Synthesis of Aminoethyl‐Substituted Piperidine Derivatives as σ1 Receptor Ligands with Antiproliferative Properties

Abstract A series of novel σ1 receptor ligands with a 4‐(2‐aminoethyl)piperidine scaffold was prepared and biologically evaluated. The underlying concept of our project was the improvement of the lipophilic ligand efficiency of previously synthesized potent σ1 ligands. The key steps of the synthesis comprise the conjugate addition of phenylboronic acid at dihydropyridin‐4(1H)‐ones 7, homologation of the ketones 8 and introduction of diverse amino moieties and piperidine N‐substituents. 1‐Methylpiperidines showed particular high σ1 receptor affinity and selectivity over the σ2 subtype, whilst piperidines with a proton, a tosyl moiety or an ethyl moiety exhibited considerably lower σ1 affinity. Molecular dynamics simulations with per‐residue binding free energy deconvolution demonstrated that different interactions of the basic piperidine‐N‐atom and its substituents (or the cyclohexane ring) with the lipophilic binding pocket consisting of Leu105, Thr181, Leu182, Ala185, Leu186, Thr202 and Tyr206 are responsible for the different σ1 receptor affinities. Recorded logD7.4 and calculated clogP values of 4a and 18a indicate low lipophilicity and thus high lipophilic ligand efficiency. Piperidine 4a inhibited the growth of human non‐small cell lung cancer cells A427 to a similar extent as the σ1 antagonist haloperidol. 1‐Methylpiperidines 20a, 21a and 22a showed stronger antiproliferative effects on androgen negative human prostate cancer cells DU145 than the σ1 ligands NE100 and S1RA.


Preparation of membrane homogenates from guinea pig brain
Five guinea pig brains were homogenized with the potter (500-800 rpm, 10 up and down strokes) in 6 volumes of cold 0.32 M sucrose. The suspension was centrifuged at 1,200 x g for 10 min at 4 °C. The supernatant was separated and centrifuged at 23,500 x g for 20 min at 4 °C. The pellet was resuspended in 5-6 volumes of buffer (50 mM TRIS, pH 7.4) and centrifuged again at 23,500 x g (20 min, 4 °C). This procedure was repeated twice. The final pellet was resuspended in 5-6 volumes of buffer and frozen (-80 °C) in 1.5 mL portions containing about 1.5 mg protein/mL.

Preparation of membrane homogenates from rat liver
Two rat livers were cut into small pieces and homogenized with the potter (500-800 rpm, 10 up and down strokes) in 6 volumes of cold 0.32 M sucrose. The suspension was centrifuged at 1,200 x g for 10 min at 4 °C. The supernatant was separated and centrifuged at 31,000 x g for 20 min at 4 °C. The pellet was resuspended in 5-6 volumes of buffer (50 mM TRIS, pH 8.0) and incubated at rt for 30 min. After the incubation, the S6 suspension was centrifuged again at 31,000 x g for 20 min at 4 °C. The final pellet was resuspended in 5-6 volumes of buffer and stored at -80 °C in 1.5 mL portions containing about 2 mg protein/mL.

Determination of protein concentration
The protein concentration was determined by the method of Bradford, 2 modified by Stoscheck. 3 The Bradford solution was prepared by dissolving 5 mg of Coomassie Brilliant Blue G 250 in 2.5 mL of EtOH (95 %, v/v). 10 mL deionized H 2 O and 5 mL phosphoric acid (85 %, m/v) were added to this solution, the mixture was stirred and filled to a total volume of 50 mL with deionized water. The calibration was carried out using bovine serum albumin as a standard in 9 concentrations (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0 and 4.0 mg /mL). In a 96 well standard multiplate, 10 µL of the calibration solution or 10 µL of the membrane receptor preparation were mixed with 190 µL of the Bradford solution, respectively. After 5 min, the UV absorption of the protein-dye complex at  = 595 nm was measured with a plate reader (Tecan Genios ® , Tecan, Crailsheim, Germany).

General procedures for the binding assays
The test compound solutions were prepared from the 10 mM stock solution. To obtain the required test solutions for the assay, the DMSO stock solution was diluted with the respective assay buffer. The filtermats were presoaked in 0.5 % aqueous polyethyleneimine solution for 2 h at room temperature before use. All binding experiments were carried out in duplicates in the 96-well multiplates. Generally, the assays were performed by addition of 50 µL of the respective assay buffer, 50 µL of test compound solution in various concentrations (10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 and 10 -10 mol/L), 50 µL of the corresponding radioligand solution and 50 µL of the respective receptor preparation into each well of the multiplate (total volume 200 µL). The receptor preparation was always added last. During the incubation, the multiplates were shaken at a speed of 500-600 rpm at the specified temperature. Unless otherwise noted, the assays were terminated after 120 min by rapid filtration using the harvester. During the filtration, each well was washed five times with 300 µL of water. Subsequently, the S7 filtermats were dried at 95 °C. The solid scintillator was melted on the dried filtermats at a temperature of 95 °C for 5 min. After solidifying of the scintillator at room temperature, the trapped radioactivity in the filtermats was measured with the scintillation analyzer.
Each position on the filtermat corresponding to one well of the multiplate was measured for 5 min with the [ 3 H]-counting protocol. The overall counting efficiency was 20 %. 3 The IC 50 values were calculated with the program GraphPad Prism ® 3.0 (GraphPad Software, San Diego, CA, USA) by non-linear regression analysis. Subsequently, the IC50 values were transformed into K i values using the equation of Cheng and Prusoff. 4 The K i values are given as mean value ± SEM from three independent experiments.

1 Receptor affinity
The assay was performed with the radioligand

Computational details
The molecular structures of  1 receptor was obtained starting from the available Protein Data Bank file (pdb code: 5HK1) 7 and following a procedure previously described. [8][9][10] The optimized structure of the new piperidine derivatives was docked into the identified binding pocket using Autodock 4.2.6/Autodock Tools 1.4.6 11 on a win64 platform. The resulting docked conformations were clustered and visualized; then, the structure of each resulting complex characterized by the lowest Autodock interaction energy in the prevailing cluster was selected for further modelling. Each compound/ 1 complex obtained from the docking procedure was refined in Amber 20 12 following a well validated procedure. 8-10 Briefly, the system density and volume were relaxed in NPT ensemble maintaining the Berendsen barostat for 20 ns. After this step, 50 ns of unrestrained NVT production simulation was run for each system. The free energy of binding (Gbind) between each compound and the 1 receptor was estimated by resorting to the well validated Molecular Mechanics/Poisson-Boltzmann Surface Area (MM/PBSA) approach 13 implemented in Amber 20. The per residue binding free energy decomposition (PRBFED) was carried out using the Molecular Mechanics/Generalized Boltzmann Surface Area (MM/GBSA) approach, 14 and was based on the same snapshots used in the binding free energy calculation. Molecular graphics images were produced using the UCSF Chimera package (v.1.15). 15 Chimera is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311). All other graphs were obtained using GraphPad Prism (v. 8.0). 16      The medium, PBS Dulbecco and trypsin were warmed to 37 °C in the water bath. The S12 medium was removed from the flask and the cells were rinsed with 12 mL of PBS. After the removal of the PBS, 4 mL of trypsin were added. The cells were incubated in the incubator at 37 °C for 5-10 min until approx. 80 % of the cells were detached from the surface. Then, 15 mL of medium were added. The mixture was centrifuged at 800 × g for 10 min at 4 °C. The supernatant was discarded and the pellet was resuspended in 12 mL of medium by manual vortexing. The medium with the cells was equally distributed over three culture flasks (VWR) and filled up with medium to a total volume of 12 mL. The cultivation conditions were the same as described above.

Live Cell imaging using the IncuCyte ® S3 Live Cell Analysis System
The measurement of the cell confluence and the determination of the IC50 values were performed with the IncuCyte ® S3 Live Cell Analysis System (Essen BioScience, Ltd., Royston, Hertfordshire, UK)

Measurement of cell confluence
The cell confluence was measured using the IncuCyte® S3. For the scanning with the company's own program (IncuCyte S3 2017A), the HD phase imaging mode was chosen

Competitive confluence assay
The plates were prepared as described above with the exception that the cells were grown in 100 µL instead of 150 µL of medium. After the cell confluence reached 30-40 %, the cells were treated with the respective test compounds and the agonist (+)-pentazocine (final concentration: 10 µM).

Calculation of IC50 values
The Eq. 2: 1 S14 in inhibition (top (right) asymptote), A1<A2, logx0 being the center of the curve, x being the common logarithm of the concentration and p being the hill slope. At an inhibition of 50 % the x value is equivalent to the log10(IC50) which then had to be transformed to the IC50 value. From the obtained values, the mean value and the corresponding standard deviation (SD) were calculated.

Results
Tumor cells with high σ 1 receptor expression, such as the non-small cell lung cancer cell line A427, 17   (+)-pentazocine 5.4 ± 0.5 no effect -A strong inhibition of the A427 cell proliferation was observed for 4a resulting in an IC50 value of 17 µM (Table S3). The  1 antagonist haloperidol showed a reduction of cell proliferation in the same concentration range (IC50 = 16 µM), whereas the selective 1 agonist (+)pentazocine did not affect A427 cell proliferation. In order to prove that the inhibition of cell growth was mediated by the σ1 receptor, A427 cells were co-incubated S15 with 4a and (+)-pentazocine (10 µM) or haloperidol and (+)-pentazocine (10 µM), respectively. In the presence of the agonist (+)-pentazocine, the inhibitory effects of 4a and haloperidol on cell proliferation were considerably reduced. The IC50 value of 4a and haloperidol was shifted from 18 µM to 30 µM and from 16 µM to 24 µM, respectively.
Altogether, the observed in vitro effects of 4a on A427 tumor cells are comparable with those of the 1 antagonist haloperidol. Moreover, they can be reduced by co-incubation of a  1 receptor agonist. Therefore, it can be concluded that 4a behaves as  1 receptor antagonist in this A427 tumor cell proliferation assay.  Figure   S2 shows the growth inhibition of the DU145 cell line by three piperidines (20a, 21a, 22a) and two reference 1 receptor antagonists. S17 Figure S2: Inhibition of the growth of the androgen negative human prostate tumor cell line DU145 by the piperidines 20a, 21a and 22a (bottom panels) and the reference  1 antagonists NE100 and S1RA (top panels). Data are mean ± SD (n = 3). *p ≤ 0.05 vs control.