Haloperidol Metabolite II Valproate Ester - ACS Publications

Oct 14, 2016 - Antiangiogenic Effect of (±)-Haloperidol Metabolite II Valproate. Ester [(±)-MRJF22] in Human Microvascular Retinal Endothelial Cells...
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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*,† †

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



S Supporting Information *

ABSTRACT: (±)-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.



colorectal cancer.11,12 Due to its antiangiogenic properties, bevacizumab is used for the treatment of several diseases linked with aberrant angiogenesis, including macular degeneration.13,14 Sigma (σ) receptors have been implicated in a myriad of cellular functions, biological processes, and diseases.15 Two σ receptor subtypes are recognized and termed sigma-1 (σ1) and sigma-2 (σ2).16,17 The σ1 receptor is an intracellular membraneassociated protein whose function is neuroprotective and neuroregulatory.18,19 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 ligands as a molecular probe for cancer imaging.15,21,22 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 voltagedependent 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) (Figure 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

INTRODUCTION Angiogenic processes play a critical role in the development of cancer, heart diseases, atherosclerosis, and various eye diseases such as wet age-related macular degeneration and proliferative diabetic retinopathy, which are two of the most common causes of irreversible visual loss.1 In particular, tumor growth is dependent on the capacity of neoplastic cells to induce vascular ingrowth.2,3 In physiological conditions, the blood−retinal barrier (BRB) is established by tight junctions between neighboring retinal endothelial cells. The retinal continuous endothelium forms the main structure of the BRB and rests on a basal lamina that is covered by the astrocyte feet, Müller cells, and pericytes. The integrity of BRB is essential for proper vision, and its breakdown contributes to retinal disorders.4 Cancer cells communicate with endothelial cells (ECs) and could activate them by cell contact or by releasing soluble factors.5,6 On the other hand, ECs 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 β, and the increase of VEGF-A levels.9 Cancers require VEGF for their survival, and so far, antiangiogenic therapies rely on neutralizing angiogenic factors such as VEGF or on blocking the receptor tyrosine kinase intracellularly.10 The monoclonal antibody bevacizumab is the first approved inhibitor of VEGF clinically available in the U.S. for the treatment of cancers in metastatic phases, such as © 2016 American Chemical Society

Received: July 13, 2016 Published: October 14, 2016 9960

DOI: 10.1021/acs.jmedchem.6b01039 J. Med. Chem. 2016, 59, 9960−9966

Journal of Medicinal Chemistry

Brief Article

Scheme 1. Synthesis of Compound (±)-2a

Figure 1. Chemical structures of HP, (±)-HP-mII, (±)-1, (−)-1, (+)-1, and (±)-2.

condensation, and fragmentation, all events indicating apoptosis.25 Different from HP, (±)-haloperidol metabolite II [(±)-HPmII) (Figure 1) displays a preferential activity on σ receptors compared to dopamine receptors.26,27 (±)-HP-mII is endowed with a σ1 antagonist and σ2 agonist functional profile, and it inhibits the proliferation of various cancer cells by depleting Ca2+ from the cellular stores, thus triggering apoptosis.28 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 cells derived from glioblastoma multiforme.29,30 These works culminated in the development of (±)-haloperidol metabolite II butyrate ester [(±)-MRJF4] and its enantiomers (+)-MRJF4 and (−)-MRJF4 (Figure 1), three derivatives obtained by esterification of (±)-HP-mII with phenylbutyric acid, a histone deacetylase inhibitor (HDACi). 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 compounds was σ receptors mediated.29,30 Here we report the synthesis of (±)-haloperidol metabolite II valproate ester prodrug [(±)-MRJF22] (Figure 1) and the evaluation of its biological activity on VEGF-A stimulated human retinal endothelial cells (HRECs), which offers an in vitro model of BRB in a proangiogenic tumoral environment. Moreover, (±)-2, being sensitive to esterases hydrolysis, can release valproic acid (VPA), a well-known HDACi31 that has been clinically investigated as an anticancer drug.32,33 Recently VPA has been implicated in antiangiogenic processes. Indeed VPA significantly inhibits the in vitro angiogenic potential and VEGF expression in human cervical cancer HeLa and SiHa cells and has antiangiogenic and antitumor activities mediated by increasing the expression levels of vascular endothelial growth inhibitor (VEGI) and death receptor 3 (DR3) in human osteosarcoma cells.34,35

a Reagents 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.

(Table S2). As could be deduced from the rate of hydrolysis (kobs), the compound undergoes a fast hydrolysis in rat plasma, whereas in human plasma it was fairly stable. The in vitro receptor binding affinity of (±)-2, (±)-HP-mII, HP, and VPA has been measured toward σ1, σ2, and dopaminergic D2 and D3 receptors (Table 1). Table 1. σ1, σ2, D2 and D3 binding assays of (±)-2, (±)-HPmII, HP, and VPA Ki ± SEM,a nM compd

σ1

σ2

D2

D3

(±)-2 (±)-HP-mII HP VPA

13 ± 0.4 2.9 ± 0.8 2.7 ± 0.5 >10000

124 ± 11 2.4 ± 0.5 17.0 ± 1.5 >10000

>5000 241 ± 38 2.5 ± 0.7 ndb

>5000 1024 ± 217 6.1 ± 1.5 ndb

a Each value is the mean ± SEM of three determinations. determined.

b

Not

(±)-2 exhibits high affinity at the σ1 receptor and appreciable 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 binding affinity for both σ receptors. However, this modification significantly reduces the affinity for D2 and D3 receptors (Ki > 5000 nM at both receptors). (±)-HP-mII showed single-digit nanomolar affinity at both σ receptor subtypes (Ki of 2.9 and 2.4 nM for σ1 and σ2 receptors, respectively), but in contrast with (±)-2 it keeps a significant binding with D2 and D3 receptors (Ki of 241 and 1024 nM, respectively). HP, in accordance with the literature, has shown a high σ1 and σ2 (Ki of 2.7 and 17.0 nM, respectively) and dopaminergic D2 and D3 (Ki of 2.5 and 6.1 nM) receptors affinity.29 Finally, VPA does not show binding ability toward σ1 and σ2 receptors (Ki > 10 000 nM at both receptors). 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 compared to VPA, (±)-HP-mII, and HP at four different time periods (24, 48, 72, and 96 h).



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 racemic mixture (Scheme 1).36 The water solubility and chemical stability of (±)-2 were determined, while log P was theoretically calculated (Table S1). (±)-2 displayed low water solubility (1.4 mg/mL) and relatively high lipophilicity values (theoretically calculated log P of 7.04). The chemical stability of (±)-2 was checked in vitro at 37 °C in aqueous buffer solutions of pH 1.3 (nonenzymatic simulated gastric fluid) and pH 7.4 (Table S2). The compound showed good stability at both pH 1.3 and pH 7.4 (129 and 134 h, respectively). The enzymatic stability of (±)-2 was also studied at 37 °C in rat and human plasma 9961

DOI: 10.1021/acs.jmedchem.6b01039 J. Med. Chem. 2016, 59, 9960−9966

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receptors on cell viability (Figure 3). Results showed that both σ receptors are involved in the cell viability effects of (±)-HP-

Table 2. MTT Viability Test on HREC: IC50 at Four Different Time Periods of (±)-2, HP-mII, HP, and VPA compd c

VPA

(±)-HP-mIIc

(±)-2c

HPd

period (h)

IC50 (μM)a (pIC50 ± SE)b

24 48 72 96 24 48 72 96 24 48 72 96 24 48 72 96

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)

a

IC50 values have been calculated with GraphPad Prism 5 for Windows using a nonlinear fit transform sigmoidal dose-response (variable slope). bpIC50 is 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.

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 h respectively). (±)-HP-mII showed a time-dependent decrease in HREC viability. Indeed, at 24 h (±)-HP-mII induced 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. HP was the most potent compound in reducing cell viability, with IC50 spanning from 2.3 to 3.7 μM. (±)-2 determined a decrease in cell viability, with IC50 ranging from 10 to 11 μM. Moreover, 5 μM (±)-2 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). (±)-HP-mII and (±)-2 have also been used in combination 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

Figure 3. Effects of HP-mII (A) and (±)-2 (B) in combination with the selective σ1 agonist (+)-PTZ and σ receptors antagonist AC927 on HREC viability by MTT test. RT-PCR was for the identification of σ1 receptor expression on HREC. The mRNA extract of the whole retina was a positive control (C).

mII (Figure 3A). Specifically, HREC incubation with (±)-HPmII 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 restored 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 induced by (±)-HP-mII. These results outline a σ1 and σ2 receptors involvement in the reduction of cell viability induced by (±)-HP-mII. 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 induced 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 affinity of (±)-2 toward σ1 and σ2 receptors. (+)-PTZ (1 μM) at 72 h induced an improvement of HREC

Figure 2. Effect of (±)-2 (5 μM) and (±)-HP-mII plus VPA (5 μM, equimolar concentrations) on HREC viability by MTT test. 9962

DOI: 10.1021/acs.jmedchem.6b01039 J. Med. Chem. 2016, 59, 9960−9966

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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 significant proangiogenic stimulus, increasing the alive cell number of 52% at 24 h and 42% at 72 h (Figure 4B). Incubation of (±)-2 at 5 μM with VEGF-A resulted in a reduction of the cell number of 28% and 30% at 24 and 72 h, respectively, 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. Endothelial cell migration is a critical process for wound healing and angiogenesis. Figure 5 shows the percentages of wound edge cell advancement 24 and 72 h after the scratch on the HREC confluent monolayer. 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 incubation of

viability while compound AC927 (1 μM) alone at 24 and 72 h did not induce any effect in cells viability. (+)-PTZ (1 μM) and AC927 (1 μM) in combination at 24 and 72 h determined an improvement in cell viability. 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 effects 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, HRECs 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, Figure S1). In the second set of experiments (Figure 4), at the chosen concentrations, the effects of VPA, (±)-HP-mII, (±)-2, and

Figure 4. Effects of (±)-2, (±)-HP-mII, VPA, (±)-HP-mII plus VPA, and bevacizumab in the presence (B) or absence (A) of 80 ng/mL VEGF-A on alive HREC count. Bars refer to alive cells excluding the dye in Trypan blue exclusion test.

bevacizumab on HREC viability were examined in basal conditions (Figure 4A) and in the presence of VEGF-A (Figure 4B) 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). Neither VPA nor (±)-HP-mII alone or

Figure 5. Effects 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. 9963

DOI: 10.1021/acs.jmedchem.6b01039 J. Med. Chem. 2016, 59, 9960−9966

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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) with respect to nonstimulated cells, and a faster migration of HREC (panels A−C). Interestingly, in VEGF-A stimulated HREC, the presence of (±)-2 slowed 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 response 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 subtoxic concentration (2 μM) did not affect the HREC migration 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 characteristic behavior of HP will be further evaluated in order to clarify this experimental observation. The presence of (+)-PTZ, a prototypical selective σ1 agonist, attenuated the antimigratory effect exerted by (±)-2 on VEGFA-stimulated cells in a dose-dependent manner (1 and 2 μM) (panel C). These data demonstrate that the effects on cell migration induced by (±)-2 are mainly mediated by the antagonism at the σ1 receptor. AC927, 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 of the fact that the σ2 receptor is not involved in the antiangiogenic effect exerted by (±)-2. The representative contrast phase images of HREC after the scratch wound are shown in Supporting Information (Figure S2). In Figure 6 the quantifications of the emerging branch points from HREC in Matrigel tube formation assays are reported.

presence of (±)-HP-mII plus VPA caused a decrease in the ramification number only by 22% in VEGF-A treated cells, demonstrating the significant antiangiogenic effect by (±)-2 with respect to the individual components taken into account in co-incubation conditions. The tube formation in vitro was strongly reduced (by 67%) when HRECs were incubated with (±)-2. HP did not affect the biological effect in VEGF-A stimulated HREC. Again, unlike AC927, which had no effect, (+)-PTZ reverted 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 organization from HREC cell bodies on Matrigel are shown in Supporting Information (Figure S3). Finally, HDAC activity inhibition of (±)-2 (5 μM) was measured in HeLa extracts as an HDAC source using a fluorescently labeled HDAC substrate. (±)-2 at 5 μM did not inhibit HDAC activity (see Supporting Informationfor details, Figure S4).



CONCLUSIONS We describe the synthesis and the pharmacological characterization of (±)-HP-mII valproate ester, (±)-2. Taken together, our results indicate that (±)-2 exhibits good antiangiogenic activity, comparable to bevacizumab. Moreover, we have shown that (±)-2 was able to significantly reduce the viable cell count, endothelial cell migration, and tube formation in VEGF-A stimulated HREC cultures and that these effects are σ1 receptor mediated. Finally, we have demonstrated the presence of σ1 receptor in HREC by RT-PCR. On the basis of our results, the prodrug (±)-2 shows significant antiangiogenic 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.



EXPERIMENTAL SECTION

General Remarks. Reagent grade chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Merck (Darmstadt, Germany) and were used without further 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 determined on a Büchi B-450 apparatus and are uncorrected. Nuclear magnetic resonance spectra (1H NMR and 13C 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 performed on a Carlo Erba instrument model E1110; all the results agreed within ±0.4% of the theoretical values. Thin-layer chromatography (TLC) was performed on silica gel Merck 60 F254 plates; the spots were visualized by UV light. 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-fluorophenyl)butan-1-one (HP) (1 g, 1.36 mmol) in EtOH (50 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 × 50 mL). The combined 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 g, 90% of yield) as a white solid. Mp 144−146 °C. 1H 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 =

Figure 6. Tube formation on Matrigel. The quantification of emerging branch points from HREC bodies was performed by using the Angiogenesis analyzer tool for ImageJ software.

Cell adhesion and growth in VEGF-A conditioning environment were heavily magnified, with an increase in the formation of branch points by almost 5-fold. In VEGF-A treated cells, HP-mII (5 μM) induced a significant reduction of the ramification number by 20%. VPA (5 μM) had no significant effect (data not shown). The 9964

DOI: 10.1021/acs.jmedchem.6b01039 J. Med. Chem. 2016, 59, 9960−9966

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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. (±)-4-(4-(4-Chlorophenyl)-4-hydroxypiperidin-1-yl)-1-(4fluorophenyl)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-dimethylaminopyridine (0.063 g, 0.52 mmol) was added while stirring continuously. A solution of 2-propylpentanoyl chloride (0.177 mL, 1.03 mmol) in THF was added dropwise at 0 °C, and the reaction mixture was stirred at room temperature for 24 h. The reaction was quenched with a saturated solution of Na2CO3 (10 mL), the organic solvent was then evaporated under vacuum, and the aqueous phase was extracted with CHCl3 (3 × 50 mL). The organic layers were dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure to obtain a crude product. Purification by flash chromatography (1:9 EtOH/CHCl3) yielded (±)-2 (0.21 g, 80% of yield). The compound was converted into the oxalic acid salt. Mp 156−158 °C (oxalate). 1H NMR (500 MHz, CDCl3): δ 7.41 (d, J = 8.31 Hz, 2H), 7.27−7.31 (m, 4H), 7.01 (t, J = 8.80 Hz, 2H), 5.71 (t, J = 7.09 Hz, 1H), 2.76 (br d, J = 10.76 Hz, 2H), 2.40−2.45 (m, 4H), 2.07−2.12 (m, 2H), 1.88−1.95 (m, 1H), 1.80−1.83 (m, 2H), 1.68 (br d, J = 12.72 Hz, 2H), 1.12−1.61 (m, 10H), 0.87 (t, J = 7.34 Hz, 3H), 0.82 (t, J = 7.34 Hz, 3H). 13C NMR (200 MHz, CDCl3): δ 180.83, 162.30 (d, JCF = 245.00 Hz), 146.57, 136.46 (d, JCF = 3.30 Hz), 132.88, 128.45, 128.34 (d, JCF = 8.05 Hz), 126.04, 115.28 (d, JCF = 21.25 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; H, 6.88; N, 2.30.



REFERENCES

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01039. Tables S1 and S2, Figures S1−S4, and procedures for in vitro biological and pharmacokinetic assays (PDF) Molecular formula strings and some data (CSV)



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Corresponding Authors

*C.D.A.: phone, (+39) 0957384070; e-mail, [email protected]. *A.M.: phone, (+39) 0957384250; e-mail, [email protected]. Author Contributions §

M.O. and E.A. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Research Funding for University (FIR) 2014. ABBREVIATIONS USED VEGF-A, vascular endothelial growth factor A; BRB, blood− retinal barrier; EC, endothelial cell; HP, haloperidol; HREC, human retinal endothelial cell; HDACi, histone deacetylase inhibitor; (+)-PTZ, (+)-pentazocine; VPA, valproic acid; VEGI, vascular endothelial growth inhibitor; DR3, death receptor 3; FBS, fetal bovine serum; RT-PCR, reverse transcription polymerase chain reaction; TLC, thin-layer chromatography 9965

DOI: 10.1021/acs.jmedchem.6b01039 J. Med. Chem. 2016, 59, 9960−9966

Journal of Medicinal Chemistry

Brief Article

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