Synthesis of 1-naphtylpiperazine derivatives as serotoninergic ligands and their evaluation as antiproliferative agents

Article abstract of DOI:10.1016/j.ejmech.2011.03.001

Serotonin (5-hydroxytryptamine, 5-HT) is one of the most important neuromediator involved in numerous physiological and pathophysiological processes. In addition it is well established that 5-HT acts as a growth factor on several types of non-tumoral and tumoral cells, and recently it was also related to oncogenes. 5-HT_1A receptor expression was identified in prostatic tumor cell lines (PC3 cells) and in human hormone refractory prostate cancer tissue. Based on these observations, development of 5-HT_1A antagonists could be useful in inhibiting the growth of cancer cells. In order to investigate on potential use of 5-HT_1A ligands as antiproliferative agents, we have analyzed a new set of 1-naphtylpiperazine derivatives. In binding studies, several molecules showed affinity in nanomolar and subnanomolar range at 5-HT_1A and moderate to no affinity for other relevant receptors (5-HT_2A, 5-HT_2C, D₁, D₂, α₁ and α₂). All compounds were then evaluated in order to assess their antiproliferative activity using PC3 cells and the most active compounds (1 and 2) were fully characterized to define the mechanism responsible for the observed antiproliferative effect.

Full text of DOI:10.1016/j.ejmech.2011.03.001

European Journal of Medicinal Chemistry 46 (2011) 2206e2216
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of 1-naphtylpiperazine derivatives as serotoninergic ligands and their
evaluation as antiproliferative agents
a
a
a
a
a
Ferdinando Fiorino , Elisa Magli , Elisa Perissutti , Beatrice Severino , Francesco Frecentese ,
a
a
a
b
Antonella Esposito , Francesca De Angelis , Giuseppina Maria Incisivo , Paola Massarelli ,
b
c
c
c
a
Cristina Nencini , Elena Di Gennaro , Alfredo Budillon , Alessandra Di Cintio , Vincenzo Santagada ,
a,
*
Giuseppe Caliendo
a
Dipartimento di Chimica Farmaceutica e Tossicologica, Università di Napoli «Federico II», Via D. Montesano 49, 80131 Naples, Italy
Dipartimento di Scienze Neurologiche, Neurochirurgiche e del Comportamento, Università di Siena, Via delle Scotte, 6, 53100 Siena, Italy
Unità di Farmacologia Sperimentale, Istituto Nazionale dei Tumori Fondazione “G. Pascale”, Via M. Semmola, 80131 Naples, Italy
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 August 2010
Received in revised form
Serotonin (5-hydroxytryptamine, 5-HT) is one of the most important neuromediator involved in
numerous physiological and pathophysiological processes. In addition it is well established that 5-HT acts
as a growth factor on several types of non-tumoral and tumoral cells, and recently it was also related to
oncogenes. 5-HT1A receptor expression was identified in prostatic tumor cell lines (PC3 cells) and in
human hormone refractory prostate cancer tissue. Based on these observations, development of 5-HT1A
antagonists could be useful in inhibiting the growth of cancer cells. In order to investigate on potential
use of 5-HT1A ligands as antiproliferative agents, we have analyzed a new set of 1-naphtylpiperazine
derivatives. In binding studies, several molecules showed affinity in nanomolar and subnanomolar range
11 February 2011
Accepted 1 March 2011
Available online 8 March 2011
Keywords:
Naphtylpiperazine
1 2 1 2
at 5-HT1A and moderate to no affinity for other relevant receptors (5-HT2A, 5-HT2C, D , D , a and a ). All
5-HT1A ligands
Antiproliferative agents
PC3 cells
compounds were then evaluated in order to assess their antiproliferative activity using PC3 cells and the
most active compounds (1 and 2) were fully characterized to define the mechanism responsible for the
observed antiproliferative effect.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
identified, based on functional, structural, and pharmacological
3
characterization. With the exception of 5-HT , which is a ligand-
Serotonin (5-hydroxytryptamine, 5-HT) has been the subject of
intense biological research since its synthesis in 1951 [1]. Among
several mediators released by neuroendocrine cells, 5-HT is one of
the most important targets for medicinal chemistry because of its
involvement in numerous physiological and pathophysiological
processes [2e5]. Serotonin receptors (5-HTRs) may be involved in
impulsivity and alcoholism [6,7], and in the different phases of sleep
gated ion channel, all of these are known to be G-protein coupled
receptors (GPCRs) with structural, pharmacological, and trans-
ductional characteristics [12]. Besides the already known involve-
ment of 5-HTRs in the regulation of several psychiatric and
neurological disorders related to serotoninergic neurotransmission,
specific receptor subtypes have been recently associated with tumor
growth. In particular, 5-HT1A receptor is the most widely studied
subtype even if signal transduction of this receptor is complex and
the exact mechanism of action is yet to be determinated. 5-HT1A
receptor, as a member of the group of GPCRs [13], shows high
similarity with other members of the family; in particular it shows
[
8], sexual behavior, appetite control, thermoregulation, and
cardiovascular function [9,10]. In addition it is well established that
-HT plays a fundamental role in regulation of growth, differentia-
5
tion, andgeneexpression [11]. Moreover serotoninis knowntoact as
a growth factor on several types of non-tumoral and tumoral cells,
and recently it was also related to oncogenes [11]. 5-HTRs comprise
a large and complex family of receptors. Seven classes have been
a high degree of homology (45%) with
1
a -adrenoreceptor [14]. In
addition to its effects on a wide range of psychiatric disorders, 5-
HT1AR is also involved in the proliferation of human tumor cells. In
fact 5-HTRs expression has been identified in PC3 cells and in human
hormone refractory prostate cancer tissue [15]. Based on these
observations, as already reported, the development of 5-HT1A
antagonists and/or serotonin-uptake inhibitors could be useful to
*
Corresponding author. Tel./fax: þ39 81 678649.
E-mail address: caliendo@unina.it (G. Caliendo).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.001

F. Fiorino et al. / European Journal of Medicinal Chemistry 46 (2011) 2206e2216
2207
4(3H)-one with 1-bromo-2-chloroethane or 1-bromo-3-chlor-
opropane, in presence of K CO in DMF, gave the corresponding
2
3
chloroalkoxy benzotriazinone derivatives (4a and 4b). The obtained
intermediates were condensed with 1-naphtylpiperazine in DMF in
presence of K CO , NaI, under reflux to give the final products 5 and 6.
2 3
Compounds 9 and 10 were synthesized following the general proce-
dure summarized in Scheme 2. Alkylation of the starting heterocycle
endo-N-hydroxy-5-norbornene-2,3-dicarboximide with 1-bromo-
2-chloroethane or 1-bromo-3-chloropropane, inpresence of NaOH in
absolute ethanol, gave the corresponding chloro-alkyl norbornene
derivatives 8a and 8b. Subsequent condensation of intermediates 8a
and 8b with 1-naphtylpiperazine, performed in CH
CO and NaI, under reflux, provided the final compounds 9 and 10,
respectively.
The synthetic strategy employed for the preparation of the
3
CN in presence of
K
2
3
0
N -cyano-N-(3-(4-(naphthalen-1-yl)piperazin-1-yl)propyl)isoni-
cotinamidine (14) is summarized in Scheme 3: isopropyl-
N-cyano-4-pyridinecarboximidate (12) was prepared starting
from 4-cyanopyridine (11) by base-promoted reaction with iso-
propanol followed by treatment with cyanamide in aqueous
Fig. 1. General structures.
inhibit the growth of prostatic tumor cell lines (PC3) [11,15]. Our
research group has been involving for a long time in developing new
-HT1A ligands [16e23] in order to have novel pharmacological tools
that could improve our knowledge of the 5-HT signal transduction
mechanism and lead to compounds with high affinity and selec-
tivity. In continuation of our research program and in order to shed
more light on potential use of 5-HT1A ligands as antiproliferative
agents, we have analyzed a new set of derivatives where a 1-naph-
5
phosphate buffer (NaH
2
PO
4
$2H
2
O:Na
2
HPO
4
¼ 4:1). Compound
0
(
12) was converted to N-(3-bromopropyl)-N -cyanoisonicotina-
midine (13) by reaction with 3-bromopropylamine$HBr and
NaOMe in methanol. Subsequent condensation of compound
(
13) with 1-naphtylpiperazine, performed in CH
3
CN in presence
of K CO and NaI, under reflux, provided the final compound 14.
2
3
tylpiperazine moiety has been linked to a 1,2,3-benzotriazinone,
-hydroxy-1,2,3-benzotriazione, norbornene, N -cyanoisonicotina-
0
Finally the synthesis of the N -cyano-N-(3-(4-(naphthalen-1-yl)
piperazin-1-yl)propyl)picolinamidine (15) is summarized in
Scheme 4: methyl-N-cyano-2-pyridinecarboximidate (16) was
prepared starting from 2-cyanopyridine (15) by base-promoted
reaction with anhydrous methanol followed by treatment with
0
3
0
midine and N -cyanopicolinamidine fragment respectively. These
nuclei were linked via two or three methylene spacing units to the 1-
naphtylpiperazine moiety (Fig. 1). The naphtylpiperazine scaffold
was selected on the basis of previous investigations demonstrating
how 1-naphtylpiperazines, acting as 5-HT2A receptor antagonists,
were also able to block skin cancer induction, probably because of an
apoptotic mechanism [24]. Instead the choice of the different nuclei
was performed on the basis of our previous investigations [16e23],
where, as a general trend, different compounds with a 1,2,3-ben-
cyanamide in aqueous phosphate buffer (NaH
Na HPO
¼ 4:1). Compound (16) was converted to N-(3-bro-
mopropyl)-N -cyanopicolinamidine (17) by reaction with
-bromopropylamine$HBr and triethylamine in anhydrous
methanol. Subsequent condensation of compound (17) with
-naphtylpiperazine, performed in CH CN in presence of K CO
and NaI, under reflux, provided the final compound 18.
2 4 2
PO $2H O:
2
4
0
3
1
3
2
3
0
zotriazinone, 3-hydroxy-1,2,3-benzotriazione, norbornene, N -cya-
noisonicotinamidine fragment, linked to different 4-substituted
piperazines through different alkyl chain length, showed good and
preferential affinity for the 5-HT1A receptor. All the new compounds
were initially tested for their affinity on 5-HT1A, 5-HT2A and 5-HT2C
receptors. Moreover, the multireceptor profiles of promising deriv-
atives were also evaluated in terms of binding affinities for dopa-
minergic (D , D ) and adrenergic (a , a ) receptors. Successively all
1 2 1 2
compounds were evaluated for their antiproliferative activity using
prostate cancer derived cell line PC3.
Purification of each final product was performed by chroma-
tography on silica gel column followed by crystallization from the
appropriate solvent. All newcompounds gave satisfactoryelemental
1
analyses and were characterized by H-NMR and mass spectrometry
1
(
API 2000 Applied Biosystem). H-NMR and MS data for all final
compounds were consistent with the proposed structures. As
already reported in literature, due to tautomeric equilibrium about
2
sp carbon, cyanoamidine derivatives 14 and 18 may exist as
amixtureof twotautomers. The NMR spectraof thefinalcompounds
14 and 18, confirmed that these compounds exist predominantly as
2
. Chemistry
the cyanoimino form (I). Moreover, about the configurational
determination of geometrical isomers (E/Z) on the amidine bond
C]N, the N-cyanoamidine derivatives 14 and 18 exhibited only one
set of relevant H-NMR signals, implying the existence of either
a single geometric form or, more probably, a fast equilibrium of two
isomeric forms as already described in literature [23].
The synthesis of compounds 1 and 2 was already reported in
1
a previous paper [21]. The synthetic strategy employed for the prep-
aration of final compounds 5 and 6 is summarized in Scheme 1.
Alkylation of the starting heterocycle 3-hydroxy-1,2,3-benzotriazin-
2 n 2 3 2 3
Scheme 1. Reagents and conditions: (i) Br(CH ) Cl, K CO , DMF; (ii) 1-naphtylpiperazine, K CO , NaI, DMF.

2208
F. Fiorino et al. / European Journal of Medicinal Chemistry 46 (2011) 2206e2216
ꢀ
Scheme 2. Reagents and conditions: (i) Br(CH
2
)
n
Cl, NaOH, absolute EtOH, 70 C, 24 h;
(
ii)1-naphtylpiperazine, K CO , NaI, CH CN, reflux, 24 h.
2
3
3
Scheme 4. Reagents and conditions: (i) CH
3
ONa,
H
2
NCN, anhydrous MeOH; (ii)
CO , NaI, CH CN,
Br(CH
2
)
3
NH
2
, CH
3
ONa, anhydrous MeOH; (iii) 1-naphtylpiperazine, K
2
3
3
3
. Results and discussion
Among the reported naphtylpiperazine derivatives, six
ꢀ
70
C, 4 h.
compounds. Results are summarized in Table 2. All the compounds
compounds (5, 6, 9, 10, 14 and 18) represent new derivatives that
were synthesized and fully evaluated for affinity and selectivity on
proved highly selective against dopaminergic receptors with K
values of above 10 nM except for compound 6, which exhibited K
i
i
4
5
-HT1A, 5-HT2A and 5-HT2C receptors and successively tested as
2 1 2
values of 85.4 nM on D receptor. Regarding a and a adrenergic
receptors, only compounds 1 and 10 showed quite moderate
affinities (112 and 189 nM respectively). The high selectivity toward
a receptors, exhibited by 1-naphtylpiperazine derivatives, is very
1
interesting considering that the amino acid sequence of the
transmembrane part of 5-HT1AR is highly homologous to that of the
antiproliferative agents. Other two derivatives (1 and 2) were
already reported in our previous paper [21] as potent and selective
5
-HT1A receptor ligands and now they were selected in order to
investigate the antiproliferative activity on PC3 cell lines. Intro-
duction of different heterocyclic nuclei as terminal part, slight
modifications concerning the alkyl or alkoxy spacer chain length
a
1
adrenergic receptor.
(
two or three units) are depicted in Fig. 1 and were performed in an
effort to obtain compounds with high affinity and selectivity for
-HT1A over other serotoninergic receptors, as well as dopami-
Growth inhibition assay was performed in order to define the
biological profile of the synthesized compounds. It is well known
that 5-HT1A receptor exhibits a high degree of homology with
ARs ( 1D- and 1B-ARs) [14] that are expressed in PC3 prostate
5
1
a -
nergic and adrenergic receptors. As anticipated on the basis of our
previous investigations, several of these compounds were potent
a
a
cancer cells and are involved in modulation of cell proliferation and
apoptosis [35]. Moreover, it has been demonstrated that 5-HT plays
a fundamental role in regulation of growth, differentiation and
gene expression. On the basis of these observations, all compounds
were examined for their capability to inhibit the growth of PC3
prostate cancer cells. Results, depicted in Fig. 2, showed that four of
the eight analyzed compounds (1, 2, 6, 10 and 18) induced, after
5
-HT1A receptor ligands. In fact, they showed nanomolar or even
subnanomolar 5-HT1A receptor affinities (Table 1). Besides the
already reported [21] 5-HT1A receptor affinity of compound 1
(K
i
¼ 0.000178 nM), most K
i
values were clustered in a relatively
narrow range from 0.293 nM (10) to 7.79 nM (2). Only compounds
5
4
i
, 6 and 14 were inactive with K values > 10 nM. Therefore the
influence of the alkyl chain length, observed for our compounds,
does not seem to be decisive in determining a general trend but the
affinity/selectivity profile is more influenced by the particular
heterocyclic nucleus linked on the naphtylpiperazine moiety.
The 5-HT2A and 5-HT2C receptor affinities of the tested
compounds were always lower than those observed for 5-HT1A
9
6 h of treatment, a dose-dependent growth inhibition of PC3 cells
with IC50 ranging between 10.5 and 23.75 M, as shown in Table 3.
Compound 9 induced growth inhibition but with higher IC50 value
47.7 M), while compounds 5 and 14, but also the 5-HT1A antag-
m
(
m
onist NAN-190 and the 5-HT1A agonist 8-OH-DPAT (data not
shown) did not show antiproliferative effect in these conditions.
Successively, in order to define the mechanism of the anti-
proliferative effect observed for two of the most active compounds
receptors and ranged from 5.29 (1) to 1320 nM (5) for 5-HT2A and
>
4
10 nM or no affinity for 5-HT2C receptors.
Additionally, the affinity of all compounds on several other
(1 and 2), we analyzed their ability to induce apoptosis or cell cycle
receptors (
1 2 1 2
a and a adrenergic and D and D dopaminergic
perturbation. Apoptosis was evaluated by measuring the exter-
nalization of phosphatidylserine (PS) on the outer layer of the
plasma membrane, one of the first characteristic features of
apoptotic cell death, by staining cells with Annexin-V-FITC and
performing flow cytometry analysis. As shown in Fig. 3 of neither 1
receptors) was examined in order to verify the selectivity of these
nor 2 (using for both 15 mM) induced after 24 or 48 h any apoptotic
effect in PC3 cells compared with cells treated with the positive
control represented by the topoisomerase I inhibitor Topotecan
(1
mM).
On the other hand, any significant perturbation of cell cycle was
observed when cells were treated with either 1 or 2. In details both
compounds induced only a slight increase of percentage of cells in
phase G2-M after 24, 48 and 72 h of treatment (Fig. 4aec). However,
similar results were obtained with NAN-190 and 8-OH-DPAT, two
compounds that did not induce any growth inhibition or apoptotic
effects in PC3 cells. On the contrary, Topotecan-treatment induced
after 24 h an increase of cells in G2-M phase and the appearance of
a sub-G1, peak indicating a subpopulation of apoptotic cells, with
Scheme 3. Reagents and conditions: (i) CH
CH ONa, anhydrous MeOH; (iii) 1-naphtylpiperazine, K
3
3
ONa, H
2
NCN, iPrOH; (ii) Br(CH
2
)
3
NH
2
,
ꢀ
2 3
CO , NaI, CH
3
CN, 70 C, 4 h.

F. Fiorino et al. / European Journal of Medicinal Chemistry 46 (2011) 2206e2216
2209
Table 1
Affinities of 1-naphtylpiperazine derivatives for 5-HT1A, 5-HT2A and 5-HT2C receptors.
a
Compd
Substituent
X
Receptor affinity Ki ꢁ SD (nM)
5-HT1A [3H]8-OHDPAT
5-HT2A [3H]Ketanserin
5-HT2C [3H]Mesulergine
O
N
N
N
1
2
5
6
0.000178 ꢁ 0.00003
7.79 ꢁ 0.06
>104
5.29 ꢁ 0.3
>104
O
N
N
127 ꢁ 17
>104
N
O
O
N
N
1320 ꢁ 119
721 ꢁ 33.7
no affinity
no affinity
N
O
N
O
N
N
>104
O
N
O
9
1.58 ꢁ 0.134
0.293 ꢁ 0.025
113 ꢁ 49.8
91.8 ꢁ 5.1
no affinity
no affinity
O
O
N
O
1
1
0
O
CN
N
4
N
H
>104
172 ꢁ 11.5
224 ꢁ 12.8
no affinity
no affinity
N
CN
N
N
18
N
H
5.85 ꢁ 0.187
a
For purpose of comparison, 8-OH-DPAT, Ketanserine and Mesulergine binds 5-HT1A, 5-HT2A and 5-HT2C receptors with values of 0.80, 0.85 and 1.90 nM, respectively,
under these assay conditions.
a complete disruption of cell cycle kinetic and many cells in sub-G1
after 48 and 72 h of treatment.
We demonstrated that 48 h of treatment with compounds 1, 2
and topotecan did not reduce basal MAPK phosphorylation while
all tree agents induced a complete inhibition of AKT phosphoryla-
tion (Fig. 5). The constitutive activation of MAPK signal due to KRAS
mutation in PC3 cells might explain the lack of effect on the
phosphorylation of MAPK. On the other hand the inhibition of AKT
activity, which has been associated with pro-survival signaling in
cancer cells, may explain at least in part the growth inhibitory
activity induced by both compounds 1 and 2. In contrast with data
It has been previously shown that the intracellular signaling by
which serotonin induces mitogenesis is quite complex and involves
the stimulation of several signaling pathways that are only partially
described. Among these pathways MAPK and AKT pathways are
apparently the most relevant to regulate proliferation. On these bases
we evaluated in PC3 cells the effects of 1, 2, NAN-190, 8-OH-DPATand
topotecan on the activation of MAPK and AKT by western blotting.

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F. Fiorino et al. / European Journal of Medicinal Chemistry 46 (2011) 2206e2216
Table 2
Table 3
Affinities of 1-naphtylpiperazine derivatives for D
1
, D
2
,
a
1
and
a
2
receptors.
IC50 values computed at 96 h of treatment (IC5096 h) in
PC3 cell lines.
Compd
Receptor affinity Ki ꢁ SD (nM)
Compounds
IC50 (mM)
D
1
D
2
a
1
a
2
1
2
5
6
10.6 ꢁ 2.06
10.5 ꢁ 1.66
N.D
12.35 ꢁ 1.6
47.7 ꢁ 5.9
13 ꢁ 1.4
N.D
[3
H]SCH-23390
[3H]spiperone
[3H]prazosin
[3H]yohimbine
>104
>104
>104
>104
>10
>10
>10
4
112 ꢁ 47
1880 ꢁ 107
3600 ꢁ 458
1
2
5
6
9
4
562 ꢁ 170
4
4
4
>10
>10
>10
9
4
85.4 ꢁ 0.41
800 ꢁ 6.3
10
14
18
4
4
4
>10
>10
>10
1200 ꢁ 175
189 ꢁ 21.8
878 ꢁ 28.2
10
14
18
>104
>104
>104
263 ꢁ 5.8
>10
>10
>10
4
23.8 ꢁ 2.47
4
1600 ꢁ 137
Note: Mean ꢁ SD of at least three different experiments
4
4
4
>10
>10
done in quadruplicate.
apoptosis or cell cycle perturbation. On these basis we have eval-
uated in PC3 cells the effects of compounds 1 and 2 on the acti-
vation of MAPK and AKT as important signaling pathways, that are
apparently the most relevant to regulate cell proliferation and
directly connected to the signaling pathways of 5-HT1A receptors.
The evidence that compounds 1 and 2 are able to determine inhi-
bition of AKT activity, which has been associated with pro-survival
signaling in cancer cells, may explain the growth inhibitory activity
induced by these derivatives. Finally, further studies could be really
useful to shed more light on the mechanism of action of the
reported by Dizeyi et al. [36], we did not observe any change in the
phosphorylation of MAPK or AKT in cells treated with NAN-190 and
8
-OH-DPAT. This discrepancy can be probably explained by the
different timing of the treatment since the authors evaluated the
effect on the signaling after few hours of treatment while our
experiment was performed at 48 h. Further studies are needed to
better define at molecular level the mechanism of the anti-
proliferative effects we have observed.
4
. Conclusion
We have described the synthesis of new series of 1-naph-
A
tylpiperazine derivatives, containing different heterocyclic frag-
ments, as serotoninergic ligands with an antiproliferative activity
on PC3 cells.
Some of the newly synthesized compounds showed high in vitro
affinity and selectivity toward 5-HT1A receptor. Moreover, the most
interesting derivatives (1, 2, 6, 10 and 18) induced, after 96 h of
treatment, a dose-dependent growth inhibition of PC3 cells with
IC50 values ranging between 10.5 and 23.75
mM, as shown in
Table 3. In order to define the mechanism of the antiproliferative
effects observed for two of the most active compounds (1 and 2) we
have demonstrated as these derivatives are not able to induce
140
120
100
B
80
60
40
20
0
compound 1
compound 2
compound 5
compound 6
compound 9
compound 10
compound 14
compound 18
1
10
100
concentration,
M
Fig. 2. Antiproliferative effect of compounds 1, 2, 5, 6, 9, 10, 14, 18 in PC3 cell lines. PC3
cells were seeded at 900 per well in 96-multiwell and 24 h after 10%-FBS RPMI
medium was replaced with 1%-FBS RPMI medium and compounds were added at the
indicated concentrations. Cell growth assessment was done by sulforhodamine B
colorimetric assay after 96 h of treatment (see Materials and Methods). Cell growth is
expressed as percentage of control for each time point. Points, mean of quadruplicates.
Standard deviation (SD) was always less than 10%.
Fig. 3. Apoptosis was evaluated by flow cytometry analysis with Annexin-V-FITC. 24 h
after seeding of PC3 cells, 10%-FBS RPMI medium was replaced with 1%-FBS RPMI
medium and cells were untreated or treated for 24 h (A) or 48 h (B) with compounds 1,
2, NAN-190, 8-OH-DPAT (15 mM) and Topotecan (2 mM).

F. Fiorino et al. / European Journal of Medicinal Chemistry 46 (2011) 2206e2216
2211
Fig. 4. Cell cycle analysis was performed after PI staining. 24 h after seeding of PC3 cells, 10%-FBS RPMI medium was replaced with 1%-FBS RPMI medium and cells were untreated
or treated with compounds 1, 2, NAN-190, 8-OH-DPAT (15 M) and Topotecan (2 M) for 24 h (A), 48 h (B) or 72 h (C).
m
m

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F. Fiorino et al. / European Journal of Medicinal Chemistry 46 (2011) 2206e2216
concentrated and chromatographed on silica gel column (diethyl
ether/methanol, 9:1 v/v) to give compounds 4a and 4b as solids
1
(
obtained yields: 4a 80%; 4b 94%). H-NMR spectra for all inter-
mediates were consistent with the proposed structures.
5
.1.3. General procedure for preparation of 3-[2 or 3-(4-
naphthalen-piperazin-1-yl)-alkoxy]-1,2,3-benzotriazin-4(3H)-one
5e6)
A mixture of appropriate 3-(n-chloroalkoxy)-1,2,3-benzotriazin-
(3H)-one 4a or 4b (0.03 mol) and NaI (0.05 mol) in DMF (50 mL)
was stirred under reflux for 30 min. Then 1-naphtylpiperazine
0.03 mol) and K CO (0.05 mol) were added. The reaction mixture
(
4
(
2
3
was stirred under reflux for 24 h. After cooling, the mixture was
concentrated to dryness and the residue was dissolved in water
(50 mL). The solution was extracted several times with CH
2 2
Cl . The
organic phase was dried on anhydrous Na SO , concentrated and
2
4
chromatographed on silica gel column (diethyl ether/ethanol 9:1
v/v) to give the final compounds (5e6) as white crystalline solids.
5.1.4. 3-(2-(4-(naphthalen-1-yl)piperazin-1-yl)ethoxy)benzo[d]
[
1,2,3]triazin-4(3H)-one (5)
Fig. 5. MAPK and AKT activations were determined by western blotting. 24 h after
seeding of PC3 cells, 10%-FBS RPMI medium was replaced with 1%-FBS RPMI medium
and cells were untreated or treated with compounds 1, 2, NAN-190, 8-OH-DPAT
ꢀ
1
Yield: 30%. Mp: 85e87 C. H-NMR (CDCl
m, 4H), 3.06 (t, 2H, J ¼ 6.8 Hz, CH -N-pip), 4.67 (t, 2H, J ¼ 6.9 Hz,
-N-benz), 6.93(d,1H, J ¼ 8.1 Hz, ArH), 7.34 (t,1H, J ¼ 7.6 Hz, ArH),
3
) d 2.85 (m, 4H), 3.00
(
CH
7
(
2
(
15
mM) and Topotecan (2 mM) for 48 h 70 mg of whole cell lysates were resolved by
2
10%-SDS-PAGE, and then immuoblotted with pMAPK, MAPK, pAKT and AKT antibodies.
.45 (m, 2H, ArH), 7.51(d,1H, J ¼ 8.1 Hz, ArH), 7.79 (m, 2H, ArH), 7.95
Ponceau staining ensured the equal loading of samples in each lane.
t,1H, J ¼ 7.6 Hz, ArH), 8.17 (m, 2H, ArH), 8.38 (d,1H, J ¼ 8.1 Hz, ArH).
þ
ESI-MS: 402.1 [M þ H] .
analyzed compounds that could represent new interesting leads as
antiproliferative agents on prostatic cancer.
23 5
Anal. (C23H N ), C, H, N.
5
.1.5. 3-(3-(4-(naphthalen-1-yl)piperazin-1-yl)propoxy)benzo[d]
[1,2,3]triazin-4(3H)-one (6)
Yield: 45%. Mp: 90e92 C 1H-NMR (CDCl
J ¼ 6.9 Hz, eCH e), 2.78 (t, 2H, J ¼ 6.8 Hz, CH -N-pip), 2.90 (m, 4H),
-N-benz), 7.07 (d, 1H,
5
5
5
. Experimental
ꢀ
3
) d 2.14 (q, 2H,
.1. Synthesis
2
2
3
.15 (m, 4H), 4.56 (t, 2H, J ¼ 6.9 Hz, CH
2
.1.1. General procedures
J ¼ 8.1 Hz, ArH), 7.37 (t, 1H, J ¼ 7.6 Hz, ArH), 7.45 (m, 2H, ArH), 7.46
(d,1H, J ¼ 8.1 Hz, ArH), 7.80 (m, 2H, ArH), 7.96 (t, 1H, J ¼ 7.6 Hz, ArH),
8.20 (m, 2H, ArH), 8.39 (d,1H, J ¼ 8.1 Hz, ArH). ESI-MS: 416.9
All reagents and substituted piperazines were commercial
products purchased from Aldrich. Melting points, determined using
a Buchi Melting Point B-540 instrument, are uncorrected and
represent values obtained on recrystallized or chromatographically
þ
[M þ H] .
25 5
Anal. (C24H N ), C, H, N.
1
purified material. H-NMR spectra were recorded on Varian
Mercury Plus 400 MHz instrument. Unless otherwise stated, all
5.1.6. General procedure for preparation of 4-(2 or 3 chloroalkoxy)-
4-aza-tricyclo[5.2.1.02,6]dec-8-ene-3,5-dione (8a and 8b)
spectra were recorded in CDCl
3
. Chemical shifts are reported in
ppm using Me Si as internal standard. The following abbreviations
are used to describe peak patterns when appropriate: s (singlet),
d (doublet), t (triplet), m (multiplet), q (quartet), qt (quintet), dd
4
A solution of absolute ethanol (50 mL) and sodium hydroxide
0.72 g (0.018 mol) was reacted with 3.22 g (0.018 mol) of
commercially available endo-N-hydroxy-5-norbornene-2,3-dicar-
boximide (7) and 2.58 g (0.018 mol) of 1-bromo-2-chloroethane or
1-bromo-3-chloropropane at 70 C for 24 h. Afterward the mixture
was cooled to room temperature, concentrated to dryness and the
residue diluted in water (40 mL). The solution was extracted several
(
double doublet), ddd (double double doublet), bs (broad singlet),
ꢀ
m (multiplet). Mass spectra of the final products were performed
on API 2000 Applied Biosystem mass spectrometer. Elemental
analyses were carried out on a Carlo Erba model 1106; analyses
indicated by the symbols of the elements were within ꢁ0.4% of the
theoretical values. All reactions were followed by TLC, carried out
on Merck silica gel 60 F254 plates with fluorescent indicator and the
plates were visualized with UV light (254 nm). Preparative chro-
matographic purifications were performed using silica gel column
times with CH
anhydrous Na
2
Cl2. The combined organic layers were dried on
SO and concentrated in vacuo. The residue was
2
4
purified by column chromatography (diethyl ether/ethanol 9:1
(v/v)). The combined and evaporated product fractions were crys-
tallizedfromdiethylether/hexane, togivethefinalcompounds8aand
1
(
Kieselgel 60). Solutions were dried over Na
2
SO
4
and concentrated
8b aswhite solid(obtained yields8a80%;8b94%). H-NMR spectra for
with a Buchi R-114 rotary evaporator at low pressure.
all intermediates were consistent with the proposed structures.
5
.1.2. General procedure for preparation of 3-(2 or 3 chloroalkoxy)-
5.1.7. General procedure for the preparation of 4-[2 or 3-[4-
(naphthalen)piperazin-1-yl]alkoxy]-4-aza-tricyclo[5.2.1.02,6]dec-8-
ene-3,5-dione (9e10)
1,2,3-benzotriazin-4(3H)-one (4a and 4b)
A mixture of 1-bromo-2-chloroethane or 1-bromo-3-chlor-
opropane (0.09 mol), 3-hydroxy-1,2,3-benzotriazin-4H-one (3)
A mixture of 4-(2-chloroethoxy)-4-aza-tricyclo[5.2.1.02,6]dec-
8-ene-3,5-dione (8a) or 4-(3-chloro-propoxy)-4-aza-tricyclo
[5.2.1.02,6]dec-8-ene-3,5-dione (8b) (0.006 mol), and NaI
(0.009 mol) in acetonitrile was stirred under reflux for 30 min.
ꢀ
(
0.03 mol), NaOH (0.05 mol) in DMF (70 mL) was stirred at 70 C for
2
4 h. After cooling, the mixture was concentrated to dryness and
the residue was dissolved in water (50 mL); the solution was
extracted several times with CH Cl2. The organic phase was dried,
2
2 3
Then 1-naphtylpiperazine (0.03 mol) and anhydrous K CO

F. Fiorino et al. / European Journal of Medicinal Chemistry 46 (2011) 2206e2216
2213
0
(
0.009 mol) were added. The reaction mixture was stirred under
5.1.12. N -cyano-N-(3-(4-(naphthalen-1-yl)piperazin-1-yl)propyl)
reflux for 24 h. After cooling, the mixture was filtered, concen-
trated to dryness and the residue was dissolved in water (50 mL).
isonicotinamidine (14)
0
A mixture of N-(3-bromopropyl)-N -cyanoisonicotinamidine
(13) (0.006 mol), and NaI (0.009 mol) in acetonitrile was stirred
under reflux for 30 min. Then 1-naphtylpiperazine (0.03 mol) and
The solution was extracted several times with CH
combined organic layers were dried on anhydrous Na SO
2
Cl2. The
4
and the
2
solvent removed in vacuo. The crude mixtures were purified by
2 3
anhydrous K CO (0.009 mol) were added. The reaction mixture
silica gel column chromatography using diethyl ether/methanol
was stirred under reflux for 4 h. After cooling, the mixture was
filtered, concentrated to dryness and the residue was dissolved in
water (50 mL). The solution was extracted several times with
8
:2 (v/v) as eluent. The crude products were recrystallized from
diethyl ether.
CH
Na
2
Cl
SO
2
. The combined organic layers were dried on anhydrous
4
and the solvent removed in vacuo. The crude mixture was
5.1.8. 4-[2-[4-(naphthalen)piperazin-1-yl]ethoxy]-4-aza-tricyclo
2
[
5.2.1.02,6]dec-8-ene-3,5-dione (9)
purified by silica gel column chromatography using diethyl ether/
methanol 8:2 (v/v) as eluent. The crude product was recrystallized
from diethyl ether.
ꢀ
1
Yield: 50%; mp 126e128 C; H-NMR (500 MHz, CDCl
pip.); 2.82 (t,
pip.); 3.19 (s, 2H); 3.43 (s,
3
) d: 1.49
(
2
2
d, 1H, J ¼ 8.9); 1.76 (d, 1H, J ¼ 8.9); 2.80 (bs, 4H, 2CH
2
1
H, N eCH
2
, J ¼ 7.3); 3.16 (bs, 4H, 2CH
2
0
H); 4.15 (t, 2H, OeCH
2
, J ¼ 6.5); 6.17 (s, 2H); 7.07(d, 1H, J ¼ 8.1 Hz,
5.1.13. N -cyano-N-(3-(4-(naphthalen-1-yl)piperazin-1-yl)propyl)
ArH), 7.37 (t, 1H, J ¼ 7.6 Hz, ArH), 7.44 (m, 2H, ArH), 7.53(d, 1H,
isonicotinamidine (14)
Yield: 25%; mp 158e160 C; H-NMR (500 MHz, CDCl
(m, 2H); 1.89 (bs, 4H, 2CH
ꢀ
1
J ¼ 8.1 Hz, ArH), 7.80 (m, 1H, ArH), 8.17 (m, 1H, ArH). ESI-MS: 418.0
3
)
d
: 1.63
þ
1
[
M þ H] .
2 2
pip.); 2.70 (t, 2H, N eCH
, J¼5.1); 2.76
Anal. (C25
H
27
N
3
), C, H, N.
(bs, 4H, 2CH pip.); 3.67 (bs, 2H, NeCH
2
2
); 6.77 (d, 1H, J ¼ 8.1 Hz,
ArH); 7.37 (t, 1H, J ¼ 7.6 Hz, ArH); 7.45 (m, 2H, ArH); 7.54 (m, 1H,
ArH); 7.56 (d, 2H, J¼5.4); 7.81 (bs, 1H, ArH); 8.09 (bs, 1H, ArH); 8.82
5
[
.1.9. 4-[3-[4-(naphthalen)piperazin-1-yl]propoxy]-4-aza-tricyclo
þ
5.2.1.02,6]dec-8-ene-3,5-dione (10)
(d, 2H, J ¼ 5.4); 9.74 (s, 1H). ESI-MS: 399.1 [M þ H] ; 421.2
ꢀ
1
þ
þ
Yield: 58%; mp 125e127 C; H-NMR (500 MHz, CDCl
d, 1H, J ¼ 8.9); 1.75 (d, 1H, J ¼ 8.9); 1.89 (q, 2H, eCH e, J ¼ 7.3); 2.62
pip.); 3.13 (bs, 4H, 2CH
2
3
)
d
: 1.49
[M þ Na] ; 437.1[M þ K] .
(
(
2
26 6
Anal. (C24H N ), C, H, N.
1
t, 2H, N eCH
2
, J ¼ 7.3); 2.74 (bs, 4H, 2CH
2
pip.); 3.18 (s, 2H); 3.43 (s, 2H); 4.05 (t, 2H, OeCH
2
2
ArH). ESI-MS: 432.9 [M þ H] .
2
, J ¼ 6.5); 6.18 (s,
5.1.14. Methyl-N-cyano-2-pyridinecarboximidate (16)
H); 7.07(d,1H, J ¼ 8.1 Hz, ArH), 7.37 (t, 1H, J ¼ 7.6 Hz, ArH), 7.44 (m,
A mixture of 2-cyanopyridine (15) (15 g, 0.192 mol) and NaOMe
(0.24 g, 0.0096 mol) in anhydrous methanol (180 mL) was stirred
overnight at room temperature. AcOH (0.0106 mol) was then added
with stirring and the solution was evaporated in vacuo. n-Hexane
H, ArH), 7.52 (d, 1H, J ¼ 8.1 Hz, ArH), 7.80 (m, 1H, ArH), 8.17 (m, 1H,
þ
29 3
Anal. (C26H N ), C, H, N.
(150 mL) was added to the residue and the resultant precipitate was
5.1.10. Isopropyl-N-cyano-4-pyridinecarboximidate (12)
filtered off. The filtrate was evaporated to give crude methyl-2-
A mixture of 4-cyanopyridine (11) (15 g, 0.144 mol) and NaOMe
pyridinecarboximidate (26.3 g; 0.193 mol) as an oil. Crude
(
0.24 g, 0.0044 mol) in anhydrous isopropanol (180 mL) was stirred
compound was then added to a mixture of NH
NaH PO $2H O (68.60 g) and Na HPO (15.62 g) in water (140 mL).
After vigorous stirring for 6 h at room temperature, the reaction
mixture was extracted several times with CH Cl . The combined
organic layers were dried on anhydrous Na SO and concentrated
2
CN (0.384 mol),
ꢀ
overnight at 0 C. AcOH (0.25 g, 0.0042 mol) was then added with
stirring and the solution was evaporated in vacuo. n-Hexane
2
4
2
2
4
(
150 mL) was added to the residue and the resultant precipitate was
2
2
filtered off. The filtrate was evaporated to give crude isopropyl-4-
2
4
pyridinecarboximidate (18.6 g) as an oil. Crude compound was then
in vacuo to yield crude methyl-N-cyano-2-pyridinecarboximidate
(16) (19 g), which was used directly in the following reaction: H-
1
added to a mixture of NH
65.55 g) and Na HPO (14.9 g) in water (112.5 mL). After vigorous
stirring for 6 h at room temperature, the reaction mixture was
extracted several times with CH Cl . The combined organic layers
were dried on anhydrous Na SO and concentrated in vacuo to yield
2
CN (8.83 g, 0.21 mol), NaH
2
PO
4
$2H
2
O
(
2
4
NMR (400 MHz, CDCl
3
)
d
: 4.16 (s, 3H); 7.63 (dd, 1H, J ¼ 9.4, 7.3); 7.94
(d, 1H, J ¼ 3.4); 7.98 (dd, 1H, J ¼ 7.3, 2.4); 8.83 (ddd, 1H, J ¼ 9.4,
2
2
3.4, 2.4).
2
4
0
crude isopropyl-N-cyano-4-pyridinecarboximidate (12) (22 g),
5.1.15. N-(3-bromopropyl)-N -cyanopicolinamidine (17)
1
which was used directly in the following reaction: H-NMR
To a solution of methyl-N-cyano-2-pyridinecarboximidate (16)
(0.118 mol) in anhydrous MeOH (120 mL), 3-bromopropylami-
ne$HBr (28.24 g, 0.129 mol) and triethylamine (0.129 mol) were
successively added and the reaction mixture was stirred at room
temperature for 18 h. After evaporation, the residue was dissolved
(
2
400 MHz, CDCl
H); 8.9e8.7 (m, 2H).
3
)
d
: 1.50 (d, 6H J ¼ 6.1); 5.42 (m, 1H); 8.0e7.8 (m,
0
5
.1.11. N-(3-bromopropyl)-N -cyanoisonicotinamidine (13)
To a solution of isopropyl-N-cyano-4-pyridinecarboximidate
in CHCl
3
. The solution was washed with water, dried on anhydrous
4
and concentrated in vacuo. The residue was purified by
(
21.89 g, 0.116 mol) in anhydrous MeOH (120 mL), 3-bromopro-
pylamine$HBr (28.24 g, 0.129 mol) and NaOMe (6.77 g,
.125 mol) were successively added and the reaction mixture was
stirred at room temperature for 18 h. After evaporation, the
residue was dissolved in CHCl . The solution was washed with
water, dried on anhydrous Na SO and concentrated in vacuo. The
Na SO
2
column chromatography (diethyl ether/ethanol 9:1 (v/v)). The
combined and evaporated product fractions were crystallized from
diethyl ether/hexane, yielding 14.2 g (45%) of the desired product as
0
ꢀ
1
3
a white solid: mp: 230e231 C; H-NMR (400 MHz, CDCl ) d: 2.10
3
2
4
(qt, 2H, J ¼ 6.2); 2.73 (t, 2H, J ¼ 6.2); 3.89 (q, 2H, J ¼ 6.2); 7.47 (t, 1H,
J ¼ 7.3); 7.86 (t, 1H, J ¼ 7.3); 8.5 (d, 1H, J ¼ 7.3); 8.64 (d, 1H, J ¼ 7.3);
9.80 (s, 1H).
residue was purified by column chromatography (diethyl ether/
ethanol 9:1 (v/v)). The combined and evaporated product frac-
tions were crystallized from diethyl ether/hexane, yielding 12.5 g
ꢀ
0
(
40%) of the desired product as a white solid: mp: 201e203 C;
5.1.16. N -cyano-N-(3-(4-(naphthalen-1-yl)piperazin-1-yl)propyl)
1
H-NMR (400 MHz, CDCl
3
)
d
: 2.30 (qt, 2H, J ¼ 6.2); 3.51 (t, 2H
picolinamidine (18)
A mixture of N-(3-bromopropyl)-N -cyanoisonicotinamidine
(17) (0.006 mol), and NaI (0.009 mol) in acetonitrile was stirred
0
J ¼ 6.2); 3.72 (q, 2H, J ¼ 6.2); 7.49 (d, 2H, J ¼ 5.5); 8.83 (d, 2H,
J ¼ 5.5); 9.60 (s, 1H).

2214
F. Fiorino et al. / European Journal of Medicinal Chemistry 46 (2011) 2206e2216
under reflux for 30 min. Then 1-naphtylpiperazine (0.03 mol) and
anhydrous K CO (0.009 mol) were added. The reaction mixture
was stirred under reflux for 4 h. After cooling, the mixture was
filtered, concentrated to dryness and the residue was dissolved in
water (50 mL). The solution was extracted several times with
above to achieve a final assay concentration of 0.1 nM, and 0.8 mL of
2
3
resuspended membranes. The tubes were incubated for 30 min at
ꢀ
37 C, and the incubations were terminated by vacuum filtration
through Whatman GF/B filters (Brandel Biomedical Research and
Laboratories Inc., Gaithersburg, MD, USA). The filters were washed
twice with 5 mL of ice-cold TriseHCl buffer, and the radioactivity
bound to the filters was measured by liquid scintillation spec-
CH
Na
2
Cl
SO
2
. The combined organic layers were dried on anhydrous
4
and the solvent removed in vacuo. The crude mixture was
2
Ò
purified by silica gel column chromatography using diethyl ether/
methanol 8:2 (v/v) as eluent. The crude product was recrystallized
from diethyl ether.
trometer (Packard TRI-CARB 2000CA e Packard BioScience s.r.l.,
3
Pero, Milan, Italy). Specific [ H]-8-OH-DPAT binding was defined as
the difference between binding in the absence and presence of 5-
HT (10 mM).
0
5
.1.17. N -cyano-N-(3-(4-(naphthalen-1-yl)piperazin-1-yl)propyl)
picolinamidine (18)
Yield: 45%; mp 148e150 C; H-NMR (400 MHz, CDCl
qt, 2H, J ¼ 6.2); 2.71 (t, 2H, J ¼ 6.2); 2.82 (bs, 4H, 2CH pip.); 3.21
bs, 4H, 2CH
pip.); 3.89 (q, 2H, J ¼ 6.2); 7.04 (d, 1H, J ¼ 8.1); 7.41 (t,
5.2.3. 5-HT2A and 5-HT2C binding assays
ꢀ
1
3
)
d
: 1.94
Radioligand binding assays were performed as previously
reported by Herndon et al. [29]. Briefly, frontal cortical regions of
male SpragueeDawley rats (180e220 g) were dissected on ice and
homogenized (1:10 w/v) in ice-cold buffer solution (50 mM Tris
HCl, 0.5 mM EDTA, and 10 mM MgCl at pH 7.4) with a Polytron
2
PT10 (setting 5 for 15 s) and centrifuged at 3000 g for 15 min. The
(
(
1
2
2
H, J ¼ 8.1); 7.46 (m,3H, ArH); 7.56 (d,1H, J ¼ 8.1 Hz, ArH); 7.82
(
m,1H, ArH); 7.86 (t, 1H, J ¼ 7.3); 8.16 (m,1H, ArH); 8.52 (d, 1H,
þ
J ¼ 7.3); 8.61 (d, 1H, J ¼ 7.3); 9.95 (s, 1H). ESI-MS: 399.1 [M þ H] .
ꢀ
26
Anal. (C24H N
6
), C, H, N.
pellet was resuspended in buffer (1:30 w/v), incubated at 37 C for
15 min and then centrifuged twice more at 3000 g for 10 min (with
5
5
.2. In vitro receptor binding
resuspension between centrifugations). The final pellet was
resuspended in buffer that also contained 0.1% ascorbate and
ꢂ5
.2.1. General procedures
10 M pargyline.
The newly synthesized compounds were tested for in vitro
Assays were performed in triplicate in a 2.0 mL volume con-
taining 5 mg wet weight of tissue and 0.4 nM [ H]ketanserin
3
affinity for serotonin 5-HT1A, 5-HT2A and 5-HT2C receptors by radi-
oligand binding assays. All the compounds weredissolved in ethanol
or in 5% DMSO. The following specific radioligands and tissue
hydrochloride (88.0 Ci/mmol; Perkin Elmer Life Sciences, Boston,
MA, USA) for 5-HT2A receptor assays, and 10 mg wet weight of
3
3
sources were used: (a) serotonin 5-HT1A receptor, [ H]-8-OH-DPAT,
tissue and 1 nM [ H]mesulergine (87.0 Ci/mmol; Amersham
3
rat brain cortex; (b) serotonin 5-HT2A receptor, [ H]ketanserin, rat
Biosciences Europe GmbH) for 5-HT2C receptor assays. Cinanserin
3
brain cortex; (c) serotonin 5-HT2C receptor, [ H]mesulergine, rat
(1.0
m
M) was used to define nonspecific binding in the 5-HT2A assay.
M) was used to define
3
brain cortex; (d) dopamine D
tum; (e) dopamine D
1
receptor [ H]SCH-23390, rat stria-
In the 5-HT2C assays, mianserin (1.0 m
3
2
receptor [ H]spiperone, rat striatum; (f)
adrenergic receptor [ H]prazosin, rat brain cortex; (g)
a
1
nonspecific binding, and 100 nM spiperone was added to all tubes
3
a
2
adrenergic
to block binding to 5-HT2A receptors. Tubes were incubated for
3
ꢀ
receptor [ H]yohimbine, rat brain cortex.
15 min at 37 C, filtered on Schliecher and Schuell (Keene, NH, USA)
Non-specific binding was determined as described in the
experimental section, and specific binding as the difference
between total and non-specific binding. Blank experiments were
carried out to determine the effect of 5% DMSO on the binding and
no effects were observed. Competition experiments were analyzed
by the “Easy Fit” program [25] to obtain the concentration of
unlabeled drug that caused 50% inhibition of ligand binding (IC50),
with six concentrations of test compounds, each performed in
triplicate. The IC50 values obtained were used to calculate apparent
glass fiber filters presoaked in polyethylene imine, and washed
with 10 mL of ice-cold buffer. Filters were counted at an efficiency
of 50%.
5.2.4. D
1
dopaminergic binding assay
dopaminergic receptors was that
The binding assay for D
1
described by Billard et al. [30]. Corpora striata were homogenized
ꢀ
in 30 vol. (w/v) ice-cold 50 mM TriseHCl buffer (pH 7.7 at 25 C)
using a Polytron PT10 (setting 5 for 20 s). Homogenates were
centrifuged twice for 10 min at 50000 g with resuspension of the
pellet in fresh buffer. The final pellet was resuspended in 50 mM
inhibition constants (K
from the following equation: K
sents the concentration of the hot ligand used and K
dissociation constant (K values, obtained by Scatchard analysis
27], were calculated for each labeled ligand).
i
) by the method of Cheng and Prussoff [26],
¼ IC50/(1 þ S/K ) where S repre-
its receptor
i
D
D
2
ice-cold TriseHCl containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl ,
D
1 mM MgCl
37 C). Each assay tube contained 50
2
, 0.1% ascorbic acid and 10
m
M pargyline (pH 7.1 at
ꢀ
3
[
mL [ H]SCH-23390 (85.0 Ci/
mmol, Perkin Elmer Life Sciences, Boston, MA, USA) to achieve
final concentration of 0.4 nM, and 900 L resuspended
5.2.2. 5-HT1A binding assay
a
m
Radioligand binding assays were performed following a pub-
membranes (3 mg fresh tissue). The tubes were incubated for
15 min at 37 C and the incubation was terminated by rapid
ꢀ
lished procedure [28]. Cerebral cortex from male SpragueeDawley
rats (180e220 g) was homogenized in 20 volumes of ice-cold
filtration under vacuum through Whatman GF/B glass fiber filters.
The filters were washed three times with 5 mL ice-cold 50 mM
TriseHCl buffer (pH 7.7 at 25 C). The radioactivity bound to the
ꢀ
TriseHCl buffer (50 mM, pH 7.7 at 22 C) with a Polytron PT10,
Brinkmann Instruments (setting 5 for 15 s), and the homogenate
3
was centrifuged at 50000 g for 10 min. The resulting pellet was
filters was measured by a liquid scintillation counter. Specific [ H]
ꢀ
then resuspended in the same buffer, incubated for 10 min at 37 C,
SCH-23390 binding was defined as the difference between binding
and centrifuged at 50000 g for 10 min. The final pellet was resus-
in the absence or in the presence of 0.1
mM piflutixol.
pended in 80 volumes of the TriseHCl buffer containing 10
mM
pargyline, 4 mM CaCl , and 0.1% ascorbate. To each assay tube was
2
5.2.5. D dopaminergic binding assay
2
added the following: 0.1 mL of the drug dilution (0.1 mL of distilled
The procedure used in the radioligand binding assay was
reported in detail by Creese et al. [31]. Corpora striata were
homogenized in 30 vol. (w/v) ice-cold 50 mM TriseHCl buffer (pH
3
water if no competing drug was added), 0.1 mL of [ H]-8-hydroxy-
3
2
-(di-n-propylamino)tetralin ([ H]-8-OH-DPAT) (170.0 Ci/mmol,
ꢀ
Perkin Elmer Life Sciences, Boston, MA, USA) in the same buffer as
7.7 at 25 C) using a Polytron PT10 (setting 5 for 20 s).

F. Fiorino et al. / European Journal of Medicinal Chemistry 46 (2011) 2206e2216
2215
Ò
Homogenates were centrifuged twice for 10 min at 50000 g with
resuspension of the pellet in fresh buffer. The final pellet was
resuspended in 50 mM ice-cold TriseHCl containing 120 mM NaCl,
Topotecan (Hycamtin ) was from GlaxoSmithKline S.p.A. e Brent-
ford, UK. Sulforhodamine B (SRB) was from ICN Biomedicals (Irvine,
CA, USA). Primary antibodies pMAPK, MAPK, pAKT, AKT were from
5
mM KCl, 2 mM CaCl
2
, 1 mM MgCl
2
, 0.1% ascorbic acid and 10
m
M
Cell Signaling Technology, Inc. (Boston, MA, USA); gH2AX was from
ꢀ
3
pargyline (pH 7.1 at 37 C). Each assay tube contained 50
spiperone (15.7 Ci/mmol, Perkin Elmer Life Sciences, Boston, MA,
USA) to achieve a final concentration of 0.4 nM, and 900
resuspended membranes (3 mg fresh tissue). The tubes were
incubated for 15 min at 37 C and the incubation was terminated
mL [ H]
Upstate (Lake Placid, NY, USA). Secondary horseradish peroxidase-
linked antibodies were from Bio-Rad (Richmond, CA, USA).
Enhanced chemiluminescence (ECL) immunodetection reagents
were from GE Healthcare (Buckinghamshire, UK). Annexin-V-FITC
was from Becton Dickinson (San Jose, CA, USA).
m
L
ꢀ
by rapid filtration under vacuum through Whatman GF/B glass
fiber filters. The filters were washed three times with 5 mL ice-
cold 50 mM TriseHCl buffer (pH 7.7 at 25 C). The radioactivity
5.3.2. Cell culture and cell proliferation assay
PC3 cell line was grown in RPMI 1640 medium supplemented
with 10% heat-inactivated fetal bovine serum, 50 units/mL peni-
ꢀ
bound to the filters was measured by a liquid scintillation counter.
3
Specific [ H]spiperone binding was defined as the difference
cillin, 500
mg/ml streptomycin, and 4 mmol/L glutamine in
ꢀ
between binding in the absence or in the presence of 1
butaclamol.
mM (þ)ꢂ
a humidified atmosphere of 95% air and 5% CO
2
at 37 C. For cell
growth assays, 1000 cells were seeded, in medium containing 10%-
FBS, in quadruplicate in 96-multiwell plates (Falcon, Becton
Dickinson Labware, Franklin Lakes, NJ, USA), after 24 h medium
was replace with a medium containing 10%-FBS or 1%-FBS as
indicated and cells were treated with increasing concentrations of
the indicated compounds. Cell survival/proliferation was
measured by a spectrophotometric dye incorporation assay using
sulforhodamine B (SRB) after 96 h from treatment as described
before [34].
5.2.6.
1
a adrenergic binding assay
The procedure used in the radioligand binding assay has been
reported in detail by Greengrass and Brenner [32]. Brain cortex was
homogenized in 30 vol. (w/v) ice-cold 50 mM TriseHCl buffer, (pH
ꢀ
7
.2 at 25 C) using a Polytron PT10 (setting 5 for 20 s). Homogenates
were centrifuged twice for 10 min at 50000 g with resuspension of
the pellet in fresh buffer. The final pellet was resuspended in 50 mM
ꢀ
ice-cold TriseHCl, (pH 7.4 at 25 C). Each assay tube contained 50
mL
3
drug solution, 50
m
L [ H]prazosin (80.5 Ci/mmol, Perkin Elmer Life
5.3.3. Protein extraction and western blotting
Sciences, Boston, MA, USA) to achieve a final concentration of
Cells grown and treated for 48 h with 1, 2, NAN-190, 8-OH-
DPAT and Topotecan were washed once with ice-cold phosphate-
buffered saline (PBS) and scraped in ice-cold PBS, and the cell
pellets were lysed for 1 h at 4 C in NP-40 lysis buffer (0.5% NP-40,
50 mM HEPES pH 7, 250 mM NaCl, 5 mM EDTA, 0.5 mM Na
pyrophosphate, 0.5 mM Na orthovanadate, 50 mM NaF, 1 mM DTT,
0
.4 nM, and 900
m
L resuspended membranes (10 mg fresh tissue).
ꢀ
The tubes were incubated for 30 min at 25 C and the incubation
was terminated by rapid filtration under vacuum through What-
man GF/B glass fiber filters. The filters were washed three times
ꢀ
ꢀ
with 5 mL ice-cold 50 mM TriseHCl, buffer (pH 7.2 at 25 C). The
radioactivity bound to the filters was measured by a liquid scintil-
lation counter. Specific [ H]prazosin binding was defined as the
0.5 mM PMSF, 5
mg/ml aprotinin, 5 mg/ml leupeptin, 5 mg/ml
3
benzamidine, 1 g/ml pepstatin) and clarified by centrifugation.
m
difference between binding in the absence or in the presence of
10 mM phentolamine.
Equal amounts of proteins were separated by SDS-PAGE. Proteins
were then transferred to nitrocellulose paper, immunoblotted
with the appropriate antibody probed with the appropriate
horseradish peroxidase-linked IgG and immunoreactive bands
were detected by ECL.
5.2.7.
2
a adrenergic binding assay
The procedure used in the radioligand binding assay was
reported in detail by Perry and U’Prichard [33]. Brain cortex was
homogenized in 30 vol. (w/v) ice-cold 5 mM triseHCl, 5 mM EDTA
buffer (pH 7.3 at 25 C) using a polytron PT10 (setting 5 for 20 s).
5.3.4. Analysis of cell cycle kinetics
ꢀ
Analysis of cell cycle kinetics was performed on PC3 cells treated
for 24 and 48 h with 1, 2, NAN-190, 8-OH-DPAT and Topotecan.
Briefly, cells were harvested, fixed in 70% ethanol and stored at
Homogenates were centrifuged three times for 10 min at 50000 g
with resuspension of the pellet in fresh buffer. The final pellet was
resuspended in 50 mM ice-cold TriseHCl, 0.5 mM EDTA (pH 7.5 at
ꢀ
ꢂ20 C until analysis. After nuclear DNA staining with propidium
ꢀ
3
2
5 C). Each assay tube contained 50
yohimbine (80.5 Ci/mmol, Perkin Elmer Life Sciences, Boston, MA,
USA) to achieve a final concentration of 1 nM, and 900 L resus-
m
L drug solution, 50
m
L [ H]
iodide (PI), flow cytometry was performed in duplicate by a FACS-
can flow cytometer (Becton Dickinson, San Jose, CA, USA). For each
sample, 20,000 events were stored and cell cycle analysis was
performed by the ModFit LT software (Verity Software House, Inc.,
Topsham, ME, USA). FL2 area versus FL2 width gating was done to
exclude doublets from the G2-M region. The percentage of
apoptotic cells was calculated in the sub-diploid region of the DNA
content, registered as FL2 signals in linear scale. To avoid cell debris
contamination due to necrotic cell death, cells were selected by
side-scatter (SSC) versus DNA signals (FL2) gating.
m
pended membranes (10 mg fresh tissue). The tubes were incubated
ꢀ
for 30 min at 25 C and the incubation was terminated by rapid
filtration under vacuum through Whatman GF/B glass fiber filters.
The filters were washed three times with 5 mL ice-cold 50 mM
ꢀ
TriseHCl, 0.5 mM EDTA buffer (pH 7.5 at 25 C). The radioactivity
bound to the filters was measured by a liquid scintillation counter.
3
Specific [ H]yohimbine binding was defined as the difference
between binding in the absence or in the presence of 10
phentolamine.
mM
5.3.5. Annexin-V assay
Annexin-V binding was identified by flow cytometry using
Annexin-V-FITC staining as described previously [35]. Briefly, cells
were harvested, centrifuged and washed twice with PBS, then
5
5
.3. Biology
6
.3.1. Reagents
resuspended in 1X binding buffer at a concentration of 10 cells/ml.
All media, serum, antibiotics and glutamine were purchased
The cell suspension was incubated with 5
m
L of Annexin-V-FITC for
ꢀ
from Cambrex Bio Science (Verviers, Belgium). NAN-190 hydro-
bromide (NAN-190) and S(ꢂ)-8-hydroxy-DPAT hydrobromide (8-
OH-DPAT) were from SigmaeAldrich Co (St. Louis, MO, USA),
10 min at room temperature (20e25 C) in the dark and then
analyzed by CellquestÔ data analysis software (Becton Dickinson,
San Jose, CA, USA).

2216
F. Fiorino et al. / European Journal of Medicinal Chemistry 46 (2011) 2206e2216
Acknowledgments
saccharine derivatives as 5-HT(1A) receptor ligands, Eur. J. Pharm. Sci. 16
2002) 15e28.
(
[
20] F. Fiorino, E. Perissutti, B. Severino, V. Santagada, D. Cirillo, S. Terracciano,
This work was supported by a grant from Regione Campania ai
sensi della L.R. n 5 del 28.03.2002. The NMR spectral data were
provided by Centro di Ricerca Interdipartimentale di Analisi Stru-
mentale, Università degli Studi di Napoli “Federico II”. The assis-
tance of the staff is gratefully appreciated.
P. Massarelli, G. Bruni, E. Collavoli, C. Renner, G. Caliendo, New 5-hydrox-
ꢀ
ytryptamine(1A) receptor ligands containing
synthesis and in vitro pharmacological evaluation, J. Med. Chem. 48 (2005)
495e5503.
a
norbornene nucleus:
5
[
21] F. Fiorino, B. Severino, F. De Angelis, E. Perissutti, F. Frecentese, P. Massarelli,
G. Bruni, E. Collavoli, V. Santagada, G. Caliendo, Synthesis and in-vitro phar-
macological evaluation of new 5-HT1A receptor ligands containing a benzo-
triazinone nucleus, Arch. Pharm. 341 (2008) 20e27 (Weinheim).
References
[
22] F. Fiorino, B. Severino, F. De Angelis, E. Perissutti, E. Magli, F. Frecentese,
A. Esposito, P. Massarelli, C. Nencini, B. Viti, V. Santagada, G. Caliendo,
[
[
[
1] M. Lesurtel, C. Soll, P.A. Claivien, Role of serotonin in the hepato-gastrointestinal
tract:an old moleculefornew perspectives, Cell. Mol. Life Sci. 65(2008) 940e952.
2] H.G. Baumgarten, M. Gother, Serotoninergic neurons and 5-HT receptors in
the CNS, Handb. Exp. Pharm. 129 (1997) Springer-Verlag Berlin.
3] G.R. Martin, R.M. Eglen, D. Hoyer, M.W. Hamblin, F. Yocca, Advances in
serotonin receptor research; molecular biology, signal transmission, and
therapeutics, Ann. N.Y. Acad. Sci. (1998) New York.
Synthesis and in vitro pharmacological evaluation of a new series of 5-HT1A
-HT2A and 5-HT2C receptor ligands containing a norbornene nucleus, Phar-
mazie 64 (9) (2009) 555e564.
,
5
[
23] F. Fiorino, B. Severino, F. De Angelis, E. Perissutti, E. Magli, F. Frecentese,
A. Esposito, P. Massarelli, C. Nencini, V. Santagada, G. Caliendo, New 5-HT1A
receptor ligands containing a N’-cyanoisonicotinamidine nucleus: synthesis
and in vitro pharmacological evaluation, Bioorg. Med. Chem. Lett. 20 (9)
[
[
[
[
[
4] N.M. Barnes, T. Sharp, A review of central 5-HT receptors and their function,
(2010) 2978e2982.
Neuropharmacology 38 (1999) 1083e1152.
[
24] C.S. Sreevidya, N.M. Khaskhely, A. Fukunaga, P. Khaskina, S.E. Ullrich, Inhibi-
tion of photocarcinogenesis by platelet-activating factor or serotonin receptor
antagonists, Cancer Res. 68 (10) (2008) 3978e3984.
25] EasyFit 1.4. Matteo Vaccari and Mario Negri Institute, Milan, Italy, 1989e1991.
1
26] Y.C. Cheng, W.H. Prusoff, Relationship between the inhibition constant (K )
5] D. Hoyer, J.P. Hannon, G.R. Martin, Molecular, pharmacological and functional
diversity of 5-HT receptors, Pharmacol. Biochem. Behav. 71 (2002) 533e554.
6] J. De Vry, 5-HT1A receptor agonists: recent developments and controversial
issues, Psychopharmacology 121 (1995) 1e26.
7] S.E. File, Recent developments in anxiety, stress, and depression, Pharmacol.
Biochem. Behav. 54 (1996) 3e12.
8] D. Neckelmann, A.A. Bjorkum, B. Bjorvatn, R. Ursin, Sleep and EEG power
spectral effects of the 5-HT1A agonist/antagonist NAN 190 alone and in
combination with citalopram, Behav. Brain Res. 75 (1996) 159e168.
9] A.J. Sleight, P.A. Pierce, A.W. Schmidt, C.R. Hekmatpanah, S.J. Peroutka, in:
S.J. Peroutka (Ed.), Serotonin Receptor Subtypes, Wiley-Liss, New York, 1991, pp.
[
[
and the concentration of inhibitor which causes 50 per cent inhibition (I50) of
an enzymatic reaction, Biochem. Pharmacol. 22 (1973) 3099e3108.
27] G. Scatchard, The attraction of proteins for small molecules and ions, Ann. N.Y.
Acad. Sci. 51 (1949) 660e672.
28] J.R. Schlegel, S.J. Peroutka, Nucleotide interactions with 5-HT1A binding sites
directly labeled by [3H]-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]-8-OH-
DPAT), Biochem. Pharmacol. 35 (1986) 1943e1949.
[
[
[
211e227.
[29] J.L. Herndon, A. Ismaiel, S.P. Ingher, M. Teitler, R.A. Glennon, Ketanserin
[
[
10] P.R. Saxena, Serotonin receptors: subtypes, functional responses and thera-
peutic relevance, Pharmacol. Ther. 66 (1995) 339e368.
analogues: structure-affinity relationships for 5-HT
2
and 5-HT1C serotonin
receptor binding, J. Med. Chem. 35 (1992) 4903e4910.
11] N. Dizeyi, A. Bjaartell, E. Nilsson, J. Hansson, V. Gadaleanu, N. Cross,
P.A. Abrahamsson, Expression of serotonin receptors and role of serotonin in
human prostate cancer tissue and cell lines, The Prostate 59 (2004) 328e336.
12] D. Hoyer, D.E. Clarke, J.R. Fozard, P.R. Hartig, G.R. Martin, E.J. Mylecharane,
P.R. Saxena, P.P. Humphrey, International union of pharmacology classification
of receptors for 5-hydroxytryptamine, Pharmacol. Rev. 46 (2) (1994) 157e203.
13] J.A. Bikker, S. Trump-Kallmeyer, C. Humblet, G-protein coupled receptors:
models, mutagenesis, and drug design, J. Med. Chem. 41 (1998) 2911e2927.
14] S. Trumpp-Kallmeyer, J. Hoflack, A. Bruinvels, M. Hibert, Modeling of G-protein-
coupled receptors: application to dopamine, adrenaline, serotonin, acetylcho-
line, and mammalian opsin receptors, J. Med. Chem. 35 (1992) 3448e3462.
15] E.J. Siddiqui, M. Shabbir, D.P. Mikhailidis, C.S. Thompson, F.H. Mumtaz, The
role of serotonin (5-hydroxytryptamine1A and 1B) receptors in prostate cancer
cell proliferation, J. Urol. 176 (2006) 1648e1653.
[
30] W. Billard, V. Ruperto, G. Crosby, L.C. Iorio Barnett, Characterization of the
binding of 3H-SCH 23390, a selective D-1 receptor antagonist ligand, in rat
striatum, Life Sci. 35 (18) (1984) 1885e1893.
31] I. Creese, R. Schneider, S.H. Snyder, 3H-spiroperidol labels dopamine receptors
in pituitary and brain, Eur. J. Pharmacol. 46 (4) (1977) 377e381.
[
[
[
[
32] P. Greengrass, R. Bremner, Binding characteristics of 3H-prazosin to rat brain
alpha-adrenergic receptors, Eur. J. Pharmacol. 55 (3) (1979) 323e326.
33] B.D. Perry, D.C. U’Prichard, [3H]rauwolscine (alpha-yohimbine): a specific
antagonist radioligand for brain alpha 2-adrenergic receptors, Eur. J. Phar-
macol. 76 (4) (1981) 461e464.
34] F. Bruzzese, M. Rocco, S. Castelli, E. Di Gennaro, A. Desideri, A. Budillon,
Synergistic antitumor effect between vorinostat and topotecan in small cell
lung cancer cells is mediated by generation of reactive oxygen species and
DNA damage-induced apoptosis, Mol. Cancer Ther. 8 (2009) 3075e3087.
35] W. Quaglia, G. Santoni, M. Pigini, A. Piergentili, F. Gentili, M. Buccioni,
M. Mosca, R. Lucciarini, C. Amantini, M.I. Nabissi, P. Ballarini, E. Poggesi,
A. Leonardi, M. Giannella, Structure-activity relationships in 1,4-benzodioxan-
related compounds. 8.1 {2-[2-(4-chlorobenzyloxy)phenoxy]ethyl}-[2-(2,6-
dimethoxyphenoxy)ethyl]amine (clopenphendioxan) as a tool to highlight
[
[
[
[
[
16] G. Caliendo, F. Fiorino, P. Grieco, E. Perissutti, V. Santagada, S. Albrizio, L. Spadola,
G. Bruni, M.R. Romeo, Synthesis and binding affinities for 5-HT1A, 5-HT2A and 5-
HT2C receptors of a series of 1- and 2-(4-arylpiperazinylalkyl)-4-(benzoyl)-
[
1,2,3-triazole derivatives, Eur. J. Med. Chem. 34 (9) (1999) 719e727.
[
[
[
17] G. Caliendo, F. Fiorino, P. Grieco, E. Perissutti, V. Santagada, B. Severino, G. Bruni,
M.R. Romeo, Synthesis of new 1,2,3-benzotriazin-4-one-arylpiperazine deriva-
tives as5-HT1A serotonin receptorligands, Bioorg.Med. Chem. 8 (2000)533e538.
18] G. Caliendo, F. Fiorino, E. Perissutti, B. Severino, S. Gessi, E. Cattabriga, P.A. Borea,
V. Santagada, Synthesis by microwave irradiation and binding properties of
novel 5-HT1A receptor ligands, Eur. J. Med. Chem. 36 (11e12) (2001) 873e886.
19] G. Caliendo, F. Fiorino, E. Perissutti, B. Severino, D. Scolaro, S. Gessi,
E. Cattabriga, P.A. Borea, V. Santagada, A convenient synthesis by microwave
heating and pharmacological evaluation of novel benzoyltriazole and
the involvement of
human PC-3 prostate cancer cell apoptosis and proliferation, J. Med. Chem. 48
2005) 7750e7763.
a1D- and a1B -adrenoreceptor subtypes in the regulation of
(
[
36] N. Dizeyi, P. Hedlund, A. Bjartell, M. Tinzl, K. Austild-Taskén, P.A. Abra-
hamsson. Serotonin activates MAP kinase and PI3K/Akt signaling pathways in
prostate cancer cell lines, Urol. Oncol., in press, doi:10.1016/j.urolonc.2009.09.
013