The influence of modifications in imide fragment structure on 5-HT1A and 5-HT7 receptor affinity and in vivo pharmacological properties of some new 1-(m-trifluoromethylphenyl)piperazines

Article abstract of DOI:10.1016/j.bmc.2007.07.029

New, flexible (7, 9, 11 and 13) and rigid (8, 10, 12 and 14) imides with a 1-(m-trifluorophenyl)piperazine fragment and a tetramethylene or a 1e,4e-cyclohexylene spacer, respectively, showed very high affinity (K_i = 0.3-34 nM) and agonistic in vivo activity for 5-HT_1A receptors. Flexible new compounds and the previously described 5 also bound to 5-HT₇ receptors (K_i = 21-134 nM). Selected glutarimide derivatives, that is, the most potent postsynaptic 5-HT_1A receptor agonist rigid compound 8 and its flexible analogue 7, as well as the previously described full agonist-rigid compound 6 and the partial agonist-its flexible counterpart 5 exhibited moderate affinity for α₁-adrenoceptors (K_i = 85-268 nM), but were practically devoid of any affinity for dopamine D₂ sites. Those glutarimides demonstrated anxiolytic- (5 and 7) and antidepressant-like (5, 6 and 8) activity in the four-plate and the swim tests in mice, respectively; at the same time, however, they inhibited the locomotor activity of mice. The antidepressant-like effect of 8 was significantly stronger than that induced by imipramine used as a reference antidepressant.

Full text of DOI:10.1016/j.bmc.2007.07.029

Bioorganic & Medicinal Chemistry 15 (2007) 7116–7125
The influence of modifications in imide fragment structure
on 5-HT1A and 5-HT7 receptor affinity and in vivo
pharmacological properties of some new
1-(m-trifluoromethylphenyl)piperazines
a
a
b
Maria H. Paluchowska,a, Ryszard Bugno, Beata Duszynska, Ewa Tatarczynska,
*
´
´
b
c
b
´
Agnieszka Nikiforuk, Tomasz Lenda and Ewa Chojnacka-Wojcik
aDepartment of Medicinal Chemistry, Polish Academy of Science, Sme˛tna 12, 31-343 Krako´w, Poland
bDepartment of New Drugs Research, Polish Academy of Science, Sme˛tna 12, 31-343 Krako´w, Poland
cDepartment of Neuropsychopharmacology, Polish Academy of Science, Sme˛tna 12, 31-343 Krako´w, Poland
Received 26 February 2007; revised 4 July 2007; accepted 10 July 2007
Available online 22 August 2007
Abstract—New, flexible (7, 9, 11 and 13) and rigid (8, 10, 12 and 14) imides with a 1-(m-trifluorophenyl)piperazine fragment and a
tetramethylene or a 1e,4e-cyclohexylene spacer, respectively, showed very high affinity (Ki = 0.3–34 nM) and agonistic in vivo activ-
ity for 5-HT1A receptors. Flexible new compounds and the previously described 5 also bound to 5-HT7 receptors (Ki = 21–134 nM).
Selected glutarimide derivatives, that is, the most potent postsynaptic 5-HT1A receptor agonist rigid compound 8 and its flexible
analogue 7, as well as the previously described full agonist—rigid compound 6 and the partial agonist—its flexible counterpart 5
exhibited moderate affinity for a1-adrenoceptors (Ki = 85–268 nM), but were practically devoid of any affinity for dopamine D2 sites.
Those glutarimides demonstrated anxiolytic- (5 and 7) and antidepressant-like (5, 6 and 8) activity in the four-plate and the swim
tests in mice, respectively; at the same time, however, they inhibited the locomotor activity of mice. The antidepressant-like effect of
8 was significantly stronger than that induced by imipramine used as a reference antidepressant.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Additionally, it has been found that compounds with a
tetramethylene spacer reveal high 5-HT7 receptor affin-
ity, while their constrained analogue practically do not
bind to these sites. Indeed, compound MM 77 (1) and
its rigid analogue MP 349 (2) (Chart 1), either having
an o-OCH3-phenyl fragment, show features of 5-HT1A
receptor antagonists, and 1 (but not 2) has significant
affinity for 5-HT7 sites.2
Structure–intrinsic activity relationship studies into a
group of 1-imido/amido substituted 4-(4-arylpiperazin-
1-yl)butane or cyclohexane derivatives have indicated
that these compounds show high affinity but diversified
functional activity for 5-HT1A receptors. However, the
same 5-HT1A receptor intrinsic activity is observed for
the majority of investigated pairs that is, flexible butane
and rigid cyclohexane counterparts.1–4 The rigid mole-
cules with restricted conformational freedom possess a
defined 3D structure and may thus serve as tools for
investigating ligand–5-HT1A receptor interactions.5
Interestingly, both these ligands demonstrate activity
characteristic of anxiolytics.6 It has also been observed
O
OCH3
N
N
N
R1 R2
O
Keywords: Arylpiperazines; 5-HT1A and 5-HT7 receptors ligands;
Structure–intrinsic activity relationships studies; 5-HT1A receptor
agonists; Anxiolytic- and antidepressant-like activity.
1
MM 77
MP 349
R1 = R2 = H
R1, R2
=
2
(CH2)2
*
Corresponding author. Tel.: +48 12 66263319; fax: +48 12
6374500; e-mail: paluchm@if-pan.krakow.pl
Chart 1.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.029

M. H. Paluchowska et al. / Bioorg. Med. Chem. 15 (2007) 7116–7125
7117
7
´
that other ligands with an o-OCH3 group in the aryl
moiety and with a cyclic amide system in the opposite
Lopez-Rodriquez et al. found two such m-CF3 deriva-
tives with agonistic activity toward postsynaptic 5-
HT1A receptors.
terminal show
a tendency to block postsynaptic
5-HT1A receptors.4 The replacement of the o-OCH3
substituent in structures 1 and 2 with an m-CF3 group
results in a change in 5-HT1A receptor activity; in fact,
these modified ligands 3 and 4 are classified as partial
agonists of postsynaptic 5-HT1A receptors2 (Table 1).
Furthermore, it has been shown that the m-CF3 aryl-
piperazines 5 and 6 containing a 3,3-dimethyl-glutari-
To carry on investigations with this group of com-
pounds, we synthesized a new series of derivatives whose
imide moiety was connected to an m-trifluoromethylsub-
stituted 1-phenylpiperazine fragment via a tetramethyl-
ene (7, 9, 11, 13) or a conformationally defined 1e,
4e-cyclohexylene (8, 10, 12, 14) spacer in order to deter-
mine the influence of imide fragment modifications on
the affinity and functional 5-HT1A receptor activity.
For all new compounds (7–14), as well as for the
previously described 5 and 63 5-HT7 receptor and
a1-adrenoceptor affinity was also determined. More-
over, for new derivatives 7–14 D2 dopamine receptor
affinity was estimated. 5-HT1A pre- and postsynaptic
mide fragment (present in gepirone,
a
5-HT1A
receptor agonist with antianxiety properties) in place
of a succinimide moiety behave like a 5-HT1A receptor
partial and a full agonist, respectively.3 It is notewor-
thy that there are a limited number of data on the role
of the m-CF3 group in the functional activity of
arylpiperazine 5-HT1A receptor ligands. In fact, only
Table 1. Structure and affinity data on serotonin (5-HT1A and 5-HT7), a1-adrenergic and dopaminergic D2 receptors of investigated compounds
CF3
R
N
N
R1 R2
Compound
R
R1
R2
Ki (nM) SEM
a1
5-HT1A
5-HT7
128
D2
O
3a
4b
H
H
H
H
H
21
4
1
0.5
6
483 36
505 62
>30000
>25000
N
–(CH2)2–
–(CH2)2–
–(CH2)2–
–(CH2)2–
1820 38
O
O
N
O
H3C
H3C
5
6
H
H
H
4
9
1b
1b
46
221 17
3
127 14
268 12
2340b
>10000b
O
N
O
7
8
3
2.6 0.3
1
21
1200 70
2
101
85
9
6
>5000c
>5000c
O
9
10
5.9 0.5
0.3 0.1
134 11
>5000
30
4
4
1
>5000c
2640c
N
O
O
11
12
H
H
H
H
34
1.5 0.3
9
31 4
335 22
73
43
7
3
2720c
4680c
N
–(CH2)2–
O
O
H3C
H3C
13
14
2.2 0.6
1.3 0.2
107
>5000
8
48
24
4
2
4370c
3600c
N
–(CH2)2–
O
a Data from Ref. 2.
b Data from Ref. 3.
c The binding experiments to D2 receptors were carried out at two compound concentrations (each run in triplicate) and ligand affinity was expressed
as estimated Ki values.

7118
M. H. Paluchowska et al. / Bioorg. Med. Chem. 15 (2007) 7116–7125
O
CF3
receptor functional activity was evaluated for 7–14 in
in vivo models. Compounds 7 and 8, as well as the
previously investigated by us gepirone analogue 5 and
6, were additionally tested in animal models used for
evaluating potential anxiolytic and antidepressant
activity.
O
O
+
H2N
N
N
R1 R2
CF3
R
N
N
R1 R2
2. Chemistry
7 - 14
The structures of the compounds under study are shown
in Table 1, and their syntheses are illustrated in
Scheme 1.
Scheme 1.
Potential anxiolytic and antidepressant activity was
tested in the four-plate16 and the forced swim tests17 in
mice, respectively.
The flexible target compounds 7, 9, 11 and 13 and the
constrained derivatives 10 and 14 were synthesized from
4-[1-(m-trifluoromethylphenyl)piperazin-4-yl]-butyl-or
4-[1-(m-trifluoromethylphenyl)piperazin-4-yl]cyclohexyl-
amine and the appropriate anhydrides by heating in xy-
lene. In the case of compounds 8 and 12, intermediate
non-cyclic aminoacids were obtained, which were then
cyclized in an acetic anhydride according to the proce-
dure described previously.3 The structures of the newly
synthesized compounds were confirmed by 1H NMR
spectra and an elemental analysis. In the 1H NMR spec-
tra of the rigid compounds 8, 10, 12 and 14, the ob-
served coupling constants in the cyclohexane ring were
consistent with those previously assigned by us to the
1e,4e-diequatorial chair conformation of 1-(2-methoxy-
phenyl)-4-[4-(2-phthal-imido)cyclohexyl]piperazine1 and
1-(2-methoxy-phenyl)-4-[4-(2-succinimido)cyclohexyl]
piperazine.8
4. Results and discussion
Like the previously described 3–6,2,3 all the newly syn-
thesized compounds (7–14) were characterized by a very
high 5-HT1A receptor affinity (Ki = 0.3–34 nM). There-
fore it seems that structural changes in both the imide
pharmacophore and the spacer constitution are not of
importance for the formation of ligand–5-HT1A recep-
tor complexes in the investigated series of compounds.
Regarding 5-HT7 receptors, new flexible derivatives 7,
9, 11 and 13, as well as the previously described 5,3
but not their constrained analogue, showed distinct
affinity; all the same, 9 and 13 were at least 4–5 times less
active. The above results confirm our earlier observa-
tions that introduction of rigidity into the spacer of
imide/amide arylopiperazine derivatives leads to a sig-
nificant decrease or a complete loss of 5-HT7 receptor
affinity. Compounds 5–14 exhibited a high or moderate
a1-adrenoceptor affinity (Ki = 4–268 nM)—higher than
that described earlier for pair 3/4 though. Like pair 3/
4, compounds 5–14 did not bind to D2 receptors.
3. Pharmacology
The compounds were tested in competition binding
experiments for serotonin 5-HT1A and 5-HT7, a1-adren-
ergic and dopamine D2 receptors. Affinity data are col-
lected in Table 1.
At a successive stage of our study, we investigated the
newly synthesized receptor ligands in in vivo models
used for evaluation of pre- and postsynaptic 5-HT1A
receptor functional activity. As has been mentioned in
the Introduction, in our previous study, the parent com-
pounds 3/4 (with fragment V) and 5/6 (with fragment
VI) were characterized by diverse 5-HT1A receptor func-
tional activity; we described 3/4 as partial agonists of
postsynaptic 5-HT1A receptors, 5 as an agonist of pre-
and a partial agonist of postsynaptic 5-HT1A receptors,
and 6 as a full agonist of 5-HT1A sites.2,3 The investiga-
tions described in this paper were conducted mainly in
order to determine whether structural changes in the
imide termini affected 5-HT1A intrinsic activity. The ob-
tained results indicate that of the new tested derivatives,
pairs: 9/10 and 11/12 (both with the bicyclic imide frag-
ment) and the restricted 14 (with two methyl substitu-
ents in imide moiety) show features of agonists of
presynaptic 5-HT1A receptors and of partial agonists
of postsynaptic ones. Indeed, like the 5-HT1A receptor
agonist 8-OH-DPAT, 9–12 and 14 evoked a decrease
in mouse body temperature (Table 2), which was sensi-
The functional activity of the investigated compounds at
pre- and postsynaptic 5-HT1A receptors was tested in
several commonly used in vivo models. It was previously
demonstrated that the hypothermia induced by the 5-
HT1A receptor agonist 8-OH-DPAT [8-hydroxy-2-(di-
n-propylamino)tetralin] in mice was connected with acti-
vation of presynaptic 5-HT1A receptors9,10 and was
abolished by 5-HT1A receptor antagonists such as, for
example, WAY 100635 (N-{2-[4-(o-methoxyphenyl)-1-
piperazinyl]ethyl}-N-(2- pyridinyl)-cyclohexanecarboxa-
mide)11 or MP 3022 (4-[3-(benzotriazol-1-yl)propyl]-1-
(o-methoxyphenyl)piperazine).12 Hence the hypother-
mia produced by the compounds tested in mice (and
reduced by WAY 100635) was regarded as a measure
of presynaptic 5-HT1A receptor agonistic activity. To
determine the postsynaptic 5-HT1A receptor agonistic
effect of the tested 5-HT1A ligands, their ability to induce
lower lip retraction (LLR) in rats was assessed. The 8-
OH-DPAT-induced LLR in rats depended on stimula-
tion of postsynaptic 5-HT1A receptors;13,14 moreover,
it was shown that the latter symptom was sensitive to
5-HT1A receptor antagonists.8,11,15

M. H. Paluchowska et al. / Bioorg. Med. Chem. 15 (2007) 7116–7125
Table 2. The effect of the tested compounds on the body temperature in mice
7119
Treatment
Dose (mg/kg)
Dt SEM (ꢁC)
90 min
30 min
0.1 0.1
60 min
0.1 0.1
120 min
Vehicle
7
—
5
À0.1 0.1
À0.4 0.2
À1.0 0.2b
À0.1 0.1
À0.1 0.2
À0.9 0.1b
À0.5 0.2
À0.4 0.1
10
À1.3 0.3b
À1.1 0.3b
8
2.5
5
À0.7 0.1a
À1.6 0.2b
À0.5 0.2
À1.5 0.1b
À0.5 0.1
À0.3 0.1
À1.5 0.2b
À1.3 0.2b
Vehicle
9
—
10
20
À0.1 0.1
0.2 0.1
À0.1 0.1
0.0 0.2
0.0 0.1
0.0 .0.2
0.1 0.1
0.0 0.2
À1.1 0.2b
0.1 0.1
À1.8 0.2b
À1.3 0.3b
À1.3 0.2b
10
2.5
5
À0.4 0.1
À0.5 0.1
À0.4 0.1
À1.2 0.2b
À1.4 0.2b
À0.7 0.1a
À0.3 0.2
Vehicle
11
—
10
20
À0.1 0.1
À0.3 0.2
À1.5 0.3b
0.0 0.1
À0.2 0.2
À1.3 0.3b
0.0 0.2
À0.9 0.3a
À1.6 0.3b
À0.1 0.2
À0.6 0.2
À1.5 0.2b
12
5
10
À0.7 0.2a
À0.4 0.1
À0.4 0.1
À0.1 0.2
À1.7 0.3b
À1.3 0.3b
À1.1 0.2b
À1.0 0.2b
Vehicle
13
—
5
0.0 0.1
À0.6 0.1a
À0.1 0.1
À0.1 0.1
À0.4 0.1
À0.2 0.1
À0.1 0.1
À0.2 0.1
À0.4 0.1
0.1 0.1
À0.5 0.1
0.1 0.1
À0.1 0.1
0.0 0.1
10
À0.2 0.2
0.1 0.1
14
5
10
À0.2 0.1
À0.1 0.1
À1.0 0.2b
À0.7 0.2a
0.1 0.1
À0.5 0.1
À0.1 0.1
WAY 100635
0.1
À0.1 0.1
The tested compounds were administered 30 min before the test. Absolute initial mean body temperatures were within the range of 36.2 0.6 ꢁC;
n = 6–8 mice per group.
a P < 0.05.
b P < 0.01 versus respective vehicle group.
tive to the 5-HT1A receptor antagonist WAY 100635
(Table 3).
Therefore a contribution of 5-HT1A receptors to this
effect should be excluded.
Moreover, they produced LLR in rats (Table 4); at the
same time, however, the 8-OH-DPAT-induced LLR in
rats was reduced by 9–12 and 14 (Table 4). Unexpect-
edly, compound 13 (a flexible analogue of 14) practically
did not change the body temperature of mice (Table 2),
but—like WAY 100635—reduced the hypothermia in-
duced by 8-OH-DPAT in a statistically significant man-
ner (data not shown). In the LLR test, 13 behaved like a
partial 5-HT1A agonist (Table 4).
On the basis of the results of in vivo functional studies
presented in this paper, it may be concluded that the
glutarimide moiety (I), which is connected to 1-(m-
CF3-phenyl)piperazine by a tetramethylene and in par-
ticular a 1e,4e-cyclohexylene spacer, is beneficial for
postsynaptic 5-HT1A receptor agonistic activity. Such
a modification yields compounds 7/8, potent and very
active in vivo agonists of postsynaptic 5-HT1A recep-
tors. Moreover, it seems that the presence of an addi-
tional ring (II or III) or substituents (IV) in the
imide fragment causes a decrease in postsynaptic 5-
HT1A receptor intrinsic activity; the pairs of com-
pounds 9/10, 11/12 and 13/14 behave like partial
agonists of 5-HT1A receptors (Table 5). The functional
profile of the investigated 5-HT1A receptor ligands sug-
gests that some of them may show anxiolytic- and/or
antidepressant-like activity, since it has been well estab-
lished that activation of 5-HT1A receptors leads to dis-
closure of such activity.
The pair 7/8 (with imide moiety I) can be classified as
agonists of postsynaptic 5-HT1A receptors; like 8-OH-
DPAT, both these compounds induced a marked LLR
(a maximal possible effect) in rats, and did affect the
LLR evoked by 8-OH-DPAT (Table 4). It is noteworthy
that rigid compound 8 produced that symptom at a very
low range of doses (0.1–1 mg/kg). Postsynaptic 5-HT1A
receptor agonistic activity of the previously investigated
analogs (5/6) in the LLR test was observed after admin-
istration of doses higher than those of compounds 7/8;
moreover, the intensity of symptoms induced by pair
5/6 did not reach the maximal possible effect.3 In the
used model of hypothermia (for the evaluation of pre-
synaptic 5-HT1A activity), derivatives 7 and 8 decreased
the body temperature of mice (Table 2), which was not
affected by WAY 100635 (Table 3).
Although the relative contribution of pre- and postsyn-
aptic 5-HT1A receptors to the anxiolytic- and antide-
pressant-like effects of 5-HT1A agonists has not been
explicitly determined,18–20 on the basis of our earlier
study we concluded that the anxiolytic- and antidepres-
sant-like effects of azapirones (i.e. ipsapirone and gepi-

7120
M. H. Paluchowska et al. / Bioorg. Med. Chem. 15 (2007) 7116–7125
Table 3. The effect of WAY 100635 on the hypothermia induced by
the tested compounds in mice
Table 4. Induction of lower lip retraction (LLR) by the investigated
compounds (A) and their effect on the 8-OH-DPAT-induced LLR (B)
in rats
Treatment and dose (mg/kg)
Dt SEM (ꢁC)
Treatment
Dose (mg/kg)
Mean SEM LLR score
30 min
60 min
A
B
Vehicle + vehicle
Vehicle + 7 (10)
WAY 100635 (0.1) + 7 (10)
À0.1 0.1
À0.1 0.1
À1.3 0.2b À1.2 0.3b
Vehicle
7
—
2.5
5
0.1 0.1
1.6 0.1
2.6 0.2b
2.8 0.3b
2.8 0.1
2.2 0.2
NT
À1.0 0.2b À0.8 0.3a
Vehicle + vehicle
Vehicle + 8 (5)
WAY 100635 (0.1) + 8 (5)
0.1 0.1
0.0 0.1
10
NT
À1.6 0.2b À1.5 0.1b
À1.2 0.1b À1.2 0.1b
8
0.1
0.3
1
1.3 0.2b
2.4 0.3b
3.0 0.0b
2.6 0.2
NT
NT
Vehicle + vehicle
Vehicle + 9 (20)
WAY 100635 (0.1) + 9 (20)
0.0 0.1
À1.4 0.2b À1.4 0.1
À0.1 0.1
À0.6 0.2A,a À0.2 0.2B
Vehicle
9
—
2.5
5
0.1 0.1
1.9 0.3b
2.2 0.2b
2.2 0.2b
2.9 0.1
2.3 0.3
1.5 0.0b
1.1 0.2b
Vehicle + vehicle
Vehicle + 10 (5)
WAY 100635 (0.1) + 10 (5)
À0.1 0.1
À0.1 0.1
À1.2 0.2b À1.4 0.2b
0.2 0.1B
0.1 0.2B
10
10
5
1.8 0.3b
2.0 0.2b
2.0 0.2b
2.5 0.3
1.9 0.3b
1.8 0.2b
Vehicle + vehicle
Vehicle + 11 (20)
WAY 100635 (0.1) + 11 (20)
0.1 0.1
0.0 0.1
À1.3 0.2b À1.6 0.2b
À1.2 0.2b À0.6 0.2B
10
20
Vehicle + vehicle
Vehicle + 12 (10)
À0.1 0.1
À0.1 0.1
Vehicle
11
—
10
20
0.1 0.1
1.4 0.2b
1.8 0.1b
2.9 0.1
1.9 0.3b
0.5 0.2b
À1.4 0.1b À1.1 0.2b
À0.5 0.1B À0.3 0.1B
WAY 100635 (0.1) + 12 (10)
Vehicle + vehicle
À0.1 0.1
À0.1 0.1
12
10
20
0.7 0.1a
1.3 0.3b
2.1 0.3
1.9 0.2b
Vehicle + 14 (10)
À1.5 0.2b À1.2 0.2b
WAY 100635 (0.1) + 14 (10)
Vehicle + vehicle
Vehicle + 8-OH-DPAT (5)
WAY 100635 (0.1) + 8-OH-DPAT (5) À0.1 0.1B
À0.8 0.3A À0.5 0.2A
0.1 0.1
0.1 0.1
Vehicle
13
—
10
20
0.1 0.1
1.3 0.1b
1.3 0.2b
2.8 0.1
1.4 0.1b
0.2 0.1b
À1.0 0.1b À0.2 0.1
0.2 0.1
WAY 100635 w as administered 15 min before the compounds studied.
Body temperature was recorded 30 and 60 min after injection of the
tested compounds. Absolute initial mean body temperatures were
within the range of 36.2 0.5 ꢁC; n = 7–8 mice per group.
A P < 0.05.
14
10
20
1.4 0.1b
1.5 0.0b
1.8 0.2b
1.1 0.2b
WAY 100635
0.1
0.0 0.0
0.2 0.2b
The investigated compounds were administered 15 min before the test
(A), or 45 min before 8-OH-DPAT (1 mg/kg); n = 6 rats per group.
B P < 0.01 versus respective vehicle + tested compound group.
a P < 0.05.
NT, not tested.
a P < 0.05.
b P < 0.01 versus respective vehicle + vehicle group.
b P < 0.01 versus vehicle group (A) or versus vehicle + 8-OH-DPAT
group (B).
rone) stem from stimulation of 5-HT1A receptors local-
ized postsynaptically.21,22 Taking into account the func-
tional activity of new derivatives, compounds 7/8, with
the strongest postsynaptic 5-HT1A receptor agonistic
activity were selected for further in vivo preclinical stud-
ies as potential anxiolytics and/or antidepressants. Addi-
tionally, we tested the pattern compounds 5/6, which in
our previous study were described as a partial agonist
and an agonist of postsynaptic 5-HT1A receptors,
respectively.3 Using the four-plate test in mice (an ani-
mal model of anxiety based on spontaneous re-
sponses),16 we showed that 5 and 7 (but not 6 and 8)
induced anxiolytic-like effects (Table 6). In that test,
compound 5 induced a strong anti-punishment effect
comparable—in terms of its potency and active dose
(5 mg/kg)—with the effect evoked by diazepam, used
as a reference anxiolytic drug (Table 6). Potential anti-
depressant activity of 5–8 was tested in the forced swim
test in mice.17 It was demonstrated that 5, 6 and 8 (but
not 7) exerted an effect characteristic of antidepressants
(Table 7).
Table 5. Functional in vivo 5-HT1A receptor activity of the investi-
gated compounds
Compound
5-HT1A activity
Postsynaptic
Presynaptic
7
8
—
—
Agonist
Agonist
9
Agonist
Agonist
Agonist
Agonist
Antagonist
Agonist
Partial agonist
Partial agonist
Partial agonist
Partial agonist
Partial agonist
Partial agonist
10
11
12
13
14
ity did not stimulate the locomotor activity of mice
(Table 8). It is noteworthy that the anti-immobility effect
of 6 was comparable to that induced by imipramine,
whereas 8 (0.3 and 1 mg/kg) shortened immobility time
in a significantly more potent manner than did imipra-
mine (10–20 mg/kg) (Table 7). A question arises whether
the anxiolytic- and/or antidepressant-like effects of 5 and
That effect seemed to be specific, since the tested com-
pounds used in doses evoking antidepressant-like activ-

M. H. Paluchowska et al. / Bioorg. Med. Chem. 15 (2007) 7116–7125
7121
Table 6. The effect of 5–8 and diazepam in the four-plate test in mice
Table 7. The effect of 5–8 and imipramine in the forced swim test in
mice
Treatment
Dose mg/kg
Number of punished
crossings mean SEM
Treatment
Dose mg/kg
Immobility time (s) mean SEM
Vehicle
5
—
2.5
5
4.3 0.4
Vehicle
5
—
5
169.2 11.0
157.7 10.7
112.1 13.5b
F(3,32) = 4.892
P < 0.01
5.3 0.5
6.9 0.7b
7.6 0.9b
F(3,32) = 4.787
P < 0.01
10
10
Vehicle
6
—
161.9 11.6
150.4 9.6
98.2 13.7b
113.3 11.7a
F(3,32) = 6.748
P < 0.01
Vehicle
6
—
3.8 0.3
4.4 0.3
4.6 0.4
4.6 0.4
F(3,32) = 1.112
Ns
1.25
2.5
5
1.25
2.5
5
Vehicle
7
—
5
161.2 7.1
183.9 11.4
196.7 5.6a
F(2,24) = 4.534
P < 0.05
Vehicle
7
—
2.5
5
3.7 0.3
4.3 0.5
10
5.8 0.5a
5.2 0.6
10
F(3,32) = 3.449
P < 0.005
Vehicle
8
—
0.1
0.3
1
167.8 7.2
128.3 9.3
Vehicle
8
—
0.3
1
3.6 0.4
3.9 0.4
3.9 0.3
4.2 0.3
F(3,32) = 0.539
Ns
70.1 11.6b
77.7 16.1b
F(3,32) = 15.782
P < 0.0001
5
Vehicle
Imipramine
—
10
20
167.1 6.7
149.1 10.7
107.8 12.4b
F(2,27) = 8.760
P < 0.01
Vehicle*
Diazepam
—
3.5 0.4
1.25
2.5
5
5.5 0.5a
6.8 0.6b
6.7 0.6b
F(3,36) = 9.514
P < 0.001
The investigated compounds and imipramine were administered
30 min before the test; n = 9–10 mice per group.
a P < 0.05.
b P < 0.01 versus vehicle (Dunnett’s test).
The investigated compounds were administered 30 min before the test;
n = 9–10 mice per group.
a P < 0.05.
b P < 0.01 versus vehicle (Dunnett’s test), ns: not significant.
* Data from Ref. 23.
induced by this compound in the swim test. The motor
activity of mice seems to be partly regulated by postsyn-
aptic noradrenaline receptors in the brain, thus it cannot
be excluded that the sedation of mice induced by the
tested compounds 5–8 is connected with an inhibition
of the noradrenaline system function. The latter com-
pounds are ligands of a1-adrenoceptors, hence it cannot
be excluded that—like in the case of prazosin, an a1-
adrenoceptor blocker28—they may reduce the spontane-
ous locomotor activity of mice due to blockade of
postsynaptic a1-receptors.
7 depend exclusively on a 5-HT1A receptor mechanism,
since these compounds bind to 5-HT7 receptors. Re-
cently the data obtained in our laboratory have shown
that blockade of 5-HT7 receptors produces antianxiety-
and antidepressant-like effects.24,25 It is noteworthy that
1, an antagonist of postsynaptic 5-HT1A and 5-HT7
(Tokarski K., unpublished data) receptors, induces an
anxiolytic-like effect.6,26,27
12
Until now we have not had information about func-
tional 5-HT7 receptor activity of 5 and 7, but participa-
tion of these sites in psychotropic-like properties of these
5-HT1A/5-HT7 ligands cannot be excluded. It has also
been shown that compounds 5–8 reduce the locomotor
activity of mice at all the doses producing statistically
significant effect in the four-plate test or the forced swim
test. Diazepam shows a weak sedative effect in mice at a
dose twice as high as this inducing a minimal anxiolytic-
like effect, whereas imipramine at a dose active in the
forced swim test slightly reduces the locomotor activity
of mice. Strong inhibition of the locomotor activity of
mice, observed after administration of compound 7,
may be the cause of immobility time prolongation
Summing up, it has been found that the newly synthe-
sized flexible and rigid imides containing the 1-(m-
CF3-phenyl)piperazine fragment (7–14) are potent
ligands of 5-HT1A receptors, and in general—like the
previously tested 5–6—show features of agonists of
these sites in in vivo studies (hypothermia and LLR
models). Therefore, it seems that the m-CF3 substituent
in phenylpiperazine, but not structure of the imide frag-
ment, is pivotal for 5-HT1A receptor agonism. Flexible
(but not rigid) imides also bind to 5-HT7 receptors.
The selected glutarimides 5–8 have properties character-
istic of anxiolytics and/or antidepressants, indeed, they
increased number of punished crossings in the four-plate
test and shortened immobility time in the forced swim

7122
M. H. Paluchowska et al. / Bioorg. Med. Chem. 15 (2007) 7116–7125
Table 8. The effect of 5–8 and imipramine on the locomotor activity of
mice
5.1.1. General procedure for the preparation of Com-
pounds 7, 9–11, 13 and 14. Equimolar amounts (2 mmol)
of 4-[4-(m-trifluoromethylphenyl)piperazin-1-yl]butyl-
amine or 4-[4-(m-trifluoromethylphenyl)piperazin-1-
yl)cyclohexylamine and proper anhydride were refluxed
in xylene (20 mL) for 5 h. The solvent was evaporated
under reduced pressure and the residue was purified by
column chromatography. For pharmacological assays
free bases were converted into the hydrochloride salts
in acetone solutions by the treatment with excess of
Et2O saturated with gaseous HCl.
Treatment
Dose mg/kg
Locomotor activity: number of
crossings during
6 min
30 min
Vehicle
5
—
5
393.0 27.12
121.7 18.0b
23.0 6.9b
1054.9 35.8
730.8 88.0b
265.1 42.6b
10
F(2,24) = 99.312 F(2,24) = 43.577
P < 0.0001
P < 0.0001
Vehicle
6
—
0.3
1
406.0 27.3
212.0 33.9b
211.7 35.6b
1092.1 33.1
630.1 99.9b
535.3 80.0b
5.1.1.1. 1-{4-[4-(m-Trifluoromethylphenyl)piperazin-1-
yl]butyl}piperidine-2,6-dione (7). The compound was
prepared by the general procedure from 4-[4-(m-trifluo-
romethylphenyl)piperazin-1-yl]butylamine and glutaric
anhydride in 43% yield as a pale yellow oil, Rf = 0.42
(SiO2, CHCl3/CH3OH = 19/1); 1H NMR (60 MHz) d
7.5–6.8 (m, 4H, Ar-H), 4.0–3.6 (m, 2H, CH2–imide),
3.4–3.0 (m, 4H, piperazine 2CH2), 2.8–2.2 (cluster,
10H), 2.2–1.2 (cluster, 6H). 7Æ2HCl: colourless crystals,
mp 191–193 ꢁC. Anal. (C20H26N3O2F3Æ2HCl) C, H, N.
F(2,24) = 11.925 F(2,24) = 15.213
P < 0.001
P < 0.0001
Vehicle
7
—
5
405.4 43.1
217.1 30.0b
16.3 3.4b
948.6 53.9
526.2 76.9b
179.3 23.3b
F(2,24) = 47.535
P < 0.0001
10
F(2,24) = 41.06
P < 0.0001
Vehicle
8
—
0.3
1
405.4 43.1
208.2 20.4b
121.6 19.7b
948.6 53.9
537.7 43.3b
374.3 52.2b
F(2,24) = 23.872 F(2,24) = 34.999
P < 0.0001
340.6 34.1
338.0 36.4
314.9 24.1
P < 0.0001
5.1.1.2. 2-{4-[4-(m-Trifluoromethylphenyl)piperazin-1-
yl]butyl}-2,3,4,5,6,7-hexahydro-1H-isoindole-1,3-dione (9).
The compound was prepared by the general procedure
from 4-[4-(m-trifluoromethylphenyl)piperazin-1-yl]butyl-
amine and 3,4,5,6-tetrahydrophthalic anhydride in 84%
yield as a pale yellow oil, Rf = 0.60 (SiO2, CHCl3/
Vehicle
—
892.7 76.6
876.8 67.0
625.3 53.6a
Imipramine 10
20
F(2,27) = 0.1945 F(2,27) = 4.930
ns P < 0.05
1
The investigated compounds and imipramine were administered
30 min before the test. n = 9–10 mice per group.
a P < 0.05.
b P < 0.01 versus vehicle (Dunnett’s test).
CH3OH = 19/1); H NMR (60 MHz) d 7.6–6.9 (m, 4H,
Ar–H), 3.7–3.4 (m, 2H, –CH2–imide), 3.4–3.1 (m, 4H,
piperazine 2CH2), 2.7–2.1 (cluster, 10H), 2.1–1.4 (clus-
ter, 8H). 9Æ2HCl: colourless crystals, mp 186–188 ꢁC.
Anal. (C23H28N3O2F3Æ2HCl) C, H, N.
test in mice. Compounds 5 and 8 exhibit the most
potent anxiolytic- and antidepressant-like activity,
respectively, but at the same time they also induce a
sedative effect.
5.1.1.3. trans-2-{4-[4-(m-Trifluoromethylphenyl)pip-
erazin-1-yl]cyclohexyl}-2,3,4,5,6,7-hexahydro-1H-isoindole-1,
3-dione (10). The compound was prepared by the general
procedure from 4-[4-(m-trifluoromethylphenyl)pipera-
zin-1-yl]cyclohexylamine and 3,4,5,6-tetrahydrophthalic
anhydride in 61% yield as colourless crystals, mp 165–
167 ꢁC, Rf = 0.38 (SiO2, CHCl3/CH3OH = 49/1); 1H
NMR (300 MHz) d 7.33 (t, J = 7.9 Hz, 1H, aryl H-5),
7.13–7.01 (m, 3H, aryl H-2, H-4 and H-6), 3.88 (tt,
J = 12.3, 4.0 Hz, 1H, cyclohexane axial H-1), 3.34–3.20
(m, 4H, piperazine 2CH2), 2.84–2.68 (m, 4H, piperazine
2CH2), 2.58–2.42 (m, 1H, cyclohexane axial H-4), 2.36–
2.25 (m, 4H, 2CH2 in tetrahydroisoindole-1,3-dione),
2.25–2.08 (m, 2H, cyclohexane axial H’s), 2.09–1.96
(m, 2H, cyclohexane equatorial H’s), 1.80–1.68 (m,
6H, 2CH2 in tetrahydroisoindole-1,3-dione and cyclo-
hexane equatorial H’s), 1.50–1.32 (m, 2H, cyclohexane
axial H’s). 10Æ2HCl: colourless crystals, mp 248–
250 ꢁC. Anal. (C25H30N3O2F3Æ2HCl) C, H, N.
5. Experimental
5.1. Chemistry
Melting points (mp) were determined with a Boetius
apparatus and are uncorrected. H NMR spectra were
1
taken with a Varian EM-360L (60 MHz) or a Varian
Mercury-VX (300 MHz) spectrophotometer in CDCl3
solutions with TMS as an internal standard. The spec-
tral data of new compounds refer to their free bases.
Chemical shifts were expressed in d (ppm) and the cou-
pling constants J in hertz (Hz). All compounds were
routinely checked by TLC using Merck silica gel 60
F254 plates (detection at 254 nm). Column chromatogra-
phy separations were carried out on Merck Kieselgel 60.
Elemental analyses were found within 0.4% of the the-
oretical values.
5.1.1.4. 2-{4-[4-(m-Trifluoromethylphenyl)piperazin-1-
yl]butyl}-cis-2,3,3a,4,7,7a-hexahydro-1H-isoindole-1,3-dione
(11). The compound was prepared by the general proce-
dure from 4-[4-(m-trifluoromethylphenyl)piperazin-1-
yl]butylamine and cis-1,2,3,6-tetrahydrophthalic anhy-
dride in 65% yield as a pale yellow oil, Rf = 0.45
(SiO2, CHCl3/CH3OH = 19/1); 1H NMR (60 MHz) d
7.6–6.9 (m, 4H, Ar–H); 6.1–5.8 (m, 2H, –CH=CH–
The starting 4-[4-(m-trifluoromethylphenyl)piperazin-
1-yl]butylamine and 4-[4-(m-trifluoromethylphenyl)pip-
erazin-1-yl)cyclohexylamine were synthesized by
published procedures.2 The preparation of compounds
5 and 6 had been previously published.3

M. H. Paluchowska et al. / Bioorg. Med. Chem. 15 (2007) 7116–7125
7123
in tetrahydroisoindole-1,3-dione), 3.8–3.5 (m, 2H, –
CH2–imide), 3.5–2.9 (m, 6H, piperazine 2CH2 and –
(CH2)3–CH2–piperazine), 2.9–1.9 (cluster, 10H), 1.9–
1.2 (m, 4H, –CH2–(CH2)2–CH2–). 11Æ2HCl: colourless
crystals, mp 166–168 ꢁC. Anal. (C23H28N3O2F3Æ2HCl)
C, H, N.
1H, cyclohexane axial H-1), 3.34–3.18 (m, 4H, pipera-
zine 2CH2), 2.84–2.70 (m, 4H, piperazine 2CH2), 2.62
(t, J = 6.5 Hz, 4H, 2CH2CO in piperidine-2,6-dione),
2.58–2.46 (m, 1H, cyclohexane axial H-4), 2.46–2.28
(m, 2H, cyclohexane axial H’s), 2.10–1.96 (m, 2H,
cyclohexane equatorial H’s), 1.96–1.84 (m, 2H,
–CH2–CH2–CH2– in piperidine-2,6-dione), 1.70–1.58
(m, 2H, cyclohexane equatorial H’s), 1.50–1.32 (m,
2H, cyclohexane axial H’s). 8ÆHClÆ0.25H2O: colourless
crystals, mp 244–246 ꢁC. Anal. (C22H28N3O2F3ÆH-
ClÆ0.25H2O) C, H, N.
5.1.1.5. 3,4-Dimethyl-1-{4-[4-(m-trifluoromethylphe-
nyl)piperazin-1-yl]butyl}-3-pyrroline-2,5-dione (13). The
compound was prepared by the general procedure from
4-[4-(m-trifluoromethylphenyl)piperazin-1-yl]butylamine
and 2,3-dimethylmaleic anhydride in 100% yield as a
colourless oil, Rf = 0.57 (SiO2, CHCl3/CH3OH = 19/1);
1H NMR (60 MHz) d 7.6–6.9 (m, 4H, Ar–H), 3.8–3.4
(m, 2H, –CH2–imide), 3.4–3.1 (m, 4H, piperazine
2CH2), 2.8–2.2 (m, 6H, piperazine 2CH2 and-(CH2)3–
CH2–piperazine), 2.0 (s, 6H, 2CH3), 1.8–1.4 (m,
5.1.2.2. trans-2-{4-[4-(m-Trifluoromethylphenyl)pip-
erazin-1-yl]cyclohexyl}-cis-2,3,3a,4,7,7a-hexahydro-1H-
isoindole-1,3-dione (12). The compound was prepared by
the general procedure in 69% yield as colourless crystals,
mp 151–153 ꢁC, Rf = 0.31 (SiO2, CHCl3/CH3OH = 49/
1
4H,
–CH2–(CH2)2–CH2–).
13Æ2HCl:
colourless
1); H NMR (300 MHz) d 7.33 (t, J = 7.9 Hz, 1H, aryl
crystals, mp 177–178 ꢁC. Anal. (C21H26N3O2F3Æ2HCl)
C, H, N.
H-5), 7.12–7.00 (m, 3H, aryl H-2, H-4 and H-6), 5.93–
5.82 (m, 2H, –CH=CH– in tetrahydroisoindole-1,3-
dione), 3.93 (tt, J = 12.4, 3.9 Hz, 1H, cyclohexane axial
H-1), 3.32–3.14 (m, 4H, piperazine 2CH2), 3.05–2.94
(m, 2H, CH–CH in tetrahydroisoindole-1,3-dione),
2.82–2.66 (m, 4H, piperazine 2CH2), 2.64–2.38 (m, 3H,
methylene 2H’s in tetrahydroisoindole-1,3-dione and
cyclohexane axial H-4), 2.34–2.12 (m, 4H, methylene
2H’s in tetrahydroisoindole-1,3-dione and cyclohexane
axial 2H’s), 2.08–1.92 (m, 2H, cyclohexane equatorial
2H’s), 1.69–1.55 (m, 2H, cyclohexane equatorial H’s),
1.48–1.28 (m, 2H, cyclohexane axial H’s). 12ÆHCl: col-
ourless crystals, mp 248–250 ꢁC. Anal. (C25H30N3O2-
F3ÆHCl) C, H, N.
5.1.1.6. trans-3,4-Dimethyl-1-{4-[4-(m-trifluoromethyl-
phenyl)piperazin-1-yl]cyclohexyl}-3-pyrroline-2,5-dione (14).
The compound was prepared by the general procedure
from 4-[4-(m-trifluoromethylphenyl)piperazin-1-yl]cyclo-
hexylamine and 2,3-dimethylmaleic anhydride in 55% yield
as colourless crystals: mp 171–173 ꢁC, Rf = 0.55 (SiO2,
1
CHCl3/CH3OH = 19/1); H NMR (300 MHz) d 7.34 (t,
J = 7.9 Hz, 1H, aryl H-5), 7.15–7.00 (m, 3H, aryl H-2,
H-4 and H-6), 3.89 (tt, J = 12.3, 4.0 Hz, 1H, cyclohexane
axial H-1), 3.42–3.14 (m, 4H, piperazine 2CH2), 2.90–
2.66 (m, 4H, piperazine 2CH2), 2.60–2.42 (m, 1H, cyclo-
hexane axial H-4), 2.26–1.98 (m, 4H, cyclohexane), 1.93
(s, 6H, 2CH3), 1.82–1.68 (m, 2H, cyclohexane equatorial
H’s), 1.54–1.32 (m, 2H, cyclohexane axial H’s).
14Æ0.5HCl: colourless crystals, mp 277–279 ꢁC. Anal.
(C23H28N3O2F3Æ0.5HCl) C, H, N.
5.2. In vitro radioligand binding assays
For all the assays inhibition constants (Ki) were
determined from at least three separate experiments
in which 7–9 drug concentrations, run in triplicate,
were used. The binding reaction was terminated by
rapid filtration through Whatman GF/B filters fol-
lowed by three 4-mL washes with ice-cold incubation
buffer.
5.1.2. General Procedure for the Preparation of
Compounds 8 and 12. Equimolar amounts (2 mmol) of
4-[4-(m-trifluoromethylphenyl)piperazin-1-yl]cyclohex-
ylamine and glutaric or cis-1,2,3,6-tetrahydrophthalic
anhydride were refluxed in xylene (20 mL) for 5 h. The
resulting precipitate of non-cyclic amidoacid was filtered
off and then was heated in acetic anhydride (20 mL) in
the presence of anhydrous sodium acetate (30% excess)
for 5 h. After cooling the reaction mixture was poured
into ice-water, neutralized with 10% NaOH and ex-
tracted with CHCl3 (3 · 30 mL). The combined extracts
were dried (K2CO3) and evaporated to give the oily res-
idue, which was purified by column chromatography.
For pharmacological assays free bases were converted
into the hydrochloride salts in acetone solutions by the
treatment with excess of Et2O saturated with gaseous
HCl.
The radioactivity retained on the filters was measured by
liquid scintillation counting (Beckman LS 6500 appara-
tus) in 4 ml scintillation fluid (Akwascynt, BioCare).
Binding isotherms of the tested compounds were ana-
lyzed by non-linear regression (Prism, GrafPad Software
Inc., San Diego), using the Cheng-Prusoff equation29 to
calculate Ki values.
5.2.1. Serotonin 5-HT1A, dopamine D2 and a1-adrenergic
binding assays. Radioligand studies with native 5-HT1A
,
D2 and a1-adrenergic receptors were conducted accord-
ing to the methods previously described by us,2,30,31
Briefly: 5-HT1A assays used rat hippocampal mem-
branes, [3H]-8-OH-DPAT (106 Ci/mmol, NEN Chemi-
cals) and 5-HT, for nonspecific binding; dopamine D2
assays used rat striatal membranes, [3H]-spiperone
(15.0 Ci/mmol, Perkin-Elmer) and butaclamol, for non-
specific binding; a1 assays used rat cortical membranes,
[3H]-Prazosin (25.0 Ci/mmol, Amersham) and phentol-
amine, for non-specific binding.
5.1.2.1. trans-1-{4-[4-(m-Trifluoromethylphenyl)pip-
erazin-1-yl]cyclohexyl}piperidine-2,6-dione (8). The com-
pound was prepared by the general procedure in 25%
yield as colourless crystals, mp 170–172 ꢁC, Rf = 0.24
(SiO2, CHCl3/CH3OH = 19/1);1H NMR (300 MHz) d
7.33 (t, J = 7.9 Hz, 1H, aryl H-5), 7.14–7.00 (m, 3H,
aryl H-2, H-4 and H-6), 4.54 (tt, J = 12.2, 3.8 Hz,

7124
M. H. Paluchowska et al. / Bioorg. Med. Chem. 15 (2007) 7116–7125
5.2.2. Expression of the gene for the human 5-HT7(b)
receptor. The full length human 5-HTR7(b) cDNA
cloned into mammalian expression vector pcDNA3.1(+)
was purchased from UMR cDNA Resource Center
(www.cdna.org). The receptor cDNA was stably trans-
fected into human embryonic kidney cells (HEK293,
ATCC) with use of Lipofectamine 2000 (Invitrogen).
A clone yielding high expression level of 5-HTR7(b)
was selected during preliminary experiments including
Western blot analysis, [3H]-CT saturation binding stud-
ies as well as cAMP accumulation assays.
(hydrobromide, 8-OH-DPAT, Tocris, Cookson Ltd.,
UK) was dissolved in saline, N-{2-[4-(o-methoxyphe-
nyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl)cyclohexanecarb-
oxamide (trihydrochloride, WAY 100635, synthesized
byBoksa, Institute of Pharmacology, Polish Academy
´
of Sciences, Krakow, Poland) and imipramine (hydro-
chloride, Polfa-Starogard, Poland) were dissolved in
´
distilled water. Diazepam (Polfa-Poznan, Poland) and
the investigated compounds were suspended in a 1%
aqueous solution of Tween 80. 8-OH-DPAT and
WAY 100635 were injected subcutaneously (sc), diaze-
pam, imipramine and the tested compounds were given
intraperitoneally (ip) in a volume of 2 ml/kg (rats) or
10 ml/kg (mice). Each experimental group consisted of
six to ten animals, and all the animals were used only
once. The obtained data were analyzed by one-way
analysis of variance followed by Dunnett’s test (when
only one drug was given) or by Newman–Keuls test
(when two drugs were administered).
5.2.3. Cell culture and preparation of cell membranes.
HEK293 cells with stable expression of 5-HTR7(b) were
maintained at 37 ꢁC in a humidified atmosphere with
5% CO2 and were grown in Dulbeco’s Modifier Eagle’s
Medium (Gibco BRL) containing 5% dialysed foetal bo-
vine serum (Gibco BRL) and 500 lg/mL G418 sulfate
(Sigma–Aldrich). For membranes preparations, cells
were subcultured in 10 cm diameter dishes, grown to
90% confluence, washed twice with prewarmed to 37 ꢁC
phosphate-buffered saline (PBS) and pelleted by centrifu-
gation (1000g) in PBS containing 0.1 mM EDTA and
1 mM dithiothreitol. Prior to membrane preparations
pellets were stored at À80 ꢁC. Membranes were prepared
from frozen cell pellets by homogenization (Polytron, set-
ting 5 for 15 s) and centrifugation (50,000g, 15 min., 4 ꢁC)
in 20 volumes of Tris–HCl buffer (50 mM, pH 7.4) con-
taining EDTA (0.1 mM). The pellets were then re-sus-
pended in buffer, incubated 20 min. in 37 ꢁC, and once
again centrifuged as described above. The membranes
were stored in aliquots at À80 ꢁC until use for binding as-
says. The protein concentration was determined with
bicinchoninic acid protein assay kit (Pierce, USA).
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 re-
corded 30, 60, 90 and 120 min after their administration.
In a separate experiment, the effect of WAY 100635
(0.1 mg/kg) on the hypothermia induced by compounds
7–12 and 14 or 8-OH-DPAT was tested. WAY 100635
was administered 15 min before the compounds or 8-
OH-DPAT and rectal body temperature was recorded
30 and 60 min after injection of the tested compounds.
In another experiment, effect of 13 (which did not
change mouse body temperature) on the 8-OH-DPAT
(5 mg/kg)-induced hypothermia was assessed. The tested
compounds were administered 45 min before 8-OH-
DPAT and rectal body temperature was measured 15,
30, 45 and 60 min after 8-OH-DPAT injection. The re-
sults were expressed as a change in body temperature
(Dt) with respect to the basal body temperature, as mea-
sured at the beginning of the experiment.
5.2.4. 5-HT7 Receptor binding assay. Binding assays on
membranes from HEK 293 cells stably expressing hu-
man 5-HT7(b) receptor were performed according to
procedure described by Thomas et al.32 Briefly, the
membranes (10 lg protein per tube) were incubated in
50 mM Tris–HCl buffer (pH 7.4) containing 4 mM
MgCl2, 0.1 mM pargyline and 0.1% ascorbic acid, in
the presence of 7–9 concentrations of test drug and
0.5 nM [3H]-5-CT (93.0 Ci/mmol, Amersham). Nonspe-
cific binding was defined in the presence of 10 lM of 5-
HT. After a 1-h incubation at 37 ꢁC, the assay samples
were rapidly filtered through Whatman GF/B filters
and subsequently washed with ice-cold 50 mM Tris buf-
fer (pH 7.4) using a Brandel harvester.
5.3.2. Lower lip retraction (LLR) in rats. LLR was as-
sessed according to the method described by Berendsen
et al.13 The rats were individually placed in cages
(30 · 25 · 25 cm) and they were scored three times (at
15, 30 and 45 min) after the administration of the tested
compounds or 8-OH-DPAT as follows: 0 = lower inci-
sors not visible, 0.5 = partly visible, 1 = completely visi-
ble. The total 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 45 min and
15 min, respectively, before 8-OH-DPAT and the ani-
mals were scored 15, 30 and 45 min after 8-OH-DPAT
administration.
5.3. In vivo experiments
The experiments were performed on male Wistar rats
(290–310 g) or male Albino Swiss mice (24–28 g). The
animals were kept at a room temperature of 20 1 ꢁC,
and had free access to food (standard laboratory pellets)
and tap water before the experiment. All the investiga-
tions were conducted in the light phase, on a natural
day-night cycle (from February to May), between 9
a.m. and 2 p.m. All the experimental procedures were
approved by the local Bioethics Commission at the
Institute of Pharmacology, Polish Academy of Sciences
5.3.3. Four-plate test in mice. The experiment was carried
out according to the method of Aron et al.16 The box
was made of an opaque plastic and was rectangular
(25 cm · 18 cm · 16 cm) in shape. The floor was covered
with four rectangular metal plates (11 cm · 8 cm), sepa-
rated by a 4 mm gap. The plates were connected to a
source of continuous current which enabled a 120 V dif-
´
in Krakow. 8-Hydroxy-2-(di-n-propylamino)tetralin

M. H. Paluchowska et al. / Bioorg. Med. Chem. 15 (2007) 7116–7125
7125
8. Paluchowska, M. H.; Bojarski, A. J.; Charakchieva-
Minol, S.; Wesołowska, A. Eur. J. Med. Chem. 2002, 37,
273.
9. Goodwin, G. M.; De Souza, R. J.; Green, A. R.
Neuropharmacology 1985, 24, 1187.
10. Martin, K. F.; Heal, D. J. In Molecular Biology, Receptors
and Functional Effects; Fozard, J. R., Saxena, P. R., Eds.;
Birkha¨user: Basel, 1991; pp 483–490.
11. Forster, E. A.; Cliffe, I. A.; Bill, D. J.; Dover, G. M.;
Jones, D.; Reilly, Y.; Fletcher, A. Eur. J. Pharmacol. 1995,
281, 81.
ference of potential 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. Afterwards, each time a mouse
passed from one plate to another, the experimenter elec-
trified the whole floor which evoked a visible flight reac-
tion of the animal. If the animal continued running, it
received no new shocks for the following 3 s. The epi-
sodes of punished crossing were counted for 60 s.
´
12. Mokrosz, J. L.; Paluchowska, M. H.; Chojnacka-Wojcik,
E.; Filip, M.; Charakchieva-Minol, S.; Deren-Wesołek,
A.; Mokrosz, M. J. J. Med. Chem. 1994, 37, 2754.
13. Berendsen, H. H. G.; Jenck, F.; Broekkamp, C. L. E.
Pharmacol. Biochem. Behav. 1989, 33, 821.
5.3.4. Forced swim test in mice. The experiment was car-
ried out according to the method of Porsolt et al.17
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 there for
6 min. A mouse was regarded as immobile when it re-
mained floating on the water, making only small move-
ments to keeps its head above it. The total duration of
immobility was recorded during the last 4 min of a 6-
min test session.
´
14. Berendsen, H. H. G.; Broekkamp, C. L. E.; Van Delft, A.
M. Behav. Neural. Biol. 1991, 55, 214.
´
15. Przegalinski, E.; Filip, M.; Budziszewska, B.; Chojnacka-
´
Wojcik, E. Pol. J. Pharmacol. 1994, 46, 21.
16. Aron, C.; Simon, P.; Larousse, C.; Boissier, J. R.
Neuropharmacology 1971, 10, 459.
17. Porsolt, R. D.; Bertin, A.; Jalfre, M. Arch. Int. Pharmac-
odyn. Ther. 1977, 229, 327.
18. De Vry J. Psychopharmacol. 1995, 121, 1.
19. Handley, S. L. Pharmacol. Ther. 1995, 66, 103.
5.3.5. Locomotor activity in mice. The spontaneous loco-
motor activity of mice was recorded by Opto-M3 multi-
channel activity monitor (MultiDevice Software v. 1.30,
Columbus Instruments). The mice were placed individu-
ally in plastic cages, and the number of crossing for each
channel (ambulation) was counted twice: during the first
6 min, that is, at the time equal to the observation period
in the forced swimming test, and during 30-min experi-
mental sessions.
´
20. Lopez-Rubalcava, C. Pharmacol. Biochem. Behav. 1996,
54, 677.
´
21. Przegalinski, E.; Tatarczynska, E.; Kłodzinska, A.;
Chojnacka-Wojcik, E. Neuropharmacology 1994, 33,
´
´
´
1109.
22. Chojnacka-Wojcik, E.; Tatarczynska, E.; Gołembiows-
´
ka, K.; Przegalinski, E. Neuropharmacology 1991, 30,
´
´
711.
23. Tatarczynska, E.; Kłodzinska, A.; Stachowicz, K.; Cho-
´
jnacka-Wojcik, E. Behav. Pharmacol. 2004, 15, 523.
´
´
24. Wesołowska, A.; Nikiforuk, A.; Stachowicz, K; Tata-
References and notes
´
rczynska, E. Neuropharmacology 2006, 51, 578.
1. Paluchowska, M. H.; Mokrosz, M. J.; Bojarski, A.;
Wesołowska, A.; Borycz, J.; Charakchieva-Minol, S.;
25. Wesołowska, A.; Nikiforuk, A.; Stachowicz, K. Eur. J.
Pharmacol. 2006, 553, 185.
´
Chojnacka-Wojcik, E. J. Med. Chem. 1999, 42, 4952.
26. Griebel, G.; Rodgers, R. J.; Perrault, G.; Sanger, D. J.
Psychopharmacology 1999, 121, 121.
27. Griebel, G.; Rodgers, R. J.; Perrault, G.; Sanger, D. J.
Neuropharmacology 2000, 39, 1848.
28. Anden, N. E.; Pauksens, K.; Svensson, K. J. Neural.
Transm. 1982, 55, 111.
29. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22,
3099.
30. Bojarski, A. J.; Cegła, M. T.; Charakchieva-Minol, S.;
´
2. Bojarski, A. J.; Paluchowska, M. H.; Duszynska, B.;
Kłodzinska, A.; Tatarczynska, E.; Chojnacka-Wojcik, E.
Bioorg. Med. Chem. 2005, 13, 2293.
3. Paluchowska, M. H.; Bugno, R.; Bojarski, A. J.; Charak-
´
´
´
´
chieva-Minol, S.; Duszynska, B.; Tatarczynska, E.;
Kłodzinska, A.; Stachowicz, K.; Chojnacka-Wojcik, E.
´
´
´
Bioorg. Med. Chem. 2005, 13, 1195.
4. Bojarski, A. J.; Paluchowska, M. H.; Duszynska, B.;
´
Bugno, R.; Kłodzinska, A.; Tatarczynska, E.; Chojnacka-
´
Wojcik, E. Bioorg. Med. Chem. 2006, 14, 1391.
´
´
Mokrosz, M. J.; Mackowiak, M.; Mokrosz, J. L. Phar-
mazie 1993, 48, 289.
31. Mokrosz, J. L.; Duszynska, B.; Charakchieva-Minol, S.;
´
5. Nowak, M.; Kołaczkowski, M.; Pawłowski, M.; Bojarski,
´
A. J. J. Med. Chem. 2006, 49, 205.
6. Wesołowska, A.; Paluchowska, M. H.; Chojnacka-Woj-
cik, E. Eur. J. Pharmacol. 2003, 471, 27.
Bojarski, A. J.; Mokrosz, M. J.; Wydra, R. L.; Janda, L.;
Strekowski, L. Eur. J. Med. Chem. 1996, 31, 973.
32. Thomas, D. R.; Gittins, S. A.; Collin, L. L.; Middle-
miss, D. N.; Riley, G.; Hagan, J.; Gloger, I.; Ellis, C.
E.; Forbes, I. T.; Brown, A. M. Br. J. Pharmacol.
1998, 124, 1300.
´
´
7. Lopez-Rodrıguez, M. L.; Morcillo, M. J.; Rovat, T. K.;
Fernandez, E.; Vicente, B.; Sanz, A. M.; Hernandez, M.;
´
´
´
Orensanz, L. J. Med. Chem. 1999, 42, 36.