Novel class of arylpiperazines containing N-acylated amino acids: Their synthesis, 5-HT1A, 5-HT2A receptor affinity, and in vivo pharmacological evaluation

Article abstract of DOI:10.1016/j.bmc.2007.02.018

Novel arylpiperazines with N-acylated amino acids, selected on the basis of a preliminary screening of two libraries previously synthesized on SynPhase Lanterns, were prepared in solution and their affinity for 5-HT_1A, 5-HT_2A

Full text of DOI:10.1016/j.bmc.2007.02.018

Bioorganic & Medicinal Chemistry 15 (2007) 2907–2919
Novel class of arylpiperazines containing N-acylated amino acids:
Their synthesis, 5-HT1A, 5-HT2A receptor affinity,
and in vivo pharmacological evaluation
a
c
c
Paweł Zajdel,a,b, Gilles Subra, Andrzej J. Bojarski, Beata Duszynska,
*
´
d
d
d
´
´
Ewa Tatarczynska, Agnieszka Nikiforuk, Ewa Chojnacka-Wojcik,
Maciej Pawłowskib and Jean Martineza
aLaboratoire des Aminoacides, Peptides et Prote´ines (LAPP), UMR 5810, Faculte´ de Pharmacie, Universite´ Montpellier I et II,
15 Avenue Charles Flahault, 34060 Montpellier, France
bDepartment of Medicinal Chemistry, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Krako´w, Poland
cDepartment of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences, 12 Sme˛tna Street,
31-343 Krako´w, Poland
dDepartment of New Drugs Research, Institute of Pharmacology, Polish Academy of Sciences, 12 Sme˛tna Street,
31-343 Krako´w, Poland
Received 31 August 2006; revised 5 February 2007; accepted 9 February 2007
Available online 13 February 2007
Abstract—Novel arylpiperazines with N-acylated amino acids, selected on the basis of a preliminary screening of two libraries pre-
TM
viously synthesized on SynPhase Lanterns, were prepared in solution and their affinity for 5-HT1A, 5-HT2A, and D2 receptors was
evaluated. The compounds bearing (3-acylamino)pyrrolidine-2,5-dione (19–26) and N-acylprolinamide (29–34) moieties showed
high affinity for 5-HT1A (Ki = 3–47 nM), high-to-low for 5-HT2A (Ki = 4.2–990 nM), and low for D2 receptors (Ki = 0.77–
21.19 lM). All the new o-methoxy derivatives of (3-acylamino)pyrrolidine-2,5-diones tested in vivo revealed agonistic activity at
postsynaptic 5-HT1A receptors, while m-chloro derivatives were classified as antagonists of these sites; similar relations were
observed for o-methoxy (29) and m-chlorophenylpiperazine derivatives of N-acylprolinamides. The reported results show that
the amino acid-derived terminal fragment modified the in vivo functional profile. Finally, the selected compounds 19 and 20, a
5-HT1A partial agonist and a full agonist, respectively, and 26, a mixed 5-HT1A/5-HT2A antagonist, were evaluated in preclinical
animal models of depression and anxiety. The project allowed selecting the lead compound 20 which exhibited an anxiolytic-like
effect in the four-plate test in mice and revealed distinct antidepressant-like effects in the forced swimming and tail suspension tests
in mice.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
tion of the compound libraries and thus has offered un-
ique opportunities for accelerating lengthy processes
revolving around hit and lead identification as well as
stages of lead optimization.1,2
Among several innovative synthetic technologies the
advent of combinatorial chemistry was one of the devel-
opments that significantly contributed to the progress in
drug discovery process. By introduction of split-and-mix
and parallel synthesis methods it has facilitated genera-
Long-chain arylpiperazines have long been focusing
great interest as a source of both neuropsychiatric drugs,
for example, Buspirone,3 Ziprasidone,4 Aripiprazol,5
and compounds with a high therapeutical potential
(Adatanserin,6 Mazapertine,7 Flesinoxan,8 Lecozotan,9
Bifeprunox10) which—among others—exert their action
via 5-HT1A and 5-HT2A receptors. Their diversified
receptor binding profile and intrinsic activity,11–13
depending on either the kind of substituent at the phenyl
Keywords: Long-chain arylpiperazines; Succinimides; Pyrrolidine-2,5-
dione; Prolinamides; Solid-phase synthesis; 5-HT1A/5-HT2A receptor
ligands; 5-HT1A receptor agonists; Four-plate test; Forced swimming
test; Tail suspension test.
*
Corresponding author. Tel.: +48 12 657 05 60; fax: +48 12 657 02
62; e-mail: mfzajdel@cyf-kr.edu.pl
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.02.018

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P. Zajdel et al. / Bioorg. Med. Chem. 15 (2007) 2907–2919
ring at piperazine or the nature of an amide/imide
terminal fragment, opens possibility for discovery of
new potent therapeutics.14–16
selected to be pharmacologically tested in order to
determine their potential CNS activity.
In the present paper, we described the pre-final stage of
our research project: the synthesis of 14 compounds
bearing (3-acylamino)pyrrolidine-2,5-diones (19–26)
and N-acylprolinamides (29–34) and containing an
o-methoxy- and an m-chlorophenylpiperazine-butyl
fragments; their 5-HT1A, 5-HT2A, dopamine D2 receptor
affinities, as well as the successive determination of their
functional profile at 5-HT1A (pre- and postsynaptic) and
5-HT2A sites. Finally, the results of preclinical evalua-
tion (animal models of anxiety and depression) of
compounds 19 and 20—the most interesting in terms
of 5-HT1A intrinsic activity—were presented.
While searching for new agents for a possible treatment
of anxiety and depressive disorder, we designed a novel
class of arylpiperazine derivatives containing N-acylated
amino acids in the amide fragment (Fig. 1). Modifica-
tions consisted in introducing 3-aminopyrrolidine-2,5-
dione (set 9) and pyrrolidine-5-one-2-carboxamide (set
10), which originated from the cyclization of aspartic
and glutamic acid, respectively. Moreover, prolinamides
(set 17) used, as deoxo-analogs of pyroglutamyl moiety,
and asparaginyl amides (set 19) as open counterparts of
set 9 were introduced. Further modifications involved
diversifying the nature of an N-acyl substituent and
the length of an alkyl chain connecting the amide/imide
fragment to the basic nitrogen of arylpiperazine.
2. Chemistry
In the initial stage of our research project, in order to get
a quick access to the above compounds, we developed a
new solid-supported approach that yielded two libraries
containing 72 members bearing phenylpiperazines and
m-chlorophenylpiperazines,17 and 60 o-methoxyphenyl-
piperazines (o-OCH3-PhP).18 Those two libraries with
three points of diversity were generated by a five-step
protocol using BAL linker functionalized SynPhase
Lanterns19,20 which included one-pot cleavage/cycliza-
tion for the generation of succinimide and pyroglutamyl
derivatives. Of the synthesized compounds, 51 library
representatives were tested in a preliminary screening
for 5-HT1A and 5-HT2A receptors. Subsequently, struc-
ture–activity relationships within the library were ana-
lyzed by the Free-Wilson and Fujita-Ban methods.21
In the structure of the compounds, three fragments
related with the building blocks used for generating
library were distinguished, and the influence of the
respective substructures on receptor affinity was deter-
mined (Fig. 1). It was found that prolyl amides (set
17), R substituents of high molecular bulk, and four-
member alkyl chain connected with o-OCH3-PhP had
the highest incremental effect on 5-HT1A affinity, while
3-aminopyrrolidine-2,5-dione (set 9), cyclohexyl, nor-
born-2-ylmethyl substituents, and a 4-(3-chlorophe-
nyl)-1-piperazinylbutyl fragment were the most
beneficial to 5-HT2A binding. On the basis of a prelimin-
ary screening, some potent 5-HT1A and/or 5-HT2A
receptor ligands (with 5-HT1A affinity <50 nM) were
2.1. Synthesis of N-((3S)-1-{4-[4-arylpiperazin-1-yl]bu-
tyl}pyrrolidin-2,5-dion-3-yl)cycloalkanecarboxamides
(19–26)
The multi-step synthesis consisted in the parallel prepa-
ration of 4-[4-arylpiperazin-1-yl]butylamine according
to the Gabriel method described elsewhere22 and the
respective N-acyl aspartic acid derivatives (Scheme 1).
Esterification of 2-(9H-fluoren-9-ylmethoxycarbonyl)
aspartic acid 4-tert-butyl ester (1) in a two-phase medi-
um yielded compound 2. After Fmoc removal, it was
coupled with the respective carboxylic acids to give
N-acyl aspartic acid derivatives (3–6) using BOP as an
activating agent.23 The latter were then subjected to
catalytic hydrogenation with a 10% Pd/C, resulting in
quantitative transformation into the respective free acids
7–10. The key intermediates were coupled to an appro-
priate butylamine to yield the protected amido-acids
11–18. The cyclization of the latter products was accom-
plished by a two-step procedure: the removal of t-Bu
protection upon TFA treatment and the subsequent
closing of amido-acids to the imides 19–26 in an acetic
anhydride in the presence of sodium acetate.
2.2. Synthesis of 1-(cycloalkylcarbonyl)-N-{4-[4-arylpi-
perazin-1-yl]butyl}-L-prolinamide (29–34)
Direct coupling of tert-butoxycarbonyl-proline to an
appropriate 4-[4-arylpiperazin-1-yl]butylamine afforded
O
H
O
N
N
N
O
ArP
O
O
O
R1
R
R
set 10
set 9
N
NH2
N
N
N
N
n
O
H
O
O
N
n = 1, 2
R 1 = H,
o-OCH3, m-Cl
O
R
O
O
R
O
R
R = methyl, cyclopentyl, cyclohexyl,
phenyl, norborn-2-ylmethyl, adamantyl
set 17
set 19
Figure 1. General structure of the investigated compounds with the marked fragments used for the Free-Wilson and the Fujita-Ban analyses.17,18

P. Zajdel et al. / Bioorg. Med. Chem. 15 (2007) 2907–2919
2909
O
O
O
O
O
N
H
N
H
i
O
ii, iii
O
Fmoc-Asp-(O
tBu)-OH
O
R
O
O
O
1
3 - 6
2
iv
O
O
O
O
N
O
N
H
H
H
N
v
OH
N
R
R
O
O
O
N
R1
11 - 18
7 - 10
vi, vii
O
H
N
N
N
R
R = cyclopentyl, cyclohexyl,
norborn-2-ylmethyl, adamantyl
O
O
N
R1
R1
=
o
-OCH3, -Cl
m
19 - 26
Scheme 1. Reagents and conditions: (i) benzyl bromide, TBAB, K2CO3, CH3CN, rt, 12 h; (ii) 20% piperidine/DMF, rt, 40 min; (iii) cycloalkane
carboxylic acid, BOP, TEA, CH2Cl2, rt, 8 h; (iv) 10% Pd/C, H2 (gas), EtOH; (v) 4-[4-arylpiperazin-1-yl]butylamine, BOP, TEA, CH2Cl2, rt, 12 h; (vi)
TFA, rt, 60 min; (vii) Ac2O, AcONa, 60 ꢁC, 2 h.
the prolyl amides 27 and 28. After Boc removal, the lat-
ter were coupled to cycloalkane carboxylic acids to yield
the final prolyl amides 29–34 (Scheme 2).
measure of presynaptic 5-HT1A receptor agonistic
activity. Similarly, the ability of those compounds to
inhibit the 8-OH-DPAT-induced hypothermia was
taken as a measure of presynaptic 5-HT1A receptor antag-
onistic activity. To determine the postsynaptic 5-HT1A
receptor agonistic effects of the tested compounds, their
ability to induce lower lip retraction (LLR) in rats was
tested. It is commonly accepted that the 8-OH-DPAT-in-
duced LLR in rats depends on stimulation of postsynaptic
5-HT1A receptors27,28 and is inhibited by WAY 100635.29
Hence the ability of the tested compounds to inhibit the
8-OH-DPAT-induced LLR was regarded as postsynaptic
5-HT1A receptor antagonistic activity.
For pharmacological in vitro and in vivo investigations,
all the final products were converted into water-soluble
hydrochlorides.
3. Pharmacology
The new compounds were tested in vitro in full displace-
ment experiments for 5-HT1A and 5-HT2A receptor
affinities, and according to the screening protocol for
dopamine D2 receptor affinity.
The central 5-HT2A antagonistic properties of com-
pounds showing 5-HT2A receptor affinity <50 nM were
assessed by their ability to antagonize head twitches
induced by the 5-HT2A receptor agonist ( )-1-(2,5-dime-
thoxy-4-iodophenyl)-2-aminopropane (( )-DOI) in
mice.30,31
The functional activity of the investigated compounds at
5-HT1A and 5-HT2A receptors was tested in commonly
used in vivo models. It was previously demonstrated
that the hypothermia induced by the 5-HT1A receptor
agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-
DPAT) in mice was connected with activation of presyn-
aptic 5-HT1A receptors,24,25 and was abolished by the
selective 5-HT1A receptor antagonist N-{2-[4-(2-meth-
oxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclo-
hexane carboxamide (WAY 100635).26 Thus the
hypothermia produced by the tested compounds in
mice (reduced by WAY 100635) was regarded as a
The potential anxiolytic activity of compounds (19,
20, and 26) was evaluated by the four-plate test in
mice,32 and the antidepressant-like activity (19 and
20) by the forced swimming33 and tail suspension tests
in mice.34 The effect of compounds 19 and 20 on the
spontaneous locomotor activity of mice was also
tested.

2910
P. Zajdel et al. / Bioorg. Med. Chem. 15 (2007) 2907–2919
O
O
O
O
N
i
N
H
N
N
OH
O
O
N
27 - 28
ii, iii
R1
R
O
N
R = cyclohexyl, norborn-2-ylmethyl,
adamantyl
= o-OCH3, m-Cl
H
N
R1
N
N
O
R1
29 - 34
Scheme 2. Reagents and conditions: (i) 4-[4-arylpiperazin-1-yl]butylamine, BOP, TEA, CH2Cl2, rt, 12 h; (ii) TFA, rt, 60 min; (iii) cycloalkane
carboxylic acid, BOP, TEA, CH2Cl2, rt, 12 h.
4. Results and discussion
tested was composed of a sufficient number of structural
analogs, and the results of biological evaluation fit the
general structure–activity relationships pattern. In al-
most all the cases the receptor affinity values obtained
for crude products synthesized on a solid support were
lower than the affinity data obtained in full-displace-
ment experiments for the same pure compounds synthe-
sized in a solution (e.g., the 5-HT1A receptor affinities of
o-OCH3-PhP derivatives were 1.5- to 3-fold higher). It
seems of great importance that for all 5-HT1A receptor
investigated compounds the initially determined recep-
tor affinity rank order remained unchanged. A similar
tendency was observed for compound 5-HT2A receptor
affinity, except for compounds 24 and 25 classified by
a preliminary screening as highly potent 5-HT2A recep-
tor ligands (Ki = 2 and 1 nM, respectively), which
appeared to be slightly less active in a full-displacement
experiment, their Ki values being 6.5 and 4.2 nM, respec-
tively. Nevertheless, the 5-HT2A receptor affinities of 24
and 25 were still the highest in the series—only slightly
lower than those reported for the reference drug (ritans-
erin, Ki = 1.1 nM).17 Despite the above-listed deviations,
ligand classification after full binding assays is in line
with the data obtained from a preliminary screening.
All the compounds tested, that is, (3-acylamino)pyrroli-
dine-2,5-diones (19–26), and N-acylprolinamide deriva-
tives (29–34), showed high affinity for 5-HT1A
receptors (3–47 nM), whereas their 5-HT2A receptor
affinities were diversified and ranged from 4.2 (25) to
990 nM (29) (Tables 1 and 2). On the basis of their bind-
ing profiles, compounds 21, 23–26, 32, and 33 were clas-
sified as dual 5-HT1A/5-HT2A receptor ligands. It is
worth noting that of the dual ligands only compound
21 had an o-OCH3-PhP fragment; the remainder of
them possessed an m-Cl substituent at the phenyl ring.
The compounds under study demonstrated weak or
negligible affinity for D2 receptor sites, which may be
explained by the absence of an aromatic moiety in the
amide fragment, commonly present within the structure
of highly potent D2 receptor ligands.35,36
As has been mentioned in the Introduction, the 5-HT
receptor activity of the compounds synthesized on a sol-
id support was determined according to the pre-screen-
ing protocol. Such quick tests of library members
without their prior purification have been discussed in
several papers and remain controversial.37 Hence one
of the goals of our investigation was to determine the
accuracy and productiveness of the preliminary screen-
ing protocol applied to estimate the 5-HT1A and
5-HT2A receptor affinities of the compounds synthesized
on a solid support and tested as crude products. Some
problems may arise due to the fact that the analyzed
samples are likely to contain impurities which do not ab-
sorb UV light (not detectable by a LC/MS analysis), and
thus alter the final response, giving false biological
results. In our experiments, compounds were synthe-
sized on a solid support by a sort-and-combine
approach, yielding an individual compound per vial.
Although in all the cases compound purity did not
exceed 95% standard, the set of library representatives
The synthesized arylpiperazines came from the introduc-
tion of N-acylated amino acid moieties into an amide
fragment. Among them, (3-acylamino)pyrrolidine-2,5-
diones (19–26) resulted from cyclization of N-acyl
aspartic acids and may be regarded as analogs of the
succinimide ligands of 5-HT1A receptors, that is, MM
7738 and I39 (Table 1), modified by a cycloalkanecarbox-
amide moiety. The other set tested, that is, N-acylproli-
namides (29–34, Table 2), may be classified as
(3-acylamino)pyrrolidine-2,5-dione analogs possessing
an exocyclic amide bond connected directly to an alky-
len spacer.
The radioligand binding studies show that within the
evaluated compounds, receptor affinity and selectivity

P. Zajdel et al. / Bioorg. Med. Chem. 15 (2007) 2907–2919
2911
Table 1. The structure and binding affinity data on serotonin (5-HT1A, 5-HT2A) and dopaminergic D2 receptors for (3-acylamino)pyrrolidine-2,5-
dione derivatives (19–26)
O
O
H
N
N
N
N
N
R
O
O
N
O
N
R1
R1
MM 77, I
19 - 26
Compound
R
R1
Ki (nM) SEM
5-HT1A activity
5-HT2A activity
a
5-HT1A
5-HT2A
D2
Presynaptic
Postsynaptic
Antagonist
MM 77b
Ic
—
—
o-OCH3
m-Cl
6.4 0.3
1510 95
121 14
480 38
183 12
490
7800
905
Non active
Agonist
Agonist
nd
nd
32
23
19
9
2
4
3
2
2
3
4
4
3
Partial agonist
Partial agonist
Agonist
19
20
21
22
23
24
25
26
Cyclopentyl
Cyclohexyl
o-OCH3
o-OCH3
o-OCH3
o-OCH3
m-Cl
nd
nd
2080
880
Agonist
Norborn-2-ylmethyl
Adamantyl
Cyclopentyl
47
173 17
6
Non active
Non active
Non active
Non active
Non active
Antagonist
Agonist
Antagonist
nd
4
1775
Partial agonist
Antagonist
Antagonist
Antagonist
Antagonist
28
26
21
47
47
6.51
8
1
12450
2130
776
Antagonist
Antagonist
Antagonist
Antagonist
Cyclohexyl
Norborn-2-ylmethyl
Adamantyl
m-Cl
m-Cl
4.2 0.5
25
m-Cl
2
21190
nd, not determined.
a Binding experiment run with two compound concentrations, expressed as an estimated Ki.
b Ref. 38.
c Ref. 39.
Table 2. The structure and binding affinity data on serotonin (5-HT1A, 5-HT2A) and dopaminergic D2 receptors for N-acylprolinamides (29–34)
R
O
N
H
N
N
N
O
R1
Compound
R
R1
Ki (nM) SEM
5-HT2A
5-HT1A activity
5-HT2A activity
a
5-HT1A
D2
Presynaptic
Postsynaptic
29
30
31
32
33
34
Cyclohexyl
o-OCH3
o-OCH3
o-OCH3
m-Cl
19 3
14.5 0.7
990 17
547 48
3595
1365
1114
1050
1550
1780
Non active
Agonist
Agonist
nd
nd
Norborn-2-ylmethyl
Adamantyl
Cyclohexyl
Non active
Non active
Antagonist
Antagonist
Antagonist
3
31
37
13
0.2
4
503
69
8
4
3
7
Non active
Agonist
Agonist
nd
nd
Norborn-2-ylmethyl
Adamantyl
m-Cl
m-Cl
3
35
140
Antagonist
nd
1
Agonist
nd, not determined.
a Binding experiment run with two compound concentrations, expressed as an estimated Ki.
depend on the nature of the amino acid moiety, as
well as the size and hydrophobic properties of R sub-
stituents at the amide fragment and on arylpiperazine
substitution mode (R1). As a general trend, N-acylpr-
olinamides showed preferential affinity for the 5-HT1A
receptors. Among them, compound 31 bearing an ada-
mantyl moiety is characterized by the highest selectiv-
ity factor (S2A/1A = 167). For comparison, the highest
selectivity ratio within the succinimide set (19–26)
equaled 90.
Further, the 5-HT1A receptor affinity of the o-methoxy-
phenylpiperazine derivatives of (3-acylamino)pyrroli-
dine-2,5-diones and N-acylprolinamides has been
found to depend on the volume of a cycloalkyl substitu-
ent; the higher molecular bulk, the more potent 5-HT1A
ligands, that is, a change of a cyclopentyl into an ada-
mantyl leads to a 6-fold increase in 5-HT1A affinity (19
vs 22). Similar affinity increase (6-fold) was also
observed in case of replacement of a cyclohexyl into
an adamantyl (29 vs 31). Such a clear influence of the

2912
P. Zajdel et al. / Bioorg. Med. Chem. 15 (2007) 2907–2919
kind of hydrocarbon moiety on 5-HT1A affinity was not
observed within m-chloro derivatives. Regarding
5-HT2A affinity, it was found that in almost all the cases
introduction of norborn-2-ylmethyl moiety, with more
conformational freedom than adamantyl, gave the most
potent 5-HT2A receptor ligands in the series. Finally,
compounds 23–26 and 32–34 bearing m-chloro substitu-
ent at the phenyl ring at the piperazine moiety were
more potent 5-HT2A receptor ligands than were o-meth-
oxy counterparts (19–22 and 29–31). The above-quoted
results confirm the general preference of a chloro substi-
tuent in the meta position of the phenyl ring for 5-HT2A
binding sites.
the ( )-DOI-induced head twitches in mice (see Supple-
mentary Data—Table 5); hence these compounds can
also be regarded as potential 5-HT2A receptor
antagonists.
It was recently reported by Bojarski et al.39 that the sub-
stitution mode in the aromatic ring of piperazine con-
trolled the postsynaptic 5-HT1A receptor functional
activity of a group of aryl-4-succinimidobutylpiperazines.
Among them, MM 77, a potent 5-HT1A ligand bearing an
o-methoxy substituent, was classified as a postsynaptic
5-HT1A antagonist,38 while its m-chloro analog (I)
showed properties of a partial agonist of postsynaptic
5-HT1A sites39 (Table 1). Unlike the model compounds
MM 7738 and I,39 in functional in vivo tests all the new
o-methoxy (3-acylamino)pyrrolidine-2,5-diones showed
properties of full (20, 21) or partial (19 and 22) agonists
at postsynaptic 5-HT1A receptors, while their m-chloro
counterparts (23–26) were classified as 5-HT1A receptor
postsynaptic antagonists. The above results indicate that
introduction of N-acyl amino acid-derived moiety into
the arylpiperazine fragment altered the in vivo functional
profile compared to the parent molecules.
The results of our in vivo study demonstrate that the
investigated compounds possess diversified 5-HT1A
receptor functional activity (Tables 1 and 2). Of the
compounds tested, 19–23, 29–34 (like 8-OH-DPAT, a
5-HT1A receptor agonist) decreased body temperature
in mice, whereas compounds 24–26 (like WAY 100635,
a 5-HT1A receptor antagonist) did not change it (see
Supplementary Data—Table 1). The hypothermia in-
duced by 19, 20, 30, 32, 33 or 34 (like that induced by
8-OH-DPAT) was reduced or abolished by WAY
100635 (see Supplementary Data—Table 2); therefore
it seems that these compounds have features of presyn-
aptic 5-HT1A receptor agonists. At the same time, the
hypothermia induced by compounds 21, 22, 23, 29 or
31 (in contrast to that induced by 8-OH-DPAT) was
not changed by WAY 100635 (see Supplementary
Data—Table 2); thus a mechanism different from stimu-
lation of 5-HT1A receptors seems to be responsible for
the hypothermic effect of these compounds. Of com-
pounds 24, 25, and 26, which did not affect mouse body
temperature, only 26 (like WAY 100635) attenuated the
8-OH-DPAT-induced hypothermia in mice (see Supple-
mentary Data—Table 3). Therefore, 26 may be classified
as a presynaptic 5-HT1A receptor antagonist, whereas
the functional activity of 24 and 25 at those receptors
was negligible in that test.
Of the o-methoxy derivatives of N-acylprolinamides
(29–34, Table 2), compound 29 was regarded as an ago-
nist of postsynaptic 5-HT1A sites, while 30 and 31 were
devoid of any postsynaptic activity. Parallel to the suc-
cinimide set (19–26, Table 1), all the m-chloro deriva-
tives (32–34) were classified as postsynaptic 5-HT1A
antagonists.
Regarding the presynaptic 5-HT1A receptor intrinsic
properties, the compounds tested showed agonistic (19,
20, 30, and 32–34) or antagonistic (26) activity or were
inactive (21–25, 29, and 31); thus a close relationship be-
tween the chemical modifications and their presynaptic
intrinsic activity could not be demonstrated. It is diffi-
cult to explain why compound 31, which in vitro showed
the highest 5-HT1A receptor affinity, appeared to be
inactive at both pre- and postsynaptic 5-HT1A sites in
functional in vivo studies. Interestingly, an earlier report
showed that a compound with high 5-HT1A potency
in vitro was not always found to be the most potent in
functional in vivo models.40
In the experiment designed to evaluate postsynaptic
5-HT1A receptor activity, compounds 19–22 and 29, given
alone, induced LLR in rats, the latter effect being consid-
erably weaker than that after 8-OH-DPAT administra-
tion, though the remaining compounds did not mimic
the effect of 8-OH-DPAT in that test (see Supplementary
Data—Table 4). The LLR induced by 8-OH-DPAT was
inhibited by compounds 19, 22–26, and 32–34, while
WAY 100635 almost completely blocked the effect of 8-
OH-DPAT (see Supplementary Data—Table 4). The
above-quoted results indicated that in the LLR model in
rats compounds 20, 21, and 29 showed features character-
istic of weak agonists of postsynaptic 5-HT1A receptors,
compounds 19 and 22 could be classified as partial ago-
nists, and compounds 23–26 and 32–34 behaved like
antagonists of postsynaptic 5-HT1A receptors. Com-
pounds 30 and 31 were practically inactive in that model.
Unexpectedly, compounds 29–31, administered in a dose
of 20 mg/kg, caused the death of rats.
Unsurprisingly, all the compounds tested by the 5-HT2A
receptor functional model showed antagonistic
properties.
The functional profile of the investigated compounds sug-
gests that some of them may exhibit anxiolytic- and/or
antidepressant-like effects. It has been demonstrated that
5-HT1A receptor agonists and partial agonists exert such
activity,41–43 and mixed 5-HT1A/5-HT2A receptor antago-
nists may also be of interest as potential anxiolytics.44 For
this reason we selected compounds 19 (a partial 5-HT1A
receptor agonist), 20 (a full 5-HT1A receptor agonist),
and 26 (a 5-HT1A/5-HT2A receptor antagonist) for further
in vivo preclinical studies.
Moreover, compounds 21, 23–26, and 33 (like ketanser-
in, a 5-HT2A receptor antagonist) effectively inhibited
Our results indicate that compounds 19 and 20 produce
an anxiolytic-like effect in the four-plate test in mice

P. Zajdel et al. / Bioorg. Med. Chem. 15 (2007) 2907–2919
2913
Table 3. Effects of compounds 19, 20, 26, and diazepam in the four-
plate test in mice
Table 4. Effects of compounds 19, 20, and imipramine in the forced
swimming (A) and the tail suspension (B) tests in mice
Treatment
Dose (mg/kg)
No. of punished
crossings means SEM
Treatment
Dose
(mg/kg)
Immobility time (s), means SEM
A
B
Vehicle 19
—
5
3.9 0.5
3.4 0.4
Vehicle 19
—
5
166.0 4.8
NT
133.4 11.2
123.3 15.7a
102.9 11.5b
F(3, 36) = 5.240
P < 0.01
10
20
5.2 0.4
10
20
6.0 0.5a
F(3, 36) = 7.189
P < 0.001
Vehicle 20
—
2.5
5
4.0 0.3
Vehicle 20
—
2.5
5
166.0 4.8
178.2 7.2
NT
4.7 0.5
131.6 12.5
110.0 9.0b
102.0 12.7b
128.1 8.1a
F(4, 45) = 6.297
P < 0.001
6.6 0.3a
7.0 0.3a
7.5 0.5a
F(4, 45) = 14.723
P < 0.001
147.9 10.6
129.1 6.9b
116.1 10.6a
F(3, 36) = 8.986
P < 0.001
10
20
10
20
Vehicle 26
—
5
4.1 1.3
4.6 0.5
5.1 0.5
5.6 0.6
F(3, 36) = 1.462
ns
Vehicle
imipramine
—
5
167.1 6.7
NT
149.1 10.7a
107.8 12.4b
F(2, 27) = 8.760
P < 0.01
164.6 11.0
147.4 9.9
102.5 10.3b
90.2 10.7b
F(3, 36) = 13.495
P < 0.001
10
20
10
20
Vehicle
diazepam
—
3.5 0.4
Compounds 19 and 20 were administered 30 min, while imipramine
60 min before the test. n = 9–10 mice per group. NT, not tested.
a P < 0.05.
b P < 0.01 versus vehicle (Dunnett’s test).
1.25
2.5
5
5.5 0.5
6.8 0.6a
6.7 0.6a
F(3, 36) = 9.514
P < 0.001
Compounds 19 and 20 were administered 30 min, while 26 and diaz-
epam 60 min before the test. n = 10 mice per group. ns, not significant.
a P < 0.01 versus vehicle (Dunnett’s test).
techniques for the generation of compound 20 from
the designed new class of arylpiperazines containing
N-acylated amino acid residues targeted on 5-HT1A
and 5-HT2A receptors. Several representatives of two
libraries (132 members) were screened as crude prod-
ucts; then 14 derivatives were re-synthesized (19–26
and 29–34) and their receptor affinity was determined
in full-displacement experiments. The advantage of the
approach was a quick identification of potent 5-HT1A
receptor ligands and several dual 5-HT1A/5-HT2A li-
gands, which—evaluated further by in vivo functional
and preclinical tests—allowed us to establish strong
structure–activity relationships between diverse factors
affecting compound activity. The results of the present
preclinical study show that compound 20 (a pre- and
postsynaptic 5-HT1A agonist) produces potential anxio-
lytic/antidepressant effects in the preclinical models
used. It is worth noting that its effects are comparable
to those produced by standard drugs, and that active
doses of 20 are devoid of a sedative effect. Further stud-
ies with this lead compound will be undertaken and
reported in due course. This report also suggests that
concurrent application of parallel solid-phase synthesis
and screening methods may be helpful for academic
and industry laboratories in discovering new lead drugs
in a time- and cost-effective manner.
(Table 3). It is worth noting that the effect of 20 is com-
parable, in terms of its potency and active doses, with
that produced by diazepam (used as a reference drug),
whereas the effect of 19 is found to be weaker. Com-
pound 26 is ineffective in this test (Table 3).
The results of our successive experiments also show
that—like the typical antidepressant imipramine—com-
pounds 19 and 20 exert a distinct antidepressant-like ef-
fect in a mouse forced swimming test. It is worth noting
that both these compounds in doses lower than that of
imipramine shorten the immobility time of mice; in the
case of 20 the minimal effective dose is 5 mg/kg (Table
4). For this reason compound 20 was additionally stud-
ied in the tail suspension test in mice. In that model, 20
used in the same doses (10–20 mg/kg) as imipramine
shortened the immobility time of mice (Table 4).
The anti-immobility effect of 19 and 20 seems to be spe-
cific, since—like imipramine—neither compound chan-
ged the spontaneous locomotor activity of mice during
the initial 6-min experimental sessions (i.e., at the time
equal to the observation period in the forced swimming
test). When locomotor activity was recorded for 30 min,
19 and imipramine reduced it, while 20 was without
effect (see Supplementary Data—Table 6).
5. Experimental
5.1. Chemistry
Summing up, the study presents a research project on
the successful application of combinatorial chemistry
All reagents and solvents were from Aldrich and
Lancaster and were used without further purification.

2914
P. Zajdel et al. / Bioorg. Med. Chem. 15 (2007) 2907–2919
Protected amino acids and BOP were purchased from
Senn Chemicals. The purity of the products was con-
firmed by an ascending TLC method on pre-coated Kie-
selgel 60 F254 plates (Merck) by applying the following
solvents (by volume): S1: CH2Cl2/MeOH, 9:1; S2:
CH2Cl2/MeOH, 95:5; S3: CHCl3/acetone, 1:1; S4:
CHCl3/MeOH/AcOH, 180:10:5; S5: EtOAc/n-hexane,
3:7. After the development of chromatograms, the plates
were inspected under UV light (k = 254 nm); additional-
ly, spots were visualized with ninhydrine; Rf values are
given for the sake of guidance. Analytical HPLC were
run on a Waters Alliance HPLC instrument, equipped
with a Chromolith SpeedROD column (4.6 · 50 mm).
Standard conditions were eluent system A (water/0.1%
TFA), system B (acetonitrile/0.1% TFA). A flow rate
of 5 mL/min and a gradient of (0–100)% B over 5 min
were used, detection 214 nm. Retention times (tR) are
given in minutes. All melting points were determined
5.1.2. General procedure A: preparation of 1-benzyl 4-
tert-butyl N-(cycloalkylcarbonyl)-L-aspartates 3–6.
1-Benzyl 4-tert-butyl N-(9H-fluoren-9-ylmethoxycar-
bonyl)-L-aspartate (2) was stirred at a room temperature
in a mixture of piperidine and dimethylformamide
(DMF) (20:80, v/v) for 60 min. After evaporation of
the deprotection solution, 1-benzyl 4-tert-butyl L-aspar-
tate (2a) was characterized by LC/MS and was used
without further purification. A commercially available
carboxylic acid (16.5 mmol) was dissolved in 60 mL
dichloromethane (CH2Cl2), and benzotriazol-l-yl-
oxy-tris-(dimethylamino)phosphonium
hexa-fluoro-
phosphate (BOP) (7.3 g, 16.5 mmol) was added under
stirring. Then triethylamine (TEA) (4.2 mL, 30 mmol)
was added dropwise, followed by a solution of H-Asp
(OtBu)-OBzl, 2a (4.19 g, 15 mmol) in CH2Cl2 (15 mL).
The reaction mixture was stirred for 8 h at a room tem-
perature. Then the solvent was evaporated and the oily
residue was dissolved in EtOAc (100 mL) and washed
with a saturated NaHCO3 solution (3·50 mL), 1 M
KHSO4 solution (3·50 mL), water (3·50 mL), and
brine; it was then dried over Na2SO4, and finally concen-
trated in a vacuum. The crude amides were purified by a
flash chromatography. Spectral data for new intermedi-
ates 3–6 are presented in Supplementary Data.
with Buchi 353 capillary apparatus and remain uncor-
¨
rected. H NMR spectra were obtained using a Varian
1
BB 200 (300 MHz) spectrometer; chemical shifts (d)
are expressed in ppm downfield from the internal TMS
as a reference; J values are in Hertz, and splitting
patterns are designated as follows: s (singlet), d
(doublet), t (triplet), m (multiplet). ESI + mass spectra
were obtained on the LC–MS system consisting of
Waters Alliance 2690 HPLC coupled to a Micromass
(Manchester, UK) Platform II spectrometer (an electro-
spray ionization mode, ESI+). All the analyses were
5.1.3. General procedure B: preparation of 4-tert-butyl N-
(cycloalkylcarbonyl)-L-aspartates 7–10. To a solution of
the corresponding esters 3–6 (7 mmol) in methanol, 0.1 g
of 10% Pd/C was added and the mixture was stirred for
4 h under atmosphere of H2. Then it was filtered over
Celite, and the filtrate was concentrated to yield the de-
sired products as a white solid. Spectral data for new
intermediates 7–10 are presented in Supplementary
Data.
carried out using a C18 Xterra MS, 2.1 · 30 mm
20
column. Optical rotations ð½aꢀ Þ for the final
D
compounds were measured with a Jasco DIP-1000
Digital Polarimeter (5 cm cell). Elemental analyses were
carried out using an Elementar Vario EL III, and were
within 0.4% of the theoretical values. Silica gel 60
230–400 mesh, purchased from Merck, was used for
preparative chromatographic purification.
5.1.4. General procedure C: preparation of 4-tert-butyl N-
(cycloalkylcarbonyl)-1-{4-[4-arylpiperazin-1-yl]butyl}-L-aspart-
amides 11–18. To a mixture of 4-tert-butyl N-(cyclo-
alkylcarbonyl)-L-aspartate (4 mmol) in 50 mL CH2Cl2,
BOP (1.94 g, 4.4 mmol) was added under stirring. Then
triethylamine (1.12 mL, 8 mmol) was added dropwise
followed by a solution of the respective 4-[4-arylpipera-
zin-1-yl]butylamine (3.6 mmol) in CH2Cl2 (5 mL). The
mixture was stirred for 6–12 h at a room temperature
until the reaction was completed (TLC control). The
solvent was evaporated and the oily residue was dis-
solved in EtOAc (100 mL). It was then washed with a
saturated NaHCO3 (3·50 mL), water (3·50 mL) and
brine, dried over Na2SO4, and was finally concentrated
in a vacuum. The crude amides were purified by a
column chromatography (silica gel, CH2Cl2/MeOH,
9:1 or CH2Cl2/MeOH, 90:7). Spectral data for new inter-
mediates 11–18 are presented in Supplementary Data.
5.1.1. 1-Benzyl 4-tert-butyl N-(9H-fluoren-9-ylmethoxy-
carbonyl)-L-aspartate (2). A mixture of Fmoc-L-Asp
(OtBu)-OH (26.7 g, 65 mmol), tetra-butylammonium
bromide (2.09 g, 65 mmol), and anhydrous K2CO3
(9.8 g, 71 mmol) in acetonitrile (160 mL) was stirred at
room temperature for 20 min. Benzyl bromide
(8.4 mL, 71 mmol) in acetonitrile (10 mL) was then add-
ed dropwise with intensive stirring. The mixture was stir-
red for 12 h at room temperature. Then the precipitate
was filtered off, and a filtrate was evaporated to dryness.
The resulting crude material was dissolved in EtOAc
(200 mL), and the organic layer was washed with a sat-
urated
NaHCO3
solution
(3·100 mL),
water
(3·100 mL), and brine, dried over Na2SO4, and finally
was concentrated in a vacuum. The residue was crystal-
lized from EtOAc/n-hexane (2/8, v/v) to afford 2 as a
white precipitate (27.8 g, 86% yield). Mp: 108–109 ꢁC;
1
Rf = 0.73 (S5); HPLC (tR, 3.69 min); H NMR (CDCl3)
5.1.5. General procedure D: preparation of N-((S)-1-{4-
[4-arylpiperazin-1-yl]butyl}pyrrolidin-2,5-dion-3-yl)cyclo-
alkanecarboxamides 19–26. The 4-tert-butyl N-(cyclo-
alkylcarbonyl)-1-{4-[4-arylpiperazin-1-yl]butyl}-L-aspar-
tamide (11–18) (2.5 mmol) was dissolved in an 8 mL
mixture of TFA/CH2Cl2 (9/1, v/v) and was stirred at a
room temperature. After complete deprotection (TLC
control), an excess of TFA was removed under reduced
d (ppm) 1.39 (s, 9H, C(CH3)3), 2.73–2.80 (dd, 1H, Hb
Asp, J = 16.9, 4.4 Hz), 2.92–2.99 (dd, 1H, Hb Asp,
J = 16.9, 4.6 Hz), 4.19–4.43 (m, 3H, CHCH2O,
CHCH2O), 4.60–4.66 (m, 1H, Ha Asp), 5.13–5.25 (m,
2H, COCH2Ph), 5.81–5.83 (d, 1H, NH, J = 8.6 Hz),
7.24–7.76 (m, 13H, Ph); ESI-MS (M+H+) 502.2. Anal.
(C30H31NO6) C, H, N.

P. Zajdel et al. / Bioorg. Med. Chem. 15 (2007) 2907–2919
2915
pressure and the residue was evaporated to dryness by
repeated co-evaporation with CH2Cl2 and ether to yield
a yellowish foam. The obtained acid was left to drying in
a desiccator overnight, and was further used without
analytical identification. Afterward, it was dissolved in
acetic anhydride (4 mL), and sodium acetate (0.2 g,
2.5 mmol) was added. The mixture was heated at 60 ꢁC
for 2 h under stirring. When the cyclization was accom-
plished, the mixture was concentrated under reduced
pressure and the residue was dissolved in EtOAc
(50 mL). The organic phase was washed with a saturated
NaHCO3 (2·50 mL) and water (2·50 mL), and was
dried over MgSO4, and finally concentrated in a vacu-
um. Further purification by a column chromatography
over silica gel afforded a pure product in the form of a
white solid or a transparent oil.
Anal. (C28H40N4O4) C, H, N. 21ÆHClÆ3/2 H2O: mp:
209–211 ꢁC. Anal. (C28H40N4O4ÆHClÆ3/2 H2O) C, H, N.
5.1.9. N-((S)-1-{4-[4-(2-Methoxyphenyl)piperazin-1-yl]bu-
tyl}pyrrolidin-2,5-dion-yl)-1-adamantanecarboxamide (22).
Obtained from 14 as a white solid (0.89 g, a 64% yield);
chromatography (SiO2, CH2Cl2/MeOH, 9:1); mp: 126–
128 ꢁC; Rf = 0.61 (S1), 0.27 (S3); HPLC (tR 2.39 min);
20
½aꢀ ¼ ꢁ0:56ꢂ (c 0.5, MeOH); 1H NMR (CDCl3) d
D
(ppm)
1.65–1.90
(cluster,
16H,
CH2-Ada,
NCH2CH2CH2), 2.08 (s, 3H, CH-Ada), 2.54–2.54 (m,
2H, CH2CH2N(CH2)2), 2.73–2.81 (m, 5H, N(CH2)2,
Hb imide), 3.04–3.13 (q, 1H, Hb imide), 3.17 (m, 4H,
(CH2)2N), 3.60–3.65 (t, 2H, NCH2, J = 6.9 Hz), 3.89
(s, 3H, OCH3), 4.34–4.41 (m, 1H, Ha imide), 6.43–6.45
(m, 1H, NH, J = 5.8 Hz), 6.87–7.06 (m, 4H, Ph); ESI-
MS (M+H+) 523.5. Anal. (C30H42N4O4) C, H, N. 22ÆH-
ClÆ3/2 H2O: mp: 222–225 ꢁC. Anal. (C30H42N4O4ÆHClÆ3/
2 H2O) C, H, N.
5.1.6. N-((S)-1-{4-[4-(2-Methoxyphenyl)piperazin-1-yl]bu-
tyl}pyrrolidin-2,5-dion-3-yl)cyclopentanecarboxamide (19).
Obtained from 11 as a colorless oil (0.84 g, a 74% yield);
5.1.10. N-((S)-1-{4-[4-(3-Chlorophenyl)piperazin-1-yl]bu-
tyl}pyrrolidin-2,5-dion-yl)cyclopentanecarboxamide (23).
Obtained from 15 as a white solid (0.77 g, a 68 % yield);
chromatography (SiO2, CH2Cl2/MeOH, 9:1); mp: 145–
chromatography (SiO2, CH2Cl2/MeOH, 99:1); Rf = 0.61
20
(S1), 0.26 (S3); HPLC (tR 1.90 min); ½aꢀ ¼ ꢁ0:52ꢂ (c
D
0.5, MeOH); 1H NMR (DMSO) d (ppm) 1.46–1.75
(m, 13H, 9H cPt, NCH2CH2CH2), 2.06–2.48 (m, 3H,
CH2CH2N(CH2)2, Hb imide), 2.85–2.94 (m, 5H,
N(CH2)2, Hb imide), 3.31–3.39 (m, 6H, (CH2)2N,
NCH2), 3.74 (s, 3H, OCH3), 4.29–4.36 (m, 1H, Ha
imide), 6.83–6.94 (m, 4H, Ph), 8.42–8.44 (d, 1H, NH,
147 ꢁC; Rf = 0.67 (S1), 0.43 (S3); HPLC (tR 2.19 min);
20
½aꢀ ¼ ꢁ0:76ꢂ (c 0.5, MeOH); 1H NMR (DMSO) d
D
(ppm) 1.49–1.71 (m, 13H, 9H cPt, NCH2CH2CH2),
2.56–2.58 (m, 2H, CH2CH2N(CH2)2), 2.87–2.96 (q,
1H, Hb imide), 3.02–3.22 (m, 5H, N(CH2)2, Hb imide),
3.38–3.49 (m, 4H, (CH2)2N), 3.80–3.85 (d, 2H, NCH2),
4.31–4.37 (m, 1H, Ha imide), 6.83–7.02 (m, 3H, Ph),
7.21–7.27 (t,1H, Ph), 8.58–8.60 (d, 1H, NH,
J = 7.4 Hz);
ESI-MS
(M+H+)
457.3.
Anal.
(C25H36N4O4) C, H, N. 19ÆHClÆH2O: mp: 181–182 ꢁC.
Anal. (C21H36N4O4ÆHClÆH2O) C, H, N.
J = 7.4 Hz);
ESI-MS
(M+H+)
461.2.
Anal.
5.1.7. N-((S)-1-{4-[4-(2-Methoxyphenyl)piperazin-1-yl]bu-
tyl}pyrrolidine-2,5-dion-3-yl)cyclohexanecarboxamide (20).
Obtained from 12 as a white solid (0.88 g, a 76% yield);
chromatography (SiO2, CH2Cl2/MeOH, 9:1); mp: 113–
(C24H33ClN4O3) C, H, N. 23ÆHClÆH2O: mp: 152–
154 ꢁC. Anal. (C24H33ClN4O3ÆHClÆH2O) C, H, N.
5.1.11. N-((S)-1-{4-[4-(3-Chlorophenyl)piperazin-1-yl]bu-
tyl}pyrrolidin-2,5-dion-3-yl)cyclohexanecarboxamide (24).
Obtained from 16 as a white solid (0.84 g, a 71% yield);
chromatography (SiO2, CH2Cl2/MeOH, 9:1); mp: 155–
115 ꢁC; Rf = 0.53 (S1), 0.28 (S3); HPLC (tR 2.07 min);
20
½aꢀ ¼ ꢁ0:54ꢂ (c 0.5, MeOH); 1H NMR (DMSO) d
D
(ppm) 1.14–1.68 (cluster, 14H, 10H, CH2-cHex,
NCH2CH2CH2), 2.03–2.09 (m, 1H, CH-cHex), 2.26–
2.30 (m, 2H, CH2CH2N(CH2)2), 2.40–2.49 (m, 5H,
N(CH2)2, Hb imide), 2.84–2.93 (m, 5H, (CH2)2N, Hb
imide), 3.35–3.39 (m, 2H, NCH2), 3.74 (s, 3H, OCH3),
4.30–4.37 (m, 1H, Ha imide), 6.83–6.92 (m, 4H, Ph),
8.34–8.37 (d, 1H, NH, J = 7.7 Hz); ESI-MS (M+H+)
471.3. Anal. (C26H38N4O4) C, H, N. 20ÆHClÆH2O: mp:
190–192 ꢁC. Anal. (C26H38N4O4ÆHClÆH2O) C, H, N.
157 ꢁC; Rf = 0.66 (S1), 0.38 (S3); HPLC (tR 2.32 min);
20
½aꢀ ¼ ꢁ0:64ꢂ (c 0.5, MeOH); 1H NMR (CDCl3) d
D
(ppm) 1.18–1.89 (cluster, 14H, 10H, CH2-cHex,
NCH2CH2CH2), 2.09–2.20 (m, 1H, CH-cHex), 2.40–
2.45 (m, 2H, CH2CH2N(CH2)2), 2.58–2.61 (m, 4H,
N(CH2)2), 2.70–2.78 (dd, 1H, Hb imide, J = 17.9 Hz,
J = 5.9 Hz), 3.02–3.12 (q, 1H, Hb imide), 3.18–3.22 (m,
4H, (CH2)2N), 3.56–3.60 (m, 2H, NCH2, J = 6.9 Hz),
4.26–4.33 (m, 1H, Ha imide), 8.34–8.37 (d, 1H, NH),
6.75–6.87 (m, 3H, Ph), 7.12–7.18 (t, 1H, Ph,
5.1.8. N-((S)-1-{4-[4-(2-Methoxyphenyl)piperazin-1-yl]
butyl}pyrrolidin-2,5-dion-3-yl)-2-(bicyclo[2.2.1]heptan-2-
yl)acetamide (21). Obtained from 13 as a white solid
(0.91 g, a 74% yield); chromatography (SiO2, CH2Cl2/
MeOH, 9:1); mp: 110–111 ꢁC; Rf = 0.63 (S1), 0.34 (S3);
J = 8.2 Hz);
ESI-MS
(M+H+)
475.4.
Anal.
(C25H35ClN4O3) C, H, N. 24ÆHCl: mp: 169–172 ꢁC.
Anal. (C25H35ClN4O3ÆHCl) C, H, N.
20
1
HPLC (tR 2.30 min); ½aꢀ ¼ ꢁ1:28ꢂ (c 0.5, MeOH); H
5.1.12.
N-((S)-1-{4-[4-(3-Chlorophenyl)piperazin-1-
D
NMR (CDCl3) d (ppm) 1.03–1.30 (m, 6H, CH2–Nrbo),
1.42–1.66 (m, 6H, CH2–Nrbo, NCH2CH2CH2), 1.83–
2.10 (m, 3H, CH–Nrbo), 2.165–2.247 (m, 2H, CH2CO),
2.46–2.48 (m, 2H, CH2CH2N(CH2)2), 2.69–2.78 (m, 5H,
N(CH2)2, Hb imide), 3.04–3.13 (m, 5H, (CH2)2N, Hb
imide), 3.56–3.61 (t, 2H, NCH2, J = 7.0 Hz), 3.85 (s,
3H, OCH3), 4.34–4.39 (m, 1H, Ha imide), 6.27 (m, 1H,
NH), 6.84–7.02 (m, 4H, Ph); ESI-MS (M+H+) 497.4.
yl]butyl}pyrrolidin-2,5-dion-3-yl)-2-(bicyclo[2.2.1]hep-
tan-2-yl)acetamide (25). Obtained from 17 as a white
solid (0.84 g, a 67% yield); chromatography (SiO2,
CH2Cl2/MeOH, 9:1); mp: 187–189 ꢁC; Rf = 0.67 (S1),
20
0.50 (S3); HPLC (tR 2.55 min) ½aꢀ ¼ ꢁ1:06ꢂ (c 0.5,
D
1
MeOH); H NMR (CDCl3) d (ppm) 1.04–1.15 (m, 6H,
CH2–Nrbo),
NCH2CH2CH2), 1.83–2.10 (m, 3H, CH–Nrbo),
1.26–1.69
(m,
6H,
CH2–Nrbo,

2916
P. Zajdel et al. / Bioorg. Med. Chem. 15 (2007) 2907–2919
2.17–2.25 (m, 2H, CH2CO), 2.44–2.46 (m, 2H,
CH2CH2N(CH2)2), 2.61 (s, 4H, N(CH2)2), 2.72–2.79
(dd, 1H, Hb imide, J = 17.9 Hz), 3.03–3.12 (dd, 1H,
Hb imide, J = 17.9 Hz), 3.19–3.23 (m, 4H, (CH2)2N),
3.56–3.61 (t, 2H, NCH2), 4.28–4.35 (m, 1H, Ha imide),
6.17 (s, 1H, NH), 6.75–6.87 (m, 3H, Ph), 7.12–7.18 (t,
1H, Ph); ESI-MS (M+H+) 501.2. Anal. (C27H37ClN4O3)
C, H, N. 25ÆHClÆ3/2 H2O: mp: 164–166 ꢁC. Anal.
(C27H37ClN4O3ÆHClÆ3/2 H2O) C, H, N.
5.1.16. General procedure D: 1-(cycloalkylcarbonyl)-N-
{4-[4-arylpiperazin-1-yl]butyl}-L-prolinamides 29–34.
Compound 27 or 28 was allowed to react with TFA
for 60 min, and all the volatilities were removed under
reduced pressure. Then deprotected compounds trifluo-
roacetates 27a and 28a (2.3 mmol) were coupled with
cycloalkane carboxylic acid (2.5 mmol) using BOP
(2.5 mmol) in the presence of TEA (8.5 mmol) in
CH2Cl2 (40mL). Reaction mixture was stirred for 12 h
and the solvent was removed under reduced pressure.
Then the oily residue was dissolved in EtOAc, washed
with a saturated NaHCO3 solution, water, and brine,
dried over Na2SO4, and finally was concentrated under
vacuum. The crude residue was purified by column chro-
matography as detailed below to give compounds 29–34.
5.1.13. N-((S)-1-{4-[4-(3-Chlorophenyl)piperazin-1-yl]bu-
tyl}pyrrolidin-2,5-dion-yl)-1-adamantanecarboxamide (26).
Obtained from 18 as a white solid (0.94 g, a 72% yield);
chromatography (SiO2, CH2Cl2/MeOH, 9:1); mp: 201–
204 ꢁC; Rf = 0.68 (S1), 0.47 (S3); HPLC (tR 2.64 min);
20
½aꢀ ¼ ꢁ0:81ꢂ (c 0.5, MeOH); 1H NMR (CDCl3) d
D
5.1.17. 1-(Cyclohexylcarbonyl)-N-{4-[4-(2-methoxyphe-
nyl)piperazin-1-yl]butyl}-L-prolinamide (29). Obtained
from a trifluoroacetate of 27 (27a, 2.3 mmol) and cyclo-
hexane carboxylic acid as a clear oil (0.8 g, a 74% yield);
(ppm) 1.56–2.05 (cluster, 16H, 12H,CH2-Ada,
NCH2CH2CH2), 2.05 (s, 3H, CH-Ada), 2.41–2.46 (m,
2H, CH2CH2N(CH2)2), 2.60 (s, 4H, N(CH2)2), 2.69–
2.77 (dd, 1H Hb imide, J = 17.9 Hz, J = 5.9 Hz), 3.00–
3.09 (q, 1H, Hb imide), 3.19–3.22 (m, 4H, (CH2)2N),
3.56–3.60 (t, 2H, NCH2, J = 7.0 Hz), 4.22–4.29 (m, 1H,
Ha imide), 6.27–6.29 (d, 1H, NH, J = 5.4 Hz), 6.75–6.86
(m, 3H, Ph), 7.12–7.18 (t, 1H, Ph, J = 8.1 Hz); ESI-MS
(M+H+) 527.3. Anal. (C29H39ClN4O3) C, H, N. 26ÆHCl:
mp: 185–187 ꢁC. Anal. (C29H39ClN4O3ÆHCl) C, H, N.
chromatography (SiO2, CH2Cl2/MeOH, 9:1); Rf = 0.73
20
D
(S1), 0.18 (S2); HPLC (tR 2.17 min); ½aꢀ ¼ ꢁ4:6ꢂ (c
1
0.5, MeOH); H NMR (CDCl3) d (ppm) 1.19–1.30 (m,
4H, NHCH2CH2CH2), 1.46–1.54 (m, 5H, CH2-cHex,
Hc Pro), 1.71–1.82 (m, 7H, CH2-cHex, Hc Pro), 1.95–
1.98 (m, 1H, CH-cHex), 2.34–2.43 (m, 4H,
CH2CH2N(CH2)2, 2Hb Pro), 2.67 (s, 4H, N(CH2)2),
3.10 (s, 4H, (CH2)2N), 3.21–3.25 (m, 2H, NHCH2),
3.46–3.58 (m, 2H, Hd Pro), 3.85 (s, 3H, OCH3), 4.54–
4.58 (dd, 1H, Ha Pro, J = 7.95, 1.8 Hz), 6.84–7.01 (m,
4H, Ph), 7.26–7.28 (m, 1H, NH); ESI-MS (M+H+)
470.99. Anal. (C27H42N4O3) C, H, N. 29ÆHCl: mp:
120–122 ꢁC. Anal. (C27H43ClN4O3ÆHCl) C, H, N.
5.1.14. 1-(tert-Butoxycarbonyl)-N-{4-[4-(2-methoxyphen-
yl)piperazin-1-yl]butyl}-L-prolinamide (27). Obtained
from Boc-L-proline (3.44 g, 16 mmol), 4-[4-(2-methoxy-
phenyl)piperazin-1-yl]butylamine (3.82 g, 14.5 mmol),
and BOP (7.07 g, 16 mmol) in CH2Cl2 (100 mL) in the
presence of TEA (4.46 mL, 32 mmol) as a yellow oil
(5.7 g, a 76% yield); chromatography (SiO2, CH2Cl2/
MeOH, 9:1); Rf = 0.92 (S1), 0.39 (S2); HPLC (tR
2.03 min); 1H NMR (CDCl3) d (ppm) 1.32 (s, 9H,
C(CH3)3), 1.37–1.44 (m, 4H, NHCH2CH2CH2), 1.71–
1.78 (m, 3H, 1Hb Pro, 2Hc Pro), 2.06–2.08 (m, 1H, Hb
Pro), 2.52–2.69 (m, 2H, CH2CH2N(CH2)2), 2.99–3.12
(m, 6H, N(CH2)2, NHCH2), 3.21–3.36 (m, 6H,
(CH2)2N, 2Hd Pro), 3.75 (s, 3H, OCH3), 3.96–4.02 (m,
1H, Ha Pro), 6.82–6.96 (m, 4H, Ph), 7.77–7.83 (m, 1H,
NH); ESI-MS (M+H+) 460.97. Anal. (C25H40N4O4) C,
H, N.
5.1.18. 1-((Bicyclo[2.2.1]heptan-2-yl)acetyl)-N-{4-[4-(2-
methoxyphenyl)piperazin-1-yl]butyl}-L-prolinamide (30).
Obtained from 27a (2.3 mmol) and norbornane-2-acetic
acid as a white solid (0.82 g, 72% yield); chromatogra-
phy (SiO2, CH2Cl2/MeOH, 99:1); mp: 153–155 ꢁC;
Rf = 0.73 (S1), 0.18 (S2); HPLC (tR 2.45 min);
20
D
½aꢀ ¼ ꢁ6:4ꢂ (c 0.5, MeOH); 1H NMR (CDCl3) d
(ppm) 1.00–1.21 (m, 4H, NHCH2CH2CH2), 1.22–2.23
(m, 17H, cluster Nrbo, 2Hb Pro, 2Hc Pro), 3.11–3.22
(m, 2H, CH2CH2N(CH2)2), 3.43–3.67 (m, 8H,
N(CH2)2, (CH2)2N), 3.95–4.12 (m, 7H, NHCH2,
OCH3, 2Hd Pro), 4.52–4.54 (m, 1H, Ha Pro), 7.00–7.42
(m, 2H, Ph), 7.36–7.42 (m, 1H, Ph), 7.66 (m, 1H, Ph),
8.01 (s, 1H, NH); ESI-MS (M+H+) 497.1. Anal.
(C29H44N4O3) C, H, N. 30ÆHCl: mp: 162–164 ꢁC. Anal.
(C29H45ClN4O3ÆHCl) C, H, N.
5.1.15.
1-(tert-Butoxycarbonyl)-N-{4-[4-(3-chlorophe-
nyl)piperazin-1-yl]butyl}-L-prolinamide (28). Obtained
from Boc-L-proline (3.44 g, 16 mmol), 4-[4-(3-chlor-
ophenyl)piperazin-1-yl]butylamine (3.88 g, 14.5 mmol),
and BOP (7.07 g, 16 mmol) in CH2Cl2 (100 mL) in the
presence of TEA (4.46 mL, 32 mmol) as a white solid
(5.3 g, a 72% yield); chromatography (SiO2, CH2Cl2/
MeOH, 9:1); mp: 132–134 ꢁC; Rf = 0.91 (S1), 0.39 (S2);
5.1.19. 1-(Adamantylcarbonyl)-N-{4-[4-(2-methoxyphe-
nyl)piperazin-1-yl]butyl}-L-prolinamide (31). Obtained
from 27a (2.3 mmol) and adamantane 1-carboxylic acid
as a pale white oil (0.86 g, a 72% yield); chromatography
(SiO2, CH2Cl2/MeOH, 99:1); Rf = 0.76 (S1), 0.17 (S2);
1
HPLC (tR 2.32 min); H NMR (CDCl3) d (ppm) 1.46
(s, 9H, C(CH3)3), 1.54–1.56 (m, 4H, NHCH2CH2CH2),
1.87–1.95 (m, 4H, 2Hb, 2Hc Pro), 2.39–2.41 (m, 2H,
CH2CH2N(CH2)2), 2.57–2.60 (m, 4H, N(CH2)2), 3.18–
3.21 (m, 4H, (CH2)2N), 3.24–3.42 (m, 4H, NHCH2,
2Hd Pro), 4.23 (s, 1H, Ha Pro), 6.75–6.81 (m, 2H, Ph),
6.85–6.87 (t, 1H, Ph, J = 8.0 Hz), 7.12–7.18 (m, 1H,
Ph); ESI-MS (M+H+) 465.1. Anal. (C24H37ClN4O3) C,
H, N.
20
1
HPLC (tR 2.53 min); ½aꢀ ¼ ꢁ5:8ꢂ (c 0.5, MeOH); H
D
NMR (DMSO) d (ppm) 1.43 (m, 4H, NHCH2CH2CH2),
1.68–1.97 (m, 19H, cluster Ada, 2Hb Pro, 2Hc Pro),
2.30–2.32 (m, 2H, CH2CH2N(CH2)2), 2.51–2.52 (m,
4H, N(CH2)2), 2.96–3.13 (m, 6H, (CH2)2N, NHCH2),
3.70 (s, 2H, Hd Pro), 3.77 (s, 3H, OCH3), 4.31 (s, 1H,
Ha Pro), 6.87–6.97 (m, 4H, Ph), 7.65 (s, 1H, NH);

P. Zajdel et al. / Bioorg. Med. Chem. 15 (2007) 2907–2919
2917
ESI-MS (M+H+) 522.95. Anal. (C31H46N4O3) C, H, N.
31ÆHCl: mp: 172–174 ꢁC. Anal. (C31H47ClN4O3ÆHCl) C,
H, N.
receptors in the hippocampus of rat brain, and for
5-HT2A receptors in the cortex according to the pub-
lished procedures.45,46 The following radioligands were
used: [3H]-8-OH-DPAT (190 Ci/mmol, Amersham),
[3H]-ketanserin (60 Ci/mmol, NEN Chemicals) for
5-HT1A- and 5-HT2A receptors, respectively. Ki values
were determined on the basis of at least three competi-
tion experiments in which 8–10 drug concentrations
(10ꢁ10–10ꢁ3 M) were run in triplicate.
5.1.20.
1-(Cyclohexylcarbonyl)-N-{4-[4-(3-chlorophe-
nyl)piperazin-1-yl]butyl}-L-prolinamide (32). Obtained
from trifluoroacetate of 28 (28a, 2 mmol) and cyclohex-
ane carboxylic acid as a transparent oil (0.78 g, a 82%
yield); chromatography (SiO2, CH2Cl2/MeOH, 99:1);
Rf = 0.76 (S1), 0.17 (S2); HPLC (tR 2.43 min);
20
D
½aꢀ ¼ ꢁ5:6ꢂ (c 0.5, MeOH); 1H NMR (CDCl3) d
5.2.2. D2 dopaminergic receptor binding assay. Competi-
tion binding studies were performed on rat striatal mem-
branes, prepared according to the previously published
procedure. The assay was carried out in a 96-well filter
plate (containing glass fiber type C, Millipore), pre-
soaked with 100 lL of an ice-cold 50 mM potassium
phosphate buffer (pH 7.4), and filtered using a Millipore
Vacuum Manifold prior to sample addition. 150 lL ali-
quots of striatal membrane preparations, 50 lL of the
radioligand ([3H]-spiperone, 15.70 Ci/mmol, NEN
Chemicals), and either 50 lL of the buffer (for total
binding assay) or 50 lL of ( )-butaclamol (5 lM) to
determine the unspecific binding, or 50 lL of the com-
pounds to be tested, were added to each well. Addition-
ally, to prevent [3H]-spiperone binding to 5-HT2A
receptors, ketanserin (50 nM) was included in the assay
buffer. After incubation at 37 ꢁC for 30 min, the binding
reaction was terminated by vacuum filtration and
washed three times with 200 lL of the buffer. Radioac-
tivity was determined by a liquid scintillation counting
using Beckman LS 6500 apparatus.
(ppm) 1.20–1.42 (m, 4H, NHCH2CH2CH2), 1.44–1.52
(m, 4H, CH2-cHex), 1.71–1.84 (m, 6H, CH2-cHex),
1.931–2.25 (m, 3H, Hb, 2Hc Pro), 2.33–2.46 (m, 4H,
CH2CH2 N(CH2)2, Hb Pro, CH-cHex), 2.56–2.59 (m,
4H, N(CH2)2), 3.17–3.25 (m, 6H, (CH2)2N, NHCH2),
3.43–3.60 (m, 2H, Hd Pro), 4.54–4.57 (dd, 1H, Ha Pro,
J = 7.95, 1.8 Hz), 6.75–6.85 (m, 3H, Ph), 7.12–7.17 (t,
1H, Ph, J = 8.0 Hz), 7.26–7.27 (m, 1H, NH,
J = 7.4 Hz);
ESI-MS
(M+H+)
475.15.
Anal.
(C26H39ClN4O2) C, H, N. 32ÆHCl: mp: 128–130 ꢁC.
Anal. (C26H40Cl2N4O2ÆHCl) C, H, N.
5.1.21. 1-((Bicyclo[2.2.1]heptan-2-yl)acetyl)-N-{4-[4-(3-
chlorophenyl)piperazin-1-yl]butyl}-L-prolinamide (33).
Obtained from 28a (2 mmol) and norbornane-2-acetic
acid as a colorless oil (0.68 g, a 68% yield); chromato-
graphy (SiO2, CH2Cl2/MeOH, 99:1); Rf = 0.94 (S1),
20
D
0.29 (S2); HPLC (tR 2.63 min); ½aꢀ ¼ ꢁ7:4ꢂ (c 0.5,
1
MeOH); H NMR (CDCl3) d (ppm) 1.05–1.31 (m, 4H,
NHCH2CH2CH2), 1.47–1.54 (m, 8H, CH2–Nrbo),
1.74–1.84 (m, 2H, Hc Pro), 1.94–1.97 (m, 3H, CH–
Nrbo), 2.10–2.28 (m, 3H, Hb Pro, CH2CO), 2.41–2.47
(m, 3H, CH2CH2N(CH2)2, Hb Pro), 2.59–2.63 (m, 4H,
N(CH2)2), 3.19–3.54 (m, 6H, (CH2)2N, NHCH2), 3.39–
3.56 (m, 2H, Hd Pro), 4.55–4.57 (d, 1H, Ha Pro,
J = 7.4 Hz), 6.75–6.87 (m, 3H, Ph), 7.12–7.18 (t, 1H,
Ph, J = 8.2 Hz), 7.27–7.31 (d, 1H, NH); ESI-MS
(M+H+) 501.1. Anal. (C28H41ClN4O2) C, H, N. 33ÆHCl:
mp: 221–222 ꢁC. Anal. (C28H42Cl2N4O2ÆHCl) C, H, N.
The estimated Ki values were determined from three
competition binding experiments in which 2 drug con-
centrations (0.1 and 1 lM), each run in triplicate, were
used. The Cheng and Prusoff equation was used for Ki
calculations.47
5.3. In vivo experiments
The experiments were performed on male Wistar rats
(280–310 g), and male Albino Swiss (24–28 g) or
C57BL/6J (22–23 g) mice (the latter strain was used in
the tail suspension test only). The animals were kept at
a room temperature of 20 1 ꢁC, and had free access
to food (standard laboratory pellets, LSM) and tap
water before the experiment. All the investigations were
conducted in the light phase, on a natural day–night
cycle (from March to July), between 9 am and 2 pm.
All the experimental procedures were approved by the
Local Bioethics Commission at the Institute of
5.1.22.
1-(Adamantylcarbonyl)-N-{4-[4-(3-chlorophe-
nyl)piperazin-1-yl]butyl}-L-prolinamide (34). Obtained
from 28a (2 mmol) and adamantane-1-carboxylic acid
as a white solid (0.81 g, a 78% yield); chromatography
(SiO2, CH2Cl2/MeOH, 99:1); mp: 128–130 ꢁC;
Rf = 0.88 (S1), 0.38 (S2); HPLC (tR 2.80 min);
20
D
½aꢀ ¼ ꢁ7:6ꢂ (c 0.5, MeOH); 1H NMR (CDCl3) d
(ppm) 1.42–1.56 (m, 4H, NHCH2CH2CH2), 1.67–1.98
(m, 15H, cluster Ada, Hb, 2Hc Pro), 2.04 (s, 3H, CH-
Ada), 2.18–2.25 (m, 1H, Hb Pro), 2.39–2.43 (m, 2H,
CH2CH2N(CH2)2), 2.57–2.61 (m, 4H, N(CH2)2), 3.18–
3.27 (m, 6H, (CH2)2N, NHCH2), 3.66–3.85 (m, 2H, Hd
Pro), 4.59–4.63 (m, 1H, Ha Pro, J = 7.95, 3.3 Hz),
6.75–6.86 (m, 3H, Ph, J = 8.1 Hz), 7.12–7.17 (t, 1H,
Ph, J = 8.0 Hz); ESI-MS (M+H+) 526.98. Anal.
(C30H43ClN4O2) C, H, N. 34ÆHCl: mp: 241–243 ꢁC.
Anal. (C30H44Cl2N4O2ÆHCl) C, H, N.
´
Pharmacology, Polish Academy of Sciences in Krakow.
8-Hydroxy-2-(di-n-propylamino)tetralin hydrobromide
(8-OH-DPAT, Tocris) and ( )-2,5-dimethoxy-4-iod-
ophenyl)-2-aminopropane hydrochloride (( )-DOI,
Research Biochemical, Inc) were dissolved in saline, N-
(2-pyridinyl)cyclohexanecarboxamide trihydrochloride
(WAY 100635, synthesized by Dr. J. Boksa, Institute
´
of Pharmacology, Polish Academy of Sciences, Krakow,
5.2. In vitro studies
Poland) was used as aqueous solutions. The compounds
under study were suspended in a 1% aqueous solution of
Tween 80. 8-OH-DPAT and WAY 100635 were injected
subcutaneously (sc), ( )-DOI and the tested compounds
5.2.1. 5-HT1A, 5-HT2A receptor binding assays. Radioli-
gand binding experiments were conducted for 5-HT1A

2918
P. Zajdel et al. / Bioorg. Med. Chem. 15 (2007) 2907–2919
were given intraperitoneally (ip) in a volume of 2 mL/kg
(rats) and 10 mL/kg (mice). Each experimental group
consisted of six to ten animals per dose and all the ani-
mals were used only once.
which evoked a visible flight reaction of an animal. If
the animal continued running, it received no new shocks
for the following 3 s. Episodes of punished crossing were
counted for 60 s.32
5.3.1. Body temperature in mice. Effects of the tested
compounds given alone on the rectal body temperature
in mice (measured with an Ellab thermometer) were
recorded 30, 60, 90, and 120 min after their administra-
tion. In a separate experiment, the effect of WAY
100635 (0.1 mg/kg) on the hypothermia induced by com-
pounds 19–23, and 29–34 or 8-OH-DPAT, was tested.
WAY 100635 was administered 15 min before the com-
pounds or 8-OH-DPAT and rectal body temperature
was recorded 30 and 60 min after injection of the tested
compounds. In another experiment, effects of 24–26 or
WAY 100635 (which did not change mouse body tem-
perature) on the 8-OH-DPAT (5 mg/kg)-induced hypo-
thermia were assessed. The tested compounds and
WAY 100635 were administered at 45 and 15 min,
respectively, before 8-OH-DPAT, and rectal body
temperature was measured 15, 30, 45, and 60 min after
8-OH-DPAT injection. The results were expressed as a
change in body temperature (Dt) with respect to the bas-
al body temperature measured at the beginning of the
experiment.
5.3.5. Forced swimming test in mice. The experiment was
carried out according to the method of Porsolt et al.33
Briefly, mice were individually placed in a glass cylinder
(25 cm high, 10 cm in diameter) containing 6 cm of
water maintained at 23–25 ꢁC, and were left therein
for 6 min. A mouse was regarded as immobile when it
remained floating in the water, making only small move-
ments to keep its head above it. The total duration of
immobility was recorded during the last 4 min of a
6-min test session.
5.3.6. Tail suspension test in mice. The experiment was
carried out on C57BL/6J mice according to the method
of Steru et al.34 The mice were individually hung by the
tail using an adhesive tape placed approximately 1 cm
from the tip of the tail attached to a wooden stick and
hanging 75 cm above the floor. The total duration of
immobility was scored manually during a 6-min test ses-
sion. Immobility was defined as the absence of any limb
or body movements, except for those caused by
respiration.
5.3.2. Lower lip retraction (LLR) in rats. LLR was as-
sessed according to the method described by Berendsen
et al.27,28 The rats were individually placed in cages
(30 · 25 · 25 cm) and were scored three times (at 15,
30, and 45 min) after administration of the tested com-
pounds or 8-OH-DPAT as follows: 0, lower incisors
not visible; 0.5, partly visible; 1, completely visible. To-
tal maximum scores amounted to 3 for each rat. In a
separate experiment, the effect of the tested compounds
or WAY 100635 on the LLR induced by 8-OH-DPAT
(1 mg/kg) was tested. The compounds and WAY
100635 were administered at 45 and 15 min, respectively,
before 8-OH-DPAT, and the animals were scored 15,
30, and 45 min after 8-OH-DPAT administration.
5.3.7. Locomotor activity in mice. The spontaneous loco-
motor activity of mice was recorded in photoresistor
actometers (24 cm in diameter) illuminated by two light
beams, which were connected to a counter for the
recording of light-beam interruptions. The mice were
placed individually in the actometers, and the number
of crossings of the light beams was counted twice: dur-
ing the first 6 min, that is, at the time equal to the obser-
vation period in the forced swimming test, and during
30-min experimental sessions.
5.4. Statistics
The obtained data were analyzed by a one-way analysis
of variance, followed by Dunnett’s test (when only one
drug was given) or by the Newman–Keuls test (when
two drugs were administered). ID50 values were calculat-
ed by the method of Litchfield and Wilcoxon.
5.3.3. Head twitch response in mice. To habituate mice to
the experimental environment, each animal was ran-
domly transferred to
a
12 cm (diameter) · 20 cm
(height) glass cage lined with sawdust at 30 min before
treatment. Head twitches in mice were induced by ( )-
DOI (2.5 mg/kg). Immediately after the treatment, the
number of head twitches was counted throughout
20 min.30,31 The tested compounds were administered
60 min before ( )-DOI.
Acknowledgments
P.Z. thanks the French Government for the fellowship.
This study was partially supported by Grant No. 2PO5F
042 26 from the State Committee for Scientific Research
(KBN), Warszawa, Poland.
5.3.4. Four-plate test in mice. The box was made of opa-
que plastic and was rectangular (25 · 18 · 16 cm) in
shape. The floor was covered with four rectangular met-
al plates (11 · 8 cm), separated by a 4-mm gap. The
plates were connected to a source of continuous current
which enabled a 120 V difference of potentials between
two adjacent plates for 0.5 s when the experimenter
pressed the switch. Individual mice were placed gently
onto the plate and were allowed to explore for 15 s.
Afterward, each time a mouse passed from one plate
to another, the experimenter electrified the whole floor
Supplementary data
Spectroscopic data on intermediate compounds 3–18
and six tables showing the effect of the investigated com-
pounds on the body temperature of mice, the effect of
WAY 100635 on the hypothermia induced by 19–23
and 29–34 in mice, the effect of 24–26 on the 8-OH-
DPAT-induced hypothermia in mice, the effect of the

P. Zajdel et al. / Bioorg. Med. Chem. 15 (2007) 2907–2919
2919
19. Gerritz, S. W.; Norman, M. H.; Barger, L. A.; Berman, J.;
Bigham, E. C.; Bishop, M. J.; Drewry, D. H.; Garrison, D.
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Marron, B. E.; Nanthakumar, S. S.; Navas, F. J., III
J. Comb. Chem. 2003, 5, 110–117.
20. Feliu, L.; Subra, G.; Martinez, J.; Amblard, M. J. Comb.
Chem. 2003, 5, 356–361.
21. Kubinyi, H. Quant. Struct. -Act. Relat. 1988, 7, 121–133.
22. Glennon, R. A.; Naiman, N. A.; Lyon, R. A.; Titeler, M.
J. Med. Chem. 1988, 31, 1968–1971.
investigated compounds on the 8-OH-DPAT-induced
LLR in rats, the effect of 21,23–26, 33, and ketanserin
on the (( )-DOI-induced head twitch response in mice,
and the effect of 19, 20, and imipramine on the locomo-
tor activity of mice. Supplementary data associated with
this article can be found, in the online version, at
doi:10.1016/j.bmc.2007.02.018.
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