Antiangiogenic Effect of (±)-Haloperidol Metabolite II Valproate Ester [(±)-MRJF22] in Human Microvascular Retinal Endothelial Cells

Article abstract of DOI:10.1021/acs.jmedchem.6b01039

(±)-MRJF22 [(±)-2], a novel prodrug of haloperidol metabolite II (sigma-1 receptor antagonist/sigma-2 receptor agonist ligand) obtained by conjugation to valproic acid (histone deacetylase inhibitor) via an ester bond, exhibits antiangiogenic activity, being able to reduce human retinal endothelial cell (HREC) viability in a comparable manner to bevacizumab. Moreover, (±)-2 was able to significantly reduce viable cells count, endothelial cell migration, and tube formation in vascular endothelial growth factor A (VEGF-A) stimulated HREC cultures.

Full text of DOI:10.1021/acs.jmedchem.6b01039

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Brief Article
Antiangiogenic Effect of (±)-Haloperidol Metabolite II Valproate ester
[(±)-MRJF22] in Human Microvascular Retinal Endothelial Cells
Melania Olivieri, Emanuele Amata, Shila Vinciguerra, Jole Fiorito, Giovanni Giurdanella,
Filippo Drago, Nunzia Caporarello, Orazio Prezzavento, Emanuela Arena, Loredana Salerno,
Antonio Rescifina, Gabriella Lupo, Carmelina Daniela Anfuso, and Agostino Marrazzo
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01039 • Publication Date (Web): 14 Oct 2016
Downloaded from http://pubs.acs.org on October 17, 2016
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Antiangiogenic Effect of (±)-Haloperidol Metabolite II
Valproate ester [(±)-MRJF22] in Human Microvascular Retinal
Endothelial Cells
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Melania Olivieri,§,‡ Emanuele Amata,†,‡ Shila Vinciguerra,† Jole Fiorito,† Giovanni Giurdanella,§
Filippo Drago,§ Nunzia Caporarello,§ Orazio Prezzavento,† Emanuela Arena,† Loredana Salerno,†
Antonio Rescifina,† Gabriella Lupo,§ Carmelina Daniela Anfuso§,* and Agostino Marrazzo†,*
†Department of Drug Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.
§Department of Biomedical and Biotechnological Sciences, School of Medicine, University of Catania, Viale A. Doria
6, 95125 Catania, Italy.
ABSTRACT: (±)-MRJF22 [(±)-2], a novel prodrug of haloperidol metabolite II obtained by conjugation to valproic acid via
an ester bond, exhibits antiangiogenic activity being able to reduce Human Retinal Endothelial Cells (HREC) viability in a
comparable manner to Bevacizumab. Moreover, (±)-2 was able to significantly reduce viable cells count, endothelial cell
migration and tube formation in Vascular endothelial growth factor A (VEGF-A) stimulated HREC cultures.
ment of several diseases linked with aberrant angiogene-
INTRODUCTION
sis, including macular degeneration.13, 14
Angiogenic processes play a critical role in the devel-
Sigma (σ) receptors have been implicated in a myriad of
opment of cancer, heart diseases, atherosclerosis, and var-
cellular functions, biological processes, and diseases.15
ious eye diseases such as wet age-related macular degen-
eration and proliferative diabetic retinopathy, that are
Two σ receptor subtypes are recognized and termed sig-
17
ma-1 (σ1) and sigma-2 (σ2).16,
The σ1 receptor is an
two of the most common causes of irreversible visual
loss.1 In particular, tumor growth is dependent on the ca-
pacity of neoplastic cells to induce vascular ingrowth.2, 3
In physiological conditions, the blood-retinal barrier
(BRB) is established by tight junctions between neighbor-
ing retinal endothelial cells. The retinal continuous endo-
thelium forms the main structure of the BRB and rests on
a basal lamina which is covered by the astrocyte feet,
Müller cells and pericytes. The integrity of BRB is essen-
tial for proper vision and its breakdown contributes to
retinal disorders.4 Cancer cells communicate with endo-
thelial cells (EC) and could activate them by cell contact
or by releasing soluble factors.5, 6 On the other hand, EC
induce cancer cell chemotaxis and tumor transendothelial
migration through the secretion of chemoattractants and
Matrix Metallopeptidase 9.7, 8 These are critical processes
in the initiation of metastasis. In this regard, in an in vitro
model of BRB, the presence of tumor cells determined a
strong pericytal loss, the reduction of the Transforming
Growth Factor Beta and the increase of VEGF-A levels.9
Cancers require VEGF for their survival and, so far, anti-
angiogenic therapies rely on neutralizing angiogenic fac-
tors such as VEGF or on blocking the receptor tyrosine
kinase intracellularly.10 The monoclonal antibody Bevaci-
zumab is the first approved inhibitor of VEGF clinically
available in the USA for the treatment of cancers in meta-
static phases, such as colorectal cancer.11, 12 Due to its anti-
angiogenic properties, Bevacizumab is used for the treat-
intracellular membrane-associated protein whose func-
19
tion is neuroprotective and neuroregulatory.18, Recent
data support the use of σ1 receptor antagonists in the
treatment of cancer due to cytotoxic and antiproliferative
effects of these ligands.20 Moreover, both σ receptors are
found in high density in a wide variety of human tumors
such as breast, colon, ovaries, lung and prostate, and this
feature has been used for the development of σ receptor
21, 22
ligands as a molecular probe for cancer imaging.15,
Clinical trials revealed that σ ligands can modulate EC
proliferation and control or inhibit angiogenesis.15, 23
Recently, the σ1 receptor has been found to dynamically
control the membrane expression of the human voltage-
dependent K+ channel hERG in different cancer cell lines.
As a consequence, the presence of σ1 receptor in cancer
cells increases VEGF secretion.24
The conventional antipsychotic haloperidol (HP) (Fig-
ure 1), shows cytotoxic and antiproliferative effects in
light of its σ1 antagonist and σ2 agonist activity. In human
brain microvascular EC, HP increases caspase activities,
chromatin condensation, and fragmentation, all events
indicating apoptosis.25
Differently from HP, (±)-haloperidol metabolite II [(±)-
HP-mII) (Figure 1), displays a preferential activity on σ
27
receptors compared to dopamine receptors.26, (±)-HP-
mII is endowed with a σ1 antagonist and σ2 agonist func-
tional profile and it inhibits the proliferation of various
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fast hydrolysis in rat plasma, whereas in human plasma it
cancer cells, by depleting Ca2+ from the cellular stores
thus triggering apoptosis.28
was fairly stable.
Scheme 1. Synthesis of compound (±)-2.a
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Figure 1. Chemical structures of compound HP, (±)-HP-mII,
(±)-1, (–)- and (+)-1 and (±)-2.
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In our previous works, we used a prodrug approach to
improve the (±)-HP-mII antiproliferative activity on
LNCaP and PC3 prostate cancer cells and in rat C6 glioma
30
cells derived from glioblastoma multiforme.29,
These
works culminated in the development of (±)-haloperidol
metabolite II butyrate ester [(±)-MRJF4] and its enantio-
mers (+)-MRJF4 and (–)-MRJF4 (Figure 1), three deriva-
tives obtained by esterification of (±)-HP-mII with
phenylbutyric acid, a Histone deacetylase inhibitors
(HDACIs). Compounds (±)-MRJF4 [(±)-1], (+)-MRJF4 [(+)-
1] and (–)-MRJF4 [(–)-1] showed a greater antiproliferative
activity respect (±)-HP-mII.30 Moreover, we demonstrated
that the antiproliferative activity induced by these com-
pounds was σ receptors mediated.29, 30 Here we report the
synthesis (±)-haloperidol metabolite II valproate ester
prodrug [(±)-MRJF22] (Figure 1) and the evaluation of its
biological activity on VEGF-A stimulated Human Retinal
Endothelial Cells (HREC), which offers an in vitro model
of BRB in a pro-angiogenic tumoral environment.
aReagents and conditions: (i) absolute EtOH and NaBH4 at 0
°C, then room temperature for 12 h; (ii) dry THF, 4-N,N-
dimethylaminopyridine, 2-propylpentanoyl chloride at 0 °C,
then room temperature for 24 h.
The in vitro receptor binding affinity of (±)-2, (±)-HP-
mII, HP, and VPA have been measured toward σ1, σ2 and
dopaminergic D2 and D3 receptors (Table 1).
Table 1. σ1, σ2, D2 and D3 binding assays of (±)-2, (±)-
HP-mII, HP, and VPA
Compounds
Ki (nM) ±SEMa
σ1
σ2
D2
D3
(±)-2
(±)-HP-mII
HP
13 ±0.4
2.9 ±0.8 2.4 ±0.5
2.7 ±0.5
>10000
124 ±11
>5000
241 ±38
2.5 ±0.7
n.d.b
>5000
1024 ±217
6.1 ±1.5
n.d.b
b
17.0 ±1.5
>10000
Moreover, (±)-2, being sensitive to esterases hydrolysis,
VPA
can release valproic acid (VPA), a well-known HDACI,31
aEach value is the mean ±SEM of three determinations; Not
determined.
that has been clinically investigated as an anticancer
33
drug.32,
Recently VPA has been implicated in anti-
angiogenic processes. Indeed VPA significantly inhibits
the in vitro angiogenic potential and VEGF expression in
human cervical cancer HeLa and SiHa cells and has anti-
angiogenic and antitumor activities mediated by increas-
ing the expression levels of vascular endothelial growth
inhibitor (VEGI) and death receptor 3 (DR3) in human
(±)-2 exhibits high affinity at the σ1 receptor and appre-
ciable affinity at σ2 receptor with Ki values of 13 and 124
nM, respectively. As expected, esterification of the
hydroxyl group of (±)-HP-mII with VPA reduces the bind-
ing affinity for both σ receptors. However, this modifica-
tion significantly reduces the affinity for D2 and D3 recep-
tors (Ki >5000 nM at both receptors). (±)-HP-mII showed
single-digit nanomolar affinity at both σ receptor sub-
types (Ki 2.9 and 2.4 nM, for σ1 and σ2 receptors respec-
tively), but in contrast with (±)-2 it keeps a significant
binding with D2 and D3 receptors (Ki 241 and 1024 nM, re-
spectively). HP, in accordance with the literature, has
shown a high σ1 and σ2 (Ki 2.7 and 17.0 nM respectively)
and dopaminergic D2 and D3 (Ki 2.5 and 6.1 nM) receptors
affinity.29 Finally, VPA does not show binding ability to-
ward σ1 and σ2 receptors (Ki >10000 nM at both receptors).
osteosarcoma cells.34, 35
.
RESULTS AND DISCUSSION
(±)-HP-mII was obtained by reduction of HP with
NaBH4 in EtOH. The reaction of (±)-HP-mII and 2-
propylpentanoyl chloride gave (±)-MRJF22 [(±)-2] as ra-
cemic mixture (Scheme 1).36
(±)-2 Water solubility and chemical stability were
determined while LogP was theoretically calculated (Ta-
ble S1). (±)-2 displayed low water solubility (1.4 mg/mL)
and relatively high lipophilicity values (theoretically cal-
culated logP 7.04). The chemical stability of (±)-2 was
checked in vitro at 37 ºC in aqueous buffer solutions of pH
1.3 (non-enzymatic simulated gastric fluid) and pH 7.4
(Table S2). The compound showed good stability both at
pH 1.3 and 7.4 (129 and 134 hours, respectively). The en-
zymatic stability of (±)-2 was also studied at 37 ºC in rat
and human plasma (Table S2). As could be deduced from
the rate of hydrolysis (kobs) the compound undergoes a
In order to assess the involvement in cell viability of
VPA, (±)-HP-mII, (±)-2 and HP, time and concentration
dependent HREC responses have been assayed by MTT
viability test. Table 2 shows the IC50 values of (±)-2 com-
pared to VPA, (±)-HP-mII and HP at four different time
periods (24, 48, 72 and 96 h).
VPA determined a reduction in HREC viability in the
mM range (1.22, 1.45, 1.39 and 1.48 mM at 24, 48, 72 and 96
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h respectively). (±)-HP-mII showed a time-dependent de- viability effects of (±)-HP-mII (Figure 3A). Specifically,
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crease in HREC viability. Indeed, at 24 h (±)-HP-mII in-
duced a reduction in cell viability lower than 50% at 200
μM, while at 48, 72 and 96 h (±)-HP-mII demonstrated a
moderate ability in reducing the HREC viability, with IC50
values of 128, 69 and 57 μM, respectively.
HREC incubation with (±)-HP-mII at 20 μM induced a
loss of cell viability of about 29% at 72 h, while treatment
with (±)-HP-mII and (+)-PTZ, as expected, completely re-
stored the loss of cell viability induced by (±)-HP-mII
alone. Incubation with (±)-HP-mII and AC927, similarly,
completely restored at 72 h the loss of cell viability in-
duced by (±)-HP-mII. These results outline a σ1 and σ2 re-
ceptors involvement in the reduction of cell viability in-
duced by (±)-HP-mII.
Table 2. MTT viability test on HREC. IC50 at four dif-
ferent time periods of (±)-2, HP-mII, HP and VPA
IC50 (μM)a (pIC50 ±SE)b
1217 (2.914 ±0.007)
1449 (2.839 ±0.026)
1393 (2.856 ±0.013)
1479 (2.830 ±0.013)
>200e
128.2 (3.892 ±0.037)
68.7 (4.163 ±0.028)
56.6 (4.247 ±0.096)
10.5 (4.980 ±0.019)
11.1 (4.956 ±0.035)
10.1 (4.996 ±0.037)
10.6 (4.973 ±0.071)
3.68 (5.434 ±0.041)
2.91 (5.536 ±0.082)
2.59 (5.586 ±0.072)
2.28 (5.641 ±0.0046)
9
Compounds
VPAc
Period (h)
24
48
72
96
24
48
72
96
24
48
72
96
24
48
72
96
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(±)-HP-mIIc
(±)-2c
HPd
aIC50 have been calculated with GraphPad prism 5 for win-
dows using a nonlinear fit transform sigmoidal dose-
response (variable slope). bpIC50 are defined as the -log(IC50).
cIC50 values are averaged from multiple determinations (n =
6). dIC50 values are averaged from multiple determinations (n
= 3). eCell viability reduction lower than 50% at 200 μM.
HP was the most potent compound in reducing cell vi-
ability, with IC50 spanning from 2.3 to 3.7 μM. (±)-2 de-
termined a decrease in cell viability, with IC50 values rang-
ing from 10 to 11 μM. Moreover, (±)-2, 5 μM induced, at 24
and 72 h, a significant decrease in cell viability, evaluated
by MTT test, (28% and 32%, respectively) compared to
the individual (±)-HP-mII and VPA incubated with HREC
in equimolar concentrations (5 μM) (Figure 2).
Figure 3. Effects of HP-mII (Panel A) and (±)-2 (panel B) in
combination with the selective σ1 agonist (+)-PTZ and σ re-
ceptors antagonist AC927 on HREC viability by MTT test.
RT-PCR for the identification of σ1 receptor expression on
HREC. The mRNA extract of the whole retina was a positive
control (Panel C).
On the other hand (Figure 3B), results showed that the
σ1 receptor is mainly involved in the cell viability effects of
(±)-2. Specifically, HREC treatment with (±)-2 at 5 μM in-
duced a loss of cell viability of about 25% at 72 h, while
treatment with (±)-2 and (+)-PTZ, completely restored
the loss of cell viability induced by (±)-2, assuming a σ1
antagonist profile for (±)-2. Whereas, treatment with (±)-
2 and AC927 caused a loss of cell viability of about 18% at
72 h. This result could be explained by the different affini-
ty of (±)-2 toward σ1 and σ2 receptors. (+)-PTZ (1 μM) at 72
h induced an improvement of HREC viability while com-
pound AC927 (1 μM) alone at 24 and 72 h did not induce
any effect in cells viability. (+)-PTZ (1 μM) and AC927 (1
Figure 2. Effect of (±)-2 (5 μM) and (±)-HP-mII plus VPA (5
μM, equimolar concentrations) on HREC viability by MTT
test.
(±)-HP-mII and (±)-2 have also been used in combina-
tion with the σ1 selective agonist (+)-Pentazocine [(+)-
PTZ] (1 μM) and σ receptors selective antagonist 1-
phenethylpiperidine (AC927) (1 μM), to evaluate by MTT
test the role of σ receptors on cell viability (Figure 3). Re-
sults showed that both σ receptors are involved in the cell
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μM) in combination at 24 and 72 h determined an im- nificant pro-angiogenic stimulus, increasing the alive cell
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provement in cells viability.
number of 52% at 24 h and 42% at 72 h (Figure 4B). Incu-
bation of (±)-2 at 5 μM with VEGF-A resulted in a reduc-
tion of the cell number of 28 and 30% at 24 and 72 h re-
spectively and this effect was comparable to Bevacizumab
200 µg/mL. In contrast, VPA and (±)-HP-mII alone or in
combination at 5 μM, did not affect the increased cell
number induced by VEGF-A.
The σ1 receptor was checked on HREC by the reverse
transcription polymerase chain reaction (RT-PCR) assay.
For the first time, we showed the considerable σ1 receptor
expression, which could explain (±)-HP-mII and (±)-2 ef-
fects on HREC (Figure 3C).
Trypan blue exclusion test experiments were performed
to investigate the cell viability after incubation with (±)-
HP-mII and (±)-2. In the first set of experiments, HREC
were grown in the presence of (±)-HP-mII (5, 20 and 50
μM) or (±)-2 (0.5, 1, 5 and 10 μM) for 24 and 72 h, in 1% of
fetal bovine serum (FBS) medium to determine optimal
concentration for assay (See Supporting Information, Fig-
ure S1).
Endothelial cell migration is a critical process for
wound healing and angiogenesis. Figure 5 shows the per-
centages of wound edge cell advancement 24 h and 72 h
after the scratch on the HREC confluent monolayer.
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When HREC monolayers were wounded and incubated
in medium containing 1% FBS, the closure of the wound,
as expected, was not complete even at 72 h. The incuba-
tion of HREC with VPA (5 μM) and HP-mII (5 μM) alone
or in combination (5 μM, equimolar concentration) and
(±)-2 (5 μM) did not change the response of cells in the
sense of migration (data not shown). VEGF-A induced a
significant enhancement of crossing cells (by almost 63%
at 24 h and 26% at 72 h) respect non-stimulated cells, and
a faster migration of HREC (panels A, B, and C). Interest-
ingly, in VEGF-A stimulated HREC, the presence of (±)-2
slowed down the wound closure values at levels similar to
the control in the absence of VEGF-A, both at 24 and 72 h
time points by 55% and 24% (panel A). In the presence of
VEGF-A, VPA or HP-mII at 5 µM did not change the re-
sponse of cells in the sense of migration (data not shown).
Also the equimolar combination of VPA and (±)-HP-mII
(5 µM) placed in the presence of the growth factor, in the
sense of migration, showed a very similar behavior of
VEGF-A treated HREC (panel A). The addition of HP at
sub-toxic concentration (2 µM) did not affect the HREC
migration either under basal conditions (control) or in
the presence of the VEGF-A 80 ng/mL (panel B), showing
that at this concentration, HP is not able to reverse the
effect on wound closure induced by VEGF-A. This charac-
teristic behavior of HP will be further evaluated in order
to clarify this experimental observation.
The presence of (+)-PTZ, a prototypical selective σ1 ag-
onist, attenuated the anti-migratory effect exerted by (±)-
2 on VEGF-A-stimulated cells in a dose-dependent man-
ner (1 and 2 µM) (panel C). This data, demonstrate that
the effect on cell migration induced by (±)-2 are mainly
mediated by the antagonism at the σ1 receptor. AC927,
both at 1 and 2 µM, did not affect the response of the
growth factor-stimulated HREC in the presence of (±)-2
(panel c), providing evidence on the fact that the σ2
receptor is not involved in the anti-angiogenic effect ex-
erted by (±)-2. The representative contrast phase images
of HREC after the scratch wound are showed in Support-
ing Information (Figure S2).
Figure 4. Effect of (±)-2, (±)-HP-mII, VPA, (±)-HP-mII plus
VPA, and Bevacizumab in the presence (panel B) or not
(panel A) of 80 ng/mL VEGF-A on alive HREC count. Bars
refer to alive cells excluding the dye in Trypan blue exclusion
test.
In the second set of experiments (Figure 4), at the cho-
sen concentrations, the effects of VPA, (±)-HP-mII, (±)-2,
and Bevacizumab on HREC viability were examined in
basal conditions (Figure 4A) and in the presence of VEGF-
A (Figure 4B), in order to mimic an in vitro angiogenic
stimulus. Under basal conditions, (±)-2 at 5 μM resulted
in a decrease in the number of the cells able to exclude
the dye (32% and 52% at 24 and 72 h, respectively). Nor
VPA neither (±)-HP-mII alone or in combination at 5 μM
and Bevacizumab at 200 µg/mL affected the cell counts
(panel A). As expected, VEGF-A induced in HREC a sig-
In Figure 6 the quantifications of the emerging branch
points from HREC in Matrigel tube formation assays are
reported. Cell adhesion and growth in VEGF-A condition-
ing environment were heavily magnified, with an increase
in the formation of branch points by almost 5-fold.
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ganization from HREC cell bodies on Matrigel are showed
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in Supporting Information (Figure S3). Finally, HDAC ac-
tivity inhibition of (±)-2 (5 μM) was measured in HeLa
extracts as an HDACs source using a fluorescently-labeled
HDAC substrate. (±)-2 at 5 μM concentration did not in-
hibit HDAC activity (see Supporting Information for de-
tails, Figure S4).
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Figure 6. Tube formation on Matrigel. The quantification of
emerging branch points from HREC bodies was performed
by using Angiogenesis analyzer tool for ImageJ software.
CONCLUSIONS
We describe the synthesis and the pharmacological
characterization of (±)-HP-mII Valproate ester, (±)-2.
Taken together our results indicate that (±)-2 exhibits
good anti-angiogenic activity, comparable to Bevaci-
zumab. Moreover, we have shown that (±)-2 was able to
significantly reduce the viable cells count, endothelial cell
migration and tube formation in VEGF-A stimulated
HREC cultures and that these effects are σ1 receptor me-
diated. Finally, we have demonstrated the presence of σ1
receptor in HREC by RT-PCR. Based on our results, the
prodrug (±)-2 shows significant anti-angiogenic activity
compared to (±)-HP-mII and HP, and for this reason, it
could be a promising candidate for the development of a
therapeutic strategy for the treatment of diseases related
to angiogenesis.
Figure 5. Effect of (±)-2, (±)-HP-mII plus VPA, HP, (+)-PTZ
and AC927 on VEGF-A-stimulated HREC migration after
wound healing assay and quantified as a percentage of
wound closure by using ImageJ software.
EXPERIMENTAL SECTION
In VEGF-A treated cells, HP-mII (5 μM) induced a sig-
nificant reduction of the ramification number by 20%.
VPA (5 μM) had no significant effect (data not shown).
The presence of (±)-HP-mII plus VPA caused a decrease
in the ramification number only by 22% in VEGF-A
treated cells, demonstrating the significant anti-
angiogenic effect by (±)-2 respect to the individual com-
ponents taken into account in co-incubation conditions.
The tube formation in vitro was strongly reduced (by
67%) when HREC were incubated with (±)-2. HP did not
affect the biological effect in VEGF-A stimulated HREC.
Again, unlike AC927, which had no effect, (+)-PTZ revert-
ed almost completely the (±)-2 inhibitory effect on the
VEGF-A-stimulated tube formation, confirming the data
obtained for the wound closure assay. The representative
contrast phase images of tube formation and spatial or-
General Remarks. Reagent grade chemicals were pur-
chased from Sigma-Aldrich (St. Louis, MO, USA) or
Merck (Darmstadt, Germany) and were used without fur-
ther purification. Flash chromatography purification was
performed on a Merck silica gel 60 0.040‒0.063 mm
(230‒400 mesh) stationary phase. Melting points were de-
termined on a Büchi B-450 apparatus and are uncorrect-
13
ed. Nuclear magnetic resonance spectra (1H NMR and C
NMR recorded at 200 and 500 MHz) were obtained on
VARIAN INOVA spectrometers using the CDCl3. TMS was
used as an internal standard. Coupling constants (J) are
reported in hertz. Purities of all tested compounds,
whether synthesized or purchased, reached at least 95%
as determined by microanalysis (C, H, N) that was per-
formed on a Carlo Erba instrument model E1110; all the
results agreed within ±0.4% of the theoretical values.
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Journal of Medicinal Chemistry
Thin-layer chromatography (TLC) was performed on sili-
ca gel Merck 60 F254 plates; the spots were visualized by
UV light.
ASSOCIATED CONTENT
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
Tables S1,2, Figure S1,2,3,4, procedures for in vitro biological
and pharmacokinetic assays are reported in Supporting In-
formation.
1
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4-(4-chlorophenyl)-1-(4-(4-fluorophenyl)-4-
hydroxybutyl)piperidin-4-ol [(±)-HP-mII]. NaBH4
(0.051 g, 1.36 mmol) was added to a solution of 4-(4-(4-
chlorophenyl)-4-hydroxypiperidin-1-yl)-1-(4-
AUTHOR INFORMATION
fluorophenyl)butan-1-one (HP) (1 g, 1.36 mmol) in EtOH
(50 mL) at 0 ºC. The mixture was stirred at room tempera-
ture for 12 h. The reaction mixture was quenched with wa-
ter (20 mL) and evaporated to remove the organic por-
tion. The residue was diluted in saturated Na2CO3 solu-
tion and extracted using CHCl3 (3 × 50 mL). The com-
bined organic layers were dried over anhydrous Na2SO4,
filtered and evaporated in vacuum. Purification by flash
chromatography (1:9 EtOH/CHCl3) yielded HP-mII (0.904
Corresponding Author
9
* Agostino Marrazzo, Tel: (+39) 0957384250. E-mail: marraz-
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zo@unict.it.
* Carmelina Daniela Anfuso, Tel: (+39)
0957384070. E-mail: anfudan@unict.it.
Author Contributions
The manuscript was written through contributions of all au-
thors. / All authors have given approval to the final version of
the manuscript. / ‡These authors contributed equally.
1
g, 90% of yield) as a white solid. mp 144‒146 °C. H NMR
(500 MHz, CDCl3): δ 7.43 (d, J = 8.31 Hz, 2H), 7.24‒7.37
(m, 4H), 6.99 (t, J = 8.56 Hz, 2H), 4.63 (d, J = 6.85 Hz, 1H),
3.00 (br. d, J = 10.76 Hz, 1H), 2.79 (br. d, J = 10.76 Hz, 1H),
2.59 (t, J = 11.00 Hz, 1H), 2.42‒2.53 (m, 3H), 2.12‒2.24 (m,
3H), 1.91‒2.00 (m, 1H), 1.64‒1.85 (m, 4H). 13C NMR (500
MHz, CDCl3): δ 161.73 (d, JCF = 244.31 Hz), 146.39, 141.61 (d,
JCF = 3.25 Hz), 132.94, 128.39, 127.19 (d, JCF = 8.12 Hz),
126.16, 114.86 (d, JCF = 21.10 Hz), 73.20, 70.84, 58.83, 50.09,
48.43, 40.28, 38.05, 37.79, 24.18. Anal. calcd for:
C21H25ClFNO2: C, 66.75; H, 6.67; N, 3.71. Found: C, 66.88;
H, 6.56; N, 3.62.
ACKNOWLEDGMENTS
This work was supported by Research Funding for University
(FIR) 2014.
ABBREVIATION USED
VEGF-A, vascular endothelial growth factor A; BRB, blood-
retinal barrier; EC, endothelial cells; HP, haloperidol; HREC,
human retinal endothelial cells; HDACIs, histone deacetylase
inhibitors; (+)-PTZ, (+)-Pentazocine, VPA, valproic acid;
VEGI, vascular endothelial growth inhibitor; DR3, death re-
ceptor 3; FBS, fetal bovine serum; RT-PCR, reverse transcrip-
tion polymerase chain reaction; TLC, thin-layer chromatog-
raphy.
(±)-4-(4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl)-
1-(4-fluorophenyl)butyl 2-propylpentanoate [(±)-2]. 4-
(4-chlorophenyl)-1-(4-(4-fluorophenyl)-4-
hydroxybutyl)piperidin-4-ol [(±)-HP-mII) (0.2 g, 0.52
mmol) was dissolved in anhydrous THF (10 mL) and 4-
N,N-dimethylaminopiridin (0.063 g, 0.52 mmol) was add-
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A solution of 2-
propylpentanoyl chloride (0.177 mL, 1.03 mmol) in THF
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1
salt. mp 156‒158 °C (Oxalate). H NMR (500 MHz, CDCl3):
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8.80 Hz, 2H), 5.71 (t, J = 7.09 Hz, 1H), 2.76 (br. d, J = 10.76
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13
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Hz), 74.70, 70.88, 57.77, 48.99, 45.36, 37.90, 35.04, 34.60,
34.18, 22.42, 20.92, 20.64, 14.01. Anal. calcd for:
C31H41ClFNO7: C, 62.67; H, 6.96; N, 2.36. Found: C, 62.94;
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Figure 5. Effect of (±)-2, (±)-HP-mII plus VPA, HP, (+)-PTZ and AC927 on VEGF-A-stimulated HREC
migration after wound healing assay and quantified as a percentage of wound closure by using ImageJ
software.
Figure_5
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