trans-4-[4-(methoxyphenyl)cyclohexyl]-1-arylpiperazines: A new class of potent and selective 5-HT1A receptor ligands as conformationally constrained analogues of 4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-y1)propyl]-1-arylpiperazines

Article abstract of DOI:10.1021/jm010866v

The present paper concerns the influence of conformational parameters on the recognition by rat 5-HT_1A receptors of derivatives 4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]-1- (2-pyridinyl)piperazine (1a) and 3-(5-methoxy-1,2,3,4-t

Full text of DOI:10.1021/jm010866v

J . Med. Chem. 2001, 44, 4431-4442
4431
tr a n s-4-[4-(Meth oxyp h en yl)cycloh exyl]-1-a r ylp ip er a zin es: A New Cla ss of P oten t
a n d Selective 5-HT1A Recep tor Liga n d s a s Con for m a tion a lly Con str a in ed
An a logu es of 4-[3-(5-Meth oxy-1,2,3,4-tetr a h yd r on a p h th a len -1-yl)p r op yl]-1-
a r ylp ip er a zin es
Roberto Perrone,* Francesco Berardi, Nicola A. Colabufo, Marcello Leopoldo, Enza Lacivita, and
Vincenzo Tortorella
Dipartimento Farmaco-Chimico, via Orabona, 4, 70126, Bari, Italy
Amedeo Leonardi, Elena Poggesi, and Rodolfo Testa
Research & Development Department, Recordati S.p.A., via Civitali, 1, 20148, Milan, Italy
Received March 2, 2001
The present paper concerns the influence of conformational parameters on the recognition by
rat 5-HT1A receptors of derivatives 4-[3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)propyl]-
1-(2-pyridinyl)piperazine (1a ) and 3-(5-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)-N-[2-(2-
pyridyloxy)ethyl]propanamine (3b), two highly potent and selective 5-HT1A receptor ligands.
Fifteen corresponding flexible and rigid analogues were prepared following several synthetic
routes and were tested in binding assays with radioligands at 5-HT1A, D2, and R1 receptors
from rat brain membranes. Among the new derivatives emerged trans-4-[4-(3-methoxyphenyl)-
cyclohexyl]-1-(2-pyridinyl)piperazine (trans-8a ) and trans-N-[4-(3-methoxyphenyl)cyclohexyl]-
2-(2-pyridyloxy)ethylamine (trans-8b). These compounds can be considered as conformationally
constrained analogues of compounds 1a and 3a , respectively. In fact, compounds trans-8a and
trans-8b showed a marked enhancement in 5-HT1A receptor affinity when compared to the
corresponding cis isomers. Because compound trans-8a was a potent and selective 5-HT1A ligand
(Ki, nM: 5-HT1A ) 0.028, D2 ) 2194, R1 ) 767), it was chosen as a lead to prepare other
analogues that were tested at 5-HT1A, D2, and R1 receptors from rat brain membranes, showing
high affinity at the 5-HT1A and selectivity vs D2 and R1 receptors. Selected compounds were
tested for their affinity at the human cloned 5-HT1A, R1a, R1b, R1d receptor subtypes. They were
also submitted to the [35S]GTPγS binding assay stimulating the 5-HT1A receptor-mediated
G-protein activation, therefore behaving as full or as partial agonists. Finally, the ability of iv
administration of trans-8a to induce fore-paw treading in rats was evaluated in comparison
with 8-OH-DPAT. Although the affinity (Ki) and in vitro activity (pD′2) of trans-8a at the 5-HT1A
receptor were higher than those of 8-OH-DPAT, the compound was less potent than the
reference standard in inducing the symptom.
Serotonin (5-HT) is involved in various physiological
and pathological process by interaction with seven
classes of receptors (5-HT1-7) containing 14 distinct
receptors grouped on the basis of amino acid sequence,
pharmacology, and signal transduction pathways.1,2 The
5-HT1A receptor subtype has been the target of consid-
erable research because of its involvement in psychiatric
disorders such as anxiety and depression.3,4 Earlier
studies were focused on the development of 5-HT1A
receptor agonists such as buspirone, the first 5-HT1A
agent launched in the market. More recently new
therapeutic perspectives have been proposed: 5-HT1A
agonists may be useful as antidepressants5,6 and as
neuroprotective agents.7 The full elucidation of the
involvement of 5-HT1A receptors in the above-mentioned
diseases still awaits the proper pharmacological tools.
Although a large number of compounds with high
affinity for 5-HT1A receptors have been described in the
past, few of them are both selective and highly effica-
cious at the receptor. During the past years many
1-arylpiperazines (Chart 1) have been synthesized, and
our research group has been interested in some arylpi-
perazine derivatives containing the tetralin nucleus
(structure I) as 5-HT1A receptor ligands. SAFIR studies
have allowed us to establish optimal structural features
for the interaction with 5-HT1A receptors and to obtain
highly potent and selective compounds.8-12 The funda-
mental elements were the 1-aryl-4-propylpiperazinyl
moiety directly linked to the 5-methoxy-1-tetralinyl
nucleus; chirality did not play any role in 5-HT1A
receptor affinity.13 The best 1-aryl group on the pipera-
zine moiety in terms of both affinity and selectivity for
the 5-HT1A receptor was 2-pyridinyl, so compounds of
structure 1a 9 (Table 1) represent the most relevant
member of that series. Furthermore, we discovered that
the 1-(2-pyridinyl)piperazine nucleus can be successfully
replaced by a 2-(2-pyridyloxy)ethylamino moiety (com-
pound 1b).14
Although a large number of papers have been pub-
lished on “long-chain” arylpiperazines as 5-HT1A ligands,
* To whom correspondence should be addressed. Phone: +39-080-
5442752. Fax: +39-080-5442231. E-mail: perrone@farmchim.uniba.it.
10.1021/jm010866v CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/07/2001

4432 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Perrone et al.
Ch a r t 1
Sch em e 1a
a
Reagents: (A) (i) NBS, (ii) triethylamine; (B) 1-(2-pyridi-
nyl)piperazine; (C) 2-(2-pyridyloxy)ethylamine.
replaced by a phenyl, 2-methoxyphenyl, or 1-naphatha-
lenyl (compounds 8c-e). The role of the methoxy group
position on the aromatic ring of compounds related to
cis-8a and trans-8a was also investigated (derivatives
9a and 10a ).
Finally, selected compounds were tested for their
affinity at the human cloned 5-HT1A, R1a, R1b, R1d
receptor subtypes, and they were submitted to the [35S]-
GTPγS binding assay for the evaluation of their intrinsic
activity on the 5-HT1A receptor. The ClogP values of all
compounds were calculated as a predictive parameter
for membrane permeation.
only a few dealt with the conformation of the poly-
methylene chain of these ligands. Crystallographic data
Ch em istr y
1
of buspirone,15 gepirone,15 and mazapertine16 and a H
The preparation of the target compounds required
several synthetic routes.
NMR study on tandospirone in water solution17 indi-
cated that these compounds adopted conformations
between bent and extended that are believed to be
responsible for the biological activity at the 5-HT1A
receptor. However, these studies do not give information
about the conformational changes in the ligand caused
by the interaction with 5-HT1A receptors, so the struc-
tural requirements of the receptor may force the mol-
ecule to bind in a conformation that would not be
favored in the nonbonded state. One way to circumvent
this uncertainty is to evaluate the pharmacological
activity of conformationally rigid analogues. This ap-
proach has been used in two related cases: Mokrosz et
al.18 prepared some rigid analogues of NAN-190; Romero
and co-workers19 studied the semirigid analogues of
ipsapirone. Both studies indicated that these ligands
bind at the 5-HT1A receptor in an extended linear
conformation.
In the present paper we address the influence of
conformational parameters on the recognition of deriva-
tives 1a and 1b by the 5-HT1A receptor. We prepared
and tested some corresponding more flexible and more
rigid analogues; in particular, the 5-HT1A receptor
affinity of compound 1a has been compared with those
of two analogues having similar conformational freedom
(compounds 2a and 3a ) and with the flexible analogue
4a obtained by removal of the saturated ring of the
tetralin nucleus. More conformationally constrained
analogues were obtained by blocking the spacer within
a cyclic structure (compounds 5a , 6a , 7a , 8a ). These
modifications have been also extended to the above-
mentioned 2-(2-pyridyloxy)ethylamino derivative 1b. All
these compounds were tested in vitro for their receptor
binding affinity at 5-HT1A, D2, and R1 receptors from
rat brain membranes: among them emerged compound
trans-8a , which was chosen for further modification that
led to compounds where the 2-pyridinyl group was
Compounds 3a ,b and 6a (Scheme 1) were prepared
by alkylating 1-(2-pyridinyl)piperazine or 2-(2-pyridyl-
oxy)ethylamine with bromopropyl derivative 12, ob-
tained from the aromatization of 1-(3-bromopropyl)-5-
methoxy-1,2,3,4-tetrahydronaphthalene (11)9 with N-bro-
mosuccinimide (NBS) and triethylamine; bromoethyl
derivative 1311 and 1-(2-pyridinyl)piperazine reacted to
afford compound 6a .
The synthesis of compounds 4a and 4b (Scheme 2)
started from stannate 14,20 which underwent a cross-
coupling reaction21 with methyl 4-bromocrotonate in the
presence of Pd(0), generated from triphenylphosphine-
palladium(II) chloride and diisobutylaluminum hydride
(DIBAL-H), to give the unsaturated ester 15. Reduction
of the latter with lithium aluminum hydride afforded
alcohol 16, which was reacted with methanesulfonyl
chloride to give the key intermediate 17. The reaction
between 1-(2-pyridinyl)piperazine and mesilate 17 gave
the final compound 4a , whereas the reaction of 2-(2-
pyridinyloxy)ethylamine with compound 17 gave the
corresponding N,N-disubstituted amine. Therefore, to
prevent this shortcoming, mesylate 17 was reacted with
N-benzyl derivative 19, prepared from commercially
available N-benzylethanolamine (18) and 2-chloropyri-
dine under phase-transfer catalysis22 to give amine 20.
Hydrogenolysis of the latter gave the final compound
4b.
The preparation of compounds 5a and 5b was ac-
complished as depicted in Scheme 3. Carboxylic acid
2123 was refluxed with ethanol in the presence of a
catalytic amount of sulfuric acid to give ester 22, which
was reduced to alcohol 23 with LiAlH4. This compound
was transformed into its mesylate derivative 24. The
reaction of the latter with 1-(2-pyridinyl)piperazine or
2-(2-pyridyloxy)ethylamine afforded the final com-
pounds 5a and 5b.

5-HT1A Receptor Ligands
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4433
Ta ble 1. Binding Affinities Determined on Rat Brain Membranes
a
See ref 14.
The synthetic route followed to prepare derivatives
7a , 7c-e, 8a -e, 9a , and 10a parallels a method
reported for the synthesis of similar compounds24
(Scheme 4). Ketone 25 was reacted with the appropriate
amine in the presence of a catalytic amount of p-
toluenesulfonic acid to give the intermediate enamines.
These were first transformed into their hydrochlo-
ride salts, then reduced with NaBH3CN.25 Deprotec-
tion of ketals 26a -e with HCl gave 4-substituted
cyclohexanones 27a -e. Reaction of these ketones with
the appropriate Grignard reagent gave the inter-
mediate alcohols, which were dehydrated to afford
alkenes 7a -e, 28a , and 29a . Final compounds 8a -e,
9a , and 10a were obtained by reduction of the double
bond with H2 in the presence of Pd/C. Trans and cis
isomers were easily separated by column chromatogra-

4434 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Perrone et al.
Sch em e 2a
a
Reagents: (A) (i) DIBAL-H, triphenylphosphinepalladium(II) chloride, (ii) methyl 4-bromocrotonate; (B) LiAlH4; (C) methanesulfonyl
chloride, triethylamine; (D) 1-(2-pyridinyl)piperazine; (E) 2-chloropyridine, KOH, 18-crown-6; (F) H2, 10% Pd/C.
Sch em e 3a
a
Reagents: (A) 96% H2SO4, EtOH; (B) LiAlH4; (C) methanesulfonyl chloride, triethylamine; (D) 1-(2-pyridinyl)piperazine or 2-(2-
pyridinyloxy)ethylamine.
phy. Stereochemical assignment is described in detail
in Experimental Section. When necessary, final com-
pounds were transformed into their hydrochloride or
hydrogen oxalate salts in the usual manner. Physical
properties of target compounds are listed in Table 2.
and trans-8a . For all these compounds the alkyl chain
is blocked in an extended conformation, but their
affinities for the 5-HT1A receptor were markedly differ-
ent. First of all, trans-8a is more potent and selective
than the reference compound 1a . Subsequently, trans-
8a is 29 000-fold more potent than the isomer cis-8a at
the 5-HT1A receptor, being strikingly selective over D2
and R1 receptors. The unsaturated derivative 7a dis-
played the same binding profile as cis-8a . These results
highlight the stringent requirements for these com-
pounds to interact with the 5-HT1A receptor. The bio-
active conformation of the alkyl chain in derivative 1a
seems to be in an extended manner bearing the 1-(2-
pyridinyl)piperazine moiety and the aromatic ring of the
tetralin nucleus in the same relative position reached
by 1-(2-pyridinyl)piperazine and the 3-methoxyphenyl
group in trans-8a .
Resu lts a n d Discu ssion
Binding affinities determined on rat brain membranes
are listed in Table 1. Considering first the 1-(2-pyridi-
nyl)piperazine derivatives, there were not great differ-
ences in affinities at the 5-HT1A receptor for compounds
2a and 3a compared with the reference compound 1a .
However, the limited blocking of the alkyl chain affects
the selectivity; compounds 2a and 3a are more selective
than the reference compound 1a over D2 and R1 recep-
tors. The removal of the saturated ring of tetralin in
compound 1a gave the analogue 4a ; the 5-HT1A receptor
affinity value indicates that the flexibility of the mol-
ecule is detrimental for a high affinity but still allows
the molecule to bind at the receptor. Derivatives 5a and
6a represent two different ways to block the alkyl chain
in a semirigid conformation. The 5-HT1A receptor affin-
ity of compound 5a clearly indicated that the blocking
of the alkyl chain in a folded manner caused a dramati-
cal loss in affinity. On the other hand, the extended
conformation reached by the alkyl chain in compound
6a seemed to favor a good interaction with the receptor.
More complete information in this sense can be achieved
by considering the cyclohexane derivatives 7a , cis-8a ,
The same modifications have been effected on the
2-(2-pyridyloxy)ethylamino derivative 1b. The 5-HT1A
receptor affinity of compounds 1-5b, cis-8b, and trans-
8b displayed the same trend observed for the corre-
sponding 1-(2-pyridinyl)piperazines. However, it can be
noted that trans-8b is less potent than the reference 1b
and the difference in affinity from the affinity of the cis
isomer is less marked than that found between the
corresponding 1-(2-pyridinyl)piperazine derivatives cis-
8a and trans-8a . This behavior could be due to the
flexibility of the 2-(2-pyridyloxy)ethylamino moiety that

5-HT1A Receptor Ligands
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4435
Sch em e 4a
a
Reagents: (A) 1-arylpiperazine or amine 19, p-toluenesulfonic acid; (B) (i) HCl (g), (ii) NaBH3CN; (C) 3 N HCl; (D) methoxyphenyl-
MgBr; (E) 20% H2SO4; (F) H2, 10% Pd/C.
can assume a unfavorable conformation for the binding
at 5-HT1A receptor.
noted that cis-9a and cis-10a are less potent than the
corresponding trans isomers, confirming the trend
discussed above.
Starting from these results, we considered some
modifications on the structure of compound 8a . In
particular, compounds 8c-e were obtained by replacing
the 2-pyridinyl group with other aromatic rings, such
as phenyl, 2-methoxyphenyl, 1-naphthalenyl, already
studied in the tetralinyl series of 5-HT1A ligands;
compounds 9a and 10a were obtained by shifting the
methoxy group of compound 8a from the 3-position to
the 2- and 4-position, respectively.
Seven of the compounds studied were selected for
further in vitro pharmacological evaluation on the basis
of their binding profile: compounds 1a ,b, 3a ,b, and
trans-8a ,b,d , displaying quite different structural fea-
tures, were tested for their affinity at human cloned
5-HT1A, R1a, R1b, and R1d receptors and in the [35S]GTPγS
binding at the human cloned 5-HT1A receptor.
All these compounds showed nano- or subnanomolar
affinity values at the human 5-HT1A receptor (Table 3)
but displayed different selectivity versus the human R1-
adrenoceptor subtypes: compounds 1a and 1b were
endowed with similar affinity for 5-HT1A serotonergic
receptor and R1a-adrenoceptor subtype; compound 3a
showed the same affinity for 5-HT1A receptor and
R-adrenoceptor subtypes; compounds 3b and trans-8a
showed good selectivity versus the R1a, R1b, R1d receptors
because trans-8a is more than 100-fold selective over
the R-adrenoceptor subtypes.
The 5-HT1A receptor affinity values of these deriva-
tives are in the same range as those of compounds 8a ,
revealing the same behavior: the trans isomers 8c-e
are always more potent than the corresponding cis, the
latter isomers being nearly equipotent to the cyclohex-
ene precursors 7c-e. The presence of a moderate D2
receptor affinity in compounds trans-8d ,e reflected in
a lowering in selectivity versus D2 receptor, whereas the
selectivity for R1 receptor is always greater than 1000-
fold. The phenyl derivative trans-8c demonstrated the
same affinity and selectivity as trans-8a .
Compounds 1a ,b, 3a ,b, and trans-8a ,b,d stimulated
5-HT1A receptor-mediated G-protein activation, as mea-
sured by [35S]GTPγS binding, behaving as the full
agonists 5-HT and 8-OH-DPAT (e.g., trans-8a , 1a ) or
as partial agonists (Table 3). This behavior indicated
that the structural differences in this group of com-
pounds did not play any role in their activity.
The position of the methoxy group on the aromatic
ring of trans-8a seemed to play a marginal role in the
interaction with the 5-HT1A receptor. In fact, the shifting
of the methoxy group to the 2-position (trans-9a ) caused
only a slight decrease in 5-HT1A receptor affinity,
whereas the 4-substituted analogue trans-10a presented
the same binding profile as trans-8a . Finally, it can be

4436 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Ta ble 2. Physical Properties of Target Compounds
Perrone et al.
compound
formulaa
mp, °C
recryst solv
ClogP
1a b
4.59
4.96
4.28
4.65
4.17
4.54
3.52
3.89
4.06
4.43
4.06
3.56
4.51
4.53
5.68
3.87
3.87
4.33
4.33
4.82
4.82
4.84
4.84
5.99
5.99
3.87
3.87
3.87
3.87
4.01
1.22
3.85
3.81
1bb
2a b
2bb
3a
3b
4a
4b
5a
5b
6a
7a
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
23H27N3O‚3HCl
228-230
206-208
226-228
177-178
233-234
207-208
222
MeOH/Et2O
MeOH
MeOH/Et2O
MeOH/Et2O
MeOH/Et2O
MeOH
MeOH/Et2O
CHCl3/n-hexane
CHCl3/n-hexane
CHCl3/n-hexane
CHCl3/n-hexane
MeOH/Et2O
MeOH/Et2O
MeOH/Et2O
MeOH/Et2O
CHCl3/n-hexane
MeOH/Et2O
21H24N2O2‚(COOH)2‚1/2H2O
20H27N3O‚2HCl
18H24N2O2‚(COOH)2‚H2O
21H27N3O‚3HCl
19H24N2O2‚2(COOH)2‚1/2H2O
22H29N3O‚2HCl‚1/2H2O
22H27N3O
137-138
135-136
127-129
120-121
239-241
234-235
162-164
169-171
101-103
250-253
107-108
101-102
246-247
250-252
123-124
99-100
7c
7d
7e
23H28N2O
24H30N2O2
27H30N2O
22H29N3O‚2HCl‚H2O
22H29N3O‚2HCl‚1/3H2O
20H26N2O2‚(COOH)2
20H26N2O2‚(COOH)2
23H30N2O
23H30N2O‚2HCl
24H32N2O2
24H32N2O2
27H32N2O‚HCl
27H32N2O‚HCl
22H29N3O
22H29N3O
22H29N3O
cis-8a
trans-8a
cis-8b
trans-8b
cis-8c
trans-8c
cis-8d
trans-8d
cis-8e
trans-8e
cis-9a
trans-9a
cis-10a
trans-10a
8-OH-DPAT
buspirone
haloperidol
phentolamine
CHCl3/n-hexane
CHCl3/n-hexane
MeOH/Et2O
MeOH/Et2O
CHCl3/n-hexane
CHCl3/n-hexane
CHCl3/n-hexane
CHCl3/n-hexane
105-106
112-113
22H29N3O
a
b
Analysis for C, H, N; results were within (0.4% of the theoretical values for the formulas given. See ref 14.
Ta ble 3. Affinity for Human Recombinant 5-HT1A Receptor and R1-Adrenoceptor Subtypes and Potency (pD2) and Relative
Effectiveness Values (Emax: Maximal Stimulation Achieved Expressed as a Percentage of the Maximal 5-HT Response) in the
[
35S]GTPγS Binding Assay at 5-HT1A Human Cloned Receptors of Selected Compounds
binding affinity (Ki, nM)a
R1a R1b
0.33 7.88
26.4
4.21
270
53.1
332
43.3
5975
selectivity vs 5-HT1A
[
35S]GTPγS binding
compound
1a
1b
3a
R1d
5.95
12.65
12.57
83.24
R1a
R1b
R1d
pD2
% max
0.08
0.23
3.0
0.86
0.2
3.77
0.21
3.44
4
2
98
115
1
74
55
4
97
590
37
21
10.22
10.24
7.87
8.2
98.2
86.8
86.3
48
0.55
1.62
0.5
3b
14.6
33.0
201
4.98
1757
17
165
53
314
265
88
trans-8a
trans-8b
trans-8d
8-OH-DPAT
118
142
4.49
>1000
9.28
7.46
9.91
7.6
95.9
82
26.5
100
24
206
a
Standard error of the mean was less than 10% of the mean.
The lipophilicity of all compounds (Table 2) was
ing appears to be the most closely associated with
activation of 5-HT1A postsynaptic receptors.28
estimated by calculations using the ClogP program,26
and it was used as a guide for selecting compounds for
further pharmacological evaluations. ClogP values of
compounds 1a ,b, 3a ,b, and trans-8b were greater than
4.0, predicting fair blood-brain barrier penetration,
whereas trans-8a and trans-8d displayed a ClogP of
3.87, suggesting reasonable blood-brain barrier per-
meation;27 for trans-8a the log P value was experimen-
tally determined to be 3.68 at pH 7.4. Therefore, this
evidence made trans-8a suitable for in vivo testing.
The agonistic interaction of trans-8a at the 5-HT1A
receptor was confirmed in vivo. The intravenous or
subcutaneous injection of a full 5-HT1A agonist (namely,
8-OH-DPAT) in rats induces, usually within 1-2 min,
a dose-dependent increase of locomotion, fore-paw tread-
ing, head weaving, and flat body posture. This “5-HT
syndrome” in rats is mediated by postsynaptic 5-HT1A
receptors. Among the symptoms evoked, fore-paw tread-
The ability of iv administration of trans-8a to induce
fore-paw treading in rats was evaluated in comparison
with 8-OH-DPAT. Although the affinity (Ki) and the
agonist activity (pD2) of trans-8a at the 5-HT1A receptor
were higher than those of 8-OH-DPAT (Table 3), the
compound assayed was less potent than the reference
standard in inducing the symptom (Table 4). This
finding was confirmed after subcutaneous administra-
tion of both reference and tested compounds.
In conclusion, this study provides further insight
about the conformations of the polymethylene chain of
“long-chain” arylpiperazines in the interaction with the
5-HT1A receptor. Moreover, trans-4-[4-(methoxyphenyl)-
cyclohexyl]-1-arylpiperazines have been identified as a
new class of potent 5-HT1A receptor ligands, highly
selective over the D2 and R1 receptors. Two members of
this class (trans-8a and trans-8d ) were found to display

5-HT1A Receptor Ligands
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4437
Ta ble 4. Agonistic Activity (ED50 ( SEa in µg/kg iv) of
Compounds trans-8a Tested on Postsynaptic 5-HT1A Receptors
(Induction of Fore-paw Treading in Rats)
agonistic properties on the human cloned 5-HT1A recep-
tor measured by the [35S]GTPγS binding assay.
Exp er im en ta l Section
ED50 ( SE for fore-paw treading induction in rats
compound
iv
subcutaneous
Ch em istr y. Column chromatography was performed with
1:30 ICN silica gel 60A (63-200 µm) as the stationary phase.
Melting points were determined in open capillaries on a
Gallenkamp electrothermal apparatus. Elemental analyses (C,
H, N) were performed on a Carlo Erba model 1106 analyzer;
the analytical results were within (0.4% of the theoretical
trans-8a
8-OH-DPAT
0.721 ( 0.005
0.084 ( 0.012
1.957 ( 0.203
0.137 ( 0.011
a
SE ≡ standard error.
1
values for the formula given. H NMR spectra were recorded
first washed with a saturated aqueous solution of NaHCO3
and then with 3 N HCl. The separated organic phase was dried
over Na2SO4 and concentrated under reduced pressure, yield-
ing a crude residue. Pure 17 was obtained after chromatog-
raphy (petroleum ether/AcOEt, 4:1, as eluent) as a colorless
oil in 44% yield. 1H NMR (90 MHz): δ 1.60-1.90 [m, 4H, CH2-
(CH2)2CH2], 2.50-2.75 (m, 2H, ArCH2), 2.95 (s, 3H, SO2CH3),
3.78 (s, 3H, OCH3), 4.21 (m, 2H, CH2O), 6.60-7.35 (m, 4H,
aromatic). GC/MS: m/z 260 (M+ + 2, 2), 259 (M+ + 1, 5), 258
(M+, 39), 161 (49), 134 (100), 121 (72).
N-Ben zyl-2-(2-p yr id yloxy)eth yla m in e (19). A mixture of
2-chloropyridine (4.00 g, 35.2 mmol), N-benzylethanolamine
(18) (12.70 g, 84.0 mmol), powdered KOH (3.95 g, 70.4 mmol),
and 18-crown-6 (3.70 g, 14.0 mmol) in toluene (100 mL) was
vigorously stirred under reflux. After 6 h, the reaction mixture
was cooled and washed with H2O (40 mL). The separated
aqueous phase was extracted with Et2O (40 mL), and the
combined organic layers were dried over Na2SO4 and concen-
trated under reduced pressure. The crude residue was chro-
matographed (CHCl3/CH3OH, 19:1, as eluent) to give amine
19 as a yellow oil (7.08 g, 88% yield). 1H NMR (90 MHz): δ
1.91 (s, 1H, NH, D2O exchanged), 2.98 (t, 2H, J ) 6.0 Hz, CH2-
CH2O), 3.87 (s, 2H, benzylic), 4.45 (t, 2H, J ) 6.0 Hz, CH2O),
6.65-8.25 (m, 9H, aromatic). GC/MS: m/z 133 (84), 132 (54),
120 (22), 91 (100).
either on a Varian EM-390 at 90 MHz where indicated (TMS
as internal standard) or on a Bruker AM 300 WB instrument,
with CDCl3 as solvent. All values are reported in ppm (δ). 2-D
NMR experiments (COSY and HETCOR) were performed on
a
Varian NMR 300 Mercury-VX (300 MHz) instrument.
Recording of mass spectra was done on an HP6990-5973 MSD
gas chromatograph/mass spectrometer; only significant m/z
peaks, with their percentage of relative intensity in paren-
theses, are reported. All spectra were in accordance with the
assigned structures. HPLC analyses were carried out using a
Perkin-Elmer series 200 LC pump. UV absorbance was
monitored with a Perkin-Elmer 785A UV/vis detector. 1-(1-
Naphthalenyl)piperazine was synthesized according to a pub-
lished procedure.29
1-(3-Br om op r op yl)-5-m eth oxyn a p h th a len e (12). N-Bro-
mosuccinimide (4.66 g, 26.2 mmol) and benzoylperoxide (0.073
g, 0.3 mmol) were added to a solution of 1-(3-bromopropyl)-5-
methoxy-1,2,3,4-tetrahydronaphthalene (11) (2.97 g, 10.5 mmol)
in CCl4. The mixture was refluxed for 15 h and then was
cooled, and the obtained suspension was filtered. The filtrate
was concentrated under reduced pressure, and the residue was
refluxed for 1 h in ethanol/triethylamine (1:1, v/v, 50 mL). Then
the solvent was evaporated in vacuo and the residue was taken
up with CH2Cl2 and washed with 3 N HCl. The organic phase
was separated, dried over Na2SO4, and concentrated under
reduced pressure. The crude residue was chromatographed
(petroleum ether/CHCl3, 4:1, as eluent) to give compound 12
Eth yl 6-Meth oxy-1,2,3,4-tetr a h yd r o-2-n a p h th a len eca r -
boxylate (22). A solution of 6-methoxy-1,2,3,4-tetrahydronaph-
thalene-2-carboxylic acid (21) (1.75 g, 8.5 mmol) in anhydrous
ethanol (50 mL) was refluxed overnight in the presence of
concentrated H2SO4 (0.5 mL). Then the solvent was removed
under reduced pressure, and the residue was partitioned
between CHCl3 and 20% aqueous Na2CO3. The separated
organic layer was dried over Na2SO4 and concentrated in
vacuo. The crude residue was chromatographed (petroleum
ether/AcOEt, 4:1, as eluent) to give ester 22 as a colorless oil
1
as a pale-yellow oil (0.95 g, 32% yield). H NMR (90 MHz): δ
2.05-2.45 (m, 2H, CH2CH2Br), 3.05-3.60 (m, 4H, ArCH2, CH2-
Br), 3.95 (s, 3H, CH3), 6.75-8.35 (m, 6H, aromatic). GC/MS:
m/z 280 (M+ + 2, 56), 279 (M+ + 1, 10), 278 (M+, 58), 199 (69),
171 (100), 128 (35).
Meth yl 4-(3-m eth oxyp h en yl)-2-bu ten oa te (15). Diisobu-
tylaluminum hydride (1.0 M in toluene, 0.3 mL, 0.3 mmol) was
added by syringe to a suspension of bis(triphenylphosphine)-
palladium(II) chloride (0.093 g, 0.14 mmol) in anhydrous THF
(30 mL). The mixture was stirred for 5 min at room temper-
ature under N2, then a solution of methyl 4-bromocrotonate
(mixture of isomers, 3.13 g, 17.5 mmol) and (3-methoxyphenyl)-
tributyl stannate (14) (6.95 g, 17.5 mmol) in the same solvent
(15 mL) was added dropwise. The mixture was refluxed
overnight, then the solvent was removed under reduced
pressure. The crude residue was chromatographed (petroleum
ether/AcOEt, 9:1, as eluent) to give ester 15 (mixture of
isomers) as a colorless oil (2.06 g, 57% yield). GC/MS: m/z 207
(M+ + 1, 5), 206 (M+, 35), 147 (47), 146 (100), 91 (40).
4-(3-Meth oxyp h en yl)-1-bu ta n ol (16). A solution of ester
15 (2.04 g, 9.9 mmol) in anhydrous THF was added dropwise
to a cooled suspension of LiAlH4 (0.75 g, 19.8 mmol) in the
same solvent. The resulting suspension was stirred for 3 h at
room temperature. Then the mixture was cooled at 0 °C, and
a few drops of H2O were added to destroy the excess hydride.
The mixture was filtered, and the solvent was evaporated
under reduced pressure to give a crude residue that was
chromatographed (petroleum ether/AcOEt, 1:1, as eluent).
Pure 16 was obtained as a colorless oil in 56% yield. Spectral
properties of this compound were fully consistent with those
reported in the literature.30
1
(1.76 g, 88% yield). H NMR (90 MHz): δ 1.23 (t, 3H, J ) 6.0
Hz, CH2CH3), 1.50-3.05 (m, 7H, tetralinic), 3.75 (s, 3H, OCH3),
4.15 (q, 2H, J ) 6.0 Hz, CH2CH3), 6.50-7.15 (m, 3H, aromatic).
6-Met h oxy-1,2,3,4-t et r a h yd r o-2-n a p h t h a len em et h a -
n ol (23). Alcohol 23 was prepared from ester 22 by reduction
with LiAlH4 through the same procedure reported for the
synthesis of compound 16. Pure 23 was obtained by column
chromatography (petroleum ether/AcOEt, 3:2, as eluent) as a
colorless oil in 76% yield. Spectral properties of this compound
were fully consistent with those reported in the literature.31
6-Met h oxy-1,2,3,4-t et r a h yd r o-2-n a p h t h a len em et h yl
Meth a n esu lfon a te (24). Title compound was prepared from
alcohol 23 following the same procedure described above for
compound 17. Pure 24 was obtained after workup as a colorless
oil in 80% yield. 1H NMR (90 MHz): δ 1.85-2.90 (m, 7H,
tetralinic), 3.00 (s, 3H, SO2CH3), 3.77 (s, 3H, OCH3), 4.18 (d,
2H, J ) 6.0 Hz, CH2O), 6.55-7.15 (m, 3H, aromatic). GC/MS:
m/z 272 (M+ + 2, 4), 271 (M+ + 1, 10), 270 (M+, 59), 174 (59),
159 (100).
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
3a , 3b, 4a , 5a , 5b, 6a , a n d 20. A stirred mixture of alkylating
agent (2.0 mmol), amine (4.0 mmol), and a slight excess of K2-
CO3 in acetonitrile was refluxed overnight. After cooling, the
mixture was evaporated to dryness and H2O was added to the
residue. The aqueous phase was extracted twice with AcOEt.
The collected organic layers were dried over Na2SO4 and
evaporated under reduced pressure. The crude residue was
chromatographed as indicated below to give the target com-
pounds.
4-(3-Met h oxyp h en yl)-1-b u t yl Met h a n esu lfon a t e (17).
Triethylamine (1.1 mL, 8.0 mmol) and methanesulfonyl chlo-
ride (0.5 mL, 6.5 mmol) were added to a solution of 16 (0.95 g,
5.3 mmol) in CH2Cl2 cooled at -10 °C. The mixture was stirred
at room temperature for 6 h. Then the reaction mixture was

4438 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
Perrone et al.
4-[3-(5-Meth oxy-1-n aph th alen yl)pr opyl]-1-(2-pyr idin yl)-
p ip er a zin e (3a ). Eluted with CHCl3/AcOEt, 1:1; 44% yield.
1H NMR: δ 1.92-2.03 (m, 2H, CH2CH2CH2), 2.46-2.58 [m,
6H, benzylic, CH2N(CH2)2], 3.09 [t, 2H, J ) 7.7, CH2N(CH2)2],
3.55 [br t, 4H, (CH2)2NAr], 3.98 (s, 3H, CH3), 6.57-7.64 (m,
9H, aromatic), 8.13-8.18 (m, 1H, aromatic NdCH). GC/MS:
m/z 362 (M+ + 1, 3), 361 (M+, 12), 267 (39), 254 (44), 252 (34),
242 (22), 240 (35), 107 (100).
3-(5-Meth oxy-1-n a p h th a len yl)-N-[2-(2-p yr id yloxy)eth -
yl]p r op a n a m in e (3b). Eluted with CHCl3/CH3OH, 19:1; 60%
yield. 1H NMR: δ 1.88-2.01 (m, 3H, CH2CH2CH2, NH, 1H D2O
exchanged), 2.78 (t, 2H, J ) 7.2 Hz, benzylic), 2.98-3.02 (m,
2H, CH2CH2O), 3.09 (br t, 2H, CH2HN), 3.98 (s, 3H, CH3),
4.38-4.42 (m, 2H, CH2CH2O), 6.71-7.62 (m, 9H, aromatic),
8.10-8.17 (m, 1H, aromatic NdCH). GC/MS: m/z 336 (M+,
1), 241 (100), 226 (42), 198 (38), 171 (30).
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
26a -e. A mixture of 1,4-cyclohexanedione mono-ethylene ketal
(25) (20 mmol) and the appropriate amine (24 mmol) in
anhydrous toluene (100 mL) was refluxed overnight in the
presence of a catalytic amount of p-toluenesolfonic acid, and
the formed water was azeotropically distilled off and collected
by a Dean-Stark trap. After the mixture was cooled, the
solvent was evaporated, the crude enamine was solubilized in
anhydrous THF, and gaseous HCl was added until precipita-
tion of enamine hydrochloride was complete. Then to the
suspension was added in one portion a solution of NaBH3CN
(19 mmol) in 10 mL of absolute CH3OH under stirring. The
resulting solution was stirred for 30 min at 25 °C. Then the
solvent was concentrated in vacuo and the residue was taken
up with 0.1 N KOH (60 mL). The aqueous suspension was
extracted with AcOEt (2 × 30 mL). The combined organic
phases were dried over Na2SO4 and concentrated under
reduced pressure. The crude residue was chromatographed
(CHCl3/AcOEt, 1:1, as eluent) to give compounds 26a -e as
semisolids in 70-80% yield.
4-[4-(3-Met h oxyp h en yl)b u t yl]-1-(2-p yr id in yl)p ip er a -
zin e (4a ). Eluted with CHCl3/AcOEt, 1:1; 83% yield. 1H
NMR: δ 1.50-1.73 [m, 4H, CH2(CH2)2CH2], 2.39 (t, 2H, J )
7.4 Hz, benzylic), 2.51-2.55 [br t, 4H, CH2N(CH2)2], 2.60 [t,
2H, J ) 7.3 Hz, CH2N(CH2)2], 3.53 [br t, 4H, (CH2)2NAr], 3.78
(s, 3H, CH3), 6.57-7.48 (m, 7H, aromatic), 8.15-8.18 (m, 1H,
aromatic NdCH). GC/MS: m/z 326 (M+ + 1), 325 (M+, 16),
231 (42), 218 (32), 206 (28), 204 (53), 121 (44), 107 (100).
4-[(6-Met h oxy-1,2,3,4-t et r a h yd r o-2-n a p h t h a len yl)m e-
th yl]-1-(2-p yr id in yl)p ip er a zin e (5a ). Eluted with CHCl3/
8-[4-(2-P yr id in yl)p ip er a zin -1-yl]-1,4-d ioxa sp ir o-[4,5]-
d eca n e (26a ). 1H NMR: δ 1.45-2.00 (m, 8H, cyclohexyl CH2),
2.13 (br s, 1H, CH), 2.73 [br t, 4H, CHN(CH2)2], 3.60 [m, 4H,
(CH2)2NAr], 3.95 [s, 4H, O(CH2)2], 6.53-7.63 (m, 3H, aromatic),
8.20-8.33 (m, 1H, aromatic NdCH). GC/MS: m/z 305 (M+
+
2, 1), 304 (M+ + 1, 9), 303 (M+, 41), 209 (41), 196 (47), 184
1
(62), 107 (100).
AcOEt, 1:1; 60% yield. H NMR: δ 1.39-1.41 (m, 1H, CHH-
CHCHHCH2), 1.95-2.02 (m, 2H, CHHCHCHHCH2), 2.32-
2.41 [m, 3H, CHHCHCHHCH2, CH2N(CH2)2], 2.52-2.59 [m,
4H, CH2N(CH2)2], 2.77-2.91 (m, 3H, CHHCHCHHCH2), 3.55
[br t, 4H, (CH2)2NAr], 3.75 (s, 3H, CH3), 6.57-7.48 (m, 6H,
aromatic), 8.16-8.19 (m, 1H, aromatic NdCH). GC/MS: m/z
338 (M+ + 1, 5), 337 (M+, 19), 243 (73), 230 (74), 176 (66), 147
(40), 121 (49), 107 (100).
8-[N-Ben zyl-2-(2-pyr idyloxy)eth ylam in o]-1,4-dioxaspir o-
[4,5]-d eca n e (26b). H NMR (90 MHz): δ 1.40-1.95 (m, 8H,
cyclohexyl CH2), 2.53-2.80 (m, 1H, CH), 2.95 (t, 2H, J ) 6.0
Hz, NCH2CH2), 3.80 (s, 2H, benzylic), 3.95 [s, 4H, O(CH2)2],
4.30 (t, 2H, J ) 6.0 Hz, NCH2CH2), 6.60-7.67 (m, 9H,
aromatic), 8.00-8.25 (m, 1H, aromatic NdCH). GC/MS: m/z
273 (79), 272 (29), 260 (91), 134 (24), 91 (100).
1
N-[(6-Meth oxy-1,2,3,4-tetr a h yd r o-2-n a p h th a len yl)m e-
th yl]-2-(2-p yr id yloxy)eth yla m in e (5b). Eluted with CHCl3/
CH3OH, 19:1; 20% yield. 1H NMR: δ 1.34-1.45 (m, 1H,
CHHCHCHHCH2), 1.92-1.99 (m, 3H, CHHCHCHHCH2, NH,
1H D2O exchanged), 2.33-2.42 (m, 1H, CHHCHCHHCH2),
2.68 (d, 2H, J ) 6.7 Hz, CH2NH), 2.76-2.87 (m, 3H, CHH-
CHCHHCH2), 3.01-3.05 (m, 2H, CH2CH2O), 3.74 (s, 3H, CH3),
4.40-4.44 (m, 2H, CH2CH2O), 6.59-7.59 (m, 6H, aromatic),
8-(4-P h e n ylp ip e r a zin -1-yl)-1,4-d ioxa sp ir o-[4,5]-d e -
ca n e (26c). 1H NMR (90 MHz): δ 1.50-2.00 (m, 8H, cyclo-
hexyl CH2), 2.43 (br s, 1H, CH), 2.66-2.85 [m, 4H, CHN-
(CH2)2], 3.30-3.33 [m, 4H, (CH2)2NAr], 3.95 [s, 4H, O(CH2)2],
6.80-7.47 (m, 5H, aromatic). GC/MS: m/z 304 (M+ + 2, 3),
303 (M+ + 1, 27), 302 (M+, 81), 209 (41), 201 (49), 132 (33),
101 (100).
8-[4-(2-Meth oxyp h en yl)p ip er a zin -1-yl]-1,4-d ioxa sp ir o-
8.11-8.14 (m, 1H, aromatic NdCH). GC/MS: m/z 313 (M+
+
1
[4,5]-d eca n e (26d ). H NMR (90 MHz): δ 1.55-2.15 (m, 8H,
1, 2), 312 (M+, 7), 217 (47), 174 (100), 159 (67), 151 (54), 122
(48).
cyclohexyl CH2), 2.70 (br s, 1H, CH), 2.90-3.10 [m, 4H, CHN-
(CH2)2], 3.18-3.35 [m, 4H, (CH2)2NAr], 3.90 (s, 3H, CH3), 4.00
[s, 4H, O(CH2)2], 7.03 (br s, 4H, aromatic). GC/MS: m/z 334
(M+ + 2, 4), 333 (M+ + 1, 28), 332 (M+, 100), 231 (96), 162
(33), 149 (35).
4-[2-(7-Met h oxy-1,2,3,4-t et r a h yd r o-2-n a p h t h a len yl)-
eth yl]-1-(2-p yr id in yl)p ip er a zin e (6a ). Eluted with CHCl3/
AcOEt, 1:1; 72% yield. 1H NMR: δ 1.34-1.95 (m, 5H, CH2-
CHCH2), 2.39-2.86 [m, 10H, CH2N(CH2)2, benzylic], 3.55 (br
t, 4H, (CH2)2NAr], 3.75 (s, 3H, CH3), 6.58-7.48 (m, 6H,
aromatic), 8.16-8.18 (m, 1H, aromatic NdCH). GC/MS: m/z
352 (M+ + 1, 3), 351 (M+, 10), 257 (33), 244 (51), 121 (29), 107
(100).
8-[4-(1-Na p h t h a len yl)p ip er a zin -1-yl]-1,4-d ioxa sp ir o-
1
[4,5]-d eca n e (26e). H NMR (90 MHz): δ 1.45-2.15 (m, 8H,
cyclohexyl CH2), 2.50 (br s, 1H, CH), 2.75-2.95 [m, 4H, CHN-
(CH2)2], 3.03-3.28 [m, 4H, (CH2)2NAr], 3.95 [s, 4H, O(CH2)2],
7.00-8.23 (m, 7H, aromatic). GC/MS: m/z 354 (M+ + 2, 3),
353 (M+ + 1, 26), 352 (M+, 99), 251 (52), 154 (31), 101 (100).
4-(3-Meth oxyp h en yl)-N-ben zyl-N-[2-(2-p yr id yloxy)eth -
yl]bu ta n a m in e (20). Eluted with CHCl3/AcOEt, 1:1; 30%
yield. 1H NMR (90 MHz): δ 1.35-1.85 [m, 4H, CH2(CH2)2CH2],
2.00 [br s, 2H, benzylic], 2.35-2.73 (m, 2H, CH2N), 2.83 (t,
2H, J ) 6.0 Hz, OCH2CH2), 3.70 (s, 2H, CH2Ph), 3.78 (s, 3H,
CH3), 4.30 (t, 2H, J ) 6.0 Hz, OCH2CH2), 6.55-8.25 (m, 13H,
aromatic). GC/MS: m/z 282 (40), 280 (86), 91 (100).
4-(3-Meth oxyp h en yl)-N-[2-(2-p yr id yloxy)eth yl]bu ta n -
a m in e (4b). The compound 20 (0.51 g, 1.3 mmol) was dissolved
in ethanol and hydrogenated at normal pressure and room
temperature in the presence of 10% Pd/C (0.1 g) until the
uptake ceased. The catalyst was removed by filtration through
Celite, and the solvent was evaporated in vacuo to give a crude
residue that was chromatographed (CHCl3/CH3OH, 19:1, as
eluent) to provide pure compound 4a as a pale-yellow oil (0.12
Gen er a l P r oced u r e for th e P r ep a r a tion of Keton es
27a -e. In a typical run, one of compounds 26a -e (10 mmol)
was refluxed for 3 h with 3 N HCl (50 mL) in acetone. Then
the mixture was concentrated under reduced pressure and the
aqueous residue was alkalized with 20% aqueous Na2CO3. The
aqueous phase was extracted with AcOEt (2 × 30 mL). The
organic phase was separated, dried over Na2SO4, and concen-
trated under reduced pressure. The crude residue was chro-
matographed (CHCl3/AcOEt, 1:1, as eluent) to give the corre-
sponding ketone 27 as a white semisolid in 90% yield.
4-[4-(2-P yr idin yl)piper azin -1-yl]cycloh exan -1-on e (27a).
1H NMR (90 MHz): δ 1.65-2.60 (m, 9H, cyclohexyl), 2.73 [br
t, 4H, CHN(CH2)2], 3.60 [br t, 4H, (CH2)2NAr], 6.55-7.70 (m,
3H, aromatic), 8.15-8.35 (m, 1H, aromatic NdCH). GC/MS:
m/z 260 (M+ + 1, 2), 259 (M+, 10), 107 (100), 140 (24).
1
g, 31% yield). H NMR: δ 1.51-1.76 [m, 4H, CH2(CH2)2CH2],
1.80 (s, 1H, NH, D2O exchanged), 2.59 (t, 2H, J ) 7.4 Hz,
benzylic), 2.68 (t, 2H, J ) 7.1 Hz, CH2HN), 2.96-3.00 (m, 2H,
CH2CH2O), 3.77 (s, 3H, CH3), 4.38 (t, 2H, J ) 5.2, CH2CH2O),
6.63-7.57 (m, 7H, aromatic), 8.09-8.12 (m, 1H, aromatic Nd
CH). GC/MS: m/z 121 (43), 78 (34), 58 (100).
4-[N-Ben zyl-2-(2-p yr id yloxy)et h yla m in o]cycloh exa n -
1-on e (27b). 1H NMR (90 MHz): δ 1.55-2.60 (m, 9H,
cyclohexyl), 2.93 (t, 2H, J ) 6.0 Hz, NCH2CH2), 3.80 (s, 2H,
benzylic), 4.35 (t, 2H, J ) 6.0 Hz, NCH2CH2), 6.60-7.70 (m,

5-HT1A Receptor Ligands
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4439
8H, aromatic), 8.07-8.27 (m, 1H, aromatic NdCH). GC/MS:
m/z 229 (42), 216 (35), 91 (100).
4-[1-(2-Meth oxyph en yl)cycloh exen -4-yl]-1-(2-pyr idin yl)-
1
p ip er a zin e (28a ). H NMR (90 MHz): δ 1.45-2.90 [m, 11H,
4-(4-P h en ylp ip er a zin -1-yl)cycloh exa n -1-on e (27c). 1H
NMR (90 MHz): δ 1.65-2.55 (m, 9H, cyclohexyl), 2.80 [br t,
4H, CHN(CH2)2], 3.27 [br t, 4H, (CH2)2NAr], 6.80-7.50 (m, 5H,
aromatic). GC/MS: m/z 260 (M+ + 2, 1), 259 (M+ + 1, 14), 258
(M+, 68), 201 (100), 173 (64), 132 (47).
cyclohexene, CHN(CH2)2], 3.55 [br t, 4H, (CH2)2NAr], 3.80 (s,
3H, CH3), 5.77 (br s, 1H, vinylic), 6.50-7.68 (m, 7H, aromatic),
8.17-8.30 (m, 1H, aromatic NdCH). GC/MS: m/z 351 (M+
2, 1), 350 (M+ + 1, 4), 349 (M+, 14), 242 (38), 107 (100).
+
4-[1-(4-Meth oxyph en yl)cycloh exen -4-yl]-1-(2-pyr idin yl)-
1
p ip er a zin e (29a ). H NMR (90 MHz): δ 1.45-2.90 [m, 11H,
4-[4-(2-Met h oxyp h en yl)p ip er a zin -1-yl]cycloh exa n -1-
on e (27d ). 1H NMR (90 MHz): δ 1.70-2.67 (m, 9H, cyclo-
hexyl), 2.80 [br t, 4H, CHN(CH2)2], 3.15 [br t, 4H, (CH2)2NAr],
3.87 (s, 3H, CH3), 6.97 (br s, 4H, aromatic). GC/MS: m/z 290
(M+ + 2, 3), 289 (M+ + 1, 22), 288 (M+, 95), 231 (100), 203
(66), 162 (48), 136 (68).
cyclohexene, CHN(CH2)2], 3.60 [br t, 4H, (CH2)2NAr], 3.83 (s,
3H, CH3), 6.03 (br s, 1H, vinylic), 6.50-7.17 (m, 7H, aromatic),
8.18-8.33 (m, 1H, aromatic NdCH). GC/MS: m/z 350 (M+
+
1, 2), 349 (M+, 9), 242 (24), 189 (28), 120 (25), 107 (100).
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
8a -e, 9a , a n d 10a . One of the alkenes 7a -e, 28a , or 29a (1.3
mmol) was dissolved in ethanol and hydrogenated at normal
pressure and room temperature in the presence of 10% Pd/C
(0.1 g) until the uptake ceased. The catalyst was removed by
filtration through Celite, and the solvent was evaporated in
vacuo to give a crude residue. The latter was chromatographed
using CH2Cl2/AcOEt, 1:1, as eluent containing 0.1% ammonia
to give the cis isomer as the faster-moving component (fraction
A) and the trans isomer as the slower-moving component
(fraction B).32 Each amine was obtained in 25-30% yield.
cis-4-[4-(3-Meth oxyp h en yl)cycloh exyl]-1-(2-p yr id in yl)-
p ip er a zin e (cis-8a ). 1H NMR: δ 1.52-1.63 [m, 4H, CH-
(CHHCHH)2], 1.97-2.05 [m, 4H, CH(CHHCHH)2], 2.31 [br s,
1H, CHN(CH2)2], 2.61-2.71 [m, 5H, benzylic CH, CHN(CH2)2],
3.55 [br s, 4H, (CH2)2NAr], 3.78 (s, 3H, CH3), 6.58-7.49 (m,
7H, aromatic), 8.16-8.19 (m, 1H, aromatic NdCH). GC/MS:
m/z 352 (M+ + 1, 4), 351 (M+, 16), 257 (33), 232 (62), 188 (52),
107 (100).
tr a n s-4-[4-(3-Met h oxyp h en yl)cycloh exyl]-1-(2-p yr id i-
n yl)p ip er a zin e (tr a n s-8a ). 1H NMR: δ 1.41-1.56 [m, 4H,
CH(CHHCHH)2], 1.92-2.08 [m, 4H, CH(CHHCHH)2], 2.42-
2.48 [m, 2H, benzylic CH, CHN(CH2)2], 2.73 [br t, 4H, CHN-
(CH2)2], 3.58 [br t, 4H, (CH2)2NAr], 3.78 (s, 3H, CH3), 6.58-
7.45 (m, 7H, aromatic), 8.16-8.19 (m, 1H, aromatic NdCH).
GC/MS: m/z 352 (M+ + 1, 5), 351 (M+, 20), 257 (35), 244 (41),
232 (59), 230 (28), 188 (31), 121 (35), 107 (100).
cis-N-[4-(3-Meth oxyph en yl)cycloh exyl]-2-(2-pyr idyloxy)-
eth yla m in e (cis-8b). 1H NMR: δ 1.55-1.88 [m, 9H, CH-
(CHHCHH)2, NH, 1H D2O exchanged], 2.48-2.58 (m, 1H,
CHNH), 2.94 (br t, 1H, benzylic CH), 2.99 (t, 2H, J ) 5.4 Hz,
CH2CH2O), 3.78 (s, 3H, CH3), 4.42 (t, 2H, J ) 5.3 Hz,
CH2CH2O), 6.68-7.58 (m, 8H, aromatic), 8.11-8.14 (m, 1H,
aromatic NdCH). GC/MS: m/z 231 (61), 188 (100), 134 (52),
124 (38).
4-[4-(1-Na p h t h a le n yl)p ip e r a zin -1-yl]cycloh e xa n -1-
on e (27e). 1H NMR (90 MHz): δ 1.55-2.75 (m, 9H, cyclo-
hexyl), 2.90 [br t, 4H, CHN(CH2)2], 3.23 [br t, 4H, (CH2)2NAr],
7.05-8.40 (m, 7H, aromatic). GC/MS: m/z 310 (M+ + 2, 3),
309 (M+ + 1, 25), 308 (M+, 100), 251 (96), 223 (53), 154 (45).
Gen er a l P r oced u r e for th e Syn th esis of Alk en es 7a -
e, 28a , a n d 29a . In a typical run, to a stirred solution of
Grignard reagent prepared from Mg turnings (24 mmol) and
the appropriate bromoanisole (15.9 mmol) in anhydrous THF
(30 mL) was added dropwise one of ketones 27a -e (11.6 mmol)
in the same solvent (15 mL), and the mixture was refluxed
for 7-8 h. After the mixture was cooled at room temperature,
a cooled saturated aqueous solution of NH4Cl (40 mL) was
added to the reaction mixture. Extraction with Et2O and
evaporation of the organic layer gave a crude residue that was
warmed at 70 °C for 6 h with 20% H2SO4 (30 mL). After the
mixture was cooled, the aqueous solution was alkalized with
10% aqueous NaOH and the suspension was extracted with
AcOEt (2 × 10 mL). The collected organic layers were dried
over Na2SO4 and concentrated under reduced pressure. The
crude residue was chromatographed (CHCl3/AcOEt, 1:1, as
eluent) to give the corresponding title compound as a white
solid in 10-15% yield.
4-[1-(3-Meth oxyph en yl)cycloh exen -4-yl]-1-(2-pyr idin yl)-
p ip er a zin e (7a ). 1H NMR: δ 1.57-1.69, 2.13-2.29, and 2.39-
2.81 [m, 11H, cyclohexene, CHN(CH2)2], 3.58 [br t, 4H,
(CH2)2NAr], 3.79 (s, 3H, CH3), 6.06 (t, 1H, J ) 2.7 Hz, vinylic),
6.57-7.49 (m, 7H, aromatic), 8.17-8.19 (m, 1H, aromatic Nd
CH). GC/MS: m/z 350 (M+ + 1, 5), 349 (M+, 18), 242 (32), 107
(100).
N-Ben zyl-N-[1-(3-m eth oxyp h en yl)cycloexen -4-yl]-2-(2-
p yr id yloxy)eth yla m in e (7b). 1H NMR (90 MHz): δ 1.55-
2.65 (m, 7H, cyclohexene), 2.97 (t, 2H, J ) 6.0 Hz, CH2CH2O),
3.75 (s, 2H, benzylic), 3.80 (s, 3H, CH3), 4.33 (t, 2H, J ) 6.0
Hz, CH2CH2O), 6.00-6.25 (m, 1H, vinylic), 6.75-7.65 (m, 12H,
aromatic), 8.17-8.19 (m, 1H, aromatic NdCH). GC/MS: m/z
415 (M+ + 1, 2), 414 (M+, 5), 319 (34), 306 (27), 278 (23), 159
(100), 158 (32).
tr a n s-N-[4-(3-Met h oxyp h en yl)cycloh exyl]-2-(2-p yr i-
1
d oxy)eth yla m in e (tr a n s-8b). H NMR: δ 1.27 [dq, 2H, J )
11.2, 2.9 Hz, axial ArCH(CHHCHH)2], 1.49 [dq, 2H, J ) 12.9,
2.6 Hz, axial ArCH(CHHCHH)2], 1.79 (br s, 1H, NH, D2O
exchanged), 1.92 [app br d, 2H, equatorial ArCH(CHHCHH)2],
1.92 [app br d, 2H, equatorial ArCH(CHHCHH)2], 2.07 [app
br d, 2H, equatorial ArCH(CHHCHH)2], 2.48 [dt, J ) 12.0,
3.4 Hz, 1H, axial CHN(CH2)2], 2.59 [dt, J ) 11.0, 3.7 Hz, 1H,
benzylic CH], 3.06 (t, 2H, J ) 5.2 Hz, CH2CH2O), 3.78 (s, 3H,
CH3), 4.41 (t, 2H, J ) 5.2 Hz, CH2CH2O), 6.69-7.58 (m, 8H,
aromatic), 8.11-8.14 (m, 1H, aromatic NdCH). GC/MS: m/z
231 (54), 188 (100), 134 (54), 121 (39).
4-[1-(3-Met h oxyp h en yl)cycloh exen -1-yl]-1-p h en ylp i-
1
p er a zin e (7c). H NMR: δ 1.55-1.69, 2.14-2.27, and 2.29-
2.85 [m, 11H, cyclohexene, CHN(CH2)2], 3.23 [br t, 4H, (CH2)2-
NAr], 6.08 (t, 1H, J ) 2.6 Hz, vinylic), 6.75-7.28 (m, 9H,
aromatic). GC/MS: m/z 350 (M+ + 2, 2), 349 (M+ + 1, 13), 348
(M+, 48), 188 (100), 132 (95).
4-[1-(3-Meth oxyp h en yl)cycloh exen -4-yl]-1-(2-m eth oxy-
cis-4-[4-(3-Meth oxyp h en yl)cycloh exyl]-1-p h en ylp ip er -
a zin e (cis-8c). 1H NMR: δ 1.53-1.61 [m, 4H, CH(CHH-
CHH)2], 1.96-2.15 [m, 4H, CH(CHHCHH)2], 2.32-2.34 [m,
1H, CHN(CH2)2], 2.64-2.71 [m, 5H, benzylic CH, CHN(CH2)2],
3.21 [br t, 4H, (CH2)2NAr], 3.79 (s, 3H, CH3), 6.69-7.28 (m,
9H, aromatic). GC/MS: m/z 352 (M+ + 2, 2), 351 (M+ + 1, 16),
350 (M+, 58), 201 (100), 132 (22).
1
p h en yl)p ip er a zin e (7d ). H NMR: δ 1.57-1.71, 2.13-2.46,
and 2.50-2.87 [m, 11H, cyclohexene, CHN(CH2)2], 3.15 [br s,
4H, (CH2)2NAr], 3.80 and 3.86 (2 s, 6H, 2 CH3), 6.07 (t, 1H, J
) 2.5 Hz, vinylic), 6.74-7.24 (m, 8H, aromatic). GC/MS: m/z
380 (M+ + 2, 2), 379 (M+ + 1, 16), 378 (M+, 58), 218 (97), 162
(100), 149 (47).
4-[1-(3-Meth oxyp h en yl)cycloh exen -4-yl]-1-(1-n a p h th a -
len yl)p ip er a zin e (7e). 1H NMR: δ 1.65-1.75, 2.22-2.37, and
2.49-2.94 [m, 11H, cyclohexene, CHN(CH2)2], 3.20 [br s, 4H,
(CH2)2NAr], 3.81 (s, 3H, CH3), 6.11 (t, 1H, J ) 2.4 Hz, vinylic),
6.76-8.23 (m, 11H, aromatic). GC/MS: m/z 400 (M+ + 2, 3),
399 (M+ + 1, 13), 398 (M+, 42), 238 (100), 237 (30), 182 (69),
169 (35).
tr a n s-4-[4-(3-Meth oxyp h en yl)cycloh exyl]-1-p h en ylp i-
p er a zin e (tr a n s-8c). 1H NMR: δ 1.41-1.58 [m, 4H, CH-
(CHHCHH)2], 1.97-2.10 [m, 4H, CH(CHHCHH)2], 2.42-2.50
[m, 2H, benzylic CH, CHN(CH2)2], 2.79 [br t, 4H, CHN(CH2)2],
3.23 [br t, 4H, (CH2)2NAr], 3.78 (s, 3H, CH3), 6.70-7.28 (m,
9H, aromatic). GC/MS: m/z 352 (M+ + 2, 2), 351 (M+ + 1, 14),
350 (M+, 53), 201 (100), 132 (19).

4440 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25
cis-4-[4-(3-Met h oxyp h en yl)cycloh exyl]-1-(2-m et h oxy-
Perrone et al.
in the aqueous layer was quantified by HPLC (column Phe-
nomenex Prodigy ODS-3 RP-18 5 µm, 4.6 mm × 250 mm;
mobile phase CH3OH/H2O/EtN3, 9:1:0.05; λ ) 249 nm; flow
rate, 1 mL/min). The concentration of the compound in the
1-octanol layer was assumed to be unchanged after shaking.
The mean log P value was determined after three separate
determinations.
Biologica l Meth od s. 1. Gen er a l. 8-OH-DPAT‚HBr was
from RBI (Research Biochemicals International, Natick, MA).
GDP and GTPγS were from SIGMA-Aldrich (Milan, Italy).
5-HT was from Merck (Milan, Italy). [3H]Prazosin, [3H]8-OH-
DPAT, and [35S]guanosine-5′-(γ-thio)triphosphate ([35S]GTPγS)
were obtained from NEN Life Science Products (Milan, Italy).
For receptor binding studies, the compounds were dissolved
in absolute alcohol. For iv administration in rats and mice,
trans-8a was dissolved in N,N-dimethylformamide (20% v/v)
and TWEEN 80 (10% v/v) in demineralized water, 1a was
dissolved in N,N-dimethylformamide (4% v/v) and TWEEN 80
(8% v/v) in demineralized water, and 8-OH-DPAT was dis-
solved in demineralized water.
Male Wistar Hannover rats (200-250 g) from Harlan, Italy,
were used for the binding experiments on rat brain homoge-
nates. Male Sprague Dawley rats (Crl:CD(SD)BR, 175-200 g
body weight (bw)), and male mice (Crl:CD-1(ICR)BR, 28-38
g bw) from Charles River, Italy, were used for the in vivo
experiments. Animals were housed with free access to food and
water and were maintained on a forced 12 h light-dark cycle
at 22-24 °C for at least 1 week before the experiments were
carried out. The animals were handled according to interna-
tionally accepted principles for care of laboratory animals
(E.E.C. Council Directive 86/609, O.J . No. L358, December 18,
1986).
2. Ra d ioliga n d Bin d in g Assa y a t Ra t Hip p oca m p a l
Mem br a n es 5-HT1A Recep tor s. Binding experiments were
performed according to Borsini et al.34 with minor modifica-
tions. Rats were killed by decapitation, the brain was quickly
removed, and the hippocampus was dissected. The hippocam-
pus (1.0 g) was homogenized with a Brinkman polytron
(setting 5 for 3 × 15 s) in 25 mL of 50 mM Tris buffer, pH 7.6.
The homogenate was centrifuged at 48 000g for 15 min at 4
°C. The surnatant was discarded, and the pellet was resus-
pended in 25 mL of buffer, then preincubated for 10 min at
37 °C. The homogenate was centrifuged at 48 000g for 15 min
at 4 °C. The supernatant was discarded, and the final pellet
was stored at -80 °C until used. Each tube received in a final
volume of 1 mL of 50 mM Tris (pH 7.6) hippocampus
membranes suspension and 1 nM [3H]-8-OH-DPAT. For
competitive inhibition experiments various concentrations of
drugs studied were incubated. Nonspecific binding was defined
using 1 µM 8-OH-DPAT. Samples were incubated at 37 °C for
20 min and then filtered on Whatman GF/B glass microfiber
filters. The Kd value determined for 8-OH-DPAT was 8.8 nM.
3. Ra d ioliga n d Bin d in g Assa y a t Ra t Str ia ta l Mem -
br a n es D2 Recep tor s. Binding experiments were performed
according to Creese and co-workers35 with minor modifications.
Rats were killed by decapitation, the brain was quickly
removed, and the corpora striata was dissected. The corpora
striata (1.0 g) was homogenized with a Brinkman polytron
(setting 5 for 3 × 15 s) in 25 mL of 50 mM Tris buffer, pH 7.4.
The surnatant was discarded, and the pellet was washed once.
The final pellet was stored at -80 °C until used. Each tube
received in a final volume of 3 mL of incubation buffer (50
mM Tris, 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2,
and 5.7 mM ascorbic acid, pH 7.4), rat striatal membranes
suspension, and 0.2 nM [3H]spiroperidol. For competitive
inhibition experiments various concentrations of drugs studied
were incubated. Nonspecific binding was defined using 1 µM
haloperidol. Samples were incubated at 37 °C for 20 min and
then filtered on Whatman GF/B glass microfiber filters. The
Kd value determined for spiroperidol was 0.05 nM.
1
p h en yl)p ip er a zin e (cis-8d ). H NMR: δ 1.52-1.60 [m, 4H,
CH(CHHCHH)2], 1.97-2.05 [m, 4H, CH(CHHCHH)2], 2.34 [br
s, 1H, CHN(CH2)2], 2.64-2.70 [m, 5H, benzylic CH, CHN-
(CH2)2], 3.09 [br s, 4H, (CH2)2NAr], 3.79 and 3.85 (2 s, 6H, 2
CH3), 6.69-7.24 (m, 8H, aromatic). GC/MS: m/z 382 (M+ + 2,
4), 381 (M+ + 1, 26), 380 (M+, 80), 232 (23), 231 (100), 162
(26), 149 (28).
tr a n s-4-[4-(3-Met h oxyp h en yl)cycloh exyl]-1-(2-m et h -
oxyp h en yl)p ip er a zin e (tr a n s-8d ). 1H NMR: δ 1.38-1.58
[m, 4H, CH(CHHCHH)2], 1.97-2.11 [m, 4H, CH(CHHCHH)2],
2.42-2.49 [m, 2H, benzylic CH, CHN(CH2)2], 2.82 [br t, 4H,
CHN(CH2)2], 3.12 [br s, 4H, (CH2)2NAr], 3.78 and 3.85 (2 s,
6H, 2 CH3), 6.70-7.24 (m, 8H, aromatic). GC/MS: m/z 382
(M+ + 2, 3), 381 (M+ + 1, 18), 380 (M+, 68), 232 (18), 231 (100),
162 (21), 149 (23).
cis-4-[4-(3-Meth oxyp h en yl)cycloh exyl]-1-(1-n a p h th a le-
n yl)p ip er a zin e (cis-8e). 1H NMR: δ 1.63-1.75 [m, 4H, CH-
(CHHCHH)2], 1.99-2.12 [m, 4H, CH(CHHCHH)2], 2.45 [br s,
1H CHN(CH2)2], 2.65-2.82 [m, 5H, benzylic CH, CHN(CH2)2],
3.17 [br s, 4H, (CH2)2NAr], 3.80 (s, 3H, CH3), 6.70-8.20 (m,
11H, aromatic). GC/MS: m/z 402 (M+ + 2, 3), 401 (M+ + 1,
19), 400 (M+, 63), 252 (20), 251 (100).
tr a n s-4-[4-(3-Met h oxyp h en yl)cycloh exyl]-1-(1-n a p h -
th a len yl)p ip er a zin e (tr a n s-8e). 1H NMR: δ 1.48-1.5 [m,
4H, CH(CHHCHH)2], 2.00-2.10 [m, 4H, CH(CHHCHH)2],
2.45-2.55 [m, 2H, benzylic CH, CHN(CH2)2], 2.92 [br s, 4H,
CHN(CH2)2], 3.18 [br s, 4H, (CH2)2NAr], 3.80 (s, 3H, CH3),
6.71-8.22 (m, 11H, aromatic). GC/MS: m/z 402 (M+ + 2, 3),
401 (M+ + 1, 18), 400 (M+, 58), 252 (19), 251 (100).
cis-4-[4-(2-Meth oxyp h en yl)cycloh exyl]-1-(2-p yr id in yl)-
p ip er a zin e (cis-9a ). 1H NMR: δ 1.52-1.62 [m, 4H, CH-
(CHHCHH)2], 1.83-2.10 [m, 4H, CH(CHHCHH)2], 2.28 [br s,
1H, CHN(CH2)2], 2.61 [br t, 4H, CHN(CH2)2], 3.04-3.14 (m,
1H, benzylic CH), 3.54 [br s, 4H, (CH2)2NAr], 3.80 (s, 3H, CH3),
6.57-7.49 (m, 7H, aromatic), 8.17-8.19 (m, 1H, aromatic Nd
CH). GC/MS: m/z 353 (M+ + 2, 1), 352 (M+ + 1, 4), 351 (M+,
17), 257 (34), 244 (56), 232 (80), 188 (39), 134 (33), 121 (59),
107 (100).
tr a n s-4-[4-(2-Met h oxyp h en yl)cycloh exyl]-1-(2-p yr id i-
n yl)p ip er a zin e (tr a n s-9a ). 1H NMR: δ 1.44-1.56 [m, 4H,
CH(CHHCHH)2], 1.90-2.07 [m, 4H, CH(CHHCHH)2], 2.47 [br
t, 1H, CHN(CH2)2], 2.76 [br t, 4H, CHN(CH2)2], 2.87-2.92 (m,
1H, benzylic CH), 3.58 [br t, 4H, (CH2)2NAr], 3.80 (s, 3H, CH3),
6.58-7.49 (m, 7H, aromatic), 8.17-8.19 (m, 1H, aromatic Nd
CH). GC/MS: m/z 353 (M+ + 2, 1), 352 (M+ + 1, 5), 351 (M+,
19), 257 (30), 244 (48), 232 (71), 230 (32), 188 (27), 134 (25),
121 (55), 107 (100).
cis-4-[4-(4-Meth oxyp h en yl)cycloh exyl]-1-(2-p yr id in yl)-
p ip er a zin e (cis-10a ). 1H NMR 1.51-1.63 [m, 4H, CH-
(CHHCHH)2], 1.90-2.00 [m, 4H, CH(CHHCHH)2], 2.29 [br s,
1H, CHN(CH2)2], 2.58-2.66 [m, 5H, benzylic CH, CHN(CH2)2],
3.53 [br t, 4H, (CH2)2NAr], 3.77 (s, 3H, CH3), 6.57-7.48 (m,
7H, aromatic), 8.17-8.19 (m, 1H, aromatic NdCH). GC/MS:
m/z 353 (M+ + 2, 1), 352 (M+ + 1, 4), 351 (M+, 15), 257 (31),
244 (39), 232 (60), 188 (30), 134 (45), 121 (45), 107 (100).
tr a n s-4-[4-(4-Met h oxyp h en yl)cycloh exyl]-1-(2-p yr id i-
1
n yl)p ip er a zin e (tr a n s-10a ). H NMR: δ 1.39-1.49 [m, 4H,
CH(CHHCHH)2], 1.94-2.15 [m, 4H, CH(CHHCHH)2], 2.38-
2.42 [m, 2H, benzylic CH, CHN(CH2)2], 2.72 [br t, 4H, CHN-
(CH2)2], 3.55 [br t, 4H, (CH2)2NAr], 3.76 (s, 3H, CH3), 6.58-
7.48 (m, 7H, aromatic), 8.16-8.18 (m, 1H, aromatic NdCH).
GC/MS: m/z 353 (M+ + 2, 1), 352 (M+ + 1, 4), 351 (M+, 15),
257 (29), 244 (34), 232 (51), 230 (21), 134 (30), 121 (42), 107
(100).
P a r tition Coefficien t. The experimental partition coef-
ficient of trans-8a was measured with the shake-flask method
as described in the literature.33 1-Octanol saturated with water
buffer and water buffer (50 mM sodium phosphate buffer, pH
7.4) saturated with 1-octanol were used. The sample (8-17
mg, free base) was shaken overnight with a mixture of
1-octanol (4 mL) and water buffer (4 mL). After centrifugation
(3000 rpm × 10 min) of the mixture, the two layers were
separated and the concentration of the partitioned substance
4. Ra d ioliga n d Bin d in g Assa y a t Ra t Cor tica l Mem -
br a n es r1-a d r en ocep tor s. Binding experiments were per-
formed according to Glossman and Hornung36 with minor
modifications. Rats were killed by decapitation, the brain was

5-HT1A Receptor Ligands
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 25 4441
quickly removed, and the cerebral cortex was dissected. The
cerebral cortex (1.0 g) was homogenized with a Brinkman
polytron (setting 5 for 3 × 15 s) in 25 mL of buffer (50 mM
Tris, 0.1 mM PMSF, pH 7.4). The homogenated was centri-
fuged at 1000g for 15 min at 4 °C. The surnatant was recovered
and centrifuged at 50 000g for 30 min at 4 °C. The final pellet
was stored at -80 °C until used. Each tube received in a final
volume 1 mL of 50 mM Tris (pH 7.4) rat cerebral cortical
membranes suspension and 1 nM [3H]prazosin. For competi-
tive inhibition experiments various concentrations of drugs
studied were incubated. Nonspecific binding was defined using
10 µM phentolamine. Samples were incubated at 25 °C for 50
min and then filtered on Whatman GF/B glass microfiber
filters. The filters were presoaked for 50 min in Tris-HCl-
polyethylenimine 0.5%. The Kd value determined for prazosin
was 0.5 nM.
8. In Vivo Activity a t P ostsyn a p tic 5-HT1A Recep tor s.
The in vivo activity on postsynaptic 5-HT1A receptors was
evaluated as an induction of fore-paw treading in rats.28 On
the day of the experiment, rats were placed singly in clear
plastic boxes, 10-15 min before intravenous or subcutaneous
injection of test compounds. Groups of four to eight rats per
dose of test compounds were used. Only the major component
of the 5-HT1A syndrome was evaluated by an observer “blind”
to drug pretreatments: the fore-paw treading (FT). Observa-
tion sessions of 30 s began 3 min after treatment and were
repeated every 3 min over a period of 15 min (five observation
sessions). The appearance of fore-paw treading was noted, and
its intensity was scored using the following ranked intensity
scale: 0 ) absent; 1 ) equivocal; 2 ) present; 3 ) intense.
The maximal cumulative score attainable was 15 per rat.
9. Sta tistica l An a lysis. The inhibition curves on the
different binding sites of the compounds reported in Table 1
were analyzed by nonlinear curve fitting utilizing the Graph-
Pad Prism program.
5. Ra d ioliga n d Bin d in g Assa y a t Hu m a n Clon ed 5HT1A
Ser oton er gic Recep tor s. Genomic clone G-21 coding for the
human 5HT1A serotonergic receptor is stably transfected in a
human cell line (HeLa).37 HeLa cells were grown as monolayers
in Dulbecco’s modified Eagle’s medium (DMEM), supple-
mented with 10% fetal calf serum and gentamicin (100 µg/
mL) amd 7% CO2 at 37 °C. Cells were detached from the
growth flask at 95% confluence by a cell scraper and were lised
in ice-cold Tris 5 mM and EDTA 5 mM buffer (pH 7.4).
Homogenates were centrifuged at 40000g for 20 min, and
pellets were resuspended in a small volume of ice-cold Tris 5
mM and EDTA 5 mM buffer (pH 7.4) and immediately frozen
and stored at -70 °C until use. On the experimental day, cell
membranes were resuspended in binding buffer 50 mM Tris
(pH 7.4), 2.5 mM MgCl2, and 10 µM pargiline.38 Membranes
were incubated in a final volume of 1 mL for 30 min at 30 °C
with 1.2 nM [3H]8-OH-DPAT, in the absence or presence of
competing drugs; nonspecific binding was determined in the
presence of 10 µM 5-HT. The incubation was stopped by
addition of ice-cold Tris buffer and rapid filtration through
0.2% polyethyleneimine pretreated Schleicher & Schuell GF52
filters.
The inhibition curves on the different binding sites of
compounds reported in Table 3 were analyzed by nonlinear
curve fitting of the logistic equation according to the method
reported by De Lean et al.,41 utilizing the ALLFIT program
(from N.I.H.). The IC50 values and pseudo-Hill slope coef-
ficients were estimated by the program.
The value for the inhibition constant, Ki, was calculated by
using the Cheng-Prusoff equation.42
Stimulation of [35S]GTPγS binding induced by the com-
pounds tested was expressed as the percentage increase in
binding above the basal value because the maximal stimula-
tion observed with 5-HT was 100%. The concentration-
response curves of the agonistic activity were analyzed by
ALLFIT as reported above. The maximal percent stimulation
of [35S]GTPγS binding (Emax) achieved for each compound and
the concentration required to obtain 50% of Emax (pD2
-log EC50 value) were evaluated.
)
In in vivo models, the sigmoidal dose-response curves were
analyzed by the nonlinear curve fitting of the logistic equation
as reported above. Emax was always considered to be 100%.
The extrapolated doses corresponding to 50% Emax were also
evaluated.
6. Ra d ioliga n d Bin d in g Assa y a t Hu m a n Clon ed r1-
Ad r en ocep tor s. Binding to cloned human R1-adrenoceptor
subtypes was performed in membranes from Chinese hamster
ovary (CHO) cells transfected by electroporation with DNA
expressing the gene encoding each R1-adrenoceptor subtype.
Cloning and stable expression of the human R1-adrenoceptor
gene was performed as previously described.39 CHO cell
membranes were incubated in 50 mM Tris, pH 7.4, with 0.2
nM [3H]prazosin in a final volume of 1.0 mL for 30 min at 25
°C in the absence or presence of competing drugs (1 pM to 10
µM). Nonspecific binding was determined in the presence of
10 µM phentolamine. The incubation was stopped by addition
of ice-cold Tris buffer and rapid filtration through 0.2%
polyethyleneimine pretreated Whatman GF/B or Schleicher
& Schuell GF52 filters.
Ack n ow led gm en t. This study was supported by
Research Grant No. 9903108895-005 from Universita`
degli Studi di Bari and MURST (Italy) for the scientific
program in CO7X field (2000-2002).
Su p p or tin g In for m a tion Ava ila ble: Two figures show-
ing the induction of fore-paw treading in rats. This mater-
ial is available free of charge via the Internet at http://
pubs.acs.org.
7. Stim u la tion of [35S]GTP γS bin d in g a t Clon ed 5-HT1A
Recep tor s. The effects of the different compounds tested on
[35S]GTPγS binding were evaluated according to the method
of Stanton and Beer40 with minor modifications. On the
experimental day, cell membranes from HeLa cells transfected
with human cloned 5-HT1A receptors were resuspended in
buffer containing 20 mM HEPES, 3 mM MgCl2, and 120 mM
NaCl (pH 7.4). The membranes were incubated with 30 µM
GDP and decreasing concentrations (from 100 µM to 0.1 nM)
of test drugs or 5-HT (reference curve) for 20 min at 30 °C in
a final volume of about 0.5 mL. Samples were then transferred
to ice, with [35S]GTPγS (200-250 pM) added, and then
incubated for further 30 min at 30 °C. Nonspecific binding was
determined in the presence of 10 µM GTPγS. The incubation
was stopped by addition of ice-cold HEPES buffer and rapid
filtration on Schleicher & Schuell GF52 filters, using a Brandel
cell harvester. The filters were washed three times with a total
of 5 mL of the same buffer.
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