Development of sigma-1 (σ₁) receptor fluorescent ligands as versatile tools to study σ₁ receptors

Abstract

Despite their controversial physiology, sigma-1 (σ₁) receptors are intriguing targets for the development of therapeutic agents for central nervous system diseases. With the aim of providing versatile pharmacological tools to study σ₁ receptors, we developed three σ₁ fluorescent tracers by functionalizing three well characterized σ₁ ligands with a fluorescent tag. A good compromise between σ₁ binding affinity and fluorescent properties was reached, and the σ₁ specific targeting of the novel tracers was demonstrated by confocal microscopy and flow cytometry. These novel ligands were also successfully used in competition binding studies by flow cytometry, showing their utility in nonradioactive binding assays as an alternative strategy to the more classical radioligand binding assays. To the best of our knowledge these are the first σ₁ fluorescent ligands to be developed and successfully employed in living cells, representing promising tools to strengthen σ₁ receptors related studies.

1. Introduction

The existence of sigma (σ) receptors was first reported in 1976 when they were considered as subtypes of opiate receptors [1]. Later studies clarified that they are a unique set of proteins divided into two subtypes, namely σ₁ and σ₂ [2]. The σ₂ subtype is the lesser known: it has not yet been cloned, and different hypotheses about its identity have been formulated [3–5]. Despite this paucity of information, interest in σ₂ receptor research is on the increase because this subtype is overexpressed in several cancers and its activation through ligands leads cancer cells to death [6–9]. Worthy of note is also the efficacy of σ₂ ligands in resistant tumors that shed light on novel strategies for tumor treatments [10]. On the other hand, σ₁ receptor was cloned right after the two subtypes were discovered [11]. Several structures were proposed for this 223 amino-acids protein and the most accepted model represents the σ₁ receptor made of three hydrophobic regions two of which are trans-membrane spanning segments. The NH₂ and the COOH termini of the σ₁ protein are on the same side of the cell compartment where a possible endogenous ligand, not yet identified, regulates its function [12]. Progesterone, sphingolipid-derived amines (D-erythro-sphingosine) and more recently N,N-dimethyltryptamines have been proposed as σ₁ endogenous ligands [13–15]. Because of the important therapeutic potentials (e.g. treatment of anxiety, depression, schizophrenia, etc) linked to σ₁ receptor targeting, interest in σ₁ receptor research is on the increase [16]. Recent evidence has shown that σ₁ receptor can naturally form dimers and/or oligomer and that heteromers between σ₁ and D₂ receptor play an important role in cocaine addiction [17,18]. These dimers/oligomers may lead to different functional forms of the σ₁ receptor (such as agonist or antagonist activity). All these lines of evidence make novel pharmacological tools appealing for the study of the still controversial σ₁ receptor physiology. With this aim, we produced three σ₁ receptor fluorescent tracers starting from previously well characterized σ₁ receptor ligands. We adopted the same strategy that was shown to be successful for σ₂ fluorescent ligands: the appropriate σ₁ pharmacophore was linked through a spacer to a green emitting fluorescent tag [19–21]. In particular, as σ₁ pharmacophores we selected 4-methyl-1-[4-(6-methoxy-1,2,3,4-tetrahydronaphthalen-1-yl)butyl]piperidine PB190 and 4-methyl-1-[4-(6-methoxynaphthalen-1-yl)butyl]piperidine PB212 (Fig. 1) which were shown to be respectively a σ₁ selective agonist and a σ₁ selective antagonist by both in vitro and in vivo assays [22–26]. We also selected 4-cyclohexyl-1-[3-(5-methoxy-1,2,3,4-tetrahydronaph thalen-1-yl)propionyl]piperazine (Fig. 1) which showed excellent σ₁ affinity and σ₁ vs σ₂ selectivity [27]. The methoxy group present in these σ₁ ligands was replaced by a hexamethylenoxy chain carrying in the ω position a 4-N,N-dimethylphthalimide moiety. These choices were made on the basis of data obtained from σ₂ receptor fluorescent ligands: the position on the naphthalene or tetralin ring and the nature of the linker showed to be well tolerated with 4-N,N-dimethylphthalimide conferring optimal fluorescent properties that allowed successful experiments in living cells by flow cytometry and confocal microscopy.

Fig. 1.

σ₁ Receptor lead compounds exploited for the development of σ₁ fluorescent ligands.

2. Results and discussion

2.1. Chemistry

The synthetic pathway for final compounds 5, 6 and 9, is depicted in Scheme 1. The already known piperidines PB190 and PB212 [22] and the piperazine 1 [27] were demethylated by reaction with BBr₃ to provide the corresponding phenolic intermediates 2, 3 and 8, although phenol 3 was previously obtained through another synthetic pathway [28]. Alkylation of the phenolic intermediates 2, 3 and 8 with the already known mesyl derivative 4 [20] provided final fluorescent compounds 5, 6 and 9. Final compounds 6 and 9 were converted into the corresponding hydrochloride salts with gaseous HCl, whereas 5 was converted into the corresponding oxalate salt.

Scheme 1.

Reagents and Conditions: (a) BBr₃, CH₂Cl₂, from −78 °C to room temperature, overnight; (b) K₂CO₃, DMF, 150 °C, overnight.

2.2. σ Receptors radioligand binding

Results from binding assays are expressed as inhibition constants (K_i values) in Table 1. As in the σ₂ receptors fluorescent ligands, the introduction of the fluorescent tag connected through the hexamethylenoxy linker at the 2- or 5-position of the naphthalene and tetralin ring respectively, produced a decrease in the σ₁ receptor affinity with respect to the corresponding lead compounds. Only compound 5 presented affinity values at both σ receptors (K_i = 3.61 nM at the σ₁, and K_i = 48.3 nM at the σ₂) comparable to those of lead compound PB190 (K_i = 1.01 nM at the σ₁, and K_i = 48.7 nM at the σ₂). Final compounds 6 and 9 presented a 100-fold drop in the σ₁ affinity (K_i = 5.20 nM and K_i = 13.1 nM, respectively) in comparison to the lead compounds PB212 and 1, respectively (K_i = 0.03 nM and K_i = 0.11 nM, respectively). By contrast, the affinity at the σ₂ receptors only slightly decreased for 6 (2.5-fold) in comparison to its lead compound PB212, whereas σ₂ receptor affinity of 9 was 5-fold higher than σ₂ receptor affinity displayed by 1. Despite the decrease in the σ₁ affinity compared to the lead compounds, the three fluorescent ligands still demonstrated strong nanomolar σ₁ binding, and compounds 5 and 6 also kept a certain σ₁ versus σ₂ receptor selectivity (10-fold), which was lower (3-fold) in 9. Taking these data together, the strategy adopted to obtain σ₂ receptor fluorescent ligands showed to be successful also for σ₁ receptor fluorescent ligands.

Table 1.

σ Receptor affinities of final and lead-compounds and fluorescence properties.

	Ki nM ± SEM (nM)a
Compound	σ1	σ2	λex nmb	λem nmb	QY EtOHc	QY CHCl3c
PB190d	1.01 ± 0.41	48.7 ± 9.2
PB212d	0.03 ± 0.013	17.9 ± 5.3
1e	0.11 ± 0.01	179 ± 56
5	3.61 ± 0.4	48.3 ± 6.0	390	510	0.02	0.5
6	5.20 ± 0.17	45.6 ± 4.6	390	520	0.02	0.4
9	13.1 ± 5.02	38.4 ± 10.9	390	520	0.02	0.4
DTG		35.4 ± 5.8
(+)-Pentazocine	4.52 ± 0.7

^aValues are the means of n ≥ 3 separate experiments, in duplicate.

^bFluorescence properties of compounds were evaluated on compound as free base in EtOH solutions.

^cQY was evaluated as in [20].

^dData from Ref. [22].

^eData from Ref. [27].

2.3. Fluorescent ligands studies

2.3.1. Fluorescent properties

The fluorescent properties are listed in Table 1. The three compounds displayed very similar maximum excitation wavelength (λex = 390 nm) and maximum emission wavelength (λem ~ 520 nm), with an important difference between λex and λem (i.e., Stokes shift) in accordance to the spectra recorded for the σ₂ ligands with the same fluorescent tag [20,21]. Measurement of quantum yields (QY) demonstrated the environment sensitive properties (low fluorescence intensity in polar solvent such as EtOH, but high fluorescence in nonpolar solvents such as CHCl₃), proper for the 4-N,N-dimethylphthalimide fluorescent tag, as recorded for σ₂ ligands.

2.3.2. Imaging of ARPE19 cells with compound 9 by confocal microscopy

In Fig. 2 the labeling of human retinal pigment epithelia ARPE19 cells with compound 9 shows fluorescent signals in intracellular membranes. The fluorescent signals were protected by PB190, indicating the specificity of binding to σ receptors. In these same cells, the cytoprotective properties of compound 9 were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) viability assay upon toxicity mediated by paraquat (Fig. 1, in the supporting information), making clear that compound 9 protects against such toxicity and therefore may be useful for oxidative stress treatments.

Fig. 2. Labeling of ARPE19 Cells with compound 9.

Signal is localized to intracellular membranes. Lower intensity of labeling is detected when cells were prelabeled with PB190 100 µM. Scale bar = 20 µm.

2.3.3. Detection of σ₁ receptors by flow cytometry

Cellular uptake of compounds 5 and 6 was studied by flow cytometry in two human breast tumor MCF7 cell lines: MCF7wt and MCF7σ₁. Both these cell lines were thoroughly and previously characterized: the former over expresses σ₂ receptors (B_max = 2.02 pmol/mg protein) [5,20] whereas σ₁ receptors' expression is very low (B_max = 0.17 pmol/mg protein) [5,29]; the latter is obtained by stable transfection of MCF7 cells with σ₁ c-DNA to obtain a good expression of σ₁ receptors (B_max = 3.45 pmol/mg protein) [21,29]. All of the experiments were performed by incubation of the two cell lines for 45 and 75 min with either compound 5 or 6 at 100 nM, as the fluorescence signal increased in a dose-dependent manner, reaching a plateau at 100 nM. Despite their 10-fold σ₁ vs σ₂ selectivity, compounds 5 and 6 still display σ₂ receptor affinity (K_i = 48.3 nM for 5 and K_i = 45.6 nM for 6). Therefore, the σ₂ selective ligand 2-(3-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)-5-methoxy-3,4-dihydroisoquinolin-1(2H)-one 10 [30] (K_i = 2435 nM at the σ₁, and K_i = 4.24 nM at the σ₂) was used at 10 µM to mask the possible fluorescent signal generated by the σ₂ targeting of the fluorescent compounds. The σ₁ specificity of the fluorescent tracers (100 nM) was demonstrated by comparison of the fluorescent signals by MCF7σ₁ and MCF7wt, (Fig. 3) upon treatment with (+)-pentazocine and 10. In MCF7wt cells, where the fluorescence intensity was very low in each experimental condition, no change in the fluorescent signal by either 5 or 6 was detected upon administration of (+)-pentazocine or 10 (Fig. 3, right panels). In MCF7σ₁ cells the administration of (+)-pentazocine (10 µM) strongly decreased the fluorescent signal by both 5 and 6 (with the signal by 5 completely abated) whereas, treatment with compound 10 (10 µM) only slightly decreased fluorescence by both the tracers. As, expected, co-administration of both (+)-pentazocine and 10 completely abated the fluorescent signals by both the tracers (Fig. 3, left panels). These results reflected the expression of σ₁ receptors in the MCF7 cell lines and suggested that these tracers, administered at 100 nM, bind σ₁ receptors rather specifically, with a negligible interference by σ₂ receptors. In MCF7σ₁, either 5 (100 nM) or 6 (100 nM) were dose dependently displaced (as demonstrated by the progressive decrease of fluorescence) by pre-incubation with increasing concentrations of either PB190 or PB212 (Figs. 4 and 5A and B; left panels; basal fluorescence of MCF7 cells in the presence of PB190 or PB212 reported in Fig. 2 in the supporting information), which were used as σ₁ reference non-fluorescent compounds (from 1 nM to 10 µM). The same experiment was performed with (+)-pentazocine and a dose-dependent displacement were again recorded, with more linear results obtained by 45 min incubation time (Fig. 6). By contrast, no differences were shown between the two incubation times studied (45 min or 75 min) when 5 or 6 were displaced by PB190 and PB212. As expected from a cell line with very low amount of σ₁ receptors, the displacement was far more modest in MCFwt (Figs. 4–6, A and B; right panels). The fluorescence intensity results obtained by either 5 or 6 in MCF7σ₁ cells by dose-dependent displacement of the σ₁ receptor ligands studied, served to generate binding curves for PB190, PB212 and (+)-pentazocine allowing the measurement of the IC₅₀ values of these compounds for the human σ₁ receptors in MCF7 cells (Table 2). Differences between radioligand binding assays performed on animal tissues and human cells homogenates have already been reported for σ₁ receptors, with slightly lower binding affinities from the latter experiment [31]. The IC₅₀ values obtained by flow cytometry on MCF7σ₁ cells were in accordance with such trend, demonstrating the utility of σ₁ receptors fluorescent ligand as tools to detect their presence in living cells and to conveniently performσ₁ binding assays of novel ligands instead of the radioligand binding assays.

Fig. 3.

Flow Cytometry analysis (cell associated fluorescence vs cell count) of MCF7σ₁ and MCF7wt, exposed to compound 5 or 6 and treated with compound 10 (10 µM) to mask σ₂ receptors, or (+)-pentazocine (10 µM) to mask σ₁ receptors, or both 10 (10 µM) with (+)-pentazocine (10 µM). A) Displacement of 5 (100 nM, yellow curve): black curve: control; red curve: 10 µM 10; green curve: 10 µM (+)-pentazocine; violet curve: 10 µM 10 with (+)-pentazocine 10 µM. B) Displacement of 6 (100 nM, yellow curve): black curve: control; red curve: 10 µM 10; green curve: 10 µM (+)-pentazocine; violet curve: 10 µM 10 with (+)-pentazocine 10 µM.

Fig. 4.

Flow Cytometry analysis (cell associated fluorescence vs cell count) of MCF7σ₁ and MCF7wt, exposed to 5 and treated with 10 (10 µM) to mask σ₂ receptors. A) Displacement of 5 (100 nM, yellow curve) with increasing concentrations of PB190: black curve: control; orange curve: 1 nM PB190; red curve: 10 nM PB190; green curve: 100 nM PB190; blue curve: 1 µM PB190; violet curve: 10 µM PB190. B) Displacement of 5 (100 nM, yellow curve) with increasing concentrations of PB212: black curve: control; orange curve: 1 nM PB212; red curve: 10 nM PB212; green curve: 100 nM PB212; blue curve: 1 µM PB212; violet curve: 10 µM PB212.

Fig. 5.

Flow cytometry analysis (cell associated fluorescence vs cell count) of MCF7σ₁ and MCF7wt, exposed to 6 and treated with 10 (10 µM) to mask σ₂ receptors. A) Displacement of 6 (100 nM, yellow curve) with increasing concentrations of PB190: black curve: control; orange curve: 1 nM PB190; red curve: 10 nM PB190; green curve: 100 nM PB190; blue curve: 1 µM PB190; violet curve: 10 µM PB190. B) Displacement of 6 (100 nM, yellow curve) with increasing concentrations of PB212: black curve: control; orange curve: 1 nM PB212; red curve: 10 nM PB212; green curve: 100 nM PB212; blue curve: 1 µM PB212; violet curve: 10 µM PB212.

Fig. 6.

Flow cytometry analysis (cell associated fluorescence vs cell count) of MCF7σ₁ and MCF7wt, exposed to 5 or 6 and treated with 10 (10 µM) to mask σ₂ receptors. A) Displacement of 5 (100 nM, yellow curve) with increasing concentrations of (+)-pentazocine: black curve: control; orange curve: 1 nM (+)-pentazocine; red curve: 10 nM (+)-pentazocine; green curve: 100 nM (+)-pentazocine; blue curve: 1 µM (+)-pentazocine; violet curve: 10 µM (+)-pentazocine. B) Displacement of 6 (100 nM, yellow curve) with increasing concentrations of (+)-pentazocine: black curve: control; orange curve: 1 nM (+)-pentazocine; red curve: 10 nM (+)-pentazocine; green curve: 100 nM (+)-pentazocine; blue curve: 1 µM (+)-pentazocine; violet curve: 10 µM (+)-pentazocine.

Table 2.

σ₁ Receptor affinities of reference σ₁ receptor ligands by flow-cytometry with novel σ₁ fluorescent ligands.

	σ1; IC50 nM ± SEMa
compound	5b	6b
PB190c	11.3 ± 2.4	12.1 ± 1.4
PB212c	15.5 ± 2.3	10.6 ± 1.2
(+)-Pentazocined	8.24 ± 1.2	7.59 ± 0.8

^aValues are the means of n ≥ 2 separate experiments, in duplicate.

^bIC₅₀ values from binding curves obtained by dose dependent displacement of 5 (100 nM) or 6 (100 nM) by σ₁ reference compounds (PB190, PB212 and (+)-pentazocine) in MCF7σ₁ cells by flow cytometry.

^cData from 75 min incubation time.

^dData from 45 min incubation time. The experiments were performed in the presence of the σ₂ masking agent 10 (10 µM).

3. Conclusions

Three σ₁ fluorescent tracers were synthesized by functionalization of three well characterized σ₁ ligands with a fluorescent tag. A good compromise between binding affinity at the σ₁ subtype and fluorescent properties was obtained, with all the compounds displaying nanomolar σ₁ receptor affinity. Their σ₁ receptor specificities were demonstrated by protection with σ₁-specific compounds in different cell lines through different techniques: confocal microscopy (compound 9) and flow cytometry (compounds 5 and 6). We also demonstrated that through the use of these fluorescent tracers in flow cytometry, σ₁ binding assays of novel ligands can be conveniently performed in spite of the more classical radioligand binding assays that involves the use of radioligands such as [³H]-(+)-pentazocine. To the best of our knowledge, these compounds are the first σ₁ fluorescent ligands to be produced and successfully employed, and they emerge as useful tools to empower σ₁ receptors studies.

4. Experimental section

4.1. Chemistry

Both column chromatography and flash column chromatography were performed with 60 Å pore size silica gel as the stationary 300 phase (1:30 w/w, 63–200 µm particle size, from ICN and 1:15 w/w, 15–40 µm particle size, from Merck, respectively). Melting points were determined in open capillaries on a Gallenkamp electrothermal apparatus. Purity of tested compounds was established by combustion analysis, confirming a purity >95%. Elemental analyses (C, H, N) were performed on an Eurovector Euro EA 3,000 analyzer; the analytical results were within ±0.4% of the theoretical values, unless otherwise indicated.¹H NMR spectra were recorded on a Mercury Varian 300 MHz using CDCl₃ as solvent, unless otherwise reported. The following data were reported: chemical shift (δ) in ppm, multiplicity (s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet), integration and coupling constants in hertz. Recording of mass spectra was done on an Agilent 6890-5973 MSD gas chromatograph/mass spectrometer and on an Agilent 1100 series LC–MSD trap system VL mass spectrometer; only significant m/z peaks, with their percentages are reported in parentheses. Chemicals were from Aldrich, TCI and Alfa Aesar and were used without any further purification.

4.2. General procedure for the synthesis of phenolic intermediates (2, 3, 8)

A solution of one among the methoxy derivatives PB190, PB212 and 1 (1.0 mmol) in anhydrous CH₂Cl₂ (10 mL) and under Argon was cooled to −78 °C and then added in a dropwise manner with BBr₃ (3.0 mmol, 0.3 mL). The mixture was stirred overnight under argon while it slowly reached room temperature. After cooling in an ice bath, H₂O was added to the mixture which was basified with Na₂CO₃. The resulting mixture was extracted with CH₂Cl₂ (3 × 10 mL) and the organic phases collected, dried (Na₂SO₄) and concentrated under reduced pressure to afford crude oils which provided the title compounds upon purification through column chromatography (CH₂Cl₂/MeOH, 95:5).

4.2.1. 5-[4-(4-Methylpiperidine-1-yl)butyl]-5,6,7,8-tetrahydronaphthalen-2-ol (2)

was obtained in 69% yield (0.204 g) as beige solid: mp = 184–186 °C; GC–MS m/z: 301 (M⁺, 20), 112 (100); LC-MS (ESI⁻) m/z: 300 [M−H]⁻.

4.2.2. 5-[4-(4-Methylpiperidine-1-yl)butyl]naphthalen-2-ol (3)

was obtained in 69% yield (0.202 g) as beige solid: mp = 182–184 °C (literature [28] mp = 183–185 °C); GC–MS m/z: 297 (M⁺, 20), 112 (100); LC-MS (ESI⁻) m/z: 296 [M−H]⁻; LC-MS-MS 296: 156.

4.2.3. 4-Cyclohexyl-1-[3-(5-hydroxy-1,2,3,4-tetrahydronaphthalen-1-yl)propionyl]piperazine (8)

was obtained in 90% yield (0.33 g) as sticky solid.¹H NMR δ 1.05–1.35 [m, 6H, cyclohexyl), 1.55–2.05 (m, 10H, COCH₂CH₂CHCH₂CH₂ and cyclohexyl CH₂), 2.20–2.45 (m, 3H, benzyl CH and CH₂), 2.50–2.58 (m, 4H, CH₂ piperazine), 2.60–2.75 (m, 2H, CH₂CO), 2.80–2.85 (m, 1H, CHN), 4.38–4.70 [m, 4H, CON(CH₂)₂], 5.20 (broad s, 1H, OH), 6.60 (d, J = 7.15 Hz, 1H aromatic), 6.78 (d, J = 7.7 Hz, 1H, aromatic), 7.00 (t, J = 7.8 Hz, 1H, aromatic); GC–MS m/z: 370 (M⁺, 30), 138 (70), 125 (100), 112 (80).

4.3. General procedure for the synthesis of final fluorescent compounds (5, 6 and 9)

A solution in DMF (10 mL) of one among the phenol intermediates 2 or 3 or 8 (1.0 mmol) was added with the mesyl derivative 4 (1.0 mmol, 0.37 g) and K₂CO₃ (1.2 mmol, 0.16 g) to provide a mixture which was stirred at 150 °C overnight. After concentration under reduced pressure the crude residue which was taken up with water was extracted with CH₂Cl₂ (3 × 10 mL) and the organic phases collected were dried (Na₂SO₄) and evaporated under reduced pressure to provide a crude oil which was purified by column chromatography (CH₂Cl₂/MeOH, 95:5) to afford the title compounds.

4.3.1. 5-(Dimethylamino)-2-(6-((5-(4-(4-methylpiperidin-1-yl) butyl)-5,6,7,8-tetrahydronaphthalen-2-yl)oxy)hexyl)isoindoline-1,3-dione (5)

The title compound was obtained in a 50% yield (0.286 g) as a bright yellow oil.¹H NMR δ 0.98 (d, J = 5.7 Hz, 3H, CHCH₃), 1.25–2.00 [m, 19H, N(CH₂)₂, CH₂(CH₂)₄, CH(CH₂)₃ e NH], 2.25–2.40 (m, 3H, benzyl CH and CH₂), 2.60–2.70 (m, 4H, N(CHH)₂ and CH₂CH₂CH₂N), 2.85–2.95 (m, 2H, N(CHH)₂), 3.10 [s, 6H, N(CH₃)₂], 3.62 (t, J = 7.15 Hz, 2H, (CO)₂NCH₂), 3.88 (t, J = 6.6 Hz, 2H, OCH₂), 6.50–7.65 (m, 6H, aromatic); LC–MS (ESI⁺) m/z: 574 [M+H]⁺; LC–MS–MS 574: 203. Anal. (C₃₆H₅₁N₃O₃·C₂O₄H₂·½H₂O) C, H, N.

4.3.2. 5-(Dimethylamino)-2-{6-[(5-(4-(4-methylpiperidin-1-yl) butyl)naphthalen-2-yl)oxy]hexyl}isoindole-1,3-dione (6)

The title compound was obtained in a 50% yield (0.284 g) as a bright yellow oil.¹H NMR δ 0.95–1.00 (m, 3H, NHCH₃), 1.15–2.05 [m, 17H, CH(CH₂)₂, (CO)₂NCH₂(CH₂)₄CH₂ and ArCH₂(CH₂)₂], 2.60–2.75 [m, 2H, N(CHH)₂], 2.85–2.95 [m, 2H, N(CHH)₂], 2.98–3.05 [m, 4H, Ar(CH₂)₃CH₂N], 3.10 [s, 6H, N(CH₃)₂], 3.65 (t, J = 7.15 Hz, 2H, (CO)₂NCH₂), 4.05 (t, J = 6.6 Hz, 2H, OCH₂), 6.70–8.05 (m, 9H, aromatic); LC–MS (ESI⁺) m/z: 570 [M+H]⁺; LC–MS–MS 570: 203. Anal. (C₃₆H₄₇N₃O₃·HCl·1.75H₂O) C, H, N.

4.3.3. 2-(6-((5-(3-(4-Cyclohexylpiperazin-1-yl)-3-oxopropyl)-5,6,7,8-tetrahydronaphthalen-1-yl)oxy)hexyl)-5-(dimethylamino) isoindole-1,3-dione (8)

The title compound was obtained in a 57% yield as a yellow oil (0.366 g).¹H NMR δ 1.05–2.05 [m, 24H, piperazine CH₂, OCH₂(CH₂)₄, CH(CH₂)₃], 2.20–2.85 [m, 10H, CHN(CH₂)₂, CH₂CO, benzyl CH and CH₂], 3.11 [s, 6H, (NCH₃)₂], 3.38–3.42 [m, 2H, CON(CHH)₂], 3.58-3.45 [m, 4H, (CO)₂NCH₂ and CON(CHH)₂], 3.90 (t, 2H, J = 6.1 Hz, OCH₂), 6.60–6.65 (m, 6H, aromatic); LC–MS (ESI⁺) m/z: 665 [M+Na]⁺; LC–MS–MS 665: 497, 393. Anal. (C₃₉H₅₄N₄O₄·2HCl) C, H, N.

4.4. Biology

4.4.1. Materials

(+)-Pentazocine was purchased from Sigma–Aldrich (Milan, Italy), and 1,3-di-2-tolylguanidine (DTG) from Tocris Cookson Ltd., U.K. [³H]-DTG (50 Ci/mmol), and (+)-[³H]-pentazocine (30 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). Male Dunkin guinea pigs and Wistar Hannover rats (250–300 g) were from Harlan, Italy. Cell culture reagents were purchased from EuroClone (Milan, Italy).

4.4.2. Competition binding assays

All the procedures for the binding assays were previously described. σ₁ And σ₂ receptor binding were carried out according to Berardi et al. [27]. [³H]-DTG, 1,3-di-2-tolylguanidine (30 Ci/mmol) and (+)-[³H]-pentazocine (34 Ci/mmol) were purchased from PerkinElmer Life Sciences (Zavantem, Belgium). DTG was purchased from Tocris Cookson Ltd. (UK). (+)-Pentazocine was obtained from Sigma–Aldrich-RBI s.r.l. (Milan, Italy). Male Dunkin guinea pigs and Wistar Hannover rats (250–300 g) were from Harlan (Italy). The specific radioligands and tissue sources were, respectively: (a) σ₁ receptor, (+)-[³H]-pentazocine, guinea pig brain membranes without cerebellum; (b) σ₂ receptor, [³H]-DTG in the presence of 1 µm (+)-pentazocine to mask σ₁ receptors, rat liver membranes. The following compounds were used to define the specific binding reported in parentheses: (a) (+)-pentazocine (73–87%), (b) DTG (85–96%). Concentrations required to inhibit 50% of radioligand specific binding (IC50) were determined using six to nine different concentrations of the drug studied in two or three experiments with samples in duplicate. Scatchard parameters (K_d and B_max) and apparent inhibition constants (Ki values) were determined by nonlinear curve fitting, using Prism GraphPad software (version 3.0) [32].

4.4.3. Cell culture

Human breast adenocarcinoma cell line (MCF7) was purchased from ICLC (Genoa, Italy), and were stably transfected with σ₁-receptor c-DNA (MCF7σ₁) as already described [29]. Cells were grown in DMEM high-glucose medium supplemented with fetal bovine serum (10%), and penicillin-streptomycin (1%) in a humidified incubator at 37 °C with a CO₂ atmosphere. Human retinal pigment epithelia ARPE19 cells were purchased from ATCC and grown in complete growth medium (DMEM supplemented with fetal bovine serum 10%) and penicillin-streptomycin (1%) in cell culture incubator with 5% CO₂.

4.4.4. ARPE19 cell imaging

Drug treatment and imaging of ARPE cells was done in HBSS. For control, some wells were pretreated with 100 µMPB190 for 40 min. Cells were further treated with compound 9 0.5 µMfor 40 min, then rinsed twice in HBSS and imaged upon excitation wavelength of 408 nm and emission detection of 525 nm under a Nikon A1R laser confocal microscope (Nikon, Tokyo, Japan) supplied with green 488 nm Argon laser, red 561 nm DPSS laser through a Apo60X VC oil-immersion objective with NIS elements software.

4.4.5. MTT cell viability assay

ARPE19 cells were seeded to 96 well plates to a density of 30,000 cells/well and grown in complete growth medium to reach 100% confluency. Then 1 mM toxin Paraquat was applied with or without various concentrations of compound 9 and cells were incubated for 24 h in complete growth media. Then wells were quickly aspirated and MTT (Sigma–Aldrich M2128) was applied under the dim light at concentration 0.5 mg/mL in phenol red free DMEM (100 µL/well) supplemented with fetal bovine serum 0.5%. After 3 h of incubation 100 µL of 10% Sodium Dodecyl Sulfate (SDS) was added per each well. Plates were left overnight in cell culture incubator to dissolve precipitate. Absorbance at λ = 540 nm was detected in 96 well plate reader.

4.4.6. Flow cytometry studies

MCF7 and MCF7σ₁ cells were incubated with increasing concentrations (0.1,1,10, and 100 nmol/L and 1 and 10 µmol/L) of either PB190 or PB212 followed by 100 nmol/L of either compounds 5 or 6 for 45 or 75 min at 37 °C. To mask σ₂ receptors, compound 10 (10 µmol/L) was co-incubated. When indicated, cells were treated with increasing concentrations (1, 10, and 100 nmol/L and 1 and 10 µmol/L) of (+)-pentazocine or PB190 or PB212, followed by 100 nmol/L of compounds 5 or 6 for 45 or 75 min at 37 °C. At the end of the incubation periods, cells were washed twice with PBS, detached with 200 µL of Cell Dissociation Solution (Sigma Chemical Co.) for 10 min at 37 °C, centrifuged at 13,000 g for 5 min and resuspended in 500 µL of PBS. The fluorescence was recorded using a Bio-Guava^® easyCyte™ 5 Flow Cytometry System (Millipore, Billerica, MA), with a 530 nm band pass filter. For each analysis, 50,000 events were collected and analyzed with the InCyte software (Millipore).

4.5. Fluorescence spectroscopy

Emission and excitation spectra of compounds 5, 6 and 9, were determined in CHCl₃ and in PBS buffer solution as previously reported. Fluorescence quantum yields were calculated with respect to quinine sulfate as previously reported [19].

Abbreviations

DTG

1,3-di-2-tolylguanidine

DMEM

Dulbecco's modified Eagle's medium

PBS

Phosphate buffered saline

SDS

Sodium dodecyl sulfate