Novel indole-based sigma-2 receptor ligands: synthesis, structure – affinity relationship and antiproliferative activity †

,


Introduction
Two subtypes of sigma (σ) receptors, termed σ 1 and σ 2 , have been identified. 1 Both subtypes display different distributions in the central nervous system and peripheral organs.The σ 1 receptor contains 223 amino acids with two transmembrane regions. 2,3It functions as "ligand-operated receptor chaperone" and regulates various ion channels, G protein-coupled receptors, lipids, and other signaling proteins. 4,5In contrast, the σ 2 receptor has not been cloned so far, and its molecular weight was estimated to be 21.5 kD.Recently, progesterone receptor membrane component 1 (PGRMC1) was reported as the putative σ 2 receptor binding site. 6t is interesting that both subtypes are expressed in a variety of human and rodent tumor cell lines. 7,8However, the expression of the σ 2 receptor was found to be higher than that of the σ 1 receptor.3][14][15] Thus, the σ 2 receptor may both serve as a receptor-based biomarker to distinguish different proliferative states of solid tumors and as a promising target for the treatment of cancer. 16n the past decades, morphans, indoles (siramesine analogues), granatanes, flexible benzamides and N-cyclohexylpiperazines have been reported to serve as selective σ 2 receptor ligands. 17mong these ligands, siramesine (also known as Lu-28-179) and its analogues, conformationally flexible amines such as RHM-1, and PB28 analogues were more extensively investigated. 17Their structures are presented in Fig. 1.Although clinical trials of siramesine for the treatment of depression and anxiety were paused in 2002, it proved to be non-toxic and well tolerated in humans.14]18 To obtain selective σ 2 receptor ligands with antiproliferative activity, we used siramesine as the lead compound to design a series of novel indole-based compounds.It was reported that the indole residue and the butyl chain between the indole and the spirocyclic piperidine moieties were important to maintain the σ 2 receptor selectivity for siramesine derivatives. 17We introduced different functional groups to develop new σ 2 receptor ligands.Moreover, we also introduced the substituents with fluorine atom to find PET radiotracers for σ 2 receptor tumor imaging.The design concept is shown in Fig. 2. First, by keeping the 4-fluorophenyl ring at the indole N-atom and the butyl chain constant, 3H-spiroĲ2-benzofuran-1,4′-piperidinyl) moiety was replaced by different pharmacophores including σ 1 preferred group C and σ 2 preferred group A, B, or D (1).Secondly, the 4-fluorophenyl ring at the indole N-atom was replaced by a 2-fluoroalkyl group (2).As a third approach, both the 4-fluorophenyl ring at the indole N-atom and the 3H-spiroĲ2-benzofuran-1,4′-piperidinyl) moiety were modified (3).Fourth, 4-fluorophenyl was replaced by the 4-iodophenyl group, while the 3H-spiroĲ2-benzofuran-1,4′piperidinyl) moiety was replaced by C (4).Finally, the indole core was replaced by 4-fluoro-benzophenone (5).Moreover, the structure-affinity relationships (SAR) of these analogues for σ 2 receptors were analyzed.The 3-Ĳ4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to investigate the antiproliferative activity of the most potent ligands.In order to further support this, cell cycle analysis was carried out to examine the effects of these potent compounds on the cell cycle progression using flow cytometry in DU145 cells.

In vitro radioligand competition studies and structureaffinity relationship analyses
The affinities of the indole-based analogues for the σ 1 and σ 2 receptors were determined by radioligand competition binding assays as reported previously. 23(+)-[ 3 H]Pentazocine and ĳ 3 H]1,3di-o-tolyl-guanidine (in the presence of 10 μM dextrallorphan) were used as radioligands for the σ 1 and σ 2 receptors, respectively.The results are listed in Table 1.
To evaluate a suitable approach for future fluorine-18 radiotracer development for imaging of σ 2 receptors by positron emission tomography, a fluoroalkyl group was introduced.Replacement of the 4-fluorophenyl ring at the indole N-atom with a 2-fluoroethyl residue decreased the affinity for σ 2 receptors but slightly increased the subtype selectivity (2 vs. siramesine).Compounds 3a and 3b with the σ 2 preferred group A displayed comparable affinity for σ 2 receptors to ISO-1. 27However, compound 3b with a 3-fluoropropyl group showed decreased selectivity in comparison to compound 3a with a 2-fluoroethyl group.Replacement of σ 2 preferred group A with 3H-spiroĲ2-benzofuran-1,4′-piperidinyl) moiety increased the σ 1 affinity significantly and thus decreased the selectivity (3c vs. 3a).Moreover, replacement of 1-Ĳ4-fluorophenyl) moiety with 1-Ĳ4-iodophenyl) group slightly decreased the affinity for σ 2 receptors but increased the selectivity (4 vs. 1c).Replacement of the whole indole moiety with 1-Ĳ4-fluorophenyl)carbonyl group dramatically increased the affinity for σ 1 receptors (5 vs. 1a, 5 vs. 3a, 5 vs. 3b), indicating the high importance of the indole moiety to retain the selectivity for σ 2 receptors.

Antiproliferative activity
Recently, a series of compounds with the indole moiety were reported to display antiproliferative activity in MCF7 and MCF7/adr cells. 25In order to find new scaffolds and new σ 2 receptor ligands as potent antitumor agents, antiproliferative activity of compounds 1a and 1b was evaluated in MCF7 (breast cancer), DU145 (androgen-independent human prostate cancer) and C6 (rat glioma) cells using the MTT assay.Antiproliferative activity of siramesine was also determined in these cells as comparison.The effects of these compounds on cellular viability were analyzed using different concentrations between 100 nM and 100 μM.The results expressed as EC 50 values are shown in Table 2.All EC 50 values were found to be in the micromolar range.Compound 1a and siramesine showed notable antiproliferative effects in MCF7 cells with EC 50 values of 20.9 and 23.6 μM, respectively, which are consistent with that reported in the literature (with EC 50 values of 17.8 and 12.3 μM, respectively). 25It is interesting to note that the new compound 1b exhibited the highest activity in MCF7 cells.Moreover, compound 1b displayed notable and comparable antiproliferative effects to compound 1a and siramesine in DU145 cells.However, all of the three compounds displayed a higher EC 50 value in C6 cells than those in the human DU145 and MCF7 tumor cells.Besides compound 1a and siramesine, the indole-based compound 1b with the 5,6-dimethoxyisoindoline moiety seems to be promising as an anti-tumor agent and warrants further evaluation.

Cell cycle analysis
To further examine the antitumor activity of compounds 1a and 1b, their effects on the cell cycle progression were analyzed by flow cytometry in DU145 cells.Cell cycle phase distribution in control DU145 cells and cells treated with different concentrations of 1a, 1b and siramesine at 24 h time point is presented in Fig. 3.The percentages of G 1 , S and G 2 phases of the untreated DU145 cells (control) are 58.2%,38.6% and 3.25%, respectively.Treatment with compound 1a or 1b or siramesine increased the percentage of G 1 cells in a dose-dependent manner.After treatment with 40 μM 1a or 30 μM 1b, the percentages of G 1 cells increased to 84.1% and 80.5%, respectively.At the same time, the percentages of S cells decreased to 15.9% and 19.3%, respectively.The percentage of G 1 phase cells was maintained at 75.7-77.2%after treatment with 15 to 25 μM siramesine.These data suggest that compounds 1a and 1b and siramesine could induce cell cycle delay and arrest the cell cycle progression predominantly at the G 1 phase in DU145 cells.It was reported that σ 2  ligands can induce the tumor cell death by multiple signaling pathways. 1510 μM siramesine decreased the expression levels of cyclin D1, which are responsible for progression through the G 1 phase in MDA-MB-435 cells in a time-dependent manner.Thus, siramesine may block G 1 -phase progression by decreasing cyclin D1 expression.In addition, siramesine also mainly decreased cyclin B1 and pRb in MDA-MB-435 cells.
The investigation of the detailed mechanism in which compounds 1a and 1b impair the G 1 phase of the cell cycle progression in DU145 cells is in progress.

Conclusion
We have developed a series of indole-based σ 2 receptor ligands derived from siramesine.Structure-affinity relationship analyses indicated the high importance of the indole moiety and σ 2 preferred group to improve the selectivity for σ 2 receptors.In the MTT experiments, compound 1b displayed notable and comparable antiproliferative effects to compound 1a and siramesine in DU145 cells and exhibited the highest activity in MCF7 cells.Cell cycle analysis by flow cytometry demonstrated that compounds 1a, 1b and siramesine impaired the cell cycle progression predominantly at the G 1 phase in DU145 cells.The indole-based compound 1b with the 5,6-dimethoxyisoindoline moiety shows potential as an antitumor agent and warrants further evaluation.
4.1.4.General procedure for the syntheses of compounds 12, 13, 14, and 15.The Boc-protected group of compound Boc-12, Boc-13, Boc-14, or Boc-15 was cleaved using TFA in dichloromethane solution at 0 °C for 1 h.Compounds 12-15 were obtained in nearly quantitative yields and used for the next step without further purification.

In vitro radioligand competition studies
Competition assays of σ 1 and σ 2 receptors were performed as previous reported in the literature. 28,29The detailed procedures are provided in the ESI.†

Cell culture and antiproliferative assay
The cancer cell lines MCF7 (human mammary carcinoma), DU145 (human prostate carcinoma) and C6 (rat glioma) were routinely cultured in Beijing Normal University.The MTT assay was used to determine the antiproliferative activity of compounds 1a and 1b and siramesine in these cell lines as described previously. 30,31The procedures are shown in the ESI.† 4.5 Flow cytometry cell cycle analysis 1a, 1b and siramesine were cultured in DU145 cell line for 24 h to examine cell cycle arrest as described previously. 32he detailed procedures are shown in the ESI.†

Fig. 2
Fig. 2 Design concept of the indole-based compounds.

Fig. 3
Fig. 3 Cell cycle phase distribution in control DU145 cells and cells treated with different concentrations of 1a (A), 1b (B) and siramesine (C) at 24 h time point.