Synthesis, Biological Evaluation, and Molecular Modeling match our pharmacophore model For each molecule in [S]GTP,S binding assays the database, a maximum of 250 conformations with an The[ SGTP, S binding assay was performed at 30C for energy threshold of 20 kcal/mol were generated using 30 min with 10 ug of membrane protein in a final volume FAST algorithm Only compounds with a fit value greater of 100 AL with various concentration of the compound than three were retained. Then Lipinski's Rule of Five was The antagonism effects of the compounds were tested in applied to reject non-drug-like compounds. The hits the existence of 10 HM haloperidol for the D3R. The bind obtained were overlaid on the active site of the D3R and ing buffer contains 50 mM Tris(pH 7.5), 5 mM MgCl2 those creating steric clashes were discarded. GoldScore 1 mM ethylenediaminetetraacetic acid (EDTA), 100 mM was used to rank the hits. The interaction analyses in NaCl, 1 mM DL-dithiothreitol(DTD, and 40 uM guanosine combination with scoring function was used to guide the triphosphate. The reaction was initiated by adding of s final selection GTP,S ( final concentration of 0. 1 nM). Non-specific binding was measured in the presence of 100 HM 5-guanylimid diphosphate(Gpp(NH)p Molecular docking Molecular docking was carried out using GOLD 5.0.1(16) The binding site was defined to include all residues within Experimental Section a 15.0 A radius of the conserved D3.32Cy carbon atom. A hydrogen-bond constraint was set between the protonated Chemicals and solvents were purchased and used without nitrogen atom(N1)of ligand and D3. 32 side chain. Ten further purification. 'H and 3C NMR spectra were conformations were produced for each ligand, and Gold ecorded on a Bruker AMX-400 instrument. The chemical Score was used as scoring function. Other parameters shifts were referenced to the solvent peak, namely were set as standard default. High-scoring complexes 8=7.26 ppm for CDCl3 using TMs as an intemal stan- were inspected visually to select the most reasonable solu- dard. Proton-coupling pattems were described as singlet tion doublet, triplet, quartet, multiplet, and broad. Mass spectra were given with an electric ionization(ESI) produced by HP5973 N analytical mass spectrometer. All tested com- Biological evaluation pounds had a minimal purity of 95% assessed by HPLC method (Schemes 1 and 2) Binding assays All the synthesized new compounds were subjected to competitive binding assays for the human dopamine(D1, General procedures for the preparation of D2, and D3)receptors, using membrane preparation compounds 11a-11q obtained from HEK293 cells stably transfected respective receptor. H SCH23390(D1)and IH-Spiperone(D2 N-cyclohexyl-2-(4-(3-(5-fluoro-1H-indol-3-yl)propy) and D3) were used as standard radioligands. The per- piperazin-1-yl)-N-phenylacetamide(11a) centage displacement of radioligand and Ki values of o Chloroacetyl chloride(1. 47 mL, 18.43 mmol) was added these compounds is reported in Table 1. Duplicated to a solution of N-cyclohexylaniline (3.23 g, 18.43 mmol) tubes were incubated at 30C for 50 min with increas- and EtaN(1.86 g, 18.43 mmol) in anhydrous CH2Cl2 at ing concentrations (1 nM--100 uM)of respective com- 0C under N2 atmosphere and then stirred at room tem- Spiperone(for D2R and DaR) in a final volume of 200 gL washed with brine, and the organic layer was dried de a pound and with 0.7 nM PHISCH23390(for D1R), or PH perature for 5 h. The reaction was diluted with CH2Cl2 binding buffer containing 50 mM Tris, 4 mM MgCl2, pH Na2SO4, evaporated, and purified by flash chromatography 7. 4. Non-specific binding was determined by parallel(PE/EtOAC, 10: 1)to yield 2-chloro-N-cyclohexyl-N-pheny incubations with either 10 AM SCH23390 for D1 or Spip- lactamide 8 as an off-white solid(3.9 g, yield 84.2%),() erone for D2, D3 dopamine receptors, respectively. The To a suspension of compound 8(3.0 g, 11.95 mmol) ICso and Ki values were calculated by non-linear regres- K2CO3(2.48 g, 17.94 mmon) and a catalytic amount of KI ion(PRISM; Graphpad, San Diego, CA, USA) using a(40 mg) in acetonitrile(40 mL) was added tert-butyl pipe igmoidal function azine-1-carboxylate(2.22 g, 11.95 mmol). The reaction Table 1: The results of virtual screening and corresponding binding assays Compound MW HBA HBD Fit value Gold score Binding affinity Ki+ SEM(nM) 11a 766 5 5.84 3.51 2161±25 2203±9 2.93 2814±35 048±0.1 Chem Bio/ Drug Des 2013: 82: 326-335 327match our pharmacophore model. For each molecule in the database, a maximum of 250 conformations with an energy threshold of 20 kcal/mol were generated using FAST algorithm. Only compounds with a fit value greater than three were retained. Then Lipinski’s Rule of Five was applied to reject non-drug-like compounds. The hits obtained were overlaid on the active site of the D3R and those creating steric clashes were discarded. GoldScore was used to rank the hits. The interaction analyses in combination with scoring function was used to guide the final selection. Molecular docking Molecular docking was carried out using GOLD 5.0.1(16). The binding site was defined to include all residues within a 15.0
A radius of the conserved D3.32Cc carbon atom. A hydrogen-bond constraint was set between the protonated nitrogen atom (N1) of ligand and D3.32 side chain. Ten conformations were produced for each ligand, and Gold￾Score was used as scoring function. Other parameters were set as standard default. High-scoring complexes were inspected visually to select the most reasonable solu￾tion. Biological evaluation Binding assays All the synthesized new compounds were subjected to competitive binding assays for the human dopamine (D1, D2, and D3) receptors, using membrane preparation obtained from HEK293 cells stably transfected respective receptor. [3 H] SCH23390 (D1) and [3 H]-Spiperone (D2 and D3) were used as standard radioligands. The per￾centage displacement of radioligand and Ki values of these compounds is reported in Table 1. Duplicated tubes were incubated at 30 °C for 50 min with increas￾ing concentrations (1 nM–100 lM) of respective com￾pound and with 0.7 nM [ 3 H]SCH23390 (for D1R), or [3 H] Spiperone (for D2R and D3R) in a final volume of 200 lL binding buffer containing 50 mM Tris, 4 mM MgCl2, pH 7.4. Non-specific binding was determined by parallel incubations with either 10 lM SCH23390 for D1 or Spip￾erone for D2, D3 dopamine receptors, respectively. The IC50 and Ki values were calculated by non-linear regres￾sion (PRISM; Graphpad, San Diego, CA, USA) using a sigmoidal function. [ 35S]GTPcS binding assays The [35S]GTPcS binding assay was performed at 30 °C for 30 min with 10 lg of membrane protein in a final volume of 100 lL with various concentration of the compound. The antagonism effects of the compounds were tested in the existence of 10 lM haloperidol for the D3R. The bind￾ing buffer contains 50 mM Tris (pH 7.5), 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaCl, 1 mM DL-dithiothreitol (DTT), and 40 lM guanosine triphosphate. The reaction was initiated by adding of [35S] GTPcS (final concentration of 0.1 nM). Non-specific binding was measured in the presence of 100 lM 5′-guanylimid￾odiphosphate (Gpp(NH)p). Experimental Section Chemicals and solvents were purchased and used without further purification. 1 H and 13C NMR spectra were recorded on a Bruker AMX-400 instrument. The chemical shifts were referenced to the solvent peak, namely d = 7.26 ppm for CDCl3 using TMS as an internal stan￾dard. Proton-coupling patterns were described as singlet, doublet, triplet, quartet, multiplet, and broad. Mass spectra were given with an electric ionization (ESI) produced by HP5973 N analytical mass spectrometer. All tested com￾pounds had a minimal purity of 95% assessed by HPLC method (Schemes 1 and 2). General procedures for the preparation of compounds 11a–11q N-cyclohexyl-2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl) piperazin-1-yl)-N-phenylacetamide (11a) (i) Chloroacetyl chloride (1.47 mL, 18.43 mmol) was added to a solution of N-cyclohexylaniline (3.23 g, 18.43 mmol) and Et3N (1.86 g, 18.43 mmol) in anhydrous CH2Cl2 at 0 °C under N2 atmosphere and then stirred at room tem￾perature for 5 h. The reaction was diluted with CH2Cl2 and washed with brine, and the organic layer was dried over Na2SO4, evaporated, and purified by flash chromatography (PE/EtOAc, 10:1) to yield 2-chloro-N-cyclohexyl-N-pheny￾lacetamide 8 as an off-white solid (3.9 g, yield 84.2%), (ii) To a suspension of compound 8 (3.0 g, 11.95 mmol), K2CO3 (2.48 g, 17.94 mmol) and a catalytic amount of KI (40 mg) in acetonitrile (40 mL) was added tert-butyl piper￾azine-1-carboxylate (2.22 g, 11.95 mmol). The reaction Table 1: The results of virtual screening and corresponding binding assays Compound MW HBA HBD AlogP Fit value Gold score Binding affinity Ki SEM (nM) 11a 476.6 5 1 5.84 3.51 58.5 2161 25 12 496.0 4 1 3.73 3.29 69.0 2203 9 13 364.5 3 2 2.93 3.30 59.1 2814 35 Spiperone 0.48 0.1 Chem Biol Drug Des 2013; 82: 326–335 327 Synthesis, Biological Evaluation, and Molecular Modeling