Active conformation of some arylpiperazine postsynaptic 5-HT1A receptor antagonists

Article abstract of DOI:10.1016/S0223-5234(01)01312-5

The synthesis and pharmacological properties of novel conformationally restricted arylpiperazine (2b-4b) or 1,2,3,4-tetrahydroisoquinoline (5b and 6b) derivatives of the known, flexible 5-HT_1A receptor ligands 2a-6a (K_i=0.95-7 nM) with different intrinsic activities are reported. Replacement of a tetramethylene chain with a 1e,4e-disubstituted cyclohexane ring in the structure of flexible ligands resulted in insignificant diminution of the 5-HT_1A receptor affinity in the case of 2b-4b (K_i=15-52 nM), whereas derivatives 5b and 6b were practically inactive (K_i>1354 nM). The results of in vivo behavioural tests showed that 2a and 3a acted as postsynaptic 5-HT_1A receptor partial agonists. Like the flexible 4a, the new rigid compounds 2b-4b showed features of postsynaptic 5-HT_1A receptor antagonists. Since all possible conformations of the constrained compounds belong to an extended family - as indicated by molecular modelling studies - our hypothesis that such conformations are responsible for the blockade of postsynaptic 5-HT_1A receptors has been confirmed. Determination of regions explored by terminal amide, or imide and hydrocarbon groups of the restricted compounds as well as the results of in vitro and in vivo studies allowed us to discuss the bioactive conformations of flexible ligands.

Full text of DOI:10.1016/S0223-5234(01)01312-5

Eur. J. Med. Chem. 37 (2002) 273–283
www.elsevier.com/locate/ejmech
Original article
Active conformation of some arylpiperazine postsynaptic 5-HT1A
receptor antagonists
Maria Halina Paluchowska a,*, Andrzej Jacek Bojarski a, Sijka Charakchieva-Minol a,
Anna Wesołowska b
a Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences, 12 Sme˛tna Street, 31-343 Krako´w, Poland
b Department of New Drug Research, Institute of Pharmacology, Polish Academy of Sciences, 12 Sme˛tna Street, 31-343 Krako´w, Poland
Received 7 July 2001; received in revised form 6 November 2001; accepted 6 November 2001
Abstract
The synthesis and pharmacological properties of novel conformationally restricted arylpiperazine (2b–4b) or 1,2,3,4-tetrahy-
droisoquinoline (5b and 6b) derivatives of the known, flexible 5-HT1A receptor ligands 2a–6a (Ki=0.95–7 nM) with different
intrinsic activities are reported. Replacement of a tetramethylene chain with a 1e,4e-disubstituted cyclohexane ring in the structure
of flexible ligands resulted in insignificant diminution of the 5-HT1A receptor affinity in the case of 2b–4b (Ki=15–52 nM),
whereas derivatives 5b and 6b were practically inactive (Ki\1354 nM). The results of in vivo behavioural tests showed that 2a
and 3a acted as postsynaptic 5-HT1A receptor partial agonists. Like the flexible 4a, the new rigid compounds 2b–4b showed
features of postsynaptic 5-HT1A receptor antagonists. Since all possible conformations of the constrained compounds belong to
an extended family—as indicated by molecular modelling studies—our hypothesis that such conformations are responsible for the
blockade of postsynaptic 5-HT1A receptors has been confirmed. Determination of regions explored by terminal amide, or imide
and hydrocarbon groups of the restricted compounds as well as the results of in vitro and in vivo studies allowed us to discuss
´
the bioactive conformations of flexible ligands. © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: postsynaptic 5-HT1A antagonists; rigid analogs of arylpiperazine 5-HT1A receptor ligands; conformational analysis
papers [1–6] and reviews [7]. Aliphatic chain modifica-
tions have also been extensively studied. At first, the
influence of the aliphatic chain length of some simple
4-alkyl-1-arylpiperazines on their 5-HT1A receptor ac-
tivity has been discussed [8,9]. Additionally, in a num-
ber of series of arylpiperazine ligands, the aliphatic
chain length has been optimized with respect to the
relative position of the above mentioned basic pharma-
cophoric groups [6]. Moreover, groups other than
methylene, i.e. heteroatoms (O, NH, S) [10–13], car-
bonyl or the amide fragment [14–20], and multiple
bonds [21] have also been introduced to the spacer.
Although some of these modifications increase the
rigidity of a link chain, the whole molecule still remains
flexible, so a prediction of its conformation within the
receptor pocket is highly speculative. In other classes of
5-HT1A receptor ligands (ergolines, tryptamines,
aminotetralins with the exception of aryloxyalky-
lamines) such problem does not exist, and the confor-
mationally constrained model ligands have already
been described [22–25].
1. Introduction
Arylpiperazines are of great importance to many
different biological targets, especially central nervous
system receptors. In the case of serotonin (5-HT) recep-
tors, compounds containing this moiety represent the
biggest and thoroughly examined class of 5-HT1A re-
ceptor ligands. However, simple arylpiperazines are
classified as non-selective 5-HT receptors (and other
ones) ligands, 4-substituted derivatives show good se-
lectivity and high affinity for 5-HT1A receptors. The
majority of them contain a flexible aliphatic chain of
different length, which connects the arylpiperazine frag-
ment with the second terminal pharmacophoric group.
Structural modifications within long-chain arylpiperazi-
nes (LCAPs) occur mainly at the two opposite ends of
a molecule and have been described in many original
* Correspondence and reprints.
E-mail address: paluchm@if-pan.krakow.pl (M.H. Paluchowska).
´
0223-5234/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0223-5234(01)01312-5

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M.H. Paluchowska et al. / European Journal of Medicinal Chemistry 37 (2002) 273–283
Among LCAPs there are compounds which show
replacing the aliphatic chain with a 1,4-substituted cy-
clohexane ring. Our earlier studies [31] allowed us to
determine a bioactive conformation of 1a, a well-
known antagonist of postsynaptic 5-HT1A receptors,
using this constrained analogues approach. These fin-
dings encouraged us to extend our studies to other
5-HT1A receptor ligands.
At present we wish to report a synthesis of conforma-
tionally restricted derivatives (2b–6b, Table 1) of the
five, previously described, active in vivo 5-HT1A recep-
tor ligands (2a–6a). We chose two postsynaptic antago-
nists (2a and 4a) [32,33] and three partial agonists (3a,
different 5-HT1A receptor functional activities, i.e. ago-
nistic, partial agonistic or antagonistic one. The most
frequently investigated member of the LCAPs is bus-
pirone (Buspar®, Bristol–Myers Squibb), used in the
treatment of anxiety (a second generation anxiolytic).
Arylpiperazine 5-HT1A receptor agents such as
gepirone, ipsapirone, tandospirone, flesinoxan and
many others, in various phases of clinical studies are
regarded as potential drugs in the therapy of anxiety,
depression, Alzheimer’s disease, learning and memory
disfunctions etc. [26,27]. Moreover, several other
agents, such as, e.g. NAN-190 (1a), WAY 100635 or
MP 3022, are frequently used as pharmacological tools
[28–30]. It is noteworthy that the majority of the above
mentioned compounds contain a flexible, 4-membered
aliphatic spacer. Hence our general concept was to
generate rigid analogues of well-known 5-HT1A recep-
tor agents with limited conformational freedom by
5a and 6a) [15,34,35] which contain
a
4-(2-
methoxyphenyl)-1-piperazinyl (oMPP) fragment (2a, 3a
and 4a) or a 1,2,3,4-tetrahydroisoquinolin-2-yl (THIQ)
moiety (5a and 6a)—a tool system recently used for
studying the ligand–5-HT1A receptor interactions [35].
The aim of the present study was to determine the
influence of conformational constraints in biologically
Table 1
Structure and binding data on the 5-HT1A receptors of the investigated compounds

M.H. Paluchowska et al. / European Journal of Medicinal Chemistry 37 (2002) 273–283
275
Fig. 1. Reagents and conditions: (a) Ph3P, NBS, Et3N, CH2Cl2, 0–25 °C; (b) BOP, Et3N, CH3CN, room temperature; (c) 20% K2CO3–CHCl3,
room temperature; (d) xylene, reflux; (e) pyridine, reflux.
active structures on their 5-HT1A receptor binding and
intrinsic activity. On the basis of pharmacological and
conformational analysis results we were able to discuss
bioactive conformations of the investigated compounds.
of the appropriate substituted cyclohexylamine with
succinic or 3,3-tetramethyleneglutaric anhydride. The
structures of all those new compounds were elucidated
from their analytical and spectroscopic data. 2D
1H-NMR spectra and decoupling experiments allowed
us to calculate the appropriate coupling constants in
the cyclohexane ring, and to assign a diequatorial
1e,4e-chair conformation for compounds 2b–6b. For
pharmacological assays, the investigated compounds
were converted into hydrochloride or fumarate salts.
2. Chemistry
Synthetic routes to the target compounds, i.e. flexible
2a and 3a, as well as the new constrained
arylpiperazines 2b–6b, prepared in this work, are
showed in Fig. 1. The synthesis of starting amines had
been previously described [1,31] (see Section 6). Amides
2a and 2b were generated in high yields under mild
conditions from 1-adamantanecarboxylic acid and the
corresponding amine in the presence of the equivalent
amounts of triphenylphosphine, N-bromosuccinimide
and triethylamine. Amide 5b was obtained by a direct
reaction between 4-[2-(1,2,3,4-tetrahydroisoquinolinyl)]-
cyclohexylamine and 1-adamantanecarboxylic acid in
the presence of benzotriazol-1-yloxy-tris(dimethyl-
3. Pharmacological results
3.1. In 6itro experiments
The affinity at 5-HT1A receptors for all the newly
synthesized compounds 2b–6b, as well as for the pre-
viously described reference arylpiperazines 2a [32] and
3a [15] was determined by standard competitive dis-
placement assays using rat brain hippocampus with
[3H]-8-OH-DPAT, a 5-HT1A receptor agonist, as a
competitive ligand. The results of those assays and the
previously published data from our laboratory on com-
pounds 1a, 1b and 4a–6a are displayed in Table 1.
amino)phosphonium
hexafluorophosphate
(BOP).
Compounds 3a and 3b were prepared by simple acyla-
tion of the respective amine in an alkaline medium.
Imides 4b and 6b were yielded upon a prolonged reflux

276
M.H. Paluchowska et al. / European Journal of Medicinal Chemistry 37 (2002) 273–283
Flexible arylpiperazines 2a and 3a showed a high
3.2.1. Lower lip retraction in rats
nanomolar affinity (Ki=7 and 4 nM, respectively);
however, the Ki values for those compounds reported
by Glennon and coworkers [15,32] were slightly lower
(0.4 and 0.6 nM, respectively). Rigid arylpiperazines
2b–4b demonstrated a good 5-HT1A receptor affinity
(Ki=15–52 nM), whereas tetrahydroisoquinoline
derivatives 5b and 6b were practically inactive (Ki\
1354 nM).
Compounds 2a and 3a, given alone in doses of 8–16
mg kg−1, induced LLR in rats, the maximum score
being 30 and 67%, respectively, at the highest doses
used. The remaining compounds 2b (8–16 mg kg−1),
3b (4–8 mg kg−1), the previously described 4a (4–16
mg kg−1) [33] and 4b (0.125–0.5 mg kg−1), given
alone, showed no activity in that test (Table 2A). At the
same time, 2a, 2b, 3a (8–16 mg kg−1), 3b (4–8 mg
kg−1), the previously investigated 4a (4–16 mg kg−1
)
3.2. In 6i6o experiments
[33] and 4b (0.125–0.5 mg kg−1) inhibited the LLR
induced by 8-OH-DPAT in rats in a dose-dependent
manner. At the highest doses used, those compounds
attenuated the effect of 8-OH-DPAT by 59 (3a)–89%
(4a) (Table 2B).
Compounds which showed a 5-HT1A receptor affinity
up to Ki=52 nM were tested in vivo. To determine
postsynaptic 5-HT1A agonistic effects of the tested com-
pounds, their ability to induce lower lip retraction
(LLR) in rats and behavioural syndrome, i.e. flat body
posture (FBP) and forepaw treading (FT), in reser-
pinized rats was tested [36,37]. The ability of the inves-
tigated compounds to inhibit those symptoms produced
by the 8-hydroxy-2-(di-n-propylamino)tetralin hydro-
bromide (8-OH-DPAT) was regarded as a postsynaptic
antagonistic activity.
3.2.2. Beha6ioural syndrome in reserpinized rats
Of the tested compounds, 2a (8–16 mg kg−1) and 3a
(4–8 mg kg−1) administered alone induced FBP (but
not FT) in reserpine-pretreated rats, the maximum
score being 62 and 36%, respectively, after the highest
doses used. Compounds 2b (8–16 mg kg−1), 3b (4–8
mg kg−1), the previously investigated 4a (4–16 mg
kg−1) [33] and 4b (0.125–0.5 mg kg−1) did not evoke
changes in the behaviour in reserpinized rats (Table
3A). The 8-OH-DPAT-induced FBP and FT were dose-
dependently reduced by 2a (8–16 mg kg−1), 3a, 3b
(4–8 mg kg−1), the previously described 4a (4–16 mg
kg−1) [33] and 4b (0.125–0.5 mg kg−1). An almost
complete blockade of the behavioural symptoms in-
duced by 8-OH-DPAT was observed after administra-
tion of 4a and 4b at the highest doses (Table 3B).
Table 2
Induction of lower lip retraction (LLR) by the investigated com-
pounds (A) and their effect on the 8-OH-DPAT-induced LLR (B) in
rats
Treatment
Dose (mg kg−1
)
Mean9S.E.M. LLR score
A
B
Vehicle
2a
0.190.1
0.690.4
0.990.2 b
2.890.1
8
16
1.190.2 a
0.690.2 a
4. Molecular modelling and discussion
Vehicle
2b
0.190.1
0.090.0
0.090.0
2.890.1
8
16
1.590.1 a
0.990.3 a
In order to better understand structural requirements
for a compound to interact with the 5-HT1A receptor,
in the present paper we have described new, con-
strained analogues of five known 5-HT1A agents. The
synthesis of these compounds extends the group of
5-HT1A receptor ligands which may be used for stu-
dying ligand–receptor interactions. Introduction of
a 1,4-disubstituted cyclohexane ring in place of the
flexible and stereochemically difficult to define te-
tramethylene chain significantly limits the spatial ar-
rangement of pharmacophoric fragments in ligands;
however, some conformational freedom still exists (Fig.
2). In Fig. 3, possible rotations in the molecule were
marked by the respective dihedral angles ~1, ~2, ~3 or ~4.
It is generally accepted that bioactive conformation of
the oMPP fragment is perpendicular; therefore, like in
our previous paper, we fixed angle ~1=180°. The re-
maining rotations were studied by a semi-empirical
AM1 method, and the resultant energy profiles are
presented in Fig. 3. All possible minimum energy
Vehicle
3a
0.190.1
2.790.1
8
16
1.390.1 c
2.090.3 c
1.590.2 a
1.190.2 a
Vehicle
3b
0.190.1
0.290.1
0.190.1
2.990.1
4
8
1.190.2 a
0.690.3 a
Vehicle
0.190.1
0.190.1
0.190.1
0.090.0
2.790.1
4a d
4
8
16
1.690.1 a
1.190.2 a
0.390.1 b
Vehicle
4b
0.190.1
0.190.1
0.090.0
0.190.1
2.890.1
0.125
0.25
0.5
1.890.8 e
1.290.2 a
0.790.1 b
a PB0.01 versus vehicle+8-OH-DPAT.
b PB0.05 versus vehicle.
c PB0.01 versus vehicle.
d Data from Ref. [33].
e PB0.05 versus vehicle+8-OH-DPAT.

M.H. Paluchowska et al. / European Journal of Medicinal Chemistry 37 (2002) 273–283
277
Table 3
Induction of behavioural syndrome by the investigated compounds (A) and their effect on the 8-OH-DPAT-induced behavioural syndrome (B)
in reserpine-pretreated rats
Treatment
Dose (mg kg−1
)
Mean9S.E.M. behavioural score
A
B
Flat body posture
Forepaw treading
Flat body posture
Forepaw treading
Vehicle
2a
0.290.1
0.290.2
0.390.2
0.890.3
15.090.0
6.891.1 b
4.091.4 b
13.090.7
8.591.4 b
3.290.5 b
8
16
5.091.5 a
9.391.7 a
Vehicle
2b
0.290.1
0.090.0
0.090.0
0.290.2
0.090.0
0.090.0
15.090.0
12.291.4
11.491.5
13.090.7
11.890.7
13.690.7
8
16
Vehicle
3a
0.290.1
0.290.2
0.190.1
0.090.0
13.490.8
11.591.1
8.091.4 c
12.890.7
7.590.8 b
3.891.3 b
4
8
5.290.9 a
5.490.7 a
Vehicle
3b
0.290.1
0.190.1
0.190.1
0.290.2
0.190.1
0.090.0
13.490.8
12.490.4
6.091.1 b
12.890.7
6.691.0 b
2.890.7 b
4
8
Vehicle
0.290.1
0.290.2
0.190.1
0.190.1
0.290.1
0.190.1
0.190.1
0.190.1
15.090.0
11.891.3 c
2.391.0 b
0.090.0 b
12.391.0
8.391.5
4a d
4
8
16
6.891.6 c
1.390.7 b
Vehicle
4b
0.290.1
0.090.0
0.190.2
0.190.1
0.290.2
0.190.1
0.190.1
0.190.1
14.890.2
13.090.5
11.790.7 c
2.090.8 b
13.090.9
7.591.4 b
4.890.7 b
1.890.8 b
0.125
0.25
0.5
a PB0.01 versus vehicle.
b PB0.01 versus vehicle+8-OH-DPAT.
c PB0.05 versus vehicle+8-OH-DPAT.
d Data from Ref. [33].
(compounds 1a–4a) results in a few-fold diminution of
affinity (compounds 1b–4b). Nevertheless, the con-
strained analogues are still good 5-HT1A receptor li-
gands. The same modification of ligands with the THIQ
fragment (compounds 5a and 6a) leads to an almost
complete loss of the 5-HT1A receptor affinity of com-
pounds 5b and 6b. Their non-constrained analogues 5a
and 6a showed a high 5-HT1A receptor affinity, which
can be explained by a different mode of complex li-
gand–receptor formation. A comparison between the
5-HT1A receptor affinities of arylpiperazine 2b (Ki=52
nM) and tetrahydroisoquinoline 5b (Ki=1354 nM)
suggests that such a big difference in their binding
properties may stem from the spatial arrangement of
the aromatic ring of heterocyclic amine moieties. Super-
imposition of both these amine fragments using a com-
mon basic nitrogen atom reveals that planes of their
benzene rings are perpendicular (Fig. 3). Compound 2b
with a 1-adamantyl group shows binding affinity com-
parable to that of compound 3b, which contains a
cyclohexyl fragment. As indicated by molecular mo-
delling, regions accessible to both adamantyl and cyclo-
conformations were constructed and optimized on the
basis of those calculations (see Table 4).
Although the number of predominant conformations
of compounds 2b and 3b are 16 and 24, respectively, all
of them, as well as these of compounds 4b–6b, belong
to a family of extended conformations. Since neither
differences in the energy of individual conformers
(1.96–4.27 kcal mol−1) nor energy barriers of the
analysed rotatable bonds (up to 4 kcal mol−1) are very
high, we assumed that each of the presented conforma-
tions of the investigated compounds may interact with
the 5-HT1A receptor. Hence the entire group of low-ener-
gy conformers for each investigated compound was
overlapped by oMPP or THIQ fragments. Finally, we
summed up the van der Waals’ volumes of terminal
hydrocarbons and amide/imide moieties (Table 4); in
Fig. 3 these regions are marked in cyan and magenta,
respectively. Thus we have determined the maximum
possible space explored by the pharmacophores men-
tioned above.
The 5-HT1A receptor binding data presented in Table
1 indicate that introduction of structural rigidity to the
aliphatic spacer of ligands containing the oMPP moiety
hexyl substituents are almost the same (341.1 and 320.1
3
,
A , respectively), though their own volumes differ

278
M.H. Paluchowska et al. / European Journal of Medicinal Chemistry 37 (2002) 273–283
3
,
significantly (135 and 83 A , respectively). Thus we may
state, that inspite of a large hydrophobic receptor
pocket in which an adamantyl group may be placed,
the cyclohexyl substituent is big enough to stabilize the
ligand–5-HT1A receptor complex. On the other hand,
rigid imide 4b with the smallest hydrophobic part of the
3
,
molecule represented by an ethylene fragment (78 A )
demonstrates the highest 5-HT1A receptor affinity. It
may be concluded that in the presently described series
of constrained arylpiperazines the terminal hydropho-
bic substituent slightly disturbs formation of the li-
gand–5-HT1A receptor complex. Although the calcu-
lated volumes accessible to an amide or an imide part
of the molecule are practically the same (Table 4), the
results of binding studies suggest that the imide group
is preferred to the amide one at the 5-HT1A receptor
site probably due to additional electrostatic interactions
involving the second carbonyl group (compare 1b and
4b vs. 2b and 3b, Table 1).
The described results of in vivo experiments clearly
show that the investigated arylpiperazine derivatives
have a different intrinsic activity at postsynaptic 5-
HT1A receptors. Flexible compounds 2a and 3a may be
classified as partial agonists of these receptors. These
data are somewhat at variance with the findings of
Fig. 3. Global energy minima of compounds 2b and 4b and a
superimposition mode of the THIQ group (in green) with the oMPP
fragment. The dihedral angles ~1–~4 are defined, and the respective
rotation energy profiles are shown. Regions penetrated by an amide
(or imide) group and a terminal hydrophobic part of a molecule
during rotations of ~2, ~3 and ~4 are marked in magenta and cyan,
respectively.
Raghupathi et al. [32] who pointed to an antagonistic
activity of 2a in a 5-HT1A postsynaptic coupled adeny-
lyl cyclase assay (rat hippocampal membranes). The
reason for such a discrepancy is difficult to explain,
though certain differences between our experimental
conditions and those provided by Raghupathi et al. [32]
consisting in the use of substantially different func-
tional models (in vivo tests vs. in vitro assays), should
be taken into account. On the other hand, our results
on 3a are coherent with the previous findings of
Fig. 2. Results of a conformational analysis (a random search proce-
dure) applied to the flexible compound 2a (A) and its constrained
analogue 2b (B). In both cases conformers were superimposed using
oMPP fragments. For simplification, hydrogen atoms have been
omitted and full structures are shown only for a global energy
minima. Local energy minimum conformers are represented solely by
NꢀCOꢀC fragments.

M.H. Paluchowska et al. / European Journal of Medicinal Chemistry 37 (2002) 273–283
279
5. Conclusions
El-Bermawy et al. [15] which showed that 3a acted as a
postsynaptic 5-HT1A receptor partial agonist in an in
vitro functional assay (i.e. the forskolin-stimulated
adenylate cyclase, using rat hippocampal membrane
homogenates). The previously described flexible com-
pound 4a exhibited features of a postsynaptic 5-HT1A
antagonist [33] in in vivo behavioural tests.
Summing up, we have found that all the three new
rigid oMPP derivatives (2b–4b) are postsynaptic 5-
HT1A receptor antagonists, whereas their flexible ana-
logues have features of partial agonists 2a and 3a or
antagonist 4a of these receptors. On the basis of present
studies it may be anticipated that the imide fragment of
antagonist 4a can be found within the volumes defined
in Fig. 3, whereas the terminal amide and hydrocarbon
groups of partial agonists 2a and 3a are located outside
these regions. These findings may create a basis for
further receptor modelling and docking studies with
other flexible 5-HT1A ligands of the arylpiperazine type.
Moreover, on the grounds of conformational searches
we have managed to confirm our hypothesis that ex-
tended conformations are responsible for the blockade
of postsynaptic 5-HT1A receptors.
Restriction of conformational freedom in the struc-
ture of partial agonists 2a and 3a affected their func-
tional profiles. Rigid analogues 2b and 3b demonstrated
a postsynaptic 5-HT1A antagonistic activity. Contrari-
wise, in the case of the postsynaptic 5-HT1A receptor
antagonist 4a, the introduced structural modification
did not change the intrinsic activity of the constrained
compound 4b. In our previous study, a similar struc-
tural modification of 1a, a postsynaptic 5-HT1A recep-
tor antagonist, afforded its rigid analogue 1b which
represented very well a bioactive conformation of the
parent compound [31]. To compare directly in vivo
activities of both pairs of compounds 1a versus 1b and
4a versus 4b, we also present their ED50 values for
inhibition of the 8-OH-DPAT-induced effects (Table 5).
The ED50 values for 1a were somewhat lower than
those determined for 1b, but were of the same order of
magnitude [31,33]. In contrast, the ED50 values for 4a
[33] were distinctly higher than those for 4b. Moreover,
the ED50 values for the latter were extremely low; thus
rigid compound 4b turned out to be a very interesting
new 5-HT1A antagonist with high in vitro and in vivo
potency.
6. Experimental
6.1. Chemistry
Melting points were determined using an Electrother-
mal Digital Melting Point IA9000 apparatus and are
uncorrected. 1H-NMR spectra were recorded on a
Brucker AMX 500 (500 MHz), Varian Mercury 300
(300 MHz) or Varian EM-360L (60 MHz) spectrome-
ters, in CDCl3 solutions. The chemical shifts values (l)
Table 4
Conformation analysis results for compounds 2b–6b and volumes of amide and hydrophobic regions
b
Compound Number of rotatable Number of
DE (kcal
Volume of amide or imide region
Volume of hydrophobic region
3
3
bonds a
conformers
mol−1
)
(A )
(A )
,
,
2b
3b
4b
5b
6b
3
3
2
3
2
16
24
4
12
6
4.04
4.27
1.96
4.12
2.15
121.6
126.9
123.1
106.9
112.2
341.1
320.1
78.1
337.6
272.3
a Excluding rotation in the oMPP fragment.
b Range of differences in the energy of individual conformers.
Table 5
Inhibition of the 8-OH-DPAT-induced effects by 1a, 1b, 4a and 4b
Effect
ED50 (mg kg−1
)
1a a
1b a
4a b
4b
Flat body posture
Forepaw treading
LLR
2.0 (1.2–3.2) c
2.5 (1.7–4.3)
1.7 (0.9–3.1)
6.3 (3.6–13.9)
2.3 (1.4–3.9)
7.5 (4.8–11.6)
5.4 (3.4–8.6)
7.0 (4.2–11.6)
6.0 (3.6–9.9)
0.28 (0.18–0.45)
0.20 (0.14–0.28)
0.24 (0.17–0.34)
a Data from Ref. [31].
b Data from Ref. [33].
c Confidence limits (95%) are given in parentheses.

280
M.H. Paluchowska et al. / European Journal of Medicinal Chemistry 37 (2002) 273–283
were expressed in ppm relative to Me4Si as internal
standard and the coupling constants J in Hertz (Hz).
When necessary, the signals were unambiguously as-
signed by 2D NMR (1H–1H) COSY technique or de-
coupled by irradiation of the NH signal frequency. The
spectral data for amines refer to their free bases. All
compounds were routinely checked by TLC using
Merck Kieselgel 60-F254 sheets. Column chromatogra-
phy separations were carried out on Merck Kieselgel 60
or aluminium oxide 90, neutral (70–230 mesh). Elemen-
tal analysis were performed in the Institute of Organic
Chemistry, Polish Academy of Sciences (Warsaw,
Poland), and were within 90.4% of the theoretical
values. The syntheses of 4-(4-aminobutyl)-1-(2-
methoxyphenyl)piperazine [1], 4-[4-(2-methoxyphenyl)-
1-piperazinyl]cyclohexylamine [31] and 4-[2-(1,2,3,4-
tetrahydroisoquinolinyl)]cyclohexylamine [31] had been
previously reported.
H-4), 3.09 (br s, 4H, piperazine 2CH2), 2.76 (app br t,
4H, piperazine 2CH2), 2.31 (td, J=11.6, 3.0, 1H, cyclo-
hexane axial H-1), 2.04 (cluster, 5H, cyclohexane equa-
torial H-3 and H-3% and adamantane 3CH), 1.98 (app
br d, 2H, cyclohexane H-2 and H-2%), 1.83 (d, J=2.6,
6H, adamantane H’s), 1.71 (q, J=12.2, 6H, adaman-
tane H’s), 1.43 (dddd, J=12.9, 12.8, 3.1, 3.0, 2H,
cyclohexane axial H-2 and H-2%), 1.14 (dddd, J=12.7,
12.6, 3.1, 2.9, 2H, cyclohexane axial H-3 and H-3%).
Complex 2b·2HCl: m.p. 289–291 °C (EtOH–Et2O).
Anal. C28H41N3O2·2HCl (C, H, N).
6.1.2. General procedure for the preparation of 3a and
3b
To a vigorously stirred solution of 4-(4-aminobutyl)-
1-(2-methoxyphenyl)piperazine or 4-[4-(2-methoxy-
phenyl)-1-piperazinyl]cyclohexylamine (2 mmol) in
CHCl3 (25 mL) and a 20% aq. solution of K2CO3 (25
mL), cyclohexanecarbonyl chloride (3 mmol) was
added dropwise. The reaction mixture was stirred at a
r.t. for 4 h. The organic layer was separated, washed
with water until neutral, and afterwards the product
was extracted with a 10% aq. solution of HCl. The
water phase was made alkaline, extracted with CHCl3
and dried (K2CO3). After evaporation of the solvent,
the residue was crystallized to give a pure product.
6.1.1. General procedure for the preparation of 2a and 2b
1-Adamantanecarboxylic acid (2 mmol) was dis-
solved in CH2Cl2 (5 mL) and triphenylphosphine (2
mmol) was added under stirring. After 5 min, N-bro-
mosuccinimide (2.1 mmol) was added in portions and
afterwards the mixture was stirred for 0.5 h at a room
temperature (r.t.). A solution of 4-(4-aminobutyl)-1-(2-
methoxyphenyl)piperazine or 4-[4-(2-methoxyphenyl)-1-
piperazinyl]cyclohexylamine (2 mmol) and Et3N (2.2
mmol) in CH2Cl2 (3 mL) was then added dropwise. The
reaction mixture was stirred for 3 h at a r.t. and left
overnight. Then, CHCl3 (20 mL) was added and the
solution was washed with a 20% aq. solution of NaOH
(10 mL), water (10 mL) and was dried over anhydrous
MgSO4. The inorganic precipitate was filtered off, the
solvents were evaporated and the amides were sepa-
rated by column chromatography using SiO2 and
CHCl3 followed by CHCl3–MeOH=49:1, as eluents.
6.1.2.1. 4 - [4 - (Cyclohexanecarboxamido)butyl] - 1 - (2-
methoxyphenyl)piperazine (3a). The title compound was
prepared by the general procedure in 80% yield as
colourless crystals: m.p. 128–129 °C (CHCl3–C6H14);
1H-NMR (60 MHz): l 7.0 (s, 4H), 6.2 (br s, 1H), 3.9 (s,
3H), 3.5–2.9 (m, 6H), 2.9–2.3 (m, 6H), 2.3–1.0 (clus-
ter, 15H). Complex 3a·0.5H4C4O4·1.5H2O: m.p. 143–
145 °C (C3H6O), lit. m.p. (for hydrochloride salt)
190–192 °C (EtOH–Et2O) [15]. Anal. C22H35N3-
O2·0.5H4C4O4·1.5H2O (C, H, N).
6.1.1.1.
4-[4-(1-Adamantanecarboxamido)butyl]-1-(2-
methoxyphenyl)piperazine (2a). The title compound was
6.1.2.2. trans - 4 - [4(Cyclohexanecarboxamido)cyclo -
hexyl]-1-(2-methoxyphenyl)piperazine (3b). The title
compound was prepared by the general procedure in
73% yield as colourless crystals: m.p. 237–239 °C
(CHCl3–MeOH); 1H-NMR (200 MHz): l 7.01–6.82
(m, 4H, ArH), 5.24 (d, J=8.2, 1H, NHCO), 3.85 (s,
3H, OCH3), 3.70 (m, decoupled leaves td, J=11.5, 4.1,
1H, cyclohexane axial H-4), 3.08 (br s, 4H, piperazine
2CH2), 2.75 (app br t, 4H, piperazine 2CH2), 2.29 (td,
J=11.5, 3.3, 1H, cyclohexane axial H-1), 2.06–1.94
(cluster, 6H, cyclohexane H’s), 1.84–1.76 (m, 4H, cy-
clohexane H’s), 1.66 (br s, 1H, cyclohexane H-1%), 1.47–
1.35 (m, 4H, cyclohexane H’s), 1.31–1.07 (cluster, 4H,
cyclohexane H’s). Complex 3b·2HCl·2.2H2O: m.p.
275–277 °C (C3H6O). Anal. C24H37N3O2·2HCl·2.2H2O
(C, H, N).
prepared by the general procedure in 70% yield as pale
1
yellow oil: H-NMR (60 MHz): l 7.0 (s, 4H), 5.9 (br s,
1H), 3.8 (s, 3H), 3.6–2.9 (m, 6H), 2.9–2.2 (m, 6H),
2.2–1.4 (cluster, 19H). Complex 2a·H4C4O4·H2O: m.p.
95–97 °C (C3H6O), lit. m.p. 112–117 °C (THF) [32].
Anal. C26H39N3O2·H4C4O4·H2O (C, H, N).
6.1.1.2. trans-4-[4-(1-Adamantanecarboxamido)cyclo-
hexyl]-1-(2-methoxyphenyl)piperazine (2b). The title
compound was prepared by the general procedure in
55% yield as colourless crystals: m.p. 210–212 °C
1
(CHCl3); H-NMR (500 MHz): l 7.02–6.90 (m, 3H,
ArH), 6.85 (dd, J=8.0, 1.2, 1H, ArH), 5.36 (d, J=8.0,
1H, NHCO), 3.86 (s, 3H, OCH3), 3.76–3.68 (m, decou-
pled leaves td, J=11.6, 4.0, 1H, cyclohexane axial

M.H. Paluchowska et al. / European Journal of Medicinal Chemistry 37 (2002) 273–283
281
6.1.3. trans-1-(2-Methoxyphenyl)-4-(4-succinimidocy-
clohexyl)piperazine (4b)
mL) were refluxed for 22 h. The solvent was removed
under reduced pressure and the residue was treated
with water and extracted with CHCl3. The extract was
dried over MgSO4, the solvent was evaporated and the
residue was purified by column chromatography (Al2O3
neutral, CHCl3–C6H14=1:1). The product was ob-
A
mixture of 4-[4-(2-methoxyphenyl)-1-pipera-
zinyl]cyclohexylamine (0.5 g, 1.7 mmol) and succinic
anhydride (0.19 g, 1.9 mmol) and xylene (20 mL) was
refluxed for 3 h. Then the solvent was evaporated under
reduced pressure and the residue was purified by
column chromatography (SiO2, CHCl3–MeOH=49:1)
to afford 4b (0.33 g, 52%) as colourless crystals: m.p.
1
tained as a pale yellow oil (0.08 g, 11%); H-NMR (300
MHz): l 7.11–7.07 (m, 3H, THIQ aromatic H’s), 7.02–
6.99 (m, 1H, THIQ aromatic H), 4.56 (tt, J=12.1, 3.8,
1H, cyclohexane axial H-4), 3.78 (s, 2H, THIQ H’s-1),
2.93–2.82 (m, 4H, THIQ H’s-3 and H’s-4), 2.63 (tt,
J=11.8, 3.3, 1H, cyclohexane axial H-1), 2.57 (s, 4H,
2CH2CO), 2.39 (dq, J=12.6, 3.3, 2H, cyclohexane
axial H’s), 2.02 (app br d, 2H, cyclohexane equatorial
H’s), 1.73–1.60 (cluster, 6H, cyclohexane equatorial
2H’s and cyclopentane 2CH2), 1.55–1.42 (cluster, 6H,
cyclohexane axial 2H’s and cyclopentane 2CH2). Com-
plex 6b·HCl·2H2O: m.p. 229–230 °C (C3H6O). Anal.
C24H32N2O2·HCl·2H2O (C, H, N).
1
190–192 °C (CHCl3–MeOH); H-NMR (300 MHz): l
7.25–6.83 (m, 4H, ArH), 3.98 (tt, J=12.4, 3.8, 1H,
cyclohexane axial H-4), 3.85 (s, 3H, OCH3), 3.09 (app
br s, 4H, piperazine 2CH2), 2.76 (app br t, 4H, pipe-
razine 2CH2), 2.65 (s, 4H, −CH2CH2− in succin-
imide), 2.46 (tt, J=11.5, 3.0, 1H, cyclohexane axial
H-1), 2.27 (dddd, J=12.9, 12.6, 3.0, 2.8, 2H, cyclo-
hexane axial H’s), 2.03 (app br d, 2H, cyclohexane
equatorial H’s), 1.68 (app br d, 2H, cyclohexane equa-
torial H’s), 1.39 (dddd, J=12.9, 12.6, 3.3, 3.0, 2H,
cyclohexane axial H’s). Complex 4b·2HCl: m.p. 294–
296 °C (EtOH–C3H6O). Anal. C21H29N3O3·2HCl (C,
H, N).
6.2. Molecular modelling
All the molecular modelling procedures and compu-
tations were performed using SYBYL package version
6.6 (Tripos Associates Inc., St. Louis, USA) run on a
Silicon Graphic Indigo II workstation. For the study of
the dihedral angles ~2–~4 by the Mopac/AM1 method
the structures of compounds 2b–6b were minimized
(precise, nomm, gnorm=0.1) over all the bonds and
angles except for the respective torsion which was con-
strained of values between 0 and 360° with a 10°
increment. Exploration of the conformational space of
compounds 2a and 2b was carried out by a standard
6.1.4. trans-2-[4-(Adamantanecarboxamido)cyclohexyl]-
1,2,3,4-tetrahydroisoquinoline (5b)
1-Adamantanecarboxylic acid (0.18 g, 1 mmol) was
dissolved in MeCN (15 mL) and BOP (1 mmol) was
added under stirring. To the homogenous solution
Et3N (2 mmol) was added, followed by solution of
4-[2-(1,2,3,4-tetrahydroisoquinolinyl)]cyclohexylamine
(0.23 g, 1 mmol) in MeCN (15 mL). The stirring was
continued for 6 h at a r.t. and the mixture was left
overnight. The solvent was evaporated and the residue
was purified by column chromatography (SiO2,
CHCl3–MeOH=19:1). The imide 5b (0.37 g, 95%) was
obtained as colourless crystals: m.p. 195–197 °C
(CHCl3–MeOH); 1H-NMR (500 MHz): l 7.12–7.07
(m, 3H, THIQ aromatic H’s), 7.02–7.00 (m, 1H, THIQ
aromatic H), 5.38 (d, J=8.0, 1H, NHCO), 3.77 (s, 2H,
THIQ H’s-1), 3.77–3.71 (m, decoupled leaves td, J=
11.7, 4.0, 1H, cyclohexane axial H-4), 2.89–2.81 ( m,
4H, THIQ H’s-3 and H’s-4), 2.48 (tt, J=11.6, 3.4, 1H,
cyclohexane axial H-1), 2.08–1.98 (cluster, 7H, 4 cyclo-
hexane equatorial H’s and 3 adamantane H’s), 1.83 (d,
J=2.6, 6H, adamantane H’s), 1.72 (q, J=12.1, 6H,
adamantane H’s), 1.34 (dddd, J=12.9, 12.8, 3.1, 3.0,
2H, cyclohexane axial H’s), 1.16 ( dddd, J=12.7, 12.6,
3.2, 3.1, 2H, cyclohexane axial H’s). Complex
5b·HCl·0.4H2O: m.p. 294–296 °C (C3H6O). Anal.
C26H36N2O·HCl·0.4 H2O (C, H, N).
random search method (energy cutoff: 3 kcal mol−1
)
and for maximin2 optimization (Tripos force field) a
conjugated gradient method was chosen. Volumes ac-
cessible by terminal amide (or imide) and hydrocarbon
groups of restricted compounds were generated using a
mvolume command.
6.3. Radioligand binding studies
The affinity of the investigated compounds for 5-
HT1A receptors was assessed on the basis of their ability
to displace [3H]-8-OH-DPAT (220 Ci mmol−1, Amer-
sham). The experiments were carried out in the
hippocampus of rat brain, according to the published
procedures [38]. Ki values were determined from at least
three competition binding experiments in which 10–14
drug concentrations, run in triplicate, were used. The
Cheng and Prusoff [39] equation was used for Ki
calculations.
6.1.5. trans-8-{4-[2-(1,2,3,4-Tetrahydroisoquinolinyl)]-
cyclohexyl}-8-azaspiro [4,5]decane-7,9-dione (6b)
4 - [2 - (1,2,3,4 - Tetrahydroisoquinolinyl)cyclohexyl-
amine (0.46 g, 2 mmol) and 3,3-tetramethyleneglutaric
anhydride (0.34 g, 2 mmol) in anhydrous pyridine (10
6.4. In 6i6o studies
The experiments were carried out on male Wistar
rats (260–300 g). The animals were kept at an ambient

282
M.H. Paluchowska et al. / European Journal of Medicinal Chemistry 37 (2002) 273–283
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temperature of 2091 °C throughout the experiment,
and had free access to food (standard laboratory pel-
lets, LSM) and tap water. All experiments were con-
ducted in the light phase on a natural light–dark cycle
(from September to October), between 9:00 and 14:00 h
8-OH-DPAT (Research Biochemical Inc.) and reserpine
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form of freshly prepared suspensions in a 1% Tween 80.
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