New N- and O-arylpiperazinylalkyl pyrimidines and 2-methylquinazolines derivatives as 5-HT7and 5-HT1Areceptor ligands: Synthesis, structure-activity relationships, and molecular modeling studies

Article abstract of DOI:10.1016/j.bmc.2016.12.039

Based on our earlier studies of structure activity relationships on 4-substituted piperazine derivatives, in this work we synthesized a novel set of long-chain arylpiperazines with the purpose of elucidating if some structural modifications in the terminal fragment could affect the binding affinity for the 5-HT₇and 5-HT_1Areceptors. In this new series, the quinazolinone system of the previous derivatives was replaced by a 6-phenylpyrimidine or a 2-methylquinazoline, which were used as versatile building blocks for the preparation of new compounds. A 4-arylpiperazine moiety through a five methylene chain was anchored at the nitrogen or oxygen atom of the heterocyclic scaffolds. The substituents borne by the piperazine nucleus were phenyl, phenylmethyl, 3- or 4-chlorophenyl, and 2-ethoxyphenyl. Binding tests, performed on human cloned 5-HT₇and 5-HT_1Areceptors, showed that, among the newly synthesized derivatives, 4-[5-[4-(2-ethoxyphenyl)-1-piperazinyl]pentoxy]-6-phenyl-pyrimidine (13) and 3-[5-[4-(2-ethoxyphenyl)-1-piperazinyl]pentyl]-2-methyl-4(3H)-quinazolinone (20) displayed the best affinity values, K_i?=?23.5 and 8.42?nM for 5-HT₇and 6.96 and 2.99?nM for 5-HT_1Areceptors, respectively. Moreover, the functional properties for both compounds were further evaluated using the cAMP assay. Finally, a molecular modeling study has been performed for 5-HT₇and 5-HT_1Areceptor homology models to investigate the binding mode of N- and O-alkylated pyrimidinones/pyrimidines 4–13, 2-methylquinazolinones/quinazolines 17–22, and previously reported 2- and 3-substituted quinazolinones 23–30.

Full text of DOI:10.1016/j.bmc.2016.12.039

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New N- and O-arylpiperazinylalkyl pyrimidines and
2-methylquinazolines derivatives as 5-HT7 and 5-HT1A receptor ligands:
Synthesis, structure-activity relationships, and molecular modeling
studies
Sebastiano Intagliata a, ,1, Maria N. Modica a,1, Valeria Pittalà a, Loredana Salerno a, Maria A. Siracusa a,
⇑
Alfredo Cagnotto b, Mario Salmona b, Rafał Kurczab c, Giuseppe Romeo a
a Dipartimento di Scienze del Farmaco, Università di Catania, viale A. Doria 6, Catania 95125, Italy
b IRCCS-Istituto di Ricerche Farmacologiche ‘‘Mario Negri”, via La Masa 19, Milano 20156, Italy
c Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343 Krakow, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on our earlier studies of structure activity relationships on 4-substituted piperazine derivatives, in
this work we synthesized a novel set of long-chain arylpiperazines with the purpose of elucidating if
some structural modifications in the terminal fragment could affect the binding affinity for the 5-HT7
and 5-HT1A receptors. In this new series, the quinazolinone system of the previous derivatives was
replaced by a 6-phenylpyrimidine or a 2-methylquinazoline, which were used as versatile building blocks
for the preparation of new compounds. A 4-arylpiperazine moiety through a five methylene chain was
anchored at the nitrogen or oxygen atom of the heterocyclic scaffolds. The substituents borne by the
piperazine nucleus were phenyl, phenylmethyl, 3- or 4-chlorophenyl, and 2-ethoxyphenyl. Binding tests,
performed on human cloned 5-HT7 and 5-HT1A receptors, showed that, among the newly synthesized
derivatives, 4-[5-[4-(2-ethoxyphenyl)-1-piperazinyl]pentoxy]-6-phenyl-pyrimidine (13) and 3-[5-[4-(2-
ethoxyphenyl)-1-piperazinyl]pentyl]-2-methyl-4(3H)-quinazolinone (20) displayed the best affinity val-
ues, Ki = 23.5 and 8.42 nM for 5-HT7 and 6.96 and 2.99 nM for 5-HT1A receptors, respectively. Moreover,
the functional properties for both compounds were further evaluated using the cAMP assay. Finally, a
molecular modeling study has been performed for 5-HT7 and 5-HT1A receptor homology models to inves-
tigate the binding mode of N- and O-alkylated pyrimidinones/pyrimidines 4–13, 2-methylquinazoli-
nones/quinazolines 17–22, and previously reported 2- and 3-substituted quinazolinones 23–30.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 24 July 2016
Revised 20 December 2016
Accepted 23 December 2016
Available online xxxx
Keywords:
5-HT7 receptor ligands
5-HT1A receptor ligands
Long-chain arylpiperazines
Molecular modeling
1. Introduction
present in an extensively studied class of serotoninergic ligands,
called long-chain arylpiperazines (LCAPs).12–16 The serotonin neu-
The heterocyclic quinazoline and quinazolinone are frequently
encountered as scaffolds in medicinal chemistry. They possess
wide pharmacological applications such as antibacterial, antidia-
betic, anti-inflammatory, and many others.1 Besides, the quinazo-
line system has been found in natural products, such as
alkaloids,2,3 which showed medicinal applications.4–6 Accordingly,
a lot of comprehensive studies regarding the quinazolines/quina-
zolinones functionalization to synthesize new effective drugs have
been done.7–9 Furthermore, quinazoline derivatives were reported
as CNS depressants10 and anticonvulsants,11 and this system is also
rotransmitter interacts with a large number of receptors which,
following the IUPHAR classification17 are classified into seven fam-
ilies (5-HT1–7). Except 5-HT3 receptor, they all belong to a G pro-
tein-coupled receptor (GPCR) family, the largest and most
versatile group of cell membrane signaling proteins. The 5-HT1A
receptor (5-HT1AR) was the first of the 5-HTRs to be cloned. It is
coupled to adenylyl cyclase via Gi/0 and is widely distributed in
CNS and in peripheral tissues. Probably, it was one of the most
studied of the serotonin receptor family and is still a current one.
It is generally believed to be involved in anxiety, depression, epi-
lepsy, and disorders of mood, learning, and memory.18 The 5-HT7
receptor (5-HT7R) discovered in 1993 is positively coupled to
adenylyl cyclase via a Gs protein.19 This receptor is located in the
brain and peripheral tissues. In particular, it is largely distributed
⇑
Corresponding author.
E-mail address: s.intagliata@studium.unict.it (S. Intagliata).
1
These authors contributed equally to the work.
http://dx.doi.org/10.1016/j.bmc.2016.12.039
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Intagliata S., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2016.12.039

2
S. Intagliata et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
in hippocampus and thalamus suggesting its involvement in CNS
disorders as well.12,19,20 In light of the fact that both receptors
are co-expressed in the same brain structures and modulate the
same second messenger but exerting opposite roles, it is little won-
der that exist a cross-talk between them. From the functional point
of view, agents that act as partial agonist at 5-HT1AR and antago-
nist at 5-HT7R might hold antidepressant-like properties.21 So,
the study of interaction between these receptors is going to be of
increasing importance as clinical interest for the treatment of
depression and anxiety as well as in emotional learning and
memory.22,23
(53% and 47%, respectively), and in addition, it was also possible to
isolate with a low yield derivatives 3 and 16 (10% and 23%, respec-
tively), alkylated at the oxygen atom as confirmed by 1H NMR and
IR spectral data (Table 1). 1H NMR spectra of compounds 2 and 15
show a signal of two protons of a methylene unit at d 3.92 and 4.03,
respectively, attributable to a N-alkylation (CONCH2). The shift of
these signals for compounds 3 and 16 at d 4.53 and 4.39, respec-
tively, is due to an O-alkylation (OCH2). IR spectra further con-
firmed such findings; compounds 2 and 15 display peaks at 1671
and 1673 cmÀ1, respectively, characteristic for the C@O stretching,
which were absent in compounds 3 and 16.
Recently, we have reported new LCAPs bearing a quinazolinone
system as a terminal fragment, which displayed high-to-low affin-
ity for 5-HT7R (Ki = 6.88–1082 nM, compounds 23–30, Table 2),
and high-to-moderate affinity for 5-HT1AR (Ki = 1.04–268 nM, com-
pounds 23–30, Table 2).24,25 Here we present the synthesis of a
novel series of LCAPs. This work deals with the modification on
the quinazolinone scaffold as terminal fragment, in order to eluci-
date how these changes influence both the affinity for 5-HT7R and
5-HT1AR, and the binding modes in receptor homology models. In
particular, the quinazolinone system was replaced by 6-phenyl-4
(3H)-pyrimidinone as a result of splitting bicyclic quinazolinone
system (benzo cracking).26 To the best of our knowledge, this is
the first time that a 6-phenylpyrimidine is used as building block
scaffold for the preparation of 5-HT7 and 5-HT1A receptor ligands.
In confirmation of this, no hit was found within sets of 4166 5-
HT1AR and 720 5-HT7R ligands with Ki < 100 nM stored in ChEMBL
v. 11 database27 for 4-oxy-pyrimidine/pyrimidinone substractural
query (using Instant JChem).28 Furthermore, the pyrimidines, sim-
ple or fused with other heterocycles, represent versatile synthetic
intermediates and constitute an important class of compounds
for new drug development.29–31
It has been well-established that regioselectivity varies from
one scaffold to another and within the same scaffold. Different syn-
thetic outcomes can be obtained depending on solvent, nature of
electrophiles, and other conditions such as temperature, base used
for deprotonation, and nature of the substituent at the 2-position
of the heterocyclic compound.32–35 For these reasons, it is very dif-
ficult establishing predictable and robust protocols for N- versus O-
alkylation reactions.36,37 The different percentage in the alkylated
product mixtures depends on the competing pathways when these
lead to different products. Prevalence of thermodynamic or kinetic
control determines the final composition of the product.38 Due to
the poor regioselectivity of the ambident nucleophiles in the alky-
lation reaction, 4-(5-chloropenthoxy)-6-phenyl-pyrimidine (3) and
4-(5-bromopenthoxy)-2-methylquinazoline (16) were more diffi-
cult to be obtained. Nevertheless, they were isolated from the reac-
tion mixture and used in preparation of the final compounds.
Compounds 4–13 and 17–22 were prepared from halo deriva-
tives 2, 3, 15, and 16 by reaction with an excess of the substituted
piperazine, in the presence of potassium carbonate and a catalytic
amount of potassium iodide using traditional heating or by micro-
wave irradiation (Scheme 1).
Further changes to the quinazolinone system regarding, the
methylation in the 2-position which gave the 2-methyl-4(3H)-
quinazolinone and the shift of the anchoring point of the arylpiper-
azinylalkyl moiety to the nitrogen or oxygen atom. Following the
results from the previous investigation only derivatives with five
methylene unit spacer were prepared and the same arylpiperazine
fragments (i.e. phenyl, phenylmethyl, 3-chloro-, 4-chloro-, and 2-
ethoxyphenyl) were used (Fig. 1).
Herein we present the synthesis and the binding properties of
new N- and O-arylpiperazinylalkyl pyrimidine and 2-methylquina-
zoline derivatives towards 5-HT7 and 5-HT1A receptors, followed
with in vitro evaluation of antagonistic properties of selected com-
pounds 13 and 20. Furthermore, a molecular modeling study has
been performed for 5-HT7 and 5-HT1A receptor homology models
to investigate the binding mode of N- and O-alkylated pyrimidi-
nones/pyrimidines 4–13, 2-methylquinazolinones/quinazolines
17–22, and previously reported 2- and 3-substituted quinazoli-
nones 23–30.
2.2. Structure–affinity relationship studies
New derivatives 4–13 and 17–22 were tested on human cloned
5-HT7 and 5-HT1A serotonin receptors expressed in CHO-K1 cells
following a previously reported procedure.39 Binding assays on 5-
HT7 and 5-HT1A receptors were carried out by using [3H]-5-HT
and [3H]-8-OH-DPAT as radioligands, respectively. Results,
expressed as Ki (nM), are summarized in Table 2.
The structure-affinity relationship (SAR) analysis in regard to
the aromatic part of the arylpiperazine moiety showed that like
in our previous study, 3-ClC6H4 (6, 11, and 18) and 2-EtOC6H4 (8,
13, and 20) were the most active derivatives at both receptors,
whereas analogous with 4-ClC6H4 (7, 12, and 19) and CH2C6H5 (5
and 10) displayed the lowest affinity. Unsubstituted arylpiperazi-
nes (4, 9, 17, and 21) showed moderate affinity for 5-HT7
(Ki = 53.7–613 nM) and higher for 5-HT1A (Ki = 18.6–111 nM).
The influence of the terminal fragment on 5-HT7 and 5-HT1A
receptor affinity can be directly traced in N-alkylated derivatives,
comparing appropriately substituted arylpiperazines from quina-
zolinone series 26–30 with 2-methylquinazolinones 17–20 and
6-phenylpyrimidinones 4–8 (Table 2). The introduction of the
methyl substituent at the 2-position of the quinazolinone system
did not significantly change affinities for both receptors, as can
be seen from a comparison of the affinities of 2-EtOC6H4 deriva-
tives 20 and 30 which are similar and the highest for both series.
An exception was observed for the phenyl derivative 17, which
showed a 6-fold increase of affinity for the 5-HT7R compared to
26 (Ki = 53.7 vs 307 nM); and the 4-chlorophenyl derivatives 19
vs 29, that conversely, demonstrated a 25-fold decrease of affinity
for this receptor (Ki = 327 and 12.9 nM, respectively). On the other
hand, the benzo cracking strategy (compounds 4–8) caused a
decrease in affinity for both receptors.
2. Results and discussion
2.1. Chemistry
The synthetic procedure adopted for the preparation of the new
pyrimidine 4–13 and quinazoline 17–22 derivatives is outlined in
Scheme 1. The 6-phenyl-4(3H)-pyrimidinone (1) and the 2-
methyl-4(3H)-quinazolinone (14) reacted with an excess of 1,5-
dibromopentane or 1-chloro-5-bromopentane in the presence of
potassium carbonate and a catalytic amount of potassium iodide
to obtain derivatives 2, 3, 15, and 16. Halo derivatives 2 and 3 were
prepared by using traditional heating method, 15 and 16 by using
microwave irradiation. From the reaction mixture, derivatives 2
and 15 (alkylated at the nitrogen atom) were isolated in fair yields
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S. Intagliata et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
LCAPs three main structural features:
Terminal
fragment
Spacer
N
N R
N
Spacer = -(CH2)5- R = C6H5
CH2C6H5
2
3
NH
3-ClC6H4
4-ClC6H4
2-EtOC6H4
O
Previously reported
quinazolinone
derivatives
N
2
3
23-30
N
O
N
N
N
O
Spacer = -(CH2)5- R = C6H5
CH2C6H5
3-ClC6H4
4-ClC6H4
2-EtOC6H4
N
N
New
pyrimidine/pyrimidinone
and
quinazoline/quinazolinone
derivatives
O
N
N
N
O
4-13
17-22
and
O
Fig. 1. Structural features of previously and newly synthesized LCAPs.
Shifting the anchoring point of the arylpiperazinylalkyl moiety
from the nitrogen (4–8 and 17–20) to the oxygen atom (9–13
and 21, 22) determined a general slight increase of affinity in the
pyrimidine derivatives (4–8 vs 9–13), whereas no homogenous
results was obtained for 2-methylquinazolinone derivatives (17,
19 vs 21, 22).
(Table 2) was performed with the use of recently developed 5-
HT7 and 5-HT1ARs homology models based on dopaminergic D3
receptor template (PDB ID: 3PBL) and trained on long-chain
arylpiperazine derivatives.39 For each compound, five top-scored
complexes were considered, of which the best with a binding mode
coherent to the previously described39 was analyzed.
Binding modes and interacting residues of the analyzed com-
pounds were similar, despite the adopted ligand conformations
for 5-HT7R and 5-HT1AR were slightly different. Generally, the com-
pounds preferred an L-shape conformation when bound to 5-HT7R
and an extended conformation if complexed with 5-HT1AR. Accord-
ing to our previous study,39 this result is attributed to the different
size of the cavity within transmembrane helices (TMHs) 2, 3, 7, and
the first extracellular loop 1 (EL1), which was smaller in 5-HT7R
than in 5-HT1AR. Comparison of the docking poses of ligand 20,
which had the highest affinity for the 5-HT7R and 5-HT1AR in this
new series, shows different orientations for the terminal
2-methyl-4(3H)-quinazolinone fragment (Fig. 2A). In the case of
5-HT7R this moiety points towards the extracellular loops and
interacts with Thr2.64, Arg7.36, and Cys146 of the EL2 (Fig. 2A,
left), whereas in the 5-HT1AR it interacts with Tyr2.64, Phe3.28,
Asn7.39, and Trp7.40 (Fig. 2A, right). The 2-ethoxyphenylpiper-
azine moiety of 20 is hosted in a small cavity between TMHs 5
2.3. In vitro functional evaluation
Compounds 13 and 20 which possess the best 5-HT7/5-HT1A
binding properties, were selected in order to investigate the func-
tional activity. The cAMP functional assays were performed in
HEK293 cell lines with stable expression of human 5-HT7R and
5-HT1AR. The results were reported as% inhibition of control ago-
nist response at 10À6 M and using as reference the antagonist SB-
269970 (97% at 10À6 M). The results of the functional assays are
shown in Table 3. Compounds 13 and 20 produced a weak inhibi-
tion of cAMP on 5-HT7R (5.5% and 19.5%, respectively), suggesting a
functional behavior as weak antagonists. On the other hand com-
pound 30 showed stronger antagonistic properties producing at
the same concentration a reduction of cAMP accumulation to
42% (Table 3).24
Interestingly, the O-arylpiperazinylalkyl 6-phenyl-pyrimidine
derivative (13) showed a significant decrease of inhibition of cAMP
levels on 5-HT7R compared to N-arylpiperazinylalkyl 4(3H)-quina-
zolinone derivative (30). Additionally, the antagonistic properties
of compounds 13 and 20 were also evaluated against the 5-HT1AR
(6% and 18%, respectively).
and 6, in which the aromatic ring formed a CH-p or p-p interaction
with the Phe6.51 in both receptors (Fig. 2B). The orientation of the
2-ethoxygroup is, however, different; in the 5-HT7R the ortho
substituent seems to interact with Cys3.36 (Fig. 2B, left), whereas
in the 5-HT1AR with Ser5.42 and Thr5.43 (Fig. 2B, right).
From the SAR studies it was found that the introduction of a
chloro atom at the meta position of the phenylpiperazine ring
increases the affinity for both receptors with respect to the para-
chloro analogous. The increased affinity could be justified by the
binding pose of the 3-chlorophenylpiperazine derivative (see 6 vs
7), which shows a favourable orientation towards the Cys3.36, by
2.4. Molecular modeling studies
Molecular docking of N- and O-alkylated pyrimidinones/pyrim-
idines 4–13, 2-methyl quinazolinones/quinazolines 17–22, and
previously reported 2- and 3-substituted quinazolinones 23–3024
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4
S. Intagliata et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
N
N
O
R
N
b
N
Cl
N
N
( )
( )
5
5
O
2
4, 9 R = C6H5
5, 10
N
O
N
O
4-8
R = CH2C6H5
a
NH
N
6, 11 R = 3-ClC6H4
7, 12 R = 4-ClC6H4
8, 13
R = 2-EtOC6H4
1
N
N
O
b
N
N
( )
R
N
O
Cl
N
( )
5
5
3
9-13
N
N
R
N
b
b
N
Br
N
N
( )
( )
5
5
O
15
O
N
N
O
17, 21 R = C6H5
17-20
a
NH
18
19, 22
20
R = 3-ClC6H4
R = 4-ClC6H4
R = 2-EtOC6H4
N
O
N
O
N
14
N
( )
N
R
N
Br
O
N
( )
5
5
16
21-22
Scheme 1. Reagents and conditions: (a) X(CH2)5Br, K2CO3, KI catalytic amount, CH3CN or CO(CH3)2, microwave or reflux; (b) substituted piperazines, K2CO3, KI catalytic
amount, CH3CN, microwave or reflux.
Table 1
Spectral data of halo derivatives 2, 3, 15, and 16.
Compound
1H NMR, d (ppm)
CONCH2
IR, (cmÀ1) C@O
OCH2
–
2
3
3.92
1673
N
N
Cl
( )
5
O
N
–
4.53
–
N
O
N
Cl
Br
( )
5
15
16
4.03
–
1671
N
( )
5
O
N
–
4.39
–
N
( )
O
Br
5
establishing an additional halogen bond interaction (Fig. 2C).40 As a
general trend, similar behavior was found for the
chlorophenylpiperazine derivatives (11 vs 12 and 18 vs 19) along
the series (Table 2).
a
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S. Intagliata et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
Table 2
Table 3
Binding properties of derivatives 4–13, 17–22, 23–30, reference compounds SB-
Antagonist activity of selected compounds for 5-HT7R and 5-HT1AR.
269970 and 8-OH-DPAT.
Comp.
% inha
5-HT7
% inhb
5-HT1A
13
5.5
19.5
42
6
20
18
NTd
–
N
30c
N
O
R
R
N
N
SB-269970
97
N
( )
R
N
N
N
( )
a
b
c
5
% inhibition of control agonist response (5-CT 10 nM) at 10À6 M.
% inhibition of control agonist response (8-OH-DPAT 1
Data from Ref. 24.
N
O
l
M) at 10À6 M.
5
4 8
-
9 13
-
d
NT = not tested.
N
N
N
regards 5-HT1AR, due to limited space between TMH2 and 7, the
more distant aromatic rings of terminal fragments are well aligned
at the expense of differences in linker conformations.
N
N
N
R
N
( )
N
5
O
O N
( )
5
17 20
21, 22
-
3. Conclusions
( )
N
N
N
R
In conclusion, we described the synthesis of new LCAPs with
structural modifications in the terminal fragment and in the
anchoring position of the arylpiperazinylalkyl moiety. We reported
the simultaneous preparation and isolation of N- and O-alkylated
pyrimidine and 2-methylquinazoline derivatives. The newly syn-
thesized piperazine derivatives have been evaluated for binding
affinities at the human cloned 5-HT7R and 5-HT1AR and the main
structure-affinity relationships were outlined.
5
NH
N
N
( )
R
5
O
O
23-25
26 30
-
Preliminary data on functional activity showed partial inhibi-
tion of cAMP levels for 4(3H)-quinazolinone derivative (30), while
derivatives 6-phenyl-pyrimidine (13) and 2-methyl-4(3H)quinazo-
line (20) showed only weak inhibition values. These results suggest
a possible correlation between scaffold types and intrinsic activity
of the new ligands.
Docking studies revealed coherent binding mode for all com-
pounds in both receptors. This observation is well matched with
our previous model and confirmed that L-shape is more suitable
than extended conformation for 5-HT7R. In addition, it was out-
lined that planar bicyclic system is preferable with respect to a sin-
gle heterocyclic ring with a bulky substituent in the interaction
with both receptors.
Comp.
R
Kia (nM)
5-HT7
5-HT1A
4
5
6
7
8
9
10
11
12
13
17
18
19
20
21
C6H5
613 68
111 21
CH2C6H5
3-ClC6H4
4-ClC6H4
2-EtOC6H4
C6H5
CH2C6H5
3-ClC6H4
4-ClC6H4
2-EtOC6H4
C6H5
3-ClC6H4
4-ClC6H4
2-EtOC6H4
C6H5
4-ClC6H4
C6H5
3-ClC6H4
4-ClC6H4
C6H5
CH2C6H5
3-ClC6H4
4-ClC6H4
2-EtOC6H4
1328 192
94.7 10
1310 179
45.1 4.6
143 17
990 177
44.8 7.8
1377 252
23.5 2.9
53.7 12
35.1 6.9
327 47
8.42 0.78
276 27
181 20
228 12
11.9 3.2
101 26
772 156
17.8 2.8
167 20
25.9 3.7
33.5 4.1
1032 114
17.4 2.0
90.3 6.5
6.96 0.76
18.6 2.1
8.21 1.1
100 14
2.99 0.26
24.7 2.0
52.1 4.4
43.5 5.4
7.33 0.77
116 20
4. Experimental
22
4.1. Chemistry
23b
24b
25b
Melting points were determined in an Electrothermal IA9200
apparatus using glass capillary tubes and are uncorrected. Infrared
spectra were recorded on a Perkin Elmer series FTIR 1600 spec-
trometer in KBr disks. Elemental analyses for C, H, and N were
within 0.4% of theoretical values and were obtained with a Carlo
Erba Elemental Analyzer Mod. 1108 apparatus. 1H NMR spectra
26b
307 57
28.9 4.6
268 25
27b
1082 100
35.8 7.0
12.9 0.85
6.88 0.66
0.71 0.06
388 58
28b
6.28 0.86
51.5 11
1.04 0.13
9024 181
2.65 0.10
29b
30b
SB-269970
8-OH-DPAT
were performed on
a Varian Inova Unity 200 spectrometer
(200 MHz) in DMSO-d6 or CDCl3 solution. Tetramethylsilane was
used as the internal standard; chemical shifts and coupling con-
stants (J) are given in d values (ppm) and in Hertz (Hz), respec-
tively. Signal multiplicities are abbreviated as follow: s (singlet),
d (doublet), t (triplet), q (quartet), m (multiplet). Microwave
irradiation experiments were carried out with a CEM Discover
instrument using closed Pyrex glass tubes (ca. 10 mL) with
Teflon-coated septa. Thin-layer chromatography was utilized to
monitor the progress of reactions and to test the purity of all the
synthesized compounds, using Merck aluminium sheet coated
with silica gel 60 F254 and detection with ultraviolet light at 254
and 366 nm of wavelength. Purification of synthesized compounds
was performed by flash column chromatography using Merck silica
a
Each value is the mean SD of the data from three separate experiments.
Data from Ref. 24.
b
Relative positions of terminal fragments can be compared in
Fig. 2D which shows the binding modes of unsubstituted
phenylpiperazines (4, 9, 17, 21, 23, and 26; Table 2). Generally,
the subset of analogs is docked in similar conformations (L-shape
for the 5-HT7R and extended for 5-HT1A, Fig. 2D) making explana-
tion of 12- and 6-fold differences between the most and the least
actives, for 5-HT7R and 5-HT1AR, respectively. Interestingly, in case
of 5-HT7R, terminal fragments of phenylpyrimidines (4, 9), 2-
methylquinazolines (17, 21) and quinazolines (23, 26), despite dif-
ferent linker anchoring points, occupied similar positions. As
Please cite this article in press as: Intagliata S., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2016.12.039

6
S. Intagliata et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 2. Panel illustrating molecular modeling results of the new N-/O-alkyl derivatives and of the previously reported analogs for 5-HT7 (left) and 5-HT1A receptors (right). (A)
Docking pose of the high-affinity ligand 20 (green) in 5-HT7 and 5-HT1A binding sites. (B) Details of the 2-ethoxyphenyl moiety orientation within the binding pocket
(compound 20). (C) Orientation of the 3-chloro 6 (yellow) and 4-chlorophenylpiperazine 7 derivatives (orange). (D) Comparison of the binding modes of phenylpiperazine
derivatives 4 (cyan), 9 (grey), 17 (magenta), 21 (purple blue), 23 (limegreen), and 26 (red).
Please cite this article in press as: Intagliata S., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2016.12.039

S. Intagliata et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
gel (0.040–0.063 mm). All chemicals and solvents were reagent
grade and were purchased from commercial source.
4.1.5. 3-[5-[4-(3-Chlorophenyl)-1-piperazinyl]pentyl]-6-phenyl-4
(3H)-pyrimidinone (6)
The title compound was obtained by recrystallization from ace-
tonitrile (14%), mp 112.2–112.7 °C. IR (KBr, selected lines) cmÀ1
2938, 1666, 1596, 1489, 1449, 1245, 692. 1H NMR (DMSO-d6) d
1.22–1.42 (m, 2H, CH2), 1.42–1.62 (m, 2H, CH2), 1.62–1.82 (m,
2H, CH2), 2.31 (t, J = 6.8 Hz, 2H, CH2N), 2.38–2.49 (m, 4H, piper-
azine), 3.18–3.22 (m, 4H, piperazine), 3.93 (t, J = 7.0 Hz, 2H,
CONCH2), 6.75–6.80 (m, 1H, aromatic), 6.80–6.97 (m, 2H, aro-
matic), 6.98 (s, 1H, NCH), 7.18–7.25 (m, 1H, aromatic), 7.42–7.78
(m, 3H, aromatic), 8.00–8.15 (m, 2H, aromatic), 8.61 (s, 1H, CCH).
Anal. (C25H29ClN4O) C, H, N.
4.1.1. General procedure for the preparation 3-(5-chloropentyl)-6-
phenyl-4(3H)-pyrimidinone (2) and 4-(5-chloropenthoxy)-6-phenyl-
pyrimidine (3)
Acetone was added (30 mL) to a mixture of pyrimidinone 1
(2.90 mmol), 1-bromo-5-chloro-pentane (5.80 mmol), potassium
carbonate (4.34 mmol), and of a catalytic amount of potassium
iodide and the mixture was refluxed under stirring for 9 h. After
being cooled, the solid was removed by filtration and the solution
was concentrated to dryness. Recrystallization with cyclohexane
gave pure compound 2. From the solution compound 3 was iso-
lated by flash chromatography using a mixture of cyclohexane/
ethyl acetate (9:1, v/v).
4.1.6. 3-[5-[4-(4-Chlorophenyl)-1-piperazinyl]pentyl]-6-phenyl-4
(3H)-pyrimidinone (7)
The title compound was obtained by recrystallization from ace-
tonitrile (10%), mp 153.6–154.0 °C. IR (KBr, selected lines) cmÀ1
2936, 1664, 1594, 1495, 1450, 1237, 812, 696. 1H NMR (DMSO-
d6) d 1.20–1.40 (m, 2H, CH2), 1.40–1.60 (m, 2H, CH2), 1.60–1.80
(m, 2H, CH2), 2.29 (t, J = 6.6 Hz, 2H, CH2N), 2.40–2.49 (m, 4H, piper-
azine), 3.02–3.15 (m, 4H, piperazine), 3.91 (t, J = 7.4 Hz, 2H,
CONCH2), 6.84–6.95 (m, 2H, aromatic), 6.96 (s, 1H, NCH), 7.15–
7.25 (m, 2H, aromatic), 7.43–7.53 (m, 3H, aromatic), 7.99–8.11
(m, 2H, aromatic), 8.59 (s, 1H, CCH). Anal. (C25H29ClN4O) C, H, N.
Compound 2: yield 53%, mp 103.9–104.5 °C. IR (KBr, selected
lines) cmÀ1 2944, 2361, 1673, 1593, 1450, 691. 1H NMR (DMSO-
d6) d 1.30–1.50 (m, 2H, CH2), 1.60–1.85 (m, 2H + 2H, CH2CH2),
3.65 (t, J = 6.6 Hz, 2H, CH2Cl), 3.92 (t, J = 7.2 Hz, 2H, CONCH2),
6.97 (s, 1H, NCH), 7.40–7.58 (m, 3H, aromatic), 7.98–8.15 (m, 2H,
aromatic), 8.59 (s, 1H, CCH). Anal. (C15H17ClN2O) C, H, N.
Compound 3: yield 10%, mp 41.0–44.0 °C. IR (KBr, selected
lines) cmÀ1 2956, 1592, 1541, 1466, 1219, 868, 696. 1H NMR
(DMSO-d6) d 1.44–1.65 (m, 2H, CH2), 1.70–1.90 (m, 2H + 2H, CH2CH2),
3.67 (t, J = 6.6 Hz, 2H, CH2Cl), 4.39 (t, J = 6.4 Hz, 2H, OCH2),
7.45–7.60 (m, 3H + 1H, aromatic + NCH), 8.15–8.25 (m, 2H,
aromatic), 8.84 (d, J = 1.2 Hz, 1H, CCH). Anal. (C15H17ClN2O) C, H, N.
4.1.7. 3-[5-[4-(2-Ethoxyphenyl)-1-piperazinyl]pentyl]-6-phenyl-4
(3H)-pyrimidinone (8)
The title compound was obtained after 16 h of reflux and was
purified by flash chromatography using a mixture of ethyl acet-
ate/methanol (9:1, v/v) as eluent (32%), mp 103.5–104.1 °C. IR
(KBr, selected lines) cmÀ1 2927, 2804, 1664, 1499, 1455, 1239,
699. 1H NMR (DMSO-d6) d 1.20–1.40 (m, 2H + 3H, CH2 + CH2CH3),
1.40–1.60 (m, 2H, CH2), 1.60–1.81 (m, 2H, CH2), 2.30 (t, J = 6.8 Hz,
2H, CH2N), 2.38–2.58 (m, 4H, piperazine), 2.85–3.04 (m, 4H, piper-
azine), 3.88–4.10 (m, 4H, CH2CH3 + CONCH2), 6.78–6.95 (m, 4H,
aromatic), 6.97 (s, 1H, NCH), 7.40–7.55 (m, 3H, aromatic), 8.00–
8.15 (m, 2H, aromatic), 8.60 (s, 1H, CCH). Anal. (C27H34N4O2) C,
H, N.
4.1.2. General procedure for the synthesis of 3-[5-(4-substituted-1-
piperazinyl)pentyl]-4(3H)-pyrimidinones (4–8) and 4-[5-(4-
substituted-1-piperazinyl)pentoxy]-pyrimidines (9–13)
Acetonitrile (4 mL) was added to a mixture of chloroderivative 2
or 3 (0.90 mmol), properly substituted piperazine (1.08 mmol),
sodium carbonate (1.08 mmol), and a catalytic amount of potas-
sium iodide. The mixture was refluxed under stirring for 9 h. After
being cooled, the solid was removed by filtration and the solution
was concentrated to dryness. The following new compounds were
obtained using this procedure.
4.1.8. 4-[5-(4-Phenyl-1-piperazinyl)pentoxy]-6-phenyl-pyrimidine (9)
The title compound was purified by flash chromatography ethyl
acetate as eluent (53%), mp 70.0–70.4 °C. IR (KBr, selected lines)
cmÀ1 2946, 2817, 1592, 1360, 1227, 1006, 756, 689. 1H NMR
(DMSO-d6) d 1.35–1.65 (m, 2H + 2H, CH2CH2), 1.70–1.90 (m, 2H,
CH2), 2.28–240 (m, 2H, CH2N), 2.40–2.60 (m, 4H, piperazine),
3.02–3.18 (m, 4H, piperazine), 4.41 (t, J = 6.4 Hz, 2H, OCH2), 6.70–
6.81 (m, 1H, aromatic), 6.83–6.98 (m, 2H, aromatic), 7.18–7.28
(m, 2H, aromatic), 7.48–7.60 (m, 1H + 3H, NCH + aromatic), 8.15–
8.25 (m, 2H, aromatic), 8.85 (d, J = 1.8 Hz, 1H, CCH). Anal.
(C25H30N4O) C, H, N.
4.1.3. 3-[5-(4-Phenyl-1-piperazinyl)pentyl]-6-phenyl-4(3H)-
pyrimidinone (4)
The title compound was obtained by recrystallization from ace-
tonitrile (51%), mp 139.6–140.4 °C. IR (KBr, selected lines) cmÀ1
2929, 2823, 1665, 1592, 1451, 1239, 750, 695. 1H NMR (CDCl3) d
1.30–1.58 (m, 2H, CH2), 1.60–1.95 (m, 2H + 2H, CH2CH2), 2.60 (t,
J = 7.2 Hz, 2H, CH2N), 2.70–2.95 (m, 4H, piperazine), 3.20–3.45
(m, 4H, piperazine), 3.98 (t, J = 7.4 Hz, 2H, CONCH2), 6.82–6.98
(m, 3H + 1H, aromatic + NCH), 7.20–7.34 (m, 2H, aromatic), 7.42–
7.52 (m, 3H, aromatic), 7.88–8.10 (m, 2H, aromatic), 8.19 (s, 1H,
CCH). Anal. (C25H30N4O) C, H, N.
4.1.9. 4-[5-[4-(Phenylmethyl)-1-piperazinyl]pentoxy]-6-phenyl-
pyrimidine (10)
4.1.4. 3-[5-[4-(Phenylmethyl)-1-piperazinyl]pentyl]-6-phenyl-4(3H)-
pyrimidinone (5)
The title compound was purified by flash chromatography using
ethyl acetate and then a mixture of ethyl acetate/methanol
(9.5:0.5, v/v)) as eluents (46%), mp 63.8–64.3 °C. IR (KBr, selected
lines) cmÀ1 2941, 2810, 1578, 1541, 1345, 1214, 1005, 696. 1H
NMR (DMSO-d6) d 1.30–1.58 (m, 2H + 2H, CH2CH2), 1.68–1.82 (m,
2H, CH2), 2.25 (t, J = 6.6 Hz, 2H, CH2N), 2.30–2.42 (m, 8H,
piperazine), 3.41 (s, 2H, CH2C6H5), 4.38 (t, J = 6.6 Hz, 2H, OCH2),
7.18–7.38 (m, 5H, aromatic), 7.49 (s, 1H, NCH), 7.50–7.60 (m, 3H,
aromatic), 8.18–8.22 (m, 2H, aromatic), 8.83 (d, J = 0.8 Hz, 1H,
CCH). Anal. (C26H32N4O) C, H, N.
The title compound was purified by flash chromatography using
a mixture of ethyl acetate/methanol (8:2, v/v) as eluent (26%), mp
106.2–106.5 °C. IR (KBr, selected lines) cmÀ1 2947, 2802, 1673,
1596, 1451, 688. 1H NMR (DMSO-d6) d 1.19–1.55 (m, 2H + 2H,
CH2CH2), 1.60–1.79 (m, 2H, CH2), 2.22 (t, J = 7.4 Hz, 2H, CH2N),
2.30–2.42 (m, 8H, piperazine), 3.39 (s, 2H, CH2C6H5), 3.90
(t, J = 7.0 Hz, 2H, CONCH2), 6.96 (s, 1H, NCH), 7.18–7.38 (m, 5H,
aromatic), 7.42–7.55 (m, 3H, aromatic), 8.00–8.15 (m, 2H,
aromatic), 8.58 (s, 1H, CCH). Anal. (C26H32N4O) C, H, N.
Please cite this article in press as: Intagliata S., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2016.12.039

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S. Intagliata et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
4.1.10. 4-[5-[4-(3-Chlorophenyl)-1-piperazinyl]pentoxy]-6-phenyl-
pyrimidine (11)
4.1.14. General procedure for the synthesis of 2-methyl-3-[5-(4-
substituted-1-piperazinyl)pentyl]-4(3H)-quinazolinones (17–20) and
2-methyl-4-[5-(4-substituted-1-piperazinyl)pentoxy]-quinazolines
(21, 22)
Acetonitrile (4 mL) was added to a mixture of bromoderivative
15 or 16 (0.73 mmol), properly substituted piperazine
(0.88 mmol), sodium carbonate (0.88 mmol), and of a catalytic
amount of potassium iodide. The mixture and a magnetic bar were
sealed in a Pyrex tube and were heated at 90 °C by microwave irra-
diation for 1 h (run time 3 min, microwave max power 150 W and
max pressure 150 psi). After being cooled, the solid was removed
by filtration and the solution was concentrated to dryness. The fol-
lowing new compounds were obtained using this procedure:
The title compound was purified by flash chromatography using
ethyl acetate as eluent to obtain an oil (64%). IR (neat, selected
lines) cmÀ1 2940, 2818, 1591, 1461, 1352, 1237, 987, 758, 694.
1H NMR (CDCl3) d 1.42–1.61 (m, 2H, CH2), 1.63–1.98 (m, 2H + 2H,
CH2CH2), 2.58 (t, J = 7.6 Hz, 2H, CH2N), 2.70–2.90 (m, 4H, piper-
azine), 3.25–3.43 (m, 4H, piperazine), 4.42 (t, J = 6.6 Hz, 2H,
CONCH2), 6.72–6.90 (m, 3H, aromatic), 7.08–7.24 (m, 1H + 1H,
NCH + aromatic), 7.42–7.53 (m, 3H, aromatic), 7.95–8.08 (m, 2H,
aromatic), 8.82 (d, J = 1.0 Hz, 1H, CCH). Anal. (C25H29ClN4O) C, H, N.
4.1.11. 4-[5-[4-(4-Chlorophenyl)-1-piperazinyl]pentoxy]-6-phenyl-
pyrimidine (12)
4.1.15. 2-Methyl-3-[5-(4-phenyl-1-piperazinyl)pentyl]-4(3H)-
quinazolinone (17)
The title compound was obtained by recrystallization from ace-
tonitrile (19%), mp 104.2–104.5 °C. IR (KBr, selected lines) cmÀ1
2943, 1590, 1496, 1354, 1236, 1003, 811. 1H NMR (DMSO-d6) d
1.38–1.61 (m, 2H + 2H, CH2CH2), 1.70–1.85 (m, 2H, CH2), 2.32 (t,
J = 6.8 Hz, 2H, CH2N), 2.41–2.49 (m, 4H, piperazine), 3.02–3.15
(m, 4H, piperazine), 4.40 (t, J = 6.6 Hz, 2H, OCH2), 6.85–6.95 (m,
2H, aromatic), 7.18–7.25 (m, 2H, aromatic), 7.44–7.58 (m, 1H
+ 3H, NCH + aromatic), 8.16–8.23 (m, 2H, aromatic), 8.84 (d,
J = 1.0 Hz, 1H, CCH). Anal. (C25H29ClN4O) C, H, N.
The crude product was purified by flash chromatography using
ethyl acetate, a mixture of ethyl acetate/methanol (9:1, v/v), and
then ethyl acetate/methanol (8:2, v/v) as eluents (37%), mp 78.0–
79.0 °C. IR (KBr, selected lines) cmÀ1 2934, 2820, 1678, 1600,
1476, 1402, 1240, 780, 762. 1H NMR (DMSO-d6) d 1.35–1.78 (m,
2H + 2H + 2H, CH2CH2CH2), 2.32 (t, J = 6.8 Hz, 2H, NCH2), 2.41–
2.58 (m, 4H, piperazine), 2.62 (s, 3H, CH3), 3.03–3.17 (m, 4H,
piperazine), 4.04 (t, J = 7.4 Hz, 2H, CONCH2), 6.71–6.82 (m, 1H,
aromatic), 6.86–6.98 (m, 2H, aromatic), 7.14–7.26 (m, 2H, aro-
matic), 7.41–7.53 (m, 1H, aromatic), 7.53–7.62 (m, 1H, aromatic),
7.72–7.84 (m, 1H, aromatic), 8.05–8.14 (m, 1H, aromatic). Anal.
(C24H30N4O) C, H, N.
4.1.12. 4-[5-[4-(2-Ethoxyphenyl)-1-piperazinyl]pentoxy]-6-phenyl-
pyrimidine (13)
The title compound was purified by flash chromatography using
ethyl acetate as eluent to obtain an oil (27%). IR (neat, selected
lines) cmÀ1 2939, 2814, 1590, 1499, 1455, 1239, 737. 1H NMR
(CDCl3) d 1.40–1.63 (m, 2H + 3H, CH2 + CH2CH3), 1.63–1.98 (m,
2H + 2H, CH2CH2), 2.65 (t, J = 6.2 Hz, 2H, CH2N), 2.78–3.08 (m,
4H, piperazine), 3.15–3.42 (m, 4H, piperazine), 4.06 (q, J = 6.8 Hz,
2H, CH2CH3), 4.42 (t, J = 6.6 Hz, 2H, OCH2), 6.80–7.08 (m, 4H, aro-
matic), 7.11 (s, 1H, NCH), 7.42–7.60 (m, 3H, aromatic), 7.98–8.15
(m, 2H, aromatic), 8.82 (d, J = 1.0 Hz, 1H, 1H, CCH). Anal.
(C27H34N4O2) C, H, N.
4.1.16. 3-[5-[4-(3-Chlorophenyl)-1-piperazinyl]pentyl]-2-methyl-4
(3H)-quinazolinone (18)
The crude product was purified by flash chromatography using
ethyl acetate, a mixture of ethyl acetate/methanol (9:1, v/v), and
then ethyl acetate/methanol (8:2, v/v) as eluents (13%), mp
113.0–114.8 °C. IR (KBr, selected lines) cmÀ1 2928, 2815, 1678,
1595, 1471, 1462, 1393, 1243, 770. 1H NMR (DMSO-d6) d 1.25–
1.78 (m, 2H + 2H + 2H, CH2CH2CH2), 2.31 (t, J = 6.8 Hz, 2H, NCH2),
2.40–2.55 (m, 4H, piperazine), 2.61 (s, 3H, CH3), 3.05–3.20 (m,
4H, piperazine), 4.03 (t, J = 7.0 Hz, 2H, CONCH2), 6.72–6.81 (m,
1H, aromatic), 6.81–6.94 (m, 2H, aromatic), 7.12–7.25 (m, 1H, aro-
matic), 7.41–7.60 (m, 2H, aromatic), 7.70–7.82 (m, 1H, aromatic),
8.04–8.15 (m, 1H, aromatic). Anal. (C24H29ClN4O) C, H, N.
4.1.13. General procedure for the preparation of 3-(5-bromopentyl)-2-
methyl-4(3H)-quinazolinone (15) and 4-(5-bromopenthoxy)-2-
methylquinazoline (16)
Acetonitrile (4 mL) was added to mixture of compound 14
(1.55 mmol), 1,5-dibromopentane (4.67 mmol), potassium carbon-
ate (2.32 mmol), and of a catalytic amount of potassium iodide. The
mixture and a magnetic bar were sealed in a Pyrex tube and were
heated at 90 °C by microwave irradiation for 90 min (run time
3 min, microwave max power 150 W and max pressure 150 psi).
After being cooled, the solid was removed by filtration and the
solution was concentrated to dryness. From the obtained residue,
compound 15 as a solid and compound 16 as an oil were isolated
by flash chromatography using ethyl acetate/cyclohexane (5:5, v/
v) as eluent.
Compound 15: yield 47%, mp 52.0–54.6 °C. IR (KBr, selected
lines) cmÀ1 3052, 1671, 1626, 1467, 1424, 1265, 737. 1H NMR
(DMSO-d6) d 1.40–1.58 (m, 2H, CH2), 1.58–1.78 (m, 2H, CH2),
1.78–1.95 (m, 2H, CH2), 2.61 (s, 3H, CH3), 3.56 (t, J = 6.6 Hz, 2H,
CH2Br), 4.03 (t, J = 7.2 Hz, 2H, CONCH2), 7.42–7.52 (m, 1H, aro-
matic), 7.52–7.60 (m, 1H, aromatic), 7.72–7.83 (m, 1H, aromatic),
8.05–8.12 (m, 1H, aromatic).
4.1.17. 3-[5-[4-(4-Chlorophenyl)-1-piperazinyl]pentyl]-2-methyl-4
(3H)-quinazolinone (19)
The crude product was recrystallized from water (49%), mp
101.2–102.2 °C. IR (KBr, selected lines) cmÀ1 2937, 1678, 1597,
1499, 1467, 1394, 1357, 1240, 768. 1H NMR (CDCl3) d 1.25–1.78
(m, 2H + 2H + 2H, CH2CH2CH2), 2.31 (t, J = 7.2 Hz, 2H, NCH2),
2.40–2.55 (m, 4H, piperazine), 2.61 (s, 3H, CH3), 3.02–3.15 (m,
4H, piperazine), 4.03 (t, J = 7.8 Hz, 2H, CONCH2), 6.91 (d,
J = 9.2 Hz, 2H, aromatic), 7.21 (d, J = 8.8 Hz, 2H, aromatic), 7.40–
7.52 (m, 1H, aromatic), 7.52–7.62 (m, 1H, aromatic), 7.70–7.82
(m, 1H, aromatic), 8.04–8.15 (m, 1H, aromatic). Anal. (C24H29ClN4O)
C, H, N.
4.1.18. 3-[5-[4-(2-Ethoxyphenyl)-1-piperazinyl]pentyl]-2-methyl-4
(3H)-quinazolinone (20)
The crude product was purified by flash chromatography using
ethyl acetate, a mixture of ethyl acetate/methanol (9:1, v/v), and
then ethyl acetate/methanol (8:2, v/v) as eluents, obtaining com-
pound 10 as a pure oil (12%). IR (neat, selected lines) cmÀ1 2940,
1671, 1594, 1500, 1474, 1394, 1266, 1241, 736. 1H NMR (CDCl3)
d 1.39–1.60 (m, 2H + 3H, CH2 + CH2CH3), 1.71–1.95 (m, 2H + 2H,
CH2CH2), 2.65 (s, 3H, CH3), 2.70 (t, J = 7.6 Hz, 2H, NCH2),
2.80–3.10 (m, 4H, piperazine), 3.25–3.45 (m, 4H, piperazine),
Compound 16: yield 23%, IR (neat, selected lines) cmÀ1 1619,
1577, 1503, 1434, 1369, 1318, 1168, 1112, 782. 1H NMR (DMSO-
d6) d 1.54–1.68 (m, 2H, CH2), 1.78–1.99 (m, 2H + 2H, CH2CH2),
2.61 (s, 3H, CH3), 3.58 (t, J = 6.8 Hz, 2H, CH2Br), 4.53 (t, J = 6.4 Hz,
2H, OCH2), 7.53–7.65 (m, 1H, aromatic), 7.76–7.95 (m, 2H, aro-
matic), 8.05–8.13 (m, 1H, aromatic). Anal. (C14H17BrN2O) C, H, N.
Please cite this article in press as: Intagliata S., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2016.12.039

S. Intagliata et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
3.98–4.20 (m, 2H + 2H, CONCH2 + CH2CH3), 6.80–7.10 (m, 4H, aro-
matic), 7.38–7.50 (m, 1H, aromatic), 7.55–7.62 (m, 1H, aromatic),
7.62–7.80 (m, 1H, aromatic), 8.18–8.27 (m, 1H, aromatic). Anal.
(C26H34N4O2) C, H, N.
maintained at 37 °C in a humidified atmosphere with 5% CO2 and
were grown in Dulbeco’s Modifier Eagle Medium containing 10%
dialysed fetal bovine serum and 500 lg/mL G418 sulphate. For
functional experiments, cells were subcultured in 25 cm2 diameter
dishes, grown to 90% confluence, washed twice with prewarmed to
37 °C phosphate buffered saline (PBS) and were centrifuged for
5 min (160g). The supernatant was aspirated, the cell pellet was
resuspended in stimulation buffer (1 Â HBSS, 5 mM HEPES,
0.5 mM IBMX, 0.1% BSA).
4.1.19. 2-Methyl-4-[5-(4-phenyl-1-piperazinyl)pentoxy]-quinazoline
(21)
The crude product was recrystallized from water (26%), mp 64.9–
66.5 °C. IR (KBr, selected lines) cmÀ1 2960, 2833, 1575, 1500, 1422,
1356, 1239, 1163, 1112, 777. 1H NMR (DMSO-d6) d 1.42–1.70 (m,
2H + 2H, CH2CH2), 1.75–1.95 (m, 2H, CH2), 2.33 (t, J = 6.4 Hz, 2H,
NCH2), 2.40–2.55 (m, 4H, piperazine), 2.61 (s, 3H, CH3), 3.00–3.15
(m, 4H, piperazine), 4.53 (t, J = 6.4 Hz, 2H, OCH2), 6.70–6.81 (m,
1H, aromatic), 6.83–6.96 (m, 2H, aromatic), 7.12–7.25 (m, 2H, aro-
matic), 7.53–7.62 (m, 1H, aromatic), 7.75–7.96 (m, 2H, aromatic),
8.02–8.18 (m, 1H, aromatic). Anal. (C24H30N4O) C, H, N.
The functional properties of compounds 13 and 20 at 5-HT1AR
and 5-HT7R were evaluated in a simplified antagonist assays, using
LANCE Ultra cAMP detection kit (PerkinElmer). In a case of
5-HT1AR, cells were stimulated with 1
l
M of forskolin (EC90) and
to determine the
1
lM
of agonist (R)-(+)-8-OH-DPAT (EC90)
antagonist induced increase in cellular cAMP levels; for 5-HT7R,
cells were stimulated with 10 nM of agonist 5-CT (EC90) and the
antagonist induced decrease in cellular production of cAMP were
measured. Each compound was tested in triplicate at the one
concentration 10À6 M.
4.1.20. 4-[5-(4-(4-Chlorophenyl)-1-piperazinyl)pentoxy]-2-methyl-
quinazoline (22)
The crude product was recrystallized from acetonitrile (12%),
mp 109.0–111.9 °C. IR (KBr, selected lines) cmÀ1 2941, 1578,
1497, 1424, 1351, 1163, 1109, 768. 1H NMR (CDCl3) d 1.48–1.70
(m, 2H, CH2), 1.70–2.00 (m, 2H + 2H, CH2CH2), 2.63 (t, J = 7.4 Hz,
2H, NCH2), 2.71 (m, 3H, CH3), 2.75–2.90 (m, 4H, piperazine),
3.25–3.39 (m, 4H, piperazine), 4.56 (t, J = 6.4 Hz, 2H, OCH2), 6.78–
6.90 (m, 2H, aromatic), 7.15–7.30 (m, 2H, aromatic), 7.42–7.58
(m, 1H, aromatic), 7.73–7.87 (m, 2H, aromatic), 8.05–8.18 (m, 1H,
aromatic). Anal. (C24H29ClN4O) C, H, N.
For quantification of cAMP levels, cells (5
lL) were incubated
with 5 L mixture of compounds (tested ligand, (R)-(+)-8-OH-
l
DPAT, and forskolin for 5-HT1AR or tested ligand and 5-CT for
5-HT7R) for 30 min at room temperature in 384-well white
opaque microtiter plate (PerkinElmer). After incubation, the
reaction was stopped and cells were lysed by the addition of
10 lL working solution (5 lL Eu-cAMP and 5 lL ULight-anti-
cAMP). The assay plate was incubated for 1 h at room temper-
ature. Time-resolved fluorescence resonance energy transfer
(TRFRET) signal was detected by an Infinite M1000 Pro (Tecan)
using instrument settings from LANCE Ultra cAMP detection kit
manual.
4.2. In vitro binding assays
Binding assays were performed using human cloned 5-HT7(a)
and 5-HT1A serotonin receptors (PerkinElmer) expressed on CHO-
K1 cells. Radioligand binding assays were carried out using the
condition reported on technical data sheet with some modifica-
tions. Briefly, 5-HT7(a) receptors41 were resuspended in Tris HCl
50 mM pH 7.4 containing 4 mM MgCl2 and incubated for 40 min
4.4. Molecular modeling
The building of homology models of 5-HT1A and 5-HT7 recep-
tors, validation of these models, and ligand-directed optimization
of the binding sites were previously described.39 The 5-HT1A and
5-HT7 receptor models selected in Induced Fit Docking (IFD) proce-
dure were used to study the binding mode of the synthesized
ligands. LigPrep was used to prepare the structures of the
molecule45 and Epik to assign the appropriate ionization states at
pH = 7.4.46 Docking was performed by using Glide at SP level.47
The spatial constrain was imposed on the creation of an ionic inter-
action between the protonated amine group of the ligand and the
Asp3.32 side chain. Next, ligand-receptor complexes were ana-
lyzed, and only those models were kept for which coherent, for
the whole set of compounds, and closest compliance with common
binding mode for monoaminergic receptor ligands was observed.48
Final figures of the docking pose in both receptors were generated
using PyMOL.49
at 27 °C in a final volume of 0.51 mL, consisting of 250
brane suspension (15 g protein/sample), 250
L of [3H]-5-HT
(final concentration 5 nM, s.a. 106 Ci/mmol, PerkinElmer) prepared
in the same buffer used for membrane suspension and 10 L of
tested compounds. Nonspecific binding was obtained in the pres-
ence of 10 M serotonin.
For 5-HT1A binding assay,42 receptors were resuspended in Tris
HCl 50 mM pH 7.4 containing 4 mM CaCl2 and incubated (10 g pro-
lL of mem-
l
l
l
l
l
tein/sample) for 1 h at 27 °C in the same volume using for 5-HT7(a)
receptors but in presence of [3H]-8-OH-DPAT (final concentration
1 nM, s.a. 137 Ci/mmol, PerkinElmer). Nonspecific binding was
obtainedinpresenceof10 lMserotoninand,forbothbindingassays,
areferencedrugwastested.Incubationswerestoppedbyrapidfiltra-
tion under vacuum, through GF/C filters (pre-soaked with 0.3% PEI)
andwashedwith12 mL(4 Â 3times)ofice-coldwashingbuffer(Tris
HCl, 50 mM, pH 7.4) using a Brandel M-48R cell harvester. The
radioactivity trapped on the filters was counted in 4 mL of Ultima
Gold MV (Packard) in a Tri-carb 2800 TR (PerkinElmer) liquid scintil-
lationspectrometerwithacountingefficiencyof60%.Allcompounds
were tested in a concentration range from 10À5 to 10À10 M in tripli-
cate and dose-inhibition curves were analyzed by the ‘‘Allfit” pro-
gram to obtain the concentration of unlabeled drug that caused 50%
inhibition of ligand binding.43 The Ki values were derived from IC50
values according to the Cheng and Prusoff equation.44
Acknowledgments
We would like to thank Prof. A.J. Bojarski, Department of Medic-
inal Chemistry, Institute of Pharmacology, Polish Academy of
Sciences, for his aid with molecular modeling studies and cAMP
assay, and Mr. Grzegorz Satała for performing the cAMP assay.
This work was supported by grants from the Italian MIUR and
the University of Catania, Italy.
4.3. Functional cAMP assay protocol
A. Supplementary material
HEK293 cell lines with stable expression of human 5-HT1AR and
5-HT7R (prepared with the use of Lipofectamine 2000) were
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.12.039.
Please cite this article in press as: Intagliata S., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2016.12.039

10
S. Intagliata et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
24. Modica MN, Intagliata S, Pittalà V, et al. Bioorg Med Chem Lett. 2015;25:1427.
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Please cite this article in press as: Intagliata S., et al. Bioorg. Med. Chem. (2017), http://dx.doi.org/10.1016/j.bmc.2016.12.039