Synthesis and characterization of selective dopamine D2 receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2005.09.008

A series of indole compounds have been prepared and evaluated for affinity at D₂-like dopamine receptors using stably transfected HEK cells expressing human D₂, D₃, or D₄ dopamine receptors. These compounds shar

Full text of DOI:10.1016/j.bmc.2005.09.008

Bioorganic & Medicinal Chemistry 14 (2006) 815–825
Synthesis and characterization of selective dopamine D2
receptor antagonists
Suwanna Vangveravong,a Elizabeth McElveen,b Michelle Taylor,b Jinbin Xu,a Zhude Tu,a
Robert R. Luedtkeb and Robert H. Macha,*
aDivision of Radiological Sciences, Washington University School of Medicine, Mallinckrodt Institute of Radiology,
510 S. Kingshighway, St. Louis, MO 63110, USA
bDepartment of Pharmacology and Neuroscience, University of North Texas Health Science Center, 3500 Camp Bowie Boulevard,
Fort Worth, TX 76107, USA
Received 12 July 2005; revised 31 August 2005; accepted 1 September 2005
Available online 8 November 2005
Abstract—A series of indole compounds have been prepared and evaluated for affinity at D2-like dopamine receptors using stably
transfected HEK cells expressing human D2, D3, or D4 dopamine receptors. These compounds share structural elements with the
classical D2-like dopamine receptor antagonists, haloperidol, N-methylspiperone, and benperidol. The compounds that share struc-
tural elements with N-methylspiperone and benperidol bind non-selectively to the D2 and D3 dopamine receptor subtypes. However,
several of the compounds structurally similar to haloperidol were found to (a) bind to the human D2 receptor subtype with nano-
molar affinity, (b) be 10- to 100-fold selective for the human D2 receptor compared to the human D3 receptor, and (c) bind with low
affinity to the human D4 dopamine receptor subtype. Binding at sigma (r) receptor subtypes, r1 and r2, was also examined and it
was found that the position of the methoxy group on the indole was pivotal in both (a) D2 versus D3 receptor selectivity and (b)
affinity at r1 receptors. Adenylyl cyclase studies indicate that our indole compounds with the greatest D2 receptor selectivity are
neutral antagonists at human D2 dopamine receptor subtypes. With stably transfected HEK cells expressing human D2 (hD2-
HEK), these compounds (a) have no intrinsic activity and (b) attenuated quinpirole inhibition of adenylyl cyclase. The D2 receptor
selective compounds that have been identified represent unique pharmacological tools that have potential for use in studies on the
relative contribution of the D2 dopamine receptor subtypes in physiological and behavioral situations where D2-like dopaminergic
receptor involvement is indicated.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
modulation of the dopaminergic pathways is thought to
occur as a consequence of acute and chronic abuse of psy-
chostimulants, including cocaine and amphetamines.9–11
There are three dopaminergic pathways that are of funda-
mental importance to the function of the normal brain:
the nigrostriatal pathway, the mesocorticolimbic path-
way and the tuberoinfundibular pathway. These neuro-
nal systems are involved in movement coordination,
cognition, emotion, affect, memory, and the regulation
of prolactin secretion by the pituitary. Alterations in
the dopaminergic pathways are thought to be involved
in the pathogenesis of neurological, neuropsychiatric,
and hormonal disorders, including Parkinsonꢀs disease,
schizophrenia, and hyperprolactinemia.1–8 In addition,
Molecular genetic studies of G-protein coupled recep-
tors have defined two types of dopamine receptors, the
D1-like (D1 and D5 receptor subtypes) and D2-like
(D2, D3, and D4 receptor subtypes) receptors based
upon structural and pharmacological similarities. For
example, D1-like receptors are structurally similar and
positively linked to the activation of adenylyl cyclase
via coupling to the Gs/Golf class of G-proteins.12 Stim-
ulation of the D2-like receptors results in coupling with
the Gi/Go class of G-proteins, leading to the inhibition
of adenylyl cyclase activity.13–15
Keywords: Indoles; D2-like dopamine receptors; Sigma receptors;
Dopamine receptor antagonists.
The D2 and D3 dopamine receptors have approximately
46% amino acid homology. However, the transmem-
brane spanning (TMS) regions of the D2 and D3
*
Corresponding author. Tel.: +1 314 362 8538; fax: +1 314 362
0039; e-mail: rhmach@mir.wustl.edu
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.09.008

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S. Vangveravong et al. / Bioorg. Med. Chem. 14 (2006) 815–825
receptors, which are thought to construct the ligand
binding site, share 78% homology.16 Despite the similar-
ities in the structure of the D2 and D3 receptors, the D2
and D3 receptors differ in their (a) neuroanatomical
localization,17 (b) levels of receptor expression, (c) effica-
cy in response to agonist stimulation,8 and (d) regula-
tion and desensitization.18 Because of the high degree
of homology between D2 and D3 receptor binding sites,
the pharmacologic properties of these two receptor sub-
types are similar and it has been difficult to obtain com-
pounds that can bind selectively to either the D2 or the
D3 dopamine receptor subtypes.8,19–21 However, D2 or
D3 dopamine receptor selective agonists and antagonists
would be useful pharmacologic tools to precisely define
the role of these two D2-like receptor subtypes in a vari-
ety of experimental physiological and behavioral situa-
tions, including the reinforcing and toxic properties of
cocaine,22,23 socialization, memory, and the regulation
of interneuronal activity in the basal ganglia.24
vation noted in the paper of Kulagowski et al.,25 but
not explored in greater detail, was the high affinity
and selectivity of compound 2 for dopamine D2 versus
D3 and D4 receptors (Fig. 1). In this communication,
we report the synthesis and in vitro binding of congen-
ers of 2 with nanomolar affinity at D2 receptors and
range from 10- to 100-fold selective for D2 compared
to the D3 receptor subtype. In vitro studies indicate
that these compounds are neutral antagonists at D2-
like dopamine receptors.
2. Chemistry
The synthesis of the target compounds is outlined in
Schemes 1 and 2. Gramine and 5-methoxygramine are
commercially available. Only 4-methoxygramine was
made in this study in quantitative yield by treatment
of 4-methoxyindole, 3, with N,N-dimethylmethyleneam-
monium iodide (Eschenmoserꢀs salt) as outlined in
Scheme 1. Reaction of 4a–c with the corresponding sec-
ondary amines gave the corresponding indoleamine ana-
logs in moderate yield (Scheme 2). The synthesis of the
40-methylthio analogs, 20 and 21, required a different
synthetic route (Scheme 3), which involved the synthesis
of the 4-piperidone intermediates, 18a and 18c, followed
by treatment with the Grignard reagent, 19, to give the
A previous study reported indole analogs, represented
by compound 1, as having a high affinity for dopamine
D4 versus D2 and D3 receptors.25 These compounds
were developed as potential atypical antipsychotic
agents based on the observation that clozapine has a
moderate affinity for D4 receptors and a relatively low
affinity for D2 and D3 receptors.26 An interesting obser-
Figure 1. Structures of the lead compounds for the current study. The in vitro binding data are Ki values reported in Ref. 25.

S. Vangveravong et al. / Bioorg. Med. Chem. 14 (2006) 815–825
817
Scheme 1.
Scheme 2.
target compounds in moderate yield. We were unable to
make the corresponding 4-methoxy analog due to the
chemical instability of the intermediate, 18b.
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.27
3. Radioligand binding studies at dopamine receptors
First, when a comparison is made of the affinity at D2
and D3 dopamine receptors of the unsubstituted indoles
which have structural elements similar to the butyrophe-
nones N-methylspiperone (13) and haloperidol (2) or
benperidol (16), only 2 exhibits pharmacological selec-
tivity at D2 receptors. Compound 2 was reported by
Competitive radioligand binding studies were performed
to determine the equilibrium dissociation constants of
each compound at human D2, D3, and D4 dopamine
receptors (Table 1). For these studies tissue homoge-
nates from stably transfected HEK 293 cells were used

818
S. Vangveravong et al. / Bioorg. Med. Chem. 14 (2006) 815–825
Scheme 3.
Table 1. In vitro binding data for the indole analogs
Compound
Kia (nM)
D3:D2 ratioe
r1
r2
LogPf
b
c
d
D2
D3
104 19
250
D4
2
5
10 2.5
4.2 0.4
4.2 0.8
4.8 0.3
2.3 0.7
2.5 0.7
23.9 5.5
5.5 0.1
134 27
449 123g
>2000
>3500
10
59
19.3 3.6
>5000
1811 569
1246 234
1900 241
2134 356
943 64
1.54
2.10
2.16
1.72
2.34
2.28
1.33
1.91
0.46
1.17
1.08
1.83
1.75
6
6
204 60
110 39
190 34
96.9 6.1
638 159
580 92
200 125
150 38
128 18
38 11
49
28
242 68
12.1 2.5
2557 334
108 17
39.6 7.1
236 13
>5000
7
652
7
8
840 197
700 80
319 58g
567 140
>5000
82
39
9
2702 696
884 32
>10,000
20
21
13
14
15
16
17
27
106
1.5
1.5
6.4
1.1
0.8
>10,000
>10,000
101
20
2
5
3
>5000
>5000
3147 1290
>5000
>5000
>10,000
>10,000
1374 159
34
756 243
133 24
203 18
153 36
331 21
a Mean SEM, Ki values were determined by at least three experiments.
b Ki values for D2 receptors were measured on human D2(long) expressed in HEK cells using [125I]IABN as the radioligand.
c Ki values for D3 receptors were measured on human D3 expressed in HEK cells using [125I]IABN as the radioligand.
d Ki values for D4 receptors were measured on human D4.4 expressed in HEK cells using [125I]IABN as the radioligand.
e Ki for D3 receptor/Ki for D2 receptor.
f Calculated value using the program ClogP.
g Reported to be 220 nM in Kulagowski et al., 1996.
Kulagowski et al.,25 to be 40-fold selective for the hu-
man D2 compared to the human D3 receptor subtype.
We found essentially the same affinity for this com-
pound at the D3 receptor subtype, but a fourfold lower
affinity at D2 receptors. Therefore, our experiments indi-
cate that compound 2 is 10-fold selective at D2 receptors
compared to D3 dopamine receptors.
group resulted in a 2- to 2.5-fold decrease in affinity at
D3 receptors with a concomitant 2-fold increase in affin-
ity at D2 receptors, resulting in compounds that were 50-
to 60-fold selective at D2 receptors. A similar modifica-
tion of the 5-position on an unsubstituted indole struc-
turally related to N-methylspiperone (15) was also
found to increase D2 receptor selectivity, indicating that
this type of substitution was important in obtaining a
compound that could discriminate pharmacologically
between D2 and D3 dopamine receptor subtypes. Sur-
prisingly, substitution of a methoxy group at the 4-posi-
tion (14) did not appear to have an effect on D2-like
receptor affinity or D3:D2 selectivity.
Based upon those results, modifications of the indole
group were prepared to determine if the addition of a
methoxy group to 2 at the 4- or 5-position (5 and 6,
respectively) would alter D2/D3 dopamine receptor
selectivity. We found that the addition of the methoxy

S. Vangveravong et al. / Bioorg. Med. Chem. 14 (2006) 815–825
819
A comparison of unsubstituted indoles structurally
related to haloperidol was made in which a Br (7) or a
SCH3 (20) was substituted for the Cl moiety (2). The
substitution with these bulkier groups increased selectiv-
ity at D2 receptors 2- to 3-fold. Although there was an
increase in D2 receptor selectivity, the substitution with
the Br group (7) increased affinity at D2 receptors 2-fold
(Ki = 5 nM), whereas the substitution with SCH3 group
(20) resulted in a 2-fold decrease in affinity.
Our analysis of the indole compounds structurally simi-
lar to haloperidol indicated that addition of a methoxy
group to the indole and the substitution of a Cl group
increased the D3:D2 affinity ratio. Therefore, a panel
of methoxy indole compounds was prepared and evalu-
ated which substituted either a Br (8 and 9) or a SCH3
group (21) for the Cl. This combination of modifications
led to the identification of two compounds (8 and 21)
which exhibited >80-fold selectivity at D2 receptors
compared to D3 dopamine receptors with nanomolar
affinity at D2 receptors (Ki values of 2.3 and 5.5 nM,
respectively).
Finally, we evaluated the compounds with the highest
selectivity for D2 receptors, 2, 6, 8, and 21, for binding
activity at D1 receptors. Concentration-dependent com-
petitive binding studies were performed using (a) the D1
receptor selective radioligand 3H-SCH 23390, (b) rat
caudate tissue, which is known to express high levels
of D1 dopamine receptors, and (c) the four indoles with
the greatest selectivity at D2 receptors or non-radioac-
tive SCH 23390, which was included as a positive con-
trol. While SCH 23390 competed for radioligand
binding with high affinity, each of the four indoles
exhibited low affinity (IC50 > 10 lM) binding at rat D1
dopamine receptors (data not shown).
4. Adenylyl cyclase studies with D2-like receptors
The pharmacological properties of a select panel of
substituted indoles with the highest selectivity for D2
dopamine receptors (5, 6, 8, and 21) were further evalu-
ated using a whole cell adenylyl cyclase assay to deter-
mine if they are agonists or antagonists at D2
dopamine receptors (Fig. 2). The data shown in Figure
2 are representative of three independent sets of experi-
ments. For these cells, 80–90% inhibition of forskolin-
dependent stimulation (100 lM) of adenylyl cyclase
activity can be achieved at high concentrations
(100 nM) of the D2-like receptor full agonist quinpirole
(Quin). For these studies 10 nM quinpirole was used.
Although the extent of the inhibition by agonist of for-
skolin (Fsk) stimulation was less than maximal, these
experimental conditions were more optimal for deter-
mining if the test compound was capable of blocking
agonist activity. Although the full agonists, such as
quinpirole or dopamine, are able to attenuate for-
skolin-dependent stimulation of adenylyl cyclase in
HEK cells expressing human D2 receptors (hD2-HEK
cells), essentially no intrinsic activity was observed for
any of the four compounds that were evaluated. In addi-
tion, each compound was able to block the forskolin-de-
Figure 2. Evaluation of intrinsic activity of D2 receptor selective
indoles at human D2 dopamine receptors expressed in HEK 293 cells.
The top panel presents representative data for compounds 5 and 6,
while the bottom panel depicts data for compounds 8 and 21. The data
presented are representative of three independent experiments for each
compound tested. The values for the bar graph are the mean of
triplicates for a single experiment.
pendent inhibition of quinipirole at D2 dopamine
receptors. These results indicate that these compounds
are antagonists at D2 dopamine receptor.
Finally, we selected compound 5 for further evaluation.
Dose–response curves were performed using increasing
concentrations of 5 with a constant amount of forskolin
(100 lM) and quinpirole (10 nM). In Figure 3, a compos-
ite of three independent inhibition studies is shown in
which 5 competitively inhibits the effect of quinpirole on
forskolin-stimulated hD2-HEK cells. Under the
conditions of these experiments quinpirole inhibited
approximately 70% of the forskolin-dependent activation

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S. Vangveravong et al. / Bioorg. Med. Chem. 14 (2006) 815–825
were found to have affinity at r1 receptors ranging from
100
90
80
70
60
50
40
30
20
10
0
Ki values of 12–19 nM, irrespective of whether they had
a Cl, Br, or SCH3 group. However, clearly the addition
and position of the methoxy groups on the indole moi-
ety contributed to both the D2/D3 dopamine receptor
subtype selectivity of the compounds and the affinity
at r1 receptors. For example, the affinity of the unsub-
stituted indole compound 2 at r1 receptors was found
to be equal to 19 nM. Addition of the methoxy group
at the 5-position (6) decreased the affinity greater than
10-fold (Ki = 242), whereas the addition of a methoxy
at the 4-position decreased r1 receptor affinity
>100-fold. Similar effects were found for the indoles
containing a Br moiety. Addition of a methoxy to the
unsubstituted indole (7) at the 5-position resulted in an
8-fold decrease in affinity at r1 receptors and addition
of a methoxy at the 4-position reduced the affinity at
r1 receptors to a Ki value of >2000 nM. These data indi-
cate that substitution of the indole ring in either the 4- or
5-position with a methoxy group has the beneficial effect
of both (a) increasing affinity at dopamine D2 receptors
and (b) decreasing affinity at r1 receptors. However, the
most advantageous strategy to reduce the r1 receptor
affinity of the substituted indoles would be to incorpo-
rate a methoxy moiety at the 4-position on the indole
ring.
Fsk Fsk -10
+
-9
-8
-7
-6
Log Concentration (Molar)
Quin
Figure 3. Dose-dependent attenuation of the inhibition of
forskolin-dependent stimulation of adenylyl cyclase by 5. The
concentration-dependent mean attenuation of quinpirole inhibition
of forskolin-dependent stimulation of adenylyl cyclase by compound
5 using hD2-HEK cells is shown. The open bar is the normalized
stimulation of adenylyl cyclase activity in hD2-HEK cells using
100 lM forskolin (Fsk). The solid bar is the mean percent inhibition
(67 4% of maximum) of forskolin-dependent stimulation using
10 nM quinpirole (Fsk + Quin). The concentration-dependent curve
is the mean percent stimulation of adenylyl cyclase activity relative
to the normalized forskolin stimulation as
a function of the
concentration of 5. The mean values and the error bars (SEM)
are for three independent experiments. The IC50 value for these
composite data is 21 nM and was fit to a one-site fit model using a
non-linear regression analysis (r2 = 0.99) where the values for
maximum percent stimulation and maximum inhibition of percent
stimulation were constrained to experimental values.
6. Discussion
The D2 and D3 dopamine receptor subtypes are struc-
turally and pharmacologically similar.16 Both receptor
subtypes regulate adenylyl cyclase, extracellular acidifi-
cation, mitogenesis, and dopamine release.28–32 Howev-
er, the neuroanatomical localization and the levels of
expression of these two receptor subtypes are quite dif-
ferent. In addition, these two subtypes differ in terms
of the magnitude of the second messenger response fol-
lowing agonist stimulation.29 Studies by Kim et al.,33
indicated that D2 and D3 receptor expression may be
regulated in a different manner and that the intracellular
trafficking properties of these two receptor subtypes may
also be different. Src, phosphatidylinositol 3-kinase, and
atypical protein kinase C are commonly involved in
D2R-/D3R-mediated ERK activation. However, ERK
activation-mediated by D3 receptors, but not D2 recep-
tors, can be blocked by ARK-CT, AG1478 epidermal
growth factor receptor (EGFR) inhibitor, and by dom-
inant negative mutants of Ras and Raf, suggesting the
involvement of the different G-proteins.34 Recent studies
also demonstrate that both D2 and D3 dopamine recep-
tors couple to inward rectifier potassium channels
(GIRK). However, the shape of the termination current
and the desensitization of the response are different for
these two D2-like dopamine receptor subtypes.18
of adenylyl cyclase. Analysis of these composite data
indicates that 5 attenuated the quinpirole inhibition with
an IC50 value equal to 21 nM and was able to completely
surmount the effect of quinpirole. Similar results were
found for compound 6 (data not shown). These studies
provide further evidence that these indole analogs are
neutral antagonists at D2 dopamine receptors.
5. Radioligand binding studies at sigma receptors
We also investigated the pharmacological properties of
these indole compounds by determining the Ki values
of these compounds at sigma-1 (r1) and sigma-2 (r2)
receptors. The tissue source for the r1 assay was guinea
pig brain and the radioligand used to measure r1 recep-
tor affinity was [3H](+)-pentazocine. The tissue source
for the r2 receptor assay was rat liver and the binding
studies were conducted using [3H]DTG as the radioli-
gand in the presence of 100 nM unlabeled (+)-pentazo-
cine to mask r1 sites.38 Although haloperidol is a
dopaminergic antagonist that is used clinically as a neu-
roleptic, its high affinity at sigma receptors has preclud-
ed its usefulness as a radioligand for in vitro or in vivo
radioligand binding studies. First, all of the compounds
in this study bound with low affinity (>800 nM) at r2
receptors. The affinity at r1 receptors varied from
12 nM to >5000 nM. Second, all of the unsubstituted in-
dole analogs that are structurally similar to haloperidol
In one sense, the D2 receptor is the predominate CNS
D2-like dopamine receptor subtype because it (a) is ex-
pressed at the highest levels in the CNS and (b) was
the first of the D2-like dopamine receptor subtypes to
be cloned and characterized pharmacologically. It has
been documented that a pharmacologic property com-
mon to the majority of clinically used antipsychotics is

S. Vangveravong et al. / Bioorg. Med. Chem. 14 (2006) 815–825
821
that they are antagonists at D2 dopamine receptors.
However, more recently it is becoming clear that the
D3 receptor subtype may also play a role in the develop-
ment of pharmacotherapeutic strategies for the treat-
ment of neuropsychiatric and neurological disorders,
as well as for the rehabilitation of individuals who abuse
psychostimulants.8 The precise physiological role of
each of these two structurally and pharmacologically
related receptor subtypes can be more precisely defined
only when selective agonists and antagonists at each of
the three D2-like dopamine receptors become available
to the neuroscience community.
These pharmacological properties make 8 and 21 good
candidates for the development of D2 receptor selective
imaging agents for the functional imaging technique,
positron emission tomography (PET).
In addition, we have identified several structurally related
compounds which bind with essentially the same affinity
at D2 receptors but with varying affinity at r1 receptors.
These compounds may prove to be valuable for pharma-
cologically dissecting the role of r1 and D2 dopamine
receptors in a variety of behavioral paradigms in which
both D2 and r1 receptors have been implicated, including
cocaine-dependent locomotor activity and toxicity.35
Over the past five years there have been a number of
structurally diverse compounds reported having a high
affinity and selectivity for dopamine D3 versus D2 recep-
tors.8,40 However, the same cannot be stated for com-
pounds having a high affinity and selectivity for D2
versus D3 receptors. As part of our ongoing effort to
identify compounds that have D2-like receptor subtype
selectivity, we have evaluated the pharmacological prop-
erties of a panel of indoles that are structurally related to
the D2-like dopamine receptor butyrophenone antago-
nists, haloperidol, and spiperone. It has been reported
previously that both spiperone and haloperidol exhibit-
ed 5- to 10-fold higher affinity at rat D2 when compared
to rat D3 receptor subtype,10,16 and that an indole ana-
log structurally related to haloperidol exhibited selective
binding at the D2 dopamine receptor subtype.25 This
information served as the rationale for the design of
the indole analogs described in this report.
7. Conclusion
Several unsubstituted and methoxy substituted indole
analogs that bind selectively and with high affinity to D2
dopamine receptor subtypes have been identified. These
compounds appear to be neutral antagonists at human
D2 receptors. These compounds bind with low affinity
at D1, D4, and r2 receptors, but with varying affinity at
r1 receptors. In conjunction with other dopaminergic
radiolabeled reagents, this family of compounds may
have utility in studies designed to quantitate the expres-
sion and regulation of the two pharmacologically related
D2-like dopamine receptor subtypes, the D2 and D3 dopa-
mine receptors. They may also prove to be useful in more
precisely defining the role of r1 and D2 dopamine recep-
tors in a variety of (a) in vitro assays and (b) in vivo behav-
ioral paradigms.
The indole analogs that we prepared were initially evalu-
ated for binding selectivity at human D2, D3, and D4
dopamine receptor subtypes. We identified two methoxy
substituted indoles, 8 and 21, which (a) are structurally
related to haloperidol, (b) bind at D2 receptors with nano-
molar affinity, and (c) have >80-fold selectivity for human
D2 receptors compared to the human D3 dopamine recep-
tor subtype. These two analogs were also found to bind
with low affinity at the D4 and D1 dopamine receptor sub-
types, as well as r1 and r2 receptors.
8. Experimental
8.1. Chemical analysis
1H NMR spectra were recorded on a Varian 300 MHz
NMR spectrometer. Chemical shifts are reported in d
values (parts per million, 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, m = multiplet, s = singlet, and t = triplet. Melting
points were determined with an electrothermal melting
point apparatus and are uncorrected. Elemental analy-
ses were performed by Atlantic Microlab, Inc., Nor-
cross, GA, and were within 0.4% of the calculated
values. All reactions were carried out under an inert
atmosphere of nitrogen. Gramine and 5-methoxygr-
amine were purchased from Sigma–Aldrich Chemicals
(St. Louis, MO); 4-methoxygramine was synthesized
using the method described by Ley and Priour.36 Lipo-
philicity measurements of the compounds were estimat-
ed using the computational program, ClogP (Advanced
Chemistry Development, Inc., Toronto, Canada).
Four of the indole analogs with the greatest D2 receptor
selectivity were evaluated for intrinsic activity at human
D2 receptors using a whole cell adenylyl cyclase assay.
None of the four compounds that were evaluated exhib-
ited agonist activity in that assay. In addition, all com-
pounds that were tested were shown to be able to
attenuate the quinpirole inhibition of forskolin-depen-
dent stimulation of adenylyl cyclase, indicating that this
series of compounds are neutral antagonists at D2 dopa-
mine receptors.
The potential utility of these compounds to distinguish
pharmacologically between the D2 and D3 receptor sub-
types in in vitro and in vivo assays remains to be estab-
lished. However, our preliminary pharmacological
analysis suggests that these D2 receptor selective com-
pounds may be valuable tools to study the expression
and regulation of both D2 and D3 dopamine receptors.
The lipophilicity (logP) of 8 and 21 (Table 1) also sug-
gests that they will readily cross the blood–brain barrier.
8.2. General procedure for the synthesis of the N-indolylm-
ethyl analogs
A mixture of gramine derivatives (1.0–7.0 mmol) and
appropriate amines (1.2–8.4 mmol, 1.2 equiv) in toluene

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S. Vangveravong et al. / Bioorg. Med. Chem. 14 (2006) 815–825
(5–25 mL) was stirred at reflux overnight. The volatile
components were evaporated and the resulting residue
was purified by silica gel column chromatography
(dichloromethane–methanol–NH4OH, 90:10:0.5) to af-
ford the target compounds. The oxalate salt was pre-
pared using 1 equiv of oxalic acid in ethyl acetate.
(d, J = 7.6 Hz, 1H), 4.04 (s, 2H), 3.93 (s, 3H), 2.95–
2.99 (m, 2H), 2.52–2.60 (m, 2H), 2.09–2.19 (m, 2H),
1.68–1.72 (m, 2H). Anal. (C21H23BrN2O2ÆC2H2O4) C,
H, N.
8.8. 4-(4-Bromophenyl)-1-((5-methoxy-1H-indol-3-yl)meth-
yl)piperidin-4-ol oxalate (9)
8.3. 1-((1H-Indol-3-yl)methyl)-4-(4-chlorophenyl)piperidin-
4-ol oxalate (2)
Yield 72% from 5-methoxygramine and 4-(4-bromophe-
nyl)-4-hydroxypiperidine. Conversion to the oxalate salt
gave 9 as a white powder, mp 158–159 °C (dec); 1H
NMR (free base, CDCl3) d 8.12 (br s, 1H), 7.17–7.47
(m, 7H), 6.87 (d, J = 8.3 Hz, 1H), 3.87 (s, 3H), 3.79 (s,
2H), 2.90–2.94 (m, 2H), 2.49–2.65 (m, 2H), 2.11–2.18
(m, 2H), 1.65–1.68 (m, 2H). Anal. (C21H23BrN2O2Æ-
C2H2O4) C, H, N.
Yield 95% from gramine and 4-(4-chlorophenyl)-4-
hydroxypiperidine. Conversion to the oxalate salt gave
2 as a white powder, mp 164–165 °C (dec); 1H NMR (free
base, CDCl3) d 8.20 (br s, 1H), 7.77 (d, J = 7.8 Hz, 1H),
7.11–7.44 (m, 8H), 3.81 (s, 2H), 2.88–2.92 (m, 2H),
2.46–2.55 (m, 2H), 2.07–2.17 (m, 2H), 1.78 (br s, 1H),
1.67–1.73 (m, 2H). Anal. (C20H21ClN2OÆC2H2O4Æ0.5-
H2O) C, H, N.
8.9. 8-((1H-Indol-3-yl)methyl)-3-methyl-1-phenyl-1,3,8-tri-
azaspiro[4.5]decan-4-one oxalate (13)
8.4. 4-(4-Chlorophenyl)-1-((4-methoxy-1H-indol-3-yl)meth-
yl)piperidin-4-ol oxalate (5)
Yield 95% from gramine and 3-methyl-1-phenyl-1,3,8-
triazaspiro[4.5]decan-4-one, 10. Conversion to the oxa-
late salt gave 13 as a white powder, mp 193–194 °C
Yield 66% from 4-methoxygramine and 4-(4-chlorophe-
nyl)-4-hydroxypiperidine. Conversion to the oxalate salt
gave 5 as a white powder, mp 127–129 °C (dec); 1H
NMR (free base, CDCl3) d 8.64 (br s, 1H), 6.96–7.42
(m, 7H), 6.51 (d, J = 7.8 Hz, 1H), 4.11 (s, 2H), 3.92 (s,
3H), 2.98–3.02 (m, 2H), 2.61–2.70 (m, 2H), 2.13–2.23
(m, 2H), 1.66–1.71 (m, 2H). Anal. (C21H23ClN2O2Æ-
C2H2O4Æ0.5H2O) C, H, N.
1
(dec); H NMR (free base, CDCl3) d 8.33 (br s, 1H),
7.85 (d, J = 7.5 Hz, 1H), 7.35–7.37 (m, 1H), 7.12–7.24
(m, 5H), 6.80–6.91 (m, 3H), 4.65 (s, 2H), 3.82 (s, 2H),
2.98 (s, 3H), 2.66–2.94 (m, 5H), 1.61–1.73 (m, 3H). Anal.
(C23H26N4OÆC2H2O4Æ0.25H2O) C, H, N.
8.10. 8-((4-Methoxy-1H-indol-3-yl)methyl)-3-methyl-1-phen-
yl-1,3,8-triazaspiro[4.5]decan-4-one oxalate (14)
8.5. 4-(4-Chlorophenyl)-1-((5-methoxy-1H-indol-3-yl)meth-
yl)piperidin-4-ol oxalate (6)
Yield 24% from 4-methoxygramine and 3-methyl-1-
phenyl-1,3,8-triazaspiro[4.5]decan-4-one, 10. Conver-
sion to the oxalate salt gave 14 as a tan powder, mp
154–155 °C (dec); H NMR (free base, CDCl3) d 9.00
(br s, 1H), 7.42 (s, 1H), 7.26–7.32 (m, 2H), 6.82–7.11
(m, 5H), 6.53 (d, J = 7.5 Hz, 1H), 4.68 (s, 2H), 4.65 (s,
2H), 3.97 (s, 3H), 3.77–3.81 (m, 2H), 3.45–3.48 (m,
2H), 3.06–3.17 (m, 2H), 3.01 (s, 3H), 1.71–1.76
(m, 2H). Anal. (C24H28N4O2ÆC2H2O4ÆH2O) C, H, N.
Yield 95% from 5-methoxygramine and 4-(4-chlorophe-
nyl)-4-hydroxypiperidine. Conversion to the oxalate salt
gave 6 as a white powder, mp 167–168 °C (dec); 1H
NMR (free base, CDCl3) d 8.23 (br s, 1H), 7.40–7.44
(m, 2H), 7.14–7.32 (m, 5H), 6.86 (dd, J = 8.5 and
2.4 Hz, 1H), 3.87 (s, 3H), 3.77 (s, 2H), 2.89–2.92 (m,
2H), 2.46–2.54 (m, 2H), 2.07–2.17 (m, 2H), 1.68–1.74
(m, 2H). Anal. (C21H23ClN2O2ÆC2H2O4) C, H, N.
1
8.11. 8-((5-Methoxy-1H-indol-3-yl)methyl)-3-methyl-1-phen-
yl-1,3,8-triazaspiro[4.5]decan-4-one oxalate (15)
8.6. 1-((1H-Indol-3-yl)methyl)-4-(4-bromophenyl)piperidin-
4-ol oxalate (7)
Yield 57% from 5-methoxygramine and 3-methyl-1-
phenyl-1,3,8-triazaspiro[4.5]decan-4-one, 10. Conver-
sion to the oxalate salt gave 15 as a white powder, mp
190–191 °C (dec); H NMR (free base, CDCl3) d 8.12
(br s, 1H), 7.22–7.31 (m, 5H), 6.80–6.92 (m, 4H), 4.66
(s, 2H), 3.90 (s, 3H), 3.79 (s, 2H), 2.99 (s, 3H), 2.71–
2.93 (m, 6H), 1.62–1.67 (m, 2H). Anal. (C24H28N4O2Æ-
C2H2O4) C, H, N.
Yield 66% from gramine and 4-(4-bromophenyl)-4-
hydroxypiperidine. Conversion to the oxalate salt gave
7 as a tan powder, mp 149–151 °C (dec); 1H NMR (free
base, CDCl3) d 8.19 (br s, 1H), 7.77 (d, J = 7.8 Hz, 1H),
7.36–7.46 (m, 4H), 7.12–7.26 (m, 4H), 3.81 (s, 2H), 2.89–
2.93 (m, 2H), 2.47–2.55 (m, 2H), 2.08–2.18 (m, 2H), 1.81
(br s, 1H), 1.68–1.72 (m, 2H). Anal. (C20H21BrN2OÆ-
C2H2O4Æ0.5H2O) C, H, N.
1
8.12. 1-(1-((1H-Indol-3-yl)methyl)piperidin-4-yl)-1H-benzo[d]-
imidazol-2(3H)-one oxalate (16)
8.7. 4-(4-Bromophenyl)-1-((4-methoxy-1H-indol-3-yl)meth-
yl)piperidin-4-ol oxalate (8)
Yield 97% from gramine and 4-(2-keto-1-benzimidazoli-
nyl)piperidine, 11. Conversion to the oxalate salt gave
16 as a white powder, mp 207–209 °C (dec); H NMR
(free base, CDCl3) d 9.74 (s, 1H), 8.28 (s, 1H), 7.79 (d,
J = 7.3 Hz, 1H), 7.38 (d, J = 8.0 Hz, 1H), 7.14–7.24
(m, 4H), 7.00–7.08 (m, 3H), 4.32–4.40 (m, 1H), 3.81 (s,
Yield 84% from 4-methoxygramine and 4-(4-bromophe-
nyl)-4-hydroxypiperidine. Conversion to the oxalate salt
gave 8 as a white powder, mp 135–136 °C (dec); 1H
NMR (free base, CDCl3) d 8.19 (br s, 1H), 7.43–7.47
(m, 2H), 7.35–7.39 (m, 2H), 6.96–7.12 (m, 3H), 6.51
1

S. Vangveravong et al. / Bioorg. Med. Chem. 14 (2006) 815–825
823
2H), 3.17 (d, J = 11.7 Hz, 2H), 2.43–2.54 (m, 2H), 2.19–
2.27 (m, 2H), 1.78–1.81 (m, 2H). Anal. (C21H22N4OÆ-
C2H2O4Æ0.25H2O) C, H, N.
7.27 (m, 5H), 6.87 (dd, J = 8.8 and 2.4 Hz, 1H), 3.87 (s,
3H), 3.77 (s, 2H), 2.87–2.93 (m, 2H), 2.48–2.57 (m, 2H),
2.47 (s, 3H), 2.08–2.19 (m, 2H), 1.71–1.75 (m, 2H). Anal.
(C22H26N2O2SÆC2H2O4Æ0.5H2O) C, H, N.
8.13. 1-(1-(1H-Indol-3-yl)methyl)-4-phenylpiperidine oxalate
(17)
9. Radioligand binding and functional assays
9.1. Dopamine receptor binding assay
Yield 89% from gramine and 4-phenylpiperidine, 12.
Conversion to the oxalate salt gave 17 as a white pow-
der, mp 147–149 °C (dec); H NMR (free base, CDCl3)
1
d 8.18 (br s, 1H), 7.77 (d, J = 7.9 Hz, 1H), 7.12–7.38 (m,
9H), 3.79 (s, 2H), 3.11–3.14 (m, 2H), 2.42–2.52 (m, 1H),
2.11–2.20 (m, 2H), 1.77–1.83 (m, 4H). Anal. (C20H22N2
C2H2O4Æ0.25H2O) C, H, N.
The method for the iodination of 125I-IABN using per-
acetic acid has been previously described.27 For radioli-
gand binding studies, membrane homogenates from
stably transfected HEK 293 cells expressing either the
human D2, D3, and D4 receptors were prepared using
a polytron tissue homogenizer (Brinkman Instruments,
Westbury, NY). The tissue was suspended in 50 mM
Tris–HCl, 150 mM NaCl, and 1 mM EDTA at pH 7.5
to approximately 5–20 lg of protein per 50 lL prior to
the assay. Assays were performed in a total volume of
150 lL. Binding reactions were carried out for 60 min
at 37 °C and the reaction was terminated by rapid filtra-
tion over glass fiber filters (Schleicher and Schuell, No.
32 filters). After washing filters with buffer, the radioac-
tivity of the 125I-labeled ligand was quantitated using a
Packard Cobra gamma counter with an efficiency of
75%. Protein concentrations were determined using a
BCA reagent (Pierce) with bovine serum albumin as
the protein standard.
8.14. 1-((1H-Indol-3-yl)methyl)-4-(4-(methylthio)phenyl)pip-
eridin-4-ol oxalate (20)
A solution of 4-piperidone monohydrate hydrochloride
(970 mg, 6.3 mmol) in 10% aqueous sodium carbonate
(1 mL) was added to a solution of gramine (1.0 g,
5.7 mmol) in 1-butanol (20 mL). The reaction mixture
was stirred at 100 °C overnight and then concentrated.
The crude residue was purified by column chromatogra-
phy on silica gel (dichloromethane–methanol–NH4OH,
90:10:0.5) to give 1-((1H-indol-3-yl)methyl)piperidin-4-
1
one, 18a (350 mg, 27%) as a white powder. H NMR
(CDCl3) d 8.14 (br s, 1H), 7.78 (d, J = 7.8 Hz, 1H),
7.38 (d, J = 7.8 Hz, 1H), 7.12–7.26 (m, 3H), 3.84 (s,
2H), 2.81 (t, J = 6.3 Hz, 4H), 2.45 (t, J = 6.3 Hz, 4H).
For competition curves using a transfected cell line
expressing D2, D3 or D4 dopamine receptors, experi-
ments were performed in triplicate with two concentra-
tions of inhibitor per decade over at least five orders
of magnitude. The concentration of the radioligand
was approximately equal to the Kd values. Controls con-
taining either no inhibitor or 2 lM (+)-butaclamol were
used to define total binding and non-specific binding,
respectively. For competition curves using dissected rat
brain tissue and the D1 receptor selective radioligand
3H-SCH 23390, two independent experiments were per-
formed in triplicate with two concentrations of inhibitor
per decade. Controls containing either no inhibitor or
10 lM (+)-butaclamol were used to define total binding
and non-specific binding, respectively. Since low levels
of D1 receptor binding were observed for the indoles,
a competition curve using non-radioactive SCH 23390
was performed simultaneously as a positive control for
the validity of the assay. Competition data for D2-like
dopamine receptors were modeled for a single-site fit
using the TableCurve program (Jandel) and the IC50
values for the competitive inhibitors were converted to
A solution of 4-bromothioanisole (685 mg, 3.4 mmol) in
dry THF (4 mL) was added to a solution of n-butyllithi-
um (3.2 mL, 5.06 mmol, 1.6 M in hexane) in dry THF
(10 mL) at ꢀ70 °C. After stirring at ꢀ70 °C for 1 h, a
solution of 18a (350 mg, 1.53 mmol) in dry THF
(5 mL) was added. The reaction mixture was stirred at
ꢀ70 °C for 4 h and then concentrated. Water (10 mL)
was added and then extracted with dichloromethane.
Purification by column chromatography on silica gel
(dichloromethane–methanol–NH4OH, 90:10:0.5) gave
20 (319 mg, 59%). Conversion to the oxalate salt gave
the compound as a tan powder, mp 118–119 °C (dec);
1H NMR (free base, CDCl3) d 8.23 (br s, 1H), 7.76 (d,
J = 7.8 Hz, 1H), 7.36–7.43 (m, 3H), 7.12–7.26 (m, 5H),
3.82 (s, 2H), 2.89–2.93 (m, 2H), 2.50–2.58 (m, 2H),
2.46 (s, 3H), 2.10–2.20 (m, 2H), 1.70–1.74 (m, 2H). Anal.
(C21H24N2OSÆC2H2O4Æ0.5H2O) C, H, N.
8.15. 1-((5-Methoxy-1H-indol-3-yl)methyl)-4-(4-(methyl-
thio)phenyl)piperidin-4-ol oxalate (21)
The reaction was carried out according to the procedure
for 20 using 5-methoxygramine (1.6 g, 7.8 mmol) to give
Ki values using the Cheng and Prusoff corrections.37
A
similar analysis was performed for SCH 23390 binding
to D1 receptors expressed in rat caudate, but the low le-
vel of binding of the test compounds to rat striatal D1
receptors precluded obtaining accurate IC50 values.
1
18c as a pale brown powder (630 mg, 31%). H NMR
(CDCl3) d 8.05 (br s, 1H), 7.27 (d, J = 8.8 Hz, 1H),
7.23 (d, J = 2.4 Hz, 1H), 7.13 (d, J = 2.4 Hz, 1H), 6.88
(dd, J = 8.8 and 2.4 Hz, 1H), 3.87 (s, 3H), 3.80 (s, 2H),
2.81 (t, J = 6.1 Hz, 4H), 2.45 (t, J = 6.1 Hz, 4H). Treat-
ment with 19 gave 21 in modest yield (530 mg, 57%).
Conversion to the oxalate salt gave the compound as a
9.2. Sigma receptor binding assays
The r1 receptor binding assay was conducted using
guinea pig brain membrane homogenates (100 lg pro-
tein). Membrane homogenates were incubated with
1
tan powder, mp 128–129 °C (dec). H NMR (free base,
CDCl3) d 8.12 (br s, 1H), 7.41 (d, J = 8.5 Hz, 2H), 7.15–

824
S. Vangveravong et al. / Bioorg. Med. Chem. 14 (2006) 815–825
3 nM [3H](+)-pentazocine (31.6 Ci/mmol) in 50 mM
Tris–HCl (pH 8.0) at 25 °C for either 120 or 240 min.
Test compounds were dissolved in ethanol and then
diluted in buffer for a total incubation volume of
0.5 mL. Test compounds were added in concentrations
ranging from 0.005 to 1000 nM. Assays were terminated
by the addition of ice-cold 10 mM Tris–HCl (pH 8.0)
followed by rapid filtration through Whatman GF/B
glass fiber filters (presoaked in 0.5% polyethylenimine)
using a Brandel cell harvester (Gaithersburg, MD). Fil-
ters were washed twice with 5 mL ice-cold buffer. Non-
specific binding was determined in the presence of
10 lM (+)-pentazocine. Liquid scintillation counting
was carried out in EcoLite(+) (ICN Radiochemicals;
Costa Mesa, CA) using a Beckman LS 6000IC spec-
trometer with a counting efficiency of 50%.
column recovery by monitoring the recovery of the cyc-
lic AMP using spectrophotometric analysis at OD
259 nm.27,39 For concentration-dependent experiments,
competition data were modeled for a single-site fit using
the TableCurve program (Jandel) and the IC50 values
for the competitive inhibitors.
Acknowledgments
We would like to acknowledge the assistance of Ms.
Suzy A. Griffin in the preparation of the stably transfec-
ted human D2 and D3 HEK cell lines and to Dr. Jona-
than Javitch for providing us with those receptor
genes. This research was funded by Grants DA 12647,
DA13584-S1, and DA 16181 awarded by the National
Institute on Drug Abuse, and NS48056 awarded by
the National Institute of Neurological Disorders and
Stroke.
The r2 receptor binding assay was conducted using rat
liver membrane homogenates (35 lg of protein). Mem-
brane homogenates were incubated with 3 nM
[3H]DTG (38.3 Ci/mmol) in the presence of 100 nM
(+)-pentazocine to block r1 sites. Incubations were car-
ried out in 50 mM Tris–HCl (pH 8.0) for 120 min at
25 °C in a total incubation volume of 0.5 mL. Test com-
pounds were added in concentrations ranging from
0.005 to 1000 nM. Assays were terminated by the addi-
tion of ice-cold 10 mM Tris–HCl (pH 8.0) followed by
rapid filtration through Whatman GF/B glass fiber fil-
ters (presoaked in 0.5% polyethylenimine) using a Bran-
del cell harvester (Gaithersburg, MD). Filters were
washed twice with 5 mL ice-cold buffer. Nonspecific
binding was determined in the presence of 5 lM DTG.
Liquid scintillation counting was carried out in Eco-
Lite(+) (ICN Radiochemicals; Costa Mesa, CA) using
a Beckman LS 6000IC spectrometer with a counting effi-
ciency of 50%.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2005.09.008.
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