evaluated for affinity at D_2-lik

Full text of DOI:10.1016/j.bmc.2011.04.021

Bioorganic & Medicinal Chemistry 19 (2011) 3502–3511
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and characterization of selective dopamine D2 receptor ligands
using aripiprazole as the lead compound
Suwanna Vangveravong a, Zhanbin Zhang a, Michelle Taylor b, Melissa Bearden b, Jinbin Xu a,
Jinquan Cui a, Wei Wang a, Robert R. Luedtke b, Robert H. Mach a,
⇑
a Division of Radiological Sciences, Washington University School of Medicine, Mallinckrodt Institute of Radiology, 510 S. Kingshighway, St. Louis, MO 63110, USA
b Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 January 2011
Revised 6 April 2011
Accepted 11 April 2011
Available online 16 April 2011
A series of compounds structurally related to aripiprazole (1), an atypical antipsychotic and antidepressant
used clinically for the treatment of schizophrenia, bipolar disorder, and depression, have been prepared and
evaluated for affinity at D2-like dopamine receptors. These compounds also share structural elements with
the classical D2-like dopamine receptor antagonists, haloperidol, N-methylspiperone, domperidone and
benperidol. Two new compounds, 7-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butoxy)-3,4-dihydroquino-
lin-2(1H)-one oxalate (6) and 7-(4-(4-(2-(2-fluoroethoxy)phenyl)piperazin-1-yl)butoxy)-3,4-dihydro-
quinolin-2(1H)-one oxalate (7) were found to (a) bind to the D2 receptor subtype with high affinity (Ki
values <0.3 nM), (b) exhibit >50-fold D2 versus D3 receptor binding selectivity and (c) be partial agonists
at both the D2 and D3 receptor subtype.
Keywords:
Dopamine D2 receptor
Aripiprazole
Dopamine partial agonist
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
that can selectively stimulate or block D2 or D3 receptors.3,6,7,9–11
This has also been true for the development of radiotracers for
Dopamine receptors belong to a large superfamily of neuro-
transmitter and hormone receptors which are coupled to their spe-
cific effectors function via guanine nucleotide regulatory (G)
proteins. Based upon genomic and cDNA cloning studies, it is cur-
rently thought that there are five functionally active dopamine
receptor subtypes expressed in mammals. These five receptor sub-
imaging dopamine D2 and D3 receptors with the functional imag-
ing technique positron emission tomography or PET. That is, all
radiotracers used in PET imaging studies bind with similar affinity
to D2 and D3 receptors, and receptor density measurements using
radiotracers such as the antagonists [11C]raclopride and [18F]fally-
pride, and the radiolabeled full agonist, [11C](+)_PHNO, are typi-
cally reported as D2/3 receptor binding potentials.12–14 There has
been a need to develop highly selective dopamine receptor ligands
capable of labeling D2 versus D3 and D3 versus D2 in order to gain a
better insight into the behavioral pharmacology and in vivo regu-
lation of these structurally-similar dopamine receptors in disor-
ders of the central nervous system.
types have been classified into two major types (D1-like and D2-like
)
based on their amino acid sequence and pharmacological proper-
ties. The D1-like receptor subtypes consist of the D1 (D1a) and the
D5 (D1b) dopamine receptors. The D2-like receptor subtypes include
the D2short (D2S), D2long (D2L), D3, and D4 receptors.1 Stimulation of
D
1-like receptors results in an activation of adenylyl cyclase.2 Stimu-
lation of D2-like receptors results in an inhibition of adenylyl cyclase
activity, mitogenesis, an increase in the release of arachidonic acid
and an increase in phosphatidylinositol hydrolysis.3
The D2 and D3 dopamine receptor subtypes are structurally and
pharmacologically similar.4 Despite these similarities, the D2 and
D3 receptors differ in the neuroanatomical localization, the levels
of receptor expression, efficacy in response to agonist stimulation,
regulation, desensitization and the intracellular trafficking proper-
ties.5–8 Because of the high degree of homology between D2 and D3
receptor binding sites, it has been difficult to develop compounds
Previously, our group synthesized a series of ((1H-indol-3-
yl)methyl)piperidin-4-ol analogs and evaluated their affinities and
intrinsic activities for dopamine D2 and D3 receptors. These com-
pounds share structural elements with the classical D2-like dopamine
receptor antagonists, haloperidol, N-methylspiperone and benperi-
dol. Several of the compounds structurally similar to haloperidol
were found to have moderate to high affinity and selectivity at D2
versus D3 receptors.15,16 Functional assays revealed that these com-
pounds were antagonists at D2 receptors.15,16
Aripiprazole, 7-[4-[4-(2,3-dichlorophenyl)-1-piperazinyl]but-
oxy]-3,4-dihydro-2(1H)-quinolinone (1, Fig. 1) has been used for
the treatment of schizophrenia and various forms of depression.
Aripiprazole has been reported to be an antagonist of postsynaptic
dopamine receptor while acting as an agonist of dopamine
⇑
Corresponding author. Tel.: +1 314 362 8538; fax: +1 314 362 0039.
E-mail address: rhmach@mir.wustl.edu (R.H. Mach).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.04.021

S. Vangveravong et al. / Bioorg. Med. Chem. 19 (2011) 3502–3511
3503
H
O
N
O
N
Cl
N
O
Cl
Aripiprazole (1)
O
N
O
NH
OH
N
N
F
F
Cl
Haloperidol
Benperidol
O
O
CH3
NH
N
O
H
N
N
N
N
O
N
N
Cl
F
Domperidone
N-methylspiperone
Figure 1. Structures of the lead compounds.
autoreceptors.17 Using aripiprazole as a lead compound, we syn-
thesized a series of new compounds and evaluated their binding
affinities and intrinsic activities at D2-like receptors. These modifi-
cations include: (a) replacing the N-2,3-dichlorophenyl piperazine
ring of aripiprazole with amine groups present in typical antipsy-
chotics (haloperidol, benperidol, domperidone and N-methyls-
piperone), (b) replacing the N-2,3-dichlorophenyl group of
aripiprazole with N-methoxyphenyl, or N-2-fluoroethoxy groups,
(c) changing the length of the carbon atom spacer between the
oxygen and nitrogen atoms, and, (d) introduction of a double bond
into the 3,4-dihydro-2(1H)-quinolinone ring. The goal of this study
is to modify the structure of aripiprazole in order to determine if it
is possible to identify ligands having a higher affinity and selectiv-
ity for D2 versus D3 receptors for behavioral pharmacology and PET
imaging studies.
3. Radioligand binding studies at dopamine receptors
Competitive radioligand binding studies were performed to
determine the equilibrium dissociation constants of each com-
pound at human D2, D3, and D4 dopamine receptors (Table 1). For
these studies tissue homogenates from stably transfected HEK
293 cells were used in conjunction with the radioligand 125I-IABN.
We have previously reported that the benzamide 125I-IABN binds
with high affinity and selectively to D2-like dopamine receptors,
but it binds non-selectively to the D2 and D3 dopamine receptor
subtypes.23
First, a comparison was made of the affinity at D2 and D3
dopamine receptors of the compounds which have structural
elements similar to haloperidol, benperidol, domperidone, and
N-methylspiperone. Surprisingly, these structural elements did
not appear to have an effect on D2-like receptor affinity or selectiv-
ity, with compounds 2–5 displaying weak affinity for D2, D3, and
D4 receptors. Next, a comparison of substituents at the piperazine
moiety of aripiprazole was made in which the 2,3-dichloro substi-
tution pattern of aripiprazole was replaced with either a 2-OCH3
(compound 6) or a 2-OCH2CH2F (compound 7) group. We found
that both methoxy and 2-fluoroethoxy groups resulted in a 2-fold
decrease in affinity at D3 receptors with a concomitant 14- and
12-fold increase in affinity at D2 receptors, with compounds 6
and 7 having a 60- and 52-fold selectivity at D2 receptors, respec-
tively. This level of binding selectivity is 25 to 30 times greater
than the lead compound aripiprazole (Table 1). Compounds 6, 7,
11, and 12, which contain a piperazine moiety and a saturated
four carbon chain, exhibited the greatest affinity (Ki values
0.07–0.26 nM, Table 1) and selectivity (39- to 60-fold) at D2
receptors compared to the D3 receptor subtype. The positions of
the substituents at the piperazine moiety were also compared.
Changing the methoxy group from the 2-position on the benzene
ring (compound 6) to the 4-position (compound 8) decreased
both D2 and D3 receptors affinity. Substitution on both 2- and
4-positions (compounds 9 and 10) decreased D3 receptor affinity
but had little effect on D2 receptor affinity. Decreasing (com-
pounds 13 and 14) or increasing (compounds 15 and 16) the
length of the four carbon spacer by one carbon atom decreased
D2 receptor affinity, while having little to no effect on D3 receptor
affinity, thereby reducing the receptor subtype binding selectivity
2. Chemistry
The syntheses of all target compounds (Fig. 2) are outlined in
Schemes 1–4. Compounds 2–10 were prepared as outlined in
Scheme 1. Starting from the commercially available 7-(4-bromo-
butoxy)-3,4-dihydroquinolin-2(1H)-one (20) and the correspond-
ing piperidines or piperazines, the desired compounds 2–6, 8–10
and the intermediate 1718 were obtained in 67–96% yields. Subse-
quently, fluoroethylation of the aromatic hydroxyl group of 17
with 1-bromo-2-fluoroethane gave 7 in 59% yield. The piperazine
19 was prepared from 2-methoxy-4-methylaniline (18) and bis-
(2-chloroethyl)amine hydrochloride in the presence of potassium
carbonate in butanol.19
Compound 11 was synthesized from 7-(4-bromobutoxy)quino-
lin-2(1H)-one (21)17 and 1-(2-methoxyphenyl)piperazine, whereas
12 was synthesized from 21 and 1-(2-(2-fluoroethoxy)phenyl)
piperazine20 (Scheme 2). Compounds 1321 and 1421 were prepared
from O-alkylation of commercially available 7-hydroxy-3,4-dihy-
droquinolin-2(1H)-one (22a) or 7-hydroxyquinolin-2(1H)-one
(22b) with 1-(3-chloropropyl)-4-(2-methoxyphenyl)piperazine22
(Scheme 3).
Reaction of 7-(5-bromopentoxy)-3,4-dihydroquinolin-2(1H)-
one (23)21 and 1-(2-methoxyphenyl)piperazine gave 15. Dehydro-
genation of 15 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ) gave 16 (Scheme 4).

3504
S. Vangveravong et al. / Bioorg. Med. Chem. 19 (2011) 3502–3511
H
N
H
O
N
O
O
O
O
O
N
N
N
N
OH
N
3
5
2
4
N
H
Cl
O
H
N
H
N
O
O
O
Cl
N CH
N
3
N
N
O
H
H
N
H
N
O
O
N
R
O
O
1
N
R
N
N
11 R = OCH
6 R = OCH , R = H
3
1
3
2
R
2
12
7
R = OCH CH F
R = OCH CH F, R = H
2
2
1
2
2
2
8 R = H, R = OCH
1
2
3
9 R = OCH , R = OCH
1
3
2
3
10 R = OCH , R = CH
1
3
2
3
N
N
H
H
N
O
O
N
O
O
N
OCH
3
O
O
O
O
N
OCH
3
14
16
13
N
N
H
N
H
N
N
OCH
3
N
OCH
3
15
Figure 2. Structures of the target compounds.
(Table 1, Fig. 2). Introduction of a double bond into the 3,4-dihy-
dro-2(1H)-quinolinone ring had little effect on both D2 and D3
receptors affinity (compounds 11, 12, 14 and 16).
the presence of 100 nM unlabeled (+)-pentazocine to mask r1
sites, or with the r2 selective ligand [3H]RHM-1, alone.24,25 The
affinity at r1 receptors varied from 150 to >20,000 nM. All of the
compounds in this study bound with low affinity (>200 nM) at
r2 receptors. The affinity of the two compounds with the greatest
D2 receptor selectivity (6 and 7) bound to sigma receptors with low
affinity (Ki values >500 nM).
4. Adenylyl cyclase studies with D2-like receptors
The pharmacological properties of the two compounds with the
highest binding selectivity for D2 dopamine receptors (6 and 7) were
further evaluated using a whole cell forskolin-dependent adenylyl
cyclase inhibition assay. The intrinsic efficacy of these compounds
were compared to the full agonist quinpirole, as previously de-
scribed.15 Compounds 6 and 7 were found to be partial agonists at
both D2 and D3 dopamine receptors. The intrinsic efficacy was found
to beslightly greater (16–75%)at D2 receptors compared to D3 recep-
tors. The intrinsic efficacy of these two compounds at human D2
receptors is similar to that found for the lead compound aripipraz-
ole, while the efficacy at D3 receptors was somewhat lower (Fig. 3).
6. Discussion
The dopaminergic system has been the most thoroughly studies
CNS receptor system using the functional imaging technique, PET.
By-and-large, most imaging studies have been conducted using the
radiotracer [11C]raclopride, which binds with similar affinity to
dopamine D2 and D3 receptors. Therefore, PET imaging studies
with this radiotracer are typically reported as dopamine D2/3
receptor binding potential, which is a measure of the density of
D2 and D3 receptors in regions of interest such as the caudate
and putamen. High affinity, low nonspecific binding radioligands
such as [18F]fallypride and [11C]FLB 45726,27 D2 and D3 antagonists
have been developed for measuring the density of extrastriatal
dopamine D2/3 receptors in the CNS.
5. Radioligand binding studies at sigma receptors
In vitro binding studies were conducted to determine the affin-
ity of the target compounds at sigma-1 (r1) and sigma-2 (r2)
A number of studies have suggested that there is a differential
regulation of dopamine D2 and D3 receptors in a variety of CNS dis-
orders. For example, using in vitro quantitative autoradiography,
Ryoo et al. reported a 45% reduction in D3 receptors in the ventral
striatum, and a 15% increase in D2 receptors in the caudate/putamen
of parkinsonian brain.28 These data were consistent with 6-OHDA
and MPTP lesioning studies in animal models of PD.29,30 Therefore,
D2 and D3 receptors appear to be regulated in an opposing manner
in response to the loss of dopaminergic input in PD. In addition, PET
studies of human subjects with a chronic history of cocaine abuse
receptors. Since many structurally-diverse ligands are capable of
binding to sigma receptors, we routinely screen our potential
CNS PET radiotracers for binding affinity to r1 and r2 receptors.
Any compound which binds with high affinity to r1 and/or r2
receptors would not be an appropriate candidate for the develop-
ment of a dopaminergic PET imaging agent since sigma receptors
are expressed ubiquitously in the CNS.
The r1 binding studies were conducted using the
r1-selective
radioligand, [3H](+)-pentazocine in guinea pig brain membranes;
r2 sites were assayed in rat liver membranes with [3H]DTG in

S. Vangveravong et al. / Bioorg. Med. Chem. 19 (2011) 3502–3511
3505
H
H
N
O
N
O
O
O
a
N
Br
R2
R1
Y
20
2, Y = C, R1
3, Y = C, R1
=
=
R
2 = OH
2 = H
Cl
N
R
N
H
O
Cl
N
R2 = H
4, Y = C, R1
=
N
O
H
O
5, Y = C, R1 R2
=
N CH3
N
OCH3
6, Y = N, R1
8, Y = N, R1
=
=
OCH3
OCH3
9, Y = N, R1
=
=
OCH3
OCH3
10, Y = N, R1
17, Y = N, R1
CH3
OH
=
F
H
N
H
N
O
O
O
O
N
OH
N
O
b
c
N
N
17
7
OCH3
HN
N
OCH3
H2N
CH3
CH3
19
18
Scheme 1. Reagents: (a) substituted piperidines or piperazines, K2CO3, KI, acetonitrile, heat; (b) 1-bromo-2-fluoroethane, K2CO3, acetone, heat; (c) bis-(2-chloroethyl)amine
hydrochloride, K2CO3, butanol, heat.
H
H
N
O
O
N
O
O
N
R
Br
N
11, R = OCH3
21
12
, R = OCH2CH2F
Scheme 2. Reagents: 1-(2-methoxyphenyl)piperazine hydrochloride or 1-(2-(2-fluoroethoxy)phenyl)piperazine, N(C2H5)3, acetonitrile.
have revealed a reduction in D2/3 receptors relative to age-matched
controls.31,32 Similar results have shown that D2/3 receptors are
reduced in autoradiography33 and PET34 imaging studies of rhesus
monkeys that have self-administered cocaine. However, autoradi-
ography studies conducted by Staley and Mash reported an upreg-
ulation of D3 receptors in human cocaine overdose victims when
compared with age-matched controls.35 More recently, Volkow
et al. reported a difference in [11C]raclopride binding in the striatum
versus thalamus in cocaine abusers versus normal controls.36 Their
data demonstrated an increased binding of raclopride in the thala-
mus, a region of brain having a high density of D3 receptors,
whereas a decrease in raclopride uptake was observed in the stria-
tum, a brain region rich in both D2 and D3 receptors. These data sug-
gest that chronic exposure to cocaine may result in a differential
regulation of D2 and D3 receptors. That is, chronic cocaine abuse
leads to an increase in D3 receptors and a decrease in density of

3506
S. Vangveravong et al. / Bioorg. Med. Chem. 19 (2011) 3502–3511
N
H
N
OH
H
O
N
OCH3
N
O
O
x
x
22a: x = CH2CH2
22b: x = CH=CH
13: x = CH2CH2
14: x = CH=CH
Scheme 3. Reagents: 1-(3-chloropropyl)-4-(2-methoxyphenyl)piperazine, NaOCH3, ethanol.
H
N
N
a
O
O
Br
H
N
O
O
N
OCH3
23
15
N
b
H
N
N
O
O
OCH3
16
Scheme 4. Reagents: (a) 1-(2-methoxyphenyl)piperazine hydrochloride, N(C2H5)3, acetonitrile; (b) DDQ, CH2Cl2.
Table 1
Binding affinities for dopamine D2/D3 and sigma r1/r2 receptors
Compound
Ki (nM)a
b
c
d
e
f
g
h
D2
D3
D4
D3:D2
D4:D2
r1
r2
log Pi
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
2604 748
145 20
309 97
611 62
0.22 0.01
0.26 0.05
102 25
25.3 5.9
4.6 1.0
0.14 0.03
0.07 0.01
4.8 0.9
2.6 0.5
7.3 1.3
1536 156
879 289
574 80
427 36
13.1 2.3
13.5 3.5
930 239
785 258
251 33
5.4 1.1
2.7 0.5
7.4 0.7
5.7 1.2
7.0 1.6
3.8 0.1
6.8 0.2
1311 100
1029 263
1352 307
865 116
212 45
185 27
359 22
286 27
27.2 1.7
30.1 7.3
31.9 9.1
38.5 6.4
52.7 16.9
234 15.4
201 7.6
168 16.6
0.6
6.1
1.9
0.7
60
52
9.1
31
54
39
39
1.5
2.2
1.0
0.5
2.2
0.5
7.1
4.4
1.4
964
712
3.5
11
5.9
215
456
8.0
20
417 105
433 33
158 55
3087 281
1176 108
13574 2463
170 49
8269 1771
36364 5623
16907 403
19930 3524
598 160
4988 837
665 36
2.25
2.54
3.58
1.17
3.63
3.89
3.43
3.12
3.97
3.67
3.93
3.29
3.34
3.94
3.98
4.50
620 64
377 56
3340 194
6393 276
1309 133
4952 412
1907 213
2838 86
231 5.85
608 17
2631 235
20904 1594
759 43
2600 464
1349 213
1614 426
NDj
32
27
54
7.4 0.5
3.1 0.5
Aripiprazole
NDj
a
b
c
d
e
f
Mean SEM, Ki values were determined by at least three experiments.
Ki values for D2 receptors were measured on human D2(long) expressed in HEK cells using [125I]ABN as the radioligand.
Ki values for D3 receptors were measured on human D3 expressed in HEK cells using [125I]ABN as the radioligand.
Ki values for D4 receptors were measured on human D4 expressed in HEK cells using [125I]ABN as the radioligand.
Ki for D3 receptors/Ki for D2 receptors.
Ki for D4 receptors/Ki for D2 receptors.
g
h
i
Ki for inhibiting the binding of [3H](+)-pentazocine to guinea pig brain homogenates.
Ki for inhibiting the binding of [3H]DTG to rat liver homogenates.
Calculated value using the program C log P.
j
Not determined.
D2 receptors. The above studies highlight the need to develop PET
radiotracers which are capable of imaging D3 versus D2 receptors
and vice versa.
Over the past decade, our group has focused on the develop-
ment of ligands having a high affinity and selectivity for D3 versus
D2 and D2 versus D3 receptors as useful probes for studying the
behavioral pharmacology of these receptors, and for studying the
differential expression of D2 and D3 receptors in the CNS with
PET. We have recently reported the development of a series of con-
formationally-flexible benzamide analogs which have the potential
for imaging D3 versus D2 receptors in the CNS.37,38 The current
study is a continuation of our effort to develop ligands having a
high affinity and selectivity for D2 versus D3 receptors which could
serves a useful D2-selective behavioral probes and lead compounds

S. Vangveravong et al. / Bioorg. Med. Chem. 19 (2011) 3502–3511
3507
The high affinity and good selectivity of compounds 6 and 7 for
D2 versus D3 receptors indicate that they should be useful probes
for studying the behavioral pharmacology of the D2 receptor. Fur-
thermore, these compounds can be easily radiolabeled with either
carbon-11 (t1/2 = 20.4 min) and fluorine-18 (t1/2 = 109.7 min) and
serve as useful PET radiotracers for measuring the density of D2
receptors in the CNS without interference from labeling dopamine
D3 receptors. The lipophilicities (log P) of 6 and 7 (Table 1) also
suggest that they will readily cross the blood brain barrier and
are good candidates for the development of D2 receptor selective
imaging agents for the functional imaging technique, PET. PET
imaging studies with [11C]6 and [18F]7 are currently ongoing in
our group and will be published separately.
100
80
60
40
20
0
D2L receptor HEK cells
D3 receptor HEK cells
In summary, a series of structural congeners of aripiprazole were
synthesized and evaluated for their affinities and efficacies at D2 and
D3 receptors. The results of the in vitro binding studies have identi-
fied two compounds, 6 and 7, which will be useful probes for study-
ing the function of the dopamine D2 versus D3 receptor in the CNS.
quin
1
6
7
Compounds
Figure 3. Intrinsic efficacy at D2 and D3 dopamine receptor subtypes for adenylyl
cyclase inhibition. Maximal inhibition for forskolin-dependent adenylyl cyclase
inhibition was evaluated using the test ligands aripiprazole (1), compounds 6 and 7
at concentrations approximately equal to 10Â the Ki values. The mean maximal
percent inhibition SEM. (n P 3) was obtained by normalizing the data to the mean
inhibition achieved using the full agonist quinpirole (quin) at a final concentration
7. Experimental
7.1. Chemical analysis
1H NMR spectra were recorded on a Varian 300 MHz NMR spec-
trometer. Chemical shifts are reported in d values (parts per mil-
lion, ppm) relative to an internal standard of tetramethylsilane
(TMS). The following abbreviations are used for multiplicity of
NMR signals: br s = broad singlet, d = doublet, dd = doublet of dou-
blets, dt = doublet of triplets, m = multiplet, s = singlet, t = triplets.
Melting points were determined on an electrothermal melting
point apparatus and are uncorrected. Elemental analyses were per-
formed by Atlantic Microlab, Inc., Norcross, GA and were within
0.4% of the calculated values. Mass spectrometry was provided
by the Washington University Mass Spectrometry Resource, an
NIH Research Resource (Grant No. P41RR0954). All reactions were
carried out under an inert atmosphere of nitrogen. Lipophilicity
measurements of the compounds were estimated using the com-
putational program, C log P (Advanced Chemistry Development,
Inc., Toronto, Canada).
of 1 l
M for D2L HEK cells (solid bars) and 100 nM for D3-HEK cells (hatched bars).42
for developing D2-selective PET radiotracers. In these earlier
studies, we prepared a number of structural analogs of the classical
D2-like dopamine receptor antagonists, haloperidol, having a high
affinity for D2 versus D3 receptors.15,16
In the current study, we focused on the atypical antipsychotic
aripiprazole because this compound was reported to have a modest
selectivity for D2 versus D3.39 We explored the possibility of prepar-
ing aripiprazole analogs that might have higher affinity and selec-
tivity for D2 versus D3 receptors than that of aripiprazole itself.
First, the N-2,3-dichlorophenyl piperazine ring of aripiprazole was
replaced with amine groups present in typical antipsychotics such
as haloperidol, benperidol, domperidone, and N-methylspiperone.
The results of this study revealed that replacing the N-2,3-dichloro-
phenyl piperazine ring of aripiprazole with these amine groups did
not result in useful ligands since compounds 2–5 had low binding
affinity at D2, D3, and D4 receptors. Second, we replaced the
N-2,3-dichlorophenyl group of aripiprazole with N-2-methoxy-
phenyl group (compound 6) or N-2-(2-fluoroethoxy)phenyl group
(compound 7). Compounds 6 and 7 bind at D2 receptors with nano-
molar affinity and have >50-fold selectivity for human D2 receptors
compared to human D3 dopamine receptor subtype. These two
analogs also bind with low affinity at the D4 dopamine receptor
subtype, as well as r1 and r2 receptors. These two analogs were
evaluated for intrinsic efficacy and found to be partial agonists at
both D2 and D3 receptors. Third, we changed the position of the
methoxy group from the 2-position to the 4-position of the pipera-
zine moiety. Compound 8 (with N-4-methoxyphenyl group) lost the
binding affinity to both D2 and D3 receptors. Substitution on both 2-
and 4-positions (compounds 9 and 10) decrease D3 receptor affinity
but had little effect on D2 receptor affinity. Fourth, we also intro-
duced a double bond into the tetrahydroisoquinoline ring of 6
and 7. Compounds 11 and 12 maintain a similar affinity for D2
receptors with a slight increase in affinity for D3 receptors. Finally,
we changed the length of the carbon spacer in the chain between
two ring systems of 6 and 11 from 4-carbon atoms to 3-carbon
atoms (compounds 13 and 14) or 5-carbon chain (compounds 15
and 16). This change resulted in compounds with nanomolar affin-
ity at both D2 and D3 receptors, resulting in a low selectivity for the
two receptor subtypes.
7.2. 7-(4-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)butoxy)-
3,4-dihydroquinolin-2(1H)-one oxalate (2)
A mixture of 7-(4-bromobutoxy)-3,4-dihydroquinolin-2(1H)-
one (20, 1.7 mmol), 4-(chlorophenyl)-4-hydroxypiperidine (1.7 m
mol), potassium carbonate (5 equiv) and potassium iodide (1 equiv)
in acetonitrile (20 mL) was stirred at reflux overnight. The reaction
mixture was evaporated. The resulting residue was suspended in
water (25 mL) and filtered. The solid was washed with water and
air driedto give the productas white powder(96% yield). The oxalate
salt was prepared using 1 equiv of oxalic acid in ethanol and recrys-
tallized from ethanol to give 2 as an off-white powder, mp 178–
179 °C; 1H NMR (free base, CDCl3 + DMSO-d6) d 9.08 (br s, 1H),
7.47 (d, J = 8.7 Hz, 2H), 7.29 (d, J = 8.7 Hz, 2H), 7.02 (d, J = 8.2 Hz,
1H), 6.49 (dd, J = 8.7 and 2.5 Hz, 1H), 6.42 (d, J = 2.5 Hz, 1H), 3.95
(t, J = 6.0 Hz, 2H), 3.50–3.60 (m, 1H), 2.78–2.89 (m, 4H), 2.43–2.62
(m, 6H), 2.02–2.10 (m, 2H), 1.70–1.80 (m, 6H). Anal. (C24H29ClN2-
O3ÁC2H2O4Á0.5H2O) C, H, N.
7.3. 7-(4-(4-(2-Oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)-
piperidin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one
oxalate (3)
Compound
3 was synthesized as described for 2, from
4-(2-keto-1-benzimidazolinyl)piperidine in 67% yield. Conversion

3508
S. Vangveravong et al. / Bioorg. Med. Chem. 19 (2011) 3502–3511
to the oxalate salt gave 3 as an off-white powder, mp 187–188 °C;
1H NMR (free base, CDCl3) d 9.88 (br s, 1H), 9.37 (s, 1H), 7.21–7.25
(m, 1H), 7.02–7.10 (m, 4H), 6.52–6.55 (m, 2H), 4.24–4.33 (m, 1H),
4.06 (t, J = 6.6 Hz, 2H), 3.11–3.15 (m, 2H), 2.87–2.92 (m, 2H),
2.50–2.64 (m, 6H), 2.17–2.24 (m, 2H), 1.71–1.89 (m, 6H). Anal.
(C25H30N4O3ÁC2H2O4Á1.25H2O) C, H, N.
off-white powder, mp 147–148 °C; 1H NMR (free base, CDCl3) d
8.07 (br s, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.95–6.99 (m, 3H),
6.85–6.88 (m, 1H), 6.53 (dd, J = 8.2 and 2.4 Hz, 1H), 6.32 (d,
J = 2.4 Hz, 1H), 4.85–4.88 (m, 1H), 4.69–4.72 (m, 1H), 4.29–4.32
(m, 1H), 4.20–4.23 (m, 1H), 3.96 (t, J = 6.2 Hz, 2H), 3.14 (br s, 4H),
2.87–2.92 (m, 2H), 2.59–2.64 (m, 6H), 2.45–2.50 (m, 2H), 1.68–
1.84 (m, 4H). Anal. (C25H32FN3O3ÁC2H2O4Á0.5H2O) C, H, N.
7.4. 7-(4-(4-(5-Chloro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-
1-yl)piperidin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one
oxalate (4)
7.9. 7-(4-(4-(4-Methoxyphenyl)piperazin-1-yl)butoxy)-3,4-di-
hydroquinolin-2(1H)-one oxalate (8)
Compound 4 was synthesized as described for 2, from 5-chloro-
1-(4-piperidyl)-2-benzimidazolinone in 70% yield. Conversion to
the oxalate salt gave 4 as an off-white powder, mp 184–185 °C;
1H NMR (free base, CDCl3) d 10.10 (br s, 1H), 9.26 (s, 1H),
6.99–7.10 (m, 4H), 6.53–6.77 (m, 2H), 4.15–4.23 (m, 1H), 4.07 (t,
J = 6.6 Hz, 2H), 3.07–3.11 (m, 2H), 2.87–2.92 (m, 2H), 2.45–2.64
(m, 6H), 2.10–2.17 (m, 2H), 1.68–1.92 (m, 6H). Anal.
(C25H29ClN4O3ÁC2H2O4) C, H, N.
Compound 8 was synthesized as described for 2, from 1-(4-
methoxyphenyl)piperazine hydrochloride in 93% yield. Conversion
to the oxalate salt gave 8 as an off-white powder, mp 152–153 °C;
1H NMR (free base, CDCl3) d 7.61 (br s, 1H), 7.05 (d, J = 8.4 Hz, 1H),
6.91 (d, J = 7.5 Hz, 2H), 6.84 (d, J = 7.5 Hz, 2H), 6.53 (d, J = 8.4 Hz,
1H), 6.29 (m, 1H), 3.93–3.98 (m, 2H), 3.77 (s, 3H), 3.10 (br s, 4H),
2.88–2.90 (m, 2H), 2.60–2.64 (m, 6H), 2.45–2.49 (m, 2H),
1.62–1.82 (m, 4H). Anal. (C24H31N3O3ÁC2H2O4Á0.5H2O) C, H, N.
7.5. 7-(4-(3Methyl-4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]decan-
7.10. 7-(4-(4-(2,4-Dimethoxyphenyl)piperazin-1-yl)butoxy)-3,4-
8-yl)butoxy)-3,4-dihydro-quinolin-2(1H)-one oxalate (5)
dihydroquinolin-2(1H)-one oxalate (9)
Compound 5 was synthesized as described for 2, from 3-
methyl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one in 72% yield.
Conversion to the oxalate salt gave 5 as an off-white powder, mp
Compound 9 was synthesized as described for 2, from 1-(2,4-
dimethoxyphenyl)piperazine hydrochloride in 71% yield. Conver-
sion to the oxalate salt gave 9 as an off-white powder, mp
167–168 °C; 1H NMR (free base, CDCl3) d 7.80 (br s, 1H), 7.05 (d,
J = 8.5 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.41–6.54 (m, 3H), 6.30 (s,
1H), 3.96 (t, J = 6.1 Hz, 2H), 3.84 (s, 3H), 3.78 (s, 3H), 3.03 (br s,
4H), 2.87–2.92 (m, 2H), 2.60–2.66 (m, 6H), 2.45–2.50 (m, 2H),
1.68–1.84 (m, 4H). Anal. (C25H33N3O4ÁC2H2O4Á0.5H2O) C, H, N.
241–242 °C; 1H NMR (free base, CDCl3)
d 7.96 (br s, 1H),
7.25–7.30 (m, 2H), 7.04 (d, J = 8.2 Hz, 1H), 6.83–6.92 (m, 3H),
6.52 (dd, J = 8.2 and 2.2 Hz, 1H), 6.33 (d, J = 2.2 Hz, 1H), 4.67 (s,
2H), 3.97 (t, J = 6.2 Hz, 2H), 3.00 (s, 3H), 2.86–2.91 (m, 6H), 2.54–
2.78 (m, 6H), 1.65–1.85 (m, 6H). Anal. (C27H34N4O3ÁC2H2O4) C, H, N.
7.6. 7-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butoxy)-3,4-di-
7.11. 7-(4-(4-(2-Methoxy-4-methylphenyl)piperazin-1-yl)but-
hydroquinolin-2(1H)-one oxalate (6)
oxy)-3,4-dihydroquinolin-2(1H)-one oxalate (10)
Compound 6 was synthesized as described for 2, from 1-(2-
methoxyphenyl)piperazine hydrochloride in 96% yield. Conversion
to the oxalate salt gave 6 as an off-white powder, mp 116–117 °C;
1H NMR (free base, CDCl3) d 7.96 (br s, 1H), 7.04 (d, J = 8.2 Hz, 1H),
6.85–7.01 (m, 4H), 6.53 (dd, J = 8.2 and 2.4 Hz, 1H), 6.32 (d,
J = 2.4 Hz, 1H), 3.96 (t, J = 6.2 Hz, 2H), 3.86 (s, 3H), 3.10 (br s, 4H),
2.87–2.92 (m, 2H), 2.59–2.67 (m, 4H), 2.45–2.50 (m, 2H),
1.70–1.84 (m, 6H). Anal. (C24H31N3O3ÁC2H2O4Á0.5H2O) C, H, N.
Compound 10 was synthesized as described for 2, from
1-(2-methoxy-4-methylphenyl)piperazine hydrochloride (19) in
95% yield. Conversion to the oxalate salt gave 10 as an off-white
powder, mp 146–147 °C; 1H NMR (free base, CDCl3) d 7.83 (br s,
1H), 6.97 (d, J = 8.1 Hz, 1H), 6.77 (d, J = 7.8 Hz, 1H), 6.64 (d,
J = 8.1 Hz, 1H), 6.61 (s, 1H), 6.45 (d, J = 8.4 Hz, 1H), 6.24 (s, 1H),
3.88 (t, J = 6.1 Hz, 2H), 3.77 (s, 3H), 2.99 (br s, 4H), 2.80–2.85 (m,
2H), 2.50–2.61 (m, 6H), 2.37–2.42 (m, 2H), 2.23 (s, 3H), 1.60–1.74
(m, 4H). Anal. (C25H33N3O3ÁC2H2O4Á1.5H2O) C, H, N.
7.7. 7-(4-(4-(2-Hydroxyphenyl)piperazin-1-yl)butoxy)-3,4-di-
hydroquinolin-2(1H)-one (17)
7.12. 1-(2-Methoxy-4-methylphenyl)piperazine hydrochloride
(19)19
Compound 17 was synthesized as described for 2, from 1-(2-
hydroxyphenyl)piperazine in 84% yield as an off-white powder;
mp 107–108 °C; 1H NMR (free base, CDCl3) d 8.15 (br s, 1H),
7.15–7.18 (m, 1H), 7.05–7.10 (m, 1H), 7.04 (d, J = 8.2 Hz, 1H),
6.83–6.96 (m, 2H), 6.53 (dd, J = 8.2 and 2.3 Hz, 1H), 6.33 (d,
J = 2.3 Hz, 1H), 3.97 (t, J = 6.0 Hz, 2H), 2.87–2.93 (m, 6H), 2.59–
2.64 (m, 6H), 2.48 (t, J = 7.4 Hz, 2H), 1.66–1.87 (m, 5H). Anal.
(C23H29N3O3) C, H, N.
A solution of 2-methoxy-4-methylaniline (18, 10.9 mmol), bis-
(2-chloroethyl)amine hydrochloride (12.0 mmol), potassium
carbonate (15.2 mmol) in 1-butanol (5 mL) was refluxed under
nitrogen overnight. The hot reaction mixture was filtered and the fil-
trate was concentrated under vacuum. The resulting residue was
triturated with acetone and filtered to give 19 as an off-white pow-
der (14% yield), mp 212–213 °C (dec); 1H NMR (free base, CDCl3) d
9.14 (s, 1H), 6.68–6.82 (m, 3H), 3.77 (s, 3H), 3.11–3.19 (m, 8H),
2.25 (s, 3H).
7.8. 7-(4-(4-(2-(2-Fluoroethoxy)phenyl)piperazin-1-yl)butoxy)-
3,4-dihydroquinolin-2(1H)-one oxalate (7)
7.13. 7-(4-(4-(2-Methoxyphenyl)piperazin-1-
1-Bromo-2-fluoroethane (5.0 mmol) and potassium carbonate
(4 equiv) were added into the solution of 17 (1.5 mmol) in acetone
(15 mL). The reaction mixture was heated at 65–70 °C for 48 h. The
solid was filtered off and washed with acetone. The filtrate was
evaporated. The resulting residue was purified by silica gel column
chromatography (5% methanol in dichloromethane) to give the
product (59% yield). Conversion to the oxalate salt gave 7 as an
yl)butoxy)quinolin-2(1H)-one oxalate (11)
A
mixture of 7-(4-bromobutoxy)quinolin-2(1H)-one17 (21,
0.78 mmol), 1-(2-methoxyphenyl)piperazine hydrochloride
(1.00 mmol), and triethylamine (0.5 mL) in acetonitrile (15 mL)
was refluxed overnight. The solvent was evaporated under reduced
pressure. The residue was dissolved in dichloromethane, washed

S. Vangveravong et al. / Bioorg. Med. Chem. 19 (2011) 3502–3511
3509
with saturated NaHCO3 solution, dried over Na2SO4, concentrated
in vacuo. The resulting residue was purified by silica gel column
chromatography (5% methanol in dichloromethane) to give the
product as an oil (62% yield). Conversion to the oxalate salt gave
11 as an off-white powder, mp 180–182 °C; 1H NMR (free base,
CDCl3) d 12.30 (s, 1H), 7.71–7.74 (m, 1H), 7.42–7.45 (m, 1H),
6.78–7.02 (m, 6H), 6.53–6.56 (m, 1H), 4.07–4.10 (m, 2H), 3.86
(s, 3H), 3.11 (br s, 4H), 2.68 (br s, 4H), 2.47–2.52 (m, 2H),
1.72–1.90 (m, 4H). HRMS calcd for C24H30N3O3 [M+H]+ 408.2287,
found: 408.2290.
J = 2.4 Hz, 1H), 3.93 (t, J = 6.5 Hz, 2H), 3.87 (s, 3H), 3.11 (br s, 4H),
2.90 (t, J = 7.5 Hz, 2H), 2.59–2.66 (m, 6H), 2.44 (t, J = 7.5 Hz, 2H),
1.50–1.83 (m, 6H). Anal. (C25H33N3O3ÁH2C2O4) C, H, N.
7.18. 7-(5-(4-(2-Methoxyphenyl)piperazin-1-
yl)pentyloxy)quinolin-2(1H)-one oxalate (16)
A mixture of 15 (0.28 mmol), and DDQ (0.34 mmol) in dichloro-
methane (5 mL) was stirred at room temperature overnight. The
mixture was washed with saturated NaHCO3 solution, dried, and
concentrated in vacuo. The residue was purified by column chro-
matography (5% methanol in dichloromethane) to give the product
as a free base in 75% yield. Conversion to oxalate salt gave 16 as a
white powder; mp 229–230 °C. 1H NMR (free base, CDCl3) d 12.05
(s, 1H), 7.72 (d, J = 9.3 Hz, 1H), 7.44 (d, J = 9.3 Hz, 1H), 6.79–7.10 (m,
6H), 6.53 (d, J = 9.3 Hz, 1H), 4.07 (t, J = 6.3 Hz, 2H), 3.86 (s, 3H), 3.11
(br s, 4H), 2.68 (br s, 4H), 2.46 (t, J = 6.6 Hz, 2H), 1.82–1.90 (m, 2H),
1.53–1.70 (m, 4H). Anal. (C25H31N3O3ÁH2C2O4) C, H, N.
7.14. 7-(4-(4-(2-(2-Fluoroethoxy)phenyl)piperazin-1-
yl)butoxy)quinolin-2(1H)-one oxalate (12)
Compound 12 was synthesized as described for 11 from 21 and
1-(2-(2-fluoroethoxy)phenyl)piperazine20 in 65% yield. Conversion
to oxalate salt gave 12 as a white powder, mp 152–154 °C; 1H NMR
(free base, CDCl3) d 12.02 (s, 1H), 7.72 (d, J = 9.4 Hz, 1H), 7.44
(d, J = 9.4 Hz, 1H), 6.79–6.96 (m, 6H), 6.53 (d, J = 9.4 Hz, 1H), 4.78
(dt, J = 47.7 and 3.9 Hz, 2H), 4.25 (dt, J = 27.6 and 3.9 Hz, 2H),
4.08–4.13 (m, 2H), 3.14 (br s, 4H), 2.67 (br s, 4H), 2.49
(t, J = 7.5 Hz, 2H), 1.72–1.90 (m, 4H). HRMS calcd for C25H31FN3O3
[M+H]+ 440.2349, found: 440.2345.
8. Radiological binding and functional assays
8.1. Dopamine receptor binding assay
The method for the iodination of 125I-IABN using peracetic acid
has been previously described.23 For radioligand binding studies,
membrane homogenates from stably transfected HEK 293 cells
expressing either the human D2, D3, or D4 receptors were prepared
using a polytron tissue homogenizer (Brinkman Instruments, West-
bury, NY). The tissue was suspended in 50 mM Tris–HCl, 150 mM
7.15. 7-(3-(4-(2-Methoxyphenyl)piperazin-1-yl)propoxy)-3,4-
dihydroquinolin-2(1H)-one oxalate (13)
1-(3-Chloropropyl)-4-(2-methoxyphenyl)piperazine22 (2.0 mmol)
was added to a solution of 7-hydroxy-3,4-dihydroquinolin-2(1H)-
one (22a, 2.0 mmol), sodium methoxide (25% in methanol,
2.0 mmol) in ethanol (10 mL). The mixture was refluxed for 5 h.
The solvent was removed in vacuo. The residue was diluted with
dichloromethane and washed with water. The organic phase was
dried and concentrated. The residue was purified by column
chromatography (5% methanol in dichloromethane) to give prod-
uct as a free base in 67% yield. Conversion to oxalate salt gave 13
as a white powder, mp 115–116 °C; 1H NMR (free base, CDCl3) d
8.07 (br s, 1H), 6.85–7.05 (m, 5H), 6.53 (dd, J = 8.1, and 2.4 Hz,
1H), 6.33 (d, J = 2.4 Hz, 1H), 4.00 (t, J = 6.3 Hz, 2H), 3.86 (s, 3H),
3.11 (br s, 4H), 2.89 (t, J = 7.2 Hz, 2H), 2.57–2.69 (m, 8H),
1.97–2.02 (m, 2H). HRMS calcd for C23H30N3O3 [M+H]+ 396.2287,
found: 396.2284.
NaCl and 1 mM EDTA at pH 7.5 to approximately 5–20
l
g of protein
L prior to the assay. Assays were performed in a total volume
L. Binding reactions were carried out for 60 min at 37 °C and
per 50
of 150
l
l
the reaction was terminated by rapid filtration over Schleicher and
Schuell No. 32 glass fiber filters (Whatman plc, Maidstone, England).
After washing filters with buffer, the radioactivity of the 125I-labeled
ligand was quantitated using a Packard Cobra gamma counter with
an efficiencyof 75%. Protein concentrations were determinedusing a
BCA reagent (Pierce, Rockford, Illinois) with bovine serum albumin
as the protein standard.
For competition curves using a transfected cell line expressing
D2, D3, or D4 dopamine receptors, experiments were performed in
triplicate with two concentrations of inhibitor per decade over at
least five orders of magnitude. The concentration of the radioligand
was approximately equal to the Kd values. Controls containing
7.16. 7-(3-(4-(2-Methoxyphenyl)piperazin-1-
yl)propoxy)quinolin-2(1H)-one oxalate (14)
either no inhibitor or 2
binding and nonspecific binding, respectively. Competition data for
2-like dopamine receptors were modeled for a single-site fit using
lM (+)-butaclamol were used to define total
Compound 14 was synthesized as described for 13 from
7-hydroxyquinolin-2(1H)-one (22b) and 1-(3-chloropropyl)-4-(2-
methoxyphenyl)piperazine22 in 52% yield. Conversion to oxalate
salt gave 14 as a white powder, mp 143–145 °C; 1H NMR (free base,
CDCl3) d 12.13 (s, 1H), 7.72 (d, J = 9.3 Hz, 1H), 7.45 (d, J = 9.3 Hz,
1H), 6.81–7.02 (m, 6H), 6.55 (d, J = 9.6 Hz, 1H), 4.15 (t, J = 6.2 Hz,
2H), 3.87 (s, 3H), 3.13 (br s, 4H), 2.71 (br s, 4H), 2.63 (t,
J = 7.4 Hz, 2H), 2.03–2.08 (m, 2H). Anal. (C23H27N3O3ÁH2C2O4ÁH2O)
C, H, N.
D
the TABLECURVE program (Jandel Scientific Software, San Rafael, Cali-
fornia); the IC50 values for the competitive inhibitors were con-
verted to Ki values using the Cheng and Prusoff corrections.40
8.2. Sigma receptor binding assays
Test compounds were dissolved in N,N-dimethylformamide
(DMF), dimethyl sulfoxide (DMSO) or ethanol and then diluted in
50 mM Tris–HCl buffer, pH 7.4, containing 150 mM NaCl and
100 mM EDTA. Membrane homogenates were made from guinea
pig brain for r1 binding assay and rat liver for r2 binding assay.
Membrane homogenates were diluted with 50 mM Tris–HCl buffer,
7.17. 7-(5-(4-(2-Methoxyphenyl)piperazin-1-yl)pentyloxy)-3,4-
dihydroquinolin-2(1H)-one oxalate (15)
Compound 15 was synthesized as described for
2 from
pH 8.0, and incubated at 25 °C in a total volume of 150
plates with the radioligand and test compounds with concentrations
ranging from 0.1 nM to 10 M. After incubation was completed, the
reactionswere terminatedby theaddition of 150 L of ice-coldwash
lL in 96-well
7-(5-bromopentyloxy)-3,4-dihydroquinolin-2(1H)-one (23)21 and
1-(2-methoxyphenyl)piperazine hydrochloride in 47% yield.
Conversion to oxalate salt gave 15 as a white powder, mp
l
l
198–200 °C; 1H NMR (free base, CDCl3)
d
7.90 (br s, 1H),
buffer (10 mM Tris–HCl, 150 mM NaCl, pH 7.4) using a 96-channel
transfer pipette (Fisher Scientific, Pittsburgh, PA), and the samples
6.85–7.06 (m, 5H), 6.52 (dd, J = 8.4 and 2.4 Hz, 1H), 6.31 (d,

3510
S. Vangveravong et al. / Bioorg. Med. Chem. 19 (2011) 3502–3511
harvested and filtered rapidly through 96-well fiber glass filter plate
(Millipore, Billerica, MA) that had been presoaked with 100 L of
50 mM Tris–HCl buffer, pH 8.0, for 1 h. Each filter was washed three
times with 200 L of ice-cold wash buffer. A Wallac 1450 MicroBeta
Appendix (continued)
l
Compound %C
%H
%N
Calcd Found Calcd Found Calcd Found
l
liquid scintillation counter (Perkin–Elmer, Boston, MA) was used to
quantitate the bound radioactivity.
9
60.21 60.47
59.99 59.60
59.87 60.09
63.14 62.91
63.39 63.27
69.85 69.72
6.74
7.08
6.23
6.87
6.50
7.39
6.72
6.79
6.21
6.79
6.48
7.25
7.80
7.77
8.38
8.18
8.21
7.70
7.41
8.28
8.00
8.07
10
14
15
16
17
The r1 receptor binding assay was conducted using guinea pig
brain membrane homogenates (ꢀ300 g protein) and ꢀ5 nM
l
[3H](+)-pentazocine (34.9 Ci/mmol, Perkin–Elmer, Boston, MA).
The incubation time was 90 min. Nonspecific binding was deter-
10.62 10.64
mined from samples that contained 10 lM of cold haloperidol.
The r2 receptor binding assays were conducted using rat liver
membrane homogenates (ꢀ300
l
g protein) and ꢀ1 nM [3H]RHM-
1 (80 Ci/mmol, American Radiolabeled Chemicals Inc., St. Louis,
References and notes
MO) alone or ꢀ5 nM [3H]DTG (58.1 Ci/mmol, Perkin–Elmer, Bos-
ton, MA) in the presence of 1 lM (+)-pentazocine to block r1 sites.
1. Liu, L.-X.; Burgess, L. H.; Gonzalez, A. M.; Sibley, D. R.; Chiodo, L. A. Synapse
1999, 31, 108.
The incubation time was 60 min for [3H]RHM-1 and 120 min for
[3H]DTG. Nonspecific binding was determined from samples that
2. Herve, D.; Le-Moine, C.; Corvol, J. C.; Belluscio, L.; Ledent, C.; Fienberg, A. A.;
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l
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Acknowledgments
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This research was funded by MH081281 and DA023957
awarded by the National Institutes of Health.
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Appendix – Elemental analyses
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Compound %C
%H
%N
Calcd Found Calcd Found Calcd Found
2
3
4
5
6
7
8
59.15 58.79
59.28 59.28
58.01 58.07
63.03 62.95
61.40 61.40
59.99 60.03
61.40 61.62
6.11
6.36
5.59
6.57
6.74
6.53
6.74
5.85
6.45
5.86
6.60
6.54
6.55
6.62
5.31
5.22
10.24 9.85
10.02 10.00
10.14 10.13
8.26
7.77
8.26
8.20
7.54
8.33
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