Synthesis of new arylpiperazinylalkylthiobenzimidazole, benzothiazole, or benzoxazole derivatives as potent and selective 5-HT1A serotonin receptor ligands

Article abstract of DOI:10.1021/jm800176x

A series of new compounds containing a benzimidazole, benzothiazole, or benzoxazole nucleus linked to an arylpiperazine by different thioalkyl chains was prepared. They were tested in radioligand binding experiments to evaluate their affinity for 5-HT_1A and 5-HT_2A serotonergic, α₁ adrenergic, D₁, and D₂ dopaminergic receptors. Many of tested compounds showed an interesting binding profile; in particular, 36 displayed very high 5-HT_1A receptor affinity and selectivity over all the other investigated receptors. Selected compounds, evaluated in functional assays, showed antagonistic or partial agonistic activity at 5-HT_1A receptor. An extensive conformational research using both NMR and modeling techniques indicated that extended conformations predominated in vacuum, in solution and during interactions with 5-HT _1A receptor. Finally, the elaborated binding mode of selected compounds at 5-HT_1A receptor was used to explain the influence of spacer length on ligands affinity.

Full text of DOI:10.1021/jm800176x

J. Med. Chem. 2008, 51, 4529–4538
4529
Synthesis of New Arylpiperazinylalkylthiobenzimidazole, Benzothiazole, or Benzoxazole
Derivatives as Potent and Selective 5-HT1A Serotonin Receptor Ligands†
Maria A. Siracusa,*,‡ Loredana Salerno,‡ Maria N. Modica,‡ Valeria Pittala`,‡ Giuseppe Romeo,‡ Maria E. Amato,§
Mateusz Nowak, Andrzej J. Bojarski, Ilario Mereghetti,| Alfredo Cagnotto,| and Tiziana Mennini|
Dipartimento di Scienze Farmaceutiche and Dipartimento di Scienze Chimiche, UniVersita` di Catania, Viale A. Doria 6, 95125 Catania, Italy,
Istituto di Ricerche Farmacologiche “Mario Negri”, Via La Masa 19, 20156 Milano, Italy, Department of Medicinal Chemistry, Institute of
Pharmacology, Polish Academy of Sciences, 12 Sme¸tna Street, 31-343 Krako´w, Poland
ReceiVed February 20, 2008
A series of new compounds containing a benzimidazole, benzothiazole, or benzoxazole nucleus linked to
an arylpiperazine by different thioalkyl chains was prepared. They were tested in radioligand binding
experiments to evaluate their affinity for 5-HT1A and 5-HT2A serotonergic, R1 adrenergic, D1, and D2
dopaminergic receptors. Many of tested compounds showed an interesting binding profile; in particular, 36
displayed very high 5-HT1A receptor affinity and selectivity over all the other investigated receptors. Selected
compounds, evaluated in functional assays, showed antagonistic or partial agonistic activity at 5-HT1A receptor.
An extensive conformational research using both NMR and modeling techniques indicated that extended
conformations predominated in vacuum, in solution and during interactions with 5-HT1A receptor. Finally,
the elaborated binding mode of selected compounds at 5-HT1A receptor was used to explain the influence
of spacer length on ligands affinity.
Introduction
Long-chain arylpiperazine (LCAP) derivatives represent one
of the most important classes of 5-HT1AR ligands. In general,
arylpiperazine moiety is a good template for many different
biological targets, especially central nervous system receptors.
As a consequence, several compounds containing arylpiperazine
portion bind with high affinity at 5-HT1AR, but few of them
show also high selectivity for 5-HT1AR over other receptors.
Buspirone (2a) (Chart 1), one of the most known member of
this class, shows high affinity for 5-HT1AR but poor selectivity
over R1-adrenergic receptors (R1-ARs). In general, LCAPs
contain an alkyl chain, constituted by two to four carbon atoms,
linked to the N-1 of piperazine moiety and to a terminal fragment
usually containing an amide or imide function. A large number
of studies have been devoted to explore the role of the terminal
part in ligand-receptor interaction, therefore several structural
modifications have been carried out in the terminal fragment.12
Serotonin (5-HT)a is an important neurotransmitter that
mediates various physiological and pathological processes in
the peripheral and central nervous system by interaction with
several different receptors. To date 14 serotonin receptor (5-
HTR) subtypes have been identified and grouped into seven
subfamilies (5-HT1-7) on the basis of molecular cloning, amino
acid sequence, pharmacology, and signal transduction.1,2 The
5-HTRs are members of the superfamily of G protein-coupled
receptors (GPCRs), with the exception of the 5-HT3, which is
a ligand-gated cation channel receptor. Among the 5-HTRs, the
5-HT1A subtype is one of the most studied and it is generally
accepted that it is involved in anxiety and depression;3,4 recently
it has been suggested that 5-HT1AR agonists have neuroprotec-
tive properties,5,6 whereas 5-HT1AR antagonists could be useful
in the treatment of Alzheimer disease.7 Several potent 5-HT1AR
ligands belong to different chemical classes8 such as aminote-
tralines,9 indolylalkylamines, ergolines, arylpiperazines,10,11
aporphines, and aryloxyalkylamines. Among aminotetralines,
8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT, 1) (Chart
1) is the most prominent member, being a potent and selective
5-HT1AR ligand, and its tritiated form is used to label the
5-HT1ARs.
For many years, our research group has been interested in
the synthesis of arylpiperazine derivatives as 5-HT1AR and R1-
AR ligands. Among them, thieno[2,3-d]pyrimidine derivatives
3 and 413 (Chart 1) showed high affinity for the 5-HT1A
R
coupled to a reduced affinity for R1-AR. More recently, we
reported structure-affinity relationship (SAR) studies on a class
of 5-phenyl[1,2,4]triazoles structurally related to 3 and 4, in
which the aryl[1,2,4]triazole replaced the 3-amino-6-ethylth-
ieno[2,3-d]pyrimidin-4(3H)-one moiety.14,15 Some of these
compounds showed Ki values in the nanomolar range, in
particular, compound 5 exhibited good selectivity for 5-HT1AR
over both the R1 and D2 receptors. These results confirmed the
hypothesis suggested in the literature that the terminal amide
function of LCAPs is not critical for binding.16,17 On the basis
of the above results and with the aim to improve affinity and
selectivity for 5-HT1AR, in this work we describe the synthesis
and the binding data for 5-HT1ARs, R1-ARs, 5-HT2A seroton-
ergic, D1, and D2 dopaminergic receptors of a new class of
arylpiperazinylthioalkyl derivatives (16-38) (Chart 1). Deriva-
tives 16-38, as the most of LCPAs, can be divided, from a
structural point of view in three principal parts: (i) a pharma-
cophoric portion constituted by a substituted arylpiperazine, (ii)
†
Part of this work was presented at the Austrian-German-Hun-
garian-Italian-Polish-Slovenian Fifth Joint Meeting on Medicinal Chem-
istry, Portoroz, Slovenia, 17-21 June, 2007.
* To whom correspondence should be addressed. Phone: +39 095
7384005. Fax: +39 095 222239. E-mail: siracusa@unict.it.
‡
Dipartimento di Scienze Farmaceutiche, Universita` di Catania.
Dipartimento di Scienze Chimiche, Universita` di Catania.
Istituto di Ricerche Farmacologiche “Mario Negri”.
§
|
Department of Medicinal Chemistry, Institute of Pharmacology, Polish
Academy of Sciences.
a Abbreviations: 5-HT, serotonin; 5-HTR, serotonin receptor; GPCRs,
G protein-coupled receptors; 8-OH-DPAT, 8-hydroxy-2-(di-n-propylami-
no)tetraline; 5-HT1AR, 5-HT1A serotonin receptor; LCAP, long-chain
arylpiperazine; R1-ARs, R1-adrenergic receptors; [35S]GTPγS, guanosine-
5′-O-(γ-thio)triphosphate; DMSO, dimethylsulfoxide; EC50, half-maximum
effective inhibitory concentration; PMF, potential mean force; IC50, median
inhibitory concentration; Ki, inhibitor constant; TMS, tetramethylsilane;
TFA, trifluoroacetic acid.
10.1021/jm800176x CCC: $40.75
2008 American Chemical Society
Published on Web 07/04/2008

4530 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Chart 1. 5-HT1A Receptor Ligands
Siracusa et al.
a terminal fragment constituted by a heterobicyclic system, (iii)
a linker between these two substructures. In these new mol-
ecules, the pharmacophoric portion was represented by a
2-nitrophenyl-, 2-methoxyphenyl-, pyridin-2-yl-, or pyrimidin-
2-yl-1-piperazine; the terminal fragment was represented by a
benzimidazole, benzothiazole, or benzoxazole ring systems
(often present in the structure of ligands for receptors of biogen
amines),18 the linker connecting arylpiperazines and bicyclic
nucleus was a flexible alkylthio chain of different length (n )
0, 1, 2, 4) (Chart 1). To get information on functional activity
at 5-HT1AR, selected compounds were also tested in [35S]GTPγS
binding assays. Furthermore, NMR experiments, conformational
analysis, and molecular dockings were conducted to understand
conformational preferences of studied compounds in different
environments. Finally, the previously elaborated full flexible
dockingmethodology19 wasusedforexplanationofligand-receptor
interactions important for affinity.
described by Mokrosz et al. for N-alkylation of benzotriazole.22
In brief, 2-mercaptobenzoxazole (7) or 2-mercaptobenzothiazole
(8) were alkylated with 1-bromo-4-chlorobutane or 1-bromo-
6-chlorohexane in acetonitrile at reflux for 1 h in the presence
of potassium fluoride/aluminum oxide. The intermediates 2-[(4-
chlorobutyl)thio]benzothiazole or benzoxazole and 2-[(6-chlo-
rohexyl)thio]benzothiazole or benzoxazole 12-15 were then
reacted with 1-(2-methoxyphenyl)piperazine in acetonitrile at
reflux for 1 h in the presence of potassium carbonate to give
desired final products 35-38.
Results and Discussion
Compounds 16-38 were tested in binding assays to evaluate
their affinity and selectivity for 5-HT1ARs over R1-ARs. For
the derivatives that showed the highest affinity and selectivity
toward the 5-HT1AR over R1-ARs, affinities for 5-HT2A sero-
tonergic, D1, and D2 dopaminergic receptors were also evaluated.
Binding data, expressed as Ki (nM), are summarized in Tables
1 and 2 along with selectivities reported as ratios of Ki values.
The first step of our investigation was the synthesis of
compounds 16-27 in which a 2-methoxy- or a 2-nitrophe-
nylpiperazine was linked to a benzoxazole, benzothiazole, or
benzimidazole system by an ethylthio or propylthio unit. In
general, derivatives 19-21 and 25-27, which possess the
2-nitrophenylpiperazine, showed lower affinity for 5-HT1AR
with respect to the 2-methoxyphenylpiperazine analogues 16-18
and 22-24, as previously described for analogues containing
5-phenyl[1,2,4]triazole nucleus.14,15 In addition, compounds
characterized by a thiopropyl chain between the terminal
fragment and the arylpiperazine portion (22-27) exhibited
higher affinity toward 5-HT1AR with respect to the analogues
16-21 containing a thioethyl chain as linker. In particular,
among thiopropyl derivatives, 23 was the most interesting
because it showed an affinity for 5-HT1AR in the subnanomolar
range (Ki ) 0.29 nM) coupled to a high selectivity over R1-AR
Chemistry
Compounds 16-38 (Table 1) were obtained by a simple one-
or two-steps condensation as outlined in Scheme 1. The
commercially available compounds 6-10 were reacted with
appropriate 1-(ω-chloroalkyl)-4-arylpiperazine in acetone at
reflux for 24 h, in the presence of potassium carbonate and
potassium iodide, to give desired products 16-34 in good yields.
Since efforts devoted to prepare compound 32 by direct
methylation of 22 with CH3I were unsuccessful, it was
synthesized using as starting material N-methylbenzimidazoline-
2-thione (11), prepared according to literature.20 Reaction of
1-(2-methoxyphenyl)piperazine with 1-bromo-4-chlorobutane
did not give the desired alkylating agent requested for the
preparation of compounds 35 and 36, but a quaternary spiro
structure21 that did not react in the standard conditions described
for the preparation of compounds 16-34. Therefore, to syn-
thesize compounds 35-38, we used the reaction condition

5-HT1A Serotonin Receptor Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4531
Table 1. 5-HT1A and R1-Adrenergic Receptors Binding Data for Compounds 16-38
Ki (nM)a
R1/
5-HT1A
b
compd
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
X
n
R
R1
5-HT1A
R1
NH
S
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
4
4
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
H
H
H
H
2-CH3OC6H4
2-CH3OC6H4
2-CH3OC6H4
2-NO2C6H4
2-NO2C6H4
2-NO2C6H4
2-CH3OC6H4
2-CH3OC6H4
2-CH3OC6H4
2-NO2C6H4
2-NO2C6H4
2-NO2C6H4
pyridin-2-yl
pyridin-2-yl
pyrimidin-2-yl
pyrimidin-2-yl
2-CH3OC6H4
2-CH3OC6H4
2-CH3OC6H4
2-CH3OC6H4
2-CH3OC6H4
2-CH3OC6H4
2-CH3OC6H4
33.1 ( 5.8
31.2 ( 3.4
67.2 ( 17.1
95.2 ( 13.0
1702 ( 398
326 ( 91
240 ( 31
207 ( 51
276 ( 34
457 ( 33
7.25
6.63
4.11
4.8
>5.87
5.87
26
O
NH
S
>10000
O
1915 ( 200
25.9 ( 5
NH
S
1.0 ( 0.1
0.29 ( 0.06
0.55 ( 0.05
16.6 ( 2.22
9.6 ( 0.6
33.2 ( 5.4
21.5 ( 4.5
77.4 ( 5.9
249 ( 30
127 ( 15
77.7 ( 7.6
98.0 ( 5.6
328 ( 44
1651 ( 122
20.0 ( 2.9
21.9 ( 0.7
46.7 ( 4.6
16.6 ( 1.4
78.0 ( 23.2
46.0 ( 7.3
52.5 ( 7.4
600
114
39
O
NH
S
4.66
26
O
S
O
S
14.1 ( 2.0
0.78 ( 0.02
1.60 ( 0.12
2.5 ( 0.1
14.9 ( 1.7
0.270 ( 0.002
1.0 ( 0.2
0.88 ( 0.01
0.27 ( 0.01
0.094 ( 0.008
1.30 ( 0.004
0.52 ( 0.01
15
9
100
61
131
111
74
22
53
61
830
53
O
32
33
34
35
36
37
38
NCH3
S
O
S
O
S
O
101
40
36
buspironec
ipsapironec
5.5
200
a Ki values were calculated as described in the Experimental Section and are the mean ( SD of three separate experiments. b Ratio Ki R1/Ki 5-HT1A
.
c Data taken from literature.31
Scheme 1a
a Reagents and conditions: (a) K2CO3, KI, CH3COCH3, reflux, 24 h; (b) 1-bromo-4-chlorobutane or 1-bromo-6-chlorohexane, CH3CN, KF/Al2O3, KI,
reflux, 1 h; (c) 1-(2-methoxyphenyl)piperazine, CH3CN, K2CO3, reflux, 1 h.
(Ki R1-AR/Ki 5-HT1AR ) 114). With regard to the terminal
fragment, compounds bearing a benzothiazole or benzoxazole
(17, 18, 20, 21, 23, 24, 26, and 27) showed higher affinity than
those bearing their isoster benzimidazole (16, 19, 22, and 25).
On the basis of these results and in order to further study SARs
within this series, we decided to carry out additional work. In
particular, considering 23 as “lead compound”, the following
structural modifications were realized: (i) replacement of the
2-methoxyphenyl with other aromatic rings, such as pyridin-
2-yl or pyrimidin-2-yl nucleus present in the buspirone and
ipsapirone, to obtain compounds 28-31; (ii) introduction of a
chlorine atom at the 5-position of the benzothiazole or benzox-
azole nucleus to obtain compounds 33 and 34; (iii) methylation
on N-1 of the benzimidazole nucleus of 22 to obtain compound
32. Compounds 28-31 showed a slight decrease in affinity at
5-HT1AR, if compared to related compounds 22-24. In par-
ticular, compounds bearing a pyridin-2-yl residue (28 and 29)
still remained potent ligands at the 5-HT1AR, whereas pyrimidin-
2-yl derivatives 30 and 31, despite their lower affinity at
5-HT1AR, maintained good selectivity over R1-AR (Ki R1-AR/
Ki 5-HT1AR ) 132 and 111, respectively); therefore, the
presence of the pyrimidin-2-yl ring in the pharmacophoric
portion seems to play an unfavorable role in the interaction of
these compounds with R1-AR binding site. The 5-HT1AR affinity

4532 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Siracusa et al.
Table 2. D1, D2, and 5-HT2A Receptors Binding Data for Selected
Compounds
Table 3. Effect of 5-HT and Compounds 4, 28, 3, 23, 36, and 38 on
[
35S]GTPγS Binding to 5-HT1ARa
Ki (nM)a
Emax
(%)
EC50
( SD, nM
Ki
D1/
D2/
5-HT2A/
b b
( SD, nM
b
compd
D1
D2
5-HT2A 5-HT1A 5-HT1A 5-HT1A
5-HT (agonist)
4 (partial agonist)
28 (partial agonist)
3 (antagonist)
23 (antagonist)
36 (antagonist)
38 (antagonist)
a
100 ( 5.6
38 ( 1.5
33 ( 2.3
323 ( 47
1210 ( 26
204 ( 45
23
28
1280 ( 161 25.8 ( 3.1 96.1 ( 7.9
5630 ( 925 462 ( 51 78.8 ( 8.8
4415
7218
6250
4000
7426
89
592
268
213
216
397
90
331
101
109
73
393
3170
398
29 >10.000
30 >10.000
35
36
38
429 ( 86 174 ( 35
532 ( 36 183 ( 18
7.4 ( 2.4
1.7 ( 0.4
6.6 ( 1.9
1.3 ( 0.4
2005 ( 388 58.3 ( 12.2 106 ( 23
1600 ( 82 37.3 ( 8.3 298 ( 44 17021
922 ( 85 46.6 ( 7.0 207 ( 19
1773
a Ki values were calculated as described in the Experimental Section
[
35S]GTPγS binding in rat hippocampus. Hippocampal homogenates
and are the mean ( SD of three separate experiments. b Ratio Ki D1/Ki
were incubated in the presence of graded concentrations of 5-HT or
compounds 4 and 28. The antagonists were tested at different concentrations
in presence of 10 µM 5-HT. IC50, EC50, and Emax were obtained using the
“Allfit” program and Ki were derived from the IC50 values using the Cheng
and Prusoff equation.29 Data are the mean ( SD of three separate
experiments.
5-HT1A, Ki D2/Ki 5-HT1A, and Ki 5-HT2A/Ki 5-HT1A
.
of N-methyl derivative 32 was greater than that of the parent
compound 22, suggesting that the presence of an unsubstituted
nitrogen at the imidazole nucleus is detrimental for affinity
toward 5-HT1AR. The introduction of a chlorine atom in the
benzoxazole or benzothiazole at the 5-position caused only a
slight decrease in the 5-HT1AR affinity. As reported in the
literature, the length of the alkyl chain greatly influences affinity
and selectivity for 5-HT1AR.11,13 Therefore, with the aim to
investigate the effect of the length of the connecting chain on
binding properties, we synthesized compounds 35-38, char-
acterized by a butylthio or a hexylthio linker, having a
connecting chain between the arylpiperazine moiety and the
bicyclic heteroaromatic system equivalent to 5 or 7 methylene
units, respectively. A comparison of the binding data of
compounds 35-38 with those of related 23 indicated that affinity
for 5-HT1AR was very similar for 35, 37, and 38, whereas
selectivity over R1-AR was reduced for 35 and 37. On the
Figure 1. Computer calculated structures of compounds 18, 24, 36,
and 38 in their fully extended conformation.
contrary,
2-[[4-[4-(2-methoxyphenyl)-1-piperazinyl]butyl]-
ylthio]benzoxazole (36) results were more potent and selective
(Ki 5-HT1AR ) 0.094 nM, Ki R1-AR/Ki 5-HT1AR ) 830) than
23, thus being the most interesting compound within this series.
Compounds 23, 28-30, 35, 36, and 38 that showed high
affinity and selectivity were tested for their affinities at D1, D2
dopaminergic, and 5-HT2A serotonergic receptors. Results clearly
demonstrated that these compounds possess a very good binding
profile, preferring 5-HT1ARs over all other evaluated receptors.
In particular, with regard to dopaminergic receptors, all the
tested compounds exhibited no affinity for D1 receptor, with Ki
values in the range 922-10000 nM; affinity for D2 receptor
was in some cases moderate (23, 35, 36, and 38), with Ki values
in the range 25-58 nM, or low (28, 29, and 30), with Ki values
in the range 429-532 nM. The 5-HT2A receptor affinity of the
tested compounds was generally lower than those observed for
5-HT1AR, and so they exhibited a favorable 5-HT2A/5-HT1A
selectivity ratio.
The functional activity of selected compounds 23, 28, 36,
and 38 along with compounds 3 and 4 was determined in
functional [35S]GTPγS binding assays in rat hippocampal
membranes, and results are reported in Table 3. Compounds 3,
23, 36, and 38 behave as 5-HT1AR antagonists being able to
block the increase of [35S]GTPγS binding induced by 10 µM
5-HT in a dose-dependent manner, with a Ki value in the
nanomolar range. Compounds 4 and 28 behave as partial
agonists because also at the high concentration tested (10 µM),
they did not reach the maximum effect elicited by 5-HT but
only a 30-40% enhancement of [35S]GTPγS binding, with EC50
values of 1210 and 204 nM, respectively.
free bases in DMSO solution and molecular modeling studies
were performed. The 500 MHz H NMR spectra consist of
1
relatively simple and well resolved patterns of resonances
straightforwardly assigned by inspection of COSY maps.
Moreover, at 300 K, the independence of chemical shifts from
concentration was verified by excluding possible aggregation
effects. 2D-NOE experiments showed the cross peaks expected
for intramolecular interactions in agreement with extended
conformations in solution. Molecular modeling studies of
compounds 18, 24, 36, and 38 were undertaken using Macro-
Model (version 8.6) with the MM2* force field.23 Conforma-
tional preferences were explored using the parameters for either
isolated “gas-phase” or water continuum. Monte Carlo searches
were conducted by allowing all flexible bonds to rotate, the
simulations collected 800 iterations each, and conformations that
were within 4 kJ of the lowest energy conformation were
examined. Among the variety of structures produced, the free
baseenergeticallyfavoredconformations(intherange178.81-187.02
kJ mol-1) showed the methylene bridging groups in an
antiperiplanar arrangement with aromatic portions enough far
each other, consistent with NMR experimental data (Figure 1).
Higher energy folded structures were also generated, which were
about 30 kJ above the extended ones, on the maximum. This
feature occurs, in particular, for compound 38, where the longest
spacer separates the pharmacophoric portion and the aromatic
terminal group.
As folded conformations could not be precluded, exploration
of the conformational properties of 38 was extended to its salt
in solution. In fact, optimized folded structures showed the space
proximity of the benzoxazole ring and piperazine, wherein the
protonation of the nitrogen might engender an intramolecular
hydrogen bond interaction with the heteroatoms of benzoxazole
To gain insight into structural properties of compounds
16-38, we selected benzoxazole derivatives 18, 24, 36, and
38, where the sole structural difference lies in the length of the
connecting chain. At this purpose, 1H NMR experiments of the

5-HT1A Serotonin Receptor Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4533
the alkyl chain and piperazine protons and between the
benzoxazole protons and the aromatic proton located on the
2-methoxyphenyl ring, suggesting that folded conformational
structures were also present in solution in equilibrium with
extended ones.
The results of conformational analysis of protonated form of
the compound 38 gave, in vacuum, the folded lowest-energy
conformation (219.7 kJ mol-1), shown in Figure 3a, where an
intramolecular hydrogen bond (2.006 Å) between the protonated
piperazine nitrogen and the properly oriented lone pair on the
nuclear oxygen of the benzoxazole ring may contribute to
stabilize the structure, whereas the two aromatic moieties were
found in an edge-to-face disposition.
Figure 2. Significant NOE signals of protonated 38 in DMSO/TFA
solution.
The continuous aqueous medium generated lowest energy
conformation (Figure 3b) showed a larger distance (4.04 Å)
between protonated piperazine and the nuclear oxygen lone pair
of the benzoxazole ring, indicating that hydrogen bonding
interaction found in “gas phase” could be overestimated with
respect to a more realistic model in a polar solvent. Despite
this finding, the folded structure (in equilibrium with extended
ones) is retained with the aromatic moieties in close contact
(ca. 3 Å), in agreement with the existence of restricted
conformations evidenced by NMR NOE data.
Figure 3. Folded low-energy conformation of the protonated form of
38 in vacuum (a) and in aqueous medium (b).
moiety, useful to stabilize folded conformations. Addition of
TFA in DMSO solution of 38 caused a sensible variation of
1
the H NMR spectrum easily interpreted with the protonation
of the piperazine nitrogen N-1. Inspection of the NOESY map
evidenced significant NOE cross peaks represented in Figure
2.
Besides the expected strong intramolecular NOE signals,
weak dipolar contacts were observed between methylenes of
To complete an examination of conformational preferences
of studied compounds (18, 24, 36, and 38), a fully flexible
docking approach with the use of 100 5-HT1AR models,
Figure 4. Top scored ligand-receptor complexes for compounds studied by means of ligand-receptor docking: (A) 18, (B) 24, (C) 36, (D) 38.
Helical bundle is presented from the extracellular side. Sticks representation depicts residues used as “active site” in FlexX docking. Amino acids
forming specific interactions with ligands were labeled. Dashed yellow line shows H-bonding.

4534 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Siracusa et al.
Table 4. 5-HT1AR Experimental Binding Affinity: PMF Score for (a)
the Top Solutions of the Entire Set of Receptor Models and for (b) the
Receptor Model 82a
top solution
receptor model no. 82a
Ki
PMF
score
receptor
model no.
PMF
score
compd 5-HT1A (nM)
rankb
18
24
36
38
67.22
0.55
0.094
0.52
-85.8
-106.2
-110.9
-111.0
82
67
82
23
-85.8
-106.0
-110.9
-102.5
1
3
1
28
a The best receptor model identified according to the lowest value of
summed PMF scores of complexes with analyzed compounds. b Position
in the ranking of the PMF scores for the complex of ligand-receptor model
82.
previously “tuned” for arylpiperazine ligands,19 was applied.
Pharmacophoric constraints imposed on 2-methoxyphenylpip-
erazine fragment facilitated reproduction of the proposed LCAPs
binding mode. The resulting 4 × 100 L-R complexes were
subjected to a consensus scoring procedure, and the PMF
(potential mean force) score was used for further analysis.
Among all the docked poses, the extended conformations
predominated and no folded arrangements were found. For the
three-unit spacer compound, however, partly bent (hockey stick-
like) conformations were also observed because even one torsion
angle in synclinal position caused such a shape of a molecule.
It has to be stressed that in all the best-scored complexes, the
analyzed molecules existed in extended conformations (Figure
4, Table 4), which is in general agreement with NMR
experimental data as well as with results of conformational
analysis for free bases (Figure 1).
A careful inspection of preferable ligand binding poses
generated in our models gives additional information that can
be discussed in regard to the observed differences in affinity.
As depicted in Figure 4, apart from principal interactions coming
from pharmacophoric arylpiperazine fragment, benzoxazole
moiety of 24, 36, and 38 formed H-bonds with Tyr7.43 and/or
Asn7.39. Additionally, for the best binder in the group (36), a
remarkable π-π stacking with Phe3.28 was observed (Figure
4C). This last finding could be considered as a possible
explanation of outstanding high affinity of this compound,
especially when compared to binding results obtained for
derivatives 24 and 38. The most visible difference in Ki values
characterized compound 18 that was at least 100-fold less active
than the others. In the scored solution for this compound, only
weak H-bond interaction was present between Tyr7.43 and
thioether fragment of the spacer (Figure 4C). In a certain number
of solutions, H-bonds between benzoxazole moiety of 18 and
residues on helix 7 were formed (results not shown) but at the
cost of weakening interactions from arylpiperazine part. On the
other hand, when this pharmacophoric portion of 18 occupied
more optimal position (common for the remaining compounds)
as was found, e.g., for the best receptor model 82, the
benzoxazole fragment has lost its specific interactions with helix
7 and is pointed toward the exterior of the binding site (Figures
4A and 5).
Figure 5. The best receptor model (no. 82) with top scored poses of
all four ligands. Arylpiperazine part was constrained to interact with
Phe6.62, Ser5.43, and Asp3.32.
compounds showed very high affinity for the 5-HT1AR (with a
Ki values in the nanomolar range) and good selectivity over
5-HT2A, R1, D1, and D2 receptors.
SAR analyses pointed out the importance of some structural
features for an optimal interaction of these molecules with the
5-HT1AR binding site. With regard to the arylpiperazine
pharmacophoric substructure, 2-methoxyphenyl and pyridin-2-
yl moieties gave compounds endowed with the best affinities;
2-nitrophenyl and pyrimidin-2-yl derivatives were poorer ligands,
although the latter still showed remarkable selectivity over the
R1-AR. Considering the terminal fragment, benzoxazole and
benzothiazole derivatives invariably were more potent ligands
than corresponding benzimidazole analogues. As far as the
length of the spacer between pharmacophoric and terminal
moieties is concerned, thiobutyl and thiopropyl chains gave the
best contribution to affinity at 5-HT1AR and selectivity over the
other receptors. Among tested compounds, 2-[[4-[4-(2-meth-
oxyphenyl)-1-piperazinyl]butyl]lthio]benzoxazole (36) displayed
very high affinity at 5-HT1AR (Ki in the subnanomolar range)
and the best selectivities over 5-HT2A, R1, D1, and D2 receptors.
This binding profile makes 36 the most striking compound
within this series and one of the most interesting 5-HT1AR ligand
so far reported. The nature of the pharmacophoric portion greatly
influences the functional activity at 5-HT1AR of these com-
pounds. In fact, in [35S]GTPγS binding assay, 2-methoxyphenyl
derivatives behaved as antagonists, whereas pyridin-2-yl and
pyrimidin-2-yl derivatives showed partial agonistic activity.
An extensive conformational research carried out for a
representative set of derivatives 18, 24, 36, and 38 showed a
strong preference of extended molecular arrangements. Although
folded conformations were detected in NMR experiments and
classic conformational analysis, they were not found during
thorough flexible docking to a set of 100 models of 5-HT1AR.
The applied approach allowed the identification of important
interactions within receptor binding site, which seems to
determine ligands affinity. Longer spacer (4-7 units) of
compounds with nanomolar activity enabled very strong interac-
tions of the terminal benzoxazole moiety with respective
residues on helix 7 or 3 and arylpiperazine pharmacophoric part
with Asp3.32, Phe6.62, and Ser5.43. On the other hand, a three-
unit spacer seems too short to form both contacts in optimal
mode, explaining significantly lower affinity of compound 18.
Because of significant simplification of scoring functions,
quantitative correlations of PMF scores with binding data are
not justified (Table 4); nevertheless, our molecular docking
experiments brought qualitative rationalization of ligand-receptor
interactions important for affinity.
Conclusions
In this study, a new series of arylpiperazine derivatives 16-38
were synthesized as 5-HT1AR ligands. Generally, most of
In the end, obtained results appear to be coherent with
observed differences in experimental binding affinities and the

5-HT1A Serotonin Receptor Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4535
piperazine), 2.79 (t, J ) 7 Hz, 2H, CH2N), 2.93-3.04 (m, 4H,
piperazine), 3.55 (t, J ) 7.0 Hz, 2H, SCH2), 7.06-7.15 (m, 1H,
aromatic), 7.27-7.39 (m, 2H, aromatic), 7.42-7.49 (m, 1H,
aromatic), 7.53-7.61 (m,1H, aromatic), 7.74-7.88 (m, 2H, aro-
matic), 7.97-8.03 (m, 1H, aromatic). Anal. (C19H20N4O2S2) C, H,
N, S.
usefulness of previously elaborated full flexible docking meth-
odology19 was additionally attested.
Experimental Section
Chemistry. Melting points were determined in a Gallemkamp
apparatus with an MFB-59 digital thermometer in glass capillary
tubes and are uncorrected. Elemental analyses for C, H, N, and S
were within ( 0.4% of theoretical values and were performed on
2-[[2-[4-(2-Nitrophenyl)-1-piperazinyl]ethyl]thio]benzoxazole (21).
1
The title compound was isolated as bright-orange oil (68%). H
NMR (DMSO-d6): δ 2.51-2.55 (m, 4H, piperazine), 2.80 (t, J )
6.6 Hz, 2H, CH2N), 2.90-3.05 (m, 4H, piperazine), 3.53 (t, J )
6.6 Hz, 2H, SCH2), 7.06-7.17 (m, 1H, aromatic), 7.26-7.39 (m,
3H, aromatic), 7.53-7.69 (m, 3H, aromatic), 7.75-7.82 (m, 1H,
aromatic). Anal. (C19H20N4O3S) C, H, N, S.
1
a Carlo Erba elemental analyzer model 1108 apparatus. H NMR
spectra were determined with a Varian Inova Unity 200 (200 MHz),
and 2D-NOE and COSY spectra were determined with a Varian
Inova Unity 500 (500 MHz) instrument in DMSO-d6. Chemical
shifts are in δ values (ppm) using tetramethylsilane as the internal
standard; coupling constants (J) are given in Hz. Signal multiplici-
ties are characterized as s (singlet), d (doublet), t (triplet), q (quartet),
qu (quintet), m (multiplet), and br (broad signal). All the synthesized
compounds were tested for purity on TLC on Merck plates
(Kieselgel 60 F254), and spots were visualized under the UV light
(λ ) 254 and 366 nm). Preparative column chromatography was
performed using Merck silica gel (0.040-0.063 mm). All chemicals
and solvents were reagent grade and were purchased from com-
mercial sources. 1-(ω-Chloroalkyl)-4-arylpiperazines were synthe-
sized according to literature.24
2-[[3-[4-(2-Methoxyphenyl)-1-piperazinyl]propyl]thio]1H-ben-
zimidazole (22). The title compound was isolated as cream powder
1
(59%); mp 153 °C. H NMR (DMSO-d6): δ 1.86-1.95 (m, 2H,
CH2CH2CH2), 2.41- 2.54 (m, 2H + 4H, CH2N + piperazine),
2.89-3.03 (m, 4H, piperazine), 3.31 (t, J ) 7.0 Hz, 2H, SCH2),
3.76 (s, 3H, CH3), 6.80-6.96 (m, 4H, aromatic), 7.05-7.18 (m,
2H, aromatic), 7.33-7.55 (m, 2H, aromatic), 12.52 (s, 1H, NH,
which exchanges with D2O). Anal. (C21H26N4OS) C, H, N, S.
2-[[3-[4-(2-Methoxyphenyl)-1-piperazinyl]propyl]thio]benzothi-
azole (23). The title compound was isolated as brown semisolid
(59%). 1H NMR (DMSO-d6): δ 1.91-2.05 (m, 2H, CH2CH2CH2),
2.42-2.55 (m, 2H + 4H, CH2N + piperazine), 2.85-3.02 (m, 4H,
piperazine), 3.41 (t, J ) 7.2 Hz, 2H, SCH2), 3.76 (s, 3H, CH3),
6.82-6.95 (m, 4H, aromatic), 7.28-7.56 (m, 2H, aromatic),
7.80-7.87 (m, 1H, aromatic) 7.90-8.05 (m, 1H, aromatic). Anal.
(C21H25N3OS2) C, H, N, S.
General Procedure for the Preparation of Compounds 16-34.
A solution of 3 mmol of suitable 1-(ω-chloroalkyl)-4-arylpiperazine
in 20 mL of acetone was slowly added dropwise to a suspension
in acetone (20 mL) of compounds 6-11 (3 mmol), potassium
carbonate (3 mmol), and a catalytic amount of potassium iodide.
The mixture was refluxed for 24 h. After this period, the mixture
was concentrated, and the residue was diluted with brine (30 mL)
and extracted with ether (4 × 20 mL). The combined ethereal phases
were washed with brine, dried, and the solvent removed in vacuo.
The obtained residue was purified by flash column chromatography
on silica gel using ethyl acetate/cyclohexane (1:1) mixtures as
eluent. Using this procedure, the following products were synthesized.
2-[[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]thio]1H-benzimi-
dazole (16). The title compound was isolated as white powder
2-[[3-[4-(2-Methoxyphenyl)-1-piperazinyl]propyl]thio]benzox-
azole (24). The title compound was isolated as light-yellow powder
(73%); mp 58-59 °C. 1H NMR 500 MHz (DMSO-d6): δ 1.99 (br
m, 2H, CH2CH2CH2), 2.45-2.59 (m, 2H + 4H, CH2N + pipera-
zine), 2.95 (m, 4H, piperazine), 3.37 (t, J ) 7.2 Hz, 2H, SCH2),
3.76 (s, 3H, CH3), 6.80-6.95 (m, 4H, aromatic), 7.26-7.38 (m,
2H, aromatic), 7.57-7.68 (m, 2H, aromatic). Anal. (C21H25N3O2S)
C, H, N, S.
1
(65%); mp 136-138 °C. H NMR (DMSO-d6): δ 2.55- 2.67 (m,
2-[[3-[4-(2-Nitrophenyl)-1-piperazinyl]propyl]thio]1H-benzimi-
dazole (25). The title compound was isolated as yellow powder
4H, piperazine), 2.74 (t, J ) 6.6 Hz, 2H, CH2N), 2.91-2.99 (m,
4H, piperazine), 3.46 (t, J ) 6.6 Hz, 2H, SCH2), 3.76 (s, 3H, CH3),
6.86-6.93 (m, 4H, aromatic), 7.08-7.12 (m, 2H, aromatic),
7.40-7.45 (m, 2H, aromatic), 12.53 (br s, 1H, NH, which exchanges
with D2O). Anal. (C20H24N4OS) C, H, N, S.
1
(84%); mp 119-121 °C. H NMR (DMSO-d6): δ 1.85-1.99 (m,
2H, CH2CH2CH2), 2.41- 2.52 (m, 2H + 4H, CH2N + piperazine),
2.95-3.05 (m, 4H, piperazine), 3.31 (t, J ) 7.2 Hz, 2H, SCH2),
7.05-7.18 (m, 3H, aromatic), 7.24-7.62 (m, 4H, aromatic),
7.75-7.82 (m, 1H, aromatic), 12.51 (br s, 1H, NH, which exchanges
with D2O). Anal. (C20H23N5O2S) C, H, N, S.
2-[[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]thio]benzothia-
zole (17). The title compound was isolated as white powder (70%);
mp 77-78 °C. 1H NMR (DMSO-d6): δ 2.58- 2.65 (m, 4H,
piperazine), 2.78 (t, J ) 6.8 Hz, 2H, CH2N), 2.93-2.99 (m, 4H,
piperazine), 3.56 (t, J ) 6.8 Hz, 2H, SCH2), 3.77 (s, 3H, CH3),
6.85-6.93 (m, 4H, aromatic), 7.31-7.50 (m, 2H, aromatic),
7.83-7.88 (m, 1H, aromatic), 7.99-8.03 (m, 1H, aromatic). Anal.
(C20H23N3OS2) C, H, N, S.
2-[[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]thio]benzox-
azole (18). The title compound was isolated as light-yellow powder
(77%); mp 75-76 °C. 1H NMR 500 MHz (DMSO-d6): δ 2.60 (br
m, 4H, piperazine), 2.79 (t, J ) 6.0 Hz, 2H, CH2N), 2.93 (br m,
4H, piperazine), 3.54 (t, J ) 6.0 Hz, 2H, SCH2), 3.76 (s, 3H, CH3),
6.85 (m, 2H, aromatic), 6.92 (m, 2H, aromatic), 7.31 (m, 2H,
aromatic), 7.63 (m, 2H, aromatic). Anal. (C20H23N3O2S) C, H, N,
S.
2-[[3-[4-(2-Nitrophenyl)-1-piperazinyl]propyl]thio]benzothi-
azole (26). The title compound was isolated as bright-orange powder
(76%); mp 73-76 °C. 1H NMR (DMSO-d6): δ 1.90-2.03 (m, 2H,
CH2CH2CH2), 2.49-2.59 (m, 2H + 4H, CH2N + piperazine),
2.85-3.10 (m, 4H, piperazine), 3.36 (t, J ) 7.2 Hz, 2H, SCH2),
7.06-7.17 (m, 1H, aromatic), 7.26-7.62 (m, 4H, aromatic),
7.51-7.84 (m, 2H, aromatic), 7.98-8.05 (m, 1H, aromatic). Anal.
(C20H22N4O2S2) C, H, N, S.
2-[[3-[4-(2-Nitrophenyl)-1-piperazinyl]propyl]thio]benzox-
azole (27). The title compound was isolated as bright-orange
semisolid (81%). 1H NMR (DMSO-d6): δ 1.90-2.05 (m, 2H,
CH2CH2CH2), 2.45-2.58 (m, 2H + 4H, CH2N + piperazine),
2.86-3.10 (m, 4H, piperazine), 3.37 (t, J ) 7.2 Hz, 2H, SCH2),
7.09-7.19 (m, 1H, aromatic), 7.26-7.38 (m, 3H, aromatic),
7.49-7.68 (m, 3H, aromatic), 7.78-7.82 (m, 1H, aromatic). Anal.
(C20H22N4O3S) C, H, N, S.
2-[[2-[4-(2-Nitrophenyl)-1-piperazinyl]ethyl]thio]1H-benzimida-
zole (19). The title compound was isolated as bright-orange powder
1
(68%); mp 174-175 °C. H NMR (DMSO-d6): δ 2.50- 2.67 (m,
4H, piperazine), 2.74 (t, J ) 7.0 Hz, 2H, CH2N), 2.92-3.05 (m,
4H, piperazine), 3.46 (t, J ) 7.0 Hz, 2H, SCH2), 7.02-7.23 (m,
3H, aromatic), 7.28-7.62 (m, 4H, aromatic), 7.71-7.82 (m, 1H,
aromatic), 12.55 (br s, 1H, NH, which exchanges with D2O). Anal.
(C19H21N5O2S) C, H, N, S.
2-[[3-[4-(Pyridin-2-yl)-1-piperazinyl]propyl]thio]benzothiazole
(28). The title compound was isolated as white powder (65%); mp
85-86 °C. 1H NMR (DMSO-d6):
δ 1.92-2.03 (m, 2H,
CH2CH2CH2), 2.42-2.50 (m, 2H + 4H, CH2N + piperazine),
3.41-3.50 (m, 2H + 4H, SCH2 + piperazine), 6.58-6.66 (m, 1H,
aromatic), 6.78-6.84 (m, 1H, aromatic), 7.31-7.57 (m, 3H,
aromatic), 7.82-7.88 (m, 1H, aromatic), 7.98-8.12 (m, 2H,
aromatic). Anal. (C19H22N4S2) C, H, N, S.
2-[[2-[4-(2-Nitrophenyl)-1-piperazinyl]ethyl]thio]benzothiazole
(20). The title compound was isolated as bright-orange powder
(68%); mp 79-81 °C. 1H NMR (DMSO-d6): δ 2.54-2.65 (m, 4H,

4536 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15
Siracusa et al.
2-[[3-[4-(Pyridin-2-yl)-1-piperazinyl]propyl]thio]benzoxazole (29).
2-[[4-[4-(2-Methoxyphenyl)-1-piperazinyl]butyl]thio]benzothia-
The title compound was isolated as cream powder (70%); mp
zole (35). The title compound was isolated as light-yellow semisolid
84-85 °C. 1H NMR (DMSO-d6):
δ
1.94-2.09 (m, 2H,
(61%). H NMR (DMSO-d6): δ 1.61-1.86 (m, 4H, CH2CH2CH2
1
CH2CH2CH2), 2.43-2.59 (m, 2H + 4H, CH2N + piperazine),
3.39-3.52 (m, 2H + 4H, SCH2 + piperazine), 6.58-6.66 (m, 1H,
aromatic), 6.78-6.84 (m, 1H, aromatic), 7.29-7.68 (m, 5H,
aromatic), 8.08-8.11 (m, 1H, aromatic). Anal. (C19H22N4OS) C,
H, N, S.
2-[[3-[4-(Pyrimidin-2-yl)-1-piperazinyl]propyl]thio]benzothi-
azole (30). The title compound was isolated as light-yellow powder
(76%); mp 70-72 °C. 1H NMR (DMSO-d6): δ 1.88-2.04 (m, 2H,
CH2CH2CH2), 2.37-2.50 (m, 2H + 4H, CH2N + piperazine), 3.41
(m, 2H, SCH2), 3.67-3.75 (m, 4H, piperazine), 6.57-6.63 (m, 1H,
aromatic), 7.30-7.50 (m, 2H, aromatic), 7.82-8.02 (m, 2H,
aromatic), 8.32-8.36 (m, 2H, aromatic). Anal. (C18H21N5S2) C, H,
N, S.
2-[[3-[4-(Pyrimidin-2-yl)-1-piperazinyl]propyl]thio]benzox-
azole (31). The title compound was isolated as white powder (74%);
mp 75-77 °C. 1H NMR (DMSO-d6): δ 1.95-2.03 (m, 2H,
CH2CH2CH2), 2.30-2.51 (m, 2H + 4H, CH2N + piperazine), 3.39
(t, J ) 7.2 Hz, 2H, SCH2), 3.69-3.75 (m, 4H, piperazine),
6.58-6.66 (m, 1H, aromatic), 7.30-7.36 (m, 2H, aromatic),
7.60-7.68 (m, 2H, aromatic), 8.32-8.37 (m, 2H, aromatic).
Anal.(C18H21N5OS) C, H, N, S.
2-[[3-[4-(2-Methoxyphenyl)-1-piperazinyl]propyl]thio]-1-methyl-
1H-benzimidazole (32). The title compound was isolated as light-
yellow powder (52%); mp 83-84 °C. 1H NMR (DMSO-d6): δ
1.87-1.98 (m, 2H, CH2CH2CH2), 2.41-2.52 (m, 2H + 4H, CH2N
+ piperazine), 2.89-3.02 (m, 4H, piperazine), 3.36 (t, J ) 7.2 Hz,
2H, SCH2), 3.67 (s, 3H, NCH3), 3.75 (s, 3H, OCH3), 6.86-6.91
(m, 4H, aromatic), 7.13-7.19 (m, 2H, aromatic), 7.43-7.56 (m,
2H, aromatic). Anal. (C22H28N4OS) C, H, N, S.
CH2), 2.33-2.50 (m, 2H + 4H, CH2N + piperazine), 2.85-2.99
(m, 4H, piperazine), 3.39 (t, J ) 7.2 Hz, 2H, SCH2), 3.76 (s, 3H,
CH3), 6.82-6.94 (m, 4H, aromatic), 7.32-7.50 (m, 2H, aromatic),
7.83-8.02 (m, 2H, aromatic). Anal. (C22H27N3OS2) C, H, N, S.
2-[[4-[4-(2-Methoxyphenyl)-1-piperazinyl]butyl]thio]benzox-
azole (36). The title compound was isolated as yellow semisolid
(69%). 1H NMR 500 MHz (DMSO-d6): δ 1.60 (qu, 2H,
CH2CH2CH2 CH2), 1.82 (qu, 2H, CH2CH2CH2CH2), 2.36 (t, J )
6.6 Hz, 2H, CH2N), 2.48 (br m, 4H, piperazine), 2.92 (br m, 4H,
piperazine), 3.37 (t, J ) 6.8 Hz, 2H, SCH2), 3.75 (s, 3H, CH3),
6.82-6.92 (m, 4H, aromatic), 7.31-7.35 (m, 2H, aromatic),
7.62-7.67 (m, 2H, aromatic). Anal. (C22H27N3O2S) C, H, N, S.
2-[[6-[4-(2-Methoxyphenyl)-1-piperazinyl]hexyl]thio]benzothi-
azole (37). The title compound was isolated as light-yellow oil
(58%). 1H NMR (DMSO-d6):
δ
1.37-1.81 (m, 8H,
CH2CH2CH2CH2CH2CH2), 2.32 (t, J ) 6.2 Hz, 2H, CH2N),
2.48-2.52 (m, 4H, piperazine), 2.89-2.91 (m, 4H, piperazine), 3.36
(t, J ) 6.2 Hz, 2H, SCH2), 3.76 (s, 3H, CH3), 6.83-6.91 (m, 4H,
aromatic), 7.32-7.52 (m, 2H, aromatic), 7.82-8.03 (m, 2H,
aromatic). Anal. (C24H31N3OS2) C, H, N, S.
2-[[6-[4-(2-Methoxyphenyl)-1-piperazinyl]hexyl]thio]benzox-
azole (38). The title compound was isolated as light-yellow semisolid
(60%). 1H NMR 500 MHz (DMSO-d6): δ 1.34 (qu, 2H,
CH2CH2CH2CH2CH2CH2), 1.44 (m, 4H, CH2CH2CH2CH2CH2CH2),
1.79 (qu, 2H, CH2CH2CH2CH2CH2CH2), 2.29 (t, 2H, CH2N), 2.46
(br m, 4H, piperazine), 2.92 (br m, 4H, piperazine), 3.33 (t, J )
7.0 Hz, 2H, SCH2), 3.76 (s, 3H, CH3), 6.85-6.93 (m, 4H, aromatic)
7.28-7.32 (m, 2H, aromatic), 7.32-7.67 (m, 2H, aromatic). Anal.
(C24H31N3O2S) C, H, N, S.
5-Chloro-2-[[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]th-
io]benzothiazole (33). The title compound was isolated as white
powder (65%); mp 77-78 °C. 1H NMR (DMSO-d6): δ 1.90-1.99
(m, 2H, CH2CH2CH2), 2.43- 2.52 (m, 2H + 4H, CH2N +
piperazine), 2.88-2.91 (m, 4H, piperazine), 3.38 (t, J ) 7.0 Hz,
2H, SCH2), 3.76 (s, 3H, CH3), 6.85-6.93 (m, 4H, aromatic),
7.39-7.45 (m, 1H, aromatic), 7.90-8.09 (m, 2H, aromatic). Anal.
(C21H24ClN3OS2) C, H, N, S.
5-Chloro-2-[[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]th-
io]benzoxazole (34). The title compound was isolated as light-yellow
powder (72%); mp 78-80 °C. 1H NMR (DMSO-d6): δ 1.93-2.02
(m, 2H, CH2CH2CH2), 2.43-2.52 (m, 2H + 4H, CH2N +
piperazine), 2.88-3.02 (m, 4H, piperazine), 3.39 (t, J ) 7.0 Hz,
2H, SCH2), 3.76 (s, 3H, CH3), 6.85-6.93 (m, 4H, aromatic),
7.32-7.39 (m, 1H, aromatic), 7.65-7.78 (m, 2H, aromatic). Anal.
(C21H24ClN3O2S) C, H, N, S.
General Procedure for the Preparation of Compounds 35-38.
Compounds 7 or 8 (10 mmol), 1-bromo-4-chlorobutane or 1-bromo-
6-chlorohexane (30 mmol), potassium fluoride/aluminum oxide (10
g), potassium iodide (0.1 g), and acetonitrile (100 mL) were refluxed
for 1 h and then allowed to stand overnight at room temperature.
The mixture was filtered to eliminate inorganic material and then
evaporated under reduced pressure to give a residue which was
taken up in water.
The solution was extracted with dichloromethane (3 × 50 mL)
and the combinated extracts were washed with water, dried, and
evaporated to give intermediates 2-(4-chlorobutyl)thiobenzothiazole
or benzoxazole and 2-(6-chlorohexyl)thiobenzothiazole or benzox-
azole that were used without further purification for the final step.
A suspension of 2-(ω-chloroalkylthio)benzothiazole or 2-(ω-
chloroalkyl)thiobenzoxazole 12-15 (10 mmol), 1-(2-methoxyphe-
nyl)piperazine (12 mmol)), and potassium carbonate (12 mmol) in
acetonitrile (30 mL) was refluxed for 24 h. After this period, the
mixture was concentrated and the residue was diluted with water
and extracted with dichloromethane (3 × 50 mL). The combined
organic layers were dried over anhydrous Na2SO4 and removed in
vacuo. The obtained residue was purified by flash column cro-
matography on silica gel using ethyl acetate/cyclohexane (1:1)
mixture as eluent.
Pharmacology: Binding Assays. Male CRL:CD(SD)BR-COBS
rats weighing about 150 g were killed by decapitation,25 and their
brains were rapidly dissected (hippocampus for 5-HT1AR; striatum
for D1 and D2 receptors; cortex for R1-AR and 5-HT2A receptor),
frozen, and stored at -80 °C until the day of assay.
Tissue was homogenized in about 50 volumes of ice-cold 50
mM Tris · HCl buffer (pH 7.4) using an Ultra Turrax TP-1810
(2 × 20 s) and centrifuged at 50000g for 10 min (Beckman model
J-21B refrigerated centrifuge). The pellet was resuspended in
the same volume of fresh buffer, incubated at 37 °C for 10 min,
and centrifuged again at 50000g for 10 min. The pellet was then
washed once by resuspension in fresh buffer and centrifuged as
before. The pellet was then resuspended in the appropriate
incubation buffer (50 mM Tris · HCl, pH 7.7 containing 10 µM
pargyline and 4 mM CaCl2 for 5-HT1ARs; 50 mM Tris · HCl,
pH 7.7 containing 10 µM pargyline and 0.1% ascorbic acid for
R1-ARs; 50 mM Tris · HCl, pH 7.4 containing 10 µM pargyline,
120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and
0.1% ascorbic acid for D1 and D2 receptors; 50 mM Tris · HCl,
pH 7.7 for 5-HT2A receptors) just before the binding assay.
Binding assays were done as previously described.26 Briefly, the
following incubation conditions were used. 5-HT1A: [3H]-8-OH-
DPAT (specific activity 157 Ci/mmol, NEN) final concentration 1
nM, 30 min, 25 °C (nonspecific binding: 5-HT 10 µM). D1:
[3H]SCH23390 (specific activity 71.1 Ci/mmol, NEN) final con-
centration 0.4 nM, 15 min, 25 °C (nonspecific binding: (-)-cis-
flupentixol 10 µM). D2: [3H]spiperone (specific activity 19.0 Ci/
mmol, NEN) final concentration 0.2 nM, 15 min, 37 °C (nonspecific
binding: (-)-sulpiride 100 µM). R1-ARs: [3H]prazosin (specific
activity 71.8 Ci/mmol, NEN) final concentration 0.2 nM, 30 min,
25 °C (nonspecific binding: phentolamine 3 µM). 5-HT2A: [3H]ket-
anserin (specific activity 63.3 Ci/mmol, Amersham) final concentra-
tion 0.35 nM, 15 min, 37 °C (nonspecific binding: methisergide 1
µM).27
For [35S]GTPγS binding assay to 5-HT1ARs, tissue was prepared
as described above for 5-HT1AR binding. Binding was performed
as follows: [35S]GTPγS (specific activity 1064 Ci/mmol, Amer-
sham) final incubation volume of 1.04 mL, consisting of 1 mL of
membrane suspension from rat hippocampus (200 µg of protein/

5-HT1A Serotonin Receptor Ligands
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 15 4537
sample), 20 µL of [35S]GTPγS (final concentration 0.1 nM), and
20 µL of drugs or solvent. Nonspecific binding was obtained in
presence of 10 µM GTPγS. Samples were preincubated for 20 min
at 37 °C without [35S]GTPγS and then for 45 min at 37 °C with
[35S]GTPγS.
Incubations were stopped by rapid filtration under vacuum
through GF/B filters, which were then washed with 12 mL of ice-
cold 50 mM Tris HCl, pH 7.4 using a Brandel M 48-R cell
harvester.
The radioactivity trapped on the filters was counted in 4 mL of
Ultima Gold MV (Packard) in a Wallac 1409 DSA liquid scintil-
lation counter with a counting efficiency of 50% for [3H] or 90%
for [35S].26 Dose-response curves were analyzed by the “Allfit”
program.28 The Ki values were derived from the IC50 values.29
Molecular Docking. The population of 100 5-HT1AR models
described previously19 was reused in the present work. Models
were constructed on the basis of slightly modified bovine
rhodopsin template. All the docking experiments were conducted
using the FlexX software (www.biosolveit.de) with FlexX-Pharm
extension. Pharmacophore constraints were applied: H-bond
acceptor at the carboxylic oxygen of Asp3.32, CH-π edge-to-
face interaction with Phe6.62 and H-bond donor at hydroxylic
group of Ser5.43. The results were rescored with 4 additional
scoring functions and subjected to consensus scoring procedure,
as implemented in SYBYL (www.tripos.com). Highest PMF
scored solutions out of those having consensus score 5 were
considered representative. PMF score was used for final scoring
of docking solutions, as it was proved to give the best enrichment
factors in virtual screening experiments.30
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Acknowledgment. This work was supported by grants from
the Italian MIUR and the University of Catania.
Supporting Information Available: Elemental analysis data of
compounds 16-36, NOESY (DMSO/TFA) of compound 38, and
1H NMR spectra at 500 MHz of compounds 18, 24, 36, and 38.
This material is available free of charge via the Internet at http://
pubs.acs.org..
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