Modification of the structure of 4,6-disubstituted 2-(4-alkyl-1-piperazinyl)pyridines: Synthesis and their 5-HT2A receptor activity

Article abstract of DOI:10.1002/ardp.200390006

Structure-activity relationship studies of a series of novel 4,6-disubstituted 2-(1-piperazinyl)pyridines were conducted to revise our model of serotonin 5-HT_2A receptor antagonist. Target compounds were synthesized using the benzotriazole-assi

Full text of DOI:10.1002/ardp.200390006

104 Paluchowska et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 2, 104–110
Maria H. Paluchowskaa,
Andrzej J. Bojarskia,
Ryszard Bugnoa,
Sijka Charakchieva-Minola,
Anna Wesolowskab
Modification of the Structure of 4,6-Disubstituted
2-(4-Alkyl-1-piperazinyl)pyridines: Synthesis and
Their 5-HT2A Receptor Activity
Structure-activity relationship studies of a series of novel 4,6-disubstituted 2-(1-pip-
erazinyl)pyridines were conducted to revise our model of serotonin 5-HT2A receptor
antagonist. Target compounds were synthesized using the benzotriazole-assisted
Katritzky method. The majority of those compounds were found to be selective
5-HT2A/5-HT1A receptor ligands, though less potent than their previously described
pyrimidine counterparts.In particular, the three compounds 6–8 showed the highest
5-HT2A receptor affinity (Ki = 34–78 nM) and were classified as 5-HT2A antagonists in
in vivo experiments. The influence of the structural modifications on the in vitro re-
sults was discussed; however, the elucidation of the role of the central core system
requires further studies.
a
Department of Medicinal
Chemistry and
Department of New Drug
Research,
Institute of Pharmacology,
Polish Academy of
Sciences, Kraków, Poland
b
Keywords: 4,6-Disubstituted pyridines; 5-HT2A/5-HT1A receptor activity; Structure-
activity relationship studies
Received: May 6, 2002 [FP697]
Introduction
5-HT2A receptors were the first population of the entire
serotoninergic family to be identified, due to an early dis-
covery of ketanserin as their selective antagonist. The
role of 5-HT2A receptors in the regulation of a number of
processes of the central nervous system (mood, appe-
tite, sexual behavior, learning, and memory, etc.) and
their dysfunctions (e.g. psychosis, depression, anxiety,
and sleep disorders) has been well documented [1, 2].
Moreover, for a large number of psychiatrically important
drugs, including atypical antipsychotics, antidepres-
sants, and anxiolytics, interactions with 5-HT2A sites are
one of the major features of their therapeutic action
[3–6].
Chart 1
Essentially the same characteristic geometry was inde-
pendently reported by Andersen [9], though for another
type of 5-HT2A receptor ligands (indole and indan
derivatives).Further support for the usefulness of such a
model comes from recent publications by Glennon and
co-workers [12, 13], who showed that compounds of
type 2 had the respective distances d1, d2, d3 consistent
with the defined ranges. At the same time, however,
9-(aminomethyl)-9,10-dihydroanthracene (AMDA, 3) did
not fit that model, since distances d1 and d2 were too
short.It was then suggested that AMDA could serve as a
template for the construction of novel 5-HT2A receptor
antagonists, as well as for the extension of a 5-HT2A
receptor model.
An important step in the drug discovery process is the
construction of various ligand-receptor interaction mod-
els on the grounds of systematic structure-activity rela-
tionship studies. The first comprehensive 5-HT2A topo-
graphic model, based on the structure of (+)-LSD (a
highly potent nonselective serotoninergic agent), was
proposed by Glennon et al. in 1991 [7]. Several other
models were later created by Höltje [8], Andersen [9],
as well as by our research group [10, 11].The last-men-
tioned model, developed from a series of 4,6-disubstitut-
ed 2-(1-piperazinyl)pyrimidines, defined three distances
between terminal nitrogen atoms and two centers of aro-
matic substituents in the 5-HT2A receptor antagonist.
The development of a model is a continuous process, so
we undertook further studies to verify our 5-HT2A phar-
macophore and to make it more general.Since our previ-
ous investigations with the pyrimidine derivatives
concentrated on the influence of 4- and/or 6-substi-
tutents, in the present paper we focused our attention on
Correspondence: Maria H. Paluchowska, Department of
Medicinal Chemistry, Institute of Pharmacology PAS,
12 Sme¸tna Str., 31-343 Kraków, Poland. Phone:
+48 12 6623365, fax: +48 12 6374500, e-mail:
paluchm@if-pan.krakow.pl.
© 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/02/0104 $ 17.50+.50/0

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 104–110
2-(4-Alkyl-1-piperazinyl)pyridines 105
the role of the central heteroaromatic ring in ligand-5-
HT2A receptor interactions.To expand the group of com-
pounds that match our 5-HT2A antagonist model, we
conducted structure-activity relationship studies with a
series of new 4,6-disubstituted 2-(1-piperazinyl)pyrid-
ines.
compounds 6–17, we applied the benzotriazole-assist-
ed Katritzky method [14] for the reaction of α,β-unsatu-
rated ketones with 2-(1-benzotriazolyl)acetonitrile and
sec-amines such as diethylamine, piperidine, pyrrolid-
ine, or morpholine in ethanol; after a 48-hour reflux, they
afforded the corresponding 4,6-disubstituted 2-(substi-
tuted amino)pyridines.In our study we used N-methyl- or
N-benzylpiperazine as a sec-amine substrate.The start-
ing α,β-unsaturated ketones were synthesized by a clas-
sical chalcone method, in the reaction of the appropriate
aldehyde and acetophenone derivative in ethanol-aque-
ous NaOH medium at the room temperature. For
the preparation of 2-(1-benzotriazolyl)acetonitrile the
Chemistry
The structures of the compounds described in this study
are shown inTable 1, and the methods of their synthesis
are outlined in Schemes 1 and 2.To synthesize the new
Table 1. Structure of the new ligands and their affinity for the 5-HT2A and 5-HT1A serotonin receptors.
Compda
X
R
R1
R2
Ki SEM [nM]
5-HT2A
5-HT1A
1b
4b
5b
N
N
N
CH3
CH3
CH3
2-thienyl
2-thienyl
2-thienyl
7.5 1.2
208 16
2095 28
9722 135
H
H
484
4
Ph
613 35
6
7
8
9
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
2-thienyl
2-thienyl
Ph
Ph
2-thienyl
Ph
2-furyl
Ph
Ph
2-thienyl
Ph
2-thienyl
Ph
CH3
CH3
Ph
2-pyridyl
p-ClC6H4
p-OCH3C6H4
64
34
78 12
144
189 29
139
125 10
222
304
168 23
292
1
1
3670
8
3150 80
3580 30
2010 20
2740 30
2130 10
1990 50
2520 50
3520 10
4
10
11
12
13
14
15
16
17
18
19
20
21b
4
1
1
Ph
2050
5
p-OCH3C6H4
p-OCH3C6H4
Ph
5
6270 14
1350 30
13000 25
5779 80
p-OCH3C6H4
440 16
180 12
437 18
H
Ph
Ph
H
Ph
Ph
H
n-C6H13
n-C6H13
n-C6H13
1452 31
10000 120
47
84 10
8
H
H
a
Compounds were tested as the hydrochloride or fumarate salts.
Data taken from ref. [10].
b

106 Paluchowska et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 104–110
aspartic acid (ASP155) of the 5-HT2A receptor. As ob-
served in the crystal structure of compounds containing
pyrimidinepiperazine fragments, and which can also be
inferred from conformational studies, both rings are co-
planar and have a relatively high (as compared to other
arylpiperazines) rotation barrier [15].The heteroaromat-
ic substituents in positions 4 and 6 of pyrimidine, which
also show a tendency to form a planar system, serve as
additional anchoring points. It has been found that their
local dipole moments are an important feature influenc-
ing 5-HT2A receptor affinity [10]. In order to verify the im-
portance of a core pyrimidine ring, one nitrogen atom
was replaced with carbon; thus a pyridine system was
used in a series of new compounds 6–20. Such a slight
structural modification leads to a differentiation between
the same substituents in positions 4 and 6 in the hetero-
aromatic ring, and changes the hydrogen-bond accept-
ing properties of the central ring and the electrostatic po-
tential of the whole molecule.
Scheme 1. a) EtOH, 48 h, reflux.
In general, 4,6-disubstituted 2-(4-methylpiperazin-1-
yl)pyridines 6–17 bind to 5-HT2A receptors with moder-
ate affinities ranging from 34 to 440 nM for 7 and 17, re-
spectively (Table 1). 4,6-Dithienyl derivative 6 (Ki =
64 nM) shows a ca. 9-fold lower 5-HT2A affinity than its
pyrimidine analog 1 (Ki = 7.5 nM) – the most active lig-
and of the previously described pyrimidine series. A dis-
parity between positions 4 and 6 in the central pyridine
ring can be seen when both pairs of thienyl, phenyl (7, 8)
and phenyl, p-methoxyphenyl (15, 16) derivatives are
compared. In each case an exchange of substituents re-
sults in a ca. 2-fold change in affinity. Consequently, di-
phenyl and di-p-methoxyphenyl analogs are the least
potent 5-HT2A ligands in both series 6–9 and 9, 15–17,
respectively. Interestingly, pyridines 10 and 11 with only
one aryl substituent in position 4 (2-thienyl and phenyl,
respectively) – and thus devoid of the second anchoring
aromatic point – bind with a higher affinity to 5-HT2A sites
than their pyrimidine counterparts 4 and 5. Since the
5-HT2A receptor affinity of 11 vs 5 rose substantially, a
subset of 4-phenyl substituted derivatives 8, 9, 11, and
13–15 was analyzed. Unexpectedly, only 6-(2-thienyl)
derivative 8 demonstrated a higher affinity than did its
methyl analogue 11.The activity of 6-phenyl substituted
compound 9 remained the same whereas the other com-
pounds (13–15) were even less active. In our earlier pa-
pers dealing with pyrimidinepiperazines [10, 11], 4,6-dia-
ryl-substituted derivatives were always more active than
monosubstituted ones. Thus it may be concluded that
the methyl group in position 6 of a pyridine ring also
brings about some interactions favorable for ligand-re-
ceptor complex formation.In a similar subset of 6-phenyl
substituted ligands (7, 9, 12, and 16), compound 7 with a
4-(2-thienyl) group also showed the highest 5-HT2A re-
ceptor affinity. Unfortunately, additive effects for both
Scheme 2.b) H2 (4 atm), Pd/C, MeOH/AcOH, 4 h, 50 °C;
c) hexanal, benzotriazol, benzene, 1 h, reflux;d) NaBH4,
THF, 3 h, room temp.
method described by Katritzky et al. [14] was used. Re-
ductive debenzylation of 2-(4-benzyl-1-piperazinyl)-4,6-
diphenylpyridine with hydrogen and palladium support-
ed on charcoal as a catalyst, in an autoclave afforded
amine 18 in a moderate yield of 20 %.n-Hexyl derivatives
19 and 20 were prepared by a two-step procedure, i.e.by
a Mannich-type reaction between benzotriazole, hex-
anal, and amine 18 or commercial N-(2-pyridyl)pipera-
zine, followed by the reduction of the obtained adducts
with NaBH4 in THF.
Results and discussion
As has been mentioned in the introduction, the majority
of model compounds used for generating a 5-HT2A an-
tagonist pharmacophore contain a pyrimidine ring as the
central core [10, 11].N-Methylpiperazine moiety in posi-
tion 2 of pyrimidine supplies a basic nitrogen atom nec-
essary for a proper ionic interaction with the respective

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 104–110
2-(4-Alkyl-1-piperazinyl)pyridines 107
thienyl substituents were not observed for compound 6,
its Ki value not being as high as expected.
cantly influence the observed affinity for those sites, hav-
ing reached the micromolar range only (Ki = 1.5–6.2 µM)
(Table 1).Thus, like the former pyrimidines, all these lig-
ands remained selective versus 5-HT1A receptors (S2A/1A
= 0.011–0.32 for 7 and 17, respectively). Further struc-
tural modifications, which involved the decrease in ba-
sicity of the N-4 piperazine atom caused by its demethyl-
ation, did not influence 5-HT2A receptor affinity (9 vs 18);
the affinity for 5-HT1A receptors was the lowest in the
tested series.
In conclusion, the presented set of pyridines showed as
a rule a weaker 5-HT2A receptor affinity than did the pre-
viously described pyrimidine series. Therefore it seems
that the central ring not only acts as a connector of three
pharmacophoric points but also directly or indirectly in-
fluences ligand-receptor complex formation. The nitro-
gen-for-carbon exchange influenced primarily the rota-
tion of the piperazine fragment with respect to the central
ring. A comparison between the rotation energy profiles
of 1-(2-pyrimidyl)- and 1-(2-pyridyl)piperazines (Fig-
ure 1) indicated that the rotation barrier of the latter was
reduced ca. 2-fold, while the positions of global energy
minima remained intact and both rings also preferred a
coplanar conformation. On the other hand, the dis-
cussed modification affected the arrangement of hetero-
aromatic substituents in position 4. While in the case of
2-thienyl moiety the s-cis conformation was preferred in
pyrimidine ligands, both the s-cis and the s-trans ar-
rangement could be adopted by that substituent with the
same probability in pyridine derivatives.Hence the direc-
tion of the local dipole moment of heteroaromatic substit-
uents in position 4 is ambiguous which can result in the
observed decrease in 5-HT2A receptor affinity.
Substitution of the N4-piperazine atom in simple aryl-
piperazines with a long hydrocarbon aliphatic chain was
found to substantially increase their 5-HT1A receptor
affinity [16], especially for n-hexyl derivatives, the most
potent and selective versus 5-HT2A receptor sites.In fact,
in our experiment, the 5-HT1A receptor affinity of such
substituted pyridine 20 was relatively high (Ki = 47 nM) –
even somewhat higher than for its pyrimidine analog 21
(Ki = 84 nM). For this reason we tried to switch the
5-HT2A/5-HT1A receptor profile of 4,6-disubstituted pyrid-
ine 18. However, the observed 5-HT1A and 5-HT2A affini-
ties of n-hexyl derivative 19, compared to those of 9, indi-
cated that such structural modifications in the structures
under discussion were not a prospect for mixed 5-HT1A/
5-HT2A receptors ligands – compounds with potential
anxiolytic and/or antidepressant properties.
On the other hand, it cannot be excluded that nitrogen
atoms are directly engaged in interactions stabilizing the
ligand receptor complex, e.g. hydrogen bonds. Further
studies using new model compounds with different core
systems are necessary to elucidate the role of the cen-
tral ring.
Our further investigation was concentrated on in vivo ef-
fects of four selected compounds which demonstrated
the highest affinity for 5-HT2A (compounds 6–8) and
5-HT1A (compound 20) receptors.It is commonly accept-
ed that the head-twitches in mice induced by ( )-1-(2,5-
dimethoxy-4-iodophenyl)-2-aminopropane hydrochlo-
ride [( )-DOI], a 5-HT2A receptor agonist, are connected
with stimulation of 5-HT2A receptors, and that the central
5-HT2A antagonistic activity can be regarded as the abili-
ty of the tested compounds to antagonize this symptom
produced by ( )-DOI in mice [17]. The results of the
As regards 5-HT1A receptors, the changes introduced in-
to the structure of model pyridines 6–17 did not signifi-
Table 2.The effect of the investigated compounds on the
inhibition of ( )-DOI-induced head twitches in mice
(2.5 mg/kg, ip).
Compound
ID50 (mg/kg, ip)a
6
7
8
9.3 (5.8–14.9)
4.5 (3.0–6.8)
10.5 (6.6–16.8)
0.14 (0.07–0.20)
Ketanserin
Figure 1. Plot of energy profiles (PM3 method) against
the dihedral angle N1-C2-N1-Nlp for 4-(2-pyrimidine)-
and 4-(2-pyridine)piperazine fragments.
a
ID50 – the dose inhibiting the head twitches in mice by
50 %; confidence limits (90 %) given in parenthesis.

108 Paluchowska et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 104–110
present study shown in Table 2 indicate that compounds
6–8, as well as the reference drug ketanserin effectively
inhibited the ( )-DOI-induced head-twitches in mice.
ID50 values of these compounds were between 4.5 (7)
and 10.5 (8) mg/kg; hence these compounds may be
classified as 5-HT2A receptor antagonists.
Experimental
The uncorrected melting points were determined using an
Electrothermal IA9000 apparatus. H NMR spectra were re-
1
corded on a Varian EM-360L (60 MHz) in CDCl3 solutions with
TMS as an internal standard.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 byTLC using Merck Kieselgel 60-F254 sheets
(detection at 254 nm). Column chromatography separations
were carried out on Merck Kieselgel 60 or aluminum oxide 90,
neutral (70–230 mesh). Commercially available reagent grade
solvents and reagents were used without further purification.
For biological experiments, free bases of the tested com-
pounds were converted into hydrochloride salts in acetone with
an excess of Et2O saturated with gaseous HCl, or into fumarate
salts in EtOH using an equimolar amount of fumaric acid.Their
molecular formulae and molecular weights were established
on the basis of an elemental analyses which were within 0.4 %
of theoretical values.
It was found previously that the lower lip retraction (LLR)
induced in rats by 8-hydroxy-2-(di-n-propylamino)tetralin
hydrobromide (8-OH-DPAT), a 5-HT1A receptor agonist,
was mediated by postsynaptic 5-HT1A receptors [18].
Therefore, in order to determine the postsynaptic 5-HT1A
receptor agonistic effect of 20 (5-HT1A ligand), its ability
to induce LLR in rats was tested, whereas its 5-HT1A an-
tagonistic activity was assessed as the inhibition of that
symptom produced by 8-OH-DPAT in rats.Compound 20
alone – likewise in both the doses used (10 and
20 mg/kg) – produced LLR in rats; the maximum score
was 63 % (p < 0.01), whereas the maximum possible
score observed after administration of 8-OH-DPAT
(1 mg/kg) was 100 %. At the same time, compound 20
(10 and 20 mg/kg) inhibited dose-dependently, by 29 %
(p < 0.05) and 46 % (p < 0.01), respectively, the 8-OH-
DPAT-induced LLR in rats.
General procedure for preparation of compounds 6–17
Equimolar amounts (3 mmol) of 2-(1-benzotriazolyl)aceto-
nitrile [14] and appropriate α,β-unsaturated ketone with N-
methylpiperazine (2 mL) were refluxed in EtOH (10 mL) for
48 h. The solvent was evaporated, and the residue was dis-
solved in CHCl3 (20 mL), washed with 10 % NaOH then with
brine, and dried over anhydrous MgSO4. After evaporation of
the solvent the residue was purified by column chromatography
as follows: 6, 9, and 12–14 – SiO2, CHCl3/MeOH = 49/1; 8, 10,
15, and 17 – SiO2, CHCl3/MeOH = 19/1; 7 and 16 – neutral
Al2O3, CHCl3/n-hexane = 1/1; 11 and 18–20 – SiO2, AcOEt/n-
hexane = 1/4.
Used as a standard compound, the well-known 5-HT1A
receptor antagonist, N-{2-[4-(2-methoxyphenyl)-1-
piperazinyl]ethyl}-N-(2-pyridyl)cyclohexanecarbox-
amide trihydrochloride (WAY 100635) [19], adminis-
tered in a dose of 0.1 mg/kg, reduced by 90 % (p < 0.01)
the LLR evoked by 8-OH-DPAT.Thus compound 20 may
be regarded as a weak partial agonist of postsynaptic
5-HT1A receptors.
2-(4-Methyl-1-piperazinyl)-4,6-di(2-thienyl)pyridine 6
Yield 43 %; pale yellow crystals, mp 92–94 °C (CHCl3/n-hex-
ane), Rf 0.2. – 1H NMR: 2.3 (s, 3 H, CH3), 2.4–2.7 (m, 4 H,
2 CH2), 3.5–3.9 (m, 4 H, 2 CH2), 6.7 (s, 1 H, H-3 in pyridine),
7.0–7.8 (cluster, 7 H, ArH). – 6 (fumarate): mp 211–213 °C
(EtOH). – Anal. (C18H19N3S2 · C4H4O4) C, H, N.
2-(4-Methyl-1-piperazinyl)-6-phenyl-4-(2-thienyl)pyridine 7
Conclusions
Yield 40 %;yellow crystals, mp 114–116 °C (CHCl3/n-hexane =
1/1), Rf 0.6.– 1H NMR:2.3 (s, 3 H, CH3), 2.4–2.8 (m, 4 H, 2 CH2),
3.5–4.0 (m, 4 H, 2 CH2), 6.8 (s, 1 H, H-3 in pyridine), 7.0–7.8
(cluster, 7 H, ArH), 7.9–8.4 (m, 2 H, H-2 and H-6 in 6-phenyl). –
The designed series of substituted pyridines originates
from our topographic model of 5-HT2A receptor antago-
nists.As can be inferred from the presented results, they
may constitute a new group of selective 5-HT2A/5-HT1A
receptor antagonists, less potent than their pyrimidine
analogs, though. It seems that introduction of the pyrid-
ine ring into the structure of a 5-HT2A receptor ligand in
place of the pyrimidine system is not a beneficial struc-
tural modification.It may be suggested, however, that the
central ring plays an important role in the ligand-receptor
interaction.Further studies are necessary to explain this
mechanism. Other core systems, such as benzene,
1,3,5-triazine and diversely substituted pyrimidine rings,
are currently under investigation.
7
(fumarate): mp 260–261 °C (EtOH/acetone).
– Anal.
(C20H21N3S · C4H4O4 · H2O) C, H, N.
2-(4-Methyl-1-piperazinyl)-4-phenyl-6-(2-thienyl)pyridine 8
Yield 47 %; yellow oil, Rf 0.3. – 1H NMR: 2.4 (s, 3 H, CH3), 2.4–
2.8 (m, 4 H, 2 CH2), 3.6–4.0 (m, 4 H, 2 CH2), 6.7 (s, 1 H, H-3 in
pyridine), 7.0–7.8 (cluster, 9 H, ArH). – 8 (fumarate): mp 217–
219 °C (EtOH). – Anal. (C20H21N3S · 1.5 C4H4O4) C, H, N.
2-(4-Methyl-1-piperazinyl)-4,6-diphenylpyridine 9
Yield 48 %; yellow oil, Rf 0.2. – 1H NMR: 2.4 (s, 3 H, CH3), 2.4–
2.8 (m, 4 H, 2 CH2), 3.6–4.0 (m, 4 H, 2 CH2), 6.8 (s, 1 H, H-3 in
pyridine), 7.2 (s, 1 H, H-5 in pyridine), 7.2–7.9 (cluster, 8 H,
ArH), 8.0–8.3 (m, 2 H, H-2 and H-6 in 6-phenyl). – 9 (fumarate):
mp 270–272 °C (EtOH/n-hexane). – Anal. (C22H23N3 · 2 HCl ·
2.5 H2O) C, H, N.
Acknowledgements
6-Methyl-2-(4-methyl-1-piperazinyl)-4-(2-thienyl)pyridine 10
This study was partially supported by the Polish State
Committee for Scientific Research (KBN), grant no. 4
P05F 005 18.
Yield 19 %;yellow oil, Rf 0.2.– 1H NMR:2.3 (s, 3 H, CH3), 2.4 (s,
3 H, CH3), 2.2–2.9 (m, 4 H, 2 CH2), 3.4–3.9 (m, 4 H, 2 CH2), 6.6

Arch. Pharm. Pharm. Med. Chem. 2003, 336, 104–110
2-(4-Alkyl-1-piperazinyl)pyridines 109
4,6-Diphenyl-2-(1-piperazinyl)pyridine 18
(s, 1 H, H-3 in pyridine), 6.7 (s, 1 H, H-5 in pyridine), 7.0–7.6 (m,
3 H, thienyl). – 10 (fumarate): mp 165–167 °C (EtOH). – Anal.
(C15H19N3S · 1.2 C4H4O4) C, H, N.
A mixture of 2-(4-benzyl-1-piperazinyl)-4,6-diphenylpyridine
(1.18 g, 2.9 mmol) – prepared according to the procedure de-
scribed above for compounds 6–17 with N-benzylpiperazine as
a substrate – and palladium supported on charcoal (0.2 g) in
methanol (50 mL) and acetic acid (5 mL) was reduced with hy-
drogen (4 atm) in an autoclave at 50 °C for 20 h. The catalyst
was filtered off and the solvent was evaporated. The oily resi-
due was treated with water (50 mL), made alkaline with aque-
ous NH3, and extracted with CHCl3 (3 × 20 mL).The combined
extracts were dried over anhydrous MgSO4 and evaporated.
The residue was purified by silica gel column chromatography
(AcOEt/n-hexane = 1/4) to give 18 (0.76 g, 83 %) as colorless
crystals, mp 146–148 °C (CHCl3/n-hexane), Rf 0.3. – 1H NMR:
2.0 (s, 1 H, NH), 2.8–3.2 (m, 4 H, 2 CH2), 3.5–3.9 (m, 4 H,
2 CH2), 6.8 (s, 1 H, H-3 in pyridine), 7.2–7.8 (m, 9 H, ArH), 7.9–
8.3 (m, 2 H, H-2 and H-6 in 6-phenyl). – 18 (hydrochloride): mp
188–190 °C (EtOH).– Anal.(C21H21N3 · 2 HCl · 1.5 H2O) C, H, N.
6-Methyl-2-(4-methyl-1-piperazinyl)-4-phenylpyridine 11
Yield 21 %;yellow oil, Rf 0.6.– 1H NMR:2.4 (s, 3 H, CH3), 2.5 (s,
3 H, CH3), 2.4–2.7 (m, 4 H), 3.4–3.8 (m, 4 H, 2 CH2), 6.6 (s, 1 H,
H-3 in pyridine), 6.8 (s, 1 H, H-5 in pyridine), 7.3–7.8 (m, 5 H,
phenyl). – 11 (hydrochloride): mp 246–248 °C (EtOH/n-hex-
ane). – Anal. (C17H21N3 · 2 HCl · 1.8 H2O) C, H, N.
4-(2-Furyl)-2-(4-methyl-1-piperazinyl)-6-phenylpyridine 12
Yield 42 %; yellow oil, Rf 0.3. – 1H NMR: 2.4 (s, 3 H, CH3), 2.1–
2.8 (m, 4 H, 2 CH2), 3.6–4.0 (m, 4 H, 2 CH2), 6.4–6.6 (m, 1 H,
H-4 in furyl), 6.8–7.0 (m, 2 H, H-3 in furyl and H-3 in pyridine),
7.2–7.7 (m, 5 H, ArH), 7.9–8.3 (m, 2 H, H-2 and H-6 in phenyl).–
12 (fumarate): mp 206–208 °C (EtOH/acetone). – Anal.
(C20H21N3O · 0.5 C4H4O4 · 0.5 H2O) C, H, N.
2-(4-Methyl-1-piperazinyl)-4-phenyl-6-(2-pyridyl)pyridine 13
General procedure for preparation of compounds 19 and 20
Yield 41 %; yellow oil, Rf 0.2. – 1H NMR: 2.4 (s, 3 H, CH3), 2.5–
2.8 (m, 4 H, 2 CH2), 3.6–4.0 (m, 4 H, 2 CH2), 6.9 (s, 1 H, H-3 in
pyridine), 7.1–8.0 (cluster, 7 H, ArH), 8.1 (s, 1 H, H-5 in pyrid-
ine), 8.3–8.9 (m, 2 H, H-3 and H-6 in 2-pyridyl).– 13 (hydrochlo-
ride): mp 185–187 °C (acetone). – Anal. (C21H22N4 · 2 HCl ·
2.2 H2O) C, H, N.
Equimolar amounts of 1-(2-pyridyl)piperazine (1 mmol) or de-
rivative 18 (2 mmol), benzotriazole and hexanal (50% excess)
were heated under reflux in benzene for 2 h with azeotropic re-
moval of water using a Dean-Stark trap. Benzene was evapo-
rated and the residue was dissolved in THF (20 mL).To this so-
lution a twofold molar excess of NaBH4 was added portionwise
and the mixture was stirred for 3 h and then left overnight at
room temperature.The solvent was evaporated and the residue
was treated with ice-water (50 mL) and extracted with benzene
(3 × 50 mL). The solvent was evaporated and the oily residue
was purified by silica gel column chromatography (AcOEt/n-
hexane = 1/4) to give expected products.
6-(4-Chlorophenyl)-2-(4-methyl-1-piperazinyl)-4-phenylpyridine
14
Yield 55 %; pale yellow crystals, mp 136–138 °C (n-hexane), Rf
0.3. – 1H NMR: 2.4 (s, 3 H, CH3), 2.4–2.8 (m, 4 H, 2 CH2), 3.5–
3.9 (m, 4 H, 2 CH2), 6.8 (s, 1 H, H-3 in pyridine), 7.2–7.8 (cluster,
8 H, ArH), 8.0 (d, J = 9 Hz, 2 H, H-2 and H-6 in 4-chlorophenyl).
– 14 (hydrochloride): mp 275–277 °C (acetone). – Anal.
(C22H22N3Cl · 3 HCl · 0.5 H2O) C, H, N.
2-(4-n-Hexyl-1-piperazinyl)-4,6-diphenylpyridine 19
Yield 61 %; pale yellow oil, Rf 0. 2. – 1H NMR: 0.7–1.0 (m, 3 H,
CH3), 1.0–1.8 (m, 8 H, 4 CH2), 2.0–2.7 (m, 6 H, 3 CH2), 3.5–3.9
(m, 4 H, 2 CH2), 6.8 (s, 1 H, H-3 in pyridine), 7.2–7.8 (cluster,
9 H, ArH), 7.9–8.3 (m, 2 H, H-2 and H-6 in 6-phenyl). – 19 (hy-
drochloride):mp 198–200 °C (EtOH).– Anal.(C27H33N3 · 2 HCl ·
H2O) C, H, N.
6-(4-Methoxyphenyl)-2-(4-methyl-1-piperazinyl)-4-phenylpyrid-
ine 15
Yield 29 %; yellow oil, Rf 0.4. – 1H NMR: 2.4 (s, 3 H, CH3), 2.4–
2.8 (m, 4 H, 2 CH2), 3.6–4.0 (m, 4 H, 2 CH2), 3.8 (s, 3 H, OCH3),
6.8 (s, 1 H, H-3 in pyridine), 7.1 (d, J = 9 Hz, 2 H, H-3 and H-5 in
4-methoxyphenyl), 7.2–7.9 (m, 6 H, ArH), 8.1 (d, J = 9 Hz, 2 H,
H-2 and H-6 in 4-methoxyphenyl). – 15 (hydrochloride): mp
233–235 C (EtOH/n-hexane).– Anal.(C23H25N3O · 2 HCl) C, H,
N.
2-(4-n-Hexyl-1-piperazinyl)pyridine 20
Yield 71 %; colorless oil, Rf 0.2. – 1H NMR: 0.6–1.0 (m, 3 H,
CH3), 1.0–1.8 (m, 8 H, 4 CH2), 2.1–2.7 (m, 6 H, 3 CH2), 3.3–3.8
(m, 4 H, 2 CH2), 6.5–6.8 (m, 2 H, H-3 and H-5 in pyridine), 7.5 (t,
J = 8 Hz, 1 H, H-4 in pyridine), 8.2 (d, J = 6 Hz, 1 H, H-6 in pyrid-
ine). – 20 (hydrochloride): mp 168–171 °C (EtOH). – Anal.
(C15H25N3 · 2 HCl · H2O) C, H, N.
4-(4-Methoxyphenyl)-2-(4-methyl-1-piperazinyl)-6-phenylpyrid-
ine 16
Yield 27 %; yellow crystals, mp 96–98 °C (CHCl3/n-hexane), Rf
0.4. – 1H NMR: 2.4 (s, 3 H, CH3), 2.4–2.7 (m, 4 H, 2 CH2), 3.3–
3.9 (m, 4 H, 2 CH2), 3.9 (s, 3 H, OCH3), 6.8 (s, 1 H, H-3 in pyrid-
ine), 7.0 (d, J = 8 Hz, 2 H, H-3 and H-5 in 4-methoxyphenyl),
7.2–7.8 (m, 6 H, ArH), 7.9–8.3 (m, 2 H, H-2 and H-6 in phenyl).–
16 (hydrochloride):mp 216–218 °C (EtOH).– Anal.(C23H25N3O
· 3 HCl · H2O) C, H, N.
Receptor binding experiments
Radioligand binding experiments were conducted in rat hip-
pocampus of the rat brain for 5-HT1A receptors, and in the cor-
tex for 5-HT2A receptors according to the published procedures
[20]. The radioligands used were [3H]-8-OH-DPAT [8-hydroxy-
2-(di-n-propylamino)tetralin, 190 Ci/mmol, Amersham] and
[3H]-ketanserin (60 Ci/mmol, NEN Chemicals) for 5-HT1A and
5-HT2A receptors, respectively.
4,6-Bis(4-methoxyphenyl)-2-(4-methyl-1-piperazinyl)pyridine 17
Yield 28 %; colorless crystals, mp 168–169 °C (CHCl3/n-hex-
ane), Rf 0.3. – 1H NMR: 2.4 (s, 3 H, CH3), 2.4–2.8 (m, 4 H,
2 CH2), 3.5–4.0 (m, 4 H, 2 CH2), 3.8 (s, 6 H, 2 OCH3), 6.8 (s, 1 H,
H-3 in pyridine), 7.0 (d, J = 9 Hz, 4 H, 2 H-3 and 2 H-5 in 4-meth-
oxyphenyl), 7.3 (s, 1 H, H-5 in pyridine), 7.6 (d, J = 9 Hz, 2 H,
H-2 and H-6 in 4-methoxyphenyl in the 4 position), 8.1 (d, J =
9 Hz, 2 H, H-2 and H-6 in 4-methoxyphenyl in the 6 position). –
17 (fumarate): mp 195–197 °C (EtOH/acetone). – Anal.
(C24H27N3O2 · C4H4O4 · H2O) C, H, N.
The Ki values were determined from at least three competition
binding experiments in which 10 drug concentrations, run in
triplicate, were used.The Cheng and Prusoff equation [21] was
used for Ki calculations.
In vivo studies
The experiments were carried out on male Wistar rats (250–
300 g) or male Albino-Swiss mice (25–30 g).The animals were

110 Paluchowska et al.
Arch. Pharm. Pharm. Med. Chem. 2003, 336, 104–110
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kept at room temperature of 20 1 °C and were housed under
standard laboratory conditions with free access to food and tap
water before the experiment. All experiments were conducted
in the light phase on a natural light-dark cycle (from September
to December), between 9 a.m. and 2 p.m. All experimental pro-
cedures were approved by the Animal Care and Use Commit-
tee at the Institute of Pharmacology, Polish Academy of Scienc-
es in Kraków. ( )-1-(2,5-Dimethoxy-4-iodophenyl)-2-amino-
propane hydrochloride [( )-DOI, Research Biochemical Inc.],
8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-
DPAT, Research Biochemical Inc.], reserpine (Ciba, ampoules)
and N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(-2-py-
ridyl)cyclohexanecarboxamide trihydrochloride (WAY 100635,
synthesized by Dr. J. Boksa, Institute of Pharmacology, PAS,
Kraków) were dissolved in saline.Diazepam (Polfa, SA) and in-
vestigated salts of tested compounds were used in the form of
freshly prepared suspensions in 1 %Tween 80.8-OH-DPAT, re-
serpine and WAY 100635 were injected subcutaneously (sc),
diazepam and tested compounds intraperitoneally (ip) in a vol-
ume of 2 mL/kg (rats) or 10 mL/kg (mice). Each group consist-
ed of 6–9 animals. The obtained data were analyzed by Dun-
nett’s test.
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[9] K. Andersen, T. Liljefors, K. Gundertofte, J. Perregaard, K.
P. Bøgesø, J. Med. Chem. 1994, 37, 950–962.
In order to habituate mice to the experimental environment,
each animal was randomly transferred to a 12 cm (diameter) ×
20 cm (height) glass cage, lined with sawdust, 30 min prior to
treatment. Head twitches were induced in mice by ( )-DOI
(2.5 mg/kg). Immediately after the treatment, the number of
head twitches was counted for 30 min.The tested compounds
or saline were administered 60 min before ( )-DOI.
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M. J. Mokrosz, A. J. Bojarski, Pharmazie 1994, 49,
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Lower lip retraction (LLR) in rats
LLR was assessed according to the method described by
Berendsen et al.[18]The rats were individually placed in cages
(30 × 25 × 25 cm) and were scored three times (at 15, 30, and
45 min after the tested compounds or 8-OH-DPAT) as follows:
0 = lower incisors invisible, 0.5 = partly visible, 1 = clearly visi-
ble. The summed up maximum scores amounted to 3 for each
rat.The effect of the tested compounds andWAY 100635 on the
LLR induced by 8-OH-DPAT (1 mg/kg) was assessed in a sep-
arate experiment. The investigated compounds were adminis-
tered 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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R. A. Glennon, Bioorg. Med. Chem. Lett. 2001, 11,
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Henderson, J. Chen, J. Org. Chem. 1997, 62, 6210–6214.
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Conformational analysis
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Both 4-methyl-1-piperazinyl derivatives of pyridine and pyrimi-
dine were sketched in Sybyl (ver. 6.7, Tripos, USA) and opti-
mized using semiempirical PM3 method (gnorm = 0.1) from a
Mopac 6.0 package. To investigate the rotational energy barri-
ers, thirty-six conformations were generated by a step-wise ro-
tation of 10 deg around the inter-ring C2-N1 bond.Next, each of
these conformations was optimized using a PM3 method over
all internal coordinates except for the torsion angle that defines
the relative orientation of both rings (N1-C2-N1-Car).
[17] N. A. Darmani, B. R. Martin, U. Pandey, R. A. Glennon,
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[19] E. A. Forster, I. A. Cliffe, D. J. Bill, G. M. Dover, D. Jones, Y.
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[20] A. J. Bojarski, M. T. Cegla, S. Charakchieva-Minol, M. J.
Mokrosz, M. Mac´kowiak, J. L. Mokrosz, Pharmazie 1993,
48, 289–294.
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