3-[[(4-aryl-1-piperazinyl)alkyl]cyclohexyl]-1H-indoles as dopamine D2 partial agonists and autoreceptor agonists

Article abstract of DOI:10.1021/jm960597m

A series of arylpiperazines and tetrahydropyridines joined to indoles by semirigid cycloalkyl spacers were prepared. Target compounds were studied for their ability to bind to the DA D2 receptor in vitro and to inhibit dopamine synthesis and spontaneous l

Full text of DOI:10.1021/jm960597m

250
J . Med. Chem. 1997, 40, 250-259
3-[[(4-Ar yl-1-p ip er a zin yl)a lk yl]cycloh exyl]-1H-in d oles a s Dop a m in e D2 P a r tia l
Agon ists a n d Au tor ecep tor Agon ists
David J . Wustrow,* William J . Smith III, Ann E. Corbin, M. Duff Davis, Lynn M. Georgic, Thomas A. Pugsley,
Steven Z. Whetzel, Thomas G. Heffner, and Lawrence D. Wise
Departments of Chemistry and Therapeutics, Parke Davis Pharmaceutical Research, Division of Warner Lambert Company,
Ann Arbor, Michigan 48105
Received August 19, 1996X
A series of arylpiperazines and tetrahydropyridines joined to indoles by semirigid cycloalkyl
spacers were prepared. Target compounds were studied for their ability to bind to the DA D2
receptor in vitro and to inhibit dopamine synthesis and spontaneous locomotor activity in rats.
Effects of tether length and relative stereochemistry were assessed for a series of 2-pyridylpip-
erazines. The cyclohexylethyl spacer was found to be the most active in the series. Further
studies explored effects of changes in the arylpiperazine and indole portions of the molecule.
From these studies trans-2-[[4-(1H-3-indolyl)cyclohexyl]ethyl]-4-(2-pyridinyl)piperazine (30a )
was selected for further evaluation. It was characterized as a partial agonist of DA D2 receptors
in vitro and caused decreases in dopamine synthesis and release as well as dopamine neuronal
firing. Compound 30a was shown to be devoid of behavioral effects associated with postsynaptic
DA D2 receptor activation. Furthermore, compound 30a was shown both to decrease DA
synthesis and to inhibit Sidman avoidance responding in squirrel monkeys. These findings
suggest that DA D2 partial agonists with the appropriate level of intrinsic activity can act to
decrease dopamine synthesis and release and may have potential utility as antipsychotic agents.
On the basis of the success of dopamine (DA) D2
antagonists in treating the symptoms of schizophrenia,
it has been proposed that hyperactive DA neurons are
involved in the pathophysiology of the disease.1 The
ability of DA agonists to decrease dopamine synthesis
and release and to inhibit the firing of these neurons
(and therefore DA neurotransmission) via activation of
presynaptic DA autoreceptors2 has led to the suggestion
of such compounds as potential antipsychotic agents.
Clinical studies with low doses of the dopamine agonists
apomorphine and N-n-propylapomorphine suggest that
indeed this hypothesis holds merit.3 In order for such
compounds to be efficacious, they must stimulate the
presynaptic DA receptors, but lack significant activity
at postsynaptic receptors since stimulation of postsyn-
aptic D2 receptors would result in exacerbation of
schizophrenic symptoms.4 Presynaptic DA receptors
appear to include both the D2 and D3 receptor sub-
types.5 It has been suggested that DA D2 autoreceptors
control DA synthesis and release while D3 receptors
control only DA release from the presynaptic terminals.6
However to date most of the behavioral effects seen in
preclinical models used to test for possible antipsychotic
activity generally have been more strongly correlated
to the D2 subtype.7 DA D2 receptors are also present
postsynaptically so differentiation of these receptor
populations by pharmacological means would appear to
be difficult.8 However, in vitro studies suggest presyn-
aptic DA D2 receptors have a higher level of receptor
reserve than postsynaptic D2 receptors9 and therefore
are more sensitive to agonists and partial agonists. Thus
it is possible that partial agonists with an appropriate
level of intrinsic agonist activity at DA D2 receptors
would preferentially stimulate presynaptic autorecep-
tors while avoiding stimulation of postsynaptic recep-
tors.10 Such a compound would decrease the synthesis
and release of dopamine as well as the firing of DA D2
neurons but would not show effects in animal models
associated with stimulation of postsynaptic dopamine
D2 receptors.
Two examples of DA D2 agonists which have been
shown to inhibit dopamine synthesis and release via a
presynaptic mechanism include roxindole11 (1) and PD
11981912 (2). This class of compounds is characterized
by an aryl cyclic amine portion linked to a distal aryl
or heterocyclic pharmacophore via an alkyl spacer.
Unlike earlier DA agonists typified by apomorphine,
7-OH DPAT, and the ergots, the flexibility of the alkyl
spacer between compounds 1 and 2 makes it difficult
to determine the required geometric alignment of the
pharmacophore units required for optimal activity.
We have designed a series of analogs which introduce
constraints in the linkage of the two pharmacophores
by replacing the linear alkyl linker with various cy-
cloalkyl moieties. This work has led to the identification
of CI-1007 (3) as a DA D2 partial agonist which is
currently being developed as an antipsychotic drug.13
In this instance the phenyl and the 4-phenyltetrahy-
dropyridine pharmacophores are separated by the rela-
tively rigid cyclohexenylmethyl spacer. A related series
X Abstract published in Advance ACS Abstracts, December 15, 1996.
S0022-2623(96)00597-3 CCC: $14.00 © 1997 American Chemical Society

Dopamine D2 Partial Agonists and Autoreceptor Agonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2 251
Sch em e 1a
Sch em e 2a
a
(a) 7, KOH, MeOH, 44%.
of autoreceptor agonists is represented by compound 4
where the phenyl and the 4-phenyltetrahydropyridine
pharmacophores are separated by a cyclohexenylethyl
spacer.14 Replacement of the phenyl functionality resi-
dent in 4 with an indole as in 1 was contemplated
because this heterocycle contained both aryl functional-
ity and the potential for additional hydrogen bonding.
It was hoped that such a feature might interact with
complementary functionality in the D2 receptor binding
pocket. Initially the cyclohexenylindole analog 6 was
prepared, and as part of these studies, we prepared the
related indole cyclohexane analogs.
a
(a) KOH, MeOH, 73-96%; (b) (i) H2, Pd/C THF; (ii) HCl, 66%
overall; (c) (i) 1-(2-pyridyl)piperazine, PTSA toluene, Dean-Stark;
(ii) NaCNBH3, 95%.
Ch em istr y
Sch em e 3a
The cyclohexenylindole 6 was prepared by condensa-
tion of ketone 514 with indole 7. Attempts to prepare
the cycloalkane analogs directly by hydrogenation re-
sulted in an inseparable mixture of cis and trans
isomers about the cyclohexane ring. An alternative
route was developed that allowed for easier separation
of the geometric isomers. Indoles 7-9 were condensed
with cyclohexanedione monoketal 10 under basic condi-
tions giving good yields of 4-(3-indolyl)-3-cyclohexenone
ketals 11-13. Reduction of the olefin and deketaliza-
tion proceeded smoothly to give the 4-(3-indolyl)-3-
cyclohexanones 14-16 as shown in Scheme 1. Ketone
14 was further elaborated via reductive amination to
the corresponding pyridylpiperazines 17a and 17b
which could be easily separated by column chromatog-
raphy (Scheme 2). The corresponding one-carbon ho-
mologs were prepared as outlined in Scheme 3. Reac-
tion of ketone 14 with the ylide derived from the
reaction of (methoxymethyl)triphenylphosphonium chlo-
ride and n-butyllithium gave enol ether 18, which was
hydrolyzed to a mixture of aldehydes 19. Following
reductive amination of aldehydes 19, the (cyclohexyl-
methyl)piperazines 20a and 20b were separated chro-
matographically. The two-carbon homologs were pre-
pared by condensation of ketones 14-16 with triethyl
phosphonoacetate to give the corresponding cyclohex-
enyl esters 21-23, which were reduced and hydrolyzed
to 2-[4-(3-indolyl)cyclohexyl]acetic acids 24-26 as ap-
proximately a 2:1 mixture of trans and cis geometric
isomers. The mixtures of carboxylic acid isomers were
converted to the corresponding amides using isobutyl
chloroformate as the coupling agent. The amide isomers
27a ,b and 28a -h were separated by column chroma-
tography. The pure amides were then reduced to their
a
(a) (Methoxymethyl)triphenylphosphonium chloride, n-BuLi,
THF, -78 to 10 °C, 57%; (b) HCl, 96%; (c) 1-(2-pyridyl)piperazine,
NaCNBH3, 36%.
respective amines 29a ,b and 30a -h with aluminum
hydride. Indole 30h could be demethylated using
pyridine hydrochloride at elevated temperature to give
the 5-hydroxy analog 31. Methylation of the indole
nitrogen of 30a was accomplished following literature
analogy giving compound 32.15 Stereochemical assign-

252 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2
Wustrow et al.
Sch em e 4a
a
(a) Triethyl phosphonoacetate, NaH, 78-83%; (b) (i) H2, Pd/C, (ii) NaOH, 83-85%; (c) isobutyl chloroformate, Et3N, chromatography;
(d) LiAlH4, AlCl3, 51-83%.
Sch em e 5a
or “low-affinity” form of the DA D2 receptor, we used
this as an initial screen for receptor binding activity
because of its ready application to high throughput
screening. Compounds were then screened for their
ability to inhibit exploratory locomotor activity after ip
injection into mice as an in vivo measure of the inhibi-
tion of DA neurotransmission and potential antipsy-
chotic activity.17 Compounds with an ED50 of less than
10 mg/kg in the mouse assay were then tested orally in
a similar paradigm in rats in order to demonstrate oral
bioavailability. Selected compounds were evaluated for
their ability to reverse γ-butyrolactone (GBL)-induced
synthesis of L-dihydroxyphenylalanine (DOPA)18 in rat
corpus striatum as a neurochemical measure of DA
autoreceptor agonist activity.
Resu lts a n d Discu ssion
The receptor binding screen showed that indeed the
indolylcyclohexene analog 6 bound with somewhat
greater affinity to the DA D2 receptor (Ki ) 270 nM)
than the corresponding phenyl analog 4 (Ki ) 412 nM).14
However, the corresponding indolylcyclohexane analog
30a showed even higher affinity for the DA D2 receptor
and thus became the focus of a larger study. A series
of [[1-(2-pyridyl)piperazinyl]cycloalkyl]indoles 17a ,b,
20a ,b, 29a , and 30a were compared in order to study
the effect of chain length and relative stereochemical
alignment required for optimal DA D2 receptor binding
(Table 1). Increasing chain length improved binding
affinity for the DA D2 receptor. For each pair of
geometric isomers, the trans isomers showed greater
binding affinity than the corresponding cis isomers. A
similar trend was observed for a pair of tetrahydropyr-
a
(a) Pyridine hydrochloride 130 °C, 56%; (b) KOH, MeI, DMSO,
56%.
ments were made on the basis of the chemical shifts
and coupling constants of the proton attached to the
carbon bearing the indole functionality.
P h a r m a cology
The affinities of compounds for DA D2 receptors were
determined in vitro by measuring their ability to
displace the DA D2 antagonist radio ligand [3H]spiper-
one ([3H]SPIP) from a rat striatal membrane prepara-
tion.16 Although this radioligand labels the antagonist

Dopamine D2 Partial Agonists and Autoreceptor Agonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2 253
Ta ble 1. Binding, Behavioral, and Neurochemical Activity of Target Compounds
X
n
NR1R2
N
Y
[3H]SPIP
binding:
Ki, nMa
inhibn LMA
inhibn of LMA
rat: ED50
mg/kg pob
% rev of rat DA
synthesis at
mouse: ED50
,
,
no.
n
stereo
cis
NR1R2
X
Y
mg/kg ipb
10 mg/kg ipc
17a
0
H
H
2596
>30
ntd
nt
N
N
N
17b
20a
20b
29a
30a
29b
0
1
1
2
2
2
trans
cis
trans
cis
trans
cis
′′
′′
′′
′′
′′
H
H
H
H
H
H
H
H
H
H
H
H
457
122
85
139
50
1.2 (0.7; 2.2)
1.2 (0.6; 2.2)
2.3 (1.4; 3.7)
4.7 (3.5; 6.5)
1.4 (0.7; 2.6)
13.7 (7.5; 25.1)
weake
8.8 (6.9; 11.1)
12.2 (7.8; 19.2)
4.8 (3.0; 7.6)
nt
38 ( 5.9
nt
81 ( 5.6
60 ( 9.9
44 ( 4.4
5 ( 5
28
10.5 (8.8; 12.7)
N
30b
30c
2
2
trans
trans
′′
H
H
H
H
8.7
124
7.2 (2.4; 21.1)
13.9 (5.7; 34.0)
>30
inc
inc
nt
N
N
N
30d
30e
2
2
trans
trans
H
H
H
H
645
161
>30
nt
nt
N
F
3.3 (2.2; 5.0)
18.6 (10.7; 32.4)
34 ( 13
N
N
N
N
S
30f
2
2
trans
trans
H
F
H
H
630
110
11.6 (4.6; 29.5)
9.0 (4.9; 16.4)
nt
nt
N
N
30g
stim
100 ( 3.0
N
N
30h
31
32
1
2
2
2
trans
trans
trans
′′
′′
′′
OMe
OH
H
H
H
Me
29
8.6
83
14
2.2 (1.8; 2.7)
0.6 (0.4; 0.9)
>30
>30
88 ( 1.3
25 ( 11
nt
>30
nt
0.25 (0.12; 0.52)
47.6 (15.7; 144.2)
70 ( 2.2
a
Competition binding studies were completed in membranes form rat striatum using [3H]spiperone (0.2 nM) to label D2 dopamine
receptors. The Ki values were determined using the Cheng-Prusoff equation following one-site analysis using nonlinear regression analysis.
b
ED50 values and 95% confidence ranges were generated using from three to six doses; 6-18 animals were used per dose. c Shown is the
per cent reversal of DOPA levels in the striatum of GBL-treated rats (n ) 4-5 animals). The control values in mg/g (SEM of the tissues
for 0% (GBL + NSD 1015) 3831 ( 149 and 100% (vehicle + NSD 1015) 1181( 65. Not tested. e 57% inhibition at 3 mg/kg ip; 1.8%
d
inhibition at 30 mg/kg.
idine diastereomers 29b and 30b. Compounds in this
group were screened for their ability to decrease spon-
taneous locomotor activity in mice when injected ip. The
pyridiylpiperazine analogs were more potent in this
assay, all having ED50 values under 5 mg/kg ip. This
series also showed activity as autoreceptor agonists
based upon their ability to decrease dopa levels in rats
pretreated with GBL. Of the compounds which had
neurochemical and behavioral activity, 30a was the
most potent at inhibiting spontaneous locomotor activity
in rats when administered orally.
Having established compound 30a with the trans-
cyclohexylethyl spacer as the most potent member of
this series, we then examined the effects of modifying
the pyridylpiperazine and indole portions of compound
30a . The 4-phenyltetrahydropyridine analog 30b had
better binding affinity for the DA D2 receptor. How-
ever, unlike 30a , it did not decrease but rather slightly
increased the DOPA levels in the striata of rats treated
with GBL. A variety of other arylpiperazine and
aryltetrahydropyridine ring systems (30c-f) had either
decreased affinity for the DA D2 receptor or less activity
in the in vivo assays.
Effects of substituents at the 5-position of the indole
portion of 30a were examined. Compound 30g, having
a fluorine substituent at the 5-position of the indole ring,
completely reversed the GBL-induced increase in DOPA
synthesis, but caused stimulation of locomotor activity
in the rat. Such a profile suggests that 30a might have
high intrinsic activity and be stimulating both pre and
postsynaptic DA receptors. The 5-hydroxyindole analog
31 had potent DA D2 receptor binding affinity and
reduced locomotor activity when administered ip in
mice; however, this compound lacked oral activity in the
rat. This may be due to the poor bioavailability of the
compound. Addition of a methyl substituent to the
indole nitrogen (compound 32) resulted in less than a
2-fold decrease in binding activity, suggesting the indole
N-hydrogen does not play a large role in the binding
affinity of 30a for the D2 receptor. Interestingly
compound 32 lacked activity in the mouse exploratory
motor activity test after ip administration.
On the basis of its profile in initial biochemical,
behavioral, and neurochemical tests, 30a was selected
for evaluation in a variety of secondary tests designed
to understand its mechanism of action and possible

254 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2
Ta ble 2. Profile of 30a
Wustrow et al.
activity was observed in the squirrel monkey as 30a
caused a 38% decrease in DA overflow in the caudate
putamen.
test
30a
DA D2 receptor binding
2.6
[3H]NPA (Ki, nM)
To determine whether the inhibitory effects on loco-
motor activity exhibited by 30a were due to antagonist
activity at postsynaptic DA D2 receptors, the compound
was evaluated for its ability to inhibit apomorphine-
induced climbing in mice.24 DA D2 antagonists such
as haloperidol potently block the climbing effects caused
by the DA agonist apomorphine. Consistent with its
partial agonist profile in vitro, compound 30a was
unable to block apomorphine-induced climbing in mice
at a dose over 10 times its ED50 for blockade of
spontaneous locomotor activity. The compound was also
evaluated for its ability to cause DA agonist related
stereotypy in rats by administering the test compound
with the DA D1 agonist SKF 38393.25 Administration
of a dose 10 times the ED50 for inhibition of locomotor
activity did not produce stereotypies, a property associ-
ated with postsynaptic receptor activation. Compound
30a was then characterized for its ability to inhibit the
Sidman avoidance responding in squirrel monkeys,17c,26
a primate test which is considered to be predictive of
antipsychotic efficacy. In this paradigm, compound 30a
was active after oral administration (ED50 1.0 mg/kg).
inhibition of 3H-thymidine uptake
intrinsic activity DA D2 receptora
EC50 (nM)
72%
2.8
29
DA D3 receptor binding
[3H]spiperone (Ki, nM)
DA D4 receptor binding
73
6.6
[3H]spiperone (Ki, nM)
DOPA accumulation in rats
after GBLb (ED50, mg/kg ip)
decrease of rat striatal dopamine
overflow (10 mg/kg, ip)c
decrease in DA neuronal firing rate in
rats (2.5 mg/kg ip)
33%
100%
>30
inhibn of APO-induced climbing in mice
(ED50, mg/kg/ip)
stereotypy in rats (ED50, mg/kg, ip)
decrease in squirrel monkey striatal
dopamine overflow (3 mg/kg ip)d
inhibn of sidman avoidance in squirrel
monkey (ED50, mg/kg po)
>24
38%
1.0
a
b
Data expressed relative to quinpirole (100%). Graphically
determined ED50 value, defining 50% reversal of the increase in
DOPA accumulation induced by GBL. c Measured via in vivo
d
microdialysis (n ) 4). Measured via in vivo microdialysis (n )
1)
Interestingly, in our hands roxindole (1) was inactive
in this paradigm of antipsychotic efficacy.27 Compound
1 has been shown to have a higher level of agonist
activity at the D2 receptor than compound 30a as 1 has
a greater intrinsic activity in stimulating [3H]thymidine
uptake in CHO cells transfected with the D2 receptor
(97%) compared to that observed for 30a (72%). Taken
together, these studies indicate that 30a is a partial
agonist which can stimulate presynaptic DA D2 recep-
tors, resulting in decreases in the synthesis and release
of dopamine. This inhibition in DA neurotransmission
decreases the observed behavioral activity in mice, rats,
and squirrel monkeys. Furthermore 30a was not active
in behavioral studies sensitive to activation of postsyn-
aptic DA D2 receptors. Thus it would appear to be
possible to prepare compounds with the appropriate
level of intrinsic activity which will selectively stimulate
presynaptic DA D2 autoreceptors (and thereby at-
tenuating dopamine neurotransmission) without acti-
vating postsynaptic DA D2 receptors which could exac-
erbate schizophrenia.
utility as an antipsychotic agent (Table 2). Compound
30a potently displaced the dopamine agonist ligand [3H]-
NPA19 from dopamine D2 receptors in a rat brain
preparation. This agonist ligand is thought to label only
DA D2 receptors in the high-affinity state. The greater
affinity of 30a for DA D2 receptors using the agonist
ligand suggests that the compound interacts more
effectively with the high-affinity state of the DA D2
receptor as would be expected of a DA agonist. Affini-
ties of 30a for the human D3 and D4 receptor subtypes
were also determined as previously outlined.20,21 As the
D3 receptor has been implicated in controling the
neuronal release of DA, affinty for this receptor may
contribute to the overall profile of the compound. The
intrinsic agonist efficacy of 30a at the DA D2 receptor
was measured by its ability to stimulate [3H]thymidine
uptake in Chinese hamster ovary (CHO-P5) cells trans-
fected with the human D2 receptor.22 In this assay 30a
stimulated [3H]thymidine uptake at 70% of the level
observed with the full DA D2 agonist quinpriole indicat-
ing compound 30a had partial agonist efficacy. The
EC50 value for this functional effect was in good agree-
ment with the Ki value obtained from the [3H]NPA
binding assay. This in vitro profile suggests 30a to be
a potent partial agonist at the DA D2 receptor.
Exp er im en ta l Section
Melting points were determined on a Thomas-Hoover capil-
lary melting point apparatus and are uncorrected. Proton
NMR were recorded on a Bruker 250 MHz NMR instrument,
a Varian 200 MHz NMR instrument, or a Bruker 400 MHz
NMR instrument. The spectra recorded were consistent with
the proposed structures. The mass spectra were obtained on
a Finnigan 4500 mass spectrometer; the spectra are described
by the molecular peak (M) and its intensity relative to the base
peak (100). Elemental analyses were performed by the
Analytical Chemistry Section at Parke-Davis, Ann Arbor, MI.
Where analyses are indicated by the symbols of the elements,
the results are within 0.4% of the theoretical values. TLC was
performed on 0.25 mm silica gel F254 (E. Merck) glass plates.
Medium-pressure chromatography (MPLC) was performed on
silica gel (E. Merck, grade 60, 230-300 mesh) with an RB-SY
pump (FMI).
The in vivo effects of compound 30a on electrophysi-
ological and neurochemical markers of DA D2 receptor
activation were determined. Compound 30a inhibited
spontaneous firing of substantia nigra DA neurons in
anesthetized rats, an effect consistent with presynaptic
DA D2 receptor activation.23 It was also effective at
inhibiting GBL-induced increases of striatal DOPA
synthesis with an ED50 of 6.6 mg/kg ip.18 In order to
determine whether compound 30a acts to reduce DA
levels in both rats and squirrel monkeys, its effects on
the release of DA in the striata of both rat and squirrel
monkey were measured by microdialysis experiments.13b
In agreement with the GBL experiments, 30a reduced
extracellular dopamine overflow in the rat. Similar
3-[4-[2-[4-(2-P yr id in yl)-1-p ip er a zin yl]et h yl]-1-cyclo-
h exen -1-yl]-1H -in d ole H yd r och lor id e (6). A mixture of

Dopamine D2 Partial Agonists and Autoreceptor Agonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2 255
indole 7 (0.82 g, 6.96 mmol), ketone 514 (2.0 g, 6.9 mmol) and
potassium hydroxide (0.194 g, 3.48 mmol) was heated to reflux
in 10 mL of methanol for 18 h. The methanol was removed
under reduced pressure, and the residue was partitioned
between water (50 mL) and methylene chloride (50 mL). The
organic layer was dried with sodium sulfate and evaporated,
and the resulting residue was purified by MPLC (2% methanol
0.1% ammonia in methylene chloride) to give the free base of
compound 6 (1.2 g 44% yield): EIMS m/z 387 (43), 386 (25),
ketal (12) to 4-(5-fluoro-1H-3-indolyl)cyclohexanone (15) was
carried out in a manner analogous to the synthesis of 14: mp
117-9 °C; EIMS m/z 231 (62), 174 (base); IR (KBr, cm-1) 3338,
1
1705, 1488, 1461; H-NMR (CDCl3, 250 MHz) δ 8.13 (br s, 1
H), 7.28 (m, 2 H), 7.04 (d, J ) 2 Hz, 1 H), 6.96 (td, J ) 9, 2 Hz,
1 H), 3.28 (tt, J ) 12, 3 Hz, 1 H), 2.53 (m, 4 H), 2.42 (m, J )
12 Hz, 2 H), 1.95 (ddd, J ) 24, 12, 6 Hz, 2 H). Anal. (C14H14
FNO) C, H, N, F.
-
4-(5-Meth oxy-1H-3-in d olyl)cycloh exa n on e (16). The
conversion of 4-(5-methoxy-1H-3-indolyl)-3-cyclohexenone eth-
ylene ketal (13) to 4-(5-methoxy-1H-3-indolyl)cyclohexanone
(16) was carried out in a manner analogous to the synthesis
1
279 (85), 107 (base); H-NMR (CDCl3, 250 MHz) δ 8.19 (d, J
) 2 Hz, 1 H), 8.09 (br s, 1 H), 7.91 (s, J ) 8 Hz, 1 H), 7.47 (t,
J ) 8 Hz, 1 H), 7.36 (d, J ) 7 Hz, 1 H), 7.25 (m, 3 H), 6.64 (d,
J ) 8 Hz, 1 H), 6.61 (t J ) 7 Hz, 1 H), 6.25 (br s, 1 H), 3.57 (t,
J ) 5 Hz, 1 H), 2.59 (t, J ) 5 Hz, 1 H), 2.51 (m, J ) 4 Hz, 4
H), 2.38 (m, 1 H), 1.91 (m, 2 H), 1.65 (m, 1 H), 1.58 (dd, J )
9.8 Hz, 2 H), 1.21 (m, 1H). The HCl salt was formed by
treating the free base (320 mg) in ethyl acetate (20 mL) with
a solution of anhydrous hydrochloric acid in 2-propanol to give
the product as a white precipitate. (370 mg): mp 258-60 °C.
Anal. (C25H30N4‚1.25HCl‚H2O) C, H, N, water.
1
of 14: mp 118-20 °C; H-NMR (CDCl3, 200 MHz) δ 7.94 (br
s, 1 H), 7.29 (d, J ) 8 Hz, 1 H), 7.08 (s, 1 H), 6.99 (s, 1 H), 6.92
(dd, J ) 8, 2 Hz, 1 H), 3.90 (s, 3 H), 3.32 (tt, J ) 12, 3 Hz, 1
H), 2.54 (m, 4 H), 2.42 (m, J ) 12 Hz, 2 H), 1.95 (ddd, J ) 24,
12, 6 Hz, 2 H). Anal. (C15H27NO2) C, H, N.
cis-3-[4-[4-(2-P yr id in yl)-1-p ip er a zin yl]cycloh exyl]-1H-
in d ole (17a ) a n d tr a n s-3-[4-[4-(2-P yr id in yl)-1-p ip er a zin -
yl]cycloh exyl]-1H-in d ole (17b). A solution of 4-(1H-3-
indolyl)cyclohexanone (14) (5.00 g, 23.4 mmol), 1-(2-pyridyl)-
piperazine (3.82 g, 23.4 mmol). and a catalytic amount of
p-toluenesufonic acid was heated to reflux in toluene (40 mL)
under a Dean-Stark trap for 8 h. The solvent was removed
under reduced pressure, and the residue was dissolved in 50
mL of methanol and 30 mL of THF. The mixture was cooled
to 0 °C and treated with sodium cyanoborohydride (1.61 g, 25.7
mmol) and a small amount of methyl red to act as a pH
indicator. The reaction was treated with aqueous 1 N HCl
(approximately 50 mL) over 1/2 h until the reaction remained
acidic. The reaction was stirred at 0 °C for 1 h, and the
solvents were then removed under reduced pressure. The
residue was partitioned between chloroform and ammonium
hydroxide. The organic layer was separated, dried over sodium
sulfate, and evaporated. The resulting diastereomers were
separated using MPLC (2% methanol, 0.1% ammonia in
chloroform) to give two fractions.
F r a ction 1: cis-3-[4-[4-(2-p yr id in yl)-1-p ip er a zin yl]cy-
cloh exyl]-1H-in d ole (17a ) (3.66 g, 43% yield): mp 132-4 °C
(ethyl acetate); EIMS m/z 360 (14), 241 (36), 107 (base); IR
(KBr, cm-1) 3248, 2920, 2807, 1595, 1411; 1H-NMR (CDCl3,
250 MHz) δ 8.19 (d, J ) 5 Hz, 1 H), 7.99 (brs, 1 H), 7.68 (d, J
) 8 Hz, 1 H), 7.47 (dt, J ) 2, 8 Hz, 1 H), 7.34 (d, J ) 8 Hz, 1
H), 7.17 (t, J ) 7 Hz, 1 H), 7.12 (t, J ) 7 Hz, 1 H), 7.05 (d, J
) 2 Hz, 1 H), 6.65 (d, J ) 8 Hz, 1 H), 6.60 (t, J ) 5 Hz, 1 H)
3.55 (t, J ) 5 Hz, 4 H), 3.14(m, 1 H), 2.65 (m, J ) 5 Hz, 4 H)
2.34 (m, 1 H), 2.15 (m, 2 H), 1.85 (m, 3 H), 1.67 (m, 3 H). Anal.
(C23H28N4) C, H, N.
F r a ction 2: tr a n s-3-[4-[4-(2-p yr id in yl)-1-p ip er a zin yl]-
cycloh exyl]-1H-in d ole (17b) (4.51 g, 53% yield): mp 156-8
°C (ethyl acetate); EIMS m/z 360 (13) 241 (30), 107 (base); IR
(KBr, cm-1) 3156, 2926, 2852, 1596, 1455; 1H-NMR (CDCl3,
250 MHz) δ 8.20 (d, J ) 5 Hz, 1 H), 7.99 (brs, 1 H), 7.64 (d, J
) 7 Hz, 1 H), 7.48 (dt, J ) 2, 8 Hz, 1 H), 7.36 (d, J ) 8 Hz, 1
H), 7.18 (t, J ) 7 Hz, 1 H), 7.10 (t, J ) 7 Hz, 1 H), 6.95 (d, J
) 2 Hz, 1 H), 6.66 (d, J ) 8 Hz, 1 H), 6.61 (t, J ) 5 Hz, 1 H),
3.57 (t, J ) 5 Hz, 4 H), 2.81 (m, 1 H), 2.75 (t, J ) 5 Hz, 4 H),
2.46 (m, 1 H), 2.22 (m, 2 H), 2.10 (m, 2 H), 1.54 (m, 4 H). Anal.
(C23H28N4) C, H, N.
3-[4-(Meth oxym eth ylen e)cycloh exyl]-1H-in d ole (18).
A mixture of (methoxymethyl)triphenylphosphonium chloride
(10 g, 21.2 mmol) in tetrahydrofuran (25 mL) was cooled to
-78 °C and treated with a 1.6 M solution of n-BuLi in hexane
(13.2 mL, 21.2 mmol). After 20 min a solution of 4-(1H-3-
indolyl)cyclohexanone (14) 2.25 g, 10.6 mmol) in tetrahydro-
furan (25 mL) was added, and the reaction mixture was
allowed to warm to 10 °C over 2 h. The reaction was quenched
with saturated ammonium chloride, and the THF was removed
under reduced pressure. The residue was partitioned between
water and chloroform and the organic layer dried with sodium
sulfate and evaporated. The residue was purified using MPLC
(1:19:80 methanol-chloroform-hexanes) to give 18 as a clear
oil (1.45 g, 57% yield): 1H-NMR (CDCl3, 200 MHz) δ 7.99 (br
s, 1 H), 7.68 (d, J ) 8 Hz, 1 H), 7.37 (d, J ) 8 Hz, 1 H), 7.23
(t, J ) 8 Hz, 1 H), 7.13 (t, J ) 8 Hz, 1 H), 6.96 (d, J ) 2 Hz,
4-(1H-3-In d olyl)-3-cycloh exen on e Eth ylen e k eta l (11).
Indole 7 (45.3 g, 0.38 mol), 1,4-cyclohexanedione monoethylene
ketal (10) (45.5 g, 0.29 mol), and potassium hydroxide (9.12 g,
0.16 mol) were heated to reflux in 100 mL of methanol for 18
h. The reaction mixture was cooled, and the product was
isolated by filtration and washed with water to give 4-(1H-3-
indolyl)-3-cyclohexenone ethylene ketal (11) as a white solid
(69.0 g, 93% yield): EIMS m/z 255 (47) 169 (base); IR (KBr,
cm-1) 3287, 2966, 2885, 1644, 1436, 1238; 1H-NMR (CDCl3,
trace DMSO-d6 250 MHz) δ 9.50 (br s, 1 H), 7.86 (d, J ) 8 Hz,
1 H), 7.36 (d, J ) 7 Hz, 1 H), 7.17 (t, J ) 7 Hz, 1 H), 7.15 (s,
1H), 7.08 (t, J ) 8 Hz, 1 H), 6.12 (t, J ) 4 Hz, 1 H), 4.02 (s, 4
H), 2.69 (m, 2 H), 2.52 (br s, 2 H), 1.94 (t, J ) 6 Hz, 1 H).
Anal. (C16H17NO2) C, H, N.
4-(5-F lu or o-1H-3-in d olyl)-3-cycloh exen on e E t h ylen e
Keta l (12). The conversion of 5-fluoroindole 8 to 4-(5-fluoro-
1H-3-indolyl)-3-cyclohexenone ethylene ketal (12) was carried
out in a manner analogous to the synthesis of 11: yield 76%;
1
CIMS m/z 274 (36) 186 (base); H-NMR (CDCl3, 250 MHz) δ
8.12 (br s, 1 H), 7.53 (dd, J ) 2, 10 Hz, 1 H), 7.24 (m, 2 H),
7.17 (d, J ) 2 Hz, 1 H), 6.94 (td, J ) 9, 2 Hz, 1 H), 6.06 (t, J
) 4 Hz, 1 H), 4.04 (s, 4 H), 2.67 (m, 2 H), 2.53 (br s, 2 H), 1.95
(t, J ) 6 Hz, 1 H). Anal. (C16H16FNO2) C, H, N.
4-(5-Meth oxy-1H-3-in d olyl)-3-cycloh exen on e Eth ylen e
Keta l (13). The conversion of 5-methoxyindole 9 to 4-(5-
methoxy-1H-3-indolyl)-3-cyclohexenone ethylene ketal (13) was
carried out in a manner analogous to the synthesis of 11: yield
93%; CIMS m/z 285 (62) 99 (base); 1H-NMR (CDCl3, 200 MHz)
δ 8.05 (br s, 1 H), 7.35 (d, J ) 2 Hz, 1 H), 7.24 (d, J ) 9 Hz,
1 H), 7.14 (d, J ) 2 Hz, 1 H), 6.87 (dd, J ) 9, 2 Hz, 1 H), 6.10
(t, J ) 4 Hz,1 H), 4.02 (s, 4 H), 3.88 (s, 3 H), 2.69 (m, 2 H),
2.55 (br s, 2 H), 1.97 (t, J ) 6 Hz, 1 H); CIMS m/z 285 (62), 99
(base).
4-(1H-3-In d olyl)cycloh exa n on e (14). 4-(1H-3-Indolyl)-3-
cyclohexenone ethylene ketal (11) (66.6 g, 0.26 mmol) was
dissolved in 500 mL of THF and 100 mL of methanol, 1.0 g of
5% palladium on carbon was added and the mixture was
placed under 60 psi of hydrogen. After 2 h the reaction
mixture was filtered and concentrated to give a tan solid which
was dissolved in 350 mL of acetone and 350 mL of 10% HCl
and allowed to stir at room temperature for 6 h. The acetone
was removed under reduced pressure, and the mixture was
made basic with concentrated ammonium hydroxide. The
mixture was extracted with chloroform. The organic fraction
was dried with sodium sulfate, and volatiles were removed
under reduced pressure. The resulting solid was taken up in
hot ethyl acetate, and upon cooling and filtration 4-(1H-3-
indolyl)cyclohexanone (14) was obtained as a crystalline solid
(36.89 g, 66% yield): mp 124-5 °C; EIMS m/z 213 (63) 156
1
(base); H-NMR (CDCl3, 250 MHz) δ 8.26 (br s, 1 H), 7.65 (d,
J ) 8 Hz, 1 H), 7.30 (d, J ) 8 Hz, 1 H), 7.19 (td, J ) 8, 1 Hz,
1 H), 7.11 (t, J ) 8 Hz, 1 H), 6.88 (d, J ) 2 Hz, 1 H), 3.29 (tt,
J ) 12, 3 Hz, 1 H), 2.47 (m, 4 H), 2.38 (dt, J ) 13, 3 Hz, 2 H),
1.92 (ddd, J ) 24, 12, 6, 2 H). Anal. (C14H15NO) C, H, N.
4-(5-F lu or o-1H-3-in d olyl)cycloh exa n on e, 15. The con-
version of 4-(5-fluoro-1H-3-indolyl)-3-cyclohexenone ethylene

256 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2
Wustrow et al.
1 H), 5.86 (s, 1 H), 3.53 (t, J ) 3 H), 2.97 (m, J ) 2 H), 2.09
(m, 4 H), 1.86 (t, J ) 12 Hz, 1 H), 1.65 (m, 2 H).
7.62 (d, J ) 7 Hz, 1 H), 7.32 (d, J ) 7 Hz, 1 H), 7.15 (t, J ) 7
Hz, 1 H), 7.08 (t, J ) 7 Hz, 1 H), 6.90 (d, J ) 2 Hz, 1 H), 5.66
(s, 1 H), 4.14 (q, J ) 7 Hz, 2 H), 3.92 (dt, J ) 17, 4 Hz, 1 H),
3.09 (tt, J ) 12, 4 Hz, 1 H), 2.39 (m, 2 H), 2.28 (m, 2 H), 2.12
(td, J ) 12, 3 Hz, 1 H), 1.65 (m, 2 H), 1.26 (t, J ) 7 Hz, 3 H).
Anal. (C18H21NO2) C, H, N.
cis- a n d tr a n s-4-(1H-In d ol-3-yl)cycloh exa n eca r ba ld e-
h yd e (19). The enol ether 18 (1.25 g, 5.17 mmol) was heated
in a refluxing solution of solution of THF (30 mL) and 10%
HCl (15 mL) for 2 h. The THF was removed under reduced
pressure, and the reaction mixture was partitioned between
methylene chloride and water. The organic layer was dried,
and solvents were evaporated under reduced pressure to obtain
a 1:2 mixture of cis- and trans-aldehydes 19 by NMR (1.15 g,
Similarly, esters 22 and 23 were prepared.
[4-(5-F lu or o-1H-in d ol-3-yl)cycloh exylid en e]a cetic a cid
eth yl ester (22): 78% yield; mp 149-151 °C; EIMS m/z 301
(base), 161 (95); 1H-NMR (CDCl3, 250 MHz) δ 8.01 (brs, 1 H),
7.28 (m, 2 H), 6.97 (d, J ) 2 Hz, 1 H), 6.91 (dd, J ) 8, 2 Hz, 1
H), 5.69 (s, 1 H), 4.16 (q, J ) 7 Hz, 2 H), 3.95 (d, J ) 14 Hz,
1 H), 3.04 (tt, J ) 12, 4 Hz, 1 H), 2.39 (m, 2 H), 2.28 (m, 2 H),
2.14 (td, J ) 12, 3 Hz, 1 H), 1.67 (m, 2 H), 1.29 (t, J ) 7 Hz,
3 H). Anal. (C18H20FNO2) C, H, N.
[4-(5-Meth oxy-1H-in dol-3-yl)cycloh exyliden e]acetic acid
eth yl ester (23): 83% yield; mp 118-20 °C; 1H-NMR (CDCl3,
250 MHz) δ 7.84 (brs, 1 H), 7.21 (d, J ) 8 Hz, 1 H), 7.03 (d, J
) 2 Hz, 1 H), 6.88 (d, J ) 2 Hz, 1 H), 6.82 (dd, J ) 8, 2 Hz, 1
H), 5.66 (s, 1 H), 4.13 (q, J ) 7 Hz, 1 H), 3.92 (dt, J ) 17, 4
Hz, 1 H), 3.84 (s, 3 H), 3.04 (tt, J ) 12, 4 Hz, 1 H), 2.39 (m, 2
H), 2.24 (m, 2 H), 2.11 (td, J ) 12, 3 Hz, 1 H), 1.62 (m, 2 H),
1.26 (t, J ) 7 Hz, 3 H). Anal. (C19H23NO3) C, H, N.
1
98% yield): key NMR resonances H-NMR (CDCl3, 200 MHz)
δ 9.81, 9.73 (s, 1 H), 8.04, 7.99 (brs, 1 H), 6.95, 6.89 (d, J ) 2
Hz, 1 H), 3.80 (m, 1 H), 2.87 (m, 1 H).
cis-1-[[4-(1H-3-In d olyl)cycloh exyl]m eth yl]-4-(2-p yr id i-
n yl)p ip er a zin e (20a ) a n d tr a n s-1-[[4-(1H-3-In d olyl)cy-
cloh exyl]m eth yl]-4-(2-p yr id in yl)p ip er a zin e (20b). A mix-
ture of cis- and trans-4-(1H-3-indolyl)cyclohexane-2-carbox-
aldehyde (19) (1.1 g, 4.93 mmol) and 2-pyridinylpiperazine
(0.81 g, 4.9 mmol) was dissolved in 35 mL of acetonitrile, cooled
to 0 °C, and treated with 0.3 mL of acetic acid. The reaction
was stirred for 15 min and then treated with sodium cy-
anoborohydride (0.33 g, 5.3 mmol). The reaction mixture was
removed from the ice bath and stirred for 3 h. The acetonitrile
was removed under reduced pressure, the residue was treated
with water (100 mL), and the mixture was adjusted to pH 10
with sodium hydroxide and extracted with chloroform. The
chloroform extracts are dried with sodium sulfate, and the
solvents are evaporated under reduced pressure. The residue
was purified using MPLC (1% methanol, 99% chloroform) to
give 20a and 20b. cis-1-[[4-(1H-3-Indolyl)cyclohexyl]methyl]-
4-(2-pyridinyl)piperazine (20a ) (0.23 g, 12% yield): mp 74-6
°C (ether); EIMS m/z (% base) 374 (M+, 19), 280 (38), 176 (57),
107 (base); IR (KBr, cm-1) 3411, 2920, 2846, 1593, 1482, 1436,
[4-(1H-In d ol-3-yl)cycloh exyl]a cetic Acid (24). A solu-
tion [4-(1H-indol-3-yl)cyclohexylidene]acetic acid ethyl ester
(21) (46.4 g, 164 mmol) in 600 mL of ethanol was treated with
5% palladium on carbon (5 g) and placed under 50 psi of
hydrogen gas. After the appropriate amount of hydrogen had
been taken up, the mixture was filtered, and the solvents were
removed under reduced pressure. The residue was redissolved
in 250 mL of ethanol, and 100 mL of 10% NaOH was added.
After 18 h the ethanol was removed under reduced pressure.
The aqueous mixture was extracted with ethyl acetate; the
aqueous layer was then was adjusted to pH 5 with potassium
biphosphate and extracted with three 300 mL portions of
methylene chloride. The combined methylene chloride extracts
were dried over sodium sulfate and evaporated to give a cream-
colored solid (35.1 g, 83% overall yield) which was a mixture
of diastereomers (trans:cis; 1:1): mp 150-154 °C; 1H-NMR
(CDCl3, 400 MHz) δ 7.92 (s, 1H), 7.63 (d, J ) 8 Hz, 1 H), 7.35
(d, J ) 8 Hz, 1 H), 7.18 (t, J ) 8 Hz, 1 H), 7.10 (t, J ) 8 Hz,
1
1242; H-NMR (CDCl3, 250 MHz) δ 8.19 (d, J ) 5 Hz, 1 H),
7.94 (br s, 1 H), 7.64 (d, J ) 8 Hz, 1 H), 7.47 (td, J ) 8, 2 Hz,
1 H), 7.35 (d, J ) 8 Hz, 1 H), 7.18 (t, J ) 7 Hz, 1 H), 7.09 (t,
J ) 7 Hz, 1 H), 7.02 (d, J ) 2 Hz, 1 H), 6.65 (d, J ) 8 Hz, 1 H),
6.64 (t, J ) 5 Hz, 1 H), 3.54 (t, J ) 5 Hz, 4 H), 3.04 (m, 1 H),
2.54 (t, J ) 5 Hz, 1 H), 2.37 (d, J ) 7 Hz, 1 H), 1.87 (m, 4 H),
1.68 (m 5 H). Anal. (C24H30N4) C, H, N. trans-1-[[4-(1H-3-
Indolyl)cyclohexyl]methyl]-4-(2-pyridinyl)piperazine (20b) (0.45
g, 24% yield): mp 169-70 °C (ethyl acetate-hexane); EIMS
m/z (% base) 374 (M+, 18), 359 (M - NH, 3), 280 (29), 267
(17), 107 (base); IR (KBr, cm-1) 3404, 2919, 1595, 1483, 1245;
1H-NMR (CDCl3, 250 MHz) δ 8.19 (d, J ) 5 Hz, 1 H), 7.97
(brs, 1 H), 7.66 (d, J ) 8 Hz, 1 H), 7.47 (td, J ) 8,2 Hz, 1 H),
7.34 (d, J ) 8 Hz, 1H), 7.18 (t, J ) 7 Hz, 1 H), 7.12 (t, J ) 7
Hz, 1 H), 6.95 (d, J ) 2 Hz, 1 H), 6.65 (d, J ) 8 Hz, 1 H), 6.62
(t, J ) 5 Hz, 1 H), 3.59 (m, 4 H), 2.82 (t, J ) 12 Hz, 1 H), 2.58
(m, 4 H), 2.29 (d, J ) 7 Hz, 2 H), 2.17 (d, J ) 11 Hz, 2 H), 2.00
(d, J ) 11 Hz, 2 H), 1.68 (m, 1 H), 1.52 (dd, J ) 20, 11 Hz, 2
H), 1.16 (dd, J ) 21, 11 Hz, 2 H). The free base 20b was
converted to its maleate salt by treatment with an equimolar
amount of maleic acid in methanol, removal of the solvent
under reduced pressure, and recrystallization from ethanol:
mp 176-7 °C. Anal. (C24H30N4‚C4H4O4) C, H, N.
1
1 H), 7.00 (d, J ) 2 Hz, /2 H, trans isomer), 6.94 (d, J ) 2 Hz,
1
1/2 H, cis isomer), 2.96 (m, /2 H cis isomer), 2.72 (tt, J ) 3, 15
1
Hz, /2 H, trans isomer), 2.44 (d, J ) 8 Hz, 1 H, trans isomer),
2.33 (d, J ) 7 Hz, 1 H, cis isomer), 2.26-1.21 (series of
multiplets, 9 H); CIMS m/z 258 (base), 257 (50).
Similarly, acids 25 and 26 were prepared.
[4-(5-Flu or o-1H-in dol-3-yl)-cycloh exyl]-acetic acid (25):
mp 166-8 °C; 1H-NMR (CDCl3, 400 MHz) δ 7.92 (s, 1H), 7.19
1
(m, 2 H), 7.05 (d, J ) 2 Hz, /2 H trans isomer), 6.99 (d, J ) 2
Hz, 1 H), 6.92 (m, 1 H), 3.07 (m, 1/2 H cis isomer), 2.81 (tt, J )
4, 12 Hz, 1/2 H, trans isomer), 2.45 (d, J ) 8 Hz, 1 H, trans
isomer), 2.32 (d, J ) 7 Hz, 1 H, cis isomer), 2.26-1.21 (series
of multiplets, 9 H); CIMS m/z 276 (base), 275 (58).
[4-(5-Meth oxy-1H-in dol-3-yl)cycloh exyl]acetic acid (26):
mp 143-5 °C; 1H-NMR (CDCl3, 400 MHz) δ 7.80 (s, 1H), 7.25
[4-(1H-In d ol-3-yl)cycloh exylid en e]a cetic Acid Eth yl
Ester (21). Sodium hydride (60% by wt in mineral oil, 13.96
g, 349 mmol) was washed with two portions of hexane (200
mL) under nitrogen. The sodium hydride was slurried in THF
(200 mL) cooled to 0 °C and treated with triethyl phospho-
noacetate (71.29 g, 318 mmol) in 100 mL of THF. The reaction
mixture was allowed to come to room temperature over 1 h
and then again cooled to 0 °C, and a solution of 4-(1H-3-
indolyl)cyclohexanone (14) (33.92 g, 159 mmol) in 200 mL of
THF was added. The reaction mixture was warmed to room
temperature over 4 h and was quenched with 200 mL of
saturated KH2PO4. The mixture was partitioned between
chloroform and water, the combined organic extracts were
washed with brine and dried over Na2SO4, and the solvents
were removed under reduced pressure. The resulting residue
was subjected to MPLC (2:1 hexane-ethyl acetate) (the
compound was loaded on the column by adding a trace of
methylene chloride) to obtain 21 as a white crystalline solid
(35.1 g, 78% yield): mp 113-5 °C; CIMS m/z 284 (base) 283
1
(d, J ) 8 Hz, 1 H), 7.05 (s, 1 H), 6.99 (d, J ) 2 Hz, /2 H, trans
isomer), 6.93 (d, J ) 2 Hz, 1/2 H, cis isomer), 6.84 (d, J ) 8 Hz,
1
1 H), 3.87 (s, 3 H), 2.98 (m, /2 H, cis isomer), 2.75 (tt, J ) 3,
1
15 Hz,
/ H, trans isomer), 2.45 (d, J ) 8 Hz, 1 H, trans
2
isomer), 2.33 (d, J ) 7 Hz, 1 H, cis isomer), 2.26-1.21 (series
of multiplets 9H), CIMS m/z 288 (base), 287 (50).
cis-2-[4-(1H-In d ol-3-yl)cycloh exyl]-1-(4-p yr id in -2-ylp ip -
er a zin -1-yl)eth a n on e (27a ) a n d tr a n s-2-[4-(1H-In d ol-3-yl)-
cycloh exyl]-1-(4-p yr id in -2-ylp ip er a zin -1-yl)-et h a n on e
(28a ). A solution of the carboxylic acid 24 (5.00 g, 19.4 mmol),
triethylamine (4.5 mL, 32 mmol), and methylene chloride (90
mL) was cooled in an ice bath and treated with isobutyl
chloroformate (2.77 mL, 21.4 mmol). After 10 min the solution
was treated with 1-(2-pyridyl)piperazine (3.49 g, 21.4 mmol),
maintained at 0 °C for 3 h, and warmed to room temperature
overnight. The reaction mixture was extracted with aqueous
sodium bicarbonate (40 mL), the organic layer was dried over
sodium sulfate and filtered, and the solvents were evaporated
under reduced pressure. The mixture was purified using
1
(91), 238 (96); H-NMR (CDCl3, 250 MHz) δ 7.92 (brs, 1 Η),

Dopamine D2 Partial Agonists and Autoreceptor Agonists
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2 257
MPLC (2% methanol, 0.1% ammonia, in chloroform) to obtain
the following.
Cis isom er 27a : H-NMR (CDCl3, 200 MHz) δ 8.19 (dd, J
) 4, 2 Hz, 1 H), 7.98 (brs, 1 H), 7.64 (d, J ) 8 Hz, 1 H), 7.53
(td, J ) 8, 2 Hz, 1 H), 7.36 (d, J ) 8 Hz, 1H), 7.19 (t, J ) 8 Hz,
1H), 7.14( t, J ) 8 Hz, 1H), 7.05 (d, J ) 2 Hz, 1 H), 6.63 (m,
2H), 3.80 (t, J ) 5 Hz, 4 H), 3.51 (t, J ) 5 Hz, 4 H), 3.08 (m,
1 H), 2.42 (d, J ) 6 Hz, 2 H), 2.42 (t, J ) 5 Hz, 1 H), 1.93 (m,
4 H), 1.81 (m, 2 H), 1.71 (m, 2 H).
δ 9.38 (brs, 1 H), 7.50 (d, J ) 8 Hz, 1 H), 7.49-7.28 (m, 5 H),
7.23 (d, J ) 7 Hz, 1 H), 7,10 (t, J ) 7 Hz, 1 H), 7.02 (t, J ) 7
Hz, 1 H), 6.94 (d, J ) 2 Hz, 1H) 6.08 (s, 1 H), 3.17 (m, 2 H),
2.72 (m, 1 H), 2.73 (m, 2 H), 2.54-2.44 (m, 4 H), 2.11 (d, J )
11 Hz, 2 H), 1.90 (d, J ) 11 Hz, 2 H), 1.64-1.34 (m, 5 H), 1.23
(J ) 24, 9 Hz, 2 H). Anal. (C27H32N2) C, H, N.
tr a n s-3-[4-[2-(4-P h en yl-1-piper azin yl)eth yl]cycloh exyl]-
1H-in d ole (30c): mp 157 °C (ethyl acetate); EIMS m/z 388
(35), 175 (base); 1H-NMR (CDCl3, 250 MHz) δ 7.96 (br s, 1 H),
7.75 (d, J ) 8 Hz, 1 H), 7.33 (d, J ) 7 Hz, 1 H), 7.26 (t, J ) 8
Hz, 2 H), 7.18 (t, J ) 7 Hz, 1 H), 7.09 (t, J ) 7 Hz, 1 H), 6.95
(d, J ) 8 Hz, 2 H), 6.93 (s, 1 H), 6.85 (t, J ) 7 Hz, 1 H), 3.23
(t, J ) 5 Hz, 4 H), 2.80 (tt, J ) 12, 3 Hz, 1 H), 2.64 (t, J ) 5
Hz, 4 H), 2.48 (t, J ) 8 Hz), 22.14 (d, J ) 11 Hz, 2 H), 1.90 (d,
J ) 11 Hz, 1 H), 1.6-1.35 (m, 5 H), 1.20 (dd, J ) 20, 12 Hz, 2
H). Anal. (C26H33N3) C, H, N.
1
Tr a n s isom er 28a : 1H-NMR (CDCl3, 200 MHz) δ 8.22 (dd,
J ) 4, 2 Hz, 1 H), 7.98 (brs, 1 H), 7.65 (d, J ) 8 Hz, 1 H), 7.53
(td, J ) 8, 2 Hz, 1 H), 7.36 (d, J ) 8 Hz, 1 H), 7.19 (t, J ) 8
Hz, 1 H), 7.14 (t, J ) 8 Hz, 1H), 6.95 (d, J ) 2 Hz, 1H), 6.64
(d, J ) 8 Hz, 1 H), 6.61 (t, J ) 7 Hz, 1 H), 3.80 (t, J ) 5 Hz,
4 H), 3.51 (t, J ) 5 Hz, 4 H), 2.82 (tt, J ) 12, 3 Hz, 1 H), 2.36
(d, J ) 6 Hz, 2 H), 2.15 (d, J )11 Hz, 2 H), 2.03 (m, 3 H), 1.60
(dd, J ) 20, 12 Hz, 2 H), 1.18 (dd, J ) 20, 12 Hz, 2 H).
cis-1-[2-[4-(1H -3-in d olyl)cycloh exyl]et h yl]-4-(2-p yr i-
d in yl)p ip er a zin e (29a ). Lithium aluminum hydride (0.19
g, 4.9 mmol) was slurried in 10 mL of THF and cooled in an
ice bath. The mixture was treated with aluminum chloride
(0.22, 1.64 mmol) in ether (10 mL) and stirred for 10 min. A
mixture of the amide 27a (0.66 g, 1.64 mmol) and 10 mL of
tetrahydrofuran were added, and the reaction mixture was
allowed to warm to room temperature overnight. The reaction
was quenched by careful addition of 0.5 mL of water and 1
mL of 25% NaOH. After 2 h of stirring the mixture was
filtered through Celite and the filtercake washed with an
additional 10 mL of THF. The solvents were removed under
reduced pressure, and the residue was triturated with hexanes
to give 29a as a white solid (0.51 g, 80% yield): mp 99-101
°C (hexanes); EIMS m/z 388 (21), 294 (30), 281 (21), 267 (31)
107 (base); IR (KBr, cm-1) 3419, 2921, 1597, 1485, 1437 1245;
1H-NMR (CDCl3, 250 MHz) δ 8.19 (dd, J ) 4, 2 Hz, 1 H), 8.01
(brs, 1 H), 7.64 (d, J ) 8 Hz, 1 H), 7.47 (td, J ) 7, 2 Hz, 1 H),
7.34 (d, J ) 8 Hz, 1H), 7.16 (t, J ) 7 Hz, 1 H), 7.09 (t, J ) 7
Hz, 1 H), 6.98 (d, J ) 2 Hz, 1 H), 6.64 (d, J ) 8 Hz, 1 H), 6.61
(t, J ) 5 Hz, 1 H), 3.56 (t, J ) 5 Hz, 4 H), 3.00 (m, 1 H), 2.57
(t, J ) 5 Hz, 4 H), 2.42 (t, J ) 8 Hz, 2 H), 1.84 (m, 4 H), 1.67
(m, 7 H). Anal. (C25H32N4) C, H, N.
tr a n s-3-[4-[2-[4-(4-F lu or op h en yl)-1-p ip er a zin yl]eth yl]-
cycloh exyl]-1-in d ole (30d ): mp 163-5 °C (ethyl acetate);
1
EIMS m/z 405 (19), 193 (base); H-NMR (CDCl3, 250 MHz) δ
9.43 (s, 1 H), 7.35 (d, J ) 8 Hz, 1 H), 6.98 (d, J ) 8 Hz, 1 H),
6.85 (dd, J ) 8, 7 Hz, 1 H), 6.79 (t, J ) 7 Hz, 1 H), 6.67 (m, 5
H), 2.90 (t, J ) 5 Hz, 4 H), 2.53 (t, J ) 12 Hz, 1 H), 2.38 (t, J
) 5 Hz, 4 H), 2.23 (t, J ) 8 Hz, 2 H), 1.86 (d, J ) 11 Hz, 2 H),
1.64 (d, J ) 11 Hz, 2 H), 1.18-1.15 (m, 5 H), 0.93 (dd, J ) 20,
12 Hz, 2 H). Anal. (C26H32FN3) C, H, N, F.
tr a n s-3-[4-[2-[4-(2-P yr im id in yl)-1-p ip er a zin yl]eth yl]cy-
cloh exyl]-1H-in d ole m on oh yd r och lor id e (30e): mp 262 °C
dec (ether-isopropyl alcohol); EIMS m/z (% base) 389 (M+, 76),
281 (66), 177 (base); IR (KBr, cm-1) 3489, 2925, 1583, 1553,
1437, 1362; 1H-NMR (DMSO-d6, 250 MHz) δ 11.12 (brs, 1 H),
10.80 (s, 1 H), 8.45 (d, J ) 5 Hz, 2 H), 7.54 (d, J ) 8 Hz, 1 H),
7.32 (d, J ) 8 Hz, 1 H), 7.04 (m, 2 H), 6.94 (t, J ) 8 Hz, 1 H),
6.77 (t, J ) 5 Hz, 1 H), 4.69 (d, J ) 15 Hz, 2 H), 4.26 (brs, 1
H), 3.57 (d, J ) 12 Hz, 2 H), 3.43 (d, J ) 12 Hz, 2 H), 3.15 (m,
2 H), 3.03 (dd, J ) 18, 10 Hz, 2 H), 2.73 (t, J ) 13 Hz, 1 H),
2.02 (d, J ) 11 Hz, 2 H), 1.84 (d, J ) 12 Hz, 2 H), 1.71 (m, 2
H), 1.47 (dd, J ) 20, 11 Hz, 2 H), 1.4 (m, 1 H), 1.16 (dd, J )
20, 11 Hz, 2 H). Anal. (C24H31N5‚HCl‚1/2H2O) C, H, N, Cl.
tr a n s-3-[4-[2-[3,6-Dih ydr o-4-(2-th ien yl)-1(2H)-pyr idin yl]-
eth yl]cycloh exyl]-1H-in d ole (30f): mp 192-4 °C (EtOAc);
t r a n s-1-[2-[4-(1H -3-In d olyl)cycloh e xyl]e t h yl]-4-(2-
p yr id in yl)p ip er a zin e (30a ). Using the same general meth-
odology the amide 28a (6.0 g, 14.9 mmol) was reduced the
amine 30a , which was purified by recrystallization from
methylene chloride (2.93 g, 51%): mp 138-9 °C (methylene
chloride); EIMS m/z 388 (14), 294 (23), 267 (25), 107 (base);
EIMS m/z (% base) 390 (M+, 56), 178 (base); IR (KBr, cm-1
)
3140, 2914, 2824, 1631, 1430, 738; 1H-NMR (DMSO-d6, 250
MHz) δ 10.69 (brs, 1 H), 7.53 (d, J ) 8 Hz, 1 H), 7.37 (d, J )
7 Hz, 1 H), 7.32 (d, J ) 8 Hz, 1 H), 7.04 (m, 4 H), 6.94 (t, J )
8 Hz, 1 H), 6.08 (brs, 1 H), 3.34 (s, 2 H), 3.04 (brs, 2 H), 2.72
(t, J ) 12 Hz, 1 H), 2.61 (t, J ) 5 Hz, 1 H), 2.44 (m, 4 H), 2.01
(d, J ) 12 Hz, 2 H), 1.86 (d, J ) 12 Hz, 2 H), 1.34 (m, 5 H),
1.16 (dd, J ) 20, 12 Hz 2 H). Anal. (C25H30N2S) C, H, N, S.
tr a n s-1-[2-[4-(1H-5-F lu or o-3-in d olyl)cycloh exyl]eth yl]-
4-(2-p yr id in yl)p ip er a zin e (30g): mp 138-9 °C (ethyl ace-
tate); EIMS m/z 406 (10.6), 312 (36), 299 (26), 285 (38), 107
1
IR (KBr, cm-1) 2913, 2848, 1594, 1483, 1436, 1244; H-NMR
(CDCl3, 250 MHz) δ 8.20 (dd, J ) 4, 2 Hz, 1 H), 8.10 (brs, 1
H), 7.65 (d, J ) 8 Hz, 1 H), 7.47 (td, J ) 8, 2 Hz, 1 H), 7.32 (d,
J ) 8 Hz, 1 H), 7.16 (t, J ) 8 Hz, 1 H), 7.09 (t, J ) 7 Hz, 1 H),
6.91 (d, J ) 2 Hz, 1 H), 6.64 (d, J ) 8 Hz, 1 H), 6.61 (t, J ) 7
Hz, 1 H), 3.56 (t, J ) 5 Hz, 4 H), 2.79 (t, J ) 12 Hz, 1 H), 2.57
(t, J ) 5 Hz, 4H), 2.46 (t, J ) 8 Hz, 2 H), 2.13 (d, J ) 11 Hz,
2 H), 1.89 (d, J ) 16 Hz, 2 H), 1.4-1.3 (m, 5 H), 1.18 (dd, J )
20, 12 Hz, 2 H). Anal. (C25H32N4) C, H, N. The maleate salt
of 30a was formed by mixing an equimolar amount of maleic
acid with the free base in methanol. Evaporation and recrys-
tallization from ethanol afforded the maleate salt: mp 167-9
°C. Anal. (C25H32N4‚C4H4O4) C, H, N.
1
(base); H-NMR (CDCl3, 250 MHz) δ 8.20 (dd, J ) 4, 2 Hz, 1
H), 7.98 (br s, 1 H), 7.48 (td, J ) 7, 2 Hz, 1 H), 7.30-7.22 (m,
2 H), 6.98 (s, J ) 2 Hz, 1 H), 6.91 (td, J ) 8, 2 Hz, 1 H), 6.65
(d, J ) 8 Hz, 1 H), 6.62 (t, J ) 4 Hz, 1 H), 3.57 (t, J ) 5 Hz,
4 H), 2.72 (t, J ) 12 Hz, 1 H), 2.58 (t, J ) 5 Hz, 1 H), 2.46 (t,
J ) 8 Hz, 2 H), 2.10 (d, J ) 11 Hz, 1 H), 1.89 (d, J ) 13 Hz,
2 H), 1.51 (m, 5 H), 1.19 (dd, J ) 20, 13 Hz, 2 H). Anal.
(C25H31FN4) C, H, N, F.
The following compounds, 29b and 30b-h , were prepared
by coupling the acid precursors 24-26 with the appropriate
amines, isolating the diastereomerically pure amides, and
reducing with AlH3 as outlined for 29a .
tr a n s-1-[2-[4-(1H-5-Meth oxy-3-in dolyl)cycloh exyl]eth yl]-
4-(2-p yr id in yl)p ip er a zin e Ma lea te (30h ): mp 181-2 °C
(ethanol); 1H-NMR (DMSO-d6, 250 MHz) δ 10.58 (s, 1 H), 8.16
(d, J ) 3 Hz, 1 H), 7.62 (t, J ) 7 Hz, 1 H), 7.21 (d, J ) 9 Hz,
1 H), 6.99 (dd, J ) 8, 2 Hz, 1 H), 6.98 (d, J ) 2 Hz, 1 H), 6.95
(d, J ) 8 Hz, 1 H), 6.72 (m, 2 H), 6.03 (s, 2 H), 3.75 (s, 3 H),
3.33 (brs, 4 H), 3.15 (brs, 4 H), 2.71 (t, J ) 12 Hz, 1 H), 2.02
(d, J ) 12 Hz, 2 H), 1.85 (d, J ) 12 Hz, 2 H), 1.62 (m, 2 H),
1.45 (m, 3 H), 1.18 (dd, J ) 20, 12 Hz, 2 H. Anal.
(C26H34N4O‚C4H4O4‚1/2H2O) C, H, N.
tr a n s-3-[4-[2-(4-P yr id in -2-ylp ip er a zin -1-yl)eth yl]cyclo-
h exyl]-1H-in d ol-5-ol (31). A mixture of the free base of 30h
(1.3 g, 3.1 mmol) and pyridine hydrochloride (2.49 g, 21.5
mmol) was heated to 130 °C in a sealed tube for 72 h. The
mixture was cooled and partitioned between 125 mL of
concentrated aqueous ammonium hydroxide and 125 mL of
chloroform. The aqueous layer was extracted with an ad-
cis-3-[4-[2-(3,6-dih ydr o-4-ph en yl-1(2H)-pyr idin yl)eth yl]-
cycloh exyl]-1H-in d ole (29b): mp 123-4 °C (ether); EIMS
m/z 384 (24), 172 (base); IR (KBr, cm-1) 3422, 3058, 2922, 1493,
1
1445, 742; H-NMR (CDCl3, 250 MHz) δ 8.10 (br s, 1H), 7.64
(d, J ) 8 Hz, 1 H), 7.49-7.25 (m, 5 H), 7.16 (t, J ) 8 Hz, 1 H),
7.11 (t, J ) 7 Hz, 1 H), 6.96 (d, J ) 2 Hz, 1 H), 6.08 (s, 1 H),
3.18 (d, J ) 3 Hz, 2 H), 2.99 (m, 1 H), 2.73 (t, J ) 6 Hz, 2 H),
2.60 (s, 2 H), 2.50 (t, J ) 6 Hz, 2 H), 1.84 (m, 4 H), 1.69 (m, 7
H). Anal. (C27H32N2) C, H, N.
tr a n s-3-[4-[2-(3,6-Dih yd r o-4-p h en yl-1(2H )-p yr id in yl)-
eth yl]cycloh exyl]-1H-in d ole (30b): mp 209-10 °C (ethyl
acetate); EIMS m/z 384 (36), 233 (8), 172 (base); IR (KBr, cm-1
)
3428, 3156, 2916, 1465, 1456, 755; 1H-NMR (CDCl3, 250 MHz)

258 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 2
Wustrow et al.
ditional 50 mL of chloroform, and the combined organic
extracts were dried over sodium sulfate. The solvent was
evaporated under reduced pressure to give a brown solid which
was washed with ca. 30 mL of 2% methanol in chloroform and
then recrystallized from ethyl acetate to give 30 (0.70 g, 56%
yield): mp 215-7 °C; EIMS m/z 404 (57), 389 (10), 283 (24),
107 (base); IR (KBr, cm-1) 3411, 2919, 2848, 1594, 1485, 1439;
1H-NMR (CDCl3, 250 MHz) δ 8.79 (brs, 1h H), 8.18 (d, J ) 4
Hz, 1 H), 7.90 (brs, 1 H), 7.51 (t, J ) 8 Hz, 1 H), 7.16 (d, J )
9 Hz, 1 H), 7.02 (d, J ) 2 Hz, 1 H), 6.89 (d, J ) 2 Hz, 1 H) 6.74
(dd, J ) 8,2 Hz, 1 H), 6.67 (d, J ) 9 Hz, 1 H), 6.62 (t, J ) 5
Hz, 1 H), 3.56 (t, J ) 5 Hz, 4 H), 2.68 (m, 1h H), 2.58 (t, J )
5 Hz, 4 H), 2.46 (t, J ) 8 Hz, 2 H), 2.09 (d, J ) 12 Hz, 2 H),
1.88 (d, J ) 12 Hz, 2 H), 1.55-1.35 (m, 5 H), 1.16 (dd, J ) 9,
25 Hz, 2 H). Anal. (C25H32N4O) C, H, N.
Data were expressed as percentage inhibition of activity
relative to vehicle-treated animals and an ED50 calculated from
various doses.
In h ibition of GBL-Stim u la ted DA Syn th esis. Com-
pounds were administered to male Long Evans rats 1 h before
sacrifice, and γ-butyrolactone (GBL) (750 mg/kg ip) and NSD
1015 (100 mg/kg ip) were administered 30 min before sacrifice.
Brain striatal levels of L-dihydroxyphenylalanine (DOPA) were
analyzed by HPLC with electrochemical detection. The control
values in mg/g ( SEM of tissue for 0% (GBL + NSD 1015)
and 100% (vehicle + NSD1015) reversal in rat striatum were
3831 ( 149 and 1181 ( 65.18
E ffect s on t h e F ir in g R a t e of Su bst a n ia Nigr a DA
Neu r on s.23 The action potential of zona compacta DA cells
was recorded in chloral-anesthetized rats using standard
extracellular recording techniques. DA cells were identified
by wave form and firing pattern, and recording sites were
varied histologically. The test compound was administered
intraperitoneally via an indwelling catheter. Base line firing
rate was calculated by averaging the rate over 2 min prior to
drug injection. Drug effects were determined by averaging the
response during the 1 min of maximal inhibition. Drug-
induced inhibition of firing was reversed with the DA antago-
nist haloperidol to confirm a DA agonist mechanism.
Ra t Str ia ta l Micr od ia lysis. Adult, male Sprague-Daw-
ley rats (200-250 g) were anesthetized with urethane and
placed in a stereotaxic frame. A 4 mm microdialysis probe
(CMA, Inc.) was surgically implanted into the striatum and
perfused with artificial cerebrospinal fluid at a flow rate of 2
µL/min. Samples were collected every 20 min and analyzed
for catecholamine content by HPLC using electrochemical
detection.
In h ibition of Ap om or p h in e-In d u ced Clim bin g.24 Mice
were injected with apomorphine (1 mg/kg sc) and returned to
their cages. Five minutes later the mice were removed and
test compounds or vehicle were injected ip. After 10 min,
climbing was scored as outlined previously.12c
1H-Meth yl-3-[4-[2-(4-p yr id in -2-ylp ip er a zin -1-yl)eth yl]-
cycloh exyl]-1H-in d ole (32). A solution of potassium hy-
droxide (0.623 g, 11.1 mmol) in 6 mL of degassed DMSO
(anhydrous) was treated with 30a (1.08 g, 2.78 mmol) and
stirred for 15 min. The mixture was treated with methyl
iodide (0.17 mL, 2.73 mmol), and the resulting solution was
stirred for 2 h at room temperature during which time a thick
precipitate formed. The reaction mixture was partitioned
between 50 mL of water and 50 mL of chloroform. The organic
extract was dried over sodium sulfate, and the solvents were
evaporated. The resulting residue was purified using MPLC
(2% MeOH, 0.1% NH3 in chloroform) to give the product which
was triturated with ether and dried to give 32 (0.63 g, 56%
yield): mp 78-9 °C (ether); EIMS m/z 402 (M+, 17), 308 (23),
1
295 (27), 281 (17), 107 (base); H-NMR (CDCl3, 250 MHz) δ
8.20 (d, J ) 2 Hz, 1H), 7.63 (d, J ) 8 Hz, 1 H), 7.48 (t, J ) 7
Hz, 1 H), 7.25 (d, J ) 8 Hz, 1 H), 7.20 (t, J ) 8 Hz, 1 H), 7.08
(t, J ) 7 Hz, 1 H), 6.79 (s, 1 H), 6.68 (d, J ) 8 Hz, 1 H), 6.62
(t, J ) 7 Hz, 1 H); 3.73 (s, 3 H), 3.57 (t, J ) 5 Hz, 4 H), 2.79
(t, J ) 12 Hz, 1h H), 2.58 (t, J ) 5 Hz, 4 H), 2.46 (t, J ) 8 Hz,
2 H), 2.12 (d, J ) 12 Hz, 2 H), 1.89 (d, J ) 13 Hz, 2 H), 1.40-
1.26 (m, 5 H), 1.19 (dd, J ) 20, 12 Hz, 2 H). Anal. (C26H34N4)
C, H, N.
Ster eotyp ed Beh a vior in Ra ts.25 Stereotyped behavior
was assessed by a trained observer who rated animals for
repetitive sniffing, licking, and gnawing. The duration of each
observation was 30 s/rat. Stereotyped behaviors were defined
by their continuous presence for a period of at least 15 s. Rats
received and injection of the test compound were observed for
1 h and then injected with the DA D1 agonist SKF 383893
and observed for an additional hour.
Mon k ey Str ia ta l Micr od ia lysis Adult male squirrel mon-
keys, Saimiri sciureus, with permanent cranial guide probes
were chair-restrained and had a microdialysis probe inserted
through the guide into the putamen nucleus. Microdialysis
samples were collected every 20 min and analyzed for cat-
echolamine content by HPLC using electrochemical detection.
Con d ition ed Avoid a n ce in Squ ir r el Mon k eys. This
procedure was carried out according to methods described
previously.26 Inhibition of conditioned avoidance was mea-
sured for 6 h after oral administration of compound. Drug
effects were expressed as a percentage of avoidance responding
relative to control performance during the 4 h of peak effect.
P h a r m a cologica l Meth od s. [3H]Sp ip er on e Bin d in g in
Ra t Str ia ta l Mem br a n es. The IC50 of compounds was
determined according to methods previously described16 using
[3H]spiperone (0.2 nM final concentration) binding to rat
striatal membranes in the presence of (+)-butaclamol (1 µM)
for nonspecific binding.
[3H]-N-P r op yln or a p om or p h in e (NP A) Bin d in g in Ra t
Str ia ta l Mem br a n es. The IC50 of compounds was deter-
mined according to methods previously described19 using [3H]-
NPA (0.35 nM final concentration) binding to rat striatal
membranes in the presence of (+)-butaclamol (2 µM) for
nonspecific binding.
Bin d in g to D3 a n d D4.2 Recep tor Isofor m s. The human
DA D3 receptor cDNA cloned in the pcDNA I/Neo plasmid was
obtained from Dr. K. O’Malley and stably transfected into CHO
K1 cells by a modified calcium phosphate precipitation tech-
nique, and transfectants were selected in G-418, isolated, and
screened for expression of human D3 receptors by radioligand
binding as previously described.20 For D4 binding, CHO K1
cells stabily transfected to express the human recombinant
DA D4.2 receptor subtype as previously described21 were used.
The Ki values were determined using [3H]spiperone (0.2 nM
final concentration) with the appropriate cell membranes.
[3H]Th ym id in e Up ta k e in D2 Tr a n sfected CHO p -5
Cells. As previously described22 CHO p-5 cells transfected
with the long form of the D2 receptor were plated and 48 h
later deprived of serum and then treated 24 h later with
standards or test compounds. Eighteen hours later [3H]-
thymidine (5 µCi/well) and then trypsin (100 µL of 0.25%) were
added. The assay was terminated by filtration using a Brandel
96-well harvester. The filters were counted for radioactivity
using the LKB plate counting system.
Ack n ow led gm en t. The authors wish to thank L.
Meltzer, F. Niniteman, K. Serpa, and J . Wiley for
pharmacological testing and D. J ohnson and N. Colbry
for performing high-pressure hydrogenation reactions.
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