Synthesis and characterization of selective dopamine D2 receptor antagonists. 2. Azaindole, benzofuran, and benzothiophene analogs of L-741,626

Article abstract of DOI:10.1016/j.bmc.2010.05.052

A series of indole, 7-azaindole, benzofuran, and benzothiophene compounds have been prepared and evaluated for affinity at D_2-like dopamine receptors. These compounds share structural elements with the classical D _2-like dopamine rec

Full text of DOI:10.1016/j.bmc.2010.05.052

Bioorganic & Medicinal Chemistry 18 (2010) 5291–5300
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and characterization of selective dopamine D2 receptor antagonists.
2. Azaindole, benzofuran, and benzothiophene analogs of L-741,626
Suwanna Vangveravong a, Michelle Taylor b, Jinbin Xu a, Jinquan Cui a, Wesley Calvin b, Sonja Babic b,
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:
A series of indole, 7-azaindole, benzofuran, and benzothiophene compounds have been prepared and
evaluated for affinity at D2-like dopamine receptors. These compounds share structural elements with
the classical D2-like dopamine receptor antagonists haloperidol, N-methylspiperone and benperidol.
Two new compounds, 4-(4-iodophenyl)-1-((4-methoxy-1H-indol-3-yl)methyl)piperidin-4-ol (6) and
4-(4-iodophenyl)-1-((5-methoxy-1H-indol-3-yl)methyl)piperidin-4-ol (7), were found to have high
affinity to and selectivity for D2 versus D3 receptors. Changing the aromatic ring system from an indole
to other heteroaromatic ring systems reduced the D2 binding affinity and the D2 versus D3 selectivity.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 10 March 2010
Revised 15 May 2010
Accepted 18 May 2010
Available online 24 May 2010
Keywords:
Dopamine D2 receptor
Indoles
Benzofurans
Benzothiophenes
1. Introduction
stimulation of D1-like receptors results in an activation of adenylyl
cyclase with increased production of cAMP.12 Stimulation of the
The neurotransmitter dopamine is synthesized by mesence-
phalic neurons of the substantia nigra and ventral tegmental area
and by hypothalamic neurons of the arcuate and periventricular
nuclei. Dopamine is present in the central nervous system and
peripheral nervous system.1 Dopamine is associated with fine
movement coordination, emotion, affect, cognition and memory.2
Regulation of dopamine plays a crucial role in mental and physical
health. Abnormal activity of the dopamine system has been impli-
cated in psychological and neurological disorders including Parkin-
son’s disease, schizophrenia, mood disorders, addiction, attention
deficit hyperactivity disorder, and Tourette syndrome.3–10
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
thought that there are five functionally active dopamine receptor
subtypes expressed in mammals. These five receptor subtypes
D2-like receptors results in an inhibition of adenylyl cyclase activity,
a decrease in intracellular levels of cAMP, an increase in the release
of arachidonic acid and an increase in phosphatidylinositol
hydrolysis.10
The D2 and D3 dopamine receptors have approximately 46%
amino acid homology. In contrast, the transmembrane spanning
(TMS) regions of the D2 and D3 receptors, which are thought to
construct the ligand binding site, share 78% homology.13 Despite
their structural similarities, D2 and D3 receptors differ in their (a)
neuroanatomical localization,14 (b) levels of receptor expres-
sion,15,16 (c) efficacy in response to agonist stimulation,10 and (d)
regulation and desensitization.17 Because of the high degree of
homology between D2 and D3 receptor binding sites, the pharma-
cologic properties of these two receptor subtypes are similar, and
it has been difficult to obtain compounds that can bind selectively
to either the D2 or the D3 dopamine receptor subtype.10,15,16,18–20
Despite this difficulty, D2 and D3 dopamine receptor selective ago-
nists and antagonists would be useful pharmacologic tools to pre-
cisely define the role of the two D2-like receptor subtypes in a
variety of experimental physiological and behavioral situations,
including the reinforcing and toxic properties of cocaine,21,22
socialization, memory, and the regulation of interneuronal activity
in the basal ganglia.23
have been classified into two major subtypes (D1-like and D2-like
)
based on their pharmacological, biochemical, and physiological
characteristics. The D1-like receptor subtypes consist of the D1
(D1a) and the D5 (D1b) dopamine receptors. The D2-like receptor sub-
types are known to include several receptor subtypes and isoforms,
including D2short (D2S), D2long (D2L), D3, and D4 receptors.11 Agonist
Previously, we reported the in vitro binding properties of a ser-
ies of indole derivatives based on haloperidol, N-methylspiperone,
and benperidol (Fig. 1). Several novel analogues of L-741,626 (1a),
* 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 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.052

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S. Vangveravong et al. / Bioorg. Med. Chem. 18 (2010) 5291–5300
O
CH3
O
O
N
OH
N
N
N
F
Cl
F
Haloperidol
N-methylspiperone
O
N
OH
O
NH
N
R
N
H
X
F
1a: R = H, X = Cl
Benperidol
1b: R = 4-OCH3, X = Cl
1c: R = 5-OCH3, X = Cl
1d: R = H, X = Br
1e: R = 4-OCH3, X = Br
1f: R = 5-OCH3, X = Br
Figure 1. Structures of the lead compounds for the development of D2-selective radiotracers.
a D2-selective antagonist,24 were made and shown to have selec-
tivity for D2 over D3 and D4 receptors25,26 (Fig. 1). The current study
involved the following strategies: (a) continuation of the previous
study by making additional structural congeners of 1a; (b) replace-
ment of the indole ring with other heteroaromatic ring systems
such as 7-azaindole, benzofuran, and benzothiophene; and (c)
the development of fluorine-containing analogs which could serve
as potential radiotracers for imaging dopamine D2 versus D3 recep-
tors with positron emission tomography (PET).
2. Chemistry
Compounds 5–15 were synthesized by treating the respective
gramine derivatives 2a–f with the appropriate pyridine deriva-
tives 3a–d and 4 in refluxing toluene (Method A, Scheme 1).
Gramine and 5-methoxygramine are commercially available. The
gramines 2d and 2e were obtained by alkylation of 4-hydrox-
yindole or 5-hydroxyindole with 1-bromo-2-fluoroethane, fol-
lowed by reacting with N,N-dimethylmethyleneammonium iodide
OH
OH
N
HN
R
N
H
X
X
3a: X = H
3b: X = I
5: R = H, X = I
6: R = 4-OCH , X = I
3
3c: X = Br
7: R = 5-OCH , X = I
3
8: R = 5-OCH , X = H
3
9: R = 4-OCH CH F, X = Br
2
2
10: R = 5-OCH CH F, X = Br
2
2
O
N
O
NH
HN
NH
N
N
CH3
CH3
R
N
toluene
reflux
4
N
H
R
N
H
Y
R:
4-OCH
5-OCH
3
3
2
2a: Y = C, R = H
2b: Y = C, R = 4-OCH
11
12
3
2c: Y = C, R = 5-OCH
3
2d: Y = C, R = 4-OCH CH F
2
2
2e: Y = C, R = 5-OCH CH F
2f: Y = N, R = H
2
2
OH
N
OH
HN
N
N
H
X
X
X:
Br
I
SCH
3b, X = I
3
3c, X = Br
3d, X = SCH
13
14
15
3
Scheme 1. Method A.

S. Vangveravong et al. / Bioorg. Med. Chem. 18 (2010) 5291–5300
5293
(Eschenmoser’s salt) (Scheme 2). The 4-methoxygramine, 2b, and
7-azagramine, 2f, were synthesized by treating 4-methoxyindole
and 7-azagramine with N,N-dimethylmethyleneammonium iodide,
respectively.
Compounds 18–27 were synthesized by reacting the respective
bromomethylbenzofurans (17a–b) or bromomethylbenzo[b]thio-
phenes (17c–d) with the appropriate piperidines in the presence
of potassium carbonate and potassium iodide in acetonitrile
CH3
CH2=N(CH3)2 I
F
N
K2CO3, acetone
reflux
HBr
+
R
HO
CH3
R
N
CH3COOH
CH3CN
N
H
N
H
H
2d: R = 4-OCH2CH2F
2e: R = 5-OCH2CH2F
4-Hydroxyindole or
5-Hydroxyindole
16a: R = 4-OCH2CH2F
16b: R = 5-OCH2CH2F
Scheme 2.
OH
N
Y
X
X
OH
Br
I
HN
_________________
Y
O
S
18
20
19
X
3b: X = I
3c: X = Br
OH
N
O
X
X
Br
I
_________________
21
22
O
O
NH
HN
N
O
NH
N
N
Br
N
CH3
R
K2CO3, KI
O
4
Y
CH3CN
CCl4
90-95 ºC
Y
Y
Y
S
________O_________
90-95 ºC
23
24
Y = O or S
17a: Y = O, R = 2-CH Br
2
17b: Y = O, R = 3-CH Br
2
17c: Y = S, R = 2-CH Br
2
17d: Y = S, R = 3-CH Br
2
O
O
X
X
N
N
N
HN
N
N
Y
28a: X = CH
28b: X = CH CH F
3
__X
______3_________
Y
CH
CH CH F
2 2
2
2
O
25
26
S
27
Scheme 3. Method B.
F
O
O
O
1.
Br
NaH, dry THF
F
(
BOC)2O
NH
NH
N
BOC
N
HN
HN
N
N
N
Et3N, CH2Cl2
2. TFA
28b
30
31
Scheme 4.

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S. Vangveravong et al. / Bioorg. Med. Chem. 18 (2010) 5291–5300
O
O
O
Br
Br
1.
N
N
NH
HN
N
BOC
N
O
NaH, dry THF
N
N
N
K2CO3, KI
CH3CN, heat
O
2. TFA
33
32
31
O
F
F
N3
N
N
N
N
N
N
CuSO4 . 5H2O
Sodium ascorbate
DMF
O
29
Scheme 5.
(Method B, Scheme 3). The required bromomethyl derivatives were
made by refluxing benzofurans or benzo[b]thiophenes with N-bro-
mosuccinimide in carbon tetrachloride. The piperidine 28b was
prepared from 1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (30).
First, the N-protected derivative 31 was synthesized and alkylated
with 1-bromo-2-fluoroethane, followed by deprotection with tri-
fluoroacetic acid to give the required piperidine 28b (Scheme 4).
Compound 29 was synthesized via alkylation of 31 with propargyl
bromide, followed by deprotection with trifluoroacetic acid to give
32. N-alkylation of 32 with freshly prepared 3-bro-
momethylbenzofuran 17b, followed by reaction with 1-azido-2-
fluoroethane in the presence of sodium ascorbate and copper sul-
fate gave the triazole analog, 29 (Scheme 5).
3. Radioligand binding studies at dopamine receptors
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 homogenates from stably transfected
Table 1
Binding affinities for dopamine D2/D3 and sigma r1
/
r2 receptors
Compound
Ki (nM)a
Log Ph
b
c
d
e
f
g
D2
D3
D4
D3/D2
r1
r2
5
6
7
8
9
10
11
12
13
14
15
18
19
20
21
22
3.8 0.8
94.4 1.2
122 2.0
100 13
53.4 15.2
60.4 18.7
129 5.1
>10,000
25
2.00 0.52
881 154
197 26
1527 158
3224 479
227 63
4129 382
1768 303
15.1 2.3
1.20 0.51
8.07 2.00
2.45 0.78
1.41 0.41
2.13 0.44
0.59 0.15
0.51 0.13
1779 256
1879 200
708 141
2200 437
970 178
1.98
2.60
2.54
1.43
2.61
2.54
2.54
2.45
2.20
2.46
1.85
3.19
3.45
4.97
3.12
3.38
3.39
4.73
2.02
2.38
3.36
1.46
4.50
1.54
2.10
2.16
1.72
2.34
2.28
0.9 0.1
0.9 0.1
1396 317
27.7 6.6
13.8 2.5
23.3 3.1
12.5 2.0
212 25
61.0 4.9
254 40
17.3 0.6
14.3 1.7
46.8 3.0
6.6 1.2
2.4 0.7
13.6 3.6
37.8 6.0
7.1 1.3
8.0 1.9
71.4 14
2.0 0.1
1.1 0.1
10.0 2.5
4.2 0.4
4.2 0.8
4.8 0.3
2.3 0.7
2.5 0.7
136
112
2.1
31
3262 387
2761 574
9510 1020
10,420 1512
16,134 2968
27,230 5205
1.90 0.15
5.53 2.37
7.75 1.24
1398 98
2927 198
849 164
188 7.1
132 32
68.2 16
2983 305
1585 380
4938 780
31.8 4.1
49.0 5.6
142 27
10.9 0.5
11.2 1.2
3.0 0.7
46.7 4.8
2.1 0.5
668 22
344 49
1390 108
2230 484
620 72
634 85
937 145
254 5.3
245 11
377 47
94.5 6.4
40.4 10.0
163 11.9
72.6 0.8
492 103
945 162
352 72
978 11.9
NDi
14
5.7
5.5
14
26
20
1.8
3.4
3.0
1.7
4.7
4.5
1.2
0.3
1.2
0.8
0.5
12
2368 52
1964 177
377 20
393 54
23
24
25
26
27
29
18,054 3531
14,575 1168
24,879 4201
10,164 641
20,248 7658
22,028 3297
24.2 3.0
1811 569
1246 234
1900 241
2134 356
943 64
9.3 2.2
60.1 17
0.9 0.2
12.7 3.9
104 19
28
8
331 87
802 227
1.45 0.33
19.3 3.6
>5000
242 68
12.1 2.5
2557 334
108 17
Haloperidol
1aj
1bj
1cj
1dj
1ej
1fj
449 123
>2000
>3500
10
59
49
28
82
39
250
6
204 60
110 39
190 34
96.9 6.1
652
7
840 197
700 80
2702 696
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 293 cells using [125I]ABN as the radioligand.
Ki values for D3 receptors were measured on human D3 expressed in HEK 293 cells using [125I]ABN as the radioligand.
Ki values for D4 receptors were measured on human D4 expressed in HEK 293 cells using [125I]ABN as the radioligand.
Ki for D3 receptors/Ki for D2 receptors.
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.
Not determined.
Data from Ref. 25.
g
h
i
j

S. Vangveravong et al. / Bioorg. Med. Chem. 18 (2010) 5291–5300
5295
HEK 293 cells were used in conjunction with the radioligand 125I-
100
80
60
40
20
0
IABN. We have previously reported that the benzamide 125I-IABN
binds with high affinity and selectively to D2-like dopamine recep-
tors, but that it binds non-selectively to D2 versus D3 dopamine
receptor subtypes.27
First, a comparison of indole analogs structurally related to
haloperidol was made in which a substituent was added at the
4- or 5-position of the indolyl moiety. We found that the addition
of the methoxy group resulted in a fourfold increase in affinity at
D2 receptors but did not change the affinity at D3 receptors, result-
ing in compounds that were 110- to 140-fold selective at D2 recep-
tors (7 and 6). The addition of 2-fluoroethoxy group resulted in a 3-
to 6-fold decrease in affinity at D2 receptors and a 2- to 8-fold de-
crease in affinity at D3 receptors; thus compounds 10 and 9 re-
sulted in no significant difference in selectivity compared to the
unsubstituted analog (1d25). The halide substituents on the phe-
nyl-piperidinol moiety are pivotal for high affinity binding at the
dopamine D2 receptors and for selectivity over D3 receptors. The
iodide analogs (6 and 7) have higher affinity and selectivity at D2
versus D3 dopamine receptors than the chloride and bromide ana-
logs (1b, 1c, 1e and 1f25). In addition, the affinity at both D2 and D3
dopamine receptors decreases dramatically in the unsubstituted
analog, 8. The 7-azaindole analogs (13–15) bind with 14- to 26-fold
selectivity for D2 over D3 receptors. The benzofuran (18, 19, 21, 22,
23, 25, 26 and 29) and benzothiophene (20, 24, and 27) analogs
bind non-selectively to the D2 over D3 dopamine receptors. Similar
results for benzofuran analogs of L-741,626 were previously re-
ported by Grundt et al.26
Figure 2. Evaluation of the intrinsic efficacy of compounds
6 and 7 using a
Forskolin-dependent adenylyl cyclase inhibition assay. The intrinsic efficacy of
compounds 6 and 7 was evaluated by determining the percent inhibition of a
forskolin-dependent adenylyl cyclase assay with human D2long receptors expressed
in stably transfected HEK 293 cells. The extent of inhibition of the two test
compounds was compared to the percent inhibition obtained using the full agonist
quinpirole (quin) at a concentration of 10 nM. At this concentration of quinpirole
the mean S.E.M. inhibition of forskolin (fsk) stimulation was 71 3.6 percent. The
test drugs were tested at a final concentration equal to 10 nM, either in the absence
or presence of quinpirole. The bar graph represents the mean the S.E.M. values
obtained for n = 4.
4. Adenylyl cyclase studies with D2-like receptors
6. Discussion
The intrinsic efficacy of the two compounds exhibiting the
greatest D2 versus D3 receptor subtype binding selectivity (6 and
7) was evaluated using a forskolin-dependent adenylyl cyclase
inhibition assay (Fig. 2). The D2-like receptor full agonist quinpirole
was used as a reference compound. At a dose of 10 nM, quinpirole
administration resulted in >70% inhibition of cAMP accumulation,
whereas at the same dose of compounds 6 and 7 no appreciable in-
crease or decrease in forskolin-dependent adenylyl cyclase activa-
tion was observed. In addition, both 6 and 7 were able to attenuate
the effect of quinpirole. Based upon these results, we categorize
compounds 6 and 7 as neutral antagonists at D2 dopamine recep-
tors. These results are consistent with our previous studies on this
class of methoxy substituted indole piperidines.25
The current study is a continuation of our effort to develop
ligands having a high affinity and selectivity for D2 versus D3 dopa-
mine receptors. In previous studies, we prepared a number of
structural analogs of the classical D2-like dopamine receptor antag-
onist, haloperidol, having a moderate to high affinity at D2 versus
D3 receptors.25 We have expanded this initial structure–activity
relationship (SAR) study and have identified two iodine-containing
derivatives of the methoxy substituted indoles (6 and 7), which (a)
bind at D2 receptors with nanomolar affinity and (b) have >100-
fold selectivity for human D2 receptors compared to the human
D3 dopamine receptor subtype. These two analogs were also found
to bind with low affinity at the r1 and r2 receptors. The binding
profiles of 6 and 7 indicate that they are the most potent and selec-
tive D2-antagonists reported to date. 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 tech-
nique, PET. The iodo substituent on the phenyl-piperidinol moiety
was found to be pivotal for high affinity binding at the dopamine
D2 receptors and for selectivity over D3 receptors. The substitution
at the 4- or 5-position on the indolyl moiety also affects the affinity
binding and selectivity. The 2-fluoroethoxy substituted indoles
(9 and 10) have lower affinity and selectivity for D2 over D3
compared to the methoxy substituted indoles (1e and 1f25). How-
ever, the minor reduction in affinity in going from the methoxy
group to the 2-fluoroethoxy group (compare 10 to 1f) suggests that
this may be a useful strategy for preparing a fluorine-18 radio-
tracer for imaging D2 versus D3 receptors.
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 (
r
1) and sigma-2 (r2
)
receptors. 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 the presence of 100 nM unlabeled (+)-pentazocine to
mask r1 sites, or with the r2 selective ligand [3H]RHM-1,
alone.28,29 Although haloperidol is a dopaminergic antagonist that
is used clinically as a neuroleptic, its high affinity at sigma recep-
tors has precluded its usefulness as a radioligand for in vitro or
in vivo radioligand binding studies. Almost all of the compounds
in this study bound with low affinity (>350 nM) at r2 receptors
with the exception of compound 13, which has a nanomolar
(nM) affinity for
sus 1 receptors. The affinity at
>4000 nM. The 7-azaindole analogs have high affinity at both r1
and 2 receptors, whereas the benzofuran analogs 21 and 22 have
a high affinity and selectivity for r1 versus r2 receptors.
r
2 receptors and a sevenfold selectivity for
r2 ver-
We have also examined heteroatom replacements in the indole
ring. The 7-azaindole analogs have lower affinity at both D2 and D3
dopamine receptors than the corresponding indole analogs. The
benzofuran and benzothiophene analogs bind non-selectively to
both D2 and D3 dopamine receptors. The interesting SAR at r1
r
r
1 receptors varied from 0.5 nM to
r

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S. Vangveravong et al. / Bioorg. Med. Chem. 18 (2010) 5291–5300
and
r
2 receptors of 13–15 and 18–22 may provide useful informa-
salt gave 6 as an off-white powder, mp 129–130 °C (dec); 1H
NMR (free base, CDCl3 + CD3OD) d 7.63 (d, J = 8.5 Hz, 2H), 7.24 (d,
J = 8.5 Hz, 2H), 6.96–7.08 (m, 3H), 6.49 (d, J = 7.5 Hz, 1H), 4.04 (s,
2H), 3.91 (s, 3H), 2.90–2.94 (m, 2H), 2.59–2.65 (m, 2H), 2.06–2.16
(m, 2H), 1.65–1.69 (m, 2H). Anal. (C21H23IN2O2ÁC2H2O4Á0.25H2O)
C, H, N.
tion for molecular modeling studies of sigma receptors.30,31
7. Experimental
7.1. Chemical analysis
7.6. 4-(4-Iodophenyl)-1-((5-methoxy-1H-indol-3-yl)methyl)-
piperidin-4-ol oxalate (7)
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. Melting
points were determined on an electrothermal melting point appa-
ratus and are uncorrected. Elemental analyses were performed by
Atlantic Microlab, Inc., Norcross, GA and were within 0.4% of the
calculated values. Mass spectrometry was provided by the Wash-
ington University Mass Spectrometry Resource, an NIH Research
Resource (Grant No. P41RR0954). All reactions were carried out
under an inert atmosphere of nitrogen. Lipophilicity measure-
ments of the compounds were estimated using the computational
program, C log P (Advanced Chemistry Development, Inc., Toronto,
Canada).
Method A. Yield 45% from 5-methoxygramine (2c) and 4-(4-
iodophenyl)-4-hydroxypiperidine (3b). Conversion to the oxalate
salt gave 7 as a tan powder, mp 121–122 °C (dec); 1H NMR (free
base, CDCl3) d 8.30 (br s, 1H), 7.65 (d, J = 8.1 Hz, 2H), 7.27 (d,
J = 8.1 Hz, 2H), 7.22 (d, J = 8.3 Hz, 2H), 7.15 (d, J = 2.3 Hz, 2H),
6.88 (dd, J = 9.0 and 2.3 Hz, 1H), 3.89 (s, 2H), 3.87 (s, 3H), 2.99–
3.03 (m, 2H), 2.63–2.71 (m, 2H), 2.13–2.29 (m, 2H), 1.69–1.73
(m, 2H). Anal. (C21H23IN2O2ÁC2H2O4Á1.25H2O) C, H, N.
7.7. 1-((5-Methoxy-1H-indol-3-yl)methyl)-4-phenylpiperidin-4-
ol oxalate (8)
Method A. Yield 93% from 5-methoxygramine (2c) and 4-hydro-
xy-4phenylpiperidine (3a). Conversion to the oxalate salt gave 8 as
an off-white powder, mp 147–148 °C (dec). 1H NMR (free base,
CDCl3) d 8.17 (br s, 1H), 7.48–7.52 (m, 2H), 7.32–7.37 (m, 2H),
7.26 (d, J = 8.8 Hz, 2H), 7.22 (d, J = 2.4 Hz, 1H), 7.17 (d, J = 2.2 Hz,
1H), 6.87 (dd, J = 8.8 and 2.4 Hz, 1H), 3.88 (s, 3H), 3.79 (s, 2H),
2.90–2.94 (m, 2H), 2.51–2.60 (m, 2H), 2.14–2.24 (m, 2H), 1.73–
1.78 (m, 2H), 1.66 (br s, 1H). Anal. (C21H24N2O2ÁC2H2O4Á0.25H2O)
C, H, N.
7.2. General procedure: Method A
A mixture of gramine derivatives (4.0 mmol) and appropriate
amines (4.8 mmol) in toluene (15 mL) was stirred at reflux over-
night. The volatile components were evaporated and the resulting
residue was purified by silica gel column chromatography (dichlo-
romethane–methanol–ammonium hydroxide, 90:10:0.5) to afford
the target compounds. The oxalate salt was prepared using one
equivalent of oxalic acid in ethyl acetate.
7.8. 4-(4-Bromophenyl)-1-((4-(2-fluoroethoxy)-1H-indol-3-yl)-
methyl)piperidin-4-ol oxalate (9)
7.3. General procedure : Method B
Method A. Yield 43% from 4-(2-fluoroethoxy)gramine (2d) and
4-(4-bromophenyl)-4-hydroxypiperidine (3c). Conversion to the
oxalate salt gave 9 as an off-white powder, mp 179–180 °C (dec);
1H NMR (free base, CDCl3 + CD3OD) d 7.44 (d, J = 8.8 Hz, 2H), 7.37
(d, J = 8.8 Hz, 2H), 7.17 (s, 1H), 7.01–7.10 (m, 2H), 6.46–6.49 (m,
1H), 4.93–4.96 (m, 1H), 4.77–4.80 (m, 1H), 4.38–4.41 (m, 1H),
4.29–4.31 (m, 1H), 4.12(s, 2H), 2.96–3.00 (m, 2H), 2.69–2.76 (m,
2H), 2.11–2.21 (m, 2H), 1.68–1.72 (m, 2H). Anal. (C22H24BrFN2O2Á-
C2H2O4Á0.5H2O) C, H, N.
A mixture of methylbenzofurans, or methylbenzo[b]thiophenes
(4.0 mmol) and N-bromosuccinimide (NBS, 4.4 mmol) in carbon
tetrachloride was heated at reflux overnight. After cooling to room
temperature, the reaction mixture was filtered and evaporated in
vacuo to give the corresponding bromomethyl analogs. These
crude bromomethyl analogs were treated with the appropriate
amine (4.0 mmol) in the presence of K2CO3 and KI in acetonitrile
at reflux overnight. After cooling to room temperature, the reaction
mixture was filtered and evaporated in vacuo. Column chromatog-
raphy (5% methanol in dichloromethane) of the resulting residue
gave the desired compounds. The oxalate salt was prepared using
one equivalent of oxalic acid in ethyl acetate.
7.9. 4-(4-Bromophenyl)-1-((5-(2-fluoroethoxy)-1H-indol-3-yl)-
methyl)piperidin-4-ol oxalate (10)
Method A. Yield 27% from 5-(2-fluoroethoxy)gramine (2e) and
4-(4-bromophenyl)-4-hydroxypiperidine (3c). Conversion to the
oxalate salt gave 10 as a tan powder, mp 163–164 °C (dec); 1H
NMR (free base, CDCl3) d 8.18 (br s, 1H), 7.45 (d, J = 8.8 Hz, 2H),
7.36 (d, J = 8.8 Hz, 2H), 7.17–7.29 (m, 3H), 6.91 (dd, J = 8.8 and
2.5 Hz, 1H), 4.79 (dt, J = 47.5 and 4.1 Hz, 2H), 4.28 (dt, J = 28.1
and 4.1 Hz, 2H), 3.77 (s, 2H), 2.88–2.92 (m, 2H), 2.48–2.56 (m,
2H), 2.08–2.18 (m, 2H), 1.68–1.72 (m, 2H). Anal. (C22H24BrFN2O2Á-
C2H2O4Á0.5H2O) C, H, N.
7.4. 1-((1H-Indol-3-yl)methyl)-4-(4-iodophenyl)piperidin-4-ol
oxalate (5)
Method A. Yield 56% from gramine (2a) and 4-(4-iodophenyl)-4-
hydroxypiperidine (3b). Conversion to the oxalate salt gave 5 as an
off-white powder, mp 149–150 °C (dec); 1H NMR (free base, CDCl3)
d 8.16 (br s, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.40
(d, J = 7.9 Hz, 1H), 7.27 (d, J = 8.4 Hz, 2H), 7.14–7.24 (m, 3H), 3.82
(s, 2H), 2.90–2.94 (m, 2H), 2.47–2.55 (m, 2H), 2.08–2.18 (m, 2H),
1.69–1.74 (m, 2H). Anal. (C20H21IN2OÁC2H2O4ÁH2O) C, H, N.
7.10. 4-(2-Fluoroethoxy)gramine (2d)
A mixture of 4-hydroxyindole (1 equiv), 1-bromo-2-fluoroe-
thane (5 equiv) and potassium carbonate (4 equiv) in acetone
was heated at reflux overnight. The reaction mixture was cooled
down, filtered and concentrated. Purification by silica gel column
chromatography (hexane–ethyl acetate, 3:1) gave 4-(2-fluoro-
ethoxy)indole (16a) (99%). 1H NMR (CDCl3) d 8.16 (br s, 1H),
7.5. 4-(4-Iodophenyl)-1-((4-methoxy-1H-indol-3-yl)methyl)-
piperidin-4-ol oxalate (6)
Method A. Yield 52% from 4-methoxygramine (2b) and 4-(4-
iodophenyl)-4-hydroxypiperidine (3b). Conversion to the oxalate

S. Vangveravong et al. / Bioorg. Med. Chem. 18 (2010) 5291–5300
5297
7.04–7.13 (m, 3H), 6.69–6.71 (m, 1H), 6.52 (d, J = 7.6 Hz, 1H), 4.90–
4.93 (m, 1H), 4.74–4.77 (m, 1H), 4.41–4.44 (m, 1H), 4.31–4.34 (m,
1H). A solution of 16a (1 equiv) in acetonitrile was slowly added to
a solution of N,N-dimethylmethyleneammonium iodide (1.2 equiv)
in acetonitrile and acetic acid (2:1). The solution was stirred for 3 h
and partitioned between 2-propanol and chloroform (1:3) and 10%
aqueous sodium hydroxide. The aqueous layer was extracted with
the same organic solvent. The organic layers were combined, dried,
and evaporated to give 2d as a tan solid (51%). This was used
without further purification. 1H NMR (CDCl3 + CD3OD) d 7.07–
7.17 (m, 3H), 6.48–6.51 (m, 1H), 4.94–4.97 (m, 1H), 4.78–4.81
(m, 1H), 4.38–4.41 (m, 1H), 4.29–4.31 (m, 1H), 4.23 (s, 2H), 2.53
(s, 6H).
3.00–3.22 (m, 4H), 2.30–2.42 (m, 2H), 1.78–1.83 (m, 2H). Anal.
(C19H20IN3OÁC2H2O4Á2.5H2O) C, H, N.
7.16. 1-((1H-Pyrrolo[2,3-b]pyridine-3-yl)methyl)-4-(4-(methyl-
thio)phenyl)piperidin-4-ol oxalate (15)
Method A. Yield 55% from 7-azagramine (2f) and 4-(4-methyl-
thiophenyl)-4-hydroxypiperidine (3d). Conversion to the oxalate
salt gave 15 as a tan powder, mp 191–192 °C (dec); 1H NMR (free
base, CDCl3 + CD3OD) d 8.21 (dd, J = 4.8 and 1.4 Hz, 1H), 8.10 (dd,
J = 8.0 and 1.4 Hz, 1H), 7.42 (d, J = 8.4 Hz, 2H), 7.41 (s, 1H), 7.24
(d, J = 8.4 Hz, 2H), 7.13 (dd, J = 8.0 and 4.8 Hz, 1H), 3.84 (s, 2H),
2.86–2.92 (m, 2H), 2.60–2.70 (m, 2H), 2.47 (s, 3H), 2.05–2.18 (m,
2H), 1.74–1.80 (m, 2H). Anal. (C20H23N3OSÁC2H2O4ÁH2O) C, H, N.
7.11. 5-(2-Fluoroethoxy)gramine (2e)
7.17. 1-(Benzofuran-2-ylmethyl)-4-(4-bromophenyl)piperidin-
4-ol oxalate (18)
Preparation according to the method described for 2d afforded
2e (83% from 5-hydroxyindole) as a yellow solid which was used
without further purification. 1H NMR (CDCl3 + CD3OD) d 7.27–
7.33 (m, 1H), 7.13–7.17 (m, 2H), 6.87–6.91 (m, 1H), 4.85–4.88
(m, 1H), 4.69–4.72 (m, 1H), 4.32–4.35 (m, 1H), 4.22–4.25 (m,
1H), 3.63 (s, 2H), 2.29 (s, 6H).
Method B. Yield 16% from 2-methylbenzofuran and 4-(4-bromo-
phenyl)-4-hydroxypiperidine (3c). Conversion to the oxalate salt
gave 18 as an off-white powder, mp 174–175 °C (dec); 1H NMR
(free base, CDCl3) d 7.49–7.54 (m, 2H), 7.47 (d, J = 8.8 Hz, 2H),
7.37 (d, J = 8.8 Hz, 2H), 7.18–7.29 (m, 2H), 6.63 (s, 1H), 3.76 (s,
2H), 2.87–2.91 (m, 2H), 2.52–2.61 (m, 2H), 2.14–2.24 (m, 2H),
1.69–1.75 (m, 2H). Anal. (C20H20BrNO2ÁC2H2O4ÁH2O) C, H, N.
7.12. 1-(1-((4-Methoxy-1H-indol-3-yl)methyl)piperidin-4-yl)-
1H-benzo[d]imidazol-2(3H)-one oxalate (11)
Method A. Yield 60% from 4-methoxygramine (2b) and 4-(2-
keto-1-benzimidazolinyl)piperidine (4). Conversion to the oxalate
salt gave 11 as an off-white powder, mp 209–210 °C (dec); 1H
NMR (free base, CDCl3) d 8.73 (s, 1H), 8.31 (s, 1H), 6.97–7.35 (m,
7H), 6.53 (d, J = 7.6 Hz, 1H), 4.34–4.42 (m, 1H), 4.11 (s, 2H), 3.95
(s, 3H), 3.26–3.30 (m, 2H), 2.54–2.60 (m, 2H), 2.38–2.44 (m, 2H),
1.78–1.83 (m, 2H). Anal. (C22H24N4OÁC2H2O4ÁH2O) C, H, N.
7.18. 1-(Benzofuran-2-ylmethyl)-4-(4-iodophenyl)piperidin-4-
ol oxalate (19)
Method B. Yield 61% from 2-methylbenzofuran and 4-(4-iodo-
phenyl)-4-hydroxypiperidine (3b). Conversion to the oxalate salt
gave 19 as an off-white powder, mp 199–200 °C (dec). 1H NMR
(free base, CDCl3) d 7.67 (d, J = 8.7 Hz, 2H), 7.48–7.55 (m, 2H),
7.26 (d, J = 8.7 Hz, 2H), 7.18–7.29 (m, 2H), 6.62 (s, 1H), 3.76 (s,
2H), 2.87–2.90 (m, 2H), 2.51–2.60 (m, 2H), 2.13–2.23 (m, 2H),
1.69–1.74 (m, 2H). Anal. (C20H20INO2ÁC2H2O4ÁH2O) C, H, N.
7.13. 1-(1-((5-Methoxy-1H-indol-3-yl)methyl)piperidin-4-yl)-
1H-benzo[d]imidazol-2(3H)-one oxalate (12)
Method A. Yield 87% from 5-methoxygramine (2c) and 4-(2-
keto-1-benzimidazolinyl)piperidine (4). Conversion to the oxalate
salt gave 12 as a tan powder, mp 197–198 °C (dec); 1H NMR (free
base, CDCl3) d 9.77 (s, 1H), 8.18 (s, 1H), 7.00–7.25 (m, 7H), 6.87 (dd,
J = 8.8 and 2.4 Hz, 1H), 4.33–4.42 (m, 1H), 3.90 (s, 3H), 3.77 (s, 2H),
3.16–3.19 (m, 2H), 2.44–2.55 (m, 2H), 2.19–2.27 (m, 2H), 1.78–1.82
(m, 2H). Anal. (C22H24N4OÁC2H2O4ÁH2O) C, H, N.
7.19. 1-(Benzo[b]thiophen-2-ylmethyl)-4-(4-bromophenyl)-
piperidin-4-ol oxalate (20)
Method B. Yield 69% from 2-methylbenzo[b]thiophene and 4-(4-
bromophenyl)-4-hydroxypiperidine (3c). Conversion to the oxalate
salt gave 20 as a white powder, mp 225–226 °C (dec); 1H NMR (free
base, CDCl3) d 7.78–7.81 (m, 1H), 7.68–7.71 (m, 1H), 7.48 (d,
J = 8.8 Hz, 2H), 7.40 (d, J = 8.8 Hz, 2H), 7.27–7.34 (m, 2H), 7.17 (s,
1H), 3.86 (s, 2H), 2.87–2.91 (m, 2H), 2.49–2.58 (m, 2H), 2.10–2.20
(m, 2H), 1.69–1.75 (m, 2H). Anal. (C20H20BrNOSÁC2H2O4Á0.5H2O)
C, H, N.
7.14. 1-((1H-Pyrrolo[2,3-b]pyridine-3-yl)methyl)-4-(4-bromo-
phenyl)piperidin-4-ol oxalate (13)
Method A. Yield 75% from 7-azagramine (2f) and 4-(4-bromo-
phenyl)-4-hydroxypiperidine (3c). Conversion to the oxalate salt
gave 13 as a off-white powder, mp 208–209 °C; 1H NMR (free base,
CDCl3 + CD3OD) d 8.22 (dd, J = 4.8 and 1.4 Hz, 1H), 8.11 (dd, J = 8.0
and 1.4 Hz, 1H), 7.45 (d, J = 8.8 Hz, 2H), 7.41 (s, 1H), 7.37 (d,
J = 8.8 Hz, 2H), 7.13 (dd, J = 8.0 and 4.8 Hz, 1H), 3.88 (s, 2H),
2.92–2.96 (m, 2H), 2.66–2.74 (m, 2H), 2.10–2.20 (m, 2H), 1.73–
1.78 (m, 2H). Anal. (C19H20BrN3OÁC2H2O4Á2H2O) C, H, N.
7.20. 1-(Benzofuran-3-ylmethyl)-4-(4-bromophenyl)piperidin-
4-ol oxalate (21)
Method B. Yield 22% from 3-methylbenzofuran and 4-(4-bromo-
phenyl)-4-hydroxypiperidine (3c). Conversion to the oxalate salt
gave 21 as an off-white powder, mp 202–203 °C (dec); 1H NMR
(free base, CDCl3) d 7.72–7.76 (m, 1H), 7.57 (s, 1H), 7.48–7.50 (m,
1H), 7.47 (d, J = 8.8 Hz, 2H), 7.38 (d, J = 8.8 Hz, 2H), 7.23–7.33 (m,
2H), 3.72 (s, 2H), 2.85–2.89 (m, 2H), 2.46–2.55 (m, 2H), 2.06–
2.16 (m, 2H), 1.69–1.74 (m, 2H). Anal. (C20H20BrNO2ÁC2H2O4) C,
H, N.
7.15. 1-((1H-Pyrrolo[2,3-b]pyridine-3-yl)methyl)-4-(4-iodo-
phenyl)piperidin-4-ol oxalate (14)
Method A. Yield 48% from 7-azagramine (2f) and 4-(4-iodo-
phenyl)-4-hydroxypiperidine (3b). Conversion to the oxalate salt
gave 14 as a white powder, mp 187–188 °C; 1H NMR (free base,
CDCl3 + CD3OD) d 8.26 (dd, J = 4.9 and 1.4 Hz, 1H), 8.14 (dd,
J = 8.0 and 1.4 Hz, 1H), 7.66 (d, J = 8.7 Hz, 2H), 7.61 (s, 1H), 7.25
(d, J = 8.7 Hz, 2H), 7.18 (dd, J = 8.0 and 4.9 Hz, 1H), 4.17 (s, 2H),
7.21. 1-(Benzofuran-3-ylmethyl)-4-(4-iodophenyl)piperidin-4-
ol oxalate (22)
Method B. Yield 33% from 3-methylbenzofuran and 4-(4-iodo-
phenyl)-4-hydroxypiperidine (3b). Conversion to the oxalate salt

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S. Vangveravong et al. / Bioorg. Med. Chem. 18 (2010) 5291–5300
gave 22 as a white powder, mp 198–199 °C; 1H NMR (free base,
CDCl3) d 7.72–7.75 (m, 1H), 7.67 (d, J = 8.7 Hz, 2H), 7.57 (s, 1H),
7.46–7.50 (m, 1H), 7.25 (d, J = 8.7 Hz, 2H), 7.24–7.33 (m, 2H),
3.72 (s, 2H), 2.84–2.88 (m, 2H), 2.45–2.54 (m, 2H), 2.05–2.16 (m,
2H), 1.68–1.74 (m, 2H). Anal. (C20H20INO2ÁC2H2O4) C, H, N.
7.27. 3-(2-Fluoroethyl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-
one (28b)
A solution of di-tert-butyl dicarbonate ((BOC)2O, 7.0 mmol) in
dichloromethane was added in a solution of 1-phenyl-1,3,8-triaza-
spiro[4.5]decan-4-one (30, 6.5 mmol) and triethylamine
(7.0 mmol) in dichloromethane. The reaction mixture was stirred
at room temperature for 3 h. After the work-up, the N-protected
compound 31 was obtained. Sodium hydride (15.8 mmol) and
1-bromo-2-fluoroethane (7.0 mmol) were added in a cold solution
of 31 (4.5 mmol) in anhydrous N,N-dimethylformamide. The reac-
tion mixture was stirred at room temperature for 4 h. The obtained
product was treated with trifluoroacetic acid to give 28b, which
was used without further purification.
7.22. 1-(1-(Benzofuran-3-ylmethyl)piperidin-4-yl)-1H-
benzo[d]imidazol-2-(3H)-one oxalate (23)
Method B. Yield 22% from 3-methylbenzofuran and 4-(2-keto-1-
benzimidazolinyl)piperidine (4). Conversion to the oxalate salt
gave 23 as a white powder, mp 244–245 °C; 1H NMR (free base,
CDCl3) d 9.72 (s, 1H), 7.76–7.79 (m, 1H), 7.58 (s, 1H), 7.48–7.51
(m, 1H), 7.28–7.35 (m, 3H), 7.03–7.12 (m, 3H), 4.33–4.41 (m,
1H), 3.72 (s, 2H), 3.12–3.16 (m, 2H), 2.42–2.54 (m, 2H), 2.20–
2.27 (m, 2H), 1.80–1.84 (m, 2H). Anal. (C21H21N3O2Á-
C2H2O4Á0.25H2O) C, H, N.
7.28. 8-(Benzofuran-3-ylmethyl)-3-((1-(2-fluoroethyl)-1H-1,2,3-
triazol-4-yl)methyl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-
one oxalate (29)
7.23. 1-(1-(Benzo[b]thiophen-3-ylmethyl)piperidin-4-yl)-1H-
benzo[d]imidazol-2-(3H)-one oxalate (24)
Sodium hydride (2.5 equiv) and propargyl bromide (1.5 equiv)
were added into a cold solution of 31 (1.0 equiv) in anhydrous
tetrahydrofuran. The reaction mixture was stirred at ambient tem-
perature for 4 h and washed with water. Evaporation of the organic
layer gave the product which was treated with trifluoroacetic acid
to give 32. Using the general method B, 33 was made from 32 and
3-methylbenzofuran. To a solution of 33 (1 equiv) and 1-azido-2-
fluoroethane (2 equiv) in anhydrous N,N-dimethylformamide was
added sodium ascorbate (5 equiv) and copper sulfate pentahydrate
(0.5 equiv). The reaction was stirred for 3 h and TLC analysis indi-
cated complete consumption of the reactants. Water was added
and the product was extracted with ethyl acetate. The combined
organic layers were washed with water, dried, and concentrated.
Column chromatography (5% methanol in dichloromethane) of
the resulting residue gave the product (4% overall yield from 3-
methybenzofuran). Conversion to the oxalate salt gave 29 as a
white powder, mp 160–161 °C; 1H NMR (free base, CDCl3) d
7.80–7.83 (m, 1H), 7.69 (s, 1H), 7.56 (s, 1H), 7.46–7.49 (m, 1H),
7.24–7.33 (m, 3H), 6.81–6.91 (m, 3H), 4.85–4.88 (m, 1H), 4.76 (s,
2H), 4.71 (s, 2H), 4.69–4.72 (m, 2H), 4.60–4.63 (m, 1H), 3.72 (s,
2H), 2.82–2.88 (m, 4H), 2.62–2.73 (m, 2H), 1.61–1.65 (m, 2H). Anal.
(C27H29FN6O2ÁC2H2O4ÁH2O) C, H, N.
Method B. Yield 63% from 3-methylbenzo[b]thiophene and 4-(2-
keto-1-benzimidazolinyl)piperidine (4). Conversion to the oxalate
salt gave 24 as a pale yellow powder, mp 242–243 °C; 1H NMR
(free base, CDCl3) d 9.92 (s, 1H), 8.01–8.04 (m, 1H), 7.86–7.89 (m,
1H), 7.34–7.46 (m, 3H), 7.23–7.26 (m, 1H), 7.03–7.12 (m, 3H),
4.36–4.44 (m, 1H), 3.82 (s, 2H), 3.12–3.16 (m, 2H), 2.43–2.54 (m,
2H), 2.21–2.29 (m, 2H), 1.79–1.84 (m, 2H). Anal. (C21H21N3OSÁ-
C2H2O4) C, H, N.
7.24. 8-(Benzofuran-3-ylmethyl)-3-methyl-1-phenyl-1,3,8-
triazaspiro[4.5]decan-4-one oxalate (25)
Method B. Yield 34% from 3-methylbenzofuran and 3-methyl-
1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (28a). Conversion to
the oxalate salt gave 25 as an off-white powder, mp 209–210 °C;
1H NMR (free base, CDCl3) d 7.81–7.84 (m, 1H), 7.57 (s, 1H),
7.46–7.49 (m, 1H), 7.27–7.33 (m, 4H), 6.82–6.92 (m, 3H), 4.66 (s,
2H), 3.72 (s, 2H), 2.99 (s, 3H), 2.87–2.90 (m, 4H), 2.65–2.73 (m,
2H), 1.62–1.67 (m, 2H). Anal. (C23H25N3O2ÁC2H2O4Á0.25H2O) C, H,
N.
7.25. 8-(Benzofuran-3-ylmethyl)-3-(2-fluoroethyl)-1-phenyl-
1,3,8-triazaspiro[4.5]decan-4-one oxalate (26)
8. Radioligand binding and functional assays
8.1. Dopamine receptor binding assays
Method B. Yield 31% from 3-methylbenzofuran and 3-(2-fluoro-
ethyl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (28b). Conver-
sion to the oxalate salt gave 26 as a white powder, mp 175–
176 °C; 1H NMR (free base, CDCl3) d 7.80–7.83 (m, 1H), 7.56 (s,
1H), 7.45–7.49 (m, 1H), 7.27–7.33 (m, 4H), 6.84–6.94 (m, 3H),
4.79 (s, 2H), 4.64 (dt, J = 47.2 and 4.6 Hz, 2H), 3.73 (dt, J = 28.6
and 4.6 Hz, 2H), 3.71 (s, 2H), 2.80–2.89 (m, 4H), 2.62–2.73
(m, 2H), 1.65–1.70 (m, 2H). Anal. (C24H26FN3O2ÁC2H2O4Á0.5H2O) C,
H, N.
The method for the iodination of 125I-IABN using peracetic acid
has been previously described.27 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,
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
l
g of protein per 50
l
L prior to the assay. Assays were
L. Binding reactions were
performed in a total volume of 150
l
7.26. 8-(Benzo[b]thiophen-3-ylmethyl)-3-methyl-1-phenyl-
1,3,8-triazaspiro[4.5]decan-4-one oxalate (27)
carried out for 60 min at 37 °C and the reaction was terminated
by rapid filtration over Schleicher and Schuell No 32 glass fiber fil-
ters (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 efficiency of 75%.
Protein concentrations were determined using a BCA reagent
(Pierce, Rockford, Illinois) with bovine serum albumin as the pro-
tein standard.
Method B. Yield 37% from 3-methylbenzo[b]thiophene and
3-methyl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (28a). Con-
version to the oxalate salt gave 27 as a white powder, mp 199–
200 °C; 1H NMR (free base, CDCl3) d 8.08–8.11 (m, 1H), 7.85–7.87
(m, 1H), 7.28–7.44 (m, 5H), 6.81–6.91 (m, 3H), 4.66 (s, 2H), 3.83
(s, 2H), 2.99(s, 3H), 2.88–2.91 (m, 4H), 2.65–2.76 (m, 2H), 1.62–
1.66 (m, 2H). Anal. (C23H25N3OSÁC2H2O4) C, H, N.
For competition curves using a transfected cell line expressing
D2, D3 or D4 dopamine receptors, experiments were performed in

S. Vangveravong et al. / Bioorg. Med. Chem. 18 (2010) 5291–5300
5299
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
trifugation, the supernatants were fractionated using Dowex AG1-
X8 and neutral alumina to separate the 3H-ATP and the 3H-cyclic
AMP. Individual samples were corrected for column recovery by
monitoring the recovery of the cyclic AMP using spectrophotomet-
ric analysis at OD 259 nm.27,33
either no inhibitor or 2 lM (+)-butaclamol were used to define to-
tal binding and nonspecific binding, respectively. Competition data
for D2-like dopamine receptors were modeled for a single-site fit
using the TableCurve program (Jandel Scientific Software, San Ra-
fael, California); the IC50 values for the competitive inhibitors were
converted to Ki values using the Cheng and Prusoff corrections.32
Acknowledgment
This research was funded by Grants MH081281 and DA023957
awarded by the National Institutes of Health.
8.2. Sigma receptor binding assays
Appendix
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 buf-
Elemental analyses
Compound
%C
%H
%N
Calc’d Found Calc’d Found Calc’d Found
5
6
7
8
9
48.90 48.50 4.66
49.61 49.92 4.62
48.05 47.95 4.82
64.10 64.07 6.20
52.76 52.96 4.98
52.76 53.13 4.98
61.53 61.13 6.02
61.53 61.50 6.02
49.23 48.83 5.11
44.38 44.32 4.79
57.25 57.63 5.90
53.45 53.66 4.89
48.81 49.17 4.47
52.70 52.97 4.62
55.48 55.25 4.66
50.49 50.31 4.24
62.51 62.61 5.36
60.91 61.05 5.11
63.89 63.93 5.90
61.65 61.84 5.77
62.35 62.22 5.65
58.38 58.16 5.58
4.44
4.98
4.55
6.08
5.03
5.25
5.74
5.72
4.80
4.39
5.62
5.18
4.63
4.57
4.69
4.28
5.22
5.21
5.75
5.62
5.64
5.41
5.18
5.03
4.87
6.50
5.13
5.13
4.76
4.63
4.46
6.37
5.09
5.25
fer, pH 8.0, and incubated at 25 °C in a total volume of 150
96-well plates with the radioligand and test compounds with con-
centrations ranging from 0.1 nM to 10 M. After incubation was
completed, the reactions were terminated by the addition of
150 L of ice-cold wash buffer (10 mM Tris–HCl, 150 mM NaCl,
lL in
l
l
10
11
12
13
14
15
18
19
20
21
22
23
24
25
26
27
29
pH 7.4) using a 96-channel transfer pipette (Fisher Scientific, Pitts-
burgh, PA), and the samples harvested and filtered rapidly through
96-well fiber glass filter plate (Millipore, Billerica, MA) that had
11.96 11.77
11.96 11.75
8.20
7.39
9.10
2.83
2.59
2.79
2.94
2.68
9.51
9.27
8.94
8.30
8.73
7.81
7.39
9.31
2.76
2.51
2.78
3.00
2.71
9.53
9.10
8.93
8.29
8.54
been presoaked with 100
for 1 h. Each filter was washed three times with 200
l
L of 50 mM Tris–HCl buffer, pH 8.0,
L of ice-cold
l
wash buffer. A Wallac 1450 MicroBeta liquid scintillation counter
(Perkin–Elmer, Boston, MA) was used to quantitate the bound
radioactivity.
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-
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,
14.09 13.69
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.
The incubation time was 60 min for [3H]RHM-1 and 120 min for
[3H]DTG. Nonspecific binding was determined from samples that
References and notes
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Data from the competitive inhibition experiments were mod-
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radioligand (IC50 value). Competitive curves were best fit to a
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were calculated using the method of Cheng and Prusoff30 and rep-
resent mean values SEM. The Kd value used for [3H](+)-pentazo-
cine with guinea pig brain homogenates was 7.89 nM; a Kd value
of 30.73 nM was used for [3H]DTG with rat liver, while 0.66 nM
was used for [3H]RHM-1 with rat liver.29
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