piperazine (PB28) - American Chemical Society

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Novel Derivatives of 1‑Cyclohexyl-4-[3-(5-methoxy-1,2,3,4tetrahydronaphthalen-1-yl)propyl]piperazine (PB28) with Improved Fluorescent and σ Receptors Binding Properties Carmen Abate,*,† Mauro Niso,† Roberta Marottoli,† Chiara Riganti,‡ Dario Ghigo,‡ Savina Ferorelli,† Giulia Ossato,§ Roberto Perrone,† Enza Lacivita,† Don C. Lamb,§ and Francesco Berardi† †

Dipartimento di Farmacia-Scienze del Farmaco, Università degli Studi di Bari ALDO MORO, Via Orabona 4, I-70125 Bari, Italy Department of Oncology, University of Turin, via Santena 5/bis, 10126, Torino, Italy § Physical Chemistry, Department of Chemistry, Center for Nanoscience (CeNS), and Center for Integrated Protein Science Munich (CiPSM), Ludwig-Maximilians-Universität München, Munich D-81377, Germany ‡

S Supporting Information *

ABSTRACT: Despite the promising potentials of σ2 receptors in cancer therapy and diagnosis, there are still ambiguities related to the nature and physiological role of the σ2 protein. With the aim of providing potent and reliable tools to be used in σ2 receptor research, we developed a novel series of fluorescent σ2 ligands on the basis of our previous work, where high-affinity σ2 ligand 1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4tetrahydronaphthalen-1-yl)-n-propyl]piperazine (1, PB28) was used as the pharmacophore. Compared to the previous compounds, these novel ligands displayed improved fluorescence and σ2 binding properties, were σ2-specifically taken up by breast tumor cells, and were successfully employed in confocal microscopy. Compound 14, which was the best compromise between pharmacological and fluorescent properties, was successfully employed in flow cytometry, demonstrating its potential to be used as a tool in nonradioactive binding assays for studying the affinity of putative σ2 receptor ligands.

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INTRODUCTION Sigma (σ) proteins were originally characterized as a subtype of the opiate receptors, but later studies clarified that they are a distinct class of receptors divided into two subtypes, σ1 and σ2.1,2 The σ1 protein, which is the better known of the two subtypes, is distributed throughout the brain, where it has been shown to modulate a number of central neurotransmitter systems. Roles in neuroprotection and neuroplasticity have been suggested for this subtype, and implication in several central nervous system (CNS) pathologies (e.g., anxiety, depression, schizophrenia, Parkinson’s and Alzheimer’s disease) has been shown.3,4 A mutation in the σ1 receptor sequence has been suggested to cause juvenile amyotrophic lateral sclerosis (JALS).5 Dimerization and oligomerization of the σ1 receptors have been recently suggested,6 and a functional form of σ1-D2 heteromer has been proposed to account for the involvement of D2 receptors in cocaine activity.7 All of these pieces of evidence keep the interest in σ1 receptors high. As for the σ2 subtypes, the evidence that they are overexpressed in a wide variety of peripheral and brain human tumors gave a great impulse to the σ2 subtype-related research. σ2 Receptors are proposed as endogenous biomarkers for the proliferative status of tumors, since they are more overexpressed in proliferative than in the corresponding quiescent tumor cells.8 Several σ2 radioligands have been developed for the imaging of tumors, and a phase I clinical trial for a promising 18F-radiolabeled σ2 ligand has just been completed.9−11 In addition, the cytotoxic properties of σ2 © 2014 American Chemical Society

agonists in tumor cells in vitro and supported by data coming from preclinical tumor models in vivo are very promising and led to the investigation of σ2 ligands as antitumor agents.12−14 Despite these encouraging data, there are still ambiguities related to the nature of σ2 receptors, their physiological role, and their mechanisms of action. This has been mainly due to the absence of the cloned gene or the purified σ2 receptor protein, and only recently the σ2 receptor binding site has been proposed to be located in the progesterone receptor membrane component 1 (PGRMC1).15 Since interest in σ2 receptor ligands grows together with the need for alternative anti-cancer strategies, powerful tools for the study of these still enigmatic receptors are required. With the aim of providing more potent and reliable tools to be used in σ2 receptor research, we synthesized novel fluorescent derivatives of 1-cyclohexyl-4-[3-(5-methoxy1,2,3,4-tetrahydronaphthalen-1-yl)-n-propyl]piperazine (1, PB28, Chart 1), one of the highest affinity σ2 ligands known.16 In previous work, we identified a position in the structure of 1 where the fluorescent tag could be conveniently attached without compromising the pharmacological properties: the 5-position on the tetraline ring, where the methoxy group could be replaced by a hexamethylenoxy linker.17 Compound 6-{5-[3-(4-cyclohexylpiperazin-1-yl)propyl]Received: December 5, 2013 Published: April 3, 2014 3314

dx.doi.org/10.1021/jm401874n | J. Med. Chem. 2014, 57, 3314−3323

Journal of Medicinal Chemistry

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2,1,3-benzoxadiazol-4-yl). Therefore, ligands emitting in the green region of the electromagnetic spectrum with higher QY were obtained. One red-emitting fluorophore was also connected to the linker to generate σ2 receptor fluorescent ligands, which fluoresce at longer wavelengths where cellular autofluorescence is significantly reduced. For all the compounds, the kinetics were studied by confocal microscopy in human breast tumor cells (MCF7 cells), where the specificity of the fluorescent tracers for σ2 receptors was also verified. The best fluorescent σ2 ligand was exploited to perform a σ2 receptor binding assay in MCF7 cells by flow cytometry.

Chart 1

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RESULTS AND DISCUSSION Chemistry. The synthetic pathway for 7 is depicted in Scheme 1. Reaction between potassium phthalimide and 1,10dibromodecane gave intermediate 4, which was used to alkylate the key phenolic intermediate 3,16 affording phthalimide derivative 5. This phthalimide underwent hydrazinolysis to afford intermediate primary amine 6, which upon reaction with NBD-chloride afforded the final compound 7. The synthetic pathway for 11 is depicted in Scheme 2. Reaction between the primary amine 817 and 4-nitro-1,8naphthalic anhydride (9) afforded the nitro derivative 10. Hydrogenation of this last compound at atmospheric pressure in the presence of 10% Pd over carbon afforded the final compound 11. The synthetic pathway for 14 is depicted in Scheme 3. 4(N,N-Dimethylamino)phthalic acid20 was condensed with 6amino-1-hexanol in the presence of carbonyldiimidazole, affording intermediate alcohol 12. Reaction of 12 with methanesulfonyl chloride afforded mesyl derivative 13 that was subsequently used to alkylate the key phenolic intermediate 3, yielding the final fluorescent compound 14. The synthetic pathway for 17 is depicted in Scheme 4. Acid 15,21 upon activation to the corresponding succinic imide 16, was reacted with amine 8 to afford the final fluorescent

5,6,7,8-tetrahydronaphthalen-5-yloxy}-N-(7-nitro-2,1,3-benzoxadiazol-4-yl)hexanamine (2, PB385, Chart 1) emerged for its best compromise between pharmacological and fluorescent properties and was successfully used in microscopy and flow cytometry studies within pancreatic cancer cells.17,18 However, both the pharmacological (σ2 receptor affinity and selectivity) and fluorescence properties (higher quantum yields, QY, and brightness) of 2 could be improved in order to obtain more powerful and reliable tools for σ2 receptor research. With this aim, we first elongated the chain from six to ten methylenes in the structure of 2. As other authors reported, this elongation resulted in significantly improved σ2 affinity and selectivity in a granatane-based pharmacophore series.19 Nevertheless, this was not the case for our 1-based series, so that subsequently the sixmethylene chain was kept as the optimal linker to connect 1 with fluorophores structurally different from NBD (7-nitroScheme 1a

a

Reagents: (a) NaI, K2CO3; (b) hydrazine hydrate; (c) NBD-chloride. 3315



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Scheme 2a

a

Reagents: (a) H2, 10% Pd on activated carbon.

Scheme 3a

a

bearing molecule 7 produced a 3-fold decrease in the affinity for σ2 receptor binding, compared to the lead compound 2. However, σ2 receptor selectivity remained unchanged since the same decrease was also recorded for σ1 receptor binding (Ki = 258 nM at σ1 and Ki = 31.2 nM at σ2 receptors). On the other hand, the six-methylene chain derivative 11 carrying the greenemitting fluorophore (4-amino-1,8-naphthylimide) displayed improved binding properties at σ2 receptors: a slightly higher affinity at σ2 (Ki = 6.57 nM) and a 2-fold lower affinity at the σ1 subtype (Ki = 156 nM), thus resulting in a convenient increase in σ 2 selectivity. Insertion of 4-(N,N-dimethylamino)phthalimide in place of NBD led to the fluorescent derivative 14, with slightly improved binding to the σ2 receptor (Ki = 6.9 nM). However, this last molecule displayed equal high affinity for the σ1 receptor (Ki = 5.37 nM). Together, these results suggested that steric hindrance of the fluorophores negatively affects binding to σ1 receptors while maintaining the high affinity for the σ2 subtype. As for the red-emitting fluorescent ligand 17, a significant drop in the affinity for both σ receptors was recorded (Ki = 235 nM at σ1 and Ki = 161 nM at σ2 receptors), suggesting that either the positive permanent charge or the more extended structure of the fluorophore, compared to the other fluorophores, hampers the interaction with both σ binding sites. Fluorescent Ligand Studies. Fluorescent Properties. The fluorescent properties of final compounds are listed in Table 1. Excitation and emission spectra were obtained from solutions of the final compounds in organic solvent (CHCl3) and in aqueous solution (phosphate-buffered saline, PBS). All of the compounds showed important differences between maximum excitation and emission wavelength (λex and λem) (i.e., Stokes shift), with 14 displaying the highest Stokes shift. QY values were determined in the above-mentioned solvents to probe the sensitivity of the final fluorescent ligands to the environment (i.e., low QY in aqueous solution but high fluorescence in

Reagents: (a) MsCl, NEt3; (b) K2CO3.

compound 17. All the final compounds were converted to the corresponding hydrochloride salts with gaseous HCl except for 17. Biology: σ Receptor Binding. Results from binding assays are expressed as inhibition constants (Ki values) in Table 1. The elongation of the linker to ten methylenes in the NBD3316



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Scheme 4a

new fluorescent compounds (11, 14, and 17) is also similar to what has been observed previously with other σ2 fluorescent ligands.17,19 At a concentration of 100 nM, the new fluorescent σ2 receptor ligands localize mostly in the cytoplasm, perinuclear region of the cell, and vesicles (Figure 1). At higher concentrations (500 nM), the fluorescence signal increases in cytosol and vesicles and fluorescence intensity was also detected in the nuclei (Figure S1 in Supporting Information). The signal from the nuclei can be clearly visualized in the intensity profile of pixels in the background and pixels in the nuclei. The presence of σ2 receptor ligands in the nuclei could be explained by the hypothesis of Colabufo et al.24 that the σ2 receptor has histone binding proprieties. However, this behavior needs further characterization since, at this high concentration (500 nM), unspecific interaction of compounds with cellular proteins other than σ2 receptors cannot be excluded. At 500 nM, ligand localization is slightly different than at 100 nM: 17 tends to accumulate more in vesicles and 11 is in the cytoplasm and concentrates in the perinuclear region, while 2 and 14 are more homogeneously distributed (Figure S1 in Supporting Information). No colocalization experiments were performed with markers for various organelles, as the high amount of fluorescence of the ligand observed throughout the cytosol would lead to an apparent colocalization signal, making the results inconclusive. Uptake of σ2 Ligands. Uptake of fluorescent compounds displaying the highest σ2 affinity (lead compound 2, 11, and 14) was studied in σ2-overexpressing MCF7 cells. Uptake of the compounds increased in a dose-dependent manner (Figure 2) at the three concentrations used, reaching a plateau between 50 and 100 nmol/L. Compound 14 showed the highest uptake, with around 2-fold higher uptake than 2, and 10-fold higher uptake than 11 (picomoles of fluorescent compound on milligram of protein). The higher intracellular fluorescence of σ ligand 14 could be due to its higher QY and/or to its peculiar chemical structure. Compound 14 displays high affinity for both σ1 and σ2 receptors, in contrast with the more σ2-receptorselective 2 and 11, making it suitable for monitoring dual nonselective uptake. However, in cell lines such as MCF7 where the density of σ1 receptors is known to be low in comparison to that of the σ2 subtypes,25 all compounds including 14 can be used as highly specific σ2 receptor ligands as demonstrated by competition assays. Pretreatment of cells with increasing concentration of reference σ2 agonists 113 or cis1-cyclohexyl-4-[4-(2,6-difluorophenyl)cyclohexyl]piperazine (18, Chart 1)17,26 (10, 25, and 50 nmol/L) was followed by administration of the fluorescent compounds 2, 11, or 14 at 25 nmol/L (Figure 2). Uptake of the three fluorescent compounds 2, 11, and 14 was reduced in a dose-dependent manner by the two σ2 agonists, confirming that these fluorescent σ2 ligands are targeting σ2 receptors. Also, in the competition assays, the absolute values of fluorescence differed for each compounds and reproduced the differences in fluorescence observed in the dose-dependence assays, due to the different QY. However, the general trendthat is, a progressive reduction of the intracellular fluorescence in the presence of increasing concentrations of compound 1 or 18strongly suggests that all the compounds are highly specific for σ2 receptors. The highest decrease in fluorescence exerted by σ2 agonists 1 or 18 (50 nmol/L) was obtained with 14, indicating that this compound is by far the most powerful ligand for σ2 receptors. Flow Cytometry Study. We then set up a σ2 receptor binding assay, by incubating nonpermeabilized MCF7 cells with

a Reagents: (a) N,N′-disuccinimidyl carbonate, 4-(dimethylamino)pyridine, NEt3; (b) Et3N.

nonpolar solvents or when bound to a hydrophobic sites). As expected, the highest QY values were those recorded in CHCl3 for all final compounds. On the other hand, all the compounds exhibited low fluorescence in PBS buffer. Compounds (11, 14, and 17) with fluorescent tags diverse from NBD displayed higher QY values and showed improved fluorescent properties compared to the so far known σ2 fluorescent ligands.17,19,22,23 These ligands were obtained through two methods: by using fluorescent moieties (β-naphthol and carbazole nuclei) as the hydrophobic portions of the σ2 ligands22 or by conjugating σ2 receptor pharmacophores with fluorescent moieties (NBD or dansyl).17,19,23 Among all the compounds tested, the greenemitting 14 displayed the highest Stokes shift and the highest QY in CHCl3 and PBS. Confocal Microscopy and in Vitro Internalization Studies. All ligands examined (2, 11, 14, and 17) were detectable by spinning disk confocal microscopy in live and fixed MCF7 cells starting from a concentration of 50 nM. Compound 7 did not undergo this study since its binding properties were worse than 2 (its inferior homologue) and the fluorescent properties were the same. Therefore, we chose to study 2 rather than 7 in order to obtain more reliable information. We observed the entry of the new σ2 receptor ligands into MCF7 cells within 40 min. The fluorescence intensity inside the cells starts increasing 20 min after treatment and reaches a plateau 40 min after treatment. This uptake kinetics agrees with the observation of Abate et al.17 for other σ2 ligands. The distribution of all the 3317



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Table 1. Receptor Affinities and Fluorescence Properties of Final and Reference Compoundsa

Values are the means of n ≥ 2 separate experiments, in duplicate. bFluorescence properties herein reported were evaluated on compounds as free bases in CHCl3 and as their corresponding hydrochloride salts in PBS solutions. cFrom ref 17. dCompound 17 displayed low solubility in CHCl3. Therefore, no fluorescence properties were calculated for this compound in this solvent.

a

increasing concentrations (from 0.1 nmol/L to 10 μmol/L) of 1, followed by 100 nmol/L 14. We detected a progressive decrease of 14 fluorescence when the concentration of 1 was increased (Figure 3A). This trend, indicative of the progressive displacement of 14 exerted by 1, suggests that fluorescent ligand 14 and compound 1 compete for binding on σ2 receptors. The binding curve (Figure 3A) obtained by this displacement shows an IC50 = 4.27 nM for 1. This data was in accordance with what we found by competition binding assay where [3H]DTG was displaced by 1 (from 0.1 nmol/L to 10 μmol/L) in MCF7 cell membranes (IC50 = 1.2 nM, Figure 3B). In order to give further support to the specificity of compound 14 for σ2 receptors in intact MCF7 cells, we performed a binding assay with the σ2 receptor reference compound 1,3di(2-tolyl)guanidine (DTG). We incubated MCF7 cells with increasing concentrations (from 0.1 nmol/L to 10 μmol/L) of DTG, followed by 100 nmol/L 14. We detected a progressive decrease of 14 fluorescence when the concentration of DTG increased (Figure S2 in Supporting Information). The experiment was performed with and without (+)-pentazocine to mask the unlikely presence of σ1 receptor, and no difference was observed, supporting the very low density of σ1 receptors in this cell line. This is the first assay showing that fluorescent ligand 14 may be a useful tool in nonradioactive binding assays, to study the affinity of putative σ2 receptors ligands and to map σ2 receptors localization and density on cells and tissues.

the compounds accumulate in cells with a distribution in line with the σ2 receptor distribution previously reported. Fluorometric experiments showed a clear and σ2-dependent uptake of the compounds, with ligand 14 (highest QY and highest σ2 affinity) showing the highest cell uptake. Therefore, the suitability of 14 as a σ2 fluorescent tracer was tested in flow cytometry where σ2 binding in MCF7 cells was successfully verified. Overall, we produced high-affinity σ2 fluorescent ligands and showed their potential to be used in σ2 receptor studies within cells. To the best of our knowledge, 14 is the first σ2 fluorescent ligand successfully employed in binding experiments in living cells by flow cytometry, demonstrating that it is a useful and safe alternative to the use of radioligands.

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EXPERIMENTAL SECTION

Chemistry. Both column chromatography and flash column chromatography were performed with 60 Å pore size silica gel as the stationary 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 or HPLC, confirming a purity >95%. Elemental analyses (C, H, N) were performed on an Eurovector Euro EA 3000 analyzer; analytical results were within 0.4% of theoretical values. High-performance liquid chromatography (HPLC) was performed on a Perkin−Elmer series 200 LC instrument with a Phenomenex Gemini RP-18 column (250 × 4.6 mm, 5 μm particle size) and equipped with a Perkin−Elmer 785A UV/vis detector set at λ = 254 nm. 1H NMR spectra were recorded on a Mercury Varian 300 MHz with CDCl3 as solvent. The following data were reported: chemical shift (δ) in parts per million (ppm), multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet), integration, and coupling constant(s) in hertz. Recording of mass spectra was done on an Agilent 1100 series LCMSD trap system VL mass spectrometer; only significant m/z peaks, with their percentage of relative intensity in

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CONCLUSIONS Novel σ2 fluorescent ligands with different structural and fluorescence properties were synthesized on the basis of 2 with the structure of 1 as the pharmacophore. Better results were obtained when fluorescent ligands carried a green-emitting tag on a six-methylene linker. Confocal microscopy showed that all 3318



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Figure 1. MCF7 cells treated for 45 min with 100 nM 2 (A), 11 (B), 14 (C), and 17 (D) before fixation with 2% paraformaldehyde (PFA). All images are background-corrected. All the compounds localize in the cytoplasm and in vesicles. The detected fluorescence from 11 and 17 is higher compared to the signals from 2 and 14. Scale bar = 25 μm. parentheses, are reported. Chemicals were from Aldrich and Across and were used without any further purification. 2-(10-{1-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-tetrahydronaphthalen-5-yloxy}decyl)-1H-isoindole-1,3(2H)-dione (5). To a solution of phenol 3 (100 mg, 0.28 mmol) in N,N-dimethylformamide (DMF) (5 mL) were added a grain of NaI, K2CO3 (52 mg, 0.38 mmol), and 4 (139 mg, 0.38 mmol). The reaction mixture was heated for 18 h at 100 °C. After cooling, the solvent was removed under reduced pressure, and then H2O (5 mL) was added to the residue and the mixture was extracted with AcOEt (3 × 5 mL). The collected organic layers were dried over Na2SO4 and evaporated to afford a residue, which was purified by column chromatography with CH2Cl2/ MeOH (95:5) as eluent to give the final compound as a yellow oil (55.67 mg, 31% yield). 1H NMR δ 1.10−1.48 [m, 10H, (CH2)5 cyclohexyl], 1.55−2.23 [m, 24H, CH2CH2CH2N, CHCH2CH2, OCH 2 (CH 2 ) 8 CH 2 N], 2.50−2.88 (m, 6H, CH cyclohexyl, CH2CH2CH2N, benzyl CH2 and CH), 3.05−3.40 (m, 8H, piperazine), 3.67 [t, 2H, J = 7.3 Hz, O(CH2)9CH2N], 3.91 [t, 2H, J = 6.3 Hz, OCH2(CH2)9N], 6.63 (d, 1H, J = 7.7 Hz, aromatic), 6.71 (d, 1H, J = 7.7 Hz, aromatic), 7.06 (t, 1H, J = 7.98 Hz, aromatic), 7.67−7.74 (m, 2H, aromatic), 7.78−7.86 (m, 2H, aromatic). LC-MS (ESI+) m/z 642 [M + H]+. 10-{1-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-tetrahydronaphthalen-5-yloxy}decylamine (6). Hydrazine hydrate 50% (0.085

mL, 0.85 mmol) was added to a solution of phthalimide 5 (179 mg, 0.28 mmol) in methanol (3 mL), and the reaction mixture was stirred at room temperature for 30 min. HCl (1.5 N, 1.2 mL) was then added and the mixture was stirred for a further 12 h. Then 3 N HCl was added until pH < 2 was obtained, and the mixture was heated at reflux for 30 min. After cooling down to room temperature, the mixture was filtered, and the solid residue was washed with cold MeOH and with Et2O and dried under vacuum. The white solid obtained was made free base with alkaline tratment to afford the target compound as a pale yellow oil (104 mg, 70% yield). 1H NMR 1.00−1.30 (m, 5H, cyclohexyl), 1.40−1.85 [m, 29H, 5H cyclohexyl, CH2CH2CH2N, CHCH2CH2, OCH2(CH2)8CH2N], 1.90−2.10 (br s, 2H, exchanged D2O), 2.20−2.68 [m, 13H, CH cyclohexyl, CH2CH2CH2N, piperazine CH2, O(CH2)9CH2N], 2.78−2.90 (m, 3H, benzyl CH2 and CH), 3.92 [t, 2H, J = 6.3 Hz, OCH2(CH2)9N], 6.61 (d, 1H, J = 7.9 Hz, aromatic), 6.75 (d, 1H, J = 7.7 Hz, aromatic), 7.05 (t, 1H, J = 7.9 Hz, aromatic). LC-MS (ESI+) m/z 512 [M + H]+. 10-{1-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-tetrahydronaphthalen-5-yloxy}-N-(7-nitro-2,1,3-benzoxadiazol-4-yl)decylamine (7). 4-Chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl, 34 mg, 0.17 mmol) was dissolved in absolute EtOH (4 mL) and added dropwise to amine 6 (87 mg, 0.17 mmol) dissolved in CH3CN/EtOH (1:1, 4 mL) and kept under N2. The reaction mixture was stirred for 2 h at room temperature. Then the solvent was removed under reduced 3319



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Figure 2. (Left panels) MCF7 cells were incubated with 25, 50, and 100 nmol/L 2, 11, or 14 for 45 min at 37 °C. (Right panels) Cells were pretreated with 10, 25, and 50 μmol/L 1 or 18 for 45 min at 37 °C followed by 25 nmol/L 2, 11, or 14 for 45 min at 37 °C. The uptake of σ2 ligands was measured fluorometrically in duplicate as reported in the Experimental Section. Data are presented as mean ± SE (n = 2). pressure and a solution of Na2CO3 was added to the residue to obtain an alkaline pH. The aqueous phase was extracted with CH2Cl2 (3 × 15 mL), and the organic layers were collected and dried with Na2SO4 and then evaporated under reduced pressure to afford a brown oil. The crude mixture was purified by column chromatography with AcOEt/ MeOH (7:3) as eluent to give the target compound as a brown oil (50.4 mg, 44% yield). 1H NMR 1.00−1.89 [m, 34H, (CH2)5 cyclohexyl, CH2CH2CH2N, CHCH2CH2, OCH2(CH2)8CH2N], 2.20−2.78 (m, 14H, CH cyclohexyl, CH2CH2CH2N, piperazine CH2, benzyl CH2 and CH), 3.40−3.58 [m, 2H, OCH2(CH2)9N], 3.92 [t and br s, 3H, J = 6.3 Hz, O(CH2)9CH2N and NH], 6.17 (d, 1H, J = 8.8 Hz, aromatic), 6.61 (d, 1H, J = 8.0 Hz, aromatic), 6.76 (d, 1H, J = 7.7 Hz, aromatic), 7.06 (t, 1H, J = 7.8 Hz, aromatic), 8.50 (d, 1H, J = 8.5 Hz, aromatic). LC-MS (ESI+) m/z 675 [M + H]+, 697 [M + Na]+. Anal. (C39H58N6O4·3HCl·0.5H2O) Calcd: 59.05, 7.88, 10.59. Found: 59.43, 7.68, 10.24. 2-(6-{1-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-tetrahydronaphthalen-5-yloxy}hexyl)-6-nitro-1H-benzo[de]isoquinoline1,3(2H)-dione (10). Under N2 atmosphere, 4-nitro-1,8-naphthalic anhydride (9) (53.5 mg, 0.22 mmol) was dissolved in absolute EtOH (5 mL) and added dropwise to hexylamine derivative 8 (102 mg, 0.22 mmol) dissolved in absolute EtOH (10 mL). The reaction mixture was refluxed for 3 h. The solvent was then removed under reduced

pressure to afford a brown oil. The crude mixture was purified by column chromatography with CH2Cl2/MeOH (98:2) as eluent to give the target compound as a brown oil (70 mg, 51% yield). 1H NMR δ 1.05−1.35 [m, 5H, (CHH)5 cyclohexyl], 1.38−2.10 [m, 21H, (CHH)5 cyclohexyl, OCH2(CH2)4CH2N, CH2CH2CH2N, CHCH2CH2], 2.25− 2.90 (m, 14H, piperazine CH2, CH2CH2CH2N, CH cyclohexyl, benzyl CH2 and CH), 3.92 [t, 2H, J = 6.1 Hz, OCH2(CH2)5N], 4.20 [t, 2H, J = 7.6 Hz, O(CH2)9CH2N], 6.60 (d, 1H, J = 8.0 Hz, aromatic), 6.74 (d, 1H, J = 7.7 Hz, aromatic), 7.04 (t, 1H, J = 7.8 Hz, aromatic), 7.98 (t, 1H, J = 8.0 Hz, aromatic), 8.40 (d, 1H, J = 8.0 Hz, aromatic), 8.68 (d, 1H, J = 8.0 Hz, aromatic), 8.73 (d, 1H, J = 7.1 Hz, aromatic), 8.84 (d, 1H, J = 8.8 Hz, aromatic). 6-Amino-2-(6-{1-[3-(4-cyclohexylpiperazin-1-yl)propyl]-1,2,3,4tetrahydronaphthalen-5-yloxy}hexyl)-1H-benzo[de]isoquinoline1,3(2H)-dione (11). Compound 10 (68 mg, 0.10 mmol) was dissolved in EtOH and hydrogenated with 10% Pd on activated carbon at atmospheric pressure for 12 h. Then the mixture was filtered off on Celite and the filtrate was concentrated under reduced pressure to afford the residue as a yellow oil. The crude mixture was purified by column chromatography with CH2Cl2/MeOH (9:1) as eluent to give the final compound as a colorless oil (22 mg, 34% yield). 1H NMR δ 1.05−1.35 [m, 5H, (CHH)5 cyclohexyl], 1.40−2.05 [m, 21H, (CHH)5 cyclohexyl, OCH2(CH2)4CH2N, CH2CH2CH2N, CHCH2CH2], 2.30− 3320



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NCH2), 4.21 (t, 2H, J = 6.5 Hz, CH2O), 6.78 (dd, 1H, J = 8.5 Hz, J′ = 2.5 Hz, aromatic), 7.06 (d 1H, J = 2.4 Hz, aromatic), 7.63 (d, 1H, J = 8.5 Hz, aromatic). LC-MS (ESI+) m/z 391 [M + Na]+. 2-(6-{1-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-tetrahydronaphthalen-5-yloxy}hexyl)-5-dimethylamino-1H-isoindole-1,3(2H)dione (14). Intermediate 13 (130 mg, 0.33 mmol), K2CO3 (45.1 mg, 0.33 mmol) and phenol 3 (103 mg. 0.26 mmol) were dissolved in DMF (10 mL), and the reaction mixture was heated at 150 °C for 24 h. After cooling, the solvent was removed under reduced pressure and H2O (10 mL) was added to the residue. The mixture was extracted with CH2Cl2 (3 × 10 mL). The collected organic layers were dried (Na2SO4) and evaporated to afford a crude residue as a yellow oil, which was purified by column chromatography with CH2Cl2/MeOH (98:2) as eluent. The final compound was obtained as a colorless oil (49 mg, 30% yield). 1H NMR δ 1.15−2.04 [m, 26H, (CH2)5 cyclohexyl, CH 2CH2CH2N, CHCH2CH2, OCH2(CH2)4CH2N], 2.35−2.82 [m, 14H, piperazine CH 2 , CH cyclohexyl, CH(CH2)2CH2N, benzyl CH2], 3.11 [s, 6H, N(CH3)2], 3.63 [t, 2H, J = 7.15 Hz, O(CH 2 ) 5 CH 2 N], 3.90 [t, 2H, J = 6.3 Hz, OCH2(CH2)5N], 6.59 (d, 1H, J = 8.0 Hz, aromatic), 6.73−6.79 (m, 2H, aromatic), 7.02−7.07 (m, 2H, aromatic), 7.63 (d, 1H, J = 8.5 Hz, aromatic). LC-MS (ESI+) m/z 629 [M + H]+, 651 [M + Na]+. Anal. (C39H56N4O3·2HCl·H2O) Calcd: 65.07, 8.40, 7.78. Found: 64.85, 8.10, 7.71. (E)-2-[2-(7-Diethylamino-2-oxo-2H-chromen-3-yl)vinyl]-3,3-dimethyl-1-[6-(2,5-dioxopyrrolidin-1-yl)-6-oxohexyl]indolium Bromide (16). To acid 15 (200 mg, 0.34 mmol) in anhydrous DMF (2 mL) were added N,N′-disuccinimidyl carbonate (205 mg, 0.80 mmol) and 4-(dimethylamino)pyridine (79 mg, 0.64 mmol), and the resulting mixture was stirred for 1.5 h at 70 °C. Upon addition of Et2O (5 mL) to the solution, crystals were formed, filtered, and dried under vacum to afford the target compound as a blue-purple solid (156 mg, 68% yield). The compound was stored at 4 °C. LC-MS (ESI+) m/z 598 [M]+; LC-MS-MS 598:500. (E)-1-[5-(6-{1-[3-(4-Cyclohexylpiperazin-1-yl)propyl]-1,2,3,4-tetrahydronaphthalen-5-yloxy}hexylaminocarbonyl)pentyl]-2-[2-(7-diethylamino-2-oxo-2H-chromen-3-yl)vinyl]-3,3-dimethyl-3H-indolinium Bromide (17). Compound 8 (68 mg, 0.15 mmol) and NEt3 (0.02 mL, 0.15 mmol) were added to a solution of 16 (90 mg, 0.15 mmol) in anhydrous DMF (3 mL). The mixture was stirred for 24 h at room temperature. The solvent was then removed under reduced pressure to afford a brown semisolid, which was purified by column chromatography with CHCl3/MeOH (95:5) as eluent to give the final compound 17 as a brown semisolid (42.2 mg, 28% yield). LC-MS (ESI+) m/z 939 [M + H]+, 974.79 [M + Na] +. HPLC analysis with MeOH/H2O/HCOOH (50 mM, pH = 5), 8:2 v/v at a flow rate 1 mL/min indicated that the compound was >98% pure. Biology. Materials. [3H]DTG (50 Ci/mmol), and (+)-[3H]pentazocine (30 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). DTG was purchased from Tocris Cookson Ltd., U.K. (+)-Pentazocine was obtained from Sigma−Aldrich (Milan, Italy). Male Dunkin guinea pigs and Wistar Hannover rats (250−300 g) were from Harlan, Italy. Cell culture reagents were purchased from EuroClone (Milan, Italy). Competition Binding Assays. All the procedures for the binding assays were previously described. σ1 and σ2 receptor binding were carried out according to Matsumoto et al.27 The specific radioligands and tissue sources were as follows: (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 by using 6−9 different concentrations of the drug studied in two or three experiments with samples in duplicate. Scatchard parameters (Kd and Bmax) and apparent inhibition constants (Ki) values were determined by nonlinear curve fitting using Prism GraphPad software (version 3.0).28

Figure 3. Binding curves of 1: (A) to MCF7 cells by flow cytometry with fluorescent ligand 14 (IC50 = 4.2 nM) or (B) to MCF7 cell membranes by radioligand binding assay (IC50 = 1.20 nM). Data shown represent the mean from two different experiments with samples in duplicate. 2.85 (m, 14H, piperazine CH2, CH2CH2CH2N, CH cyclohexyl, benzyl CH2 and CH), 3.91 [t, 2H, J = 6.2 Hz, OCH2(CH2)5N], 4.17 [t, 2H, J = 7.4 Hz, O(CH2)9CH2N], 4.99 (br s, 2H, D2O exchanged), 6.59 (d, 1H, J = 7.7 Hz, aromatic), 6.72 (d, 1H, J = 7.4 Hz, aromatic), 6.89 (d, 1H, J = 8.3 Hz, aromatic), 7.04 (t, 1H, J = 7.8 Hz, aromatic), 7.65 (t, 1H, J = 7.8 Hz, aromatic), 8.11 (d, 1H, J = 8.5 Hz, aromatic), 8.41 (d, 1H, J = 8.2 Hz, aromatic), 8.60 (d, 1H, J = 8.2 Hz, aromatic). LC-MS (ESI+) m/z 651 [M + H]+, 673 [M + Na] +. Anal. (C41H54N4O3· 3HCl·0.5H2O) Calcd: 64.01, 7.60, 7.28. Found: 63.93, 7.45, 7.32. 5-Dimethylamino-2-(6-hydroxyhexyl)-1H-isoindole-1,3(2H)dione (12). To a solution of 4-dimethylaminophthalic acid (266 mg, 1.28 mmol) in anhydrous DMF (8 mL), kept under Ar, was added 1,1carbonyldiimidazole (357 mg, 2.18 mmol). The mixture was stirred for 30 min at room temperature, and then 6-amino-1-hexanol (148 mg, 1.28 mmol) in anhydrous DMF (8 mL) was added. The reaction mixture was stirred for 12 h at room temperature. The solvent was then removed under reduced pressure and H2O was added. The aqueous phase was extracted with AcOEt (3 × 15 mL) and CH2Cl2 (3 × 15 mL). The organic layers were collected, dried over Na2SO4, and evaporated under reduced pressure to afford the crude mixture as an orange oil. Purification by column chromatography with CH2Cl2/ MeOH (9:1) as eluent gave the title compound as a yellow oil (160 mg, 43% yield). LC-MS (ESI+) m/z 291 [M + H]+. 6-[5-Dimethylamino-1,3(2H)-dioxo-1H-isoindol-2-yl]hexyl Methanesulfonate (13). To a solution of 12 (150 mg, 0.50 mmol) in CH2Cl2 (5 mL) were added methanesulfonyl chloride (0.6 mmol, 0.033 mL) and triethylamine (1.2 mmol, 0.16 mL) at 0 °C, and the mixture was stirred for 1.5 h. Afterward, H2O was added to the reaction and the mixture was extracted with CH2Cl2 (3 × 15 mL). The organic layers were then collected and dried over Na2SO4 and evaporated under reduced pressure to afford the crude mixture as a yellow oil. Upon purification by column chromatography with AcOEt/ hexane (8:2) as eluent, the target final compound was obtained as a yellow solid (130 mg, 71% yield), mp 79−81 °C. 1H NMR δ 1.28− 1.52 (m, 4H, NCH2CH2), 1.58−1.82 (m, 4H, CH2CH2CH2O), 3.0 (s, 3H, SO2CH3), 3.11 [s, 6H, N(CH3)2], 3.63 (t, 2H, J = 7.15 Hz, 3321



Article

Cell Cultures. Human MCF7 breast adenocarcinoma was purchased from ICLC (Genoa, Italy). MCF7 cells was grown in Dulbecco’s modified Eagle’s medium (DMEM) high glucose supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin, in a humidified incubator at 37 °C with a 5% CO2 atmosphere. Fluorescence Spectroscopy. Emission and excitation spectra of 7, 11, 14, and 17 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.17,29 Fluorometric Studies. Uptake of σ2 Ligands. In the dosedependence assays, MCF7 cells were incubated for 45 min at 37 °C with increasing concentrations (25, 50, and 100 nmol/L) of 2, 11, and 14. In the competition assays, cells were pretreated with 10, 25, and 50 μmol/L 1 or 18 for 45 min at 37 °C, followed by 25 nmol/L of 2, 11, and 14 for 45 min at 37 °C. All the compounds were dissolved in ethanol and prepared as 1000× stock solutions. At the end of the incubation periods, cells were washed five times with PBS, detached with trypsin/ethylenediaminetetraacetic acid (EDTA) (0.05/0.02% v/ v), centrifuged at 13000g for 5 min, resuspended in 1 mL of ethanol, and sonicated (two bursts of 10 s, amplitude 40%, Hielscher UP200S ultrasound sonicator, Hielscher Ultrasonics GmbH, Teltow, Germany). A 50 μL aliquot was used to measure the cell proteins with the bicinchoninic acid (BCA) kit (Sigma Chemical Co., St. Louis, MO). The remaining sample was analyzed for intracellular fluorescence with a LS-5 spectrofluorometer (PerkinElmer, Waltham, MA), at the following wavelengths (from spectra in EtOH): λex = 467 nm, λem = 520 nm (2); λex = 40 nm, λem = 530 nm (11); and λex = 390 nm, λem = 520 nm (14). The fluorescence of nontreated cells, considered as a blank, was subtracted from the fluorescence of each sample. Fluorescence values were normalized for the protein content and expressed as picomoles of ligand per milligram of cell proteins, according to the titration curves performed previously for each fluorescent ligand. Flow Cytometry. MCF7 cells were incubated with increasing concentrations (0.1, 1, 10, and 100 nmol/L and 1 and 10 μmol/L) of 1 followed by 100 nmol/L 14 for 45 min at 37 °C. Analogously, the same experiment was performed with increasing concentrations of DTG (0.1, 1, 10, and 100 nmol/L and 1 and 10 μmol/L) with or without (+)-pentazocine (1 μmol/L) followed by 100 nmol/L 14 for 45 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 13000g for 5 min, and resuspended in 500 μL of PBS. The fluorescence was recorded on a FACSCalibur system (Becton Dickinson Biosciences, San Jose, CA), with a 530 nm band-pass filter. For each analysis, 10 000 events were collected and analyzed with Cell Quest software (Becton Dickinson Biosciences). Microscopy Studies: σ2 Ligand Localization. Confocal fluorescence microscopy experiments were performed on a spinningdisk confocal microscope system (Revolution System; Andor Technology) that utilized a Nikon microscope base (TE2000E) and the spinning-disk unit CSU10 from Yokogawa. Measurements were performed with an oil-immersion fluorescence objective (PlanFluor 40×, NA 1.30, Nikon). The detection path was equipped with a TuCam system (Andor Technology) for dual-color detection, corresponding dichroic mirror and filter set for the green and red detection paths (BS580, HC525/50 and HC617/73; AHF Analysentechnik AG), and two DV-887 Ixon EMCCD cameras (Andor). In addition, a triple-band dichroic beam splitter was used to separate laser excitation from fluorescence emission (Di01-T405/488/568/647; Semrock). The excitation was controlled with an acousto-optic tunable filter (AOTF). The sample position was controlled with an xyz piezo stage (ProScan II, NanoScanZ; Prior Scientific). Compounds 11 and 14 were excited at 405 nm and emission was observed in the green channel (HC525/50); 2 was excited at 488 nm and emission was detected in the green channel (HC525/50); and 17 was excited at 561 nm and emission was measured in the red channel (HC617/73).

MCF7 cells were seeded onto LabTek slide chamber systems (eight-well) 24 h before the treatment with σ2 receptor ligands for 45 min. After treatment, cells were fixed with 2% PFA at 37 °C for 30 min, washed with PBS, and imaged.

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ASSOCIATED CONTENT

S Supporting Information *

Two figures showing confocal images of MCF7 cells incubated with novel fluorescent σ2 ligands (500 nM) and binding curve of reference σ2 ligand DTG by flow cytometry in MCF7 cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel +39-080-5442750; fax +39-080-5442231; e-mail carmen. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by MIUR with Grant 2009NB3E8M_002. D.C.L. gratefully acknowledges support of the Deutsche Forschungsgemeinschaft (DFG) through the SFB 1035 and the excellence cluster Nanosystems Initative Munich (NIM), and from the Ludwig-Maximilians-Universität München through the LMUInnovativ BioImaging Network and the Center for NanoScience (CeNS).

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ABBREVIATIONS USED CNS, central nervous system; JALS, juvenile amyotrophic lateral sclerosis; MCF7, human breast adenocarcinoma cells; NBD, 7-nitro-2,1,3-benzoxadiazol-4-yl; PBS, phosphate-buffered saline; PGRMC1, progesterone receptor membrane component 1; QY, quantum yield

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REFERENCES

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