Synthesis and biological characterization of novel hybrid 7-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro- naphthalen-2-ol and their heterocyclic bioisosteric analogues for dopamine D2 and D3 receptors

Article abstract of DOI:10.1016/j.bmc.2004.06.019

In a recent preliminary communication we described the development of a series of hybrid molecules for the dopamine D2 and D3 receptor subtypes. The design of these compounds was based on combining pharmacophoric elements of aminotetralin and piperazine m

Full text of DOI:10.1016/j.bmc.2004.06.019

Bioorganic & Medicinal Chemistry 12 (2004) 4361–4373
Synthesis and biological characterization of novel hybrid 7-{[2-(4-
phenyl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-
naphthalen-2-ol and their heterocyclic bioisosteric analogues
for dopamine D2 and D3 receptors
Aloke K. Dutta,a,* Sylesh K. Venkataraman,a Xiang-Shu Fei,a Rohit Kolhatkar,a
Shijun Zhanga and Maarten E. A. Reithb
aWayne State University, Department of Pharmaceutical Sciences, Applebaum College of Pharmacy and Health Science,
RM# 3128, Detroit, MI 48202, USA
bNew York University, Department of Psychiatry, New York, NY 10016, USA
Received 21 April 2004; accepted 11 June 2004
Available online 7 July 2004
Abstract—In a recent preliminary communication we described the development of a series of hybrid molecules for the dopamine
D2 and D3 receptor subtypes. The design of these compounds was based on combining pharmacophoric elements of aminotetralin
and piperazine molecular fragments derived from known dopamine receptor agonist and antagonist molecules. Molecules developed
from this approach exhibited high affinity and selectivity for the D3 receptor as judged from preliminary [3H]spiperone binding data.
In this report, we have expanded our previous finding by developing additional novel molecules and additionally evaluated func-
tional activities of these novel molecules in the [3H]thymidine incorporation mitogenesis assay. The binding results indicated highest
selectivity in the bioisosteric benzothiazole derivative N6-[2-(4-phenyl-piperazin-1-yl)-ethyl]-N6-propyl-4,5,6,7-tetrahydro-benzo-
thiazole-2,6-diamine (14) for the D3 receptor whereas the racemic compound 7-({2-[4-(2,3-dichloro-phenyl)-piperazin-1-yl]-ethyl}-
propyl-amino)-5,6,7,8-tetrahydro-naphthalen-2-ol (10c) showed the strongest potency. Mitogenesis studies to evaluate functional
activity demonstrated potent agonist properties in these novel derivatives for both D2 and D3 receptors. In this regard, compound
7-{[4-(4-phenyl-piperazin-1-yl)-butyl]-prop-2-ynyl-amino}-5,6,7,8-tetrahydro-naphthalen-2-ol (7b) exhibited the most potent ago-
nist activity at the D3 receptor, 10 times more potent than quinpirole and was also the most selective compound for the D3 receptor
in this series. Racemic compound 10a was resolved; however, little separation of activity was found between the two enantiomers of
10a. The marginally more active enantiomer ())-10a was examined in vivo using the 6-OH-DA induced unilaterally lesioned rat
model to evaluate its activity in producing contralateral rotations. The results demonstrated that in comparison to the reference
compound apomorphine, ())-10a was quite potent in inducing contralateral rotations and exhibited longer duration of action.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
classified into two main classes D1 and D2.4 This clas-
sification was based on distinct physiological and
pharmacological properties of these two receptors as
stimulation of the D1 receptor leads to activation of
adenylate cyclase, which promotes synthesis of cAMP,
whereas D2 receptor activation leads to inhibition of
adenylate cyclase activity.4 However, recent advances in
molecular biological techniques have led to the identi-
fication of additional DA receptor subtypes. So far, five
subtypes of the DA receptor have been discovered and
they are grouped into two main classes as D1-type and
D2-type. D2-type receptors include D2, D3, and D4
receptors, whereas D1-type includes D1 and D5.5–9 In
The major neurotransmitter dopamine (DA) exhibits a
variety of pharmacological actions in the central ner-
vous system (CNS) and also in the periphery. The
dopamine receptor system has been traditionally tar-
geted for drug development for treatment of psychiatric
illness, neurodegeneration, and recently, for drugs of
abuse.1–3 The receptors for DA in the CNS were initially
* Corresponding author. Tel.: +1-313-577-1064; fax: +1-313-577-2033;
e-mail: adutta@wayne.edu
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.06.019

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A. K. Dutta et al. / Bioorg. Med. Chem. 12 (2004) 4361–4373
the CNS, D1-type receptors are located post-synaptic-
ally, whereas D2-type receptors are located both pre-
and post-synaptically and have a high affinity for
DA.1;10
cell lines applied.22 However, high selectivity of 7-OH-
DPAT for the D3 receptor, found in the antagonist
radioligand binding assay, presumably
G protein
uncoupled low-affinity sites, was decreased substantially
when the binding activity was measured in the radio-
labeled agonists binding assay, a high-affinity G protein
coupled functional state.22;23 Similarly, in vitro func-
tional assay of D2/D3 agonists indicated a good corre-
lation of intrinsic activity of different agonists with their
high affinity binding values.24 Numerous other amino-
tetralin derived ligands have been synthesized and
characterized.25 Recently, a bioisosteric analog of 7-OH-
DPAT, pramipexole, was developed by replacing the
phenolic group by a more metabolically stable thiazo-
lidinium moiety. Pramipexole represents one of
the highest D3 selective compounds and is currently
being used for Parkinson’s disease (PD) therapy.26 In
another recent study, the aminotetralin moiety was
combined with substituted benzamides which led to
the development of a series of molecules as both
agonists and antagonists exhibiting high affinity for the
D2, D3, and 5HT1a receptors, for example, compound
1a (Fig. 1).27;28
The DA D3 receptor was discovered a decade ago and
was found to have different distribution in the brain
compared to the D2 receptor.5;6 The distribution of the
D3 receptor is particularly interesting given that its
location in the brain is mostly limited to the limbic re-
gions that have been implicated in a number of psychi-
atric disorders.11 In the human brain, the highest
expression of the D3 receptor was found in the area of
the ventral striatum and associated striatum.11;12 The
neuroanatomical distribution of D3 receptors is conso-
nant with their function as both presynaptic autore-
ceptors and postsynaptic receptors.13 It has also recently
been proposed that the D3 receptor-mediated motor
inhibition, unlike the D2-mediated inhibition, is post-
synaptically located.14 However, another study with
quinpirole suggested presynaptic locations of D3 auto-
receptors in the striatum.15 The D3 receptor has been
suggested as an interesting target for development of
potential atypical antipsychotic agents, antiparkinso-
nian drugs, and pharmacotherapeutics for treatment of
drug abuse.16;17 Cloning of D2 and D3 receptors re-
vealed a molecular structural sequence containing 50%
homology between these two receptors. A higher
homology of 75–80% was found in helical transmem-
brane spanning regions between these two receptor
subtypes where agonists binding sites are believed to
be located.18 As a result, development of drugs selective
for either one of these two receptors is a challenging
task.
In our effort to design and develop selective and novel
ligands for D2/D3 receptors, we have adopted a hybrid
structure approach by incorporating a disubstituted
piperazine fragment into the aminotetralin pharmaco-
phoric moiety.29 Interaction of the aminotetralin moiety
with the dopamine receptors was expected to produce
agonist activity and the piperazine fragment was
thought to impart selectivity for the D3 receptor subtype
by accessing the accessory binding sites in the receptor
molecule. In this regard, disubstituted piperazine deriv-
atives have already been demonstrated to exhibit high
selectivity for the D3 receptor.30 In our preliminary
communication of this work, we demonstrated success-
ful application of this strategy in the development of
potent compounds exhibiting preferential affinity for the
D3 receptor. We have now expanded our studies by
incorporating additional compounds and also have
evaluated functional activity of selected potent com-
pounds. Furthermore, we have incorporated bioisosteric
replacement of the phenolic moiety in one of our potent
molecules by more metabolically stable heterocyclic
moieties.
An enormous amount of work has already been per-
formed in developing selective agonists for the D3
receptor.19;20 Aminotetralin derivatives were among the
earliest molecules that were investigated for D3 activ-
ity.21 7-OH-DPAT (Fig. 1), an aminotetralin derivative,
was analyzed in various binding assays to evaluate its
binding activity for D2/D3 receptors. From these stud-
ies, 7-OH-DPAT was shown to exhibit preferential
affinity for the D3 receptor even though the selectivity
ratio varied among different laboratories largely due to
different assay conditions, radioligands, and transfected
Additionally, we have carried out an experiment with
one of the enantiomers of an active lead derivative to
observe its in vivo activity to produce contralateral
rotations in 6-OH-DA-induced unilaterally lesioned
rats. In this model, the DA neurons of one side of the
nigrostriatal DA system are selectively and completely
lesioned by intracerebral injection of the neurotoxin 6-
OH-DA, which induces a postsynaptic supersensitivity
at the lesioned side. DA agonists, when administered
systemically, will produce contralateral rotations in rats,
that is, toward the nonlesioned side.31 The results from
this experiment will indicate potency of an agonist test
compound in vivo including its blood brain barrier
crossing ability and thus, may serve as a further
screening device for developing novel D3-selective
compounds.
O
HO
N
N
HO
R-(+)-7-OH-DPAT
PD 128907
N
H N
2
N
NHCO
Ph
S
NH
Pramipexole
CN
1a
Figure 1. Molecular structures of D3 preferring agonists.

A. K. Dutta et al. / Bioorg. Med. Chem. 12 (2004) 4361–4373
4363
2. Chemistry
5a–d in good yield. Next, N-alkylation of the amines
5a–d was carried out in two different ways as shown in
Scheme 2. Reaction with propargyl chloride under basic
conditions produced targets 6a–c, which after demeth-
ylation of the methoxy group produced 7a–c in an
overall good yield. On the other hand, targets 10a–d
were produced by a series of reactions, which involved
first acylation with propionyl chloride followed by
reduction of the intermediate amides and subse-
quent demethylation to produce the final targets in
an overall good yield. Compound 10c was prepared by
Synthesis of the target molecules, shown in Schemes 1–4,
was carried out mainly in two stages which involved first
synthesis of the starting amines 4a–d from phenyl
piperazine 2a–b via N-alkylation with the appropriate
phthalimide-protected alkyl bromide followed by reac-
tion with hydrazine. Thus, the phthalimide derivatives
3a–d on reaction with hydrazine liberated free amines
4a–d. Reductive amination of the free amines with 7-
methoxytetralone produced aminotetralin derivatives
O
N
H
N
NH
(CH )n
2
2
(CH )n
N
N
2
N-(Bromoalkyl)
7-Methoxytetralone
N
phthalimide
N
O
1,2-dichloroethane
NaBH(OAc)3/ AcOH
NH2NH2
EtOH
R
N
R
K2CO3, DMF
R
R
R
R
2a, R=H
2b, R= Cl
4a; n = 1, R=H
4b; n = 3, R=H
4c; n=1, R=Cl
4d; n=3, R=Cl
3a; n = 1, R=H
3b; n = 3, R=H
3c; n=1, R=Cl
3d; n=3, R=Cl
R
R
(CH )n
2
N
N
MeO
N
H
5a-d
Scheme 1.
R
R
R
R
Propargyl Chloride
K2CO3, DMF
(CH )n
(CH )n
2
2
N
N
MeO
N
N
N
MeO
N
H
5a-d
6a; n = 1, R=H
6b; n = 3, R=H
6c; n=3, R=Cl
R
R
(CH )n
2
BBr3/CH2Cl2
N
N
HO
N
7a; n = 1, R=H
7b; n = 3, R=H
7c; n=3, R=Cl
R
R
R
R
(CH )n
2
N
N
MeO
N
(CH )n
2
CH3CH2COCl, Et3N
CH2Cl2, rt
N
N
MeO
N
H
O
5a-b, 5d
8a; n = 1, R=H
8b; n = 3, R=H
8d; n=3, R=Cl
R
R
(CH )n
2
LiAlH4, THF, reflux
BBr3/CH2Cl2
N
N
MeO
N
9a; n = 1, R=H
9b; n = 3, R=H
9d; n=3, R=Cl
1. 1-Bromopropane,
DMF, K2CO3
R
R
Cl
Cl
(CH )n
2
10c
5c
N
N
HO
N
H CO
3
N
N
N
1. BBr3/CH2Cl2
10a; n = 1, R=H
10b; n = 3, R=H
10d; n=3, R=Cl
9c
Scheme 2.

4364
A. K. Dutta et al. / Bioorg. Med. Chem. 12 (2004) 4361–4373
O
1-Bromopropane
O
O
K2CO3, DMF, 600C
O
N
N
N
N
H N
2
O
Na(OAc)3BH, ClCH2CH2Cl
N
4a
11
H
i. Pyrrolidine, PhH, Dean-
Stark
ii. S8, NH2-CN, MeOH
O
O
N
N
HCl/THF/H2O
N
N
O
N
Reflux
N
13
12
1. Tris(dimethylamino)methane
toluene, reflux
2. Guanidine carbonate/EtOH, reflux
N
S
N
N
H N
2
N
H N
2
N
N
N
N
N
14
15
Scheme 3.
Ph
N
N
Ph
Preparative HPLC
H3CO
N
N
(R)-(-)−α-methoxyphenylacetic
N
O
H3CO
N
acid, EDCI, Et3N, CH2Cl2;
H
5a
OCH3
16
Ph
Ph
N
N
N
O
N
H3CO
N
H3CO
N
O
+
OCH3
16a
OCH3
16b
Ph
N
Ph
(i) t-BuOK, THF,
(ii) MeLi, THF
N
N
16a
HO
N
N
H3CO
N
H
17a
(+)-10a
Scheme 4.
N-alkylation of 5c with bromopropane followed by
demethylation of methoxy group.
(dimethylamino)propyl)-3-ethylcarbodiimide, Scheme 4.
The two diastereomers, 16a and 16b, were separated by
preparative HPLC. Each individual diastereomer was
first hydrolyzed in the presence of potassium-t-butoxide
followed by treatment with methyl lithium to generate
optically active amine 17a which, after a series of reac-
tions as described in Scheme 2, produced the optically
pure enantiomers (+)-10a and ())-10a. We have not yet
determined the absolute configuration of (+)-10a and
())-10a.
Bioisosteric analogues 14 and 15 were prepared by fol-
lowing a different synthetic route as shown in Scheme 4.
Reductive amination of commercially available cyclo-
hexanedione monoethylene ketal with amine 4a yielded
11 in good yield. N-Propylation of 11 with 1-bromo-
propane produced 12, which on acidic hydrolysis liber-
ated keto compound 13. Thiazolidinium derivative 14
was synthesized from the keto derivative 13 by following
a two steps synthetic procedure in a reasonably good
yield.32 On the other hand, reaction of keto compound
13 with tris(dimethylamino)methane followed by treat-
ment with guanidine carbonate under refluxing condi-
tion in ethanol provided 15 in reasonably good yield.33
3. Results and discussion
In our previous short communication, we have demon-
strated that our adopted hybrid approach of combining
aminotetralin and piperazine fragments could produce
novel compounds exhibiting high affinity and selectivity
for the D3 receptor.29 Our earlier study revealed that the
length of the linker connecting the aminotetralin moiety
with the piperazine fragment plays an important role in
Resolution of the racemic compound 10a was achieved
by synthesizing diastereomers from the reaction of the
intermediate amine 5a with R-())-a-methoxyphenyl-
acetic acid in the presence of the coupling agent 1-(3-

A. K. Dutta et al. / Bioorg. Med. Chem. 12 (2004) 4361–4373
4365
exhibiting selectivity for the D3 receptor. Selectivity for
the D3 receptor was more pronounced in compounds
with a shorter methylene chain link connecting the
aminotetralin moiety with the piperazine fragment al-
though compound 7b with a longer linker size was also
quite selective. In addition, most of these compounds
exhibited high potency for the D3 receptor.
of differential activity between the two enantiomers of
10a might be due to the presence of the piperazine
fragment in this hybrid molecule.
In our effort to replace the phenolic moiety by a more
metabolically stable bioisosteric heterocyclic moiety,
compounds 14 and 15 were designed and synthesized. In
compound 14, the phenolic moiety was replaced by a
thiazolidinium moiety, which is a known bioisostere of a
phenolic group. Thiazolidinium moiety containing DA
agonists like pramipexole displayed interesting biologi-
cal activity in the past by showing preferential activity
for the D3 receptor and also exhibited potent antioxi-
dant activity.35;36 As was mentioned earlier, pramipexole
is currently being used in the clinic for the therapy of
PD.26 Binding results indicate that compound 14
exhibited high potency for the D3 receptor and was the
most selective compound in the current series for the D3
receptor (D2/D3; 502). In comparison to the reference
(+)-7-OH-DPAT, compound 14 was five-fold more
selective (D2/D3; 502 vs 108) for D3 while their D3
potencies were comparable (Table 1). On the other
hand, amino pyrimidine derivative 15 was less potent
and less selective for the D3 receptor compared to 14.
One of the more selective compounds in binding D3,
racemic 10a, was subjected to enantiomeric resolution to
isolate pure enantiomers through a diastereoisomeric
separation approach as described earlier. Both enantio-
mers were characterized for their binding to cloned D2
and D3 receptors. Results indicated a somewhat higher
D3 binding potency and selectivity in the ())-10a isomer
compared to the (+)-10a isomer (Table 1). The func-
tional assay for the two enantiomers indicated that no
appreciable separation of activity exists between these
two enantiomers (Table 2). On the contrary, the am-
inotetralin derivative 7-OH-DPAT and other related
aminotetralin-derived DA agonists displayed apprecia-
ble differential binding activity in their corresponding
enantiomers.34 It seems logical to conclude that the lack
Table 1. Inhibition constants for displacing [3H]spiperone binding to
the cloned D2L and D3 receptors expressed in HEK cells
We next evaluated functional activity of the selected
potent compounds in [3H]thymidine incorporation
mitogenesis assay in CHOp-cell lines transfected with
human D2 and D3 receptors. Agonist activity of a test
compound was measured by assessing the capability to
induce cell division by incorporation of [3H]thymidine.
Intrinsic activity of our compounds was compared
against the reference quinpirol, which is considered a
full agonist at both receptors. Intrinsic activity was
expressed as the ratio of the EC50 value of drug over
that of the reference standard compound quinpirole.
Maximum stimulation of receptor was also evaluated. It
is apparent from the results that these novel analogs are
agonists as all the compounds exhibited potent intrinsic
activity. The highest selectivity in intrinsic activity for
the D3 receptor was exhibited by 7b (Table 2), even
though 14 exhibited the highest selectivity in the binding
assay (Table 1).
Compound
Ki (nM), D2
[3H]spiperone
Ki (nM), D3
[3H]spiperone
D2/D3
7-OH-DPAT
( )-7aa
538 108
245 26
68.4 7.6
213 26
5.00 1.18
14.2 1.7
108
17
49
122
128
97
30
4
( )-7ba
1.40 0.14
1.75 0.34
1.88 0.42
2.36 0.41
3.79 0.40
2.26 0.50
3.59 0.58
1.13 0.04
4.14 0.55
22.3 2.3
( )-10a
())-10a
(+)-10a
( )-10ba
( )-10da
( )-7ca
241
229 17
114
7
8
8.78 0.81
7.37 0.27
27.4 1.0
2
( )-10ca
24
502
50
( )-14
( )-15
2080 162
1120 144
Results are means SEM for three experiments each performed in
triplicate.
a Value taken from Ref. 29.
Table 2. Evaluation of agonist potency in [3H]thymidine incorporation mitogenesis assay
Compound
D2 (EC50 drug/EC50
quinpirole)
Maximum stimula-
tion relative to quin-
pirole
D3 (EC50 drug/EC50
quinpirole)
Maximum stimula-
tion relative to quin-
pirole
D2/D3 ratio of drug
EC50
7-OH-DPATa
7aa
7ba
10a
0.42 0.22
0.7 0.00
0.3 0.1
0.30 0.2
0.41 0.1
0.10 0.03
0.57 0.17
0.24 0.03
0.21 0.03
0.41 0.19
1.85 0.45
0.92 0.26
0.2 0.02
4.6
4.9
0.87 0.02
0.85 0.06
0.84 0.01
0.78 0.02
0.86 0.36
1.20 0.26
0.80 0.08
0.86 0.09
0.90 0.04
0.94 0.06
0.72 0.10
0.83 0.03
0.76 0.03
0.73 0.03
0.75 0.09
0.86 0.01
0.90 0.15
0.86 0.03
14.2
2.73
3.78
3.60
3.5
0.465 0.015
0.285 0.005
0.245 0.015
0.415 0.08
0.785 0.10
1.125 0.235
0.315 0.025
(+)-10a
())-10a
10ba
10da
7ca
14
1.28
3.83
5.07
a Quinpirole had an EC50 of 9.98 0.68 nM at D2 and 2.91 0.25 nM at D3. These values were used to normalize the D2/D3 ratios of drug EC50 for
compounds without asterisk (see Section 6). Maximum stimulation is observed stimulation relative to that produced by the full agonist quinpirole
(defined as 1.00).

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A. K. Dutta et al. / Bioorg. Med. Chem. 12 (2004) 4361–4373
4. Contralateral turning by ())10a in 6-OH-DA
lesioned rats
high activity for producing contralateral rotations with
long duration of action. This result confirmed potent
agonist action of ())-10a and its rapid blood brain
barrier crossing ability. Further structural exploration is
ongoing to produce compounds with higher selectivity
for the D3 receptor.
After resolving racemic mixture of 10a, the slightly more
potent enantiomer ())-10a was evaluated for in vivo
activity. Agonist property of ())-10a was evaluated in
rats with a unilaterally lesioned nigrostriatal dopamine
system induced by 6-OH-DA. As expected from func-
tional studies, compound ())-10a turned out to be a
potent agonist at DA receptors as it produced rapid
contralateral rotations, which lasted over 6 h. In con-
trast, the reference compound apomorphine had a faster
onset of action but much shorter duration of action
compared to ())-10a (Fig. 2). The total number of
rotations produced by ())-10a was much greater than
that produced by apomorphine (1608 vs 345). This result
also demonstrated that the compound ())-10a pene-
trated the blood brain–barrier effectively with long
duration of action at the concentration tested and its
action in the CNS was specific. It is not known whether
this long duration of action is due to the activity of the
drug alone or a combination of the drug and an active
metabolite. Interestingly, long duration of activity
emanating from compounds containing a phenolic hy-
droxyl group has been reported in the past.37
6. Experimental
Analytical silica gel-coated TLC plates (Silica Gel 60
F254) were purchased from EM Science and were visu-
alized with UV light or by treatment with phosphomo-
lybdic acid (PMA). Flash chromatography was carried
out on Baker Silica Gel 40 mM. 1H NMR spectra were
routinely obtained on GE-300 MHz and Varian
400 MHz FT NMR. The NMR solvent used was either
CDCl3 or CD3OD as indicated. TMS was used as an
internal standard. Elemental analyses were performed
by Atlantic Microlab, Inc and were within 0.4% of the
theoretical value. Optical rotations were recorded on a
Perkin Elmer 241 polarimeter. [3H]spiperone (16.5 Ci/
mmol) was from NEN Life Sciences Products, Boston,
MA, and (+)-butaclamol from Research Biochemical
Corp. (Natick, MA).
Materials for the mitogenesis assay (D2 and D3): Cell
lines: human D2- and D3-receptor containing CHOp
cells. Growth medium: a-MEM complete contains: alfa
minimum essential medium from Gibco (Invitrogen
Corp); FBS ¼ fetal bovine serum from Atlas Biologi-
cals, Fort Collins, CO; penicillin–streptomycin from
Gibco (Invitrogen Corp.); Geneticin (G418 sulfate) from
Gibco. Assay medium: alpha MEM incomplete (same as
above, but without the FBS). Subculturing: Trypsin–
EDTA solution (10X) from Sigma. [3H]Thymidine from
NEN (NET-355), aqueous solution, specific activity
18.00 Ci/mmol. Quinpirole from Sigma/RBI. Buffer
items: NaCl, KCl, KH2PO4, NaHCO3, HEPES, glucose,
and NaOH are either from Mallinckrodt or Sigma.
5. Conclusion
In this report, we have been able to demonstrate the
development of novel potent agonists for DA D2 and
D3 receptors. In vitro [3H]spiperone binding studies
indicated preferential affinity of most of these molecules
for the D3 receptor with compound ( )-14 exhibiting
the highest selectivity. In this current series compounds
with the shorter methylene linker generally exhibited
higher selectivity for the D3 receptor. In the functional
assay, all of these derivatives exhibited higher intrinsic
activity at the D3 receptor compared to the D2 receptor,
thus, correlating well with their binding data. The pure
enantiomer, ())-10a, was tested in vivo in 6-OH-DA
induced unilaterally lesioned rats and found to display
7. Procedure A
7.1. 2-[2-(4-Phenyl-piperazin-1-yl)-ethyl]-isoindole-1,3-di-
one (3a)
A mixture of 1-phenylpiperazine 2 (2.59 g, 15.98 mmol),
N-(2-bromoethyl)phthalimide (8.12 g, 31.96 mmol),
Et3N (2.0 mL) and K2CO3 (8.0 g) in DMF (40 mL) was
stirred at 70 ꢁC overnight under a nitrogen atmosphere.
The reaction mixture was cooled, water (100 mL) was
added and the mixture extracted with diethyl ether. The
organic phase was combined dried over Na2SO4 and
evaporated under vacuo to give the crude product,
which was purified by flash chromatography (hexane/
EtOAC ¼ 1:1) to furnish 3a, 3.81 g (71% yield). 1H
NMR (CDCl3) 2.68–2.72 (m, 6H, N(CH2)3, 3.12–3.15 (t,
J ¼ 4:8 Hz, 4H, N(CH2)2), 3.84–3.89 (t, J ¼ 6:3 Hz, 2H
CONCH2), 6.80–6.82 (t, J ¼ 8:1 Hz, 1H, Ar-H), 6.88–
6.91 (d, J ¼ 8:1 Hz, 2H, Ar-H), 7.21–7.26 (t, J ¼ 8:1 Hz,
2H, Ar-H), 7.69–7.86 (m, 4H, Ar-H).
Figure 2. Effect on turning behavior of ())-10a, apomorphine, and
vehicle in unilaterally 6-OH-DA lesioned rats studied over 6 h. Each
point is the mean SEM of four rats. The analysis of variance
(ANOVA) performed with repeated measures indicated significant
effects for treatments (F ½3; 6ꢀ ¼ 26:48, p < 0:05) and intervals
(F ½11; 22ꢀ ¼ 10:03, p < 0:05).

A. K. Dutta et al. / Bioorg. Med. Chem. 12 (2004) 4361–4373
4367
7.2. 2-[4-(4-Phenyl-piperazin-1-yl)-butyl]-isoindole-1,3-di-
one (3b)
CH2N(CH2)2), 2.76–3.04 (m, 7H), 3.16–3.20 (t,
J ¼ 4:8 Hz, N(CH2)2), 3.77 (s, 3H, OCH3), 6.62–6.71
(m, 2H, Ar-H), 6.64–7.02 (m, 4H, Ar-H), 7.24–7.30 (m,
2H, Ar-H).
1-Phenylpiperazine 2 (2.21 g, 7.81 mmol) was reacted
with N-(4-bromobutyl)-phthalimide (1.26 g, 7.81 mmol),
K2CO3 (8.0 g), Et3N (1.0 mL), and DMF (20 mL) as
described in Procedure A to afford a yellow solid 3a,
9.2. (7-Methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-[4-
(4-phenyl-piperazin-1-yl)-butyl]-amine (5b)
1
2.70 (55% yield). H NMR (CDCl3) 1.55–1.82 (m, 4H,
CH2CH2), 2.40–2.45 (t, J ¼ 7:5 Hz, 2H, NCH2), 2.57–
2.61 (t, J ¼ 4:8 Hz, 4H, N(CH2)2), 3.17–3.20 (t,
J ¼ 4:8 Hz, 4H, N(CH2)2), 3.71–3.75 (t, J ¼ 6:7 Hz, 2H,
CONCH2), 6.82–6.94 (3H, Ar-H), 7.23–7.28 (m, 2H, Ar-
H), 7.70–7.74 (m, 2H, Ar-H), 7.83–7.86 (m, 2H, Ar-H).
4-(4-Aminobutyl)-1-phenylpiperazine
4b
(4.30 g,
18.30 mmol) was reacted with 7-methoxy-2-tetralone
(3.21 g, 18.24 mmol) and Na(OAc)3BH (11.40 g,
54.03 mmol) and HOAc (1.0 mL) in 1,2-dichloroethane
1
(50 mL) to give 5b 5.80 g (81% yield) (Procedure C). H
NMR (CDCl3) 1.58–1.60 (m, 4H), 2.03–2.07 (m, 2H),
2.40–2.47 (t, J ¼ 6:7 Hz, 2H), 2.59–2.60 (t, J ¼ 4:8 Hz,
m, 4H, N(CH2)2), 2.68–2.85 (m, 5H), 2.91–3.03 (m, 2H),
3.19–3.22 (t, J ¼ 4:8 Hz, 4H, N(CH2)2), 3.76 (s, 3H,
CH3O), 6.61–6.67 (m, 2H, Ar-H), 6.82–6.97 (m, 4H, Ar-
H), 7.23–7.29 (m, 2H, Ar-H).
8. Procedure B
8.1. 2-(4-Phenyl-piperazin-1-yl)-ethylamine (4a)
Hydrazine (1.0 g, 31.2 mmol) was added to a solution of
3a (1.97 g, 5.88 mmol) in EtOH (25 mL) under a N2
atmosphere and the reaction mixture was stirred at
room temperature for 24 h. The solvent was removed
under vacuo and EtOAc was added to the residue. The
solids were filtered off and the solution collected was
dried over Na2SO4 and evaporated under vacuo to give
9.3. {2-[4-(2,3-Dichloro-phenyl)-piperazin-1-yl]-ethyl}-(7-
methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-amine (5c)
Compound 5c was synthesized from 2b by following
procedures A, B, and C (73%, 3 steps). 1H NMR
(300 MHz, CD3Cl) 1.61–1.67 (m, 2H), 2.60–2.64 (m, 6H,
CH2N(CH2)2), 2.76–3.05 (m, 7H), 3.16–3.20 (t,
J ¼ 4:8 Hz, N(CH2)2), 3.78 (s, 3H, CH3O), 6.72–6.82
(m, 2H, Ar-H), 6.90–6.90 (m, 2H, Ar-H), 7.10–7.28 (m,
2H, Ar-H).
1
a white solid 4a, 1.18 g (98% yield) H NMR (CDCl3)
2.46–2.51 (t, J ¼ 6:3 Hz, 2H, CH2NH2), 2.68–2.71 (t,
J ¼ 4:8 Hz, 4H, N(CH2)2), 2.86–2.92 (t, J ¼ 6:3 Hz, 2H,
NCH2), 3.12–3.16 (t, J ¼ 4:8 Hz, 4H, N(CH2)2), 6.81–
6.83 (t, J ¼ 8:1 Hz, 1H, Ar-H), 6.88–6.91 (d, J ¼ 8:1 Hz,
2H, Ar-H), 7.21–7.26 (t, J ¼ 8:1 Hz, 2H, Ar-H).
9.4. {4-[4-(2,3-Dichloro-phenyl)-piperazin-1-yl]-butyl}-(7-
methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-amine (5d)
8.2. 4-(4-Phenyl-piperazin-1-yl)-butylamine (4b)
Compound 3b (7.70 g, 21.21 mmol) was reacted with
NH2NH2 (3.0 g, 93.75 mmol) as detailed in Procedure B
to give 4b, 4.60 g (93% yield). H NMR (CDCl3) 1.48–
1.61 (m, 4H), 2.40–2.43 (t, J ¼ 7:2 Hz, 2H, NCH2),
2.61–2.63 (t, J ¼ 4:5 Hz, 4H, N(CH2)2), 2.72–2.76 (t,
J ¼ 6:4 Hz, 2H, CH2NH2), 3.19–3.22 (t, J ¼ 4:6 Hz, 4H,
N(CH2)2), 6.33–6.94 (m, 3H, Ar-H), 7.23–7.28 (m, 2H,
Ar-H).
Compound 5d was synthesized from 2b by following the
procedures A, B, and C (70%, 3 steps). 1H NMR
(CDCl3) 1.58–1.64 (m, 4H), 2.04–2.10 (m, 2H), 2.43–
2.47 (t, J ¼ 6:6 Hz, 2H), 2.60–2.65 (m, 4H, (N(CH2)2),
2.76–2.82 (m, 5H), 2.93–2.98 (m, 2H), 3.02–3.08 (m, 4H,
N(CH2)2), 3.77 (s, 3H, CH3O), 6.62–6.71 (m, 2H, Ar-H),
6.94–7.01 (m, 2H, Ar-H), 7.14–7.16 (m, 2H, Ar-H).
1
9. Procedure C
10. Procedure D
9.1. (7-Methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-[2-
(4-phenyl-piperazin-1-yl)-ethyl]-amine (5a)
10.1. (7-Methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-[2-
(4-phenyl-piperazin-1-yl)-ethyl]-prop-2-ynyl-amine (6a)
A mixture of compound 4a (1.18 g, 5.75 mmol),
7-methoxy-2-tetralone (1.22 g, 6.93 mmol), and
Compound 5a (1.18 g, 3.04 mmol) was reacted with
propargyl chloride (1.20 g, 16.11 mmol) in the presence
of K2CO3 (4.0 g) and DMF (25 mL) at 60 ꢁC for 5 h. The
mixture was cooled and was diluted with water, ex-
tracted with diethyl ether, and washed with brine. The
combined ether fraction was dried over Na2SO4, evap-
orated, and purified by flash chromatography to furnish
Na(OAc)3BH (3.60 g, 17.26 mmol) and HOAc (1.0 mL)
in 1,2-dichloroethane (20 mL) was stirred at room tem-
perature under a N2 atmosphere overnight. The solvent
was evaporated and saturated NaHCO3/H2O (10 mL)
was added to the mixture, which was then extracted with
EtOAc. The combined organic phase was dried over
Na2SO4 and evaporated to give the crude product which
was purified by flash chromatography (EtOAc/MeOH/
Et3N ¼ 50:5:1) to give the product (5a), 1.95 g (93%).
1H NMR(CDCl3) 1.60–1.68 (m, 2H), 2.60–2.63 (m, 6H,
1
6a, 0.35 g (29% yield). H NMR (CDCl3) 1.59–1.71 (m,
2H), 2.13–2.18 (m, 2H), 2.20–2.34 (t, J ¼ 2:1 Hz, 1H,
CBCH), 2.56–2.60 (t, J ¼ 6:9 Hz, 2H, NCH2), 2.65–2.68
(t, J ¼ 4:8 Hz, 4H, N(CH2)2), 2.75–2.81 (m, 2H), 2.84–
2.89 (t, J ¼ 7:2 Hz, 2H). 2.98–3.02 (m, 3H), 3.20–3.23 (t,

4368
A. K. Dutta et al. / Bioorg. Med. Chem. 12 (2004) 4361–4373
J ¼ 4:8 Hz, 4H, N(CH2)2), 3.60 (s, 2H, NCH2CBCH),
3.77 (s, 3H, CH3O), 6.60–6.83 (m, 2H, Ar-H), 6.85–6.98
(m, 4H, Ar-H), 7.23–7.29 (m, 2H, Ar-H).
11.2. 7-{[4-(4-Phenyl-piperazin-1-yl)-butyl]-prop-2-ynyl-
amino}-5,6,7,8-tetrahydro-naphthalen-2-ol (7b)
Compound 6b (0.195 g, 0.742 mmol) was reacted with
1 M BBr3/CH2Cl2 (0.80 mL, 0.80 mmol) (Procedure E) to
give compound 7b, 0.13 g (72%). 1H NMR (CDCl3)
1.54–1.68 (m, 4H), 2.09–2.14 (m, 2H), 2.17–2.18 (t,
J ¼ 2:1 Hz, 1H), 2.41–2.45 (t, J ¼ 7:0 Hz, 2H), 2.60–2.63
(t, J ¼ 4:5 Hz, 4H, N(CH2)2), 2.67–2.71 (t, J ¼ 6:2 Hz,
2H), 2.76–2.80 (m, 4H), 2.89–2.92 (m, 1H) 3.51–3.52 (d,
J ¼ 2:1 Hz, 2H, CH2CBCH), 3.20–3.24 (t, J ¼ 4:6 Hz,
4H, N(CH2)2), 6.56–6.61 (m, 2H, Ar-H), 6.88–6.95 (m,
4H, Ar-H), 7.24–7.29 (m, 2H, Ar-H),). Free base was
converted into its HBr salt. Mp ¼ 182–186 ꢁC. Anal.
Calcd for (C27H35N3OÆ3HBrÆ0.75H2O) C, H, N.
10.2. (7-Methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-[4-
(4-phenyl-piperazin-1-yl)-butyl]-prop-2-ynyl-amine (6b)
A mixture of 5b (0.65 g, 1.65 mmol), propargyl chloride
(0.30 mL, 4.63 mmol), K2CO3 (1.0 g), and Et3N (1.0 mL)
was stirred in DMF (20 mL) as shown in Procedure D to
give a thick oil 6b, 0.33 g (46%). 1H NMR (CDCl3) 1.51–
1.56 (m, 4H), 2.11–2.15 (m, 2H), 2.17–2.19 (t,
J ¼ 2:2 Hz, 1H, CBCH), 2.40–2.44 (t, J ¼ 7:2 Hz, 2H),
2.59–2.63 (t, J ¼ 4:8 Hz, 4H, N(CH2)2), 2.67–2.83 (m,
5H), 2.93–3.01 (m, 2H), 3.19–3.23 (t, J ¼ 4:5 Hz, 4H,
N(CH2)2), 3.52–3.53 (d, J ¼ 2:1 Hz, 2H, CH2CBCH),
3.77 (s, 3H, CH3O), 6.62–6.70 (m, 2H, Ar-H), 6.83–7.00
(m, 4H, Ar-H), 7.24–7.29 (m, 2H, Ar-H).
11.3. 7-({4-[4-(2,3-Dichloro-phenyl)-piperazin-1-yl]-butyl}-
prop-2-ynyl-amino)-5,6,7,8-tetrahydro-naphthalen-2-ol
(7c)
10.3. {4-[4-(2,3-Dichloro-phenyl)-piperazin-1-yl]-butyl}-
(7-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-prop-2-
ynyl-amine (6c)
Compound 6c (0.28 g, 0.70 mmol) was reacted with
BBr3/CH2Cl2 (1.50 mL, 1.50 mmol) (Procedure E) to
give pure product 7c, 0.23 g (84%). H NMR (CDCl3)
1
1.48–1.56 (m, 4H), 2.09–2.13 (m, 2H), 2.17–2.18 (t,
J ¼ 1:8 Hz, 1H, CBCH), 2.45–2.47 (t, J ¼ 6:6 Hz, 2H),
2.66–2.68 (br s, 4H, N(CH2)2), 2.71–2.80 (m, 5H), 2.89–
2.96 (m, 2H), 3.09–3.10 (br s, 4H, N(CH2)2), 3.51–3.52
(d, J ¼ 1:5 Hz, 2H, CH2CBCH), 6.57–6.61 (m, 2H, Ar-
H), 6.92–6.98 (m, 2H, Ar-H), 7.14–7.16 (m, 2H, Ar-H).
Free base was converted into its HCl salt, mp ¼ 180–
183 ꢁC and analysis demonstrated partial hydrochloride
salt formation. Anal. Calcd for (C27H33N3OCl2Æ
2.70HClÆ0.06H2O) C, H, N.
Compound 5d was reacted with propargyl chloride
(1.50 g, 20.13 mmol) and K2CO3 (4.50 g) in DMF
(20 mL) (Procedure D) to give 6c, 0.48 g (24%). 1H
NMR (CDCl3) 1.55–1.68 (m, 4H), 2.11–2.15 (m, 2H),
2.17–2.18 (t, J ¼ 1:8 Hz, 1H, CBCH), 2.43–2.47 (t,
J ¼ 6:6 Hz, 2H), 2.49–2.66 (br s, 4H, N(CH2)2), 2.68–
2.88 (m, 8H), 2.98–3.00 (m, 1H), 3.06–3.10 (br s, 4H,
N(CH2)2), 3.52–3.53 (d, J ¼ 1:8 Hz, 2H, CH2CBCH),
3.77 (s, 3H, CH3O), 6.30–6.72 (m, 2H, Ar-H), 6.94–7.00
(m, 2H, Ar-H), 6.97–7.16 (m, 2H, Ar-H).
11.4. 7-{[2-(4-Phenyl-piperazin-1-yl)-ethyl]-propyl-
amino}-5,6,7,8-tetrahydro-naphthalen-2-ol (10a)
11. Procedure E
Compound 9a (0.60 g, 0.88 mmol) was reacted with 1 M
BBr3/CH2Cl2 (1.60 mL, 1.60 mmol) in CH2Cl2 (20 mL)
to furnish 10a, 0.29 g (83%) (Procedure E). H NMR
11.1. 7-{[2-(4-Phenyl-piperazin-1-yl)-ethyl]-prop-2-ynyl-
amino}-5,6,7,8-tetrahydro-naphthalen-2-ol (7a)
1
(CDCl3) 0.86–0.91 (t, J ¼ 7:2 Hz, 3H, CH3CH2CH2N),
1.43–1.50 (m, 4H), 1.89–1.93 (m, 2H), 2.46–2.51 (t,
J ¼ 7:5 Hz, 2H, NCH2), 2.53–2.68 (m, 6H), 2.70–2.74 (t,
J ¼ 4:8 Hz, 4H, N(CH2)2), 2.83–2.88 (m, 1H), 3.23–3.26
(t, J ¼ 4:8 Hz, 4H, N(CH2)2), 6.45–6.56 (m, 2H, Ar-H),
6.83–6.93 (m, 4H, Ar-H), 7.23–7.26 (m, 2H, Ar-H). Free
base was converted into its HCl salt, mp ¼ 143–146 ꢁC.
Anal. Calcd for (C27H35N3OÆ3HClÆ0.9H2O) C, H, N.
Borontribromide (1 M solution in dichloromethane),
(1.5 mL, 1.5 mmol) was added to a solution of 6a (0.28 g,
0.70 mmol) in anhydrous CH2Cl2 (10 mL) at )40 ꢁC
under N2 atmosphere. The reaction mixture was stirred
at )40 ꢁC for 2 h and then at overnight at room tem-
perature. The reaction was quenched by the addition of
saturated NaHCO3 solution and the mixture was ex-
tracted with CH2Cl2. The combined organic layer was
dried over Na2SO4, evaporated under vacuo, and the
crude product was purified by flash chromatography
(EtOAc/Et3N ¼ 50:1) to afford compound 7a, 0.23 g
11.5. 7-{[4-(4-Phenyl-piperazin-1-yl)-butyl]-propyl-
amino}-5,6,7,8-tetrahydro-naphthalen-2-ol (10b)
1
(85%). H NMR(CDCl3) 1.56–1.68 (m, 2H), 2.11–2.15
(m, 2H), 2.20–2.22 (t, J ¼ 2:1 Hz, 1H, CBCH), 2.57–
2.61 (t, J ¼ 6:6 Hz, 2H, NCH2), 2.65–2.69 (t, J ¼ 4:8 Hz,
4H, N(CH2)2), 2.75–2.81 (m, 2H), 2.84–2.86 (m, 5H),
2.86–2.91(t, J ¼ 4:8 Hz, 2H, N(CH2)2) 3.58 (s, 2H,
CH2CBCH), 6.55–6.61 (m, 2H, Ar-H), 6.83–6.94 (m,
4H, Ar-H). 7.23–7.27 (m, 2H, Ar-H). Free base was
converted into its HCl salt. Mp ¼ 164–166 ꢁC. Anal.
Calcd for (C27H31N3OÆ3HClÆ0.8H2O) C, H, N.
Compound 9b (0.30 g, 0.69 mmol) was reacted with 1 M
BBr3/CH2Cl2 (1.3 mL) to give 10b, 0.25 g (87%) (Proce-
dure E). 1H NMR(CDCl3) 0.86–0.91 (t, J ¼ 7:2 Hz, 3H),
1.40–1.82 (m, 7H), 1.93–2.05 (m, 1H), 2.39–2.44
(t, J ¼ 7:2 Hz, 2H), 2.44–2.50 (t, J ¼ 7:5 Hz, 2H),
2.56–2.51(t, J ¼ 6:6 Hz, 2H), 2.60–2.63 (t, J ¼ 4:2 Hz,
4H, N(CH2)2), 2.75–2.80 (m, 4H), 2.92–2.99 (m, 1H),
3.20–3.23 (t, J ¼ 4:2 Hz, 4H, N(CH2)2), 6.54–6.60 (m,

A. K. Dutta et al. / Bioorg. Med. Chem. 12 (2004) 4361–4373
4369
2H, Ar-H), 6.83–6.95 (m, 4H, Ar-H), 7.24–7.29 (m, 2H,
Ar-H). Free base was converted into its HBr salt. Mp ¼
152–156 ꢁC. Anal. Calcd for (C27H42N3OBr3Æ0.30H2O)
C, H, N.
(1.0 mL) in CH2Cl2 (10 mL) (Procedure F) to give pure
1
compound 8b, 0.32 g (99%). H NMR (CDCl3) 1.11–
1.26 (t, J ¼ 6:6 Hz, 3H), 1.52–1.74 (m, 6H), 1.89–1.98
(m, 2H), 2.35–2.45(m, 4H), 2.58–2.61 (t, J ¼ 4:5 Hz, 4H,
N(CH2)2), 2.78–3.05 (m, 4H), 3.18–3.23 (t, J ¼ 4:5 Hz,
4H, N(CH2)2), 3.15–3.56 (m, 1H), 3.76 (s, 3H, CH3O–),
6.58–6.73 (m, 2H, Ar-H), 6.85–7.03 (m, 4H, Ar-H),
7.23–7.28 (t, J ¼ 7:6 Hz, 2H, Ar-H).
11.6. 7-({2-[4-(2,3-Dichloro-phenyl)-piperazin-1-yl]-ethyl}-
propyl-amino)-5,6,7,8-tetrahydro-naphthalen-2-ol (10c)
Compound 9c (0.23 g, 0.47 mmol) in CH2Cl2 (20 mL)
was demethylated with BBr3/CH2Cl2 (2.0 mL, 2.0 mmol)
(Procedure E) to give 7c as a thick oil 0.19 g (87%). H
13. Procedure G
1
NMR (300 MHz, CDCl3) 0.86–0.91 (t, J ¼ 7:2 Hz, 3H,
CH3CH2CH2N), 1.44–1.50 (m, 4H), 1.90–1.94 (m, 2H),
2.46–2.51 (t, J ¼ 7:5 Hz, 2H, NCH2), 2.53–2.68 (m, 6H),
2.70–2.74 (t, J ¼ 4:8 Hz, 4H, N(CH2)2), 2.83–2.88 (m,
1H), 3.25–3.27 (t, J ¼ 4:8 Hz, 4H, N(CH2)2), 6.40–6.54
(m, 2H, Ar-H), 6.87–6.93 (m, 2H, Ar-H), 7.08–7.15 (m,
2H, Ar-H). Anal. Calcd for (C25H36N3OCl5Æ1.9H2O) C,
H, N.
13.1. (7-Methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-[2-
(4-phenyl-piperazin-1-yl)-ethyl]-propyl-amine (9a)
Compound 8a (0.38 g, 0.90 mmol) in anhydrous THF
(15 mL) was added dropwise into a suspension of
LiAlH4 (0.15 g, 4.41 mmol) in anhydrous THF at 0 ꢁC
under N2 atmosphere. The reaction mixture was ref-
luxed for 8 h, cooled to room temperature and saturated
NaOH/H2O (1 mL) was added dropwise. The mixture
was filtered and the solution was dried over Na2SO4.
The solvent was removed under vacuo to afford a white
11.7. 7-({4-[4-(2,3-Dichloro-phenyl)-piperazin-1-yl]-butyl}-
propyl-amino)-5,6,7,8-tetrahydro-naphthalen-2-ol (10d)
1
solid 9a, 0.36 g (97%). H NMR (CDCl3) 0.87–0.93 ( t,
J ¼ 7:2 Hz, 3H, CH3CH2CH2N), 1.46–1.53 (m, 4H),
1.88–2.07 (m, 2H), 2.51–2.55 (t, J ¼ 7:2 Hz, 2H, CH2N),
2.64–2.68 (t, J ¼ 4:8 Hz, 4H, N(CH2)2), 2.72–2.83 (m,
6H), 2.90–3.06 (m, 1H), 3.19–3.22 (t, J ¼ 4:8 Hz, 4H,
N(CH2)2), 3.77 (s, 3H, CH3O), 6.62–6.70 (m, 2H, Ar-H),
6.85-7.00 (m, 4H, Ar-H), 7.24-7.29 (m, 2H, Ar-H).
Compound 9d (0.36 g, 0.71 mmol) was reacted with 1 M
BBr3/CH2Cl2 (1.5 mL, 1.5 mmol) (Procedure E) to give
10d (0.29 g, 83%). 1H NMR (CDCl3) 0.85–0.90 (t,
J ¼ 7:2 Hz, 3H, CH3), 1.43–1.58 (m, 6H), 1.96–2.00 (m,
2H), 2.44–2.55 (m, 6H, CH2NN(CH2)2), 2.69–2.77 (m,
8H), 2.94–2.95 (m, 1H), 3.08–3.09 (br s, 4H, N(CH2)2),
6.51–6.57 (m, 2H, Ar-H), 6.88–6.93 (m, 2H, Ar-H),
7.09–7.15 (m, 2H, Ar-H). Free base was converted into
its HCl salt, mp ¼ 155–159 ꢁC. Anal. Calcd for
(C27H37N3OCl2Æ3HClÆ0.8H2O) C, H, N.
13.2. (7-Methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-[4-
(4-phenyl-piperazin-1-yl)-butyl]-propyl-amine (9b)
Compound 8b (0.30 g, 0.71 mmol) was reacted with
LiAlH4 (0.25 g, 7.57 mmol) in THF (20 mL) (Procedure
G) to give 9b 0.28 g (99%). 1H NMR (CDCl3) 0.86–0.91
(t, J ¼ 7:2 Hz, 3H), 1.40–1.66 (m, 7H), 1.98–2.05(m,
1H), 2.38–2.43 (t, J ¼ 7:2 Hz, 2H), 2.45–2.50 (t,
J ¼ 7:2 Hz, 2H), 2.52–2.56 (t, J ¼ 6:8 Hz, 2H), 2.59–
2.62 (t, J ¼ 4:8 Hz, 4H, N(CH2)2), 2.69–2.88 (m, 4H),
2.91–3.02 (m, 1H), 3.20–3.22 (t, J ¼ 4:5 Hz, 4H,
N(CH2)2), 3.77 (s, 3H, CH3O), 6.62–6.69 (m, 2H, Ar-H),
6.83–7.00 (m, 4H, Ar-H), 7.24–7.29 (m, 2H, Ar-H).
12. Procedure F
12.1. N-(7-Methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-
N-[2-(4-phenyl-piperazin-1-yl)-ethyl]-propionamide (8a)
Propionyl chloride (0.2 g, 2.16 mmol) was added to a
solution of compound 5a (0.36 g, 0.97 mmol) and Et3N
(1.0 mL) in anhydrous methylene chloride at 0 ꢁC under
N2 atmosphere and then stirred at room temperature for
2 h. The reaction was diluted with CH2Cl2, washed with
water, brine, and the organic layer was dried over
Na2SO4, evaporated, and purified by flash chromatog-
raphy. (EtOAc/MeOH/Et3N ¼ 95:5:0.5) to give 8a, 0.42 g
13.3. {2-[4-(2,3-Dichloro-phenyl)-piperazin-1-yl]-ethyl}-
(7-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-propyl-
amine (9c)
1
(93%). H NMR (CDCl3) 1.14–1.20 (t, J ¼ 6:6 Hz, 3H,
CH3CH2CO), 1.63–1.70 (m, 2H), 1.90–2.04 (m, 2H),
2.40–2.50 (m, 4H), 2.66–2.74 (t, J ¼ 4:8 Hz, 4H,
N(CH2)2) 2.76–2.91 (m, 5H), 3.20–3.22 (t, J ¼ 4:8 Hz,
4H, N(CH2)2), 3.78 (s, 3H, CH3O), 6.60–6.70 (m, 2H, Ar-
H), 6.91–7.01 (m, 4H, Ar-H), 7.25–7.29 (m, 2H, Ar-H).
A mixture of amine 5c (1.47 g, 3.39 mmol), 1-bromo-
propane (2.08 g, 15.40 mmol), and K2CO3 (3.0 g,
21.74 mmol) in dry DMF (40 mL) was stirred at 60 ꢁC
for 20 h. The reaction mixture was poured into water
(100 mL) and extracted with Et2O (3 · 200 mL). The
combined organic phase was washed with brine and
dried over Na2SO4 (Procedure D). The solvent was re-
moved by evaporation and residue was purified by flash
chromatography (EtOAc/Et3N ¼ 100:1) to a thick oil
1.32 g (82%). 1H NMR (300 MHz, CD3Cl) 0.87–0.93 ( t,
J ¼ 7:2 Hz, 3H, CH3CH2CH2N), 1.46–1.53 (m, 4H),
1.88–2.07 (m, 2H), 2.51–2.55 (t, J ¼ 7:2 Hz, 2H, CH2N),
12.2. N-(7-Methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-
N-[4-(4-phenyl- piperazin-1-yl)-butyl]-propionamide (8b)
Compound 5b (0.28 g, 0.71 mmol) was reacted with
propionyl chloride (0.25 g, 3.57 mmol) and Et3N

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A. K. Dutta et al. / Bioorg. Med. Chem. 12 (2004) 4361–4373
2.64–2.68 (t, J ¼ 4:8 Hz, 4H, N(CH2)2), 2.71–2.83 (m,
6H), 2.90–3.06 (m, 1H), 3.21–3.24 (t, J ¼ 4:8 Hz, 4H,
N(CH2)2), 3.78 (s, 3H, CH3O), 6.64–6.71 (m, 2H, Ar-H),
6.95–7.01 (m, 2H, Ar-H), 7.14–7.15 (m, 2H, Ar-H).
14.3. 4-{[2-(4-Phenyl-piperazin-1-yl)-ethyl]-propyl-
amino}-cyclohexanone (13)
A solution of ketal 12 (2.00 g, 5.17 mmol) in THF
(20 mL) and 1 N HCl (20 mL) was stirred at 80 ꢁC under
N2 for 2 h. THF was removed under vacuo and satu-
rated NaHCO3 solution was added slowly. The mixture
was extracted with EtOAc (3 · 100 mL) and the com-
bined organic layer was washed with brine, dried over
Na2SO4, and evaporated to give the crude product,
which was purified by flash chromatography (EtOAc/
Et3N ¼ 100:1) to afford 13 1.31 g (99%). 1H NMR
(300 MHz, CDCl3) 0.87–0.90 (t, J ¼ 5:7 Hz, 3H, CH3),
1.43–1.51 (dt, J ¼ 5:7, 6.0 Hz, 2H, NCH2CH2CH3),
1.70–1.79 (m, 2H), 2.04–2.07 (m, 2H), 2.30–2.52 (m,
8H), 2.64–2.70 (m, 6H), 2.97–3.03 (t, J ¼ 10:4 Hz, 1H,
NCH(CH2)2), 3.19–3.21 (t, J ¼ 4:4 Hz, 4H, N(CH2)2),
6.83–6.87 (t, J ¼ 7:2 Hz, 1H, Ar-H), 6.91–6.93 (d,
J ¼ 8:0 Hz, 2H, Ar-H), 7.24–7.28 (m, 2H, Ar-H).
13.4. {4-[4-(2,3-Dichloro-phenyl)-piperazin-1-yl]-butyl}-
(7-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-propyl-
amine (9d)
Compound 9d was synthesized from 5d by following the
Procedures F and G (92%, two steps). 1H NMR(CDCl3)
0.89–0.93 (t, J ¼ 7:2 Hz, 3H, CH3CH2), 1.47–1.61 (m,
6H), 2.02–2.05 (m, 2H), 2.44–2.49 (t, J ¼ 7:2 Hz, 2H,
NCH2), 2.52–2.65 (m, 8H), 2.76–2.87 (m, 5H), 3.07–3.08
(br s, 4H, N(CH2)2), 3.77 (s, 3H, CH3O), 6.63–6.70 (m,
2H, Ar-H), 6.95–7.00 (m, 2H, Ar-H), 7.14–7.19 (t,
J ¼ 2:7 Hz, 2H, Ar-H).
14. Synthesis of 14 and 15
14.4. N6-[2-(4-Phenyl-piperazin-1-yl)-ethyl]-N6-propyl-
4,5,6,7-tetrahydro-benzothiazole-2,6-diamine (14)
14.1. (1,4-Dioxa-spiro[4.5]dec-8-yl)-[2-(4-phenyl-pipera-
zin-1-yl)-ethyl]-amine (11)
Compound 13 (0.50 g, 1.45 mmol) and pyrrolidine
(0.13 mL, 1.52 mmol) in anhydrous benzene was heated
to reflux in a Dean–Stark apparatus for 1.5 h. The
mixture was then cooled to room temperature and
concentrated in vacuo. The residue was dissolved in
A mixture of amine 4a (2.21 g, 10.8 mmol), cyclohex-
anedione monoethylene ketal (1.68 g, 10.8 mol),
Na(OAc)3BH (3.24 g, 15.12 mmol), and HOAc(0.65 g,
10.8 mol) in ClCH2CH2Cl (40 mL) was stirred overnight
at room temperature. The reaction mixture was diluted
with EtOAc (200 mL), washed with saturated NaHCO3
solution, water, and brine. The organic layer was dried
over Na2SO4, evaporated and the crude product was
purified by flash chromatography (EtOAc/MeOH/
Et3N ¼ 100:5:1) to give 11 as a white solid 3.55 g (91%).
1H NMR (300 MHz, CDCl3) 1.52–1.56 (m, 6H), 1.74–
1.79 (m, 2H), 1.91–1.96 (m, 2H), 2.57–2.66 (m, 7H,
N(CH2)2, CHNHCH2), 2.80–2.84 (t, J ¼ 6:0 Hz, 2H),
3.14–3.18 (t, J ¼ 48 Hz, 4H, N(CH2)2), 3.90 (s, 4H,
OCH2CH2O), 6.83–6.91 (m, 3H, Ar-H), 7.21–7.25 (m,
2H, Ar-H).
anhydrous
MeOH.
Sulfur
powder
(0.046 g,
0.1812 mmol, of S8) was added at room temperature,
stirred for 20 min and then cooled to 0 ꢁC. Cyanamide
(0.061 g, 1.45 mmol) in MeOH was added to the reaction
mixture and stirred overnight at rt. Methanol was
evaporated on the rotavapor, the residue dissolved in
EtOAc, washed with water, brine, dried, concentrated,
and purified by column chromatography (0.5% MeOH
and 1% Et3N in EtOAc) to afford the title product 14
(0.385 g, 66%) as a thick yellow solid.
1H NMR (400 MHz, CDCl3): 0.88 (t, 3H, J ¼ 7:4 Hz),
1.42–1.51 (m, 2H), 1.71 (dq, 1H, J ¼ 5:6, 12.0 Hz), 1.97–
1.99 (m, 1H), 2.45–2.59 (m, 6H), 2.63 ()2.72 (m,
8H), 3.00–3.05 (m, 1H), 3.19 (t, 4H, J ¼ 4:8 Hz), 4.84
(s, 2H), 6.85 (t, 1H, J ¼ 7:2 Hz), 6.92 (d, 2H, J ¼ 8:0
Hz), 7.23–7.27 (m, 2H). The product was converted into
the corresponding trioxalate salt. Anal. Calcd for
(C28H39N5SO12Æ1.55H2O) C, H, N.
14.2. (1,4-Dioxa-spiro[4.5]dec-8-yl)-[2-(4-phenyl-pipera-
zin-1-yl)-ethyl]-propyl-amine (12)
Compound 11 (1.0 g, 2.94 mmol), 1-bromopropane
(1.45 g, 11.80 mol), and K2CO3 (1.22 g, 8.70 mmol) in
dry DMF (20 mL) was stirred at 60 ꢁC for 10 h and the
mixture was poured into water (50 mL) and extracted
with Et2O (3 · 100 mL). The combined organic layer was
washed with brine, dried over Na2SO4 evaporated and
the residue was purified by flash chromatography
(EtOAc/MeOH/Et3N ¼ 100:2:1) to give 12 as a thick oil
14.5. N6-[2-(4-Phenyl-piperazin-1-yl)-ethyl]-N6-propyl-
5,6,7,8-tetrahydro-quinazoline-2,6-diamine (15)
Into a solution of ketone 13 (0.60 g, 1.75 mmol) in dry
toluene (20 mL), tris(dimethylamino)methane (1.27 g,
8.76 mmol) was added and the mixture was stirred under
nitrogen at 90 ꢁC for 4 h. The solvent was removed un-
der vacuo and the residue was dissolved in EtOH
(25 mL). Guandine carbonate (0.78 g, 4.33 mmol) was
added next. The mixture was then refluxed for 17 h. The
solvent was evaporated in vacuo and the residue was
diluted with CH2Cl2 and washed with brine. The organic
layer was dried over Na2SO4 and evaporated to give the
1
0.80 g (70%). H NMR (300 MHz, CDCl3) 0.83–0.88 (t,
J ¼ 7:2 Hz, 3H, CH3), 1.40–1.47 (m, 2H), 1.52–1.58 (m,
4H), 1.73–1.80 (m, 4H), 2.41–2.50 (m, 4H), 2.58–2.65
(m, 7H), 3.17–3.20 (t, J ¼ 5:2 Hz, 4H, N(CH2)2), 3.17–
3.20 (t, J ¼ 5:2 Hz, 4H, N(CH2)2), 3.92 (s, 4H,
OCH2CH2O), 6.82–6.86 (t, J ¼ 7:2 Hz, 1H, Ar-H),
6.90–6.93 (d, J ¼ 7:8 Hz, 2H, Ar-H), 7.22–7.28 (m, 2H,
Ar-H).

A. K. Dutta et al. / Bioorg. Med. Chem. 12 (2004) 4361–4373
4371
crude product, which was purified by flash chromato-
graphy (EtOAc/MeOH/Et3N ¼ 25:1:1) to Give 15 as a
6.92 (d, 2H, J ¼ 8:8 Hz), 7.23–7.27 (m, 2H), 7.28–7.36
(m, 3H), 7.38–7.40 (m, 2H), 7.47–7.49 (m, 1H).
1
yellow solid, 0.61 g (88%). H NMR (300 MHz, CDCl3)
0.86–0.91 (t, J ¼ 7:2 Hz, 3H, CH3), 1.44–1.52 (dt,
J ¼ 7:2, 7.2 Hz, 2H, NCH2CH2CH3), 1.61–1.74 (m, 1H),
2.03–2.10 (m, 1H), 2.49–2.56 (m, 4H), 2.64–2.67 (t,
J ¼ 5:0 Hz, 4H, N(CH2)2), 2.69–2.75 (m, 4H), 2.79–
2.81(dd, J ¼ 2:8, 6.0 Hz, 1H), 2.85–2.95 (m, 1H), 3.18–
3.21 (t, J ¼ 5:0 Hz, 4H, N(CH2)2), 4.88 (s, 2H, NH2),
6.82–6.87 (t, J ¼ 7:2 Hz, 1H, Ar-H), 6.90–6.93 (d,
J ¼ 8:4 Hz, 2H, Ar-H), 8.01 (s, 1H, H-pyrimidine). Free
base was converted into its HCl salt. Mp ¼ 97–102 ꢁC.
Anal. Calcd for (C23H34N6Æ4HClÆ1.5H2O) C, H, N.
1H NMR (400 MHz, CDCl3) of 16b: 1.24–1.28 (m, 2H),
2.34–2.44 (m, 2H), 2.58–2.74 (m, 8H), 2.80–2.87 (m,
2H), 3.19 (t, 4H, J ¼ 4:8 Hz), 3.46 (s, 3H), 3.75 (s, 3H),
4.19–4.21 (m, 1H), 5.04 (s, 1H), 6.46 (d, 1H, J ¼ 2:4 Hz),
6.69 (dd, 1H, J ¼ 2:4, 8.0 Hz), 6.85 (t, 1H, J ¼ 7:2 Hz),
6.92 (d, 2H, J ¼ 8:8 Hz), 7.24–7.29 (m, 2H), 7.31–7.35
(m, 3H), 7.38–7.40 (m, 2H), 7.48–7.50 (m, 1H).
15.3. (7-Methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-[2-
(4-phenyl-piperazin-1-yl)ethyl]-amine: (+)-isomer-(17a)
15. Synthesis of enantiomers of 10a
Potassium t-butoxide (0.26 g, 2.34 mmol) was added to a
solution of 16a (0.20 g, 0.39 mmol) in THF at room
temperature under nitrogen atmosphere and stirred
overnight. TLC revealed complete consumption of
the starting material and formation of a mixture of the
free amine and N-formyl derivative. The solvent was
removed, the residue suspended in EtOAc, washed with
water, NH4Cl solution, brine, dried, and concentrated.
15.1. 2-Methoxy-N-(7-methoxy-1,2,3,4-tetrahydro-naph-
thalen-2-yl)-2- phenyl-N-[2-(4-phenyl-piperazin-1-yl)-
ethyl]-acetamide (16)
A
mixture of (R)-())-a-methoxyphenylacetic acid
(0.456 g, 2.74 mmol), EDCI (0.58 g, 3.02 mmol), HOBT
(0.40 g, 3.02 mmol), and Et3N (0.38 mL, 2.75 mmol) in
anhydrous methylene chloride was stirred at room
temperature under nitrogen atmosphere for 1 h. A
solution of amine 5a (0.5 g, 1.374 mmol) in anhydrous
methylene chloride was added to the reaction mixture
under N2 at room temperature and stirred for 20 h. The
mixture was diluted with CH2Cl2, washed with 5% citric
acid solution, sodium bicarbonate, and brine. The sol-
vent was removed and the residue was purified by
column chromatography (1% MeOH, 3% Et3N in
EtOAc) to afford a diastereomeric mixture of 16 (0.57 g,
81%).
Into a solution of this mixture in dry THF, a solution of
MeLi (0.56 mL, 0.78 mmol) was added at rt under N2
atmosphere and stirred for approximately 0.5 h when
TLC revealed completion of the reaction. The mixture
was quenched with water, the solvent evaporated, and
the residue suspended in EtOAc. The organic layer was
washed with water, brine, dried, concentrated, and
purified by column chromatography (10% MeOH, 1%
Et3N in EtOAc) to afford the optically pure amine
(+)17a (0.13 g, 88%).
1H NMR (400 MHz, CDCl3): 1.60–1.68 (m, 2H), 2.60–
2.63 (m, 6H), 2.76—3.04 (m, 7H), 3.18 (t, 4H,
J ¼ 4:8 Hz). 3.78 (s, 3H), 6.51 (d, 1H, J ¼ 2:4 Hz), 6.68
(dd, 1H, J ¼ 2:4, 8.0 Hz), 6.84 (t, 1H, J ¼ 7:2 Hz), 6.92
(d, 2H, J ¼ 8:8 Hz), 7.28–7.36 (m, 3H).
15.2. Separation of diastereomers
The diastereoisomers were separated by semi-pre-
parative HPLC using a normal phase column (Nova-
Pack Silica 6 lm). The mobile phase used was 3%
isopropanol in hexane with a flow rate of 18 mL/min.
The two fractions were eluted with retention time of
18.76 min for the (+)-isomer 16a and 23 min for ())-
isomer 16b. Final purity of the separated diastereoiso-
mers was checked by an analytical normal phase column
15.4. (7-Methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-[2-
(4-phenyl-piperazin-1-yl)ethyl]-amine: ())-isomer-(17b)
Amide 16b (0.200 g, 0.39 mmol) was hydrolyzed under
similar conditions as reported above to afford the opti-
cally pure amine 17b (0.13 g, 88%).
ꢀ
(Nova-Pack Silica 60 A 4 lm) using the same mobile
phase with a flow rate of 1 mL/min. Pure diastereomers
16a (+)-isomer and 16b ())-isomer were eluted at 10.96
and 13.43 min, respectively.
1H NMR identical is identical with that of 17a.
The amines (+)-17a and ())-17b were converted to the
final products (+)-10a and ())-10a in three steps as
described in Scheme 2 (Procedures F, G, and E) in
81% and 82% yield, respectively.
Compound 16a (+)-isomer was isolated in 0.20 g, 30%
yield.
Compound 16b ())-isomer was isolated in 0.20 g, 30%
yield.
7-{[2-(4-Phenyl-piperazin-1-yl)-ethyl]-propyl-amino}-
5,6,7,8-tetrahydro-naphthalen-2-ol (+)-10a
1H NMR (400 MHz, CDCl3) of 16a: 1.24–1.29 (m, 2H),
2.33–2.43 (m, 2H), 2.50–2.74 (m, 8H), 2.82–2.89 (m,
2H), 3.19 (t, 4H, J ¼ 4:8 Hz), 3.52 (s, 3H), 3.75 (s, 3H),
4.26–4.34 (m, 1H), 5.10 (s, 1H), 6.51 (d, 1H, J ¼ 2:4 Hz),
6.68 (dd, 1H, J ¼ 2:4, 8.0 Hz), 6.84 (t, 1H, J ¼ 7:2 Hz),
Optical rotation: ½aꢀ +8.21 (c 2, MeOH)
D
1H NMR (400 MHz, CDCl3): 0.90 (t, 3H, J ¼ 7:2 Hz),
1.43–1.51 (m, 4H), 1.88–1.95 (m, 2H), 2.49 (t, 2H,

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A. K. Dutta et al. / Bioorg. Med. Chem. 12 (2004) 4361–4373
J ¼ 7:2 Hz), 2.52–2.69 (m, 6H), 2.72 (t, 4H, J ¼ 4:8 Hz),
2.84–2.89 (m, 1H), 3.21 (t, 4H, J ¼ 4:8 Hz), 6.51 (d, 1H,
J ¼ 2:4 Hz), 6.68 (dd, 1H, J ¼ 2:4, 8.0 Hz), 6.85 (t, 1H,
J ¼ 7:2 Hz), 6.91 (d, 2H, J ¼ 8:8 Hz), 7.27–7.37 (m, 3H).
and 200 mg/mL of G418. After 48 h, the cells were rinsed
twice with serum-free a-MEM and incubated for 24 h at
37 ꢁC. To initiate the functional agonism assay, the
medium was removed and replaced with 90 mL of ser-
um-free a-MEM and 10 mL of test compounds in sterile
water. After another 24 h incubation at 37 ꢁC, 0.25 mCi
of [3H]thymidine was added to each well and the plates
were further incubated for 2 h at 37 ꢁC. The cells were
then trypsinized and the plates were filtered and counted
as usual. Quinpirole was run on every plate as an
internal standard. Intrinsic activity relative to quinpirole
was calculated as the ratio of the EC50 of test drug over
that of quinpirole. This was done because some test
compounds were evaluated at a much later point in time
when the test system gave higher EC50 values for
quinpirole at both D2 and D3. Computation of the
D2/D3 selectivity ratio also took into account these
quinpirole shifts occurring in the latter assays by
including normalization to the quinpirole selectivity
observed in the early assays (9.98 nM at D2 and 2.91 nM
at D3).
The product was converted into the corresponding
trihydrochloride salt. Anal. Calcd for (C25H38N3Cl3O
0.5H2O) C, H, N.
7-{[2-(4-Phenyl-piperazin-1-yl)-ethyl]-propyl-amino}-
5,6,7,8-tetrahydro-naphthalen-2-ol ())-10a
Optical rotation: ½aꢀ )11.06 (c2, MeOH)
D
1H NMR (400 MHz, CDCl3): 0.90 (t, 3H, J ¼ 7:2 Hz),
1.43–1.51 (m, 4H), 1.88–1.95 (m, 2H), 2.49 (t, 2H,
J ¼ 7:2 Hz), 2.52–2.69 (m, 6H), 2.72 (t, 4H, J ¼ 4:8 Hz),
2.84–2.89 (m, 1H), 3.21 (t, 4H, J ¼ 4:8 Hz), 6.51 (d, 1H,
J ¼ 2:4 Hz), 6.68 (dd, 1H, J ¼ 2:4, 8.0Hz), 6.85 (t, 1H,
J ¼ 7:2 Hz), 6.91 (d, 2H, J ¼ 8:8 Hz), 7.27–7.37
(m, 3H).
The product was converted into the corresponding tri-
hydrochloride salt. Anal. Calcd for (C25H38N3Cl3OÆ
0.85H2O) C, H, N.
17. Rotational experiment with 6-OH-DA lesioned rats
The lesioned rats were purchased from Taconic Bio-
technology (Rensselaer, NY) and their unilateral lesion
was checked twice by apomorphine challenge following
the surgery. The first 14 days post-lesion challenge with
apomorphine was done to observe a complete rotation
session post administration. In the second challenge
with apomorphine (0.05 mg/kg) 21 days post lesion,
contralateral rotations were recorded for 30 min; apo-
morphine produced rotations in all four rats (average
rotation >250) indicating successful unilateral lesion. In
these rats, lesion was performed on the left side with the
rotations produced upon agonist challenge occurring
clockwise. In this study, apomorphine was also used as a
reference compound. The test drugs were dissolved in
sterile water and were administered as water solution.
The number of rotations was measured over 6 h. For
control, vehicle was administered alone. Rotations were
measured in the Rotomax Rotometry System (AccuScan
Instruments, Inc. Columbus, Ohio) equipped with Ro-
tomax Analyser, high resolution sensor and animal
chambers with harnesses. Data were analyzed with
Rotomax Window software program. Test drugs ())-
10a (10 mM/kg) was dissolved in sterile water and were
administered ip. Apomorphine (0.05 mg/kg) was also
administered, in the same manner, as a reference com-
pound. The rotations were measured in a rotational
chamber immediately after administration of drugs. The
data were collected at every 30 min. Data were analyzed
with the microsoft excel program. Compound ())-10a
produced contralateral rotations in all four lesioned rats
which lasted over 6 h. The peak effect was observed
between 2 and 3 h. The reference drug apomorphine
exhibited a fast onset of action with the peak effect
occurring within the first 30 min. It exhibited a short
duration of action. Each point on the graphs represents
the mean results from four rats with the corresponding
standard mean error (SEM).
16. Biological experiments
16.1. Potencies at dopamine D2 and D3 receptors
Binding affinities were assessed according to previously
published procedures.38–40 Human embryonic kidney
(HEK) 293 cells, stably transfected with cDNA for the
human D2L and D3 receptors39 were the source for
membranes prepared as described previously.38;40
Approximately 50 (for D2L) or 135 (for D3) mg of
protein was incubated with each test compound and
[3H]spiperone (0.49 nM) for 1 h at 30 ꢁC in 50 mM Tris–
HCl (pH 7.4), with 0.9% NaCl, and 0.025% ascorbic
acid in the absence of GTP, in a total volume of 2 mL.
(+)-Butaclamol (2 mM) was used to define nonspecific
binding. As in our previous study,40 assays were termi-
nated by addition of ice-cold buffer and washing with a
24-pin Brandel harvester. IC50 values were estimated by
nonlinear regression analysis with the ALLFIT equa-
tion, which is equivalent to the logistic model in the least
squares fitting program ORIGIN, and converted to
inhibition constants (Ki) by the Cheng–Prusoff equa-
tion.41 In this conversion, the Kd values for [3H]spipe-
rone binding were 0.18 nM for D2-receptors and
0.40 nM for D3-receptors as measured in our previous
experiments.40
16.2. D2 and D3 mitogenesis functional assay
To measure D2L and D3 stimulation of mitogenesis
(agonists assay), CHOp-cells (human receptor) were
seeded in a 96-well plate at a concentration of
5,000 cells/well. The cells were incubated at 37 ꢁC in a-
MEM with 10% FBS, 0.05% penicillin–streptomycin,

A. K. Dutta et al. / Bioorg. Med. Chem. 12 (2004) 4361–4373
4373
20. Crider, A. M.; Scheideler, M. A. Mini Rev. Med. Chem.
2001, 1, 89–99.
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25. Alexander van Vliet, L.; Tepper, P. G.; Dijkstra, D.;
Damsma, G.; Wikstrom, H.; Pugsly, T. A.; Akunne, H.
C.; Heffner, T. G.; Glase, S. A.; Wise, L. D. J. Med. Chem.
1996, 39, 4233–4237.
We thank Janet Berfield for conducting the binding
assays. We are also grateful to the NIDA medication
department for carrying out the functional mitogenesis
experiments under NIDA contract no. N01DA-1-8816.
We acknowledge Dr. Hanley Abramson for careful
reading of the manuscript.
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