Haloperidol metabolite II prodrug: Asymmetric synthesis and biological evaluation on rat C6 glioma cells

Article abstract of DOI:10.1016/j.ejmech.2014.11.012

In a previous work we reported the antiproliferative effects of (±)-MRJF4, a novel haloperidol metabolite II (HP-mII) (a sigma-1 antagonist and sigma-2 agonist) prodrug, obtained through conjugation to 4-phenylbutyric acid (PhBA) [a histone deacetylase in

Full text of DOI:10.1016/j.ejmech.2014.11.012

European Journal of Medicinal Chemistry 90 (2015) 1e9
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Haloperidol metabolite II prodrug: Asymmetric synthesis and
biological evaluation on rat C6 glioma cells
,
,
,
Piera Sozio a 1, Jole Fiorito b 1, Viviana Di Giacomo a 1, Antonio Di Stefano a, Lisa Marinelli a,
Ivana Cacciatore a, Amelia Cataldi a, Stephanie Pacella a, Hasan Turkez c, Carmela Parenti d,
Antonio Rescifina d, Agostino Marrazzo d
,
*
a
ꢀ
Dipartimento di Farmacia, Universita degli Studi di Chieti Gabriele D'Annunzio, Via dei Vestini 31, 66100 Chieti, Italy
b Department of Pathology and Cell Biology and Taub Institute for Research on Alzheimer's Disease and the Aging Brain, Columbia University, 630 W 168th
St., New York, NY 10032, USA
c Department of Molecular Biology and Genetics, Faculty of Science, Erzurum Technical University, 25240 Erzurum, Turkey
d
ꢀ
Dipartimento di Scienze del Farmaco, Universita degli Studi di Catania, Viale Andrea 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:
In a previous work we reported the antiproliferative effects of ( )-MRJF4, a novel haloperidol metabolite
II (HP-mII) (a sigma-1 antagonist and sigma-2 agonist) prodrug, obtained through conjugation to 4-
phenylbutyric acid (PhBA) [a histone deacetylase inhibitor (HDACi)] via an ester bond. As a continua-
tion of this work, here we report the asymmetric synthesis of compounds (R)-(þ)-MRJF4 and (S)-
(ꢀ)-MRJF4 and the evaluation of their biological activity on rat C6 glioma cells, derived from glioblas-
toma multiforme (GBM), which is the most common and deadliest central nervous system (CNS) invasive
malignancy. Favourable physicochemical properties, high permeability in the parallel artificial membrane
permeability assay (PAMPA), good enzymatic and chemical stability, in vivo anticancer activity, associ-
ated with the capacity to reduce cell viability and to increase cell death by apoptosis, render compound
(R)-(þ)-MRJF4 a promising candidate for the development of a useful therapeutic for gliomas therapy.
© 2014 Elsevier Masson SAS. All rights reserved.
Received 1 February 2014
Received in revised form
4 November 2014
Accepted 5 November 2014
Available online 6 November 2014
Keywords:
Glioma
HDAC
Sigma receptors
Inhibitors
Medicinal chemistry
1. Introduction
signalling pathways relative to either growth or angiogenesis and
were efficient in preclinical models of gliomas [1]. Decreasing the
Malignant gliomas are the most common types of primary brain
tumours and remain one of the deadliest forms of brain cancer in
humans. New efficient chemotherapeutics for such malignant gli-
omas treatment were developed over the years and many are still
under investigation. There is evidence that the best treatment
consists of surgical resection followed by chemotherapy; combi-
nation of prednisone, lomustine and vincristine could increase
survival rate in children with gliomas, whereas temozolomide
could prolong the survival of adult patients [1]. Despite the fact that
different treatments are available, the prognosis remains poor,
particularly for glioblastoma multiforme (GBM), which has survival
rate of less than 3% at 3 years [2,3].
level of migration in various cancer cell types, including GBM,
commonly restores a certain level of sensitivity to apoptosis and/or
cytotoxic drugs [4]. Furthermore, was reported that glioma cells
tend to display an overexpression of sigma (s) receptors [5]. In this
regard, N-(1-benzylpiperidin-4-yl)-4-iodobenzamide (4-IBP), a se-
lective s1 agonist, demonstrated significant anti-migratory in vitro
activity in different analysed cancer cell lines, including the highly
motile human U373-MG GBM cell line [4,6e8]. On the other hand,
also haloperidol, a potent s1 antagonist used as anti-psychotic drug,
showed antiproliferative effects against glioma cells at low con-
centration (5 mM) [9].
Literature data reported that the prodrug approach was widely
used to improve the delivery of anticancer drugs (chlorambucil,
camptothecin, paclitaxel, doxorubicin, and vinblastine) [10]. In our
previous work, using this strategy, we synthesized ( )-MRJF4, a
novel ester prodrug of haloperidol metabolite II (HP-mII) for the
treatment of prostate cancer [10] (Fig. 1). HP-mII e endowed with
s1 antagonist and s2 agonist properties e resulted to be more
lipophilic than the parent drug following the esterification with
To date, new anticancer compounds that are currently in clinical
trial for gliomas are inspired from existing molecules selected for
other types of cancer. Mainly, these molecules target intracellular
* Corresponding author.
E-mail address: marrazzo@unict.it (A. Marrazzo).
These authors contribute equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2014.11.012
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

2
P. Sozio et al. / European Journal of Medicinal Chemistry 90 (2015) 1e9
(Scheme 1). The obtained compounds (1R)-(þ)-4-chloro-1-(4-
fluorophenyl)butan-1-ol (R)-(þ)-2 and (1S)-(ꢀ)-4-chloro-1-(4-
fluorophenyl)butan-1-ol (S)-(ꢀ)-2 were condensed with 4-(4-
methylphenyl)piperidin-4-ol, which is also reported in literature
[19], and then esterified with 4-phenylbutanoyl chloride, according
to the procedure already used by us [11], to give (R)-(þ)-MRJF4 and
(S)-(ꢀ)-MRJF4, respectively. All compounds were characterized by
their 1H and 13C NMR spectroscopic data that resulted superim-
posable with the literature ones [11,19].
Moreover, the reduction of compound 1 resulted highly enan-
tioselective and the target compounds (R)-(þ)-MRJF4 and (S)-
(ꢀ)-MRJF4 were isolated, after two recrystallization from ethyl
acetate/diisopropyl ether, almost enantiomerically pure (98% ee
and 99% ee, respectively) as ascertained by HPLC utilising a Chir-
alcel OJ[-RH] column (Fig. S1).
2.2. Biology
Major hurdle in the treatment of malignant glioma with sys-
temic chemotherapy is the restricted delivery, due to the presence
of BBB, of systemically administered agents for the therapy of brain
tumours.
Fig. 1. Chemical structures of MRJF4 enantiomers.
The physicochemical properties of drugs influence the diffusion
through the biological membranes; therefore, their evaluation may
be useful to understand the pharmacokinetics profile of drugs
employed as antineoplastic agents in CNS tumours. Indeed, the
apparent partition coefficient (log P) may be used to predict the
distribution of a drug in a biological system and can be correlated to
its adsorption, distribution, and CNS penetration. For both MRJF4
enantiomers water solubility and chemical stability were deter-
mined, while CLogP was theoretically calculated (Table 1); they
displayed low water solubility (1.2
lipophilicity values.
The chemical stability of ( )-MRJF4 was evaluated at pH 1.3 and
pH 7.4 using a 0.02 M phosphate buffer, containing 0.1% (v/v) of
Cremophor ELP at 37 ꢁC (Table 2). The compounds showed good
PhBA (an HDACi) thus facilitating its entrance into CNS. MTT cell
viability assays have highlighted a notable increase of anti-
proliferative activity of ( )-MRJF4 compared to PhBA, HP-mII, and
respective equimolar pharmacological association. ( )-MRJF4 has
also been used in combination with s1 agonist (þ)-pentazocine and
s2 antagonist AC927 to evaluate the role of
s receptor subtypes in
prostate cancer cell death [11].
In this study we report the asymmetric synthesis of prodrugs
(R)-(þ)-MRJF4 and (S)-(ꢀ)-MRJF4 (Fig. 1) and the evaluation of
their biological activity on rat C6 glioma cells, derived from GBM,
which is the most common CNS invasive malignancy [12,13]. Taken
into account that the blood brain barrier (BBB) restricts the delivery
of systemically administered agents for treating brain tumours, we
evaluated the pharmaceutical profiles of our new agents to deter-
mine their stability and the potential BBB permeability. Therefore,
the present study included the evaluation of chemical and enzy-
matic stability of (R)-(þ)-MRJF4 and (S)-(ꢀ)-MRJF4, their solubility
in Fasted State Simulated Intestinal Fluid (FASSIF), and their
respective permeability coefficient as measured by PAMPA assay.
Furthermore, we also investigated the effect of MRJF4 racemic
mixture and its two enantiomers on the molecular mechanisms,
which drive malignant C6 glioma cells proliferation and migration
since gliomas constitute nearly 60% of primary brain tumours by
inducing angiogenesis and infiltrating in the normal brain paren-
chyma. We also evaluated the ability of our prodrug ant its enan-
tiomers to inhibit HDAC3, a member of HDACs upregulated in solid
brain tumours; these prodrugs, being sensitive to esterases hy-
drolysis, can release PhBA, which is a well-known HDACi [14e16].
In fact, HDAC inhibitors (HDACis), alone or in combination with
other drugs, are emerging as a new class of anticancer agents and
were demonstrated to exert antitumour effects such as growth
arrest, differentiation, and apoptosis [17,18].
m
g mLꢀ1) and relatively high
2. Results and discussion
2.1. Chemistry
The synthesis of both enantiomers of the potential antineo-
plastic MRJF4 (4-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]-1-
(4-fluorophenyl)butyl 4-phenylbutanoate) was achieved via the
chiral reduction of 4-chloro-1-(4-fluorophenyl)-1-butanone (1)
with (þ)- or (ꢀ)-DIP-chloride, as also reported in literature [19],
Scheme 1. Synthesis of MRJF4 enantiomers. (i) (þ)-Ipc2BCl or (ꢀ)-Ipc2BCl, THF, 25 ꢁC,
(HOCH2CH2)2NH, diethyl ether; (ii) 4-(4-methylphenyl)piperidin-4-ol, NaHCO3, DMF,
80 ꢁC; (iii) 4-phenylbutanoyl chloride, THF, DMAP, r.t.

P. Sozio et al. / European Journal of Medicinal Chemistry 90 (2015) 1e9
3
Table 1
General characteristics of ( )-MRJF4 and its two enantiomers.
Table 3
s1, s2, D2 and D3 binding assays.
(
)-MRJF4 (R)-(þ)-MRJF4 (S)-(ꢀ)-MRJF4
Compound
Ki (nM) S.E.M.
Water solubility (
cLogPb
m
g/mL)a
1.2 0.2
6.95 0.45
5.25
e
e
e
e
e
e
s1
s2
D2
D3
(
)-MRJF4
162 20a
87.5 4.5
230 8.9
2.3 0.7a
2.0 0.4
3.0 0.8
2.5 0.6
70.1 3.2
105 12a
52.7 3.8
118 7.3
2.0 0.5
32.0 2.0
9.8 1.3
18.0 2.2
22.0 3.5
>5000a
>5000
>5000
>5000a
>5000
>5000
PAMPA BBB Peff (10ꢀ6 cm sꢀ1
)
c
(R)-(þ)-MRJF4
a
Values are means SD of three experiments.
n-Octanol/water partition coefficients were theoretically calculated by the
(S)-(ꢀ)-MRJF4
b
(
)-HP-mII
232 46a
256 7.4
71.0 3.5
2.3 0.7
n.d.b
1095 245a
1278 22
353 6.7
8.8 1.5
n.d.b
program ClogP for windows, version 2.0 (Biobyte Corp., Claremont, CA), according to
(R)-(þ)-HP-mII
(S)-(ꢀ)-HP-mII
Haloperidol
DTG
the methods based on the atom-additive and fragmental approaches.
c
“CNSþ” (high BBB permeation predicted), Peff ꢂ 4.0 ꢃ 10ꢀ6 cm sꢀ1; “CNS” (BBB
uncertain permeation), Peff from 4.0 to 2.0 ꢃ 10ꢀ6 cm sꢀ1; “CNSe” (low BBB
permeation predicted), Peff < 2.0 ꢃ 10ꢀ6 cm sꢀ1 [20,21].
a
Ref. [11].
Not determined.
b
stability both at pH 1.3 and 7.4 (about 5 days). The degradation
products were not characterized.
in the presence and absence of co-solvents, indicating that the
presence of Cremophor ELP did not alter the capacity of the phos-
pholipid layer to act as a barrier [21].
Our results indicate that ( )-MRJF4 showed good permeability
(Peff ꢂ4 ꢃ 10ꢀ6 cm sꢀ1) (Table 1) and, consequently, both (R)-
(þ)-MRJF4 and (S)-(ꢀ)-MRJF4 enantiomers seem to be very
promising in BBB penetration [25,26].
Considering these results, binding affinity and selectivity were
measured for both enantiomers of MRJF4 and HP-mII, by con-
ducting competitive binding assays [11,27]. In the s1, s2, dopamine
D2 and D3 assays, guinea pig membrane (for s1 and s2), rat striatum
(for D2), and rat olfactory tubercle (for D3) tissue membranes were
used as receptor sources. Moreover, [3H]-(þ)-pentazocine, [3H]-1,3-
di(2-tolyl)guanidine ([3H]-DTG) with unlabelled (þ)-pentazocine,
[3H]-spiperone and [3H]-7-OH-DPAT were used as radioactive
tracers.
The enzymatic stability of both MRJF4 enantiomers was also
studied at 37 ꢁC in rat and human plasma (Table 2). As could be
deduced from the rate of hydrolysis (kobs) both compounds un-
dergo a fast hydrolysis in rat plasma, whereas in human plasma
they were fairly stable. Taken together, these results point out that
(R)-(þ)- and (S)-(ꢀ)-MRJF4 are sufficiently stable in the acidic
environment of the stomach and are potentially absorbed by the
intestine after oral administration.
Moreover, to study the stability of ( )-MRJF4 in the presence of
glioma C6 cells we adopted a mass spectrometric approach. Glioma
C6 cells were incubated with ( )-MRJF4 (5 m
M) for 24 h at 37 ꢁC,
and then the cell lysates were analysed [22] to verify the potential
generation of metabolites within cells; in particular, a signal at m/z
214 was observed in the control cell lysates (Fig. S2, B). We iden-
tified additional prominent ions at m/z 524, 360, and 212 in the
( )-MRJF4-treated cell lysates (Fig. S2, C). The peak at m/z 524 re-
fers to MRFJ4 (Fig. S2, A), while the peak at m/z 360 was generated
from hydrolyses of ester bond followed by dehydration. We also
identified a minor product ion at m/z 212 corresponding to 4-(4-
Table 3 displays s1, s2, D2, and D3 receptor affinities of
(þ)-MRJF4, (ꢀ)-MRJF4, (þ)-HP-mII, and (ꢀ)-HP-mII relative to
respective racemic mixtures. The (þ)- and (ꢀ)-enantiomers of HP-
mII exhibited high s1 binding affinity (Ki ¼ 2.0
0.4 and
chlorophenyl)-4-hydroxypiperidine. At 24
h after ( )-MRJF4
3.0 0.8 nM, respectively). Moreover, (þ)-HP-mII showed lower
affinity to s2 receptor (Ki ¼ 32.0 2.0 nM) if compared with its
opposite isomer (ꢀ)-HP-mII (Ki ¼ 9.8 1.3 nM) and haloperidol
(Ki ¼ 18.0 2.2 nM), these data are in accord to literature ones [28].
The apparently anomalous higher affinity of ( )-HP-mII, for s2 re-
ceptors, with respect to its enantiomers, could be related to a
positive allosteric modulation of the two enantiomers, as previ-
ously reported in literature for sigma receptors [29e33]. Conversely
to haloperidol, (þ)-HP-mII and (ꢀ)-HP-mII showed a reduced af-
finity for dopamine D2 and D3 receptors. In particular, (þ)-HP-mII
displayed a 111-fold lower affinity and a 145-fold lower affinity for
treatment, we observed two main peaks originated from hydrolysis
of ester prodrug and one metabolite showing a reduced meta-
bolism within the glioma C6 cells at physiological pH.
In order to better predict the ability of drugs to diffuse through
the biological membranes, PAMPA was used as a non-cell-based
assay for measuring passive permeability of the investigated
compounds [23]. Depending on the phospholipid type, PAMPA can
mimic different adsorption/permeation environments. In partic-
ular, porcine polar brain lipid is used for BBB permeation assays
(PAMPA-BBB) [24]. Since our derivatives were not thoroughly sol-
uble in the conventional buffer used for PAMPA, the permeability
tests of new compounds were examined in the presence of co-
solvents (0.1% (v/v) Cremophor ELP). To demonstrate that co-
solvents do not change the permeability of the phospholipid layer
at the investigated concentrations, we evaluated their effect on the
permeability of Dopamine (DA), used as reference compound, due
to its inability to cross the membrane passively. After 18 h of in-
cubation, the effective permeability (Peff) of DA was irrelevant, both
D2 and D3 receptors (Ki
ꢀ
D2
¼
256
7.4 nM;
Ki D3 1278 22 nM) with respect to haloperidol
ꢀ
¼
(Ki ꢀ D2 ¼ 2.3 0.7 nM; Ki ꢀ D3 ¼ 8.8 1.5 nM), while (ꢀ)-HP-mII
displayed a Ki value of 71.0 3.5 nM and 353 6.7 nM for D2 and D3
receptors, respectively. According to our previously reported data
on ( )-MRJF4 [11], the esterification of the hydroxyl group on the
(þ)- and (ꢀ)-HP-mII enantiomers with PhBA decreased the affinity
Table 2
Chemical and enzymatic stabilities of ( )-MRJF4 and its two enantiomers.
Stability
(
)-MRJF4
(h)
(R)-(þ)-MRJF4
(h)
(S)-(ꢀ)-MRJF4
(h)
t
½
kobs (hꢀ1
)
t
kobs (hꢀ1
)
t
½
kobs (hꢀ1
)
½
Chemicala
pH 1.3
pH 7.4
Rat plasma
Human plasma
140.1 4.2
127.2 2.5
0.108 0.004
92.0 1.4
0.005 0.001
0.005 0.001
6.41 0.16
e
e
e
e
e
e
e
e
Enzymatica
0.312 0.004
84.3 1.1
2.22 0.07
0.008 0.001
0.224 0.002
90.7 1.0
3.09 0.09
0.008 0.001
0.008 0.001
a
Values are means SD of three experiments.

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P. Sozio et al. / European Journal of Medicinal Chemistry 90 (2015) 1e9
for both
s
receptor subtypes. Nevertheless, good s1 and s2 affinities
s1 87.5 4.5 nM;
3.8 nM) compared to (ꢀ)-MRJF4
Annexin-V/PI staining which allows the detection of apoptotic
were found in (þ)-MRJF4 (Ki
ꢀ
¼
features by detecting phosphatidylserine exposure at the mem-
brane level. Under treatment conditions, the amounts of Annexin
Vpos/PIpos late apoptotic cells and Annexin Vneg/PIpos necrotic ones
were significantly increased by ( )-MRJF4 and both enantiomers
(Fig. 2); this increase was more significant after 48 h of treatment
compared to 24 and 72 h samples. In particular, (R)-(þ)-MRJF4 was
the most effective compound (about 40% of late/necro) followed by
( )-MRJF4 and (S)-(ꢀ)-MRJF4 (35% and 25%, respectively).
Ki s2 52.7
ꢀ
¼
(Ki ꢀ s1 ¼ 230 8.9 nM; Ki ꢀ s2 ¼ 118 7.3 nM) and ( )-MRJF4
(Table 3). Compounds (þ)-MRJF4 and (ꢀ)-MRJF4, analogously to
racemate MRJF4, showed insignificantly affinity for dopamine D2
and D3 receptors (Ki > 5000 nM).
In the present study, we also investigated the effect of both
enantiomers of MRJF4 on the molecular mechanisms of glioma cell
migration and invasion. To assess the effects of (R)-(þ)-MRJF4, (S)-
(ꢀ)-MRJF4, and their racemic mixture on C6 cells, in terms of
apoptosis and cell death, we performed flow cytometry analysis of
The effect of all five compounds on C6 cell proliferation was
studied after 24, 48, and 72 h of treatment at different concentra-
tions ranging from 0 to 5
mM as time and dose response experiment
(Fig. 3). Cell proliferation was inhibited in
a
concentration-
Fig. 2. Annexin V/PI detection of early apoptotic and apoptotic necrotic/late cells in C6
cells treated with 5
m
M compounds ( )-MRJF4, (R)-(þ)-MRJF4, (S)-(ꢀ)-MRJF4, PhBA
and ( )-HP-mII for 24, 48, and 72 h. Early apoptotic cell populations (Annexin-Vpos
/
Fig. 3. Effects of compounds
( )-HP-mII on C6 cell proliferation. Graphs show results of MTT assay after 24, 48, and
(
)-MRJF4, (R)-(þ)-MRJF4, (S)-(ꢀ)-MRJF4, PhBA and
PIneg
)
can be discriminated from late apoptotic (Annexin-Vpos/PIpos)/necrotic cells
(AnnexinVneg/PIpos) according to their fluorescence emission. *p < 0.05; **p < 0.01
72 h of treatment with increasing concentration of all five agents. *p < 0.05; **p < 0.01
relative to control sample.
relative to control sample.

P. Sozio et al. / European Journal of Medicinal Chemistry 90 (2015) 1e9
5
dependent manner. At 24 h, concentrations higher than 0.5
m
M
with ( )-HP-mII did not show any significant decrease of cell
viability; PhBA reduced cell viability of about 20%, while the cell
viability of samples treated with the other three compounds was
around 60%. At 72 h, only ( )-MRJF4, (R)-(þ)-MRJF4, and (S)-
(ꢀ)-MRJF4 retained an antiproliferative effect with cell viability
decrease of about 40%.
induced a significant reduction of cell viability in all treated sam-
ples, with (R)-(þ)-MRJF4 being the most effective (40% of the
control sample with respect to about 50% of (S)-(ꢀ)-MRJF4 and of
( )-MRJF4) with IC50 value of 5 mM. At 48 h, the samples treated
Since ( )-MRJF4 causes a notable increase of antiproliferative
activity in LNCaP and PC3 cell lines [11], flow cytometry cell cycle
analysis on C6 cells was performed. Our analysis showed an in-
crease in the S phase when cells were treated with ( )-MRJF4 and
both enantiomers for 72 h (Fig. 4), whereas shorter treatments did
not produce any significant changes. Such effects were quite
evident in the histograms events/DNA content of 72 h that showed
an increased S phase with ( )-MRJF4 and both enantiomers, while
in samples treated with PhBA and ( )-HP-mII S phase was found
comparable to controls.
Transwell chamber assay was employed to determine the effect
of these agents on the migration capability of C6 glioma cell line. As
shown in Fig. 5, the percentage of cells passing through the inserted
filter significantly decreased when cells were treated for 24 h with
compound ( )-MRJF4 and both enantiomers (less than 10% vs
almost 60% in the control sample). On the other hand, PhBA and
( )-HP-mII had no effect on the capability of C6 glioma cell
migration.
To evaluate the inhibitory activity of our compounds, histone3
(H3) acetylation was also investigated at 6, 15, and 24 h after
treatment (Fig. 6). After
6 h the inhibition was especially
augmented by (R)-(þ)-MRJF4 (16.1% vs 4.5% of the DMSO);
( )-MRJF4 had the same activity as PhBA (about 14%) while the
other compounds did not produce any significant effects on
acetylation. After 15 h of treatment, there were no changes in all
samples with respect to the vehicle alone, whereas after 24 h (R)-
(þ)-MRJF4 showed an increase in H3 acetylation (7.2 vs 4.5 of the
control sample); this effect could probably be due to the meta-
bolic pathways involving both enantiomers. Specifically, first hy-
drolysis of ester generates HP-mII and the PhBA responsible for
acetylation of H3 at 6 h. Subsequently, as already reported in
literature, HP-mII could undergo additional metabolism gener-
ating 4-(p-fluorophenyl)-4-hydroxybutyric acid and/or haloper-
idol metabolite III able to interfere with HDAC activity only after
24 h [22,34,35].
3. Conclusions
Glioma is an aggressive cancer characterized by high mortality,
especially in children. It is known that glioma cells tend to display
an overexpression of
s receptors. This study was aimed to explore
the potential effects of haloperidol metabolite II prodrugs as useful
Fig. 4. Effects of a 5
m
M concentration of compounds ( )-MRJF4, (R)-(þ)-MRJF4, (S)-
(ꢀ)-MRJF4, PhBA, and ( )-HP-mII on cell cycle progression of C6 cells. The amount of
cells in G1, S, and G2/M phase can be detected correlating the number of events with
the fluorescence emission on FL3. Graphs show the percentage of cells found in G1, S
and G2/M phase.
Fig. 5. Effects of a 5
m
M concentration of compounds ( )-MRJF4, (R)-(þ)-MRJF4, (S)-
(ꢀ)-MRJF4, PhBA and ( )-HP-mII on C6 cell migration after 24 h. *p < 0.05; **p < 0.01;
***p < 0.001 relative to control sample.

6
P. Sozio et al. / European Journal of Medicinal Chemistry 90 (2015) 1e9
Analytical HPLC measurements were run on a Waters 600 HPLC
pump (Waters Corporation, Milford, MA, USA), equipped with a
Waters 2996 photodiode array detector, a 20 L Rheodyne injector
and a computer-integrating apparatus. HPLC was performed using
a Waters Symmetry RP-C18 column (150 ꢃ 4.6 mm, 5 m); the
m
m
mobile phase consisted in a mixture of acetonitrile, water, and
formic acid. Two channels were used: channel A with acetonitrile/
water 5/95 and 0.1% v/v of formic acid; channel B with acetonitrile
and 0.1% v/v of formic acid. The gradient used was from 100% A to
100% B over 20 min,100% B was maintained for 5 min and in the last
minute, we came back to 100% of A. The flow rate was 1 mL min.
The UV-detector was set at a length of 264 nm. Enantioselective
HPLC analyses were carried out using the same above described
apparatus and mobile phase utilising a Chiralcel OJ[-RH] column
(150 ꢃ 4.6 mm, 5
mm).
Fig. 6. Effects of a 5
m
M concentration of compounds ( )-MRJF4, (R)-(þ)-MRJF4, (S)-
(ꢀ)-MRJF4, PhBA and ( )-HP-mII on H3 acetylation in C6 cells. Graph shows the
4.3. Chemistry
percentage of acetylated H3. *p < 0.05; **p < 0.01 relative to control sample.
4.3.1. (1R)-(þ)- and (1S)-(ꢀ)-4-chloro-1-(4-fluorophenyl)-butan-
1-ol, (R)-(þ)-2 and (S)-(ꢀ)-2
therapeutic tools for gliomas therapy. Taken together our results
indicate that the racemic mixture and the two enantiomers exhibit
good anticancer activity; they are able to reduce cell viability
(measured by MTT assay), and to increase cell death by apoptosis.
The obtained data indicate that cell proliferation is inhibited in
concentration- but not in a time-dependent manner. The amounts
of Annexin Vpos/PIpos late apoptotic cells and Annexin Vneg/PIpos
necrotic ones were significantly increased with all compounds; in
particular, results obtained with (R)-(þ)-MRJF4 were more
pronounced.
Both compounds were synthesized as already reported in
literature [19], and all analytical and spectral data are consistent
with the reported ones.
4.3.2. (1R)-(þ)- and (1S)-(ꢀ)-4-(4-chlorophenyl)-1-[4-(4-
fluorophenyl)-4-hydroxybutyl]piperidin-4-ol, (R)-(þ)-HP-mII and
(S)-(ꢀ)-HP-mII
Both compounds were synthesized as already reported in
literature [19], and all analytical and spectral data are consistent
with the reported ones. Here we report only the data inherent to
purity, obtained after two recrystallization from ethyl acetate/dii-
sopropyl ether.
4. Experimental section
(R)-(þ)-HP-mII, white solid, mp: 131e132 ꢁC; 98% ee,
20
4.1. Material and methods
[
C
C
a
]
¼ þ66.2 (c ¼ 1.5 in CHCl3); HRMS-FAB: m/z [M þ H]þ calcd for
D
21H26ClFNO2: 378.1636, found: 378.1633; Anal. calcd for
21H25ClFNO2: (C, H, N, O).
(þ)- or (ꢀ)-DIP-chloride, 4-chloro-1-(4-fluorophenyl)-1-
butanone,
4-(4-methylphenyl)piperidin-4-ol,
and
4-
(S)-(ꢀ)-HP-mII, white solid, mp: 132e133 ꢁC; 99% ee,
20
phenylbutanoyl chloride were purchased from Sigma Aldrich
(Milan, Italy). Cremophor® ELP was obtained from BASF-The
chemical Company. All other chemicals were of the highest purity
commercially available.
[a
]
¼ ꢀ67.7 (c ¼ 1.5 in CHCl3); HRMS-FAB: m/z [M þ H]þ calcd for
D
C
C
21H26ClFNO2: 378.1636, found: 378.1639; Anal. calcd for
21H25ClFNO2: (C, H, N, O).
4.3.3. (1R)-(þ)- and (1S)-(ꢀ)-4-[4-(4-chlorophenyl)-4-
hydroxypiperidin-1-yl]-1-(4-fluorophenyl)butyl 4-phenylbutanoate,
(þ)-(R)-MRJF4 and (ꢀ)-(S)-MRJF4)
4.2. General
To a solution of (R)-(þ)-HP-mII or (S)-(ꢀ)-HP-mII (400 mg,
1.058 mmol) in anhydrous THF (10 mL) 4-phenylbutanoyl chloride
The identity of all new compounds was confirmed by NMR data.
Homogeneity was confirmed by TLC on silica gel Merck 60 F254 and
their purities (>98%) were quantified by HPLC and HR-MS. Solu-
tions were routinely dried over anhydrous sodium sulphate prior to
evaporation. Chromatographic purifications were performed by
Merck 60 70e230 mesh ASTM silica gel column.
(181 m
L, 1.095 mmol) was added at 0 ꢁC and under stirring. The
reaction was left for 24 h at r.t. under a nitrogen atmosphere.
Subsequently, a NaHCO3 saturated solution (20 mL) was added and
the organic solvent was evaporated. After extraction with CH2Cl2
and purification by flash chromatography the final compound (R)-
(þ)- or (S)-(ꢀ)-MRJF4 was obtained as a colourless oil (250 mg
45%): Rf ¼ 0.33 (CHCl3/MeOH 95:5); 1H NMR (300 MHz, CDCl3):
NMR spectra were recorded on a Varian VXR 300 MHz spec-
trometer. Chemical shifts are reported in parts per million (d)
downfield from the internal standard tetramethylsilane (Me4Si).
The LC-MS/MS system used consisted of an LCQ (Thermo Finnigan)
ion trap mass spectrometer (San Jose, CA, USA) equipped with an
electrospray ionization (ESI) source. The capillary temperature was
set at 300 ꢁC and the spray voltage at 4.25 kV. The fluid was
nebulized using nitrogen (N2) as both the sheath and the auxiliary
gas. Melting points were determined on a Büchi B-450 apparatus
and are uncorrected. Optical rotations were taken at 20 ꢁC with a
PerkineElmer 241 polarimeter. Microanalyses were performed on a
EA1106 Carlo Erba CHN analyser; analyses indicated by the symbols
of the elements or functions were within 0.4% of the theoretical
values.
d
7.37e6.92 (m,13H, ArH), 5.67 (t, J ¼ 6 Hz,1H, CH), 3.65 (bs,1H, OH)
2.70e2.66 (m, 2H, CH2), 2.56e2.51 (m, 2H, CH2), 2.35e2.24 (m, 6H,
3CH2), 2.06e1.60 (m, 10H, 5CH2); 13C NMR (75 MHz, CDCl3):
d
172.71 (s, 1C, CO), 159.86 (s, J ¼ 330.2 Hz, 1C, Ar), 148.74 (s, 1C, Ar),
141.28 (s, 1C, Ar), 136.39 (s, 1C, Ar), 134.12 (s, J ¼ 21 Hz, 1C, Ar),
128.44 (s, J ¼ 54 Hz, 2C, Ar), 128.38 (s, 2C, Ar), 128.19 (s, 2C, Ar),
126.06 (s, 2C, Ar), 125.98 (s, 1C, Ar), 115.58 (s, J ¼ 38 Hz, 2C, Ar),
75.08 (s, 1C, CH), 71.00 (s, 1C, C), 58.12 (s, 1C, CH2), 49.36, (s, 2C,
CH2), 38.28 (s, 2C, CH2), 35.03 (s,1C, CH2), 34.18 (s,1C, CH2), 33.81 (s,
1C, CH2), 26.47 (s, 1C, CH2), 22.90 (s, 1C, CH2).
20
(R)-(þ)-MRJF4, 97.98% ee, Rt 3.21 min, [
a]
¼ þ65.4 (c ¼ 1.2 in
D
CHCl3); HRMS-FAB: m/z [M
þ
H]þ calcd for C31H36ClFNO3:

P. Sozio et al. / European Journal of Medicinal Chemistry 90 (2015) 1e9
7
524.2368, found: 524.2372; Anal. calcd for C31H35ClFNO3: (C, H, N,
O).
4.8. Stability studies of MRJF4 in glioma C6 cells
20
(S)-(ꢀ)-MRJF4, 98.89% ee, Rt 3.48 min, [
a
]
¼ ꢀ66.9 (c ¼ 1.2 in
Glioma C6 cell lysates (2 mg) were prepared for the LC-MS
analysis as reported by Kim et al. [22]. The stability of MRJF4 in
presence and in absence of glioma C6 cells (at 24 h) was assayed
using a LCQ™ Deca XP Plus LC/MSn spectrometer (Thermo Fin-
nigan, San Jose, CA, USA). The potential at the nanospray needle was
set at 4400 V. The orifice potential was 46 V, and the curtain gas
was 15 psi (pound-force per square inch).
D
CHCl3); HRMS-FAB: m/z [M
þ
H]þ calcd for C31H36ClFNO3:
524.2368, found: 524.2371; Anal. calcd for C31H35ClFNO3: (C, H, N,
O).
4.3.4. (R)-(þ)-MRJF4 and (S)-(ꢀ)-MRJF4) oxalates
Both enantiomers were transformed into oxalate salts to best
preserve them for biological tests. All spectral data are consistent
with the reported ones for ( )-MRJF4 [11].
4.9. PAMPA method
(R)-(þ)-MRJF4 oxalate, white solid, mp: 111e113 ꢁC; 98% ee,
HRMS-FAB: m/z [M þ H]þ calcd for C31H36ClFNO3: 524.2368, found:
524.2371; Anal. calcd for C33H37ClFNO7: (C, H, N, O).
The following protocol was applied to measure the Peff through
the artificial membrane to predict oral absorption and BBB
permeation. The effective permeability of BBB was measured using
phospholipid mixture from porcine polar brain lipid extract,
composed by phosphatidylcholine (PC) 12.6%, phosphatidyletha-
nolamine (PE) 33.1%, phosphatidylserine (PS) 18.5%, phosphatidy-
linositol (PI) 4.1%, phospatidic acid (PA) 0.8% and 30.9% of other
compounds (purchased from Avantis Polar Lipids-Alabaster, AL).
Each donor filtration plate well was carefully impregnated with
(S)-(ꢀ)-MRJF4 oxalate, white solid, mp: 112e114 ꢁC; 99% ee,
HRMS-FAB: m/z [M þ H]þ calcd for C31H36ClFNO3: 524.2368, found:
524.2365; Anal. calcd for C33H37ClFNO7: (C, H, N, O).
4.4. Kinetics of chemical hydrolysis
A 0.02 M phosphate buffer of pH 7.4 or a 0.02 M chloridric buffer
of pH 1.3, containing 0.1% (v/v) Cremophor ELP, was used to eval-
uate chemical stability at physiological pH. Reaction was initiated
by adding 1 mL of 10ꢀ4 M stock solution (in acetonitrile) of the
compound to 10 mL of thermostated (37 0.5 ꢁC) aqueous buffer
solution. At appropriate time intervals (for a total period of one
5
m
L of this solution and, immediately after, 150
mL of phosphate
buffer (pH 7.4/pH 6.5), containing 500 M of each compound, and
iPrOH 20% as co-solvent was added. Then the drug-filled donor
plate was placed into the acceptor plate that was prefilled with the
m
same buffer (300
mL) as acceptor solution. After plate lid was
replaced, the resulting assembled donor-acceptor plates were
incubated at r.t. for 18 h, following which drugs concentration in
the acceptor and donor solutions were determined by HPLC [38].
Log Peff can be calculated from the equation below:
week), samples of 20 mL were withdrawn and analysed by HPLC.
Pseudo-first-order rate constants (kobs) for the hydrolysis of the
compounds were then calculated from the slopes of the linear plots
of log (% residual compound) against time. The experiments were
run in triplicate and the mean values of the rate constants were
calculated [25,26].
!
ꢀ
ꢁ
½drugꢄacceptor
½drugꢄequilibrium
VD ꢃ VA
Log Peff
¼
ꢀ ln 1 ꢀ
ðVD þ VAÞ ꢃ A ꢃ t
where Peff is the effective permeability coefficient (cm sꢀ1), VD is
volume of donor compartment (0.15 cm3) and VA is volume of
4.5. Kinetics of enzymatic hydrolysis
acceptor compartment (0.30 cm3),
A is effective filter area
Human and rat plasma were obtained by centrifugation of blood
samples containing 0.3% citric acid at 3000 ꢃ g for 15e20 min.
Plasma fractions (4 mL) were diluted with 0.02 m phosphate buffer
(pH 7.4) to give a final volume of 5 mL (80% plasma). Incubation was
performed at 37 0.5 ꢁC using a shaking water bath. The reaction
(0.28 cm2), t is incubation time for the assay (s), [drug]acceptor is the
concentration of the compound in the acceptor compartment at the
completion of the assay, and [drug]equilibrium is the concentration of
compound at theoretical equilibrium.
was initiated by adding 200
in acetonitrile) to 5 mL of preheated plasma. Aliquots (100
taken at various times and deproteinized by mixing with 200
0.01 M HCl in methanol. After centrifugation for 5 min at 5000 ꢃ g,
10 L of the supernatant layer were analysed by chromatography as
m
L of a stock solution of drug (1 mg/mL
L) were
L of
m
4.10. Receptor binding studies
m
The s1, s2, D2 and D3 receptor binding studies were performed
m
according to literature [11,27]. Briefly, guinea pig brain membranes
described above. The amounts of remaining intact compound were
plotted as a function of incubation time [36].
(500
m
g protein) were incubated with 3 nM [3H]-(þ)-pentazocine
(29 Ci/mM; (Kd) was 14 0.3 nM, n ¼ 3) and six concentrations of
tested compounds or sigma ligands (from 10ꢀ5 to 10ꢀ10 M) in 1 mL
of 50 mM TriseHCl (pH 7.4). The reaction was performed for
150 min at 37 ꢁC and terminated by filtering the solution through
Whatman GF/B glass fibre filters which were presoaked for 1 h in a
0.5% poly(ethylenimine) solution. Filters were washed with ice-
cold buffer (2 ꢃ 4 mL). Nonspecific binding was assessed in the
4.6. Water solubility
Compounds (R)-(þ)-MRJF4 and (S)-(ꢀ)-MRJF4 (50 mg) were
placed in deionized water (1 mL), shaken at 25 ꢁC for 1 h to ensure
the solubility equilibrium and then centrifuged. The supernatant
presence of 10
made according to the following protocol: Guinea pig brain mem-
branes (360
g protein) were incubated with 3 nM [3H]DTG
mM of unlabelled haloperidol. s2 binding assays were
(20 mL) was analysed by HPLC [37].
m
4.7. Lipophilicity
(53.3 Ci/mM; Kd ¼ 11 0.8 nM; n ¼ 3) and each test compound
(from 10ꢀ5 to 10ꢀ10 M) in 0.5 mL of 50 mM TriseHCl (pH 8.0) for
120 min at room temperature in the presence of 400 nM
(þ)-SKF10,047 to mask s1 sites. Nonspecific binding was evaluated
n-Octanol/water partition coefficients were theoretically calcu-
lated by the program ClogP for windows, version 2.0 (Biobyte Corp.,
Claremont, CA), according to the methods based on the atom-
additive and fragmental approaches.
with DTG (5 mM). Each sample was filtered through Whatman GF/B
glass fibres filters, which were presoaked for 1 h in a 0.5%

8
P. Sozio et al. / European Journal of Medicinal Chemistry 90 (2015) 1e9
poly(ethylenimine) solution, using a Millipore filter apparatus.
Filters were washed twice with 4 mL of ice-cold buffer.
exclusion of aneuploid cells and nuclei doublets from further
analysis. PI fluorescence gathered at linear FL3 was used to measure
DNA content.
The rat striatum and rat olfactory tubercle were used for D2 and
D3 receptors, respectively. Tissue preparations and binding assays
were carried out according to Mennini et al. [39]. After incubation,
the samples were filtered through Whatman GF/B or GF/C glass
fibre 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.
Radioactivity was counted in 4 mL of ‘Ultima Gold MV’ in a 1414
Winspectral PerkinElmer Wallac or Beckman LS6500 scintillation
counter. Inhibition constants (Ki values) were calculated using the
EBDA/LIGAND program purchased from Elsevier/Biosoft.
4.15. Migration assay
Cell migration was assayed by means of a transwell chamber
containing a polycarbonate insert with 8 mm pores placed between
the upper and lower wells (Corning, NY, USA). Cells were cultured
to 70e80% of confluency, and then starved for 24 h in serum free
condition. C6 glioma cells were then trypsinized, centrifuged and
resuspended in serum free HAM’S F12 at a concentration of 105
cells mLꢀ1. 100
chamber of the transwell and 600
added in the lower chamber. Compounds, at a final concentration of
M, were added 2 h later in order to allow cells to adhere to
mL of such suspension was added to the upper
mL of HAM’S F12 with 5% FCS was
4.11. Cell culture
5
m
membrane. After 24 h of incubation at 37 ꢁC, cells on the upper side
of the filter were removed with a cotton swab, while cells that
migrated through the pores to the lower side of the membrane
were fixed with absolute methanol and stained with DAPI. The filter
was then cut out with a scalpel, mounted on a slide and nuclei were
counted under a microscope in five random fields whose area were
known.
C6 rat glioma cell line was obtained from the American Type
Collection (ATCC) and maintained in HAM'S F12 supplemented
with 2 mM Glutamine, penicillin-streptomycin (100 m
g mLꢀ1) and
10% FBS. Cells were grown at 37 ꢁC in a humidified atmosphere of
5% CO2.
4.12. Cell viability assay
To calculate migration, the total number of cells was determined
counting and averaging the total number of cells in each of the
random fields. The number obtained was divided by area of the
microscope viewing field and multiplied by the entire area of the
transwell insert. Percentage of migration was calculated by dividing
the total number of cells by the number of cells seeded and
multiplying this value by 100 to get percent.
Cell viability was measured by MTT (3[4,5-dimethylthiazol-2-
yl]-2,5-diphenyl tetrazolium bromide) growth assay, according to
manufacturer's instruction (Sigma Aldrich, St Louis, USA). In this
assay the cell number was quantified by the amount of tetrazolium
reduction in viable mitochondria. Cultured cells were incubated
into 24-well plate at 5 ꢃ 104 cells/well and exposed to various
concentrations of all the five agents (0.1e5 mM). After 24, 48, and
72 h cells were processed and the absorbance of each well was
detected at 570 nm. Percentage of viable cells was calculated using
the equation As/A0 ꢃ 100 where As is the absorbance value obtained
for a sample containing cells in the presence of a given concen-
tration of agent, and A0 is the absorbance value of vehicle treated
control. Four independent experiments were repeated under the
same experimental conditions.
4.16. Flow cytometry detection of acetylated histone H3
C6 cells were stained for acetylated H3 as previously described
[40]. Briefly, after incubation, cell culture medium was removed
and cells were fixed for 15 min in 1% p-formaldehyde on ice. Then
cells were trypsinized and pellets were washed with 1 mL of PBS/
BSA 1% and centrifuged at 130 g for 10 min at 4 ꢁC. Cell was then
permeabilized in 200
temperature. After washing, each pellet was resuspended in 100
mL of 0.1% Triton-X in PBS for 10 min at room
4.13. Annexin-V/PI detection of apoptotic and necrotic cells in flow
cytometry
m
L
of a saturation solution (PBS without calcium and magnesium
containing 10% of goat serum) and incubated for 20 min on ice.
Anti-Acetyl H3 (Lys 9) rabbit monoclonal antibody (Thermo Sci-
entific, OH, USA) was added diluted 1:100 in the saturation solu-
tion. Samples were incubated for 1 h on ice. Primary antibody was
removed and secondary FITC goat anti-rabbit IgG antibody (Milli-
pore, MA, USA) was added (20
dark for 45 min. Secondary antibody was removed and, prior to
running on the FC500, cells were resuspended in 400 L of PBS.
About 10000 events were collected for all samples on FC500 using
488 nm laser excitation and analysed with CXP software (Bekmann
Coulter). Mean fluorescence intensity (MFI) was obtained by his-
togram statistics and are provided to quantify the H3 acetylation.
To assess apoptosis,
a commercial Annexin-V-FITC/PI Kit
(Bender Med System, Vienna, Austria) was used according to the
manufacturer instructions. Briefly, 2.5 ꢃ 105 cells were gently
resuspended in binding buffer and incubated for 10 min at room
temperature in the dark with Annexin-V-FITC. Samples were then
washed, supravitally stained with propidium iodide (PI)
m
g mLꢀ1) and incubated on ice in the
(5 m
g mLꢀ1) and analysed on a FC500 flow cytometer with the FL1
m
and FL3 detector in a log mode using the CXP analysis software
(Beckmann Coulter, FL, USA). For each sample, at least 104 events
were collected. Viable cells were Annexin-Vneg/PIneg (unlabelled),
early apoptotic cells were Annexin-Vpos/PIneg, late apoptotic and
necrotic cells were Annexin-Vpos/PIpos and Annexin-Vneg/PIpos
respectively.
,
Appendix A. Supplementary data
4.14. Cell cycle analysis
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.11.012.
Approximately 3 ꢃ 105 cells per experimental condition were
harvested, fixed in 70% (v/v) cold ethanol and kept at 4 ꢁC over-
night. Cells were then resuspended in 20
m
g mLꢀ1 PI and
References
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m
g mLꢀ1 RNAse, final concentrations. Cell cycle profiles (104
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