On the bioactive conformation of NAN-190 (1) and MP3022 (2), 5-HT(1A) receptor antagonists

Article abstract of DOI:10.1021/jm991045h

Structural modifications of 1, a postsynaptic 5-HT(1A) receptor antagonist, provided its flexible (8, 12) and rigid (7, 9, 11, 13) analogues. Compounds 7, 8, 9, and 11 showed high 5-HT(1A) receptor affinity (K(i) = 4-72 nM). They acted as 5-HT(1A) postsynaptic receptor antagonists, since, like 1, they inhibited the behavioral syndrome, i.e., flat body posture (FBP) and forepaw treading (FT), in reserpine-pretreated rats as well as the lower lip retraction (LLR) in rats, both induced by 8-hydroxy-2-(di-n- propylamino)tetralin hydrobromide (8-OH-DPAT), a 5-HT(1A) receptor agonist. Compound 12, which demonstrated high 5-HT(1A) receptor affinity (K(i) = 50 nM), revealed properties of a partial 5-HT(1A) receptor agonist: it induced LLR and, at the same time, inhibited FT in rats. Compound 13 (K(i) = 1600 nM) was not tested in a behavioral study. Restriction of the conformational freedom in 2, a full 5-HT(1A) receptor antagonist, yielded compound 14 with high 5-HT(1A) receptor affinity (K(i) = 47 nM) and partial agonist properties at postsynaptic 5-HT(1A) receptors in the above tests in vivo; i.e., it induced LLR and inhibited FBP and FT in rats. New constrained analogues of I and 2 (compounds 7 and 14, respectively) were also synthesized to recognize a bioactive conformation of those 5-HT(1A) receptor antagonists. On the basis of in vitro and in vivo investigations, binding and functional properties of compound 7 were found to reflect those of I at 5-HT(1A) receptors. On the other hand, compound 14, a rigid analogue of 2, showed a different activity in vivo in comparison with the parent compound. PM3 and MM calculations revealed the existence of three low-energy conformers of 7 and six of 14, all of them belonging to the extended family of conformations. The optimized structures of both analogues had a different angle between aromatic planes of terminal fragments; moreover, the heteroaromatic system of those molecules occupied various space regions. Our present study provides support to the hypothesis that the bioactive conformation of 1, responsible for its postsynaptic 5-HT(1A) receptor antagonism, is an extended linear structure represented by 7.

Full text of DOI:10.1021/jm991045h

4952
J . Med. Chem. 1999, 42, 4952-4960
On th e Bioa ctive Con for m a tion of NAN-190 (1) a n d MP 3022 (2), 5-HT1A Recep tor
An ta gon ists†
Maria H. Paluchowska,*,‡ Maria J . Mokrosz,‡ Andrzej Bojarski,‡ Anna Wesołowska,§ J olanta Borycz,§
Sijka Charakchieva-Minol,‡ and Ewa Chojnacka-Wo´jcik§
Departments of Medicinal Chemistry and New Drug Research, Institute of Pharmacology, Polish Academy of Sciences,
12 Sme¸tna Street, 31-343 Krako´w, Poland
Received March 22, 1999
Structural modifications of 1, a postsynaptic 5-HT1A receptor antagonist, provided its flexible
(8, 12) and rigid (7, 9, 11, 13) analogues. Compounds 7, 8, 9, and 11 showed high 5-HT1A receptor
affinity (Ki ) 4-72 nM). They acted as 5-HT1A postsynaptic receptor antagonists, since, like 1,
they inhibited the behavioral syndrome, i.e., flat body posture (FBP) and forepaw treading
(FT), in reserpine-pretreated rats as well as the lower lip retraction (LLR) in rats, both induced
by 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), a 5-HT1A receptor
agonist. Compound 12, which demonstrated high 5-HT1A receptor affinity (Ki ) 50 nM), revealed
properties of a partial 5-HT1A receptor agonist: it induced LLR and, at the same time, inhibited
FT in rats. Compound 13 (Ki ) 1600 nM) was not tested in a behavioral study. Restriction of
the conformational freedom in 2, a full 5-HT1A receptor antagonist, yielded compound 14 with
high 5-HT1A receptor affinity (Ki ) 47 nM) and partial agonist properties at postsynaptic 5-HT1A
receptors in the above tests in vivo; i.e., it induced LLR and inhibited FBP and FT in rats.
New constrained analogues of 1 and 2 (compounds 7 and 14, respectively) were also synthesized
to recognize a bioactive conformation of those 5-HT1A receptor antagonists. On the basis of in
vitro and in vivo investigations, binding and functional properties of compound 7 were found
to reflect those of 1 at 5-HT1A receptors. On the other hand, compound 14, a rigid analogue of
2, showed a different activity in vivo in comparison with the parent compound. PM3 and MM
calculations revealed the existence of three low-energy conformers of 7 and six of 14, all of
them belonging to the extended family of conformations. The optimized structures of both
analogues had a different angle between aromatic planes of terminal fragments; moreover,
the heteroaromatic system of those molecules occupied various space regions. Our present study
provides support to the hypothesis that the bioactive conformation of 1, responsible for its
postsynaptic 5-HT1A receptor antagonism, is an extended linear structure represented by 7.
Ch a r t 1
In tr od u ction
The class of arylpiperazines has been of great impor-
tance for studies into ligand-serotonin (5-HT) receptor
interactions. 1-(2-Methoxyphenyl)-4-[4-(2-phthalimido)-
butyl]piperazine (1)1 and 4-[3-(1-benzotriazolyl)propyl]-
1-(2-methoxyphenyl)piperazine (2)2 (Chart 1) represent
this group of ligands. Both have been shown to bind with
high affinity at the 5-HT1A receptor (Ki ) 0.6 and 15
nM, respectively). Compound 1 is a mixed agonist/
antagonist at central 5-HT1A receptors, since its effect
on pre- and postsynaptic 5-HT1A receptors is varied. It
behaves like a 5-HT1A receptor agonist at somatoden-
dritic 5-HT1A autoreceptors3 and like a 5-HT1A receptor
antagonist at postsynaptic ones.3-5 Compound 2 may
be regarded as a full potent, 5-HT1A receptor antagonist,
as indicated by in vivo,2 electrophysiological, and bio-
chemical studies.6,7 Like the majority of 5-HT1A receptor
ligands, these compounds are represented by linear
molecules whichsthanks to a hydrocarbon spacer con-
necting two terminal fragmentsspossess high confor-
mational freedom. The fully extended conformations
found in crystals which are in an energy minimum as
indicated by a conformational analysis are generally
believed to be responsible for the biological activity of
ligands at the 5-HT1A receptors.8,9 Several topographic
† Part 37 of the series: Structure-Activity Relationship Studies of
CNS Agents.
* To whom correspondence should be addressed. E-mail: paluchm@
if-pan.krakow.pl.
‡ Department of Medicinal Chemistry.
§ Department of New Drug Research.
10.1021/jm991045h CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/10/1999

Bioactive Conformation of NAN-190 and MP3022
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 4953
Sch em e 1a
Sch em e 2a
a
Reagents: (a) xylene, reflux.
Sch em e 3a
a
Reagents: (a) benzene, reflux; (b) NaBH4, THF; (c) HCl, H2O,
acetone; (d) NH2OH‚HCl, Na2SO4, MeOH; (e) Na, n-BuOH.
models,9-11 as well as those based on a CoMFA ap-
proach,12,13 have been developed to understand the
nature of binding properties of 5-HT1A receptor ligands.
In all these models an in vitro activity has been
correlated with specific elements of the ligand struc-
tures, but since they were based on single chemical
classes of ligands, such attempts have permitted only
limited general conclusions. The phenyl ring and the
basic nitrogen atom, pharmacophores described by
Hibert et al.,14 and at least two additional interactions
of, e.g., a lipophilic or π-π character are needed for the
high affinity of 5-HT1A receptor ligands. However, so far
no convincing explanation of their functional activity
has been offered.
a
Reagents: (a) Et2O, room temperature; (b) 4-(2-methoxyphen-
yl)piperazine, room temperature; (c) NaBH4, 1,4-dioxane, reflux.
Bt, benzotriazole.
b
We reported earlier15 that the removal of a benzene
ring from the phthalimide system in 1 provided com-
pound MM-77 (Chart 1) with the same 5-HT1A postsyn-
aptic receptor functional profile. It was also found that
the nitrogen atom in 1,2,3,4-tetrahydroquinoline (THIQ)
mimicked that in 1-arylpiperazines at 5-HT1A recep-
tors.16 These findings prompted us to conduct further
studies in order to determine how important specific
residues of that molecule were to the ligand-receptor
complex formation and to its functional activity toward
5-HT1A receptors. For this purpose the effect of satura-
tion of the phthalimide moiety and/or the substitution
of a 4-[1-(2-methoxyphenyl)]piperazinyl fragment with
a 1,2,3,4-tetrahydroquinolin-2-yl residue in the struc-
ture of 1 on the 5-HT1A receptor affinity and in vivo
activity was examined and is discussed herein. Evalu-
ation of chemical features that modulate the 5-HT1A
receptor in vitro as well as in vivo properties of 2 was
discussed previously.2,17-19 Moreover, on the basis of
X-ray studies and a conformational analysis,20 some
efforts were made to determine its bioactive conforma-
tion; however, due to the flexibility of the molecule, they
did not bring unequivocal results. An insight into
ligand-receptor interactions at the molecular level
seems to be more attainable when the number of
rotatable bonds in a molecule is limited. Replacement
of the flexible aliphatic spacer with cyclohexane ring
diminishes the possibility of a mutual spatial arrange-
ment of terminal fragments which are responsible for
a ligand activity toward, e.g., the 5-HT1A receptor. Thus,
in the present paper we would like to report for the first
time the synthesis of such conformationally constrained
analogues of 1 and 2. On the basis of their 5-HT1A
receptor binding properties and functional activity, we
tried to determine the bioactive conformation respon-
sible for the functional behavior of parent compounds
1 and 2.
Ch em istr y
The structures of the compounds described in this
study are shown in Table 1, and the methods of their
synthesis are outlined in Schemes 1-3. The key step
in the preparation of 7, 9, 11, and 13 was a multistage
synthesis of amines 5 and 6 (Scheme 1). Preparation of
ketones 3 and 4 involved condensation of benzotriazole,
amine [1-(2-methoxyphenyl)piperazine or 1,2,3,4-tet-
rahydroisoquinoline], and 1,4-cyclohexanedione mono-
ethylene ketal, followed by reduction of the obtained
adduct and hydrolysis of the ketal function. Ketones
were converted to the corresponding oximes which were
directly reduced to yield target amines 5 and 6.
Analogues of 1 were synthesized from the appropriate
anhydrides and amines (Scheme 2) by refluxing in
xylene. Benzotriazole readily underwent 1,4-addition to
2-cyclohexen-1-one to afford the respective adduct which

4954 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Paluchowska et al.
Ta ble 1. Structure and Binding Data on the 5-HT1A Receptors
of the Investigated Compounds
fragment gave compound 9, which demonstrated lower
5-HT1A receptor affinity than 1, but still fairly good (Ki
) 72 nM). Modification of both terminal fragments
yielded compound 13, in which a remarkable loss of the
5-HT1A affinity was observed (Ki ) 1600 nM). On the
other hand, compound 2 and its rigid analogue 14
exhibited comparable 5-HT1A binding properties (Ki )
15 and 47 nM, respectively).
In Vivo Exp er im en ts. Compounds 7-9, 11, 12, and
14, which showed 5-HT1A receptor affinity up to Ki )
100 nM, were tested in vivo. To determine postsynaptic
5-HT1A agonistic effects of the tested compounds, their
ability to induce a behavioral syndrome, i.e., a flat body
posture (FBP) and a forepaw treading (FT) in reserpin-
ized rats and a lower lip retraction (LLR) in rats, was
tested. The ability of the investigated compounds to
inhibit those symptoms produced by 8-OH-DPAT was
regarded as a postsynaptic 5-HT1A antagonistic activity.
In those models, compounds 1 and 2 acted as 5-HT1A
receptor antagonists.2-5
Ki ( SEM (nM)
compd
structure
R1
R2
R3
R4
5-HT1A
1a
2b
7
8
9
I
II
I
I
I
I
I
I
I
C
E
A
A
A
A
A
B
B
B
B
A
H
H
H
H
0.55 ( 0.14
15 ( 2
8 ( 2
4 ( 2
72 ( 19
355 ( 28
34 ( 4
50 ( 11
1600 ( 270
47 ( 8
C
D
D
C
C
D
D
E
-(CH2)2-
H
-(CH2)2-
H
-(CH2)2-
H
-(CH2)2-
-(CH2)3-
H
10c
11
12
13
14
H
H
II
a
Ki value according to Glennon et al. was 0.58 nM, respectively
b
In a behavioral syndrome test, compounds 7-9, 11,
and 12, analogues of 1, as well as compound 14, an
analogue of 2, given alone, like 1 and 2, did not evoke
FBP or FT in reserpine-pretreated rats (Table 2, A). On
the other hand, like 1 and 2, they strongly inhibited FBP
and/or FT induced by 8-OH-DPAT, showing a profile of
postsynaptic 5-HT1A receptor antagonists of diverse
potency. The ED50 values for 7 and 8 were similar, and
those for 9, 11, and 12 were higher than for 1; whereas
for 14 and 2 they were practically the same (Table 2,
B). In the LLR test, compounds 7-9, given alone, did
not induce this symptom in rats, while 12 and 14 did
(Table 3, A). The LLR induced by 8-OH-DPAT was
inhibited by 7-9, yet at doses higher than in the case
of 1. Compound 11 did not induce LLR, nor did it change
the LLR evoked by 8-OH-DPAT (Table 3, B).
(ref 1). Ki value from ref 2. c Compound previous reported as
hydrobromide salt (see ref 24).
was directly reacted with 1-(2-methoxyphenyl)pipera-
zine (Scheme 3). Reduction of the obtained intermediate
product (not isolated) with sodium borohydride resulted
in 14 with a 20% overall yield for a three-step reaction.
The structure of the newly synthesized compounds was
1
confirmed by H NMR and elemental analysis. In 2D
1H NMR experiments, the observed coupling constants
in the cyclohexane ring allowed us to assign a diequa-
torial conformation for 7, 9, 11, and 14. For biological
assays, the investigated compounds were converted into
hydrochloride salts.
Resu lts a n d Discu ssion
The described results of in vivo experiments showed
that, like the parent compound 1, the majority of its
analogues, i.e., 7, 8, 9, and 11, demonstrated 5-HT1A
postsynaptic receptor antagonistic properties, while
compound 12 could be classified as a partial agonist of
these receptors. Thus it may be concluded that neither
hydrogenation of the phthalimide moiety (8 and 9) nor
replacement of the 4-(2-methoxyphenyl)piperazine resi-
due with THIQ (11), nor restriction of conformational
freedom (7, 9, 11) affected the functional profile of the
investigated compounds in comparison with 1. Unex-
pectedly, compound 12, a flexible analogue of 1 with a
saturated cycloimide fragment and a THIQ residue,
behaved like a partial 5-HT1A receptor agonist; thus it
may be anticipated that in that case the structure of
the ligand-receptor complex is of a different mode. It
is noteworthy that, in contrast to the postsynaptic
5-HT1A receptor antagonistic properties of 2, its rigid
analogue 14 shows a profile of a partial agonist of these
receptors. In this case, like for 12, a different type of
the bioactive complex can be suggested.
Ra d ioliga n d Bin d in g Stu d ies. The affinity of com-
pounds 7-14 at 5-HT1A receptors was assessed on the
basis of their ability to displace [3H]-8-OH-DPAT, and
the results are summarized in Table 1. The reference
compounds 1 and 2 demonstrated very high 5-HT1A (Ki
) 0.55 and 15 nM, respectively) and R1 (Ki ) 0.4 and
69 nM, respectively) receptor affinities and pronounced
5-HT2A/5-HT1A selectivity. All investigated compounds,
except 10 and 13, showed significant affinity for 5-HT1A
receptors (Ki ) 4-72 nM), lack of R1/5-HT1A selectivity,
and diverse 5-HT2A/5-HT1A selectivity (data not shown).
First, we focused our attention on the influence of
terminal fragment modifications in 1 on the 5-HT1A
receptor affinity. Compound 8 with a hydrophobic,
saturated imide fragment demonstrated high affinity for
the 5-HT1A receptor (Ki ) 4 nM); in contrast, a change
of the arylpiperazine residue into THIQ, regardless of
the cycloimide moiety (compounds 10 and 12), caused
a decrease in the 5-HT1A receptor affinity (Ki ) 355 and
50 nM, respectively). Second, we investigated the 5-HT1A
receptor affinity of ring-constrained compounds, i.e., 7,
9, 11, and 13. Compound 7, a rigid analogue of 1,
possessed very high 5-HT1A receptor affinity, yet slightly
lower than that of the parent compound (Ki ) 8 and
0.55 nM, respectively). Additionally replacement of the
arylpiperazine moiety with THIQ yielded compound 11,
which also showed significant affinity for 5-HT1A recep-
tors (Ki ) 34 nM). Hydrogenation of the phthalimide
Mod elin g Stu d ies. As we have mentioned elsewhere,
inspection of ligand-receptor interactions leading to
specific biological effects could be more profound if
inflexible model compounds existed. Having synthesized
compounds 7 and 14 whose structures were the same
as those of 1 and 2 yet constrained, we encountered a
unique opportunity to discuss a problem of the bioactive

Bioactive Conformation of NAN-190 and MP3022
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 4955
Ta ble 2. Induction of Behavioral Syndrome by the Investigated Compounds (A) and Their Effect on the 8-OH-DPAT-Induced
Behavioral Syndrome (B) in Reserpine-Pretreated Rats
mean ( SEM behavioral score
A
B
treatment
dose (mg/kg)
FBP
FT
FBP
FT
vehicle
0.2 ( 0.1
0.1 ( 0.1
0.2 ( 0.1
0.1 ( 0.1
0.2 ( 0.2
0.2 ( 0.2
0.1 ( 0.1
0.2 ( 0.2
0.2 ( 0.1
0.1 ( 0.1
13.8 ( 0.8
11.5 ( 0.6
10.8 ( 0.6
1a
1
2
4
8
12.4 ( 0.5
6.0 ( 2.3b
5.7 ( 1.2b
2.3 ( 0.8b
6.2 ( 1.1b
2.3 ( 0.8b
1.8 ( 0.7b
ED50 ) 2.0 (1.2-3.2)
ED50 ) 2.5 (1.7-4.3)
vehicle
0.2 ( 0.1
0.2 ( 0.1
0.2 ( 0.2
0.1 ( 0.1
0.2 ( 0.2
0.2 ( 0.2
0.2 ( 0.1
0.1 ( 0.1
14.8 ( 0.2
14.3 ( 0.3
2c
4
8
16
11.5 ( 1.2
5.8 ( 1.7b
8.0 ( 2.0b
3.7 ( 1.5b
6.1 ( 0.3b
2.6 ( 0.9b
ED50 ) 10.5 (7.0-15.8)
ED50 ) 5.2 (3.2-8.6)
vehicle
7
0.2 ( 0.1
0.1 ( 0.1
0.2 ( 0.1
0.2 ( 0.2
0.2 ( 0.2
0.1 ( 0.1
0.2 ( 0.1
0.2 ( 0.2
13.3 ( 0.5
13.0 ( 0.7
2
4
8
9.8 ( 1.1b
6.2 ( 0.7b
8.8 ( 0.5b
4.2 ( 0.3b
5.6 ( 0.2b
1.0 ( 0.4b
ED50 ) 6.3 (3.6-13.9)
ED50 ) 2.3 (1.4-3.9)
vehicle
8
0.2 ( 0.1
0.1 ( 0.1
0.2 ( 0.2
0.2 ( 0.2
0.2 ( 0.2
0.2 ( 0.1
0.2 ( 0.1
0.2 ( 0.2
14.6 ( 0.2
13.2 ( 0.4
2
4
8
8.3 ( 0.7b
3.8 ( 0.3b
6.7 ( 1.0b
3.5 ( 0.6b
5.5 ( 0.6b
0.5 ( 0.3b
ED50 ) 3.2 (1.8-5.8)
ED50 ) 2.6 (1.5-4.4)
vehicle
9
0.2 ( 0.1
0.2 ( 0.2
0.1 ( 0.1
0.1 ( 0.1
0.2 ( 0.1
0.2 ( 0.2
0.2 ( 0.1
0.1 ( 0.1
13.6 ( 0.7
13.2 ( 0.2
4
8
16
11.6 ( 0.6
9.2 ( 0.5d
9.8 ( 0.4d
5.4 ( 0.8b
3.2 ( 1.1b
2.0 ( 0.4b
ED50 ) 11.0 (7.6-16.0)
ED50 ) 7.3 (5.4-9.8)
vehicle
11
0.2 ( 0.1
0.3 ( 0.1
0.5 ( 0.2
0.3 ( 0.2
0.2 ( 0.1
0.1 ( 0.1
0.1 ( 0.2
0.1 ( 0.1
13.6 ( 0.7
11.0 ( 0.4
10.3 ( 0.7
11.8 ( 0.6
ED50 > 32
13.2 ( 0.2
8
16
32
7.5 ( 1.1b
5.7 ( 0.7b
4.3 ( 0.5b
ED50 ) 13.5 (7.9-23.0)
vehicle
12
0.2 ( 0.1
0.1 ( 0.1
2.3 ( 0.2
3.7 ( 0.3
0.2 ( 0.2
0.1 ( 0.2
0.2 ( 0.2
0.2 ( 0.1
13.7 ( 0.6
14.6 ( 0.2
13.3 ( 0.6
11.7 ( 0.4
ED50 > 32
13.5 ( 0.8
8
16
32
9.8 ( 1.2d
7.7 ( 0.4b
6.0 ( 0.4b
ED50 ) 17.0 (11.3-25.5)
vehicle
14
0.2 ( 0.1
0.6 ( 0.2
0.5 ( 0.2
1.0 ( 0.4
0.2 ( 0.2
0.1 ( 0.1
0.3 ( 0.3
0.1 ( 0.1
13.3 ( 0.4
12.2 ( 1.3
4
8
16
12.5 ( 0.4
9.6 ( 0.6
7.0 ( 0.9b
3.5 ( 0.7b
6.2 ( 1.0b
0.8 ( 0.5b
ED50 ) 11.0 (7.6-16.0)
ED50 ) 7.0 (4.5-10.9)
a
b
d
Data from ref 4. p < 0.01 vs vehicle + 8-OH-DPAT. c Data from ref 2. p < 0.05 vs vehicle + 8-OH-DPAT.
conformation of the latter compounds. Taking into
account the in vitro and in vivo results, it may be
concluded that the constrained structure of 7 represents
very well the 5-HT1A receptor binding and functional
properties of 1. In contrast, the functional activity of
the rigid molecule 14 differed from that of the parent
compound 2. Such a difference in the characteristics of
7 vs 1 and 14 vs 2 might be attributed to a steric
arrangement of terminal fragments in these molecules.
Therefore, to explain this phenomenon, we analyzed in
global energy minimum according to the first method
and up to 12 kcal/mol according to the other. Obviously,
only a 1e,4e chair conformer exists, yet some rotations
may influence the relative positions of terminal frag-
ments of the molecule. Since the rotation around τ1 in
the 1-(2-methoxyphenyl)piperazine system has already
been extensively studied,21,22 we used a global energy
minimum conformation (angle τ1 ) 180°) which is
generally accepted as a bioactive conformation of this
fragment (Figure 1a). Two other rotations around bonds
in positions 1 and 4 of the cyclohexane ring were
considered. We calculated a rotation energy profile for
two separate piperazine-cyclohexane (τ2) and cyclohex-
ane-phthalimide (τ3) subunits. The symmetry of a
phthalimide moiety made its two preferred orientations
(τ3 = 0° and 180°) equivalent. On the other hand, in the
case of the piperazine subunit we observed an energy
minimum for two equivalent conformations (τ2 ) 66.3°
or -66.3°). The third possible conformation (τ2 = 180°)
was by 0.6 kcal/mol less stable than the two others, the
rotation barrier being ca. 2.5 kcal/mol. The above results
indicate that the rigid molecule 7 can exist in three
1
detail both those structures. Since the H NMR spectra
showed that compound 7 existed in a diequatorial 1e,4e
chair form with respect to the cyclohexane ring, we
performed a conformational analysis to recognize the
flexibility of the investigated structure. As generally
expected, the above conformation should correspond to
a global energy minimum of the molecule, which was
confirmed by semiempirical (PM3) and MM (MAXI-
MIN2, Tripos) calculations. The energy of the other
conformations resulting from inversion of the cyclohex-
ane ring, i.e., 1a,4e boat (1-axial, 4-equatorial), 1e,4a
boat, and 1a,4a chair, was ca. 4-8 kcal/mol above the

4956 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Paluchowska et al.
Ta ble 3. Induction of LLR by the Investigated Compounds (A)
and Their Effect on the 8-OH-DPAT-Induced LLR (B) in Rats
mean ( SEM LLR score
treat-
dose
ment (mg/kg)
A
B
vehicle
1
0.1 ( 0.1
2.8 ( 0.2
1
2
4
0.1 ( 0.1
0.1 ( 0.1
0.1 ( 0.2
1.7 ( 0.3a
1.4 ( 0.2b
0.6 ( 0.2b
ED50 ) 1.7 (0.9-3.1)
vehicle
0.1 ( 0.1
0.0 ( 0.0
0.1 ( 0.1
0.6 ( 0.2
2.6 ( 0.2
2c
4
8
16
1.1 ( 0.2b
0.7 ( 0.3b
0.9 ( 0.2b
ED50 ) 6.5 (4.2-10.1)
vehicle
7
0.1 ( 0.1
1.3 ( 0.1d
1.0 ( 0.2
1.2 ( 0.1d
2.8 ( 0.2
4
8
16
2.0 ( 0.2
1.5 ( 0.4a
0.6 ( 0.2b
ED50 ) 7.5 (4.8-11.6)
vehicle
8
0.1 ( 0.1
0.5 ( 0.1
0.5 ( 0.1
0.8 ( 0.1
2.8 ( 0.2
4
8
16
1.7 ( 0.2b
1.3 ( 0.2b
0.7 ( 0.2b
ED50 ) 7.5 (4.8-11.6)
F igu r e 1. Low-energy conformers of (a) 7 and (b) 14. Torsional
angles τ1, τ2, and τ3 and centers of aromatic rings c1, c2, and c3
are shown.
vehicle
9
0.1 ( 0.1
0.2 ( 0.1
0.3 ( 0.1
0.3 ( 0.1
2.8 ( 0.2
8
16
32
2.3 ( 0.2
1.6 ( 0.2b
1.1 ( 0.1b
Ta ble 4. Relative Energy Difference (∆E; PM3) and Geometry
Parameters of Low-Energy Conformers of 7 and 14 and
Geometry Parameters from Crystal Structures of 123 and 220
ED50 ) 20.0 (12.1-33.0)
vehicle
11
0.1 ( 0.1
0.1 ( 0.1
0.2 ( 0.1
0.3 ( 0.1
2.8 ( 0.2
2.3 ( 0.3
2.5 ( 0.2
2.3 ( 0.2
ED50 > 32
NT
NT
NT
NT
8
16
32
torsion
angle (deg)
distance
(Å)
angle
(deg)
plane
angle
∆E
no.
7a -66.3 -176.5 12.52 6.98
7b 66.3 179.3 12.53 7.00
7c 180.0 -179.9 12.67 6.99
τ2
τ3
c1-c2 N4-c2 c1-N4-c2 φ (deg) (kcal/mol)
vehicle
12
0.1 ( 0.1
162.2
162.3
178.4
144.5
132.9
144.7
144.0
132.1
143.8
146.5
165.3
67.74
67.42
179.78
121.25
62.59
93.04
117.75
66.51
91.79
67.82
2.77
0.94
0.89
0.0
0.82
0.02
0.00
1.47
0.44
0.33
2
4
8
1.0 ( 0.1d
1.9 ( 0.2e
2.5 ( 0.3e
14a 179.2
14b -66.3
2.0 11.14 6.01
3.4 10.78 6.08
3.8 11.11 5.81
ED50 ) 3.9 (2.9-5.3)
vehicle
14
0.1 ( 0.1
NT
NT
NT
NT
14c
66.9
4
8
16
0.9 ( 0.2d
14d 179.5 177.9 11.09 5.97
14e -66.3 178.9 10.69 6.02
1.3 ( 0.1e
1.6 ( 0.3e
14f
1
2
66.4 178.2 11.03 5.92
63.7 -124.5 11.04 5.87
179.8 -161.6 12.88 7.33
ED50 ) 8.0 (4.4-14.4)
a
b
p < 0.05. p < 0.01 vs vehicle + 8-OH-DPAT. c Data from ref
d
2. p < 0.05. e p < 0.01 vs vehicle. NT, not tested.
the same angle φ, its components were not equivalent
due to an unsymmetrically substituted benzotriazole
moiety which resulted in, e.g., different electrostatic
potential distribution around the molecule. The six
respective conformations of 2, optimized by an MAXI-
MIN2 method, were energetically less stable (between
0.86 and 1.85 kcal/mol) than the conformer of the
extended family in a global energy minimum, and one
of these conformers (derived from 14e) was very close
to the crystal structure of 220 (Table 4).
predominant conformations (7a ) 7b, and 7c) (Table
4) which differ in the angle between the aromatic planes
of terminal fragments (φ = 67° or 180°). Conformations
of the flexible molecule of 1, derived from favorable
conformers of 7, were found to be only 1.4 kcal/mol above
the global energy minimum which corresponds to its
fully extended structure. They were also similar to the
crystal structure of 123 (Table 4).
1
Like in the case of compound 7, a H NMR spectrum
analysis of structure 14 indicated its existence in the
1e,3e chair conformation. A conformational analysis was
based on the same procedure as previously (τ1 ) 180°),
and results for the (1R,3S)-enantiomer are given in
Table 4. Rotation of the benzotriazole-cyclohexane bond
(Figure 1b) gave two energy minima at τ3 = 0° and 180°,
which differed by 0.6 kcal/mol in favor of the former.
Regarding the three possible values of angle τ2 = 60°,
-60°, and 180°, six dominant conformations of 14 (Table
4: 14a -14f) within an energy range of 1.4 kcal/mol
should be considered. Those conformers formed three
pairs with respect to the angle between the aromatic
planes of terminal fragments (φ = 120°, 64°, and 92°,
respectively). Although each pair was characterized by
The conformational analysis has shown that predomi-
nant conformers of 7 and 14 belong to an extended
family of conformations. A closer insight into these
structures reveals that 7 is a linear molecule, whereas
compound 14 has an extended yet bent structure, as
indicated by the c1-N4-c2 angle (Table 4). Conse-
quently, compound 7 appears to be a good model of the
fully extended conformation of 1, and since 7, like 1,
displayed postsynaptic 5-HT1A antagonistic activity in
the tests used, it may be anticipated that the bioactive
conformation of 1, responsible for its postsynaptic
antagonism, is represented by the extended linear
conformer 7.

Bioactive Conformation of NAN-190 and MP3022
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 4957
F igu r e 2. Superimposition of low-energy conformers of 7a -c and 14a -c. For simplification cyclohexane rings and hydrogen
atoms in the rotating part of the structure have been omitted. Space explored by phthalimide and benzotriazole moieties are
represented by positions of c2-c3 segments. Color codes: phthalimide, red; benzotriazole, magenta; c2-c3 in phthalimide, yellow;
c2-c3 in benzotriazole, green.
A brief inspection of structures 14 and 2 suggests that
both these compounds possess the same spatial ar-
rangements of pharmacophores. However, among the
conformers of the flexible compound 2, one can find such
a conformation that fits fairly well the fully extended
conformation of 1 or 7; hence phthalimide or benzotria-
zole moieties in 1 and 2, respectively, may interact with
the same 5-HT1A receptor site, which results in the same
5-HT1A receptor functional activity. In contrast, the
respective fragments of 14 and 7 do not overlap. It is
very likely that they interact with different receptor
sites, which may be one of the reasons for their distinct
functional activity. To visualize differences in the spatial
arrangement of 7 and 14, we superimposed their
optimized structures using a common arylpiperazine
fragment (Figure 2). Then, the remaining fragments of
the molecules were allowed to rotate around τ2. For
simplification, we have presented the condensed five-
and six-membered rings as segments connecting their
centers. As can be seen, the cycloimide residue of 7 may
occupy a very limited space around the c1-N4 axis,
while the benzotriazole moiety in 14 may explore a
significantly larger region and, which is even more
noteworthy, in a different direction. Although the region
penetrated by the benzotriazole residue for both enan-
tiomers of 14 is essentially the same, their configuration
may also play a role at the receptor and, alternatively,
influence the functional activity of the compound.
However, it may be suggested that the bioactive con-
formation of 2, responsible for its postsynaptic antago-
nism at 5-HT1A receptors, is also represented by 7.
Nonetheless, further detailed studies are necessary to
solve unequivocally this problem.
5-HT1A antagonistic activity. Restriction of the confor-
mational freedom of 2 yielded 14 which revealed high
affinity and partial agonist properties at those receptors.
Conformational investigations of the flexible 1 and 2,
along with their newly synthesized constrained ana-
logues 7 and 14, allowed us to identify a bioactive
conformation of 1. We showed that the extended linear
structure, represented by 7, was responsible for the
postsynaptic 5-HT1A antagonism of the parent com-
pound.
Exp er im en ta l Section
Ch em istr y. Melting points were determined on a Boetius
1
apparatus and are uncorrected. H NMR spectra were taken
with a Bruker AMX 500 (500 MHz) or Varian EM-360 L (60
MHz) spectrometer in CDCl3 solutions with TMS as an
internal standard. 2D-NMR (H-H) COSY technique was used
to support interpretation of 1D spectra. The spectral data of
amines refer to their free bases. Chemical shifts are expressed
in δ (ppm) and the coupling constants J in hertz (Hz). All
compounds were routinely checked by TLC using Merck
Kieselgel 60-F254 sheets (detection at 254 nm). Column chro-
matography separations were carried out on Merck Kieselgel
60 or aluminum oxide 90, neutral (70-230 mesh). Elemental
analyses were performed in the Institute of Organic Chemis-
try, Polish Academy of Sciences (Warsaw, Poland), and were
within (0.4% of the theoretical values. The syntheses of 2-(4-
aminobutyl)-1,2,3,4-tetrahydroisoquinoline24 and 4-(4-ami-
nobutyl)-1-(2-methoxyphenyl)piperazine25 have been previ-
ously reported.
4-[4-(2-Me t h o x y p h e n y l)-1-p ip e r a zin y l]c y c lo h e x a -
n on e (3). Equimolar amounts (10 mmol) of 1-(2-methoxyphen-
yl)piperazine, benzotriazole, and 1,4-cyclohexanedione mono-
ethylene ketal were heated under reflux in benzene (100 mL)
for 2 h with azeotropic removal of water using a Dean-Stark
trap. The reaction mixture was cooled, washed with 15% Na2-
SO4 solution, and dried (K2CO3) and the solvent was evapo-
rated under reduced pressure. The residue solidified and
without further purification was suspended in THF (50 mL).
To this suspension was added portionwise NaBH4 (0.76 g, 20
mmol) and the mixture was stirred under reflux for 3 h. The
solvent was evaporated and the residue was treated with ice-
water (100 mL) and extracted with benzene. The organic layer
Con clu sion s
Summing up, structure modifications of 1, a postsyn-
aptic 5-HT1A receptor antagonist, provided its flexible
(8) and rigid (7, 9, 11) analogues which demonstrated
high affinity for 5-HT1A receptors and postsynaptic

4958 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24
Paluchowska et al.
was washed with water and dried over MgSO4. After evapora-
tion of the solvent the resulting oily ketal was dissolved in
100 mL of a 1:1 solution of acetone and 10% HCl and the
mixture was left at room temperature overnight. The acetone
was removed under reduced pressure and the remaining
aqueous solution was made alkaline with concentrated am-
monium hydroxide, extracted with CHCl3, and dried (MgSO4).
The extract was evaporated and the oily residue was treated
with hexane to afford the desired ketone 3 as colorless crystals
(1.5 g, 52%): mp 98-100 °C; 1H NMR (60 MHz) δ 7.0 (s, 4H),
3.9 (s, 3H), 3.3-3.0 (m, 4H), 3.0-2.6 (m, 5H), 2.6-2.2 (m, 4H),
2.2-1.5 (m, 4H).
4-[2-(1,2,3,4-T e t r a h y d r o is o q u in o lin y l)]c y c lo h e x a -
n on e (4). The title compound was prepared using 1,2,3,4-
tetrahydroisoquinoline according to the procedure described
for the preparation of 3. After evaporation of the final extract
0.6 g (26%) of a colorless oil was obtained: 1H NMR (60 MHz)
δ 7.1 (s, 4H), 3.8 (s, 2H), 3.1-2.6 (m, 1H), 2.8 (s, 4H), 2.6-1.6
(m, 8H).
4-[4-(2-Me t h oxyp h e n yl)-1-p ip e r a zin yl]cycloh e xyl-
a m in e (5). Hydroxylamine hydrochloride (0.43 g, 6.25 mmol)
was slurred in MeOH (10 mL), cooled to 0 °C, and treated with
Na2CO3 (0.33 g, 3.12 mmol). The mixture was stirred for 5 min
and a solution of ketone 3 (1.44 g, 5 mmol) in 20 mL of MeOH
was added. The reaction mixture was allowed to come to room
temperature, after stirring for 4 h water (30 mL) was added,
and stirring was continued for 30 min. Oxime of ketone 3 was
obtained as colorless crystals (1.5 g, 100%): mp 161-162 °C.
The resulting crude oxime was reduced in boiling n-BuOH (50
mL) with sodium (1.6 g, 70 mmol). The solvent was removed
under reduced pressure and the residue was treated with
water (100 mL) and extracted with CHCl3 (3 × 30 mL).
Combined extracts were washed with water and dried (K2CO3).
After evaporation of the solvent the product was purified by
column chromatography (SiO2, CHCl3/MeOH ) 9/1) to afford
the desired amine (1.0 g, 69%) as a pale yellow oil, which was
immediately used in the next step of synthesis: 1H NMR (60
MHz) δ 7.0 (s, 4H), 3.9 (s, 3H), 3.3-3.0 (m, 4H), 2.9-2.6 (m,
4H), 2.5-0.8 (cluster, 10H), 2.0 (s, 2H).
4-[2-(1,2,3,4-Te t r a h yd r oisoq u in olin yl)]cycloh e xyl-
a m in e (6). Starting with ketone 4 (0.69 g, 3 mmol) and
following the method described for the preparation of amine
5, the appropriate oxime was obtained (0.70 g, 95%) as
colorless crystals: mp 109-111 °C. Reduction of the crude
oxime (0. 64 g, 2.6 mmol) gave the amine 6, which was purified
by column chromatography (SiO2, CHCl3/MeOH ) 4/1). Com-
pound 6 was obtained as a pale yellow oil (0.48 g, 80%): 1H
NMR (60 MHz) δ 7.1 (s, 4H), 3.8 (s, 2H), 2.8 (s, 4H), 2.8-0.8
(cluster, 12H).
Gen er a l Meth od for th e P r ep a r a tion of Com p ou n d s
7-9 a n d 11-13. Equimolar amounts (1 mmol) of phthalimide
or cis-1,2-cyclohexanedicarboxylic anhydride and 4-(4-ami-
nobutyl)-1-(2-methoxyphenyl)piperazine or 2-(4-aminobutyl)-
1,2,3,4-tetrahydroisoquinoline or amines 5 and 6 were refluxed
in xylene (20 mL) for 3 h. After the mixture was allowed to
cool to room temperature, xylene was evaporated under
reduced pressure and the products were isolated on a column
with silica gel using eluents as follows: 7, CHCl3/MeOH ) 19/
1; 8 and 11, CHCl3; 9, 12, and 13, CHCl3/MeOH ) 49/1. Free
bases were converted into the hydrochloride salts in CHCl3 or
acetone solution by treatment with excess of Et2O saturated
with gaseous HCl.
tr a n s-1-(2-Meth oxyp h en yl)-4-[4-(2-p h th a lim id o)cyclo-
h exyl]p ip er a zin e (7). This compound was prepared by the
general procedure from phthalimide anhydride and amine 5
in 62% yield as colorless crystals: mp 210-212 °C (benzene);
1H NMR (500 MHz) δ 7.85-7.79 (m, 2H, phthalimide H-4 and
H-7), 7.72-7.68 (m, 2H, phthalimide H-5 and H-6), 7.03-6.90
(m, 3H, aromatic H) 6.87 (dd, J ) 8.0, 2.0, 1H, aromatic H),
4.12 (dt, J ) 12.4, 4.0, 1H, cyclohexane axial H-4), 3.88 (s, 3H,
OCH3), 3.12 (app br s, 4H, piperazine 2CH2), 2.82 (app br t,
4H, piperazine 2CH2), 2.54 (dt, J ) 11.7, 3.3, 1H, cyclohexane
axial H-1), 2.34 (dq, J ) 12.9, 3.2, 2H, cyclohexane axial H-3
and H-3′), 2.08 (app br d, 2H, cyclohexane equatorial H-2 and
H-2′), 1.84 (app br d, 2H, cyclohexane equatorial H-3 and H-3′),
1.47 (dq, J ) 12.8, 3.2, 2H, cyclohexane axial H-2 and H-2′).
7‚2HCl: mp 256-258 °C (MeOH/acetone). Anal. (C25H29N3O3‚
2HCl) C, H, N.
4-{4-[2-(cis-1,2-Cycloh exa n ed ica r boxyim id o)]bu tyl}-1-
(2-m eth oxyp h en yl)p ip er a zin e (8). This compound was
prepared by the general procedure from cis-1,2-cyclohexanedi-
carboxylic anhydride and 4-(4-aminobutyl)-1-(2-methoxyphen-
yl)piperazine in 86% yield as a pale yellow oil: 1H NMR (60
MHz) δ 7.0 (s, 4H), 3.85 (s, 3H), 3.8-3.3 (m, 2H), 3.3-3.0 (m,
4H), 3.0-2.2 (cluster, 8H), 2.1-1.1 (cluster, 12H). 8‚2HCl: mp
178-180 °C (EtOH). Anal. (C23H33N3O3‚2HCl) C, H, N.
tr a n s-4-{4-[2-(cis-1,2-Cycloh exa n ed ica r boxyim id o)]cy-
cloh exyl}-1-(2-m eth oxyp h en yl)p ip er a zin e (9). This com-
pound was prepared by the general procedure from cis-1,2-
cyclohexanedicarboxylic anhydride and amine 5 in 43% yield
as colorless crystals: mp 180-181 °C (EtOH); 1H NMR (60
MHz) δ 7.0 (s, 4H), 3.9 (s, 3H), 4.3-3.6 (m, 1H), 3.3-3.0 (m,
4H) 3.0-1.1 (cluster, 23H). 9‚2HCl: mp 249-251 °C (MeOH/
acetone). Anal. (C25H35N3O3‚2HCl) C, H, N.
tr a n s-2-[4-(2-P h th a lim id o)cycloh exyl]-1,2,3,4-tetr a h y-
d r oisoqu in olin e (11). This compound was prepared by the
general procedure from phthalimide anhydride and amine 6
in 65% yield as colorless crystals: mp 188-189 °C (CHCl3);
1H NMR (500 MHz) δ 7.85-7.77 (m, 2H, phthalimide H-4 and
H-7), 7.74-7.66 (m, 2H, phthalimide H-5 and H-6), 7.16-7.07
(m, 3H, THIQ), 7.06-7.00 (m, 1H, THIQ), 4.15 (dt, J ) 12.4,
4.0, 1H, cyclohexane axial H-4), 3.82 (s, 2H, THIQ H’s-1), 2.92-
2.82 (m, 4H, THIQ H’s-3 and H’s-4), 2.69 (dt, J ) 11.7, 3.3,
1H, cyclohexane axial H-1), 2.36 (dq, J ) 12.9, 3.3, 2H,
cyclohexane axial H-3 and H-3′), 2.11 (app br d, 2H, cyclohex-
ane equatorial H-2 and H-2′), 1.85 (app br d, 2H, cyclohexane
equatorial H-3 and H-3′), 1.54 (dq, J ) 12.8, 3.1, 2H, cyclo-
hexane axial H-2 and H-2′). 11‚HCl‚0.4H2O: mp 230 °C dec
(MeOH/acetone). Anal. (C23H24N2O2‚HCl‚0.4H2O) C, H, N.
2-{4-[2-(cis-1,2-Cycloh exa n ed ica r b oxyim id o)]b u t yl}-
1,2,3,4-tetr a h yd r oisoqu in olin e (12). This compound was
prepared by the general procedure from cis-1,2-cyclohexanedi-
carboxylic anhydride and 2-(4-aminobutyl)-1,2,3,4-tetrahy-
droisoquinoline in 89% yield as a pale yellow oil: 1H NMR (60
MHz) δ 7.1 (s, 4H), 3.6 (s, 2H), 3.7-3.3 (m, 2H), 3.1-2.3
(cluster, 8H), 2.0-1.2 (cluster, 12H). 12‚HCl‚0.5H2O: mp
178-180 °C (EtOH/acetone). Anal. (C21H28N2O2‚HCl‚0.5H2O)
C, H, N.
2-{4-[2-(ci s-1,2-C y c lo h e x a n e d ic a r b o x y im id o )]c y -
cloh exyl}-1,2,3,4-tetr a h yd r oisoqu in olin e (13). This com-
pound was prepared by the general procedure from cis-1,2-
cyclohexanedicarboxylic anhydride and amine 6 in 50% yield
as colorless crystals: mp 159-161 °C (EtOH/hexane); 1H NMR
(60 MHz) δ 7.0 (s, 4H), 4.3-3.8 (m, 1H), 3.85 (s, 2H), 3.0-1.2
(cluster, 19H), 2.9 (s, 4H). 13‚HCl: mp 235 °C dec (MeOH/
acetone). Anal. (C23H30N2O2‚HCl) C, H, N.
2-[4-(2-P h th a lim id o)bu tyl]-1,2,3,4-tetr a h yd r oisoqu in o-
lin e (10). This compound was obtained according to Mokrosz
et al.24 10‚HCl: mp 244-246 °C (acetone). Anal. (C21H22N2O2‚
HCl) C, H, N.
cis-4-[3-(1-Ben zotr iazolyl)cycloh exyl]-1-(2-m eth oxyph en -
yl)p ip er a zin e (14). A mixture of benzotriazole (1.19 g, 10
mmol) and 2-cyclohexen-1-one (0.48 g, 5 mmol) in Et2O (30
mL) was stirred at room temperature for 6 h and left overnight.
The reaction mixture was cooled in an ice-water bath, a
solution of 1-(2-methoxyphenyl)piperazine (1.06 g, 5.5 mmol)
in Et2O (10 mL) was added, and the stirring was continued
for 20 h. Afterward the intermediate product was filtered off,
washed with Et2O, and suspended in dioxane (40 mL). This
suspension was treated with NaBH4 (0.1 g, 2.5 mmol) on
stirring, refluxed for 4 h, and left overnight at room temper-
ature. The reaction mixture was poured into 10% NaOH (40
mL) and extracted with Et2O (3 × 30 mL). The combined
organic layers were washed with water and dried (MgSO4).
Then the solvent was evaporated and the residue was purified
by column chromatography (Al2O3 neutral, AcOEt/hexane )
1/2) to afford 14 (0.31 g, 16%) as a pale yellow oil: 1H NMR
(500 MHz) δ 8.07 (d, J ) 8.4, 1H, benzotriazole H-4), 7.59 (d,

Bioactive Conformation of NAN-190 and MP3022
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 24 4959
J ) 8.4, 1H, benzotriazole H-7), 7.48 (t, J ) 7.4, 1H, benzo-
triazole), 7.37 (t, J ) 7.6, 1H, benzotriazole), 7.02-6.99 (m,
1H, aromatic H), 6.95-6.90 (m, 2H, aromatic H), 6.86 (d, J )
7.9, 1H, aromatic H), 4.76 (dddd, J ) 12.0, 11.9, 4.0, 3.8, 1H,
cyclohexane H-3), 3.86 (s, 3H, OCH3), 3.14 (app br s, 4H, 2CH2),
2.89 (app br s, 4H, 2CH2), 2.76 (app br t, J = 11.3, 1H,
cyclohexane H-1), 2.50 (app br d, 1H, cyclohexane H), 2.21 (br
s, 1H, cyclohexane H), 2.18-2.11 (m, 4H, cyclohexane H),
1.61-1.46 (m, 2H, cyclohexane H). 14‚2HCl: mp 186-188 °C
(MeOH/acetone). Anal. (C23H29N5O‚2HCl) C, H, N.
Molecu la r Mod elin g. All of the compounds investigated
in molecular modeling experiments were first built with the
sketch option in the Sybyl package, version 6.4 (Tripos
Associates, Inc., St. Louis, MO). Geometry optimizations were
performed using the semiempirical PM3 (MOPAC) method as
well as molecular mechanics MAXIMIN2. The rotation barriers
between separate piperazine-cyclohexane (τ2) and cyclohex-
ane-phthalimide (τ3) subunits were studied with PM3 method.
The fragments were minimized over all bonds and angles
except the respective torsion angle, which was constrained at
values between 0° and 360° with a 10° increment. Global
energy minima of 1 and 2 were found by RANDOMSEARCH
procedure (energy cutoff: 5 kcal/mol) in which only internal
C-C bonds of the spacer were allowed to rotate.
Refer en ces
(1) Glennon, R. A.; Naiman, N. A.; Pierson, M. E.; Titeler, M.; Lyon,
R. A.; Weisberg, E. NAN-190: an Arylpiperazine Analogue That
Antagonizes the Stimulus Effects of the 5-HT1A Agonist 8-Hy-
droxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). Eur. J . Phar-
macol. 1988, 154, 339-341.
(2) Mokrosz, J . L.; Paluchowska, M. H.; Chojnacka-Wo´jcik, E.; Filip,
M.; Charakchieva-Minol, S.; Deren´-Wesołek, A.; Mokrosz, M. J .
Structure-Activity Relationship Studies of Central Nervous
System Agents. 13. 4-[3-(Benzotriazol-1-yl)propyl]-1-(2-meth-
oxyphenyl)piperazine, a New Putative 5-HT1A Receptor Antago-
nist, and Its Analogues. J . Med. Chem. 1994, 37, 2754-2760.
(3) Hjorth, S.; Sharp, T. Mixed Agonist/Antagonist Properties of
NAN-190 at 5-HT1A Receptors: Behavioural and In Vivo Brain
Microdialysis Studies. Life Sci. 1990, 46, 955-963.
(4) Przegalin´ski, E.; Ismaiel, A. M.; Chojnacka-Wo´jcik, E.; Bud-
ziszewska, B.; Tatarczyn´ska, E.; Błaszczyn´ska, E. The Behav-
ioural, but not the Hypothermic or Corticosterone, Response to
8-Hydroxy-2-(di-n-propylamino)tetralin, is Antagonized by NAN-
190 in the Rat. Neuropharmacology 1990, 29, 521-526.
(5) Moore, N. A.; Rees, G.; Sander, G.; Perrett, L. 5-HT1A-Mediated
Lower Lip Retraction: Effects of 5-HT1A Agonists and Antago-
nists. Pharmacol. Biochem. Behav. 1993, 46, 141-143.
(6) Przegalin´ski, E.; Filip, M.; Bijak, M.; We¸dzony, K.; Budziszew-
ska, B.; Tokarski, K.; Mac´kowiak, M.; Fijał, K. MP-3022, a New
Putative Antagonist at Pre- and Postsynaptic 5-HT1A Receptors.
Pol. J . Pharmacol. 1996, 48, 13-22.
(7) Filip, M.; Przegalin´ski, E. Effects of MP-3022 on the 8-OH-
DPAT-induced Discriminative Stimulus in Rats. Pol. J . Phar-
macol. 1996, 48, 397-402.
Ra d ioliga n d Bin d in g Stu d ies. The affinity of the inves-
tigated compounds for 5-HT1A receptors was assessed on the
basis of their ability to displace [3H]-8-OH-DPAT (220 Ci/mmol;
Amersham), and experiments were carried out in the hippo-
campus of rat brain, according to published procedures.26 Ki
values were determined from at least three competition
binding experiments in which 10-14 drug concentrations, run
in triplicate, were used. The Cheng and Prusoff27 equation was
used for Ki calculations.
In Vivo Stu d ies. The experiments were carried out on male
Wistar rats (260-300 g). The animals were kept at an ambient
temperature of 20 ( 1 °C throughout the experiment and had
free access to food (standard laboratory pellets, LSM) and tap
water. All experiments were conducted in the light phase on
a natural light-dark cycle (from May to August), between 9
a.m. and 2 p.m. 8-OH-DPAT (Research Biochemical, Inc.) and
reserpine (Ciba; ampules) were dissolved in saline. The
investigated salts of tested compounds were used in the form
of freshly prepared suspensions in 1% Tween 80. 8-OH-DPAT
and reserpine were injected subcutaneously (sc) and tested
compounds intraperitoneally (ip) in a volume of 2 mL/kg. Each
group consisted of 6 animals. The obtained data were analyzed
by the Newman-Keuls test.
Beh a vior a l Syn d r om e in Reser p in ized Ra ts. The rats
were individually placed in cages (30 × 25 × 25 cm) 5 min
before injection of tested compounds or 8-OH-DPAT. Observa-
tion sessions, lasting 45 s each, began 3 min after drug
administration and were repeated every 3 min. Reciprocal FT
and FBP were scored using a ranked intensity scale where 0
) absent, 1 ) equivocal, 2 ) present, and 3 ) intense. The
maximum score, summed up over 5 observation periods,
amounted to 15 for each symptom/animal.28 The effect of tested
compounds on the behavioral syndrome induced by 8-OH-
DPAT (5 mg/kg) was tested in a separate experiment. The
investigated compounds were administered 60 min before
8-OH-DPAT, and the animals were scored of 3, 6, 9, 12, and
15 min after 8-OH-DPAT treatment. Reserpine (1 mg/kg) was
administered 18 h before the test.
Low er Lip Retr a ction in Ra ts. LLR was assessed ac-
cording to the method described by Berendsen et al.29 The rats
were individually placed in cages (30 × 25 × 25 cm) and were
scored three times (at 15, 30, and 45 min after tested
compound or 8-OH-DPAT administration) as follows: 0 )
lower incisors not visible, 0.5 ) partly visible, 1 ) completely
visible. The maximum score, summed, amounted to 3 for each
rat. The effect of investigated compounds on the LLR induced
by 8-OH-DPAT (1 mg/kg) was tested in a separate experiment.
The investigated compounds were administered 45 min before
8-OH-DPAT, and the animals were scored at 15, 30, and 45
min after 8-OH-DPAT administration.
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