Synthesis and pharmacological evaluation of fluorine-containing D 3 dopamine receptor ligands

Article abstract of DOI:10.1021/jm101323b

A series of fluorine-containing N-(2-methoxyphenyl)piperazine and N-(2-fluoroethoxy)piperazine analogues were synthesized, and their affinities for human dopamine D₂, D₃, and D₄ receptors were determined. Radioligand binding studies identified five compounds, 18a, 20a, 20c, 20e, and 21e, which bind with high affinity at D₃ (K _i = 0.17-5 nM) and moderate to high selectivity for D₃ vs D₂ receptors (ranging from ～25- to 163-fold). These compounds were also evaluated for intrinsic activity at D₂ and D₃ receptors using a forskolin-dependent adenylyl cyclase assay. This panel of compounds exhibits varying receptor subtype binding selectivity and intrinsic activity at D₂ vs D₃ receptors. These compounds may be useful for behavioral pharmacology studies on the role of D₂-like dopamine receptors in neuropsychiatric and neurological disorders. Furthermore, compound 20e, which has the highest binding affinity and selectivity for the D₃ receptor (K_i = 0.17 nM for D₃, 163-fold selectivity for D₃ vs D₂ receptors), represents a candidate fluorine-18 radiotracer for in vivo PET imaging studies on the regulation of D₃ receptor expression.

Full text of DOI:10.1021/jm101323b

ARTICLE
pubs.acs.org/jmc
Synthesis and Pharmacological Evaluation of Fluorine-Containing D3
Dopamine Receptor Ligands
Zhude Tu,† Shihong Li,† Jinquan Cui,† Jinbin Xu,† Michelle Taylor,|| David Ho,|| Robert R. Luedtke,|| and
Robert H. Mach*,†,‡,§
†Department of Radiology, ‡Department of Cell Biology and Physiology, and §Department of Biochemistry and
Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, United States
Department of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth,
Texas 76107, United States
S Supporting Information
b
ABSTRACT: A series offluorine-containing N-(2-methoxyphenyl)piperazine
and N-(2-fluoroethoxy)piperazine analogues were synthesized, and their
affinities for human dopamine D2, D3, and D4 receptors were determined.
Radioligand binding studies identified five compounds, 18a, 20a, 20c, 20e,
and 21e, which bind with high affinity at D3 (Ki = 0.17-5 nM) and
moderate to high selectivity for D3 vs D2 receptors (ranging from ∼25- to
163-fold). These compounds were also evaluated for intrinsic activity at D2
and D3 receptors using a forskolin-dependent adenylyl cyclase assay. This
panel of compounds exhibits varying receptor subtype binding selectivity
and intrinsic activity at D2 vs D3 receptors. These compounds may be useful
for behavioral pharmacology studies on the role of D2-like dopamine
receptors in neuropsychiatric and neurological disorders. Furthermore,
compound 20e, which has the highest binding affinity and selectivity for the
D3 receptor (Ki = 0.17 nM for D3, 163-fold selectivity for D3 vs D2
receptors), represents a candidate fluorine-18 radiotracer for in vivo PET imaging studies on the regulation of D3 receptor
expression.
’ INTRODUCTION
a rodent model of L-DOPA-induced dyskinesia (LID); previous
studies have suggested that there is an upregulation of D3
receptors in dyskinetic animals.8-10 D3 receptor selective ligands
have been shown to be effective in attenuating L-DOPA-induced
dyskinesia in rats, suggesting that the D3 receptor may be an
important therapeutic target for the treatment of LID.11-15
Finally, the positive reinforcing effects of psychostimulants, such
as cocaine and methamphetamine, may be mediated, in part, by
the stimulation of D3 receptors. Therefore, D3 receptor selective
partial agonists and/or antagonists may be useful pharmacother-
apeutic agents for the treatment of substance abuse.16-21
Positron emission tomography (PET) is a noninvasive ima-
ging technique that has been used to study the expression of
dopamine receptors in the brain. However, the identification of
D3 receptor specific PET radioligands has been challenging
because of the high degree of amino acid sequence homology
between D2 and D3 receptor binding sites in the ligand binding
domain.1,20,3,22-25 A number of D3-selective ligands have
served as lead compounds for PET radiotracer development
(Figure 1).26-30 Unfortunately, none of the D3 receptor selective
radiotracers reported to date have shown promise in in vivo
Dopamine receptors are G protein-coupled receptors and are
classified into two major types, the D1-like and D2-like receptors.
The D1-like receptor subtypes include the D1 (rat D1a) and D5
(rat D1b) receptors, whereas the D2-like receptor subtypes
include the D2, D3, and D4 receptors. Agonist stimulation of
D1-like receptors results in an activation of adenylyl cyclase.
Stimulation of D2-like receptors results in an inhibition of
adenylyl cyclase activity, an increase in the release of arachidonic
acid, activation of G protein-coupled inwardly rectifying potas-
sium channels (GIRKs), activation of phospholipase D (PLD),
and also an increase in phosphatidylinositol hydrolysis.1 D2 and
D3 receptors have ∼46% overall amino acid sequence homology
and 78% sequence homology within the transmembrane-span-
ning segments.2
There is a large body of evidence indicating that D3 dopamine
receptors may play an important role in a number of neurological
and neuropsychiatric disorders.3 First, the high density of D3
receptors in limbic regions4-7 suggests that this receptor subtype
may play a role in the etiology of schizophrenia and that D3-
selective antagonists may exhibit an antipsychotic profile devoid
of extrapyramidal side effects.2,5,7 Second, prolonged treatment
of 6-hydroxydopamine unilaterally lesioned rats with L-DOPA is
Received: July 14, 2010
Published: February 24, 2011
r
2011 American Chemical Society
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Figure 1. Representative dopamine D3 receptor ligands.
Scheme 1a
a Reagents and conditions: (a) 98% H2SO4, methanol; (b) BrCH2CH2F, K2CO3, acetone; (c) NaOH, methanol/H2O.
imaging or brain uptake studies in rodents or nonhuman
primates. One of the main limitations of many D3-selective
ligands is their relatively high lipophilicity, which could compro-
mise their ability to cross the blood-brain barrier and label D3
receptors in vivo.
versus 11C-labeled radiotracers. In this paper, we report the
synthesis and in vitro evaluation of a series of fluorine-containing
conformationally flexible benzamide analogues, in which the
structure was altered by (1) replacing the 2-methoxyphenyl
group in the piperazinyl ring with a 2-(2-fluoroethoxy)phenyl
group, (2) introducing a 2-fluoroethoxy or 2-fluoroethyl group in
the 2- and 4-position of the benzamide moiety, and (3) compar-
ing the effect of having a double bond (trans-butenyl) within the
four carbon chain that links the arylamide with the 4-phenylpi-
perazine moiety.
Over the past decade, our group has focused on identifying
candidate ligands having the right balance between D3 receptor
affinity (1-5 nM), selectivity (>50-fold selective for D3 versus
D2 receptors), and lipophilicity (log P = 2.0-4.0) to give a
suitable signal:noise ratio in PET imaging studies. We previously
reported benzamide analogues, 1 (WC-10) (Ki = 0.8 nM for D3
receptor, D2/D3 ratio = 43) and 2 (WC-44) (Ki = 2.4 nM for D3,
D2/D3 ratio = 23), as lead compounds for radiotracer develop-
ment.24 Quantitative autoradiography studies using [3H]-1
demonstrated it has high affinity and moderate selectivity
for D3 vs D2 receptor,24,31 which is consistent with in vitro
screening data using competition binding assays.25 However,
microPET studies of [11C]-1 in rhesus brain have exhibited
high levels of variability for D3 imaging between subjects, and
similar studies using [18F]-2 have not shown good target to
nontarget ratios.25
’ RESULTS AND DISCUSSION
Chemistry. The target compounds were synthesized as de-
picted in Schemes 1-3. The synthesis of the substituted benzoic
acids (9a-e) was accomplished as outlined in Scheme 1. The
acids were first converted into the corresponding methyl esters
by Fischer esterification. O-Alkylation of 2-hydroxyl or 4-hydro-
xyl group was achieved by treatment with 1-bromo-2-fluor-
oethane in acetone using potassium carbonate as the base.
Hydrolysis of the methyl ester with sodium hydroxide in aqueous
methanol afforded the corresponding 2-fluoroethoxybenzoic
acids 9a-e. The synthesis of the 4-fluoropegylated benzoic acids
12a and12b is shown in Scheme 2. O-Alkylation of the phenol
group of 4-hydroxybenzoic acid methyl ester with either 2-(2-
chloroethoxy)ethanol or 2-(2-(2-chloroethoxy)ethoxy)ethanol
in the presence of potassium carbonate in tetrahydrofuran
afforded 10a and 12b (Scheme 2). Conversion of alcohols 10a
Our laboratory has continued to investigate the structure-
activity relationships of conformationally flexible benzamide
analogues by optimizing the structures of 1 and 2 to identify
promising candidates for imaging the D3 receptor with PET. The
longer half-life of 18F (t1/2 = 109.8 min) compared to 11C (t1/2
=
20.4 min) places fewer time constraints on radiotracer synthesis
and permits longer scan sessions for 18F-labeled radiotracers
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Scheme 2a
a Reagents and conditions: (a) 2-(2-chloroethoxy)ethanol or 2-(2-(2-chloroethoxy)ethoxy)ethanol, K2CO3, THF; (b) DAST, CH2Cl2, 0 °C; (c)
triethylamine, p-toluenesulfonyl chloride, CH2Cl2; (d) TBAF/THF, reflux, 4 h; (e) NaOH, methanol/H2O.
Scheme 3a
a Reagents and conditions: (a) (E)-1,4-dibromobut-2-ene, DMF, 0 °C; (b) 2-(4-bromobutyl)isoindoline-1,3-dione or 14, N(C2H5)3/CH2Cl2, room
temperature; (c) 2-fluoro-1-bromoethane, K2CO3, acetone, reflux; (d) hydrazine, ethanol, reflux 5 h.
and 10b to the corresponding fluoro derivatives 11a and 11b was
accomplished using two different methods. Direct conversion of
the hydroxyl group of 10a with diethylaminosulfur trifluoride
(DAST) gave 11a in modest yield (43%). Alternatively, conver-
sion of the hydroxyl group of 10b to the corresponding tosylate
group, 10c, followed by displacement with tetrabutylammonium
fluoride (TBAF) gave 11b in an overall yield of 56%. Hydrolysis
of 11a and 11b with sodium hydroxide in aqueous methanol
afforded benzoic acids 12a and 12b.
The substituted 4-(4-phenylpiperazin-1-yl)butan-1-amines
(17a,b) and the substituted 4-(4-phenylpiperazin-1-yl)-trans-
but-2-en-1-amines (17b,d) were synthesized according to
Scheme 3. Treatment of potassium 1,3-dihydro-1,3-dioxoisoindole
(13) with trans-1,4-dibromo-2-butene in N,N-dimethylforma-
mide (DMF) gave 2-(trans-4-bromobut-2-enyl)-1,3-dihydro-
1,3-dioxoisoindole, 14, in modest yield (69%). The N-alkylation
of 1-(2-methoxylphenyl)piperazine (15a) or 1-(2-hydroxylphe-
nyl)piperazine (15b) with either 2-(4-bromobutyl)-1,3-dihydro-
1,3-dioxoisoindole or 14 produced 16a-d. O-Alkylation of the
phenol group in 16b and 16d with 1-bromo-2-fluoroethane in
acetone using potassium carbonate as the base afforded com-
pounds 16e,f. Treatment of 16a,c and 16e,f with hydrazine in
refluxing ethanol (Scheme 3) afforded the corresponding amines
17a-d in variable yields.
The target benzamides 18a-c, 19a-g, 20b-d,f, and 21b-d,
were synthesized by coupling amines 17a-d with substituted
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Scheme 4
benzoic acids 9a-e, 12a,b, and 4-(2-fluoroethyl)benzoic acid25
with N,N0-dicyclohexylcarbodiimide (DCC) in dichloromethane
(Scheme 4). Benzamides 20a,e and 21a,e, were prepared by
coupling amines 17b,d with the corresponding commercially
available benzoic acid. All final compounds were converted into
the corresponding oxalic acid salt for in vitro binding studies.
In Vitro Binding Studies. Compounds were first evaluated for
affinity at human D2 and D3 dopamine receptors expressed in
stably transfected HEK cells. Analogues which exhibited high
binding affinity at D3 receptors were further evaluated for affinity
at (a) D4 dopamine receptors and (b) σ1 and σ2 sigma receptors.
The σ receptor binding studies were undertaken because of the
ubiquitous expression of sigma receptors in the CNS. Therefore,
high σ1 or σ2 receptor binding affinity would preclude the
usefulness of a D3-selective radiotracer for PET imaging studies.
The σ receptor binding studies were included to ensure that our
compounds bind with low affinity for σ receptors.
Table 1. D2, D3, and D4 Affinities (Ki ( SD, nM) of the
Benzamide Analogues
D2
D3
D4
D2/D3 ratio log Pa
1
34.4 ( 4.7
54.5 ( 4.4
27.1 ( 3.5
0.8 ( 0.1
2.4 ( 0.4
1.1 ( 0.1
6.2 ( 0.9
4.2 ( 0.2
24.9 ( 3.3
6.2 ( 0.3
896 ( 272
804 ( 46
1400 ( 320
ND
43
23
25
3.09
2.94
3.48
3.08
2.72
3.43
3.55
3.16
3.66
4.49
4.73
4.15
3.75
3.68
3.73
3.34
4.67
2.98
3.83
3.61
3.81
3.41
4.74
3.26
3.72
4.36
2
18a
18b 20.9 ( 3.7
3.4
18c
19a
52.0 ( 6.6
131 ( 13
2100 ( 380
ND
12.2
5.2
8.9
3.3
1
19b 55.3 ( 6.0
19c
ND
59.2 ( 5.8 18.2 ( 2.3
ND
19d 17.8 ( 0.8 18.5 ( 2.4
19e 13.4 ( 2.3 13.6 ( 2.0
ND
ND
1
19f
19g
20a
13.2 ( 0.8 10.9 ( 1.5
57.6 ( 3.7 13.8 ( 1.2
ND
1.2
4.2
23
3.1
29
5.7
ND
The [125I]-IABN inhibition constants (Ki) at D2 and D3
receptors are reported in Table 1. The ligand binding selectivity,
in terms of a selectivity index, is calculated as Ki(D2)/Ki(D3). For
the ensuing discussion, binding affinities are characterized as very
high (Ki < 1.0 nM), high (Ki = 1-10 nM), moderate (Ki = 11-
50 nM), or low (Ki > 50 nM).
15.1 ( 1.7
0.65 ( 0.2
6.9 ( 1.0
890 ( 100
ND
20b 21.4 ( 2.9
20c 15.1 ( 2.7
20d 14.2 ( 1.9
0.52 ( 0.03 990 ( 200
2.5 ( 0.3 ND
0.17 ( 0.01 246 ( 13
20e
20f
21a
27.7 ( 5.4
31.7 ( 2.1
35.2 ( 2.5
163
5.2 ( 0.3
3.6 ( 0.6
1.1 ( 0.2
8.4 ( 1.3
ND
6.1
9.8
16.1
4.5
3.3
64
The radioligand binding assays identified a number of poten-
tially useful structure-activity trends as well as several promising
fluorinated analogues which could serve as potential PET radio-
tracers. First, in a comparison of 1 and 18a-c, it was observed
that replacing the 4-dimethylamino group in the benzamide
moiety with a 4-(2-fluoroethoxy) group (compound 18a) re-
sulted in a D3 binding affinity (Ki = 1.1 nM) comparable to 1 (Ki
= 0.8 nM). However, there was a decrease in D3 receptor affinity
when the 4-(2-fluoroethoxy) group was homologated to the
corresponding fluoropegylated groups, 18b and 18c. Thus, the
D3 vs D2 selectivity for 18b and 18c was <15-fold, whereas the D3
vs D2 selectivity for 18a was ∼25-fold.
ND
21b 17.7 ( 2.7
21c 37.9 ( 5.0
890 ( 380
ND
21d 64.8 ( 8.4 19.6 ( 3.2
ND
21e
21f
21g
70.5 ( 9.6
1.1 ( 0.2
182 ( 5
ND
25.9 ( 2.4 11.2 ( 2.2
2.3
1.6
3.9
10.9 ( 0.4
6.7 ( 1.1
3.1 ( 0.3
ND
21h 12.0 ( 0.6
ND
a Calculated using ACD log D software, Advanced Chemistry Develop-
ment, Toronto, Canada.
To further explore the structure-activity relationships of
this series, the structures of the amides were modified by
introducing the 2-fluoroethoxy group in the 2-position compared
to 4-position of the benzamide moiety. These analogues also had
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a methyl or halogen atom (Br or I) in the 5-position and the trans
double bond 4-carbon spacer group. This approach generally
resulted in compounds with only moderate affinity for both D3
and D2 receptors (compounds 19d-f), except for compound
19g which displayed a 4-fold selectivity for D3 versus D2
receptors. Based on these results, a higher D3 receptor affinity
and selectivity were observed when the 2-fluoroethoxy is in the
4-position of the benzamide region (compound 19b) rather than
the 2-position.
The next substitution involved replacing the 2-methoxy group
with a 2-fluoroethoxy group in the N-phenylpiperazinyl moiety.
The replacement of a methoxy group with a 2-fluoroethoxy
group is a standard method for preparing a potential 18F-labeled
radiotracer. This modification generally resulted in compounds
having a slightly increased D3 binding affinity when compared
with the corresponding N-(2-methoxyphenyl)piperazinyl analo-
gues (e.g., 20a vs 1, 20c vs 18a, and 20d vs 18c). However, one
exception to this trend was noticed, with 20b having a lower D3
affinity than its corresponding 2-methoxy analogue, 2. An inter-
esting observation was in the substitution of the 4-position of the
benzamide group with a 3-thiophene ring to give compound 20e;
this analogue displayed both the highest D3 binding affinity (0.17
nM) and greatest D3 vs D2 receptor selectivity (163-fold) among
the compounds reported in this paper.
When the saturated 4-carbon spacer linking the arylamide with
the N-(4-methoxyphenyl)piperazine moiety was replaced with a
trans double bond, the binding affinity at both D2 and D3 receptors
generally decreased. However, replacing the single bond with a
trans double bond caused a larger reduction in D3 affinity rela-
tive to the reduction in D2 affinity. This trend was also observed
with the N-(2-fluoroethoxyphenyl)piperazine-containing analo-
gues shown in Table 1. These results are consistent with the
N-(2-methoxyphenyl)piperazinyl analogues described by Taylor
et al.32 but opposite to what has been reported with the correspon-
ding N-(2,3-dichlorophenyl)piperazinylbenzamide analogues.20,33
Therefore, the effect of the single vs double bond replacement
on binding selectivity appears to be governed by the type of
substitution on the N-phenylpiperazine group. The only analo-
gue showing an increase in D3 affinity when the trans double
bond was introduced was 21b, which had a 6-fold higher affinity
at D3 receptors when compared with its saturated analogue 20b.
Affinity at dopamine D4 receptors was determined on com-
pounds having a high affinity (Ki < 5 nM) for D3 receptors and
high selectivity for D3 vs D2 receptors (>10-fold). All compounds
that were tested exhibited low binding affinity at D4 receptors
(Table 1).
Since many dopamine ligands have been shown to bind to σ1 and
σ2 receptors, we determined the σ receptor binding affinities for
compounds having a high D3 receptor affinity and good selectivity
for D3 vs D2 receptors. All of the compounds tested exhibited low
binding affinities at σ1 and σ2 receptors. The affinity ratios of D3 to σ
receptors were >260-fold (Table 2). Compound 20e, which has the
highest D3 affinity and D3 vs D2 selectivity ratio, binds with low
affinity at both σ1 and σ2 receptors. This observation eliminates any
concern that σ receptor binding might interfere with the imaging
signal when a 18F-radiolabeled derivative of 20e is made for PET
imaging studies of the D3 receptor.
Table 2. Sigma Receptor Affinities (Ki ( SD, nM) of Selected
Analogues
D3
σ1
σ2
D3/σ1 ratio D3/σ2 ratio
1
0.8 ( 0.1
2.4 ( 0.4
1.1 ( 0.1
4.2 ( 0.2
1260 ( 290 1570 ( 310
3540 ( 2500 2210 ( 260
4780 ( 730 660 ( 36
4870 ( 470 1120 ( 30
1573
1476
4344
1159
3017
8377
1970
919
2
18a
601
18c
266
20a
0.65 ( 0.2 1960 ( 50
650 ( 38
1002
1527
35047
1839
1200
20c
0.52 ( 0.03 4360 ( 570 794 ( 14
20e
0.17 ( 0.01 20900 ( 5250 5960 ( 360 122706
21b
1.1 ( 0.2
1.1 ( 0.2
7200 ( 880 2020 ( 260
7780 ( 540 1320 ( 33
1.5 ( 0.3 24.2 ( 3.0
6541
7076
21e
haloperidol
Table 3. Intrinsic Efficacy of Selected Analogues at Dopa-
mine D2 and D3 Receptorsa
compd
hD2 HEK
hD3 HEK
haloperidol
-0.6 ( 1.6
33.5 ( 3.1
35.3 ( 1.0
58.6 ( 1.1
63.8 ( 4.2
66.3 ( 1.0
73.2 ( 0.7
29.3 ( 7.3
65.8 ( 0.2
21.2 ( 5.5
100
4.0 ( 5.5
18.7 ( 2.2
96.2 ( 4.2
68.8 ( 5.6
59.9 ( 7.4
64.5 ( 8.3
50.3 ( 5.2
34.5 ( 1.7
65.5 ( 7.2
55.4 ( 4.2
100
1
2
18a
18c
20a
20c
20e
21b
21e
quinpirole
a The intrinsic efficacy of the test compounds was evaluated by
determining the percent inhibition of a forskolin-dependent whole cell
adenylyl cyclase assay. The results were normalized to the percent
inhibition obtained using the full agonist quinpirole at human D2 (1 μM)
and D3 (100 nM) receptors expressed in stably transfected HEK 293
cells. For D2 receptors the maximum inhibition was >90%, and for D3
receptors the maximum inhibition ranged from 38% to 53%. The test
drug was used at a concentration equal to approximately 10 times the Ki
value that was determined from the radioligand binding analysis. The
mean ( SEM values are reported for n g 3.
expressing human D2 or D3 dopamine receptors. For each
compound, the inhibition was compared to the intrinsic efficacy
of the full agonist quinpirole and the antagonist haloperidol. The
compounds that were evaluated were all partial agonists at D3
dopamine receptors, displaying intrinsic efficacy from 34.5 (
1.7% (20e) to 68.8 ( 5.6% (18a) (Table 3). As previously
reported, the constituent at the para position of the benzamide
group plays a pivotal role in determining the intrinsic activity of
our compounds. For example, 1, 2, 18a, and 18c each contain an
4-(2-methoxyphenyl)piperazine moiety with a saturated 4-car-
bon spacer, yet their efficacy compared to quinpirole varies from
34% to 64% at D2 receptors and 18% to 96% at D3 receptors. In
addition, the structure of the 4-carbon spacer influences efficacy.
For example, substitution of a trans double bond (21e) for the
saturated spacer (20e) had minimal effect on efficacy at D2
receptors (29% vs 21% maximal efficacy), while efficacy at D3
receptors increased almost 60% (35% to 55% maximal efficacy)
(Table 3). The diverse range of D3 and D2 receptor affinities
and intrinsic activities at these receptors indicates that these
Intrinsic Activity at Dopamine Receptors. The intrinsic
activity of compounds 18a,c, 20a,c,e, and 21b,e at D3 and D2
receptors was also evaluated. This assay measures the ability of
the compounds to inhibit forskolin-dependent stimulation of
adenylyl cyclase activity in stably transfected HEK 293 cells
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compounds are useful probes for studying the behavioral phar-
macology of D3 and D2 receptors in animal models of substance
abuse, schizophrenia, and L-DOPA-induced dyskinesia. In addi-
tion, since most dopamine receptor imaging agents have been
either antagonists (e.g., [11C]raclopride and [18F]fallypride)
or full agonists (e.g., [11C]-(þ)-4-propyl-9-hydroxynaphth-
oxazine ([11C]-(þ)-PHNO) and [11C]-N-propylapomorphine
([11C]NPA)) at both D2 and D3 receptors, it will be of interest to
see if the partial agonists described here are capable of serving as
radiotracers for imaging the D3 receptor in vivo with PET.
In summary, we observed that the 2-methoxy group in the
4-(2-methoxyphenyl)piperazinyl moiety can be replaced with a
2-fluoroethoxy group, a commonly used strategy for preparing
18F-labeled PET radiotracers, without causing a significant
change in D3 receptor affinity or D3 vs D2 selectivity ratio. An
exception to this trend was the structural congeners which
contained the 4-(2-fluoroethyl)benzamide moiety: 21b had a
much higher D3 affinity than its 4-(2-methoxyphenyl)pipe-
razine analogue, 19a (Ki = 1.1 ( 0.2 nM vs 24.9 ( 3.3 nM,
respectively), and 20b, which had a lower D3 affinity and poorer
D3 vs D2 selectivity ratio than its corresponding 4-(2-methox-
yphenyl)piperazine analogue, 2. Replacing the saturated 4-car-
bon spacer that links the benzamide and the 4-phenylpiperazinyl
moieties with a trans double bond reduced the binding affinity at
D3 and D2 receptors. However, the presence of the trans double
bond can modulate the intrinsic efficacy of the analogue. Finally,
although increasing the length of the 2-fluoroethoxy side chain by
pegylation did not dramatically alter the D3 binding affinity or the
D3 vs D2 receptor binding selectivity, it did decrease the log P
value, which may facilitate the penetration of the blood-brain
barrier.
Scientific (Columbia, SC). All other synthetic intermediates were
purchased from Sigma-Aldrich and used as received unless otherwise
stated. Tetrahydrofuran (THF) was distilled from sodium hydride
immediately prior to use.
All air-sensitive reactions were carried out in oven-dried glassware
under an inert nitrogen atmosphere unless otherwise stated. Standard
handling techniques for air-sensitive materials were employed through-
out this study. Yields were not optimized. Melting points were deter-
mined on a Haake-Buchler or Mel-Temp melting point apparatus and
1
are uncorrected. H NMR spectra were recorded at 300 MHz on a
Varian Mercury-VX spectrometer with CDCl3 as the solvent and
tetramethylsilane (TMS) as the internal standard. The following ab-
breviations were used to describe peak patterns wherever appropriate: br
= broad, d = doublet, t = triplet, q = quartet, and m = multiplet. Analytical
thin-layer chromatography (TLC) was performed on Analtech GHLF
silica gel glass plates, and visualization was aided by UV. Elemental
analyses (C, H, N) were determined by Atlantic Microlab, Inc.
(Norcross, GA), and the results are within 0.4% of the calculated values
unless otherwise noted. The purity of the target compounds was
determined by elemental analysis and by HPLC methods. All of the
compounds reported in this paper have a purity g95%. The synthesis of
benzoic acid intermediates 9a-e, 12a,b, and the amine intermediates
17a-d can be found in the Supporting Information section.
General Method for Preparing the Substituted Benza-
mide Analogues. 4-(2-Fluoroethoxy)-N-(4-(4-(2-methoxyphenyl)-
piperazin-1-yl)-butyl)benzamide (18a). A mixture of compound 17a
(379 mg, 1.44 mmol) and 9a (221 mg, 1.20 mmol) in dichloromethane
(20 mL) was stirred at 0 °C (ice-water bath). Dicyclohexylcarbodii-
mide (DCC) (356 mg, 1.73 mmol) and hydroxybenzotriazole (HOBT)
(234 mg, 1.73 mmol) were added to the above solution. Then the ice
bath was removed, and the reaction mixture was stirred at ambient
temperature for 15 h. Dichloromethane (60 mL) was added into the
reaction mixture, and the solution was washed with saturated aqueous
NaHCO3 solution (3 ꢀ 10 mL). The organic layer was dried over
Na2SO4 and concentrated under reduced pressure, and the crude product
was purified by silica gel column chromatography using dichloro-
methane/methanol (20/1, v/v) as the mobile phase to give 18a (443
mg, 86%). Mp (oxalate salt): 151.5-152.6 °C. 1H NMR (300 MHz, free
base, CDCl3): δ 1.61-1.69 (m, 4H), 2.48 (t, J = 5.2 Hz, 2H), 2.66
(s, 4H), 3.08 (s, 4H), 3.48 (q, J = 5.7 Hz, 2H), 3.85 (s, 3H), 4.19 (t, J =
4.2 Hz, 1H), 4.28 (t, J = 4.2 Hz, 1H), 4.68 (t, J = 4.2 Hz, 1H), 4.84 (t, J =
4.2 Hz, 1H), 6.61 (br s, 1H), 6.82-7.04 (m, 6H), 7.74 (d, J = 8.7 Hz,
’ CONCLUSION
In the present study, we have reported the synthesis and
pharmacological evaluation of a series of benzamides which have
high binding affinity for D3 receptors and good selectivity for D3
vs D2 receptors. Within the series, five compounds exhibited high
D3 binding affinity (e5.0 nM) and/or moderate to high selec-
tivity for D3 vs D2 receptors, including 18a, 20a, 20c, 20e, and
21e. Moreover, all of these analogues contain a fluorine atom,
thus providing candidate ligands for PET imaging studies via the
corresponding 18F-labeled analogues.
Since recent studies indicate that the density of D3 receptors in
the striatal regions of brain is ∼40% that of the D2 receptor,32
ligands having a high D3 versus D2 selectivity (>50-fold) will
likely be needed in order to image D3 versus D2 receptors in the
CNS. Among the five compounds described above, 20e dis-
played the highest D3 affinity (0.17 nM) and selectivity for D3 vs
D2 receptors (163-fold). However, the high lipophilicity of this
analogue (log P = 4.67) may limit its utility as a PET radiotracer
because of its predicted low brain uptake and relatively high
level of nonspecific binding. In vivo evaluation of a number of
the fluorinated ligands described above is currently ongoing to
assess their suitability for use as PET tracers for studying the
in vivo expression and regulation of D3 dopamine receptors in
the CNS.
2H). Anal. (C24H32FN3O3 1.5H2C2O4) C, H, N.
3
4-(2-(2-Fluoroethoxy)ethoxy)-N-(4-(4-(2-methoxyphenyl)piperazin-
1-yl)butyl)benzamide (18b). 18b was made from 12a and 17a. Yield:
84%. Mp (oxalate salt): 127.9-129.0 °C. 1H NMR (free base, CDCl3):
δ 1.66-1.68 (m, 4H), 2.47 (t, J = 3.6 Hz, 2H), 2.65 (s, 4H), 3.08 (s, 4H),
3.47 (q, J = 5.4 Hz, 2H), 3.76 (t, J = 4.8 Hz, 1H), 3.85 (s, 3H), 3.91 (t, J =
3.6 Hz, 2H), 4.17 (t, J = 4.8 Hz, 2H), 4.52 (t, J = 4.2 Hz, 1H), 4.67 (t, J =
4.2 Hz, 1H), 6.58 (t, J = 10.0 Hz, 1H), 6.83-7.04 (m, 6H), 7.72 (d, J =
9.0 Hz, 2H). Anal. (C26H36FN3O4 H2C2O4) C, H, N.
3
4-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)-N-(4-(4-(2-methoxyphe-
nyl)piperazin-1-l)butyl)benzamide (18c). 18c was prepared from 12b
and 17a. Yield: 98%. Mp (oxalate salt): 103.0-103.9 °C. 1H NMR (free
base, CDCl3): δ 1.60 (s, 4H), 1.67 (t, J = 3.3 Hz, 4H), 2.47 (t, J = 4.8 Hz,
2H), 2.66 (s, 4H), 3.08 (s, 4H), 3.47 (q, J = 5.7 Hz, 2H), 3.69-3.76 (m,
4H), 3.80 (t, J = 4.2 Hz, 1H), 3.86 (s, 3H), 3.88 (t, J = 4.8 Hz, 2H), 4.16
(t, J = 4.8 Hz, 2H), 4.48 (t, J = 4.2 Hz, 1H), 4.64 (t, J = 4.2 Hz, 1H), 6.54
(t, J = 9.0 Hz, 1H), 6.83-7.04 (m, 6H), 7.72 (d, J = 9.0 Hz, 2H). Anal.
(C28H40FN3O5 H2C2O4 0.4H2O) C, H, N.
3
3
4-(2-Fluoroethyl)-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)-trans-
butyl-2-enyl)benzamide (19a). 19a was prepared from 4-(2-fluor-
oethyl)benzoic acid and 17c. Yield: (397 mg, 98%). Mp (oxalate salt):
116.7-121.3 °C. 1H NMR (free base, CDCl3): δ 2.63 (s, 4H), 3.03 (t,
’ EXPERIMENTAL SECTION
General. 4-Dimethylaminobenzoic acid was purchased from Sigma-
Aldrich (Milwaukee, WI) and 3-thienylbenzoic acid from Matrix
1560
dx.doi.org/10.1021/jm101323b |J. Med. Chem. 2011, 54, 1555–1564

Journal of Medicinal Chemistry
ARTICLE
J = 4.8 Hz, 2H), 3.08- 3.12 (m, 6H), 3.85 (s, 3H), 4.06-4.08 (t, J = 6.2
Hz, 2H), 4.57 (t, J = 4.8 Hz, 1H), 4.72 (t, J = 4.8 Hz, 1H), 5.78 (t, J = 5.4
Hz, 2H), 6.80 (t, J = 6.3 Hz, 1H), 6.82-7.04 (m, 4H), 7.31 (d, J = 8.4 Hz,
2H), 7.73 (d, J = 8.4 Hz, 2H). Anal. (C24H30FN3O2 0.5H2C2O4 H2O)
4-(2-Fluoroethyl)-N-(4-(4-(2-(2-fluoroethoxy)phenyl)piperazin-1-yl)-
butyl)benzamide (20b). 20b was prepared from 4-(2-fluor-
oethyl)benzoic acid and 17b. Yield: 95%. Mp (oxalate salt): 140.5-
142.1 °C. 1H NMR (free base, CDCl3): δ 1.60-1.78 (m, 4H), 2.46 (t, J
= 6.8 Hz, 2H), 2.63 (s, 4H), 3.00 (t, J = 6.4 Hz, 2H) 306-3.10 (m, 4H),
3.48 (q, J = 5.4 Hz, 2H), 4.20 (t, J = 4.2 Hz, 1H), 4.29 (t, J = 4.2 Hz, 1H),
4.54 (t, J = 6.4 Hz, 1H), 4.67-4.71 (m, 2H), 4.85 (t, J = 4.1 Hz, 1H), 6.72
(br s, 1H), 6.83-6.90 (m, 2H), 6.95-6.98 (m, 2H), 7.28 (d, J = 8.1 Hz,
3
3
C, H, N.
4-(2-Fluoroethoxy)-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)-trans-
butyl-2-enyl)benzamide (19b). 19b was prepared from 9a and 17c.
1
Yield: 77%. Mp (oxalate salt): 133.6-134.9 °C. H NMR (free base,
CDCl3): δ 2.67 (s, 4H), 3.08-3.09 (m, 6H), 3.86 (s, 3H), 4.09 (t,
J = 4.3 Hz, 2H), 4.22 (t, J = 4.2 Hz, 1H), 4.30 (t, J = 4.2 Hz, 1H), 4.69 (t,
J = 4.2 Hz, 1H), 4.85 (t, J = 4.2 Hz, 1H), 5.78 (t, J = 5.3 Hz, 2H), 6.13 (t,
J = 6.3 Hz, 1H), 6.83-7.04 (m, 6H), 7.75 (d, J = 9.3 Hz, 2H). Anal.
(C24H30FN3O3) C, H, N.
2H), 7.71(dd, J = 2.1, 6.3 Hz, 2H). Anal. (C25H33F2N3O2 0.5H2C2O4)
3
C, H, N.
4-(2-Fluoroethoxy)-N-(4-(4-(2-(2-fluoroethoxy)phenyl)piperazin-1-
yl)butyl)benzamide (20c). 20c was prepared from 9a and 17b. Yield:
80%. Mp (oxalate salt): 110.3-112.8 °C. 1H NMR (free base, CDCl3):
δ 1.60-1.72 (m, 4H), 2.45 (t, J = 6.8 Hz, 2H), 2.63 (s, 4H), 3.10 (s, 4H),
3.47 (q, J = 5.7 Hz, 2H), 4.20 (t, J = 4.5 Hz, 2H), 4.28 (t, J = 4.5 Hz, 2H),
4.69 (t, J = 4.5 Hz, 2H), 4.83 (t, J = 4.5 Hz, 2H), 6.59 (br s, 1H), 6.83-
4-(2-(2-Fluoroethoxy)ethoxy)-N-(4-(4-(2-methoxyphenyl)piperazin-
1-yl)-trans-butyl-2-enyl)benzamide (19c). 19c was prepared from 12a
and 17c. Yield: 99%. Mp (oxalate salt): 108.3-109.8 °C. 1H NMR (free
base, CDCl3): δ 2.67 (s, 4H), 3.07-3.10 (m, 6H), 3.77 (t, J = 4.2 Hz,
1H), 3.85 (s, 3H), 3.86-3.94 (m, 3H), 4.06-4.10 (m, 2H), 4.19 (m,
2H), 4.52 (t, J = 4.1 Hz, 1H), 4.68 (t, J = 4.1 Hz, 1H), 5.78 (t, J = 3.3 Hz,
2H), 6.10 (t, J = 5.3 Hz, 1H), 6.82-7.04 (m, 6H), 7.73 (d, J = 8.7 Hz,
7.00 (m, 6H), 7.73 (d, J = 9.0 Hz, 2H). Anal. (C25H33F2N3O3 H2C2O4)
3
C, H, N.
4-(2-(2-Fluoroethoxy)ethoxy)-N-(4-(4-(2-(2-fluoroethoxy)phenyl)
piperazin-1-yl)butyl)benzamide (20d). 20d was prepared from 12a and
1
2H). Anal. (C26H34FN3O4 H2C2O4) C, H, N.
17b. Yield: 77%. Mp (oxalate salt): 110.5-112.6 °C. H NMR (free
3
2-(2-Fluoroethoxy)-5-methyl-N-(4-(4-(2-methoxyphenyl)piperazin-
1-yl)-trans-butyl-2-enyl)benzamide (19d). 19d was prepared from 9b
base, CDCl3): δ 1.67-1.71 (m, 4H), 2.50 (t, J = 6.3 Hz, 2H), 2.68 (s,
4H), 3.13 (s, 4H), 3.47 (q, J = 5.4 Hz, 2H), 3.77 (t, J = 4.1 Hz, 1H),
3.85-3.91 (m, 3H), 4.16-4.213.47 (m, 3H), 4.29 (t, J = 4.4 Hz, 1H),
4.51 (t, J = 4.1 Hz, 1H), 4.66-4.7169 (m, 2H), 4.85 (t, J = 4.1 Hz, 1H),
6.6159 (br s, 1H), 6.83-7.04 (m, 6H), 7.73 (d, J = 9.0 Hz, 2H). Anal.
1
and 17c. Yield: 79%. Mp (oxalate salt): 151.1-152.3 °C. H NMR
(free base, CDCl3): δ 2.33 (s, 3H), 2.67 (s, 4H), 3.07-3.09 (m, 6H),
3.85 (s, 3H), 4.06-4.14 (m, 2H), 4.26 (t, J = 4.1 Hz, 1H), 4.35 (t, J =
4.1 Hz, 1H), 4.70 (t, J = 4.1 Hz, 1H), 4.86 (t, J = 4.1 Hz, 1H), 5.79 (t, J =
2.4 Hz, 2H), 6.80-7.04 (m, 5H), 7.214 (dd, J = 2.4, 8.7 Hz, 1H), 7.98
(C27H37F2N3O4 H2C2O4) C, H, N.
3
4-(Thiophen-3-yl)-N-(4-(4-(2-(2-fluoroethoxy)phenyl)piperazin-1-yl)
butyl)benzamide (20e). 20e was prepared from 4-(thiophen-3-yl)
benzoic acid and 17b. Yield: 62%. Mp (oxalate salt): 193.3-194.1 °C.
1H NMR (free base, CDCl3): δ 1.68-1.72 (m, H), 2.48 (t, J = 6.0 Hz,
2H), 2.65 (s, 4H), 2.10 (s, 4H), 3.50 (q, J = 5.7 Hz, 2H), 4.19 (t, J = 4.2
Hz, 1H), 4.28 (t, J = 4.2 Hz, 1H), 4.69 (t, J = 4.2 Hz, 1H), 4.84 (t, J = 4.2
Hz, 1H), 6.79 (br s, 1H), 6.82-7.00 (m, 4H), 7.41 (d, J = 2.7 Hz, 2H),
7.52 (t, J = 2.7 Hz, 1H), 7.64 (d, J = 8.7 Hz, 2H), 7.80 (d, J = 8.7 Hz, 2H).
(br s, 1H), 8.00 (dd, J = 2.4, 8.7 Hz, 1H), Anal. (C25H32FN3O3
H2C2O4) C, H, N.
3
5-Bromo-2-(2-fluoroethoxy)-N-(4-(4-(2-methoxyphenyl)piperazin-
1-yl)-trans-butyl-2-enyl)benzamide (19e). 19e was prepared from 9c
and 17c. Yield: 35%. Mp (oxalate salt): 157.6-158.7 °C. 1H NMR (free
base, CDCl3): δ 2.65(s, 4H), 3.06-3.10 (m, 6H), 3.85 (s, 3H), 4.09 (t,
J = 5.1 Hz, 2H), 4.27 (t, J = 4.1 Hz, 1H), 4.37 (t, J = 4.1 Hz, 1H), 4.71 (t,
J = 4.1 Hz, 1H), 4.87 (t, J = 4.1 Hz, 1H), 5.76-5.80 (m, 2H), 6.80-7.04
(m, 5H), 7.52 (dd, J = 2.4, 8.4 Hz, 1H), 7.61 (br s, 1H), 8.32 (d, J = 5.4
Hz, 1H). Anal. (C24H29BrFN3O3) C, H, N.
Anal. (C27H32FN3O2S H2C2O4) C, H, N.
3
4-(2-(2-(2-Fluoroethoxy)ethoxy)ethoxy)-N-(4-(4-(2-(2-fluoroethoxy)
phenyl)piperazin-1-yl)butyl)benzamide (20f). 20f was prepared from
12b and 17b. Yield: 82%. Mp (oxalate salt): 110.7-111.6 °C. 1H NMR
(free base, CDCl3): δ 1.67-1.69 (m, 4H), 2.48 (t, J = 5.9 Hz, 3H), 2.67
(s, 4H), 3.12 (s, 4H), 3.47 (q, J = 5.4 Hz, 2H), 3.68-3.76 (m, 4H), 3.80
(t, J = 4.2 Hz, 1H), 3.87 (t, J = 4.8 Hz, 2H), 4.12 (t, J = 4.8 Hz, 2H), 4.20
(t, J = 4.2 Hz, 1H), 4.31 (t, J = 4.2 Hz, 1H), 4.48 (t, J = 4.2 Hz, 1H), 4.64
(t, J = 4.2 Hz, 1H), 4.69 (t, J = 4.2 Hz, 1H), 4.85 (t, J = 4.2 Hz, 1H), 6.55
(br s, 1H), 6.82-7.02 (m, 6H), 7.73 (d, J = 9.0 Hz, 2H). Anal.
2-(2-Fluoroethoxy)-5-iodo-N-(4-(4-(2-methoxyphenyl)piperazin-1-
yl)-trans-butyl-2-enyl)benzamide (19f). 19f was prepared from 9d and
1
17c. Yield: 60%. Mp (oxalate salt): 162.2-163.6 °C. H NMR (free
base, CDCl3): δ 2.65 (s, 4H), 3.07-3.10 (m, 6H), 3.86 (s, 3H), 4.09 (t,
J = 5.1 Hz, 2H), 4.27 (t, J = 4.1 Hz, 1H), 4.36 (t, J = 4.1 Hz, 1H), 4.72 (t,
J = 4.1 Hz, 1H), 4.88 (t, J = 4.1 Hz, 1H), 5.74-5.82 (m, 2H), 6.71 (d, J =
8.7 Hz, 1H), 6.84-7.04 (m, 4H), 7.71 (dd, J = 2.4, 8.4 Hz, 1H), 7.83 (br
s, 1H), 8.49 (d, J = 2.1 Hz, 1H). Anal. (C24H29FIN3O3 H2C2O4
3
3
(C29H41F2N3O5 H2C2O4) C, H, N.
3
0.5H2O) C, H, N.
4-(Dimethylamino)-N-(4-(4-(2-(2-fluoroethoxy)phenyl)piperazin-1-
yl)-trans-butyl-2-enyl)benzamide (21a). 21a was prepared from 4-di-
methylaminobenzoic acid and 17d. Yield: 50%. Mp (oxalate salt): 84.9-
85.9 °C. 1H NMR (free base, CDCl3) δ 2.66 (s, 4H), 3.01 (s, 6H), 3.07
(d, J = 4.5 Hz, 2H), 3.14 (s, 4H), 4.09 (t, J = 5.4 Hz, 2H), 4.21 (t, J =
4.2 Hz, 1H), 4.30 (t, J = 4.2 Hz, 1H), 4.70 (t, J = 4.2 Hz, 1H), 4.86 (t, J =
4.2 Hz, 1H), 5.76-5.80 (m, 2H), 6.05 (s, 1H), 6.66 (d, J = 9.0 Hz, 2H),
6.82-6.87 (m, 1H), 6.94-6.97 (m, 3H), 7.68 (d, J = 9.0 Hz, 2H). Anal.
(C25H33FN4O2 H2C2O4 H2O) C, H, N.
5-Bromo-2-(2-fluoroethoxy)-3-methoxy-N-(4-(4-(2-methoxyphe-
nyl)piperazin-1-yl)-trans-butyl-2-enyl)benzamide (19g). 19g was pre-
pared from 9e and 17c. Yield: 84%. Mp (oxalate salt): 193.5-195.5 °C.
1H NMR (free base, CDCl3): δ 2.66 (s, 4H), 3.06-3.10 (m, 6H), 3.86
(s, 3H), 3.87 (s, 3H), 4.06 (t, J = 4.2 Hz, 2H), 4.27 (t, J = 4.1 Hz, 1H),
4.36 (t, J = 4.1 Hz, 1H), 4.62 (t, J = 3.9 Hz, 1H), 4.78 (t, J = 3.9 Hz, 1H),
5.76 (t, J = 3.3 Hz, 2H), 6.84-7.04 (m, 4H), 7.14 (d, J = 2.4 Hz, 1H),
7.88 (d, J = 2.1 Hz, 1H), 8.04 (br s, 1H). Anal. (C25H31BrFN3O4
H2C2O4) C, H, N.
3
3
3
4-(2-Fluoroethyl)-N-(4-(4-(2-(2-Fluoroethoxy)phenyl)piperazin-1-yl)-
trans-butyl-2-enyl)benzamide (21b). 21b was prepared from 4-(2-fluor-
oethyl)benzoic acid and 17d. Yield: 72%. Mp (oxalate salt): 155.0-
156.1 °C. 1H NMR (free base, CDCl3): δ 2.67 (s, 4H), 3.01 (t, J = 4.1 Hz,
2H), 3.07-3.13(m, 6H),4.10 (t, J=4.1Hz, 2H),4.20-4.20(t, J=4.1 Hz,
1H), 4.30 (t, J = 4.1 Hz, 1H), 4.57 (t, J = 6.3 Hz, 1H), 4.68-4.76 (m, 2H),
4.84 (t, J = 4.1 Hz, 1H), 5.76-5.80 (m, 2H), 6.18 (br s, 1H), 6.82-6.88
(m, 1H), 6.95-6.97 (m, 3H), 7.29 (d, J = 8.1 Hz, 2H), 7.704 (d, J = 8.1 Hz,
4-(Dimethylamino)-N-(4-(4-(2-(2-fluoroethoxy)phenyl)piperazin-
1-yl)butyl)benzamide (20a). 20a was prepared from 4-dimethylamino-
benzoic acid and 17b. Yield: 80%. Mp (oxalate salt): 103.4-106.3 °C. 1H
NMR (free base, CDCl3): δ 1.60-1.80 (m, 4H), 2.45 (t, J = 2.1 Hz, 2H),
2.65 (s, 4H), 3.00 (s, 6H), 3.12 (s, 4H), 3.46 (q, J = 5.4 Hz, 2H), 4.19 (t, J =
4.1 Hz, 1H), 4.32 (t, J = 4.1 Hz, 1H), 4.69 (t, J = 4.1 Hz, 1H), 4.85 (t, J = 4.1
Hz, 1H), 6.36 (br s, 1H), 6.66 (d, J = 9.2 Hz, 2H), 6.83-7.00 (m, 4H),
7.676 (d, J = 9.2 Hz, 2H). Anal. (C25H35FN4O2 H2C2O4) C, H, N.
2H). Anal. (C25H31F2N3O2 0.5H2C2O4) C, H, N.
3
3
1561
dx.doi.org/10.1021/jm101323b |J. Med. Chem. 2011, 54, 1555–1564

Journal of Medicinal Chemistry
ARTICLE
4-(2-Fluoroethoxy)-N-(4-(4-(2-(2-fluoroethoxy)phenyl)piperazin-1-
yl)-trans-butyl-2-enyl)benzamide (21c). 21c was prepared from 9a and
17d. Yield: 48%. Mp (oxalate salt): 112.8-125.1 °C. H NMR (free
base, CDCl3): δ 2.68 (s, 4H), 3.09 (s, 3H), 3.15 (s, 3H), 4.10 (t, J =
4.2 Hz, 2H), 4.20 (t, J = 4.2 Hz, 2H), 4.30 (t, J = 4.2 Hz, 2H), 4.70 (t, J =
4.2 Hz, 2H), 4.86 (t, J = 4.2 Hz, 2H), 5.70-5.90 (m, 2H), 6.170 (br s,
1H), 6.81-6.85 (m, 1H), 6.93-7.00 (m, 5H), 7.75 (d, J = 10.5 Hz, 2H).
temperature. Nonspecific binding was determined from samples that
contained 10 μM cold haloperidol. After 90 min, the reaction was
terminated by adding 150 μL of ice-cold wash buffer (10 mM Tris-HCl,
150 mM NaCl, pH = 7.4) using a 96-channel transfer pipet (Fisher
Scientific, Pittsburgh, PA). The samples were harvested and filtered
rapidly through a 96-well fiber glass filter plate (Millipore, Billerica, MA)
that had been presoaked with 100 μL of 50 mM Tris-HCl buffer at pH =
8.0 for 1 h. Each filter was washed three times with 200 μL of ice-cold
wash buffer, and the filter was counted in a Wallac 1450 MicroBeta liquid
scintillation counter (Perkin-Elmer, Boston, MA).
1
Anal. (C25H31F2N3O3 2H2C2O4) C, H, N.
3
4-(2-(2-Fluoroethoxy)ethoxy)-N-(4-(4-(2-(2-fluoroethoxy)phenyl)
piperazin-1-yl)-trans-but-2-enyl)benzamide (21d). 21d was prepared
from 12a and 17d. Yield: 76%. Mp (oxalate salt): 113.4-115.0 °C. 1H
NMR (free base, CDCl3): δ 2.67 (s, 4H), 3.08 (d, J = 2.7 Hz, 2H), 3.84-
3.14 (s, 4H), 3.77 (t, J = 4.2 Hz, 1 H), 3.86-3.96 (m, 3H), 4.08 (t, J =
4.2 Hz, 2H), 4.17-4.22 (m, 3H), 4.30 (t, J = 4.2 Hz, 1H), 4.52 (t, J =
4.2 Hz, 1H), 4.66-4.70 (m, 2H), 4.85 (t, J = 4.2 Hz, 1H), 5.77-5.80 (m,
2H), 6.11 (t, J = 8.7 Hz, 1H), 6.84-6.88 (m, 1H), 6.93-6.99 (m, 5H),
The σ2 receptor binding assays were conducted using rat liver
membrane homogenates (∼300 μg of protein) and ∼5 nM [3H]-
DTG (58.1 Ci/mmol; Perkin-Elmer, Boston, MA) in the presence of 1
μM (þ)-pentazocine to block σ1 sites. The incubation time was 2 h at
room temperature. Nonspecific binding was determined from samples
that contained 10 μM cold haloperidol. All other procedures were
identical to those described above for the σ1 receptor binding assay.
Data from the competitive inhibition experiments were modeled using
nonlinear regression analysis to determine the concentration that inhibits
50% of the specific binding of the radioligand (IC50 value). Competitive
curves were best fit to a one-site fit and gave pseudo-Hill coefficients of
0.6-1.0. Ki values were calculated using the Cheng and Prusoff method35
and were presented as the mean ( SEM. For these calculations, we used a
Kd value of 7.89 nM for (þ)-[3H]pentazocine and guinea pig brain and a
Kd value of 30.73 nM for [3H]-DTG and rat liver.24
7.74 (td, J= 2.4, 9.0Hz, 2H). Anal. (C27H35F2N3O4 1.5H2C2O4) C, H, N.
3
4-(Thiophen-3-yl)-N-(4-(4-(2-(2-fluoroethoxy)phenyl)piperazin-1-
yl)-trans-but-2-enyl)benzamide (21e). 21e was prepared from 4-
(thiophen-3-yl)benzoic acid and 17d. Yield: 62%. Mp (oxalate salt):
140.5-142.1 °C. 1H NMR (free base, CDCl3): δ 2.66 (s, 4H), 3.08 (d, J
= 4.0 Hz, 2H), 3.14 (s, 4H), 4.12 (t, J = 4.0 Hz, 2H), 4.22 (t, J = 4.0 Hz,
1H), 4.30 (t, J = 4.0 Hz, 1H), 4.70 (t, J = 4.0 Hz, 1H), 4.86 (t, J = 4.0 Hz,
1H), 5.80-5.82 (m, 2H), 6.23 (br s, 1H), 6.83-6.88 (m, 1H), 6.93-
7.00 (m, 3H), 7.42 (d, J = 10.2 Hz, 2H), 7.54 (t, J = 2.1 Hz, 1H), 7.66 (td,
J = 3.6, 10.5 Hz, 2H), 7.81 (td, J = 3.3, 13.2 Hz, 2H). The elemental
analysis was conducted on the free base of 21e. Anal. (C27H30FN3O2S)
C, H, N.
3
Whole Cell Adenylyl Cyclase Assay. The accumulation of H-cyclic
AMP in HEK cells was measured by a modification of the method of
Shimizu et al.36 Transfected HEK cells were treated with serum-free
media containing 2,8-[3H]adenine (ICN Pharmaceutical Inc., Costa
Mesa, CA), and cells were incubated at 37 °C for 75 min. Cells and drugs
diluted in serum-free media containing 0.1 mM 3-isobutyl-1-methyl-
xanthine (Sigma) were mixed to give a final volume of 500 μL, and cells
were incubated for 20 min at 37 °C. The reaction was stopped by
addition of 500 μL of 10% trichloroacetic acid and 1 mM cyclic AMP.
After centrifugation, 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 spectrophotometric
analysis at OD 259 nm.34,36
In Vitro Binding Studies. Dopamine Receptor Binding Assays.
The binding properties of membrane-associated receptors were char-
acterized by a filtration binding assay.34 For human D2 long, D3, and D4
dopamine receptors expressed in HEK 293 cells, 50 μL of membrane
homogenates was suspended in 50 mM Tris-HCl/150 mM NaCl/
10 mM EDTA buffer, pH = 7.5 and incubated with 50 μL of
[
125I]IABN34 at 37 °C for 60 min, using 20 μM (þ)-butaclamol to
define the nonspecific binding. The radioligand concentration was equal
to approximately 0.5 times the Kd value, and the concentration of the
competitive inhibitor ranged over 5 orders of magnitude for competition
experiments. For each competition curve, two concentrations of in-
hibitor per decade were used, and triplicates were performed. Binding
was terminated by the addition of the cold wash buffer (10 mM Tris-
HCl/150 mM NaCl, pH = 7.5) and filtration over a glass-fiber filter
(Schleicher and Schuell No. 32). A Packard Cobra gamma counter was
used to measure the radioactivity. The equilibrium dissociation constant
and maximum number of binding sites were generated using unweighted
nonlinear regression analysis of data modeled according to the equation
describing mass action binding. The concentration of inhibitor that
inhibits 50% of the specific binding of the radioligand (IC50 value) was
determined by using nonlinear regression analysis to analyze the data of
competitive inhibition experiments. Competition curves were modeled
for a single site, and the IC50 values were converted to equilibrium
dissociation constants (Ki values) using the Cheng and Prusoff35
correction. Mean Ki values ( SEM are reported for at least three
independent experiments.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures and
b
analytical data for compounds 9a-e, 10a-c, 11a, 11b, 12a, 12b,
14, 16a-f, and 17a-d, HPLC conditions to confirm the purity
of final compounds, and elemental analysis data on all new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (314) 362-8538. Fax: (314) 362-0039. E-mail: rhmach@
mir.wustl.edu.
Sigma Receptor Binding Assays. Before determining the σ1 and σ2
receptor binding assays, the compounds were dissolved in either DMF,
DMSO, or ethanol and then diluted in 50 mM Tris-HCl buffer containing
150 mM NaCl and 100 mM EDTA at pH = 7.4. The procedures for
isolating the membrane homogenates and performing the σ1 and σ2
receptor binding assays have been described previously.24,33
Briefly, the σ1 receptor binding assays were conducted in 96-well
plates using guinea pig brain membrane homogenates (∼300 μg of
protein) and ∼5 nM (þ)-[3H]pentazocine (34.9 Ci/mmol; Perkin-
Elmer, Boston, MA). The total incubation time was 90 min at room
’ ACKNOWLEDGMENT
This work was supported by NIH Grants DA16181 (R.H.M.),
DA23957 (R.R.L.), and DA029840 (R.H.M.).
’ ABBREVIATIONS
CIMS, chemical ionization mass spectrometry; DCC, N,
N0-dicyclohexylcarbodiimide; DMF, N,N-dimethylformamide;
DMSO, dimethyl sulfoxide;DAST, diethylaminosulfur trifluoride;
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ARTICLE
DTG, 1,3-ditolylguanidine; GIRKs, G protein-coupled inwardly
rectifying potassium channels; HEK cells, human embry-
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[
125I]-N-benzyl-5-
iodo-2,3-dimethoxy[3.3.1]azabicyclononan-3-β-ylbenzamide; LID,
L-DOPA-induced dyskinesia; PET, positron emission tomogra-
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