Conformationally-flexible benzamide analogues as dopamine D3 and σ2 receptor ligands

Article abstract of DOI:10.1016/j.bmcl.2003.09.083

A series of conformationally-flexible analogues was prepared and their affinities for D2-like dopamine (D₂, D₃ and D ₄) were determined using in vitro radioligand binding assays. The results of this structure-activity rela

Full text of DOI:10.1016/j.bmcl.2003.09.083

Bioorganic & Medicinal Chemistry Letters 14 (2004) 195–202
Conformationally-flexible benzamide analogues as dopamine D3
and ꢀ2 receptor ligands
Robert H. Mach,a,b,* Yunsheng Huang,a Rebekah A. Freeman,c Li Wu,a
Suwanna Vangveravonga and Robert R. Luedtkec
aDepartment of Radiology, PET Center, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA
bDepartment of Physiology & Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA
cDepartment of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107, USA
Received 14 July 2003; accepted 25 September 2003
Abstract—A series of conformationally-flexible analogues was prepared and their affinities for D2-like dopamine (D2, D3 and D4)
were determined using in vitro radioligand binding assays. The results of this structure–activity relationship study identified one
compound, 15, that bound with high affinity (Ki value=2 nM) and moderate selectivity (30-fold) for D3 compared to D2 receptors.
In addition, this series of compounds were also tested for affinity at s1 and s2 receptors. We evaluated the affinity of these dopa-
minergic compounds at sigma receptors because (a) several antipsychotic drugs, which are high affinity antagonists at dopamine
D2-like receptors, also bind to sigma receptors and (b) sigma receptors are expressed ubiquitously and at high levels (picomoles per
mg proteins). It was observed that a number of analogues displayed high affinity and excellent selectivity for s2 versus s1 receptors.
Consequently, these novel compounds may be useful for characterizing the functional role of s2 receptors and for imaging the s2
receptor status of tumors in vivo with PET.
# 2003 Elsevier Ltd. All rights reserved.
The dopamine receptor subtypes are members of the G
protein coupled receptor protein superfamily. Based
upon genetic and cDNA cloning studies it is currently
thought that there are five functionally active dopamine
receptor subtypes expressed in mammalian brain. These
five subtypes have been classified into two major clas-
ses, the D1-like and D2-like receptors. Stimulation of the
D1-like receptor subtypes, which include the D1 (D1a)
and the D5 (D1b) receptors, results in an activation of
adenylyl cyclase and an increase in the production of
cAMP. Agonist stimulation of the D2-like receptors,
which consist of the D2, D3 and D4 receptors, results in
an inhibition of adenylyl cyclase activity, an increase in
the release of arachadonic acid, and an increase in
phosphatidylinositol hydrolysis.
oping agents that are antagonists of the dopamine D3
receptor.1,2 This interest was largely generated by the
hypothesis that dopamine D3 receptors may play a
pivotal role in the development of a number of neuro-
logical and neuropsychiatric disorders. Receptor auto-
radiography studies have shown that both D2 and D3
receptors are widely distributed in striatal regions of
human3 and monkey4 brain. However, the higher ratio
of D3 versus D2 receptors in limbic structures indicates
that the D3 receptor may play an integral role in the
pathological abnormalities that occur in neuropsyciatric
disorders. Autoradiography studies have also revealed a
decrease of D3 receptors in the frontal cortex and an
increase in expression in the ventral striatum of schizo-
phrenics compared to normal individuals.5,6 Dopamine
D3 receptors are also believed to play a role in the dys-
kinesias associated with l-dopa treatment of patients
with Parkinson’s Disease. For example, chronic treat-
ment of squirrel monkeys with the neurotoxin, 1-
Over the past 5 years, there has been interest in devel-
methyl-4-phenyl-1,2,3,4-tetrahydropyridine
(MPTP),
Keywords: dopamine D3 receptors; atypical antipsychotics; s2 recep-
tors.
* Corresponding author at present address: Division of Radiological
Sciences, Washington University School of Medicine, Campus Box
8225, 510 S. Kingshighway Blvd., St. Louis, MO 63110, USA. Fax:
+1-314-362-0039; e-mail: rhmach@mir.wustl.edu
which causes a selective destruction of the nigrostriatal
dopaminergic system, results in a decline of D3 recep-
tors in the caudate (motor region) but not the putamen
of globus pallidus (limbic regions). However, treatment
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.083

196
R. H. Mach et al. / Bioorg. Med. Chem. Lett. 14 (2004) 195–202
with the drug levodopa led to a restoration of D3
receptor levels and a reversal of Parkinsonian symptoms
in MPTP-treated animals. Finally, the activation of
dopamine D3 receptors in the nucleus accumbens is
believed to be involved in the sensitization/rewarding
properties of psychostimulants, such as cocaine. There-
fore, agents that can block the interaction of psycho-
stimulant-induced increases in synaptic dopamine levels
with the D3 receptor have potential for the pharmaco-
logical treatment of cocaine abuse.1
selectivity for D3 versus D2 receptors. However, unlike
the naphthamide analogues, the pyrrole analogues
bound with low affinity at sigma receptors.12 As part of
a continuing effort to develop potent and selective D3
receptor ligands as potential radiotracers for studies of
the dopaminergic system with the noninvasive imaging
procedure, Positron Emission Tomography (PET), we
explored the possibility of preparing hybrid ligand
structures of the conformationally-flexible benzamide
analogues (Fig. 1) and the naphtamide and pyrrole
analogues (Fig. 2). The results of this study led to the
identification of a number of compounds possessing a
high affinity and moderate selectivity for dopamine D3
versus D2 receptors.
A number of conformationally-flexible benzamide ana-
logues displaying a high affinity and selectivity for D3
versus D2 receptors have been reported in recent years.
Examples of this include NGB 2849, NGB 2904, and
the structural congeners 1–3 (Fig. 1). A common struc-
tural feature in the conformationally-flexible benzamide
analogues is the N-2,3-dichlorophenylpiperazine ring
and the four carbon spacer group separating the benza-
mide and the basic amino moieties.7À10
In addition, the results of this structure–activity rela-
tionship study led to the identification of a number of
conformationally-flexible benzamide analogues that had
a high affinity and excellent selectivity for s2 versus s1
receptors. Sigma-2 receptors have been shown to be a
potential biomarker for determining the proliferative
status of breast tumors.13À15 Therefore, the results of
this study have led to the identification of lead com-
pounds for radiotracer development with two com-
pletely different functions: (1) D3 receptor imaging
agents for studying the change in dopamine receptor
function in a variety of neurological and neuropsychia-
tric disorders and, (2) s2 selective imaging agents for
measuring the proliferative status of breast tumors in
vivo with PET.
We have recently reported two different classes of com-
pounds, naphthamide analogues and pyrrole deriva-
tives, displaying a modest affinity and selectivity for D3
versus D2 receptors.11,12 For example, the naphthamide
analogues 4 and 5 have a moderate selectivity for D3
versus D2 receptors. Unfortunately, these compounds
were found to have a relatively high affinity for sigma
receptors (Fig. 2). Because sigma receptors are expres-
sed ubiquitously and at high levels (picomoles per mg
proteins), the high affinity of these compounds for
sigma receptors limits their utility for in vitro and in
vivo studies of dopamine D3 receptors.11 The pyrrole
analogues 6 and 7 have a high affinity and 10-fold
The strategy chosen for the current study involved the
combination of the following structural moieties of the
lead compounds: (a) the 2,3-dichlorophenylpiperazine
Figure 1.

R. H. Mach et al. / Bioorg. Med. Chem. Lett. 14 (2004) 195–202
197
Figure 2.
and the two- and four-carbon spacer groups of the
conformationally-flexible benzamide analogues shown
in Figure 1; (b) the naphthyl group of naphthamide
compounds shown in Figure 2; and, (c) the 2,3-dime-
thoxyphenyl and tetrahydroisoquinoline moieties of the
pyrrole analogues shown in Figure 2.
Table 1. Binding affinities for dopamine D2/D3 and sigma s1/s2
receptors
Compd
Ki (nM)a
b
c
d
e
D2
D3
s1
s2
11a
11b
12
13a
13b
14
131.6Æ24.6
240.5Æ19.4
81.6Æ21.28
126.5Æ42.4
15.1Æ1.7
189.1Æ2.6
1,159Æ7
47.7Æ2.5
21.2Æ0.1
17.6Æ0.7
The synthesis of the target tetrahydroisoquinoline ana-
logues is shown in Scheme 1.16 Reaction of the second-
ary amine of 8a and 8b with either bromoacetonitrile or
bromobutyronitrile gave N-alkylated products, 9a–d, in
75–85% yield. Reduction with either lithium aluminum
hydride in THF or hydrogenation over palladium on
charcoal gave the corresponding amines, 10a–d, in
quantitative yields. Condensation of amines 10a–d with
either 2-methoxy-5-bromonaphthoyl chloride12 or 5-
bromo-2,3-dimethoxybenzoic acid13 gave the corre-
sponding amide analogues, 11–14, in excess of 90%
yield. Synthesis of the 2,3-dichlorophenylpiperazine
benzamide and naphthamide analogues, 15 and 16, was
accomplished using a similar reaction sequence as out-
lined in Scheme 2.16
741.0Æ287.3 106.5Æ24.3
429.7Æ77.68.11
714.0Æ133.7
2200Æ390
Æ0.5
21.4Æ2.3
627Æ244
2.1Æ0.4
276.5Æ35.7 716.5Æ9.8
2932Æ28
12,900Æ111
809Æ66
16.4Æ2.0
8.2Æ1.4
15
16
58.8Æ13.7
75.0Æ4.1
26.4Æ1.4
107.0Æ19.0
10.2Æ5.3
751Æ6
a MeanÆSEM, Ki values were determined by at least three experiments.
b Ki values for D2 receptors were measured on rat D2(long) expressed in
Sf9 cells using [125I]IABN as the radioligand.18
c Ki values for D3 receptors were measured on rat D3 expressed in Sf9
cells using [125I]IABN as the radioligand.18
d Ki values for s1 receptors were measured on quinea pig brain mem-
branes using [3H](+)-pentazocine as the radioligand.17
e Ki values for s2 receptors were measured on rat liver membranes
using [3H]-DTG as the radioligand in the presence of (+)-pentazo-
cine.17
The naphthamide analogue 11a, which contains a two-
carbon spacer group between the amide nitrogen and
the basic amino group had a relatively low affinity for
dopamine D3 and D2 receptors (Table 1). Compound
11a also had an appreciable affinity for both s1 and s2
receptors. Introduction of a methoxy groups into the 4-
and 5-positions of the tetrahydro-isoquinoline ring
resulted in a reduction in affinity for D2, D3 and s1
receptors, and an increase in affinity for s2 receptors.
Increasing the length of the spacer group from two car-
bon units in 11b to four carbon units (i.e., 12) resulted
in no change in affinity for D3 and s2 receptors, but a
dramatic reduction in affinity for D2 and s1 receptors.
Replacement of the naphthamide group of 11a with a 5-
bromo-2,3-dimethoxy benzamide group (i.e., 13a)
resulted in an increase in affinity for dopamine D3
receptors, and a decrease in affinity for D2, s1 and s2
receptors. Introduction of the methoxy groups into the
4- and 5-positions of the tetrahydroisoquinoline ring of
13a to give 13b resulted in no change in affinity for D3
receptors and a reduction in affinity for D2 receptors.
However, this change in substitution pattern resulted in

198
R. H. Mach et al. / Bioorg. Med. Chem. Lett. 14 (2004) 195–202
Scheme 1. (a) BrCH2CN or BrCH2CH2CH2CN, Et3N, CH2Cl2, rt; (b) LiAlH4, THF or H2, Pd(c), ethanol; (c) ref 10; (d) ref 12.
a dramatic decrease in affinity for s1 receptors and a
large increase in affinity for s2 receptors. Increasing the
length of the spacer group from two carbons in 13b to
four carbons in 14 resulted in large decrease in affinity
for D2, D3, and s1 receptors and an increase in s2
receptor affinity. Compound 14 is unique in that is has
over a 1500-fold higher affinity for s2 versus s1 recep-
tors. Although many different classes of compounds
have been shown to bind with high affinity to sigma
receptors, most compounds studied to date have either
(a) a higher affinity for s1 versus s2 receptors, or (b)
equipotency with respect to binding to s1 and s2
receptors. Compound 14 is one of the most potent and
selective s2 receptor ligands reported to date.
sizes the importance in the 2,3-dichlorophenyl-piper-
azine group for binding to dopamine D3 receptors.
Compounds 17–20 were prepared with the goal of
increasing the s2 receptor affinity and reducing the
dopamine D2 and D3 receptor affinity of this class of
compounds (Scheme 3). The results of our previous
structure–activity relationship studies indicated that
both the 5-bromo and 3-methoxy groups were impor-
tant for the binding the binding of pyrrole analogues
shown in Figure 2 to dopamine D2 and D3 receptors.
Therefore, removal of the 3-methoxy group, or replace-
ment of the 5-bromo moiety with a methyl group, was
expected to result in a reduction in affinity for D2 and
D3 receptors. The results of the structure–activity rela-
tionship study are shown in Table 2. Compound 14 is
included in Table 2 for comparison. Removal of the 3-
methoxy group of 13a to give 17 resulted in a large
reduction in affinity for D2 and D3 receptors and an
increase in affinity for s1 and s2 receptors. The same
change in the structure of 13b to give compound 18 resul-
ted in a similar effect on dopamine receptor binding.
However, there was a marked decrease in affinity of 18 for
s1 receptors relative to that 13b. Compound 18 also had a
Compound 15 was prepared to determine the effect of
introducing the 5-bromo-2,3-dimethoxy benzamide
moiety into NGB 2904 and its structural congeners (Fig.
1) on D2 and D3 receptor affinity. In addition, compound
16 was prepared to assess the effect that increasing the
two-carbon spacer group of 2 (Fig. 1) to four carbons
would have on dopamine receptor affinity. Compounds
15 and 16 had the highest affinity for D3 receptors of the
analogues prepared in this study, which further empha-

R. H. Mach et al. / Bioorg. Med. Chem. Lett. 14 (2004) 195–202
199
Scheme 2. (a) BrCH2CH2CH2CN, Et3N, CH2Cl2; (b) H2, Pd(c), ethanol; (c) ref 12; (d) ref 10.
Table 2. Binding affinities of 17–20 for dopamine D2/D3 and sigma s1/s2 receptors
Compd
Ki (nM)a
b
c
d
e
D2
D3
s1
s2
s1/s2 ratio
14
17
18
19
20
2200Æ390
0.7 2190Æ35131
3570Æ796
627Æ244
Æ54.4
12,900Æ111
21.8Æ5.6
8.2Æ1.4
89.4Æ13.9
12.4Æ1.8
13.3Æ0.1783
10.3Æ1.5
1573
0.24
442
488.0Æ70.7
3760Æ618
313.0Æ141.0
5484Æ266
10,412Æ462
3078Æ87
2850Æ316
642.0Æ141.0
300
a MeanÆSEM, Ki values were determined by at least three experiments.
b Ki values for D2 receptors were measured on rat D2(long) expressed in Sf9 cells using [125I]IABN as the radioligand.
c Ki values for D3 receptors were measured on rat D3 expressed in Sf9 cells using [125I]IABN as the radioligand.
d Ki values for s1 receptors were measured on guinea pig brain membranes using [3H](+)-pentazocine as the radioligand.
e Ki values for s2 receptors were measured on rat liver membranes using [3H]-DTG as the radioligand in the presence of (+)-pentazocine.
higher affinity for s2 receptors than that of 13b. Replace-
ment of the 5-bromo moiety of 18 with a methyl group
(i.e., 19) resulted in a further reduction in affinity for D3
and s1 receptors, and no change in affinity for D2 and
s2 receptors. Increasing the length of the spacer group
of 19 from two carbons to four carbons (i.e., 20) resul-
ted in an increase in affinity for D2, D3 and s1 receptors,
and no change in affinity for s2 receptors. The high s1/s2
selectivity ratio of compounds 14, 18, 19, and 20 indicate
that they have the potential to be useful lead compounds
for the development of imaging agents for determining
the s2 receptor status of breast tumors with PET.
Table 3. In vitro binding data for dopamine D4 receptors
Compd
Ki (nM)a
b
c
d
D2
D3
D4
D2/D3 ratioe D4/D3 ratiof
13a
13b
15
429.7Æ76.71.18
714.0Æ133.7 21.4Æ2.3 265.0Æ60.0
58.8Æ13.7 2.1Æ0.4 800.0Æ330.0
107.0Æ19.0 10.2Æ5.3 1345Æ448
Æ0.5 47.9Æ11.6
24
33
28
10.5
2.7
12.4
381
132
16
a Same as Table 1.
b Same as Table 1.
c Same as Table 1.
d Ki for inhibiting the binding of [125I]IABN to human D4.4 receptors.
e Ki for D2/Ki for D3.
f
Ki for D4/Ki for D3.
Binding assays were also conducted on compounds 13a,
13b, 15 and 16 to determine their affinity for dopamine
D4.4 receptors. The results of this study are shown in
Table 3. All four compounds had a lower affinity for
D4.4 receptors relative to their binding potencies at
dopamine D3 receptors. Compound 15 had the highest
D3 receptor affinity and highest D2/D3 and D4/D3
selectivity ratios of the four compounds listed in Table
3. The presence of the methoxy groups indicates that a
carbon-11 labeled version of 15 can be prepared by

200
R. H. Mach et al. / Bioorg. Med. Chem. Lett. 14 (2004) 195–202
Scheme 3. (a) SOCl2, benzene, reflux; (b) 10a, Et3N, CH2Cl2, rt; (c) 10c, Et3N, CH2Cl2, rt; (d) 10d, CH2Cl2, Et3N, rt.
alkylation of the des-methyl precursor with 11C-methyl
iodide. We are currently exploring [11C]15 as a potential
radiotracer for imaging dopamine D3 receptors with
PET and the relatively low affinity of 15 for s1 and s2
receptors indicates that there will be minimal in vivo
binding to sigma receptors.
Defense Breast Cancer Research Program of the US
Army Medical Research and Materiel Command
Office.
References and notes
1. Luedtke, R. R.; Mach, R. H. Curr. Pharm. Des. 2003, 9,
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2. Hackling, A. E.; Stark, H. ChemBioChem. 2002, 3, 946.
3. Morissette, M.; Goulet, M.; Grodin, R.; Blancet, P.;
Bedard, P. J.; Di Paolo, T.; Levesque, D. Eur. J. Neurosci.
1998, 10, 1565.
4. Ryoo, H. L.; Pierrotti, D.; Joyce, J. N. Movement Dis-
orders 1998, 13, 788.
5. Gurevich, E. V.; Bordelon, Y.; Shapiro, R. M.; Arnold,
S. E.; Gur, R. E.; Joyce, J. N. Arch. Gen. Psych. 1997, 54,
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6. Gurevich, E. V.; Joyce, J. N. Neuropsychopharmacology
1999, 20, 60.
7. Glase, S. A.; Akunne, H. C.; Hefner, T. G.; Johnson, S. J.;
Kesten, S. R.; MacKenzie, R. G.; Manley, P. J.; Pugsley,
T. A.; Wright, J. L.; Wise, L. D. Bioorg. Med. Chem. Lett.
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9. Robarge, M. J.; Husbands, S. M.; Kielytka, A.; Brod-
beck, R.; Thurkauf, A.; Newman, A. H. J. Med. Chem.
2001, 44, 3175.
In conclusion, we have completed a structure–activity
relationship study on a series of benzamides with the
goal of identifying a potential radiotracer for imaging
dopamine D3 receptors with PET. The results of this
study revealed compound 15 as a candidate for labeling
D3 dopamine receptors in vivo. The low affinity binding
of 15 at sigma receptors subtypes servers to minimize
sigma receptor interactions as a potential source of
nonspecific/background binding. In addition, this study
has lead to the identification of a number of novel, s2-
receptor selective ligands. Based upon those findings we
are also exploring the potential of 11C-labeled versions
of 14, 18, 19 and 20, and 76Br-labeled versions of 14 and
18, as potential radiotracers for imaging the s2 receptor
status of breast tumors.
Acknowledgements
This research was funded by grants DA 12647 awarded
by the National Institute on Drug Abuse and
DAMD17-01-1-0446 awarded by the Department of
10. Bettinetti, L.; Schlotter, K.; Hubner, H.; Gmeiner, P. J.
Med. Chem. 2002, 45, 4597.

R. H. Mach et al. / Bioorg. Med. Chem. Lett. 14 (2004) 195–202
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11. Huang, Y.; Luedtke, R. R.; Freeman, R. A.; Wu, L.;
Mach, R. H. J. Med. Chem. 2001, 44, 1815.
J=8.7 Hz, 1H), 7.86 (s, 1H), 8.57 (t, J=5.4 Hz, 1H).
Calcd: C: 51.22; H: 5.05; N: 5.19. Obsvd: C: 51.23; H:
5.07; N: 5.04.
12. Mach, R. H.; Huang, Y.; Freeman, R. A.; Wu, L.; Blair,
S.; Luedtke, R. R. Bioorg. Med. Chem. 2003, 11, 225.
13. Mach, R. H.; Smith, C. R.; Al-Nabulsi, I.; Whirrett, B. R.;
Childers, S. R.; Wheeler, K. T. Cancer Res. 1997, 57, 156.
14. Al-Nabulsi, I.; Mach, R. H.; Sten, K.; Childers, S. R.;
Wheeler, K. T. Br. J. Cancer 1999, 81, 925.
15. Wheeler, K. T.; Wang, L.-M.; Wallen, C. A.; Childers,
S. R.; Cline, J. M.; Keng, P. C.; Mach, R. H. Br. J. Can-
cer 2000, 82, 1223.
16. Data for 11a. Mp 180–182 ꢀC (oxalate salt); 1H NMR
(DMSO-d6) d 3.02 (s, 2H), 3.19 (9s, 2H), 3.72–3.74 (m,
3H), 3.94 (s, 3H), 4.21(s, 3H), 7.16–7.25 (m, 4H), 7.75 (t,
J=8.1Hz, 1H), 7.81 (t, J=8.1 Hz, 1H), 8.05 (s, 1H), 8.15
(d, J=8.3 Hz, 1H), 8.24 (d, J=8.3 Hz, 1H), 8.77 (t,
J=5.4 Hz, 1H). Calcd: C: 56.72; H: 4.76; N: 5.29. Obsvd:
C: 56.50; H: 4.91; N: 5.14.
Data for 19. Mp 160–162 ꢀC (oxalate salt); 1H NMR
(DMSO-d6) d 2.27 (s, 3H), 2.94 (s, 2H), 3.16 (s, 2H), 3.65–
3.66 (m, 3H), 3.72 (s, 3H), 3.73 (s, 3H), 3.82 (s, 3H), 4.14
(s, 3H), 6.73 (s, 1H), 6.79 (s, 1H), 7.03 (d, J=8.4 Hz, 1H),
7.29 (d, J=8.4 Hz, 1H), 7.63 (s, 1H), 8.51 (s, 1H). Calcd:
C: 60.18; H: 6.42; N: 5.85. Obsvd: C: 60.32; H: 6.39; N:
5.56.
1
Data for 20. H NMR (CDCl3) d 1.70–1.79 (m, 4H), 2.32
(s, 3H), 2.59 (s, 2H), 2.73–2.76 (m, 2H), 2.81–2.84 (m,
2H), 3.51–3.52 (m, 2H), 3.58 (s, 2H), 3.81 (s, 3H), 3.83 (s,
3H), 3.89 (s, 3H), 6.50 (s, 1H), 6.58 (s, 1H), 6.86–6.96 (m,
2H), 7.11 (s, 1H), 8.00 (s, 1H). Calcd: C: 62.14; H: 6.82;
N: 5.57. Obsvd: C: 62.33; H: 6.60; N: 5.06.
17. ꢀ Receptor binding assays. The s1 receptor binding assay
was conducted using guinea pig brain membrane homo-
genates (100 mg protein). Membrane homogenates were
incubated with 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 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 fil-
ters (presoaked in 0.5% polyethylenimine) using a Bran-
del cell harvester (Gaithersburg, MD, USA). Filters were
washed twice with 5 mL of ice cold buffer. Nonspecific
binding was determined in the presence of 10 mM (+)-
pentazocine. Liquid scintillation counting was carried out
in EcoLite(+) (ICN Radiochemicals; Costa Mesa, CA,
USA) using a Beckman LS 6000IC spectrometer with a
counting efficiency of 50%.
The s2 receptor binding assay was conducted using rat
liver membrane homogenates (35 mg of protein). Mem-
brane homogenates were incubated with 3 nM [3H]DTG
(38.3 Ci/mmol) in the presence of 100 nM (+)-pentazo-
cine to block s1 sites. Incubations were carried 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 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 (Gai-
thersburg, MD, USA). Filters were washed twice with
5 mL of ice cold buffer. Nonspecific binding was deter-
mined in the presence of 5 mM DTG. Liquid scintillation
counting was carried out in EcoLite(+) (ICN Radio-
chemicals; Costa Mesa, CA, USA) using a Beckman LS
6000IC spectrometer with a counting efficiency of 50%.
The IC50 values at sigma sites were generally determined
in triplicate from non-linear regression of binding data as
analyzed by JMP (SAS Institute; Cary, NC, USA), using
eight concentrations of each compound. Ki values were
calculated using the method of Cheng–Prusoff19 and
represent mean valuesÆSEM. All curves were best fit to a
one site fit and gave Hill coefficients of 0.8–1.0. The Kd
value used for [3H]DTG in rat liver was 17.9 nM and was
4.8 nM for [3H](+)-pentazocine in guinea pig brain.11,12
18. Dopamine receptor binding assays. A filtration binding
assay was used to characterize the binding properties of
membrane-associated receptors. For rat D2Long, rat D3
receptors expressed in Sf9 cells and human D4 dopamine
receptors expressed in HEK 293 cells, tissue homogenates
(50 mL) were suspended in 50 mM Tris–HCl/150 mM
NaCl/10 mM EDTA buffer, pH 7.5 and incubated with
Data for 11b. Mp 187–189 ꢀC (oxalate salt); 1H NMR
(DMSO-d6) d 2.94 (s, 3H), 3.19 (s, 4H), 3.71 (s, 3H), 3.73
(s, 3H), 3.94 (s, 3H), 4.12 (s, 3H), 6.74 (s, 1H), 6.79 (s,
1H), 7.75 (t, J=7.6 Hz, 1H), 7.82 (t, J=7.6 Hz, 1H), 8.05
(s, 1H), 8.15 (d, J=8.3 Hz, 1H), 8.25 (d, J=8.3 Hz, 1H),
8.76 (s, 1H). Calcd: C: 55.02; H: 4.96; N: 4.75. Obsvd: C:
54.76; H: 5.04; N: 4.65.
Data for 12. Mp 165–167 ꢀC (oxalate salt); 1H NMR
(DMSO-d6) d 1.61–1.80 (m, 4H), 2.96 (s, 2H), 3.36–3.39
(m, 5H), 3.71(s, 3H), 3.73 (s, 3H), 3.95 (s, 3H), 4.19 (s,
3H), 6.77 (s, 1H), 6.80 (s, 1H), 7.75 (t, J=7.4 Hz, 1H),
7.81(t, J=7.4 Hz, 1H), 7.95 (s, 1H), 8.15 (d, J=8.3 Hz,
1H), 8.25 (d, J=8.3 Hz, 1H), 8.60 (t, J=5.6 Hz, 1H).
Calcd: C: 56.41; H: 5.39; N: 4.54. Obsvd: C: 56.32; H:
5.45; N: 4.45.
Data for 13a. Mp 142–144 ꢀC (oxalate salt); 1H NMR
(DMSO-d6) d 2.99 (s, 2H), 3.10 (s, 2H), 3.61–3.63 (m,
3H), 3.73 (s, 3H), 3.85 (s, 3H), 4.15 (s, 3H), 7.13–7.23 (m,
4H), 7.34 (s, 1H), 7.36 (s, 1H), 8.59 (t, J=5.0 Hz, 1H).
Calcd: C: 51.88; H: 4.95; N: 5.50. Obsvd: C: 51.73; H:
4.95; N: 5.51.
Data for 13b. Mp 91–93 ꢀC (oxalate salt); 1H NMR
(DMSO-d6) d 2.92 (s, 2H), 3.13 (s, 2H), 3.59–3.63 (m,
3H), 3.71(s, 3H), 3.73 (s, 3H), 3.74 (s, 3H), 3.85 (s, 3H),
4.10 (s, 3H), 6.72 (s, 1H), 6.78 (s, 1H), 7.34 (s, 1H), 7.37 (s,
1H), 8.60 (s, 1H). Calcd: C: 50.63; H: 5.13; N: 4.92.
Obsvd: C: 50.49; H: 5.30; N: 4.60.
1
Data for 14. H NMR (CDCl3) d 1.70–1.79 (m, 4H), 2.59
(s, 2H), 2.73–2.76 (m, 2H), 2.81–2.84 (m, 2H), 3.51–3.52
(m, 2H), 3.58 (s, 2H), 3.85 (s, 3H), 3.86 (s, 3H), 3.88 (s,
3H), 3.90 (s, 3H), 6.52 (s, 1H), 6.60 (s, 1H), 7.12 (d,
J=2.7 Hz, 1H), 7.78 (d, J=2.7 Hz, 1H), 8.05 (s, 1H).
Calcd: C: 52.27; H: 5.57; N: 4.69. Obsvd: C: 52.07; H:
5.40; N: 4.64.
1
Data for 15. H NMR (CDCl3) d 1.66 (s, 4H), 2.44–2.66
(m, 6H), 3.06 (s, 2H), 3.17–3.21 (m, 2H), 3.48–3.50 (m,
2H), 3.88 (s, 6H), 6.76–6.96 (m, 2H), 7.12–7.16 (m, 2H),
7.77–7.79 (m, 1H), 7.99 (s, 1H).
1
Data for 16. H NMR (CDCl3) d 1.72 (s, 4H), 2.47–2.67
(m, 6H), 3.03 (s, 2H), 3.15–3.20 (m, 2H), 3.54–3.63 (m,
2H), 3.99 (s, 3H), 6.73–6.93 (m, 2H), 7.11–7.17 (m, 1H),
7.60–7.72 (m, 2H), 8.01–8.26 (m, 3H), 8.38 (s, 1H).
Data for 17. Mp 166–168 ꢀC (oxalate salt); 1H NMR
(DMSO-d6) d 3.02 (s, 3H), 3.14 (s, 2H), 3.66 (s, 2H), 3.83
(s, 3H), 4.19 (s, 3H), 7.12–7.25 (m, 5H), 7.65–7.67 (m,
1H), 7.86 (s, 1H), 8.56 (t, J=5.4 Hz, 1H). Calcd: C: 52.62;
H: 4.84; N: 5.84. Obsvd: C: 52.38; H: 4.75; N: 5.69.
Data for 18. Mp 158–160 ꢀC (oxalate salt); 1H NMR
(DMSO-d6) d 2.93 (s, 2H), 3.16 (s, 2H), 3.60–3.66 (m,
3H), 3.71(s, 3H), 3.73 (s, 3H), 3.84 (s, 3H), 4.14 (s, 3H),
6.73 (s, 1H), 6.78 (s, 1H), 7.13 (d, J=8.9 Hz, 1H), 7.66 (d,

202
R. H. Mach et al. / Bioorg. Med. Chem. Lett. 14 (2004) 195–202
50 mL of 125I-IABN at 37 ꢀC for 60 min. Nonspecific
equation describing mass action binding.20 Data from
competitive inhibition experiments are modeled using non-
linear regression analysis to determine the concentration of
inhibitor that inhibits 50% of the specific binding of the
radioligand. Competition curves will be modeled for a single
site and the IC50 values will be converted to equilibrium
dissociation constants (Ki values) using the Cheng–Prusoff19
correction. Mean Ki valuesÆSEM are reported for at least
three independent experiments.
binding was be defined using 25 mM (+)-butaclamol. For
competition experiments the radioligand concentration is
generally equal to 0.5 times the Kd value and the concen-
tration of the competitive inhibitor ranges over five orders
of magnitude. Binding will be terminated by the addition
of 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). Filters will be washed with 10 mL of cold
buffer and the radioactivity will be measured using a
Packard Cobra gamma counter. Estimates of the equili-
brium dissociation constant and maximum number of
binding sites are obtained using unweighted nonlinear
regression analysis of data modeled according to the
19. Cheng, Y. C.; Prusoff, W. H. Biochem. Pharmacol. 1973,
22, 3099.
20. McGonigle, P.; Molinoff, P. B. In Basic Neurochemistry,
4th ed.; Siegle Agranoff, Albers, Molinoff, Eds., Raven:
1989; Chapter 9.