Haloperidol Metabolite II Valproate Ester (S)-(-)-MRJF22: Preliminary Studies as a Potential Multifunctional Agent Against Uveal Melanoma

Abstract

Increased angiogenesis and vascular endothelial growth factor (VEGF) levels contribute to higher metastasis and mortality in uveal melanoma (UM), an aggressive malignancy of the eye in adults. (±)-MRJF22, a prodrug of the sigma (σ) ligand haloperidol metabolite II conjugated with the histone deacetylase (HDAC) inhibitor valproic acid, has previously demonstrated a promising antiangiogenic activity. Herein, the asymmetric synthesis of (R)-(+)-MRJF22 and (S)-(-)-MRJF22 was performed to investigate their contribution to (±)-MRJF22 antiangiogenic effects in human retinal endothelial cells (HREC) and to assess their therapeutic potential in primary human uveal melanoma (UM) 92-1 cell line. While both enantiomers displayed almost identical capabilities to reduce cell viability than the racemic mixture, (S)-(-)-MRJF22 exhibited the highest antimigratory effects in endothelial and tumor cells. Given the fundamental contribution of cell motility to cancer progression, (S)-(-)-MRJF22 may represent a promising candidate for novel antimetastatic therapy in patients with UM.

Figure 1

Chemical structure of HP, HP-mII, and (±)-MRJF22.

Scheme 1. Enantioselective Synthesis for Compounds (+)-1 and (−)-1

Reagents and conditions: (i) (+)- or (−)-DIP-Cl, tetrahydrofuran (THF), −25 °C, 16 h; DEA, Et₂O, room temperature (rt), on; (ii) 4-(4-chlorophenyl)hydroxypiperidine, KHCO₃, anhydrous dimethylformamide (DMF), 80 °C, 24 h; (iii) 2-propylpentanoyl chloride, THF, 0 °C to rt, 3 h.

Figure 2

Antiproliferative effects of (±)-1, (+)-1, and (−)-1 in HREC stimulated with VEGF-A, assessed by the crystal violet assay. HREC were treated with 5 μM (±)-1, (+)-1, and (−)-1 in the presence or absence of 80 ng/mL VEGF-A for 24 h. Effects of the σ₂ receptor antagonist AC927 (2 μM) and the selective σ₁ receptor agonist (+)-pentazocine (PTZ) (2 μM) in HREC cotreated with 5 μM (±)-1 or (+)-1 and (−)-1 80 ng/mL of VEGF-A for 24 h. Ctrl, vehicle control (dimethyl sulfoxide, DMSO). Data are expressed as a percentage of proliferation with respect to vehicle control. *p < 0.05 vs Ctrl; ^#p < 0.05 vs VEGF-A; ^§p < 0.05 vs the same conditions without agonist or antagonist.

Figure 3

Evaluation of cell motility by wound healing assays in HREC treated with 80 ng/mL VEGF-A in the presence or absence of 5 μM (±)-1 (A), (+)-1 (B), and (−)-1 (C) at 48 h. Selective σ₁ receptor agonist PTZ (2 μM) or σ₂ receptor antagonist AC927 (2 μM) was tested in cotreated HREC with 80 ng/mL of VEGF-A and (±)-1, (+)-1, or (−)-1. Wound closure percentage was quantified by ImageJ software. Ctrl, vehicle control (DMSO). Values are expressed as a mean ± standard error of the mean (SEM) of three independent experiments, each involving three different wells per condition. Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Tukey’s test. *p < 0.05 vs Ctrl; ^#p < 0.05 vs VEGF-A; ^§p < 0.05 vs the same conditions without agonist or antagonist.

Figure 4

Effects (±)-1, (+)-1, and (−)-1 on tubelike structures formed by HREC stimulated with VEGF-A. Representative optical phase-contrast micrographs of tubelike structures (40× magnification) observed in the tube formation assays (Matrigel) at 24 h (A). Quantification of tube length was carried out using the Angiogenesis Analyzer tool for ImageJ software. HREC were treated with 80 ng/mL VEGF-A in the presence or absence of 5 μM (±)-1, (+)-1, and (−)-1 or further supplemented with selective σ₁ receptor agonist PTZ (2 μM) and σ₂ receptor antagonist AC927 (2 μM). We included HREC treated with 40 μg/mL of AFL (B). Values are expressed as mean ± SEM of three independent experiments, each conducted in triplicate. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s test. *p < 0.05 vs Ctrl; ^#p < 0.05 vs VEGF-A; ^§p < 0.05 vs the same condition without agonist or antagonist.

Figure 5

RT-PCR for the σ₁ (SIGMAR1) and σ₂ (TMEM97) receptor expression in UM 92-1 cells and MCF-7 human breast cancer cells. 45S Ribosomal pre-RNA was used as the positive control.

Figure 6

Effects of VPA (2 mM), HP (10 μM), and (±)-HP-mII (30 μM) on 92-1 cell proliferation (A). Antiproliferative effects of (±)-1 (5 μM) in combination with the selective σ₁ receptor agonist PTZ (2 μM) and σ₂ receptor antagonist AC927 (2 μM). Ctrl, vehicle control DMSO (B). Data in A and B represent the percentage of proliferation with respect to the vehicle control. Values are expressed as mean ± SEM of four independent experiments, each conducted in triplicate. Statistical analysis was performed using one-way ANOVA, followed by Tukey’s test. *p < 0.05; **p < 0.01; ***p < 0.001 vs vehicle control.

Figure 7

Effect of (±)-1, (+)-1, and (−)-1 on human 92-1 uveal melanoma cell migration. Representative images of the wound healing assay (A). Magnification, 4×. All compounds were used at 3 μM. Ctrl, vehicle control (DMSO). Concentration–response curves of the inhibition of cell migration by the indicated compounds at the 48 h time point (B). Data are shown as % inhibition of cell migration with respect to the vehicle control.