Antiproliferative activity of phenylbutyrate ester of haloperidol metabolite II [(±)-MRJF4] in prostate cancer cells

Article abstract of DOI:10.1016/j.ejmech.2010.10.012

Complex mechanisms of prostate cancer progression prompt to novel therapeutic strategies concerning a combination of drugs or of single molecules able to interact with more crucial targets. Histone deacetylase inhibitors and sigma ligands with mixed σ₁ antagonist and σ₂ agonist properties were proposed as new potential tools for treatment of prostate cancer. (±)-MRJF4 was synthesized as phenylbutyrate ester of haloperidol metabolite II, which is a molecule consisting of a histone deacetilase inhibitor (4-phenylbutyric acid) and a sigma ligand (haloperidol metabolite II). Antiproliferatives activities of 4-phenylbutyric acid, haloperidol metabolite II, equimolar mixture of both compounds and (±)-MRJF4 were evaluated in vitro on LNCaP and PC3 prostate cancer cells. Preliminary binding studies of (±)-MRJF4 for σ₁, σ₂, D₂ and D₃ receptors and inhibition HDAC activity were reported. MTT cell viability assays highlighted a notable increase of antiproliferative activity of (±)-MRJF4 (IC₅₀ = 11 and 13 μM for LNCaP and PC3, respectively) compared to 4-phenylbutyric acid, haloperidol metabolite II and the respective equimolar pharmacological association. (±)-MRJF4 was also used in combination with σ₁ agonist (+)-pentazocine and σ₂ antagonist AC927 in order to evaluate the role of σ receptor subtypes in prostate cancer cell death.

Full text of DOI:10.1016/j.ejmech.2010.10.012

European Journal of Medicinal Chemistry 46 (2011) 433e438
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Antiproliferative activity of phenylbutyrate ester of haloperidol
metabolite II [(ꢀ)-MRJF4] in prostate cancer cells
, ,
,
,
Agostino Marrazzo a 1, Jole Fiorito a 1, Laura Zappalà b 1, Orazio Prezzavento a, Simone Ronsisvalle a,
*
Lorella Pasquinucci a, Giovanna M. Scoto b, Renato Bernardini c, Giuseppe Ronsisvalle a
a Department of Pharmaceutical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy
b Department of Pharmaceutical Sciences, Pharmacology Section, University of Catania, Viale A. Doria 6, 95125 Catania, Italy
c Department of Experimental and Clinical Pharmacology, University of Catania, Viale A. Doria 6, 95125 Catania, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Complex mechanisms of prostate cancer progression prompt to novel therapeutic strategies concerning
a combination of drugs or of single molecules able to interact with more crucial targets. Histone
deacetylase inhibitors and sigma ligands with mixed s1 antagonist and s2 agonist properties were
proposed as new potential tools for treatment of prostate cancer. (ꢀ)-MRJF4 was synthesized as phe-
nylbutyrate ester of haloperidol metabolite II, which is a molecule consisting of a histone deacetilase
inhibitor (4-phenylbutyric acid) and a sigma ligand (haloperidol metabolite II). Antiproliferatives activ-
ities of 4-phenylbutyric acid, haloperidol metabolite II, equimolar mixture of both compounds and
(ꢀ)-MRJF4 were evaluated in vitro on LNCaP and PC3 prostate cancer cells. Preliminary binding studies of
Received 4 August 2010
Accepted 11 October 2010
Available online 15 October 2010
Keywords:
Histone deacetylase
Inhibitors
LNCaP
PC3
(ꢀ)-MRJF4 for s1
,
s2, D2 and D3 receptors and inhibition HDAC activity were reported. MTT cell viability
M for
assays highlighted a notable increase of antiproliferative activity of (ꢀ)-MRJF4 (IC50 ¼ 11 and 13
m
Sigma receptors
LNCaP and PC3, respectively) compared to 4-phenylbutyric acid, haloperidol metabolite II and the
respective equimolar pharmacological association. (ꢀ)-MRJF4 was also used in combination with s1
agonist (þ)-pentazocine and s2 antagonist AC927 in order to evaluate the role of
s receptor subtypes in
prostate cancer cell death.
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
activity, which results in aberrant transcription of key genes that
regulate important cellular functions such as cell proliferation, cell-
cycle regulation and apoptosis [6]. Histone acetylation is a dynamic
reversible process regulated by histone acetyltransferase (HAT) and
HDAC enzymes that are very essential to the epigenetic regulation
of gene transcription [7]. An imbalance in the equilibrium of acet-
ylation and deacetylation has been associated with carcinogenesis
and cancer progression [8]. HDAC overexpression causes a decrease
in histone acetylation. This effect is associated with an alteration of
oncogene and oncosuppressor activity as well as tumor develop-
ment [9]. Therefore, histone deacetylase inhibitors (HDACi), alone
or in combination with other drugs, are emerging as a new class of
anticancer agents and have been demonstrated to exert antitumor
effects such as growth arrest, differentiation and apoptosis [10].
Butyrate was the first HDACi discovered [11]. A related com-
pound, 4-phenylbutyric acid, has reached phase I/II clinical trials for
the treatment of refractory solid tumor malignancies. This com-
pound has good compliance and tolerability but has lesser potency
compared to current HDAC inhibitors [12,13].
Prostate cancer is one of the most common causes of death in
western countries and is the second leading cause of cancer-related
death in men [1]. Prostate cancer is a heterogeneous tumor com-
posed of multiple types of cells that have a variable response to
endocrine therapy [2]. The conversion of an androgen-sensitive
cancer into an androgen-refractory cancer consequently increases
the risk of metastasis and remains a relevant clinical issue [3].
Several pharmacological agents, alone or in combination, have been
proposed for the treatment of hormone-resistant prostate cancer.
However, complex mechanisms underlie prostate cancer progres-
sion, and novel therapeutic strategies use a combination of drugs
that interact with crucial targets involved in prostate cancer [4,5].
The development of several human cancers, including prostate
cancer, is related to an alteration of histone deacetylase (HDAC)
* Corresponding author. Tel.: þ39 095 7384019; fax: þ39 095 222239.
E-mail address: marrazzo@unict.it (A. Marrazzo).
Several studies have demonstrated that prostate cancer cell lines
overexpress sigma (
s) receptors, a class of membrane receptor
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.10.012

434
A. Marrazzo et al. / European Journal of Medicinal Chemistry 46 (2011) 433e438
proteins composed of s1 and s2 subtypes [14].
s receptors are found
in different areas of the central nervous system and of healthy
tissues, such as liver, lungs, gonads, endocrine glands and kidneys.
They are located at subcellular level in plasma membranes, mito-
chondria and endoplasmic reticulum. The two subtypes can be
distinguished pharmacologically, functionally, and by their molec-
ular size. The s1 receptors have been cloned and have shown to be
distinct from any other known receptor class [15]. The s1 receptor is
a 25 kDa, single polypeptide with two putative trans-membrane
regions. The s2 receptor is a 18e21 kDa protein which has not yet
been cloned [16]. Both receptor subtypes have been detected in
multiple types of tissue and are highly expressed in various tumor
cell lines. The high density of
s receptors, particularly the s2
receptors in cells and tumor tissue compared to normal tissue,
suggested their use in diagnostic imaging of tumors such as PET
(Positron Emission Tomography) [17] and SPECT (Single Photon
Emission Computed Tomography) [18]. In particular, some
s ligands
were used as markers for prostate cancer because of the high
expression of these receptors in hormone sensitive prostate cell lines
(LNCaP) and hormone refractory (DU-145 and PC3) [19,20]. More-
Fig. 1. Chemical structures of 4-phenylbutyric acid, haloperidol metabolite II,
(ꢀ)-MRJF4, (þ)-pentazocine, haloperidol and AC927.
over, recent studies using
s ligands that are conjugated to drug
delivery systems, such as liposomes and nanoparticles, have de-
monstrated the selective delivery of chemotherapeutic substances,
antisense oligodeoxynucletides or small interfering RNAs to tumor
3. Result and discussion
cells that overexpress
s receptors [21e23].
Among all ligands, we focused our studies on haloperidol
s
Evaluation of the s1 and s2 affinity of (ꢀ)-MRJF4 determined
that the esterification of the hydroxyl group on the haloperidol
metabolite II with 4-phenylbutyric acid decreased the affinity for s1
and s2 receptors. However, the compound maintained a low
metabolite II, which inhibits the proliferation of various cancer
cells, by depleting Ca2þ from the endoplasmatic and mitochon-
drial stores, thereby inducing apoptosis [24,25]. In contrast to the
conventional antipsychotic haloperidol, haloperidol metabolite II
micromolar affinity and the ability to interact with
s receptors
displays a preferential activity at
mine receptors [26,27]. In addition,
expression of P-glycoprotein (P-gp),
s
receptors compared to dopa-
compounds decrease the
multidrug resistance
(Table 1). In regard to dopaminergic D2 and D3 receptors, (ꢀ)-MRJF4
s
had a low or negligible binding affinity in comparison to haloperidol
a
metabolite II and haloperidol keeping a good selectivity for
receptors. As expected, 4-phenylbutyric acid did not have affinity for
the receptor subtypes.
The effects on the proliferation of LNCaP (androgen-sensitive)
s
transporter involved in the efflux transport of several cytotoxic
drugs and increase the efficacy of conventional chemotherapy and
P-gp-mediated chemosensitivity in some drug-resistant tumors
[28,29]. Recent study showed that exposure of cancer cells to
HDACi promotes drug resistance and reduces treatment efficacy
s
and PC3 (androgen-refractory) prostate cancer cells were measured
using an MTT assay. Table 2 shows the IC50 values of (ꢀ)-MRJF4
compared to 4-phenylbutyric acid, haloperidol metabolite II and an
equimolar mixture of both compounds.
[30], the inhibition of P-gp expression by
s ligands might coun-
teract the HDACi effect and provide an effective combinational
therapy.
(ꢀ)-MRJF4 was by far the most potent compound to inhibit cell
The role of different
recently evaluated with several
conclusion that ligands that have s2 agonist and s1 antagonist
s
receptor subtypes on tumor cells has been
growth in the two prostate cancer cell lines with an IC50 value of
s
ligands. These studies led to the
approximately 11 mM for LNCaP cells and 13 mM for PC3 cells at 24 h.
s
Moreover, in comparison with the equimolar mixture, (ꢀ)-MRJF4
effects may be potential antineoplastic agents [28,31].
was approximately 15-fold more potent. Superimposable results
In this report we evaluate antiproliferative effects of a novel
compound, named (ꢀ)-MRJF4, an ester of 4-phenylbutyric acid
(HDACi) and haloperidol metabolite II (s1 antagonist and s2
agonist) (Fig. 1). The antiproliferative activity of (ꢀ)-MRJF4 in
prostate cancer cell lines LNCaP and PC3 was compared to the
effects of 4-phenylbutyric acid, haloperidol metabolite II and an
equimolar mixture of both compounds. Preliminary, binding
affinity of this new compound to sigma (s1, s2), dopamine (D2, D3)
receptors as well as HDAC inhibition were reported. Moreover, with
the use of putative s1 agonist (þ)-Pentazocine and s2 antagonist
AC927 the contribution of each
activity was established.
s subtypes on antiproliferative
2. Chemistry
(ꢀ)-MRJF4 was obtained by the reduction of 4-chloro-1-(4-
fluorophenyl)-1-butanone 1 with NaBH4 in EtOH (Scheme 1). The
reaction of alcohol 2 and 4-phenylbutanoyl chloride gave ester 3,
which was treated with 4-(4-chlorophenyl)piperidin-4-ol in DMF
to achieve the final compound as racemic mixture.
Scheme 1. Synthesis of (ꢀ)-MRJF4. Reagents and conditions: (i) EtOH, NaBH4, r.t.; (ii)
Ph(CH2)3COCl, THF, 4-DMP, r.t.; (iii) 4-(4-chlorophenyl)-piperidin-4-ol, DMF, 75 ꢁC.

A. Marrazzo et al. / European Journal of Medicinal Chemistry 46 (2011) 433e438
435
Table 1
Sigma-1, sigma-2, D2 and D3 binding assays of (ꢀ)-MRJF4, haloperidol metabolite II,
haloperidol and 4-phenylbutyric acid.
Compounds
Ki (nM) ꢀ S.E.M.
s1
s2
105 ꢀ 12
D2
D3
( )-MRJF4
Haloperidol metabolite II
haloperidol
162 ꢀ 20
>5000
>5000
2.3 ꢀ 0.7 0.98 ꢀ 0.3 232 ꢀ 46 1095 ꢀ 245
2.2 ꢀ 0.5 16 ꢀ 1.7
> 10000 >10000
2.1 ꢀ 0.3
5.8 ꢀ 3.7
4-phenylbutyric acid
n.d.a
n.d.a
a
Not determined.
were obtained from the 48, 72 and 96 h treatments (data not
shown).
LNCaP and PC3 cells were treated with various concentrations of
(ꢀ)-MRJF4 (1e25
mM) and vehicle (0.1% DMSO) for 24, 48, 72 and
96 h. As shown in Fig. 2, cell growth was inhibited to a variable
degree in the presence of (ꢀ)-MRJF4 having half-maximum effects
at approximately 10 mM.
(ꢀ)-MRJF4 exerted potent antiproliferative effects in a time- and
dose-dependent manner in LNCaP cells (IC50 ¼ 10.87 mol/l at 24 h;
mol/l at 96 h). A similar result was obtained in PC3
m
IC50 ¼ 6.65
m
cells, except that these cells required a longer incubation time.
Higher IC50 values were obtained in comparison to the LNCaP cells
Fig. 2. Percent growth as compared to vehicle treated control (% cell viability) for
LNCaP and PC3 cells treated with (ꢀ)-MRJF4 for 24, 48, 72, 96 h. (n ¼ 4). *p < 0.05
relative to negative controls. Error bars were omitted for clarity; see Table 3 for an
indication of reproducibility.
(IC50 ¼ 12.86
mmol/l at 24 h; IC50 6.78 mmol/l at 96 h). The inhibitory
concentration (IC50) was calculated for each incubation time and is
reported in Table 3.
The antiproliferative effect of haloperidol metabolite II has been
related to s1 antagonist and s2 agonist properties [32,33]. There-
fore, we evaluated the contribution of each receptor subtypes to
antiproliferative activity of (ꢀ)-MRJF4 on LNCaP and PC3 cells using
prototypic selective s1 agonist (þ)-pentazocine (PT) [32] and s2
HDAC activity was also tested in LNCaP and PC3 cell lines that
were treated for 1 h with TSA (1
mM), 4-PHB (1.5 mM) and
(ꢀ)-MRJF4 (10 M) (Fig. 5). The inhibition of HDAC activity in the
m
nuclear extracts after 1 h of treatment was similar to the HDAC
inhibition induced by (ꢀ)-MRJF4 in the HeLa extracts. These results
suggest that, contrary to 4-PHB and the potent inhibitor TSA,
(ꢀ)-MRJF4 does not have direct inhibitory effects on HDAC
enzymes. Rather, the inhibition of LNCaP and PC3 cell proliferation
antagonist AC927 (AC) [31,34] (Fig. 3). Results showed that both
s
receptor subtypes were involved in the antiproliferative effects of
(ꢀ)-MRJF4 but with a prevalence of the s2 receptors. Specifically,
on LNCaP cells, treatment with (ꢀ)-MRJF4 induced a loss of cell
viability of about 45% while treatment with (ꢀ)-MRJF4 and
(þ)-pentazocine, which blocks the s1 activity of (ꢀ)-MRJF4,
induced a loss of cell viability of about 40%. Whereas, treatment
with (ꢀ)-MRFJ4 and AC927, which contrasts the s2 activity of
(ꢀ)-MRJF4, caused a loss of cell viability of about 20%. Similar
results were obtained on hormone-refractory PC3 cells but in this
case the loss of cell viability was approximately 10%.
may be related to the high expression of
s receptors in prostate
cancer cell lines [20,21], the subsequent enhancement of
(ꢀ)-MRJF4 uptake and intracellular hydrolysis into 4-phenybutyric
acid and haloperidol metabolite II. In fact, several studies [18e24]
have confirmed that the overexpression of
s receptors in cancer
cells is an important tool to selectively increase the concentration
of radiomarker ligands or cytotoxic substances in tumor cells. The
appreciable binding affinity of (ꢀ)-MRJF4 is in agreement with this
hypothesis.
The ability of (ꢀ)-MRJF4 to inhibit HDAC activity was measured
in HeLa extracts in situ using
a fluorescently-labeled HDAC
substrate. Trichostatin A (TSA) was used as a positive control for
maximal inhibition. Fig. 4 shows relative HDAC activities measured
in triplicate. TSA at a concentration of 1
mM almost completely
4. Conclusion
inhibited HDAC activity, while 4-phenylbutyric acid (4-PHB) at
a concentration of 1.5 mM induced a minimum of 60% inhibition
compared to the untreated control. (ꢀ)-MRJF4 did not inhibit HDAC
In summary, we have reported the synthesis of (ꢀ)-MRJF4
a conjugate ester of phenylbutyric acid (HDAC inhibitor) and
haloperidol metabolite II (s2 agonist/s1 antagonist ligand), as
a novel tool for treatment of prostate cancer. (ꢀ)-MRJF4 has
activity at a concentration of 10 mM.
showed good binding affinity and selectivity for
s
receptors and
Table 2
Antiproliferation activity on LNCaP and PC3 cell lines.a
Table 3
Compounds
LNCaP Cells
IC50 M) s.e.
PC3 Cells
IC50 M) s.e.
Growth inhibition of cell treated with (ꢀ)-MRJF4.a
(
m
(m
(ꢀ)-MRJF4
LNCaP Cells
PC3 Cells
( )-MRJF4
11 ꢀ 1.5
2324 ꢀ 0.1
177 ꢀ 7.6
190 ꢀ 1.7
13 ꢀ 3.3
2273 ꢀ 0.1
208 ꢀ 1.3
165 ꢀ 3.0
4-Phenylbutyric acid
Haloperidol metabolite II
4-Phenylbutyric acid D Haloperidol
metabolite II
IC50
(
m
M) ꢀ s.e.
IC50
(
m
M) ꢀ s.e.
24 h
48 h
72 h
96 h
10.87 ꢀ 1.54
8.83 ꢀ 3.83
8.13 ꢀ 7.89
6.65 ꢀ 4.04
12.86 ꢀ 3.3
11.02 ꢀ 8.1
7.8 ꢀ 8.8
a
IC50 values ꢀ s.e. of (ꢀ)-MRJF4, 4-phenylbutyric acid, haloperidol metabolite II
and equimolar pharmacological association of two drugs on LNCaP and PC3 cells for
24 h. IC50 values are averaged from multiple determinations (n ꢂ 3).
6.78 ꢀ 3.14
a
IC50 values are averaged from multiple determinations (n ꢂ 3).

436
A. Marrazzo et al. / European Journal of Medicinal Chemistry 46 (2011) 433e438
Fig. 3. Effect of (ꢀ)-MRJF4 þ PT and (ꢀ)-MRJF4 þ AC927 on LNCaP and PC3 cells after 24 h of exposure. *P < 0.05 (ANOVA followed by Fisher’s test) vs. CTRL. ꢁP < 0.05 (ANOVA
followed by Fisher’s test) vs. (ꢀ)-MRJF4.
antiproliferative activity mainly mediated by s2 subtypes.
(ꢀ)-MRJF4 in comparison with the single compounds or the equi-
molar mixture of 4-phenylbutyric acid and haloperidol metabolite
II acquired an enhanced potency to produce anticancer effects.
Consequently, we can assume that the combination in the same
molecule of HDAC inhibition activity and s2 agonist/s1 antagonist
properties could be a useful strategy to develop novel anticancer
agents. Further studies on (ꢀ)-MRJF4 and its enantiomers are in
progress in our laboratories and will be reported in the future.
[1,3-di-(2-tolyl)guanidine] (31 Ci/mmol), obtained from Per-
kineElmer, [3H]spiperone (20 Ci/mmol) was obtained from NEN and
[3H]-7OH-DPAT (139 Ci/mmol) was obtained from Amersham (U.K.).
5.2. Chemical synthesis
5.2.1. (ꢀ)-4-Chloro-1-(4-fluorophenyl)butan-1-ol (2)
NaBH4 (0.483 g, 12.78 mmol) was added to a solution of (1)
(5.0 g, 24.92 mmol) in EtOH (30 mL) at 0 ꢁC. The mixture was stirred
at room temperature for 12 h. The reaction mixture was quenched
with water (20 mL) and evaporated to remove the organic portion.
The residue was diluted in saturated Na2CO3 solution and extracted
using CHCl3 (3 ꢃ 50 mL). The combined organic layers were dried
over anhydrous Na2SO4, filtered and evaporated in vacuum to
obtain (2) (4.69 g, 93% of yield) as a yellow oil, which was used
without further purification.
5. Experimental
5.1. Materials and methods
Solvents and reagents were obtained from commercial suppliers
and were used without further purification. Flash chromatography
purification was performed on a Merck silica gel 60 0.040-0.063 mm
(230e400 mesh) stationary phase. Melting points were determined
using a Thomas Hoover apparatus and are uncorrected. Nuclear
magnetic resonance spectra (1H NMR and 13C NMR recorded at 200
and 50 MHz respectively) were obtained on VARIAN INOVA spec-
trometers using the CDCl3 or DMSO-d6 solvents. TMS was used as an
internal standard. Coupling constants (J) are reported in hertz.
Microanalysis (C, H, N) was performed on a Carlo Erba instrument
model E-1110, and the results agreed with the theoretical values
(ꢀ0.4%). Thin-layer chromatography (TLC) was performed on silica
gel plates with a fluorescence indicator of F254 (0.2 mm, E. Merck);
the spots were visualized by UV light. The following radioactive
materials were used: [3H]-(þ)-pentazocine (45 Ci/mmol), [3H]DTG
5.2.2. (ꢀ)-4-Chloro-1-(4-fluorophenyl)butyl 4-phenylbutanoate (3)
4-Chloro-1-(40-fluorophenyl)-butanol (2) (1.0 g, 4.93 mmol) was
dissolved in anhydrous THF (35 mL) and 4-N,N-dimethylaminopir-
idin (0.6 g 4.93 mol) was added while stirring continuously. A
solution of 4-phenylbutanoyl chloride (0.9 g, 4.93 mmol) in THF
was added dropwise at 0 ꢁC and the reaction mixture was stirred at
room temperature for 48 h. The reaction was quenched with water
(10 mL), the organic solvent was then evaporated under vacuum
and the residue was dissolved in AcOEt. The residue was then
treated with a solution of 1 N HCl, followed the addition of
a NaHCO3 (5%) solution. The organic layer was dried over anhydrous
Na2SO4, filtered and evaporated under reduced pressure to obtain
a crude yellow oil product. Purification by flash chromatography
Fig. 5. The HDAC activity of LNCaP and PC3 nuclear extracts after (a) 1 h of treatment
Fig. 4. The HDAC activity of HeLa extracts in the presence of positive control (TSA
with positive control (TSA 1
untreated control (DMSO). Experiments were performed in triplicate and the bars
indicate ꢀ S.D.
m
M), 4-PHB (1.5 mM) and (ꢀ)-MRJF4 (10
mM), relative to
1
m
M), 4-PHB (1.5 mM) and (ꢀ)-MRJF4 (10
mM), relative to untreated control (DMSO).
Experiments were performed in triplicate and the bars indicate ꢀ S.D.

A. Marrazzo et al. / European Journal of Medicinal Chemistry 46 (2011) 433e438
437
(10e90% AcOEt in cyclohexane) yielded (3) (1.1 g, 60% of yield) as
colorless oil. 1H NMR (200 MHz, CDCl3):
¼ 7.33-6.98 (m, 9H), 5.74
(t, J ¼ 6.0 Hz, 1H), 3.52 (m, 2H), 2.61 (t, J ¼ 6.0 Hz, 2H), 2.38-2.30 (m,
2H), 2.06-1.62 ppm (m, 6H); 13C NMR (50 MHz, CDCl3):
162.60 (d, JCF ¼ 245.5 Hz), 141.44, 136.26 (d, JCF ¼ 3.65 Hz), 128.67,
128.62, 128.43 (d, JCF ¼ 7.9 Hz), 126.24, 115.69 (d, JCF ¼ 21.9 Hz),
74.64, 44.66, 35.23, 33.94, 33.79, 28.77, 26.63 ppm.
inactivated fetal bovine serum and 50 U/ml penicillin/streptomycin,
10 nM HEPES, 1 mM sodium pyruvate and 2.5 g/L D-(þ)-glucose.
Cells were maintained at 37 ꢁC in a humidified 5% CO2 atmosphere.
Drug treatments were performed by preparing a 1:1000 dilution
from the dimethyl sulfoxide (DMSO) stock solutions in culture
medium immediately before use.
d
d
¼ 172.85,
5.5. Cell viability assay
5.2.3. (ꢀ)-4-[4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl]-
1-(4-fluorophenyl)butyl 4-phenylbutanoate [(ꢀ)-MRJF4]
Cell viability was measured using an MTT (3-[4,5-dimethylth-
iazol-2yl]-2,5-diphenyl tetrazolium bromide) growth assay
according to the manufacturer’s instructions [38]. This quantitative
colorimetric assay is based on the ability of the cells to reduce the
MTT tetrazolium salt, a pale yellow substrate, to a dark-blue for-
mazan product. In viable mitochondria, succinate dehydrogenases
cleave the tetrazolium ring into formazan crystals. DMSO is added
to dissolve the insoluble crystals into a colored solution. Cells were
plated in 96-well plates at a density of 4 ꢃ 104 cells per well and
allowed to adhere overnight. The experiments were performed in
quadruplicate and began by replacing the medium with fresh
medium containing various concentrations of the compounds
under investigation (haloperidol metabolite II, 4-phenylbutiric
acid, haloperidol metabolite II þ 4-phenylbutyric acid, (ꢀ)-MRJF4,
(þ)-pentazocine and AC927). Cells were incubated for different
time periods (24, 48, 72 and 96 h) with compounds at the optimal
concentrations without refreshing the medium to carry out the
A
mixture of 4-(4-chlorophenyl)piperidin-4-ol (0.217 g,
1.026 mmol), NaHCO3 (0.086 g, 1.026 mmol) and compound (3)
(0.179 g, 0.513 mmol) in anhydrous DMF was stirred at 75 ꢁC for
12 h. The organic solvent was evaporated under vacuum and the
residue was dissolved in water (50 mL) followed by an extraction
with CH2Cl2 (3 ꢃ 50 mL). The combined organic layers were dried
over anhydrous Na2SO4, filtered and evaporated in a vacuum to
obtain a crude yellow oil product. Purification by flash chromatog-
raphy (20e80% AcOEt in cyclohexane) yielded ( )-MRJF4 (0.113 g,
43% of yield) as colorless oil, which was transformed into an oxalate
salt: mp: 132.5e135.2 ꢁC; 1H NMR (200 MHz, DMSO-d6):
d
¼ 7.46-
7.07 (m, 13H), 6.36 (br s, 3H), 5.67 (t, J ¼ 6.0 Hz, 1H), 3.28-3.03 (m,
6H), 2.53-2.12 (m, 6H), 1.79-1.66 ppm (m, 8H); 13C NMR (50 MHz,
[D6]DMSO):
d
172.35, 164.8, 161.5 (d, JCF ¼ 242.45 Hz), 147.01, 141.35,
136.63, 131.48, 128.49, 128.33, 128.06, 126.71, 125.91, 115.50, 115-07,
73.91, 67.98, 48.03, 34.74, 34.26, 33.04, 32.84, 26.29, 19.97 ppm;
Anal. Calcd for C31H35ClFNO3$C2H2O4: C 64.54, H 6.07, N 2.28, found:
C 64.21, H 6.28, N 2.55.
tests described. Following the appropriate incubation period, 10 mL
MTT (10% total volume) was added to each well, and the cells were
incubated for an additional 4 h. The media from culture plates
containing adherent cells was then aspirated using a fine-point
Pasteur pipette and the crystals were solubilized in DMSO. The
absorbance was measured at 570 nm using a spectrophotometer.
The ED50 values (dose at which 50% of the metabolic activity
compared to the vehicle control remains) were derived from the
dose effect curves.
5.3. Radioligand binding assays
Guinea pig membranes were prepared as previously reported by
Matsumoto et al.[35] and Mach et al.[36] Sigma-1 binding affinity
was determined incubating membrane aliquots (400
mL, 500 mg
protein) to equilibrium (150 min at 37 ꢁC) with 3 nM [3H]-
(þ)-pentazocine (45 Ci/mmol) and increasing concentrations of
displacing ligands in 50 mM TriseHCl (pH 7.4) to a total volume of
5.6. Nuclear protein extraction
1 mL. Nonspecific binding was assessed in the presence of 10
haloperidol. For the sigma-2 binding assays, the membranes
(300 L, 360 g protein) were incubated (120 min at room temper-
ature) with 3 nM [3H]DTG [1,3-di-2-tolylguanidine] (31 Ci/mM) in
the presence of 0.4
mM
Total nuclear protein was extracted using the NEPER extraction
kit (Pierce). LNCaP and PC3 cells were seeded in 6 mm2 dishes at
a density of 3 ꢃ 105 cells per dish and were allowed to adhere
overnight. The following day the medium was replaced by medium
m
m
m
M (þ)-SKF10,047 to block the s1 sites. The
incubation was performed in 50 mM TriseHCl (pH 8.0) to a total
volume of 0.5 mL. Nonspecific binding was evaluated in the pres-
containing substrate and (ꢀ)-MRJF4 (10
mM), Trichostatin A (TSA)
(1 M) or 4-phenylbutiric acid (4-PHB) (1.5 mM). After 1 h and again
m
ence of 5
m
M DTG. The rat striatum and rat olfactory tubercle were
after 24 h the cells were washed with PBS and harvested in 100 mL
of lysis buffer (50 mmol/L Tris, pH 7.6, 150 mmol/L NaCl, 5 mmol/L
EDTA, 1 mmol/L phenyl methyl sulfonyl fluoride, 0.5 mg/mL leu-
peptin, 5 mg/mL aprotinin, and 1 mg/mL pepstatin). The samples
were centrifuged at 16,000 ꢃ g for 30 min at 4 ꢁC. The resulting
supernatants were isolated and protein concentration was deter-
mined by the Bradford (1976) method [39]. Subsequently, the
separation of cytoplasmic and nuclear proteins was performed.
Forty milligrams of cells were spun down into a 1.5 mL micro-
centrifuge tube at 500 ꢃ g for 3 min. After spinning, the supernatant
was discarded. Two hundred microliters of ice-cold CER I was then
added to the cell pellet. The mixture was vortexed vigorously for
15 s to fully resuspend the pellet. The tube was then incubated on
ice for 10 min. Eleven microliters of ice-cold CER II was added, and
the samples were vortexed for 5 s. Another 1-min incubation on ice
was followed by vortexing for 5 s. Next, the mixture was centrifuged
at 16,000 ꢃ g for 5 min. The supernatant (cytoplasmic extract) was
transferred to a new pre-chilled tube. For the extraction of nuclear
used for D2 and D3 receptors, respectively. Tissue preparations and
binding assays were carried out according to Mennini et al. [37].
After incubation, the samples were filtered through Whatman GF/B
or GF/C glass fiber filters, which were pre-soaked in a 0.5% poly-
ethylenimine solution, using a Millipore filter apparatus. The filters
were washed twice with 4 ml of a suitable ice-cold buffer, and
radioactivity was counted in 4 ml of “Ultima Gold MV” using a 1414
Winspectral Perkin Elmer Wallac liquid scintillation counter. Inhi-
bition constants (Ki values) for the tested compounds were calcu-
lated using the EBDALIGAND program 34 purchased from Elsevier/
Biosoft.
5.4. Cell culture
The androgen-sensitive cell line, LNCaP, and the androgen-
refractory cell line, PC3, were purchased from the American Type
Culture Collection. PC3 cells were maintained in RPMI 1640
medium supplemented with 10% v/v heat-inactivated fetal bovine
serum and 50 U/ml penicillin/streptomycin. LNCaP cells were
cultured in RPMI 1640 medium supplemented with 10% v/v heat-
proteins, the insoluble pellet was resuspended in 100 mL of ice-cold
NER and vortexed for 15 s. The sample was put on the ice for 10 min
and vortexed for 15 s. This step was repeated 4 times for a total of

438
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HDAC activity was measured using the HDAC Fluorescent
Activity assay/Drug Discovery kit according to the manufacturer’s
instructions (AK-500, BIOMOL International) [40,41]. TSA and the
fluorescent substrate were purchased with the Fluor-de-Lys kit. The
assay measures the activity of HDACs to deacetylate a substrate that
contains acetylated lysine side chains. The HeLa extracts (supplied
in the kit) were added to each well in a 96-well plate except wells
containing the untreated control samples. The assay buffer, TSA
(1
m
M), 4-phenylbutyric acid (1.5 mM) and (ꢀ)-MRJF4 (10
mM) were
added to appropriate wells. Only the assay buffer solution was
added to the LNCaP and PC3 extracts (treated as described in the
nuclear proteins extraction procedure). The HDAC reaction initiated
by adding the Fluor de Lys substrate (25 mL) to each well and mixing
thoroughly. The assay was performed at 25e37 ꢁC. The reaction was
quenced after 10 min with the Fluor the Lys developer solution
(50
mL) and the plate was incubated at room temperature for
10e15 min. The substrate is deacetylated by the HDAC1, HDAC2,
HDAC3, HDAC4, HDAC7, HDAC8, HDAC9 and SIRT1 enzymes. The
intensity of the fluorescent signal is indicative of the amount of
HDAC activity in the sample. The fluorescence of each reaction was
measured on a fluorometric reader (Perkin Elmer Wallac 1420-032
Victor2 multilaber counter) with the excitation set to 360 nm and
the emission set to 460 nm. The percent inhibition for each fluo-
rescence reading of the inhibited reaction was calculated relative to
the fluorescence of the control reaction. Experiments were per-
formed in triplicate.
Acknowledgments
Acknowledgments. This work was supported by grants (FIRB
2003, No RBNE03FH5Y and PRIN 2005 and 2007, No 2005032713)
from MIUR (Rome).
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