Synthesis, biological evaluation and docking analysis of substituted piperidines and (2-methoxyphenyl)piperazines J. Serb. Chem. Soc. 81 (4) 347–356 (2016) UDC 577.354+547.233–304.2+547.822.3+ JSCS–4851 547.828:57.02:543 Original scientific paper 347 Synthesis, biological evaluation and docking analysis of substituted piperidines and (2-methoxyphenyl)piperazines JELENA Z. PENJIŠEVIĆ1#, VLADIMIR V. ŠUKALOVIĆ1#, DEANA B. ANDRIĆ2*#, GORAN M. ROGLIĆ2#, IRENA T. NOVAKOVIĆ1#, VUKIĆ ŠOŠKIĆ3# and SLAĐANA V. KOSTIĆ-RAJAČIĆ1# 1ICTM – Center of Chemistry, University of Belgrade, Njegoševa 12, Belgrade, Serbia, 2Faculty of Chemistry, University of Belgrade, Studentski trg 12–16, Belgrade, Serbia and 3ORGENTEC Diagnostica GmbH, Carl-Zeiss-Straße 49–51, Mainz, Germany (Received 21 October, revised 25 November, accepted 26 November 2015) Abstract: A series of sixteen novel substituted piperidines and (2-methoxy- phenyl)piperazines were synthesized, starting from the key intermediates 1-(2- -methoxyphenyl)-4-(piperidin-4-yl)piperazine and 1-(2-methoxyphenyl)-4- -[(piperidin-4-yl)methyl]piperazine. Biological evaluation of the synthesized compounds was illustrated by seven compounds, of which 1-(2-methoxy- phenyl)-4-{[1-(2-nitrobenzyl)piperidin-4-yl]methyl}piperazine had the highest affinity for the dopamine D2 receptor. For all seven selected compounds, dock- ing analysis was performed in order to establish their structure-to-activity relationship. Keywords: dopamine D2 receptor; docking analysis; allosteric; orthosteric bind- ing site. INTRODUCTION G-protein-coupled receptors (GPCRs) are transmembrane receptors that mediate most of their intracellular actions through pathways involving an acti- vation of the G-protein.1 D2 Dopamine receptors (D2DAR) are members of this large protein family. Dysfunction of the dopaminergic system in CNS can lead to a number of diseases, such as Parkinson’s disease, schizophrenia, some neuro- humoral disturbances, etc.2,3 Therefore it is not surprising that the design and synthesis of new potential dopaminergic drugs is one of the main objectives of organic and medicinal chemistry. Arylpiperazines are a common structural motif included in various com- pounds that interact in a specific manner with various GPCRs.4 Within the scope * Corresponding author. E-mail: deanad@chem.bg.ac.rs # Serbian Chemical Society member. doi: 10.2298/JSC151021097P _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 348 PENJIŠEVIĆ et al. of the program aimed at the discovery of new dopaminergic (DA-ergic) ligands and in order to explore further previously published data, a series of sixteen novel arylpiperazines were synthesized.5 For all the synthesized ligands, their in vitro binding affinities at rat D2DAR were estimated and compared with the results obtained through docking analysis, using an available D2DAR molecular model. EXPERIMENTAL General Melting points were measured on a Boetius PHMK apparatus (VEB Analytic, Dresden, Germany) and are uncorrected. The 1H- and 13C-NMR (200 and 50 MHz) spectra of the com- pounds in deuterochloroform, unless otherwise stated, were recorded on a Gemini 2000 ins- trument (Varian, Oxford). The chemical shifts (δ) are reported in ppm downfield from the internal standard tetramethylsilane. The LC-MS results were acquired on a 6210 time-of-flight LC–MS system (Agilent Technologies, Germany); MassHunter workstation software was used for data analysis. The IR spectra were taken on a Thermo Scientific spectrometer. A MicroSYNTH Milestone and a Biotage Initiator 2.5 EXP were used for the microwave irradiations. Analytical TLC was performed on Polygram SIL G/UV254 plastic-backed thin- layer silica gel plates (Macherey-Nagel, Germany). The chromatographic purifications were realized on Merck-60 silica gel columns (diameter 70 mm, h = 45 mm; the same for all compounds), 230–400 mesh ASTM, medium pressure (dry column flash chromatography). The reagents and solvents (Alfa–Aesar or Sigma–Aldrich) were used without purification. The solutions were routinely dried over anhydrous Na2SO4 prior to evaporation. Chemistry Ethyl 4-[4-(2-methoxyphenyl)piperazin-1-yl]piperidine-1-carboxylate (3). To a stirred solution of N-carbethoxy-4-piperidone (1, 1.7 g, 0.01 mol) in methanol (25 mL; the pH value of the solution was adjusted to 7 by addition of CH3CO2H), 1-(2-methoxyphenyl)piperazine (2, 3.24 g, 0.02 mol) was added, followed by the addition of NaBH3CN (0.4 g, 0.0072 mol) in portions (Scheme 1). Stirring was continued at room temperature for 24 h. The pH value of the resulting solution was adjusted to 2 by the addition of 10 % HCl solution and the excess of the methanol was removed under vacuum. The pH value of the residue was adjusted to 9 by the addition of 10 % NaOH solution and extracted with dichloromethane. The organic layer was dried over anhydrous Na2SO4 and evaporated in vacuo. The product was purified by dry- flash chromatography using a gradient of CH3OH (0–10 %) in dichloromethane as the solvent. Yield: 88 %. General procedure for the hydrolysis of the carbamates 3 and 9. Carbamate 3 or 9 (0.02 mol) was suspended in cc HCl (60 mL), transferred into a sealed tube and placed into a microwave oven. Irradiation at 130 °C was completed after 90 min at an initial power of 300 W (Schemes 2 and 3). The reaction mixture was poured into water, the pH value adjusted to 9 by addition of 10 % NaOH solution and extracted with dichloromethane. The organic layer was dried over anhydrous Na2SO4 and evaporated in vacuo. The product was purified by dry- flash chromatography using a gradient of methanol (0–10 %) in dichloromethane as the solvent. 1-(Ethoxycarbonyl)piperidine-4-carboxylic acid (6). To a solution of piperidine-4-carbo- xylic acid (5, 20 g, 0.155 mol) in water (200 mL), Na2CO3 (20 g) was added, mixture stirred at room temperature for 30 min and a solution of ethyl chloroformate (25.5 g, 0.28 mol) in toluene (240 mL) was added dropwise (Scheme 2). Stirring was continued at room tempe- _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ SUBSTITUTED PIPERIDINES AND (2-METHOXYPHENYL)PIPERAZINES 349 rature for 20 h. After separation of the layers, the aqueous layer was acidified with conc. HCl to pH ≈2, and extracted with dichloromethane. The organic layer was dried over anhydrous Na2SO4 and evaporated in vacuo. Yield: 78 %. Scheme 1. Synthesis of 1-(2-methoxyphenyl)-4-(piperidin-4-yl)piperazine (4); reagents: a) NaBH3CN, MeOH, pH 7, r.t; b) conc. HCl, MW, 180 °C, 300 W. Scheme 2. Synthesis of 1-(2-methoxyphenyl)-4-[(piperidin-4-yl)methyl]piperazine (10); reagents: a) Na2CO3, ethyl chloroformate, toluene, r.t; b) thionyl chloride, CH2Cl2, 0 °C; c) triethylamine, chloroform, 5 °C; d) NaBH4, boron trifluoride diethyl etherate, diglyme, –5 °C; e) conc. HCl, MW, 180 °C, 300 W. _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 350 PENJIŠEVIĆ et al. Ethyl 4-(chlorocarbonyl)piperidine-1-carboxylate (7). A solution of 1-(ethoxycarbonyl) piperidine-4-carboxylic acid (6, 7.5 g, 0.0375 mol), thionyl chloride (5.35 g, 0.045 mol) and chloroform (200 mL) was stirred for 2 h at 0 °C (Scheme 2). The reaction mixture was evaporated in vacuo and the product was used immediately, without further purification. Yield: 67 %. Ethyl 4-{[4-(2-methoxyphenyl)piperazin-1-yl]carbonyl}piperidine-1-carboxylate (8). A solution of chloride 7 in chloroform (50 mL) was added dropwise to a solution of triethyl- amine (3.78 g, 0.0375 mol), 1-(2-methoxyphenyl)piperazine (2, 6.07 g, 0.0375 mol) in chloro- form (150 mL) at 5 °C (Scheme 2). The reaction mixture was stirred at room temperature for 20 h, the resulting mixture extracted with 10 % Na2CO3 and the organic layer was extracted with 10 % HCl solution. The organic layer was dried over anhydrous Na2SO4, and evaporated in vacuo. The product was purified by dry-flash chromatography eluting with dichloro- methane containing increasing amounts of methanol (0–10 %). Yield: 85 %. Ethyl 4-{[4-(2-methoxyphenyl)piperazin-1-yl]methyl}piperidine-1-carboxylate (9). Mix- ture of compound 8 (0.01 mol) and NaBH4 (1 g, 0.025 mol) in diglyme (1-methoxy-2-(2- -methoxyethoxy)ethane, 25 mL) was stirred for 40 min at –5 °C under argon, during which time, boron trifluoride diethyl etherate (3.9 g, 3.4 mL, 0.025 mmol) was added dropwise (Scheme 2). After stirring for 1 h at 5 °C, the reaction mixture was heated to 80–90 °C, followed by stirring for additional I h. The mixture was cooled to room temperature, carefully poured into 10 mL of water and then 20 mL cc HCl was added. Mixture was heated at 60–80 °C on aqueous bath for 3 h and concentrated under vacuo. To the residue, 10 % NaOH solution was added until pH 9 and extracted with CH2Cl2. The organic layer was dried over anhydrous Na2SO4 and evaporated in vacuo. The product was purified by dry-flash chromatography using the dichloromethane/methanol system as eluent. Yield: 85 %. General procedure for the alkylation of compounds 4 and 10. A mixture of compound 4 or 10 (0.0018 mol), benzyl halides 11–14 (0.0018 mol), K2CO3 (0.0036 mol) and acetonitrile (25 mL) was stirred at room temperature for 48 h, poured into water and extracted with dichloromethane. The organic layer was dried over anhydrous Na2SO4 and evaporated in vacuo. The product (15–22, Scheme 3) was purified by dry-flash chromatography using a gradient of methanol (0–10 %) in dichloromethane as the solvent. General procedure for the synthesis of compounds 27–30 and 31–34. A solution of benzoyl or nitrobenzoyl chloride 23–26 (0.0017 mol) and CH2Cl2 (0.34 mL) was added dropwise to a solution of 4 or 10 (0.0017 mol), CH2Cl2 (1.7 mL) and Et3N (0.24 mL, 0.0017 mol) at 0 °C. The reaction mixture was stirred at room temperature for 72 h. The resulting mixture was extracted with 10 % Na2CO3 solution, the separated organic layer was washed with 10 % K2CO3 solution, dried over anhydrous Na2SO4 and evaporated in vacuo. The product (27–34, Scheme 3) was purified by dry-flash chromatography eluting with CH2Cl2 containing increasing amounts of MeOH (0–10 %). Analytical and spectral data for the synthesized compounds are given in the Supple- mentary material to this paper. Membrane preparation, radio-ligand binding assays and data analysis Synaptosomal membranes from rat striatum were prepared for radio-ligand binding assays as previously described.6 [3H]Spiperone (specific. activity: 9.25 MBq mmol-1) used to label D2DAR were purchased from Perkin Elmer LAS GmbH, Rodgau, Germany. _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ SUBSTITUTED PIPERIDINES AND (2-METHOXYPHENYL)PIPERAZINES 351 HN N N H3CO Cl R COClR a b n N N N H3CO n R N N N H3CO n R O 4, n=0 10, n=1 15-18, n=0 19-22, n=1 27-30, n=0 31-34, n=1 Scheme 3. Synthetic route and chemical structures of the (2-methoxyphenyl)piperazine dopaminergic ligands; reagents: a) compounds 11–14 (R = H, 2-NO2, 3-NO2, 4-NO2, respectively), K2CO3, CH3CN, r.t; b) compounds 23–26 (R = H, 2-NO2, 3-NO2, 4-NO2, respectively), triethylamine, CH2Cl2, 0 °C; yields for 15–22 and 27–34: 68–89 %. [3H]Spiperone–receptor binding assay. [3H]Spiperone binding was assayed in 4 mM MgCl2, 1.5 mM CaCl2, 5 mM KCl, 120 mM NaCl, 25 mM Tris–HCl solution, pH 7.4, at a membrane protein concentration of 0.7 mg mL-1 at 37 °C for 10 min in a total volume of 0.4 mL of the incubation mixture. Binding of the radioligand to the 5-HT2 receptors was prevented by 50 μM ketanserin. The Ki values of the tested compounds were determined by competition binding at 0.2 nM of the radio-ligand and eight to ten different concentrations of each compound (10-4 to 10-10 M). Nonspecific binding was measured in the presence of 1.0 mM spiperone. The reaction was terminated by rapid filtration through Whatman GF/C filters, which were further washed three times with 5.0 mL of ice-cold incubation buffer. Each point was determined in triplicate. The retained radioactivity was measured by introducing dry filters into 10 mL of toluene-based scintillation liquid and counting in a 1219 Rackbeta Wallac scintillation counter (EG & G Wallac, Turku, Finland) at an efficiency of 51–55 % for tritium. The results were analyzed by nonlinear curve fitting of the inhibition curves of the compounds utilizing the Graph-Pad Prism program.7 Hill slope coefficients were fixed to unity during the calculation. Docking analysis Docking analysis was performed with an already available D2DAR model based on the D3DAR crystal structure.8 The binding site of the receptor was determined by combining results from experimental data9,10 and the Schrödinger Maestro receptor grid generation module.11 Amino acid residue charges were adjusted where needed, assuming physiological conditions. Selected ligands were prepared with the ligprep Maestro module and docked using the Glide module from the Schrödinger Suite 2011.11 All ligands were docked as protonated, using the OPLS_2005 force field. The initial position of the ligand in the binding site was arbitrary, while the protonated nitrogen on the ligand part was kept in close proximity to Asp 114 of the D2DAR. After initial ligand placement, no further constraints were applied and the _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 352 PENJIŠEVIĆ et al. docking procedure was performed. The obtained structures were examined and those meeting the following criteria were selected: best docking score of the complex, shortest salt bridge formed between Asp 114 of the D2DAR and ligand, chair conformation of arylpiperazine ring and aryl part of the molecule positioned in the rear hydrophobic pocket of the receptor (Phe 386, Trp 390 and Tyr 420).12 After an initial criterion was satisfied, the second step was the examination of different interactions that could be formed between the receptor and ligand (hydrogen bonds, aromatic–aromatic interactions, etc.). In that way, the best possible docking structures were selected. Structures were visualized using DS Visualize v2.5.113 and the obtained images were rendered using PovRay Raytracer v3.6.14 RESULTS AND DISCUSSION The general synthetic route and chemical structures of the novel substituted piperidine and (2-methoxyphenyl)piperazine are summarized in Schemes 1–3. Preparation of the key intermediates, 1-(2-methoxyphenyl)-4-(piperidin-4- -yl)piperazine (4) and 1-(2-methoxyphenyl)-4-[(piperidin-4-yl)methyl]piperazine (10) are described in Schemes 1 and 2. Ethyl 4-[4-(2-methoxyphenyl)piperazin- -1-yl]piperidine-1-carboxylate (3), produced by the reductive amination of the commercially available ketone 1 was further hydrolyzed, under microwave con- ditions, and intermediate 4 was obtained (Scheme 1). Commercially available piperidine-4-carboxylic acid (5) was transformed into carbamate 6 and further into chloride 7 by reaction with thionyl chloride. Acylation of (2-methoxyphenyl)piperazine with chloride 7 gave amide 8, which provided compound 9 by reduction with NaBH4/boron trifluoride ethyl etherate. The carbamate 9 was converted to the secondary amine 10 by hydrolysis with cc HCl under MW conditions (Scheme 2). Both 4 and 10 intermediates were alkylated with benzyl or nitrobenzyl halo- genide to give the final ligands 15–18 and 19–22, respectively. Ligands 27–30 and 31–34 were obtained by acylation with the corresponding acyl chloride (Scheme 3). The final products 15–22 and 27–34 were evaluated for their affinity to D2DAR by the in vitro competitive displacement assay of [3H]spiperone (Table I). As a source of D2DAR, synaptosomal membranes prepared from rat striatum were used. The compound with the highest affinity for D2DAR was 1-(2-methoxy- phenyl)-4-{[1-(2-nitrobenzyl)piperidin-4-yl]methyl}piperazine (20, Ki = 30.6 nM). Compounds 19, 21, 22 and 31–34 expressed moderate binding affinity for D2DAR, while 15–18 and 27–30 were completely inactive competitors of bound [3H]spiperone. Compounds 20–22 and 31–34 were selected for docking analysis in order to establish their structure-to-activity relationship. D2DAR model and selected compounds were prepared as described and docking analysis was performed. The obtained results showed that compound 20 binds to D2DAR via a salt bridge with Asp 114 on TM3. This is followed by _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ SUBSTITUTED PIPERIDINES AND (2-METHOXYPHENYL)PIPERAZINES 353 multiple aromatic interactions between the aryl part of the ligand and the hydro- phobic pocket (Phe 386, Trp 390 and Tyr 420).8,15,16 In this way, the ligand establishes a favorable orientation inside the receptor binding cavity that is a pre- requisite for the formation of hydrogen bonds with Ser 193 on TM5. The stated interactions are formed with D2DAR inside the orthosteric bind site (OBS). Docking analysis showed possible aromatic interactions with Phe 393 and His 397, both located inside the alosteric bind site (ABS, Fig. 1) and the listed inter- actions lead to high compound activity. TABLE I. Binding constants of the synthesized compounds for the dopamine D2 receptor N N N H3CO n R Compound n R (Ki±SEM) / nM 15 0 H 736±24 16 0 2-NO2 521.5±13 17 0 3-NO2 937.5±35 18 0 4-NO2 1512±30 19 1 H 341.5±12 20 1 2-NO2 30.6±1.2 21 1 3-NO2 258 ±11 22 1 4-NO2 200±12 Compound n R (Ki±SEM) / nM 27 0 H 1583.5±32 28 0 2-NO2 1205±19 29 0 3-NO2 755±21 30 0 4-NO2 905±28 31 1 H 189.5±12.1 32 1 2-NO2 219.5±14.4 33 1 3-NO2 300±16.2 34 1 4-NO2 334.5±17.8 In the case of compounds 21 and 22, in contrast to compound 20, the optimal hydrogen bond with the serine residues on TM5 cannot be formed. The reduced binding affinity was a clear consequence of the unfavorable orientation of ligands 21 and 22 inside the receptor binding cavity. Compounds 31–34 dock, in the _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ 354 PENJIŠEVIĆ et al. same manner, with aryl part oriented inside the hydrophobic pocket (Phe 386, Trp 390 and Tyr 420), a salt bridge with Asp 114 and hydrogen bonds with Ser 193 (Fig. 2). The only difference, compared to compound 20, is the positioning of the head part of the ligand. The reduced flexibility of the head part leads to sub-optimal positioning of the aromatic part inside the ABS and the only obs- erved aromatic interaction is with Phe 394. This leads to reduced binding affinity, with respect to ligand 20. Fig. 1. Docking of compound 20 into the D2DAR binding pocket. Fig. 2. Docking of compound 31 into the D2DAR binding pocket. The other compounds cannot form the interactions discussed above, mostly due to their rigidity. Therefore, they either cannot achieve the correct orientation inside the ABS, or cannot form any hydrogen bond with serine residues on TM5. CONCLUSIONS In order to achieve high binding affinity, a D2DAR ligand has to fulfill several requirements. The formation of a salt bridge with Asp 114 is the crucial interaction that starts the binding process, which is followed by orientation of _________________________________________________________________________________________________________________________ (CC) 2016 SCS. All rights reserved. Available on line at www.shd.org.rs/JSCS/ SUBSTITUTED PIPERIDINES AND (2-METHOXYPHENYL)PIPERAZINES 355 arylpiperazine ligand part into the OBS hydrophobic cavity. After these initial requirements are met, the ligand has to establish one or more hydrogen bonds with serine residues on TM5. Failing this, the ligand could still bind to D2DAR, but with reduced affinity. In order to establish hydrogen bonds, the ligand has to be of considerable length to span the entire OBS between Asp 114 and Ser 193 and/or 197. Since the OBS is not linear, the ligand has to adopt a slightly curved conformation in order to bind successfully. The conformation of the arylpiper- azine part is fixed at the chair conformation of the arylpiperazine ring and the rest of the ligand has to be flexible enough to fit into the OBS space. In the case of compounds 15–34, only compounds 21, 22 and 31–34 can adopt the described conformation that leads to high affinity receptor binding. Once the conform- ational requirements are fulfilled, the affinity is determined by the number and strength of particular receptor–ligand interactions. Ligand 20 has the best overall fit into D2DAR that, together with the formed interactions, leads to the highest affinity in the group of ligands. Obtained results suggest that in future studies special attention should be paid to the synthesis of the ligands with a prolonged, flexible bridge that will provide more degrees of rotational freedom of the molecules, which allows a pro- per orientation of the ligands in the OBS cavity, which is an essential prerequisite for high affinity D2DAR ligands. SUPPLEMENTARY MATERIAL Analytical and spectral data of the synthesized compounds are available electronically from http://www.shd.org.rs/JSCS/, or from the corresponding author on request. Acknowledgments. These results are part of Project 172032, supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia. И З В О Д СИНТЕЗА, БИОЛОШКО ИСПИТИВАЊЕ И ДОКИНГ АНАЛИЗА СУПСТИТУИСАНИХ ПИПЕРИДИНСКИХ И (2-МЕТОКСИФЕНИЛ)ПИПЕРАЗИНСКИХ ЛИГАНАДА ЈЕЛЕНА З. ПЕЊИШЕВИЋ1, ВЛАДИМИР В. ШУКАЛОВИЋ1, ДЕАНА Б. АНДРИЋ2, ГОРАН М. РОГЛИЋ2, ИРЕНА Т. НОВАКОВИЋ1, ВУКИЋ ШОШКИЋ3 и СЛАЂАНА В. КОСТИЋ-РАЈАЧИЋ1 1ИХТМ –Центар за хемију, Универзитет у Београду, Његошева 12, Београд, 2Хемијски факултет, Универзитет у Београду, Студентски трг 12–16, Београд и 3ORGENTEC Diagnostica Gmbh, Carl-Zeiss-Straße 49–51, Mainz, Germany Синтетисана је серија од шеснаест нових супституисаних пиперидина и (2-метокси- фенил)пиперазина, полазећи од кључних интермедијера 1-(2-метоксифенил)-4-(пипе- ридин-4-ил)пиперазина и 1-(2-метоксифенил)-4-(пиперидин-4-илметил)пиперазина. Биолошко испитивање синтетисаних једињења је истакло седам једињења, од којих 1-(2- -метоксифенил)-4-{[1-(2-нитробензил)пиперидин-4-ил]метил}пиперазин има највиши афинитет ка D2 допаминском рецептору. За свих седам једињења је урађена докинг ана- лиза у циљу утврђивања односа структуре и активности. (Примљено 21. октобра, ревидирано 25. новембра, прихваћено 26. новембра 2015) _________________________________________________________________________________________________________________________ (CC) 2016 SCS. 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