复旦大学：《药物设计学》课程教学资源（教学研究）基于配体结构的药物设计_Design, Synthesis, and Evaluation of Indolebutylamines as a Novel Class of Selective Dopamine D3 Receptor Ligands

Chem Biol Drug Des 2013: 82: 326-335 Research Article Design, Synthesis, and Evaluation of Indolebutylamines as a Novel Class of selective Dopamine D3 Receptor Ligands Peng Du',., Lili Xu',, Jiye Huang,, Kungian Yu2, high sequence homology among the D2-like dopamine Rui Zhao, Bo Gao, Hualiang Jiang, Weili receptor subtypes(D2, D3, and D4). For instance, hD3R Zhao Xuechu Zhen and Wei Fu and hD2R share 78% sequence homology within the seven transmembrane domains and 94% sequence 'Department of Medicinal Chemistry Key Laboratory of homology within the active site (10, 11). To date, most of Smart Drug Delivery, Ministry of Education PLA, School D3R selective ligands are 4-Phenylpiperazines and their f Pharmacy, Fudan University, Shanghai, 201203, Chin 2Drug Discovery and Design Center, State Key Laboratory close analogs(12, 13). Considering the importance of the of Drug Research, Shanghai Institute of MateriaMedica D3R in the treatment of addiction and other neuropsy- cho disorders, it is meaningful to discover novel chemi- Chinese Academy of Sciences, Shanghai, 201203, China Department of Pharmacology, College of Pharmaceutical cal entities to enrich the structural diversity of potent Sciences, Soochow University, Suzhou, 215123, China and selective D3R ligands. Using a strategy that com Correspondingauthor:WeiFu,wfu@fudan.edu.cn bines synthetic chemistry, binding assays, and a set of fThese authors contributed equally to this work. computational approach(integrating active site mappin pharmacophore-based virtual screening, and automate A series of indolebutylamine derivatives were designed molecular docking), we designed a series of IBA deriva- synthesized, and evaluated as a novel class of selective tives as a new type of highly selective D3R antagonists ligands for the dopamine 3 receptor. The most potent Furthermore, the molecular determinants compound 11q binds to dopamine 3 receptor with a K binding specificity and selectivity of D3R were identified value of 124 nM and displays excellent selectivity over and the structure-activity relationships(SAR) was investi he dopamine 1 receptor and dopa amine gated Investigation based on structural information indicates that site S182 located in extracellular loop 2 may Methods and Materials account for high selectivity of compounds. Interaction models of the dopamine 3 receptor-11q complex and Structure-based pharmacophore model generation structure-activity relationships were discussed by inte- grating all available experimental and computational Dopamine 3 receptor was obtained from the Protein Data data with the eventual aim to discover potent and selec Bank(PDB ID: 3PBL)(11). The GRID22 program(14)was tive ligands to dopamine 3 receptor. employed to map the active sites of the optimized X-ray structure of D3R with five types of chemical probes, that Key words: dopamine 3 receptor, indolebutylamine, pharma- IS, negative ionizable(Coo-), positive ionizable(N1+) cophore model, selectivity, structure-activity relationship hydrogen-bond acceptor(O), hydrogen-bond donor(N1) and hydrophobic probes(DRY). For each of the five Received 22 November 2012, revised 17 April 2013 and probes used in the grid calculations, grid points were accepted for publication 26 April 2013 superimposed to identify clusters of positions. The mem- bers of each identified clusters were combined into one pharmacophore feature, and the centers of each pharma- Since the discovery by Sokoloff et al. in 1990(1), dopamine cophore features were set at the geometric centers of the 3 receptor(D3R) has been proved to be a promising thera- members in each clusters(15. Finally, a four-feature phar- peutic target for drug discovery. Dopamine 3 receptor macophore model was generated antagonist was shown to play a key role in the treatment of schizophrenia(2, 3)and drug addiction(4). Although consid able efforts have been devoted to the design and develop- Virtual screening ment of D3R antagonists(5-9), function study of D3R in vivo The obtained phamacophore model was used to screen is still limited due to the lack of highly selective antagonists the Asinex GOLD and Maybridge collection database which contain 238 000 compounds. The Ligand Pharma- One of the important reasons for the difficulty in devel- cophore Mapping protocol embedded in DISCOVERY STUDIO oping selective antagonist for D3R is attributed to the 3.5 was employed to retrieve molecules, which can well e 2013 John Wiey& Sons AS. doi: 10. 1111/cbdd. 12158

Design, Synthesis, and Evaluation of Indolebutylamines as a Novel Class of Selective Dopamine D3 Receptor Ligands Peng Du1,† , Lili Xu1,† , Jiye Huang2,† , Kunqian Yu2 , Rui Zhao3 , Bo Gao3 , Hualiang Jiang2 , Weili Zhao1 , Xuechu Zhen3 and Wei Fu1,* 1 Department of Medicinal Chemistry & Key Laboratory of Smart Drug Delivery, Ministry of Education & PLA, School of Pharmacy, Fudan University, Shanghai, 201203, China 2 Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of MateriaMedica, Chinese Academy of Sciences, Shanghai, 201203, China 3 Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou, 215123, China *Corresponding author: Wei Fu, wfu@fudan.edu.cn †These authors contributed equally to this work. A series of indolebutylamine derivatives were designed, synthesized, and evaluated as a novel class of selective ligands for the dopamine 3 receptor. The most potent compound 11q binds to dopamine 3 receptor with a Ki value of 124 nM and displays excellent selectivity over the dopamine 1 receptor and dopamine 2 receptor. Investigation based on structural information indicates that site S182 located in extracellular loop 2 may account for high selectivity of compounds. Interaction models of the dopamine 3 receptor-11q complex and structure-activity relationships were discussed by inte￾grating all available experimental and computational data with the eventual aim to discover potent and selec￾tive ligands to dopamine 3 receptor. Key words: dopamine 3 receptor, indolebutylamine, pharma￾cophore model, selectivity, structure-activity relationship Received 22 November 2012, revised 17 April 2013 and accepted for publication 26 April 2013 Since the discovery by Sokoloff et al. in 1990 (1), dopamine 3 receptor (D3R) has been proved to be a promising thera￾peutic target for drug discovery. Dopamine 3 receptor antagonist was shown to play a key role in the treatment of schizophrenia (2,3) and drug addiction (4). Although consid￾erable efforts have been devoted to the design and develop￾ment of D3R antagonists (5–9), function study of D3R in vivo is still limited due to the lack of highly selective antagonists. One of the important reasons for the difficulty in devel￾oping selective antagonist for D3R is attributed to the high sequence homology among the D2-like dopamine receptor subtypes (D2, D3, and D4). For instance, hD3R and hD2R share 78% sequence homology within the seven transmembrane domains and 94% sequence homology within the active site (10,11). To date, most of D3R selective ligands are 4-Phenylpiperazines and their close analogs (12,13). Considering the importance of the D3R in the treatment of addiction and other neuropsy￾cho disorders, it is meaningful to discover novel chemi￾cal entities to enrich the structural diversity of potent and selective D3R ligands. Using a strategy that com￾bines synthetic chemistry, binding assays, and a set of computational approach (integrating active site mapping, pharmacophore-based virtual screening, and automated molecular docking), we designed a series of IBA deriva￾tives as a new type of highly selective D3R antagonists. Furthermore, the molecular determinants critical to the binding specificity and selectivity of D3R were identified and the structure-activity relationships (SAR) was investi￾gated. Methods and Materials Structure-based pharmacophore model generation Dopamine 3 receptor was obtained from the Protein Data Bank (PDB ID: 3PBL) (11). The GRID22 program (14) was employed to map the active sites of the optimized X-ray structure of D3R with five types of chemical probes, that is, negative ionizable (COO
), positive ionizable (N1+), hydrogen-bond acceptor (O), hydrogen-bond donor (N1), and hydrophobic probes (DRY). For each of the five probes used in the grid calculations, grid points were superimposed to identify clusters of positions. The mem￾bers of each identified clusters were combined into one pharmacophore feature, and the centers of each pharma￾cophore features were set at the geometric centers of the members in each clusters (15). Finally, a four-feature phar￾macophore model was generated. Virtual screening The obtained pharmacophore model was used to screen the Asinex GOLD and Maybridge collection database which contain 238 000 compounds. The Ligand Pharma￾cophore Mapping protocol embedded in DISCOVERY STUDIO 3.5a was employed to retrieve molecules, which can well 326 ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12158 Chem Biol Drug Des 2013; 82: 326–335 Research Article

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: 326-335 327

match 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

CAB mixture was refluxed for 8 h, and the reaction mixture was 2.2 Hz, 1H, Ar-H), 3.27 (s, 3H, CH3), 2.91(s, 2H evaporated to dryness. The residue was dissolved in CH2CO), 2.70(t, J=7.5 Hz, 2H, CH2), 2.47-2.38(m EtOAC(100 mL), washed with H2O(50 mL), dried, and 10H, CH2), 1.91-1.80(m, 2H, CH2).C-NMR (100 MHz, evaporated to obtain the crude product, which was puri- CDCl3 )8 169.53, 158.81, 156.49, 143.55, 132.88, 129.70 afford tert-butyl 4-(2-cyclohexyl(phenyl)amino)-2 oxethyl) 111.58, 110.26, 109.99, 103.90, 103.67, 59.61, 58.16, piperazine-1-carboxylate 9 as a yellow liquid(4.58 g, yield 53.24, 53.02, 37.49, 27.13, 22.87. ESI-MS m/z 409.2 4%),To a solution of compound 93.0 g, M+H 8.68 mmol) in CH2Cl2 (15 mL was added trifluoroacetic acid(15 mL). The mixture was stirred at room temperature for 12 h, then concentrated, washed with PE and Et2O 2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1 separately to yield N-cyclohexyl-N-phenyl-2-(piperazin-1-yl) yl)-N-isopropyl-N-phenylacetamide(11d) acetamide 10 as an off-white solid(2.68 g, yield 90%),(iv) White solid (81 mg, yield 40.5%).H NMR(400 MHZ, A solution of compound 10(0.467 g, 1.17 mmol), com- CDCl3)8 8.04(s, 1H), 7.41 (d, J=6.1 HZ, 3H), 7.26-7.20 pound30.3g,1.17mmo,andK2CO30.324g,(m,2H,7.15-7.06m,2H),7.02(s,1H,6.92td,d=9.1 2.34 mmol)in acetonitrile(20 mL was stirred at 80C for 2.4 Hz, 1H), 5. 10-4.89(m, 1H), 2.75(s, 2H),2.70(t, 12 h, then the reaction mixture was evaporated to dry- J=7.5 Hz, 2H), 2. 48-2.38(m, 10H), 1.93-1.80(m, 2H) ness, water added, and the mixture was extracted with 1.05(d, J=6.8 Hz, 6H).C-NMR(100 MHZ, CDCl3) 8 CH2 Cl2(3×30mlL, washed with brine, dried over anhy.168.72,15881,156.49,137.96,132.88,130.51,129.16 drous Na2SO4, and concentrated. The residue was puri- 128. 37, 128.01, 127.92, 123. 10, 116.44, 116.40, 111.68 fied with flash chromatography on silica gel (CH2Cl2/ 111.58, 110.25, 109.99, 103.90, 103.67, 60.45, 58.17, MeOH, 40/1)to afford compound 11a as an off-white solid 53. 24, 53.26, 53.04, 46.16, 27. 09, 22.86, 20.93 ESI-Ms (0.2 g, yield 35.7%)(17). H NMR(400 MHZ, CDCl3)8 m/z 437.2 [M+HI 8.11(s,1H,NH,7.40(d,J=5.1Hz,3H,Ar-H,7.24(d, J=13.6,8.6Hz,2H,Ar-H,7.11-7.09(m,2H,Ar-+H 7.02(s, 1H, Ar-H), 6.92(t, J=9.0 Hz, 1H, Ar-H), 4.59(t, N-cyclohexyl-2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl) J=12.0Hz,1H,NCH,2.75(s,2H,cH2CO,2.70 piperazin-1-yI)-N-(2-methoxyphenyl)acetamide J=7.5Hz,2H,CH2,2.44m,J=22.1,14.7Hz,10H,(11e) CH2), 1.91-1.81(m, 6H, CH2), 1.72(d, J=13.6 Hz, 3H), White solid(154 mg, yield 43.4%). White solid(160 mg 1.57(d,J=126Hz,1H),1.39(d,J=26.0,12.9Hz,yied433%HNMR(400MHz,cDOD)6741 2H,0.98(m,4H.CMR(100MHz,CDc)5170.38,J=7.8Hz,H,7.250d,J=9.3,4,7Hz,1H,717-7.07 159.94,15763,139.33,134.79,131.56,130.45,12986,(m,3H,7.07(s,1H,7.01(t,J=78H,1H,6.82(d 129.10,12901,124.99,11609,116.04,11296,11287,J=9.0,2.4Hz,1H,4.42(tJ=12.1,35Hz,1H),382 110.38,110.12,104.34,103.81,61.17,5927,56.03,(s,3H,2.81-267m,4H,2.48-2.01m,10H,1.92 3.73,5369,3262,27.99,26.92,26.52,2388.ES|-Ms1.1.74m,5H,1.67(d,J=11.4Hz,1H,1.57(d m/24772M+H J=129Hz,1H),1.42-1.30m,2H,0.960.80(m,3H CNMR(100MHz,CDC6170.86,159.84,157.54 157.47,134.70,132.28,131.46,129.01,128.91,127.89, 2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1 124.94,121.86,115.97,115.92,113.13,112.90,11 yl)-N-phenylacetamide(11b) 110.31,110.04,103.98,103.75,60.64,59.26 White solid(150mg,yied40.5%).HNMR(400Mz,5590,5373,5361,3310,30.82,27.96,26.89 CDc)69.13(,1H,8.06(s,1H),7.58-7.56m,2H,26.60,23.84.ES|MSm/z5074M+H 7.34(t,J=7.Hz,2H,7.27-7.23m,2H),7.11( J=7.4H,1H,7.03(,J=1.8Hz,H,6.93td J=9.1, 2. 4 Hz, 1H),3.14(, 2H), 2.75(t, J=7.5 Hz, 2(4-(3-(5-chloro-1H-indol-3-ylpropyl)piperazin-1 3H), 2.68(s, 4H), 2.56(s, 4H), 2.47(t, =8.0 HZ, 2H), yl)-N-cyclohexyl-N-phenylacetamide(11f) 1.94-1.87(m,2H).C-NMR(100 MHZ, CDCl3 8 168.42, White solid(160 mg, yield 43.3%).H NMR(400 MHZ, 15886,156.53,137.64,13288,129.05,128.03,127.94,CDCd8.50(s,1H,7.41-7.40(m,3H,727-724m 124.22,123.06,11947,116.51,116.46,111.69,111.60,2H,7.12(d,J=7.9Hz,1H,7.06-7.03(m,3H,6.90(td 110.37,110.10,103.93,103.70,61.7,58.01,53.52,J=90,1.9H,1H,4.53(,J=12.1H,1H,295-259 5340,27.24,22.79.ES-Msm/z3952M+H m,14H),2082.0m,2H,1.79(d,J=10.7Hz,2H) 1.71(d,J=13.1H,2H,1.55d,J=12.6Hz,1H 141-1.31(m,2H,1.060.84(m,3H).1CNMR 2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1 (100MHz,CDCa167.94,158.80,156.47,137.65 yl)-N-methyl-N-phenylacetamide(11c) 13283,130.07,129.49,128.78,127.47,127.37,123.55, White solid(150mg,yied39.5%).HNMR(400MHz,114.07,11200,11191,110.47,110.21,10342,103.19 717m,1H,A-HD,700(,1H,A+,.6.91t.J=9.0,2523,2429,213 ESI-MS m/2472M+6°8,2566 CDCa)806(s,1H,NH),7.45-7.30(m,3H,Ar-+H,7265938,56.91,54.47,51.87,50.61,31.34,29 328 Chem Biol Drug Des 2013: 82: 326-335

mixture was refluxed for 8 h, and the reaction mixture was evaporated to dryness. The residue was dissolved in EtOAc (100 mL), washed with H2O (50 mL), dried, and evaporated to obtain the crude product, which was puri- fied by flash chromatography (CH2Cl2/MeOH, 40:1) to afford tert-butyl 4-(2-(cyclohexyl(phenyl)amino)-2- oxoethyl) piperazine-1-carboxylate 9 as a yellow liquid (4.58 g, yield 95.4%), (iii) To a solution of compound 9 (3.0 g, 8.68 mmol) in CH2Cl2 (15 mL) was added trifluoroacetic acid (15 mL). The mixture was stirred at room temperature for 12 h, then concentrated, washed with PE and Et2O separately to yield N-cyclohexyl-N-phenyl-2-(piperazin-1-yl) acetamide10 as an off-white solid (2.68 g, yield 90%), (iv) A solution of compound 10 (0.467 g, 1.17 mmol), com￾pound 3 (0.3 g, 1.17 mmol), and K2CO3 (0.324 g, 2.34 mmol) in acetonitrile (20 mL) was stirred at 80 °C for 12 h, then the reaction mixture was evaporated to dry￾ness, water added, and the mixture was extracted with CH2Cl2 (3 9 30 mL), washed with brine, dried over anhy￾drous Na2SO4, and concentrated. The residue was puri- fied with flash chromatography on silica gel (CH2Cl2/ MeOH, 40/1) to afford compound 11a as an off-white solid (0.2 g, yield 35.7%) (17). 1 H NMR (400 MHz, CDCl3) d 8.11 (s, 1H, NH), 7.40 (d, J = 5.1 Hz, 3H, Ar-H), 7.24 (dd, J = 13.6, 8.6 Hz, 2H, Ar-H), 7.11–7.09 (m, 2H, Ar-H), 7.02 (s, 1H, Ar-H), 6.92 (t, J = 9.0 Hz, 1H, Ar-H), 4.59 (t, J = 12.0 Hz, 1H, NCH), 2.75 (s, 2H, CH2CO), 2.70 (t, J = 7.5 Hz, 2H, CH2), 2.44 (m, J = 22.1, 14.7 Hz, 10H, CH2), 1.91–1.81 (m, 6H, CH2), 1.72 (d, J = 13.6 Hz, 3H), 1.57 (d, J = 12.6 Hz, 1H), 1.39 (dd, J = 26.0, 12.9 Hz, 2H), 0.98 (m, 4H).13C-NMR (100 MHz, CDCl3) d 170.38, 159.94, 157.63, 139.33, 134.79, 131.56, 130.45, 129.86, 129.10, 129.01, 124.99, 116.09, 116.04, 112.96, 112.87, 110.38, 110.12, 104.34, 103.81, 61.17, 59.27, 56.03, 53.73, 53.69, 32.62, 27.99, 26.92, 26.52, 23.88. ESI-MS m/z 477.2 [M + H]+ . 2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1- yl)-N-phenylacetamide (11b) White solid (150 mg, yield 40.5%). 1 H NMR (400 MHz, CDCl3) d 9.13 (s, 1H), 8.06 (s, 1H), 7.58–7.56 (m, 2H), 7.34 (t, J = 7.9 Hz, 2H), 7.27–7.23 (m, 2H), 7.11 (t, J = 7.4 Hz, 1H), 7.03 (d, J = 1.8 Hz, 1H), 6.93 (td, J = 9.1, 2.4 Hz, 1H), 3.14 (s, 2H), 2.75 (t, J = 7.5 Hz, 3H), 2.68 (s, 4H), 2.56 (s, 4H), 2.47 (t, J = 8.0 Hz, 2H), 1.94–1.87 (m, 2H). 13C-NMR (100 MHz, CDCl3) d 168.42, 158.86, 156.53, 137.64, 132.88, 129.05, 128.03, 127.94, 124.22, 123.06, 119.47, 116.51, 116.46, 111.69, 111.60, 110.37, 110.10, 103.93, 103.70, 61.97, 58.01, 53.52, 53.40, 27.24, 22.79. ESI-MS m/z 395.2 [M + H]+ . 2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1- yl)-N-methyl-N-phenylacetamide (11c) White solid (150 mg, yield 39.5%). 1 H NMR (400 MHz, CDCl3) d 8.06 (s, 1H, NH), 7.45–7.30 (m, 3H, Ar-H), 7.26– 7.17 (m, 1H, Ar-H), 7.00 (s, 1H, Ar-H), 6.91 (t, J = 9.0, 2.2 Hz, 1H, Ar-H), 3.27 (s, 3H, CH3), 2.91 (s, 2H, CH2CO), 2.70 (t, J = 7.5 Hz, 2H, CH2), 2.47–2.38 (m, 10H, CH2), 1.91–1.80 (m, 2H, CH2). 13C-NMR (100 MHz, CDCl3) d 169.53, 158.81, 156.49, 143.55, 132.88, 129.70, 128.02, 127.92, 127.33, 123.09, 116.48, 116.43, 111.68, 111.58, 110.26, 109.99, 103.90, 103.67, 59.61, 58.16, 53.24, 53.02, 37.49, 27.13, 22.87. ESI-MS m/z 409.2 [M + H]+ . 2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl)piperazin-1- yl)-N-isopropyl-N-phenylacetamide (11d) White solid (81 mg, yield 40.5%). 1 H NMR (400 MHz, CDCl3) d 8.04 (s, 1H), 7.41 (d, J = 6.1 Hz, 3H), 7.26–7.20 (m, 2H), 7.15–7.06 (m, 2H), 7.02 (s, 1H), 6.92 (td, J = 9.1, 2.4 Hz, 1H), 5.10–4.89 (m, 1H), 2.75 (s, 2H), 2.70 (t, J = 7.5 Hz, 2H), 2.48–2.38 (m, 10H), 1.93–1.80 (m, 2H), 1.05 (d, J = 6.8 Hz, 6H). 13C-NMR (100 MHz, CDCl3) d 168.72, 158.81, 156.49, 137.96, 132.88, 130.51, 129.16, 128.37, 128.01, 127.92, 123.10, 116.44, 116.40, 111.68, 111.58, 110.25, 109.99, 103.90, 103.67, 60.45, 58.17, 53.24, 53.26, 53.04, 46.16, 27.09, 22.86, 20.93. ESI-MS m/z 437.2 [M + H]+ . N-cyclohexyl-2-(4-(3-(5-fluoro-1H-indol-3-yl)propyl) piperazin-1-yl)-N-(2-methoxyphenyl)acetamide (11e) White solid (154 mg, yield 43.4%). White solid (160 mg, yield 43.3%). 1 H NMR (400 MHz, CD3OD) d7.41 (t, J = 7.8 Hz, 1H), 7.25 (dd, J = 9.3, 4.7 Hz, 1H), 7.17–7.07 (m, 3H), 7.07(s, 1H), 7.01(t, J = 7.8 Hz, 1H), 6.82(td, J = 9.0, 2.4 Hz, 1H), 4.42 (tt, J = 12.1, 3.5 Hz, 1H), 3.82 (s, 3H), 2.81–2.67 (m, 4H), 2.48–2.01 (m, 10H), 1.92– 1.1.74 (m, 5H), 1.67 (d, J = 11.4 Hz, 1H), 1.57 (d, J = 12.9 Hz, 1H), 1.42–1.30(m, 2H), 0.96–0.80 (m, 3H). 13C-NMR (100 MHz, CDCl3) d 170.86, 159.84, 157.54, 157.47, 134.70, 132.28, 131.46, 129.01, 128.91, 127.89, 124.94, 121.86, 115.97, 115.92, 113.13, 112.90, 112.80, 110.31, 110.04, 103.98, 103.75, 60.64, 59.26, 56.68, 55.90, 53.73, 53.61, 33.10, 30.82, 27.96, 26.89, 26.86, 26.60, 23.84. ESI-MS m/z 507.4 [M + H]+ . 2-(4-(3-(5-chloro-1H-indol-3-yl)propyl)piperazin-1- yl)-N-cyclohexyl-N-phenylacetamide (11f) White solid (160 mg, yield 43.3%). 1 H NMR (400 MHz, CDCl3) d 8.50 (s, 1H), 7.41–7.40 (m, 3H), 7.27–7.24 (m, 2H), 7.12 (d, J = 7.9 Hz, 1H), 7.06–7.03 (m, 3H), 6.90 (td, J = 9.0, 1.9 Hz, 1H), 4.53 (t, J = 12.1 Hz, 1H), 2.95–2.59 (m, 14H), 2.08–2.0 (m, 2H), 1.79 (d, J = 10.7 Hz, 2H), 1.71 (d, J = 13.1 Hz, 2H), 1.55 (d, J = 12.6 Hz, 1H), 1.41–1.31 (m, 2H), 1.06–0.84 (m, 3H). 13C-NMR (100 MHz, CDCl3) d 167.94, 158.80, 156.47, 137.65, 132.83, 130.07, 129.49, 128.78, 127.47, 127.37, 123.55, 114.07, 112.00, 111.91, 110.47, 110.21, 103.42, 103.19, 59.38, 56.91, 54.47, 51.87, 50.61, 31.34, 29.68, 25.66, 25.23, 24.29, 22.13. ESI-MS m/z 477.2 [M + H]+ . 328 Chem Biol Drug Des 2013; 82: 326–335 Du et al

Synthesis, Biological Evaluation, and Molecular Modeling 2-4-(3-(5-chloro-1H-indol-3-yl)propyl)piperazin-1 (t,J=121,3.2H,1H,2.77-2.73m,4H,2.492.42 yl)-N-cyclohexyl-N-(2-methoxyphenyl)acetamide m,10H),1.94-1.86m,2H,1.82(d,J=11.7Hz,2H) (11g) 1.71(d,J=13.4Hz,2H,1.56(d,J=123Hz,1H White solid(137 mg, yield 28.8%).H NMR(400 MHZ, 1.43-1.38(m, 2H), 1.06-0.87(m, 4H).ESI-MS m/z CDCa)68.14⑤s,1H,7.34(td,J=9.5,1.8Hz,1H,459.4M+H 7.25-720m,2H,7.066.88m,5H,4.554.49m,1H, 3.78(s,3H),2.792.67(m,4H,2.48-2.37m,10H 1.96-1.83m,3H,1.71(d,J=14.9Hz,1H,1.63(d,2-14-(3-(1H- indol-3- yD))piperazin-1-y)-N J=13.0 Hz, 1H), 1.54(d, J=13.3 Hz, 1H), 1.42-1.27 cyclohexyl-N-(2-methoxyphenyl)acetamide(11k) 3H,1.16(ad,J=246,123,37Hz,4H,0.95- White solid(130mg,yeld444%).HNMR(400MHz 076m,2H.1CNMR(100MH,CDC169.18,cDcd7.55(,J=7.9Hz,1H,748(od,J=114 15867,156.34,156.21,13283,131.39,12968,4.4Hz,1H,7.33(,J=8.1Hz,1H.7.21(dd,J=13.7, 2788,127.78,12736,123.17,120.53,116.14,5.3H,2H,7.14-7.04m,3H,7.00(,J=7.4Hz,1H, 111.72,111.63,11152,11008,109.81,103.80,4.43(td=11.8,3.3Hz,1H),3.87(d,d=8.0Hz,13H 10356,59.80,58.22,55.19,5497,53.28,53.03,3205,3283.17m,2H),2.89(J=7.0Hz,2H,224-2.14 29.63,27.10,25.79,25.76,2549,22.85.ES|Msm2H).1.95(d,J=11.2H,1H,1.80(t,J=10.7Hz,2H 5072M+H 1.69(d,J=13.1Hz,1H,1.58(d,J=128Hz,1 1.43-1.19m,3H,1.050.80(m,2H. ESI-MS m/24894 2-(4-(3-(5-bromo-1H-indol-3-yl)propyl)piperazin-1 yl)-N-cyclohexyl-N-phenylacetamide(11h) White solid (110 mg, yield 20.5%). H NMR(400 MHZ, 2-(4-(2-(1H-indol-3-yl)ethyl)piperazin-1-yl-N- CDCl3)8 8.46(s, 1H),7.70(s, 1H), 7.34(t, J=7.8 Hz, cyclohexyl-N-phenylacetamide(111) 1H),7.22(s, 2H), 7.04 (d, J=7.4 Hz, 1H), 6.97-6.93(m, White solid(120 mg, yield 28.4%). H NMR(400 MHZ, 3H,4.53J=11.7H,1H,3.78(s,3H,279-2.67(m,CDo68.08(s,1H,7.59(d,J=7.8Hz,1H,7.40(dd, 4H,2.47-2.36m,10H,1.93-1.78m,4H,1.71(d,J=5.0,1.6Hz,3H,7.34(d,J=8.1Hz,1H,7.17(t, J=129Hz,1H,1.63(,J=12.1H,1H,1.54(d,J=7.4hz,1H,7.12-7.08m,3H,701(s,1H,4.59(t =12.1Hz,1H,1.42-1.26m,2H,1.20-1.14m,1H,J=122,35H,1H,2.992.95m,2H,2.77-2.54m, 0950.78(m,2H.1CNMR(100MHz,CDC616859 2H,1.82(,J=106H,2H,1.72(d,J=134Hz 13824,134.86,130.39,129.28,129.09,128.31,124 2H,1.56(d,J=125Hz,1H,1.43-1.33(m,3H,1.03 12253,121.49,11580,11253,11220,60.32,5809,(d,J=25.1,12.5,3.4H,2H).ES-Msm/z445.4 54.06,53.28,52.98,31.43,27.20,25.69,25.29,22.69 ESI-MS m 539.2 [M+HI 2-(4-(2-(1H-indol-3-yl)ethyl)piperazin-1-yl)-N- 2-(4-(3-(5-bromo-1H-indol-3-yl)propyl)piperazin-1 cyclohexyl-N-(2-methoxyphenyl)acetamide(11m) yI)-N-cyclohexyl-N-(2-methoxy phenylacetamide White solid (150 mg, yield 30.5%). H NMR(400 MH (11 CDca)b8.32(,1H,7.58(d,J=7.8Hz,1H,7.37(dd White solid (129 mg, yield 18.8%). H NMR(400 MHZ, J=13.6, 5.0 Hz, 2H),7.17(t,J=7.1 Hz, 1H),7.10 CDCa)68.56(s,1H),7.70(s,1H,7.38(s,3H,7.19(s,J=7.0Hz,1H,7.05-7.03m,2H,6.97(t,J=7.5H, 1H,708s,1H,7.07(,1H,6.95(s,1H,4.58(t,2H,4.51(t1J=11.7,3.3Hz,1H,3.81(s,3H,3.12 J=12.1Hz,1H,2.74(s,2H,2.68(t,J=74H,2H,3.08(m,2H,2.91-2.64m,12H,1.3(d,J=1.7Hz, 2.46-235m,10H,1.83-1.80m,4H),1.70(d,1H),1.80(d,d=125Hz,1H,1.73(d,J=13.2H,1H, J=125Hz,2H,1.55(d,J=12.9H,1H,1.42-1.321.65(d,J=13.3Hz,1H,1.55(d,J=13.1Hz,1H), m,2H,1050.83(m,3H.1CNMR(100MHz,CDC)1.38-1.30(m,2H,097-0,77(m,3H. ESI-MS m/z4754 169.18,156.18,134.92,131.35,12969,129.29,127.29,M+H 124.36,12265,121.47,120.54,115.67,112.61,112.18, 111.52,59.76,58.13,5520,54.97,53.25,52.98,32.03, 29.61,27.26,25.77,25.73,25.47,22.71.ES|MSm/z2-(4-(4-(1 H-indol-3- yl)butyl)piperazin-1-y-N 5692M+H cyclohexyl-N-phenylacetamide(11n) White solid(125 mg, yield 33.2%). H NMR(400 MHZ, CDo3)8.01(,1H,7.57(d,d=7.7Hz,1H,7.40-7.34 2-4-(3-(1H-indol-3-yl)propyl)piperazin-1-yl-N m,4H),7.17(.,J=75Hz,1H,7.13-7.03m,3H,6.97 cyclohexyl-N-phenylacetamide(11j1 s,1H,4.57(,J=120Hz,1H,2.77-2.47m,14H) White solid(115mg,yeld34.8%).HNMR(400MHz,1.81(,J=11.6Hz,2H),1.72-1.54(m,7H,1.37(ad CDCa8.12(s,1H,7.58(d,J=7.8比,1H,7.43J=26.0,127H,2H,101(dd,d=23.1,10.6Hz,2H 7.38m,3H,734(d,J=8.1Hz,1H,7.16(t,0.89(dd,J=26.1,13.1Hz,1H.Es-Msm/z47344 J=7.5H,1H,7.10-7.08m,3H,6.96(s,1H,4.58M+H Chem Bio/ Drug Des 2013: 82: 326-335 329

2-(4-(3-(5-chloro-1H-indol-3-yl)propyl)piperazin-1- yl)-N-cyclohexyl-N-(2-methoxyphenyl)acetamide (11g) White solid (137 mg, yield 28.8%). 1 H NMR (400 MHz, CDCl3) d 8.14 (s, 1H), 7.34 (td, J = 9.5, 1.8 Hz, 1H), 7.25–7.20(m, 2H), 7.06–6.88 (m, 5H), 4.55–4.49(m, 1H), 3.78 (s, 3H), 2.79–2.67 (m, 4H), 2.48–2.37 (m, 10H), 1.96–1.83 (m, 3H), 1.71 (d, J = 14.9 Hz, 1H), 1.63 (d, J = 13.0 Hz, 1H), 1.54 (d, J = 13.3 Hz, 1H), 1.42–1.27 (m, 3H), 1.16 (ddd, J = 24.6, 12.3, 3.7 Hz, 4H), 0.95– 0.76 (m, 2H). 13C-NMR (100 MHz, CDCl3) d 169.18, 158.67, 156.34, 156.21, 132.83, 131.39, 129.68, 127.88, 127.78, 127.36, 123.17, 120.53, 116.14, 111.72, 111.63, 111.52, 110.08, 109.81, 103.80, 103.56, 59.80, 58.22, 55.19, 54.97, 53.28, 53.03, 32.05, 29.63, 27.10, 25.79, 25.76, 25.49, 22.85. ESI-MS m/z 507.2 [M + H]+ . 2-(4-(3-(5-bromo-1H-indol-3-yl)propyl)piperazin-1- yl)-N-cyclohexyl-N-phenylacetamide (11h) White solid (110 mg, yield 20.5%). 1 H NMR (400 MHz, CDCl3) d 8.46 (s, 1H), 7.70 (s, 1H), 7.34 (t, J = 7.8 Hz, 1H), 7.22 (s, 2H), 7.04 (d, J = 7.4 Hz, 1H), 6.97–6.93 (m, 3H), 4.53 (t, J = 11.7 Hz, 1H), 3.78 (s, 3H), 2.79–2.67 (m, 4H), 2.47–2.36 (m, 10H), 1.93–1.78 (m, 4H), 1.71 (d, J = 12.9 Hz, 1H), 1.63 (d, J = 12.1 Hz, 1H), 1.54 (d, J = 12.1 Hz, 1H), 1.42–1.26 (m, 2H), 1.20–1.14 (m, 1H), 0.95–0.78 (m, 2H). 13C-NMR (100 MHz, CDCl3) d168.59, 138.24, 134.86, 130.39, 129.28, 129.09, 128.31, 124.43, 122.53, 121.49, 115.80, 112.53, 112.20, 60.32, 58.09, 54.06, 53.28, 52.98, 31.43, 27.20, 25.69, 25.29, 22.69. ESI-MS m/z 539.2 [M + H]+ . 2-(4-(3-(5-bromo-1H-indol-3-yl)propyl)piperazin-1- yl)-N-cyclohexyl-N-(2-methoxyphenyl)acetamide (11i) White solid (129 mg, yield 18.8%). 1 H NMR (400 MHz, CDCl3) d 8.56 (s, 1H), 7.70 (s, 1H), 7.38 (s, 3H), 7.19 (s, 1H), 7.08 (s, 1H), 7.07 (s, 1H), 6.95 (s, 1H), 4.58 (t, J = 12.1 Hz, 1H), 2.74 (s, 2H), 2.68 (t, J = 7.4 Hz, 2H), 2.46–2.35 (m, 10H), 1.83–1.80 (m, 4H), 1.70 (d, J = 12.5 Hz, 2H), 1.55 (d, J = 12.9 Hz, 1H), 1.42–1.32 (m, 2H), 1.05–0.83 (m, 3H).13C-NMR (100 MHz, CDCl3) d 169.18, 156.18, 134.92, 131.35, 129.69, 129.29, 127.29, 124.36, 122.65, 121.47, 120.54, 115.67, 112.61, 112.18, 111.52, 59.76, 58.13, 55.20, 54.97, 53.25, 52.98, 32.03, 29.61, 27.26, 25.77, 25.73, 25.47, 22.71. ESI-MS m/z 569.2 [M + H]+ . 2-(4-(3-(1H-indol-3-yl)propyl)piperazin-1-yl)-N￾cyclohexyl-N-phenylacetamide (11j) White solid (115 mg, yield 34.8%). 1 H NMR (400 MHz, CDCl3) d 8.12 (s, 1H), 7.58 (d, J = 7.8 Hz, 1H), 7.43– 7.38 (m, 3H), 7.34 (d, J = 8.1 Hz, 1H), 7.16 (t, J = 7.5 Hz, 1H), 7.10–7.08 (m, 3H), 6.96 (s, 1H), 4.58 (tt, J = 12.1, 3.2 Hz, 1H), 2.77–2.73 (m, 4H), 2.49–2.42 (m, 10H), 1.94–1.86 (m, 2H), 1.82 (d, J = 11.7 Hz, 2H), 1.71 (d, J = 13.4 Hz, 2H), 1.56 (d, J = 12.3 Hz, 1H), 1.43–1.38 (m, 2H), 1.06–0.87 (m, 4H). ESI-MS m/z 459.4 [M + H]+ . 2-(4-(3-(1H-indol-3-yl)propyl)piperazin-1-yl)-N￾cyclohexyl-N-(2-methoxyphenyl)acetamide (11k) White solid (130 mg, yield 44.4%). 1 H NMR (400 MHz, CDCl3) d 7.55 (d, J = 7.9 Hz, 1H), 7.48 (dd, J = 11.4, 4.4 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.21 (dd, J = 13.7, 5.3 Hz, 2H), 7.14–7.04 (m, 3H), 7.00 (t, J = 7.4 Hz, 1H), 4.43 (tt, J = 11.8, 3.3 Hz, 1H), 3.87 (d, J = 8.0 Hz, 13H), 3.28–3.17 (m, 2H), 2.89 (t, J = 7.0 Hz, 2H), 2.24–2.14 (m, 2H), 1.95 (d, J = 11.2 Hz, 1H), 1.80 (t, J = 10.7 Hz, 2H), 1.69 (d, J = 13.1 Hz, 1H), 1.58 (d, J = 12.8 Hz, 1H), 1.43–1.19 (m, 3H), 1.05–0.80 (m, 2H). ESI-MS m/z 489.4 [M + H]+ . 2-(4-(2-(1H-indol-3-yl)ethyl)piperazin-1-yl)-N￾cyclohexyl-N-phenylacetamide (11l) White solid (120 mg, yield 28.4%). 1 H NMR (400 MHz, CDCl3) d 8.08 (s, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.40 (dd, J = 5.0, 1.6 Hz, 3H), 7.34 (d, J = 8.1 Hz, 1H), 7.17 (t, J = 7.4 Hz, 1H), 7.12–7.08 (m, 3H), 7.01 (s, 1H), 4.59 (tt, J = 12.2, 3.5 Hz, 1H), 2.99–2.95 (m, 2H), 2.77–2.54 (m, 12H), 1.82 (d, J = 10.6 Hz, 2H), 1.72 (d, J = 13.4 Hz, 2H), 1.56 (d, J = 12.5 Hz, 1H), 1.43–1.33 (m, 3H), 1.03 (ddd, J = 25.1, 12.5, 3.4 Hz, 2H). ESI-MS m/z 445.4 [M + H]+ . 2-(4-(2-(1H-indol-3-yl)ethyl)piperazin-1-yl)-N￾cyclohexyl-N-(2-methoxyphenyl)acetamide (11m) White solid (150 mg, yield 30.5%). 1 H NMR (400 MHz, CDCl3) d 8.32 (s, 1H), 7.58 (d, J = 7.8 Hz, 1H), 7.37 (dd, J = 13.6, 5.0 Hz, 2H), 7.17 (t, J = 7.1 Hz, 1H), 7.10 (t, J = 7.0 Hz, 1H), 7.05–7.03 (m, 2H), 6.97 (t, J = 7.5 Hz, 2H), 4.51 (tt, J = 11.7, 3.3 Hz, 1H), 3.81 (s, 3H), 3.12– 3.08 (m, 2H), 2.91–2.64 (m, 12H), 1.93 (d, J = 11.7 Hz, 1H), 1.80 (d, J = 12.5 Hz, 1H), 1.73 (d, J = 13.2 Hz, 1H), 1.65 (d, J = 13.3 Hz, 1H), 1.55 (d, J = 13.1 Hz, 1H), 1.38–1.30 (m, 2H), 0.97–0.77 (m, 3H). ESI-MS m/z 475.4 [M + H]+ . 2-(4-(4-(1H-indol-3-yl)butyl)piperazin-1-yl)-N￾cyclohexyl-N-phenylacetamide (11n) White solid (125 mg, yield 33.2%). 1 H NMR (400 MHz, CDCl3) d 8.01 (s, 1H), 7.57 (d, J = 7.7 Hz, 1H), 7.40–7.34 (m, 4H), 7.17 (t, J = 7.5 Hz, 1H), 7.13–7.03 (m, 3H), 6.97 (s, 1H), 4.57 (t, J = 12.0 Hz, 1H), 2.77–2.47 (m, 14H), 1.81 (d, J = 11.6 Hz, 2H), 1.72–1.54 (m, 7H), 1.37 (dd, J = 26.0, 12.7 Hz, 2H), 1.01 (dd, J = 23.1, 10.6 Hz, 2H), 0.89 (dd, J = 26.1, 13.1 Hz, 1H). ESI-MS m/z 473.4 [M + H]+ . Chem Biol Drug Des 2013; 82: 326–335 329 Synthesis, Biological Evaluation, and Molecular Modeling

2-(4-(4-(1H-indol-3-yl)butyl)piperazin-1-yl) Results and Discussion -N-cyclohexyl-N-(2-methoxyphenyl) acetamide(11o) Pharmacophore-based virtual screening White solid (135 mg, yield 37.4%). H NMR(400 MHz, The obtained pharmacophore model was shown in CDCl3)8 8.13(, 1H),7.57(d, J=7.8 Hz, 1H),7.36- Figure 1A. As a result of our virtual screening protocol, 16 7.32(, 2H), 7.16 (t, J=7.1 Hz, 1H), 7.08 (t, compounds were selected to purchase and submitted to J=7. 4 Hz, 1H), 7.04(dd, J=7.6, 1.6 Hz, 1H), 6.97- pharmacological experiments(Tables S1 and S2). To our 6.92(m, 3H), 4.52(tt, J=12.0, 3.5 Hz, 1H), 3.78(s, delight, three of them revealed moderate D3R activities 3H), 2.78-266(m, 4H), 2.49-2. 35(m, 10H),1.94-1.91 Their chemical structures and corresponding binding (m,1h),1.80(d,j=12.3Hz,1h),1.72(d,J=7.4Hz,assaysweresummarizedinFigure1bandTable1.com 1H),1.67(d, J=7.6 Hz, 1H),1.61-1.53(m, 4H), 1.44- pound 11a, with a high fit value and a core structure of in- 1. 24(m, 3H), 1.17(ddd, J= 24.7, 12.2, 3.6 Hz, 1H), dolepropylamine and N-phenylacetamide, matches the 0.96-0.88(m, 1H),0.81(ddd, J=25.1, 12.6, 3.6 Hz, pharmacophore model quite well(Figure S1)and repre 1H).13C-NMR(100 MHz, CDCl3)8 169.10, 156. 20, sents a novel class of D3R ligands. It was identified to 136.33, 131.38, 129.69, 127.46, 127.31, 121.66, bind hD3R with 2161 nM affinity and was thus chosen as 121.26, 120.54, 118.85, 116.34, 111.53, 111.01, 59.73, the lead compound for further optimization 5848,5521,54.96,53.11,5291,48.58,32.04,29.62 28.04, 26.52, 25.80. 25.76, 25.49, 25.02. ESI-MS m/z Rational design and structure-activity 034M+H relationships The structural analysis and the ligand-receptor interaction elucidated by molecular docking were investigated to N-cyclohexyl-2-(4-(4-(5-fluoro-1H-indol-3-yl)butyl) guide the structure modification and optimization of com piperazin-1-yl)-N-phenylacetamide(11p) pound 11a. Compound 11a is characterized by an indole White solid (110 mg, yield 29.5%). H NMR(400 MHz, head, a linear alkyl linker and the N-phenylacetamide tail CDCl3)8 8.16(s, 1H), 7. 38(dd, J=5.0, 1.7 Hz, 3H), connected to a piperazine moiety To rationalize the design 7.24 (dd, J=8.8, 4.4 Hz, 1H), 7.19(dd, J=9.7, of the derivatives, the structural model of the complex 2.3 Hz, 1H), 7.09-707(m, 2H), 6.98(s, 1H), 6.90(td, D3R-11a was constructed by combining molecular dock =9.0, 2.4 Hz, 1H),4.57(tt, J=12.0, 3.3 Hz, 1H), ing and all available experimental data(Figure 2A).Three 2.73(s, 2H), 2.69 (t, J=7. 3 Hz, 2H), 2.50-2.38(m, important interactions were identified in the D3R-11a 10H),1.81(d, J=10.8 Hz, 2H), 1.72-1.63(m, 2H), model: the conserved salt bridge interaction between the 1.60-1.54(m, 3H), 1.42-1.35 (m, 3H), 1.01(ddd, protonated nitrogen atom(N1)of 11a and the carboxylate J=249,123,32Hz,3H),0.950.85(m,2H) group of D3. 32; the cation-Tt contact between the proton NMR (100 MHZ, CDCl3)8169352, 168.48, 158.71, ated nitrogen atom and F6.51; and the hydrogen bond 156.39, 138.23, 132.79, 130.39, 129.14, 128.37, formed by the oxygen atom of carbonyl group in 11a and 127.84,127.74,12308,116.52,114.73,111 Y7. 35 in D3R. It indicates that the piperazine ring and the 11.56, 110.22, 109.96, 103.85, 103.62, 60.17, 58.25, carbonyl group are critical to the activity, as these indis 54.12, 52.80, 31.44, 29.70, 27. 72, 26.13, 25.71, 25.32, pensable interactions determined the binding orientation of 2487.ES-MSm/z4914M+H the head down into the orthosteric binding site(OBS enclosed by TM-Ill, -V, -V, -vIn)and the tail up to the sec. ond binding pocket (SBP; comprised of ECL2 and the N-cyclohexyl-2-(4-(4-(5-fluoro-1H-indol-3-yl)butyl) extracellular segments of TM-lll, -)(18). The hollow piperazin-1-yl)-N-(2-methoxyphenyl)acetamide space was found in the OBS and SBP(Figure 2A), sug (11q) gesting that 11a could be optimized by appending larger White solid (135 mg, yield 37.4%). H NMR(400 MHz, groups in the head and tail or lengthening the linker CDCl3)87.34(td, J=8.1, 1.7 Hz, 1H), 7.24(dd, J=8.8, Therefore, a series of IBA derivatives were designed, syn 4.4 Hz, 1H), 7.18(dd, J=9.7, 2.1 Hz, 1H), 7.03(dd, thesized, and bioassyed for D3R activity with the aim to J=7.6, 1.7 HZ, 1H), 6.99-686(m, 4H), 4.51(tt, improve the potency of this series of ligands (Table 2) J=120.35Hz,H,3.77(s,3H,2.95(s,1H),2.88(s, 1H), 2.78-267(m, 4H), 2.53-2.38(m, 9H), 1.91(d, As our lead compound 11a already carries an aromatic J=11. 8 Hz, 1H), 1.79(d, J=12.3 Hz, 1H), 1.72-1.52 head and a bulky tail, the length of the linker was first con- 3H), 1.16(ddd, J=24.7, 12.2, sidered to be incremented to fill the hollow space in the 6.6 Hz, 1H), 0.95-0.85(m, 1H), 0.80(ddd, J=25. 1, 12.5, active site and 11p was obtained, providing a delightful 3.6 Hz, 1H). C-NMR(100 MHZ, CDCl3)8 169.02, 158.69, improvement in the binding affinity(;=636 nM). To verify 156.18, 132.82, 131.35, 129.72, 127. 73, 127.26, 123.12, our predicted binding mode, the effect of the length of the 120.56, 116.41, 111.68, 111.55, 110.16, 109.89, 103.81, linker (n= 2-4)on affinity was further examined. Indeed, 103.58, 59.62, 58.25, 55.21, 55.01, 52.79, 32.03, 29.62, the affinity of 11o with a 4-carbon linker is superior to 27.74. 26.14. 25.79. 25.75. 25.48. 24.88. ESI-Ms m/z those of 11m with 2-carbon and/or 11k with 3-carbon 521.4M+H linkers. As predicted, it proved that the longer linker could Chem Biol Drug Des 2013: 82: 326-335

2-(4-(4-(1H-indol-3-yl)butyl)piperazin-1-yl) -N-cyclohexyl-N-(2-methoxyphenyl) acetamide (11o) White solid (135 mg, yield 37.4%). 1 H NMR (400 MHz, CDCl3) d 8.13 (s, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.36– 7.32 (m, 2H), 7.16 (t, J = 7.1 Hz, 1H), 7.08 (t, J = 7.4 Hz, 1H), 7.04 (dd, J = 7.6, 1.6 Hz, 1H), 6.97– 6.92 (m, 3H), 4.52 (tt, J = 12.0, 3.5 Hz, 1H), 3.78 (s, 3H), 2.78–2.66 (m, 4H), 2.49–2.35 (m, 10H), 1.94–1.91 (m, 1H), 1.80 (d, J = 12.3 Hz, 1H), 1.72 (d, J = 7.4 Hz, 1H), 1.67 (d, J = 7.6 Hz, 1H), 1.61–1.53 (m, 4H), 1.44– 1.24 (m, 3H), 1.17 (ddd, J = 24.7, 12.2, 3.6 Hz, 1H), 0.96–0.88 (m, 1H), 0.81 (ddd, J = 25.1, 12.6, 3.6 Hz, 1H). 13C-NMR (100 MHz, CDCl3)d 169.10, 156.20, 136.33, 131.38, 129.69, 127.46, 127.31, 121.66, 121.26, 120.54, 118.85, 116.34, 111.53, 111.01, 59.73, 58.48, 55.21, 54.96, 53.11, 52.91, 48.58, 32.04, 29.62, 28.04, 26.52, 25.80, 25.76, 25.49, 25.02. ESI-MS m/z 503.4 [M + H]+ . N-cyclohexyl-2-(4-(4-(5-fluoro-1H-indol-3-yl)butyl) piperazin-1-yl)-N-phenylacetamide (11p) White solid (110 mg, yield 29.5%). 1 H NMR (400 MHz, CDCl3) d 8.16 (s, 1H), 7.38 (dd, J = 5.0, 1.7 Hz, 3H), 7.24 (dd, J = 8.8, 4.4 Hz, 1H), 7.19 (dd, J = 9.7, 2.3 Hz, 1H), 7.09–7.07 (m, 2H), 6.98 (s, 1H), 6.90 (td, J = 9.0, 2.4 Hz, 1H), 4.57 (tt, J = 12.0, 3.3 Hz, 1H), 2.73 (s, 2H), 2.69 (t, J = 7.3 Hz, 2H), 2.50–2.38 (m, 10H), 1.81 (d, J = 10.8 Hz, 2H), 1.72–1.63 (m, 2H), 1.60–1.54 (m, 3H), 1.42–1.35 (m, 3H), 1.01 (ddd, J = 24.9, 12.3, 3.2 Hz, 3H), 0.95–0.85 (m, 2H). 13C￾NMR (100 MHz, CDCl3) d169352, 168.48, 158.71, 156.39, 138.23, 132.79, 130.39, 129.14, 128.37, 127.84, 127.74, 123.08, 116.52, 114.73, 111.65, 111.56, 110.22, 109.96, 103.85, 103.62, 60.17, 58.25, 54.12, 52.80, 31.44, 29.70, 27.72, 26.13, 25.71, 25.32, 24.87. ESI-MS m/z 491.4 [M + H]+ . N-cyclohexyl-2-(4-(4-(5-fluoro-1H-indol-3-yl)butyl) piperazin-1-yl)-N-(2-methoxyphenyl)acetamide (11q) White solid (135 mg, yield 37.4%). 1 H NMR (400 MHz, CDCl3) d7.34 (td, J = 8.1, 1.7 Hz, 1H), 7.24 (dd, J = 8.8, 4.4 Hz, 1H), 7.18 (dd, J = 9.7, 2.1 Hz, 1H), 7.03 (dd, J = 7.6, 1.7 Hz, 1H), 6.99–6.86 (m, 4H), 4.51 (tt, J = 12.0, 3.5 Hz, 1H), 3.77 (s, 3H), 2.95 (s, 1H), 2.88 (s, 1H), 2.78–2.67 (m, 4H), 2.53–2.38 (m, 9H), 1.91 (d, J = 11.8 Hz, 1H), 1.79 (d, J = 12.3 Hz, 1H), 1.72–1.52 (m, 5H), 1.44–1.22 (m, 3H), 1.16 (ddd, J = 24.7, 12.2, 3.6 Hz, 1H), 0.95–0.85 (m, 1H), 0.80 (ddd, J = 25.1, 12.5, 3.6 Hz, 1H).13C-NMR (100 MHz, CDCl3)d 169.02, 158.69, 156.18, 132.82, 131.35, 129.72, 127.73, 127.26, 123.12, 120.56, 116.41, 111.68, 111.55, 110.16, 109.89, 103.81, 103.58, 59.62, 58.25, 55.21, 55.01, 52.79, 32.03, 29.62, 27.74, 26.14, 25.79, 25.75, 25.48, 24.88. ESI-MS m/z 521.4 [M + H]+ . Results and Discussion Pharmacophore-based virtual screening The obtained pharmacophore model was shown in Figure 1A. As a result of our virtual screening protocol, 16 compounds were selected to purchase and submitted to pharmacological experiments (Tables S1 and S2). To our delight, three of them revealed moderate D3R activities. Their chemical structures and corresponding binding assays were summarized in Figure 1B and Table 1. Com￾pound 11a, with a high fit value and a core structure of in￾dolepropylamine and N-phenylacetamide, matches the pharmacophore model quite well (Figure S1) and repre￾sents a novel class of D3R ligands. It was identified to bind hD3R with 2161 nM affinity and was thus chosen as the lead compound for further optimization. Rational design and structure-activity relationships The structural analysis and the ligand–receptor interaction elucidated by molecular docking were investigated to guide the structure modification and optimization of com￾pound 11a. Compound 11a is characterized by an indole head, a linear alkyl linker and the N-phenylacetamide tail connected to a piperazine moiety. To rationalize the design of the derivatives, the structural model of the complex D3R-11a was constructed by combining molecular dock￾ing and all available experimental data (Figure 2A). Three important interactions were identified in the D3R-11a model: the conserved salt bridge interaction between the protonated nitrogen atom (N1) of 11a and the carboxylate group of D3.32; the cation-p contact between the proton￾ated nitrogen atom and F6.51; and the hydrogen bond formed by the oxygen atom of carbonyl group in 11a and Y7.35 in D3R. It indicates that the piperazine ring and the carbonyl group are critical to the activity, as these indis￾pensable interactions determined the binding orientation of the head down into the orthosteric binding site (OBS; enclosed by TM-III, -V, -VI, -VII) and the tail up to the sec￾ond binding pocket (SBP; comprised of ECL2 and the extracellular segments of TM-III, -VII) (18). The hollow space was found in the OBS and SBP (Figure 2A), sug￾gesting that 11a could be optimized by appending larger groups in the head and tail or lengthening the linker. Therefore, a series of IBA derivatives were designed, syn￾thesized, and bioassyed for D3R activity with the aim to improve the potency of this series of ligands (Table 2). As our lead compound 11a already carries an aromatic head and a bulky tail, the length of the linker was first con￾sidered to be incremented to fill the hollow space in the active site and 11p was obtained, providing a delightful improvement in the binding affinity (Ki = 636 nM). To verify our predicted binding mode, the effect of the length of the linker (n = 2–4) on affinity was further examined. Indeed, the affinity of 11o with a 4-carbon linker is superior to those of 11m with 2-carbon and/or 11k with 3-carbon linkers. As predicted, it proved that the longer linker could 330 Chem Biol Drug Des 2013; 82: 326–335 Du et al

Synthesis, Biological Evaluation, and Molecular Modeling Figure 1: (A Top view of the pharmacophore model superimposed into the active pocket of the D3R structure. Two hydrophobic elements(Hydro-1 and Hydro-2) were shown in cyan spheres, the positive ionizable element POS in red, and a hydrogen-bond-acceptor element(HBA) in green. Three exclusion volumes(ExcI-1, -2, -3) are shown in black spheres, corresponding to the positions of conserved residues D3. 32, F6.51, and Y7. 35(B) Chemical structures of three active compounds were obtained from virtual screening Table 2: Binding affinities of compounds 11a-11q for D1R, D2R, and D3R (K or percentage displacement of radioligand at 10 uM) Linker R1 R Head inhibition(%)orK(±SEM, 11a 3431% 31.71% 2161±25 1808% 52.99% 11 949% 5721% 11d 1.48% 21.84% 328% 2-OMe 43.28% 37.84% 12 ±115 ±11 119 Hm33333333333224444 2-OMe ccccc 1804% 42.93% 950±81 4204% 20.13% 136±9 11 cBBHHHHHHFF 2-OMe 2457% 1513±121 3.23% 2-OMe Cyclohex 1497±8 11l Cyclohex 15.379 11m 2-OMe Cyclohex 65.49% 11n Cyclohexyl 8740% 11o 2-OM 25 240±2 11p 18.76% 35±5 2-OMe 3501% 23±3 1.69±0.12 1.08±0.09 048±0.07 aThe activity was not determined. All values are means of three separate experiments. The values of K, were given only when the inhibition rate of compound is >90% allow the molecule to extend to the hollow space in the and SBP by introducing proper functional groups to the OBS and SBP, thus the affinity was increased. In addition, molecules could improve the affinity to D3R the affinity of 11a with larger cyclohexyl group on the tai was higher than those of 11b, 11c, and 11d with smaller Compounds 11f, 11h, and 11g were next prepared to groups. It further confirms that the tight binding in OBs evaluate the influence of different halogens at 5-position of Chem Bio/ Drug Des 2013: 82: 326-335

allow the molecule to extend to the hollow space in the OBS and SBP, thus the affinity was increased. In addition, the affinity of 11a with larger cyclohexyl group on the tail was higher than those of 11b, 11c, and 11d with smaller groups. It further confirms that the tight binding in OBS and SBP by introducing proper functional groups to the molecules could improve the affinity to D3R. Compounds 11f, 11h, and 11g were next prepared to evaluate the influence of different halogens at 5-position of A B Figure 1: (A) Top view of the pharmacophore model superimposed into the active pocket of the D3R structure. Two hydrophobic elements (Hydro-1 and Hydro-2) were shown in cyan spheres, the positive ionizable element POS in red, and a hydrogen-bond-acceptor element (HBA) in green. Three exclusion volumes (Excl-1, -2, -3) are shown in black spheres, corresponding to the positions of conserved residues D3.32, F6.51, and Y7.35. (B) Chemical structures of three active compounds were obtained from virtual screening. Table 2: Binding affinities of compounds 11a–11q for D1R, D2R, and D3R (Ki or percentage displacement of radioligand at 10 lM) N R3 O N N N H R1 n Head Linker Tail R2 Compound n R1 R2 R3 Inhibition (%) or Ki (SEM, nM) D1 D2 D3 11a 3 F H Cyclohexane 34.31% 31.71% 2161 25 11b 3 F H H 18.08% 9.13% 52.99% 11c 3 F H Methyl 1.63% 9.49% 57.21% 11d 3 F H Isopropyl 1.48% 21.84% 73.28% 11e 3 F 2-OMe Cyclohexyl
2.41% 43.28% 1839 115 11f 3 Cl H Cyclohexyl 37.84% 54.25% 292 11 11g 3 Cl 2-OMe Cyclohexyl 18.04% 42.93% 950 81 11h 3 Br H Cyclohexyl 42.04% 20.13% 2136 9 11i 3 Br 2-OMe Cyclohexyl 19.29% 24.57% 1513 121 11j 3 H H Cyclohexyl 26.57% 34.68% 73.23% 11k 3 H 2-OMe Cyclohexyl 28.32% 58.69% 1497 8 11l 2 H H Cyclohexyl
3.03% 25.23% 15.37% 11m 2 H 2-OMe Cyclohexyl 21.23% 25.59% 65.49% 11n 4 H H Cyclohexyl 62.58% 17.98% 87.40% 11o 4 H 2-OMe Cyclohexyl 12.36% 35.57% 240 2 11p 4 F H Cyclohexyl 3.58% 18.76% 635 5 11q 4 F 2-OMe Cyclohexyl 8.65% 35.01% 123 3 SCH23390 1.69 0.12 –a – Spiperone – 1.08 0.09 0.48 0.07 a The activity was not determined. All values are means of three separate experiments. The values of Ki were given only when the inhibition rate of compound is >90%. Chem Biol Drug Des 2013; 82: 326–335 331 Synthesis, Biological Evaluation, and Molecular Modeling

Du et al Figure 2: Compound 11a(A) and 11q(B)bellow sticks) docked into the binding pocket of D3R. that all halogenated derivatives displayed improved binding B 93: (35S]GTP, S binding assays of compound 11q for the the indole head on binding affinity. The results indicated affinity, and the chlorinated compound performs better than the fluorinated or brominated one(11f> 11a>11h Compound EC ICso(nM) when the length of linker is 3 (n= 3). In addition, when 11q 827.63 n= 4, the binding affinity of 11p with a 5-fluorine on indole Haloperidol 248.02 is superior to 1in with a hydrogen, demonstrating the Quinpirole 25.673 important role played by halogens in D3R ligand design a SGTP S binding activity could not be detected /e noted that the o-methoxy group in the phenyl ring of the tail plays a positive role in the binding of D3R(19, 20). activity toward D3R. The D3R-11q model shows that the Therefore, a set of o-methoxy substituted derivatives interactions formed by the protonated nitrogen of pipera (11e,g, -i, -k,-m, -o, -q) of compounds 11a, -f, -h,j, -1, zine and the negatively charged d3. 32, and the amide -n,-p were designed and synthesized. Generally, introduc- moiety with Y7. 35 (Figure 2B)anchor the binding orienta tion of the o-methoxy group positively contributed to the tion of 11q. It makes the indole head down to the hydro- ffinity to D3R. Close examination indicated that the o- phobic cavity in OBS, in contact with hydrophobic methoxy substituent in the phenyl ring played an optimal residues V3. 33, V5. 39, W.48, F6.51, F6.52, H6.55 ole when a 4-carbon linker was present. Among these(Figure 3B). In the meantime, the N-phenylacetamide tail molecules, 11q emerged as the most potent ligand, dis- extends to the SBP and participates in the hydrophobic playing fourfold increase in binding affinity (K;=124 nm) interactions with 1183 in ECL2 and V2.61, L2. 64, F3. 28, compared with 11p. pSSGTP,S binding assay of com- V3.29, Y7. 43(Figure 3B). Our predicted binding mode is pound 11q showed that it produced antagonistic activity consistent with the site-directed mutagenesis studies, at D3R, and the calculated antagonistic potencies(Cso) of which indicated that the mutation of F6.51 and V2.61 11q were 828 nM for the D3R (Table 3) caused significant reduction in binding affinities of D3R ligands(22, 23 ). Thus, the hydrophobic interactions in the Molecular docking study was promptly conducted to OBS and SBP, and the hydrogen-bond interactions investigate the binding mode of 11q with D3R to reveal formed with the key residues D3. 32 and Y7. 35 mostly the role played by the o-methoxy group and the structural contribute to the activity to D3R features responsible for the increased affinity(Figure 2B) Consistent with the binding mode of D3R with the lead It is worth noting that the designed series of compound 11a( without the o-methoxy group), the salt bridge formed have high selectivity to D3R. As D2R and D3R share high with D3. 32, the cation- interaction with F6.51, and the sequence identity in the active site, we sought to identify hydrogen bond with Y7. 35 were maintained. When n the structural determinants for such a high selectivity over 11q extends to the hollow space of the oBs and SBP, the other dopamine receptor subtypes. Sequence align which makes the o-methoxy group involved in the hydro. ment of the key residues involved in the binding of 11q gen-bond interaction with Y7.43 and enables the indole was conducted within D3R, D2R, and D1R(Figure 3A). As head to forms I-i interaction with F6. 52 in a parallel dis- D1R and D3R share less sequence identity, the non-con- placed manner. The interactions illustrated by our model served residues at positions 7.35, 7.43, and 6.55 in D1R are consistent with most of the known mutagenesis stud- make collision with 11q, hence, it is not difficult to under- ies(21-23 )and well account for the higher affinity of 11q. stand that this series of compounds display no activity for hdi. Whereas in the case of d2r and d3r which have Taking the SAR analyses together, we looked deep into almost identical residues in the binding pocket, what con the D3R-11q model to find the intrinsic factor that gives tributes to their selectivity? Only three of 22 residues are his novel series of compounds a unique pharmacological not conserved in the 11q-contact binding region of D3R Chem Biol Drug Des 2013: 82: 326-335

the indole head on binding affinity. The results indicated that all halogenated derivatives displayed improved binding affinity, and the chlorinated compound performs better than the fluorinated or brominated one (11f > 11a > 11h) when the length of linker is 3 (n = 3). In addition, when n = 4, the binding affinity of 11p with a 5-fluorine on indole is superior to 11n with a hydrogen, demonstrating the important role played by halogens in D3R ligand design. We noted that the o-methoxy group in the phenyl ring of the tail plays a positive role in the binding of D3R (19,20). Therefore, a set of o-methoxy substituted derivatives (11e, -g, -i, -k, -m, -o, -q) of compounds 11a, -f, -h, -j, -l, -n, -p were designed and synthesized. Generally, introduc￾tion of the o-methoxy group positively contributed to the affinity to D3R. Close examination indicated that the o￾methoxy substituent in the phenyl ring played an optimal role when a 4-carbon linker was present. Among these molecules, 11q emerged as the most potent ligand, dis￾playing fourfold increase in binding affinity (Ki = 124 nm) compared with 11p. [35S]GTPcS binding assay of com￾pound 11q showed that it produced antagonistic activity at D3R, and the calculated antagonistic potencies (IC50) of 11q were 828 nM for the D3R (Table 3). Molecular docking study was promptly conducted to investigate the binding mode of 11q with D3R to reveal the role played by the o-methoxy group and the structural features responsible for the increased affinity (Figure 2B). Consistent with the binding mode of D3R with the lead 11a (without the o-methoxy group), the salt bridge formed with D3.32, the cation-p interaction with F6.51, and the hydrogen bond with Y7.35 were maintained. When n = 4, 11q extends to the hollow space of the OBS and SBP, which makes the o-methoxy group involved in the hydro￾gen-bond interaction with Y7.43 and enables the indole head to forms p–p interaction with F6.52 in a parallel dis￾placed manner. The interactions illustrated by our model are consistent with most of the known mutagenesis stud￾ies (21–23) and well account for the higher affinity of 11q. Taking the SAR analyses together, we looked deep into the D3R-11q model to find the intrinsic factor that gives this novel series of compounds a unique pharmacological activity toward D3R. The D3R-11q model shows that the interactions formed by the protonated nitrogen of pipera￾zine and the negatively charged D3.32, and the amide moiety with Y7.35 (Figure 2B) anchor the binding orienta￾tion of 11q. It makes the indole head down to the hydro￾phobic cavity in OBS, in contact with hydrophobic residues V3.33, V5.39, W6.48, F6.51, F6.52, H6.55 (Figure 3B). In the meantime, the N-phenylacetamide tail extends to the SBP and participates in the hydrophobic interactions with I183 in ECL2 and V2.61, L2.64, F3.28, V3.29, Y7.43 (Figure 3B). Our predicted binding mode is consistent with the site-directed mutagenesis studies, which indicated that the mutation of F6.51 and V2.61 caused significant reduction in binding affinities of D3R ligands (22,23). Thus, the hydrophobic interactions in the OBS and SBP, and the hydrogen-bond interactions formed with the key residues D3.32 and Y7.35 mostly contribute to the activity to D3R. It is worth noting that the designed series of compounds have high selectivity to D3R. As D2R and D3R share high sequence identity in the active site, we sought to identify the structural determinants for such a high selectivity over the other dopamine receptor subtypes. Sequence align￾ment of the key residues involved in the binding of 11q was conducted within D3R, D2R, and D1R (Figure 3A). As D1R and D3R share less sequence identity, the non-con￾served residues at positions 7.35, 7.43, and 6.55 in D1R make collision with 11q, hence, it is not difficult to under￾stand that this series of compounds display no activity for hD1R. Whereas in the case of D2R and D3R, which have almost identical residues in the binding pocket, what con￾tributes to their selectivity? Only three of 22 residues are not conserved in the 11q-contact binding region of D3R/ A B Figure 2: Compound 11a (A) and 11q (B) (yellow sticks) docked into the binding pocket of D3R. Table 3: [ 35S]GTPcS binding assays of compound 11q for the D3R Compound EC50 (nM) IC50 (nM) 11q –a 827.63 Haloperidol – 248.02 Quinpirole 25.673 – a [ 35S]GTPcS binding activity could not be detected. 332 Chem Biol Drug Des 2013; 82: 326–335 Du et al

Synthesis, Biological Evaluation, and Molecular Modeling selectivity of this series of D3R ligands over D2R. Heinrich et al., reported a series of indolebutylamines(BAs) and D3R VVL F VDVCCSISVSWFFHVYTY phenylpiperazine derivatives showing high activities against 5-HT1AR and D2R (24, 25). Our lead compound 11a D2R VVL F VDVCCILAVSWFFHIYT obtained from virtual screening bears N-phenylacetamide DIR VKAWVD IS CD SS ASWFF NC F W tail, and it displays different pharmacological profile from phenylpiperazine derivatives and shows high selectivity toward D3R instead. The introduction of amide group causes the lost of D2R activity F3.28 Conclusions H6.55 V2 In summary, a new type of compounds with the core high selective D3R ligands over D1R and D2R by combin- ng a series of computational approaches, the synthetic W6,4s 743V2 chemistry, and binding assays. Compound 11q displays relatively high binding affinity (Ki= 124 nM), and it was evaluated as D3R antagonist by SGTP, S binding assay The results indicated that a fluorine substituted indole head, a 4-carbon linker, an o-methoxy of the phenyl ring and a cyclohexyl substituent on the amide mainly contrib- Figure 3: (A The sequence alignment in the binding pocket of uted to the affinity to d3R. In-depth analysis of the binding D1R, D2R, and D3R;(B)Predicted binding mode of compound mode of 11q with D3R constructed by molecular docking 11q (yellow sticks) with D3R(blue cartoon), and D2R (gray not only elucidated SARs in detail, but also recognized the cartoon) is superimposed to D3R molecular determinants critical for D3R affinity as well as selectivity. The carboxylate group of D3.32 formed con- D2R(Figure 3B), namely V6.561, S184A, and S1821. The served salt bridge interaction with the protonated nitrogen side chains of both v6.56l and S184A stretch outward atom(N1) of the piperazine ring, which is a common fea from the binding pocket, indicating that the two sites may ture among D3R ligands. Y7. 43 is engaged in the hydro contribute little to the selectivity. S182, however, is facing gen-bond interaction with o-methoxy of the phenyl ring the binding pocket and might be the key factor for the F6.52 forms T-n-stacking interaction with indole ring. And electivity. We noted that 1182 in D2R acts as a cap(Fig- together with D3. 32, Y7.35, and F6.51, they were recog ure S2a)and plugs up the entrance of ligand. It probably nized as key residues for binding of the designed com prevents this series of compounds from entering the active pounds. $182 in ECL2 is involved in the ligand's entrance tes, and it may also causes collision with designed com- to D3R and was found to be the molecular structural pounds in the active site of D2R. In contrast, the counter- determinant for the selectivity of D3R over D2R. As these part of 1182 in D2R is S182 in D3R(Figure S2b), the three residues have not obtained much attention in the small-size S182 allows the smooth entrance of designed previously reported studies on selective D3R ligands, this compounds and forms favorable hydrophobic interaction study may give a hint in the design of novel selective D3R with them. Hence, we conclude that S182 in ecl2 in ligands. Further optimization of this series of compounds D3R might be the key residue that determines the high IS ongoing OH NH cde Scheme 1: Syntheses of important intermediates 3a-d and 6b. Reagents and conditions: (a) dihydropyran, DMA/%H2SO4;( b) CBra PPh3, DCM; (c)TMSCN, TBAF, THF;(d) NaoH/EtOH; (e)LiAlHa, THF; (f) CBra, PPh3 Chem Bio/ Drug Des 2013: 82: 326-335

D2R (Figure 3B), namely V6.56I, S184A, and S182I. The side chains of both V6.56I and S184A stretch outward from the binding pocket, indicating that the two sites may contribute little to the selectivity. S182I, however, is facing the binding pocket and might be the key factor for the selectivity. We noted that I182 in D2R acts as a cap (Fig￾ure S2a) and plugs up the entrance of ligand. It probably prevents this series of compounds from entering the active sites, and it may also causes collision with designed com￾pounds in the active site of D2R. In contrast, the counter￾part of I182 in D2R is S182 in D3R (Figure S2b), the small-size S182 allows the smooth entrance of designed compounds and forms favorable hydrophobic interaction with them. Hence, we conclude that S182 in ECL2 in D3R might be the key residue that determines the high selectivity of this series of D3R ligands over D2R. Heinrich et al., reported a series of indolebutylamines (IBAs) and phenylpiperazine derivatives showing high activities against 5-HT1AR and D2R (24,25). Our lead compound 11a obtained from virtual screening bears N-phenylacetamide tail, and it displays different pharmacological profile from phenylpiperazine derivatives and shows high selectivity toward D3R instead. The introduction of amide group causes the lost of D2R activity. Conclusions In summary, a new type of compounds with the core structure of IBA and N-phenylacetamide were identified as high selective D3R ligands over D1R and D2R by combin￾ing a series of computational approaches, the synthetic chemistry, and binding assays. Compound 11q displays relatively high binding affinity (Ki = 124 nM), and it was evaluated as D3R antagonist by [35S]GTPcS binding assay. The results indicated that a fluorine substituted indole head, a 4-carbon linker, an o-methoxy of the phenyl ring, and a cyclohexyl substituent on the amide mainly contrib￾uted to the affinity to D3R. In-depth analysis of the binding mode of 11q with D3R constructed by molecular docking not only elucidated SARs in detail, but also recognized the molecular determinants critical for D3R affinity as well as selectivity. The carboxylate group of D3.32 formed con￾served salt bridge interaction with the protonated nitrogen atom (N1) of the piperazine ring, which is a common fea￾ture among D3R ligands. Y7.43 is engaged in the hydro￾gen-bond interaction with o-methoxy of the phenyl ring. F6.52 forms p–p-stacking interaction with indole ring. And together with D3.32, Y7.35, and F6.51, they were recog￾nized as key residues for binding of the designed com￾pounds. S182 in ECL2 is involved in the ligand’s entrance to D3R and was found to be the molecular structural determinant for the selectivity of D3R over D2R. As these three residues have not obtained much attention in the previously reported studies on selective D3R ligands, this study may give a hint in the design of novel selective D3R ligands. Further optimization of this series of compounds is ongoing. A B Figure 3: (A) The sequence alignment in the binding pocket of D1R, D2R, and D3R; (B) Predicted binding mode of compound 11q (yellow sticks) with D3R (blue cartoon), and D2R (gray cartoon) is superimposed to D3R. R1 NH NH2 N H R1 OH N H R1 Br a b N H F Br N H F OH N H F Br c,d,e f 3a: R1=H 3b: R1=F 3c: R1=Cl 3d: R1=Br 1 2 3b 5b 6b Scheme 1: Syntheses of important intermediates 3a–d and 6b. Reagents and conditions: (a) dihydropyran, DMA/4% H2SO4; (b) CBr4, PPh3, DCM; (c) TMSCN, TBAF, THF; (d) NaOH/EtOH; (e) LiAlH4, THF; (f) CBr4, PPh3. Chem Biol Drug Des 2013; 82: 326–335 333 Synthesis, Biological Evaluation, and Molecular Modeling

Du et al R2 R2 11a-11 Scheme 2: Syntheses of compounds 11a-q. Reagents and conditions: (a) chloroacetylchloride, TEA, DCM;(b)Boc-piperazine, K2CO3, CH3CN; (c) TFADCM;(d) 3a-3d, 6b, K2CO3, CH3CN Acknowledgment 7. Banala A K, Levy B.A. Khatri S.S., Furman C A, Roof R.A., Mishra Y, Griffin S.A., Sibley D R, Luedtke R.R This work was supported by the National Natural Science Newman A H.(2011)N-(3-fluoro-4-(4-(2-methoxy or Foundation of China (No. 81172919, 81130023 2, 3-dichlorophenyl)piperazine-1-yl)butyl)arylcarboxamid 0825042)and grants from the National High Technology es as selective dopamine D3 receptor ligands: critical Research and Development Program of China(863 Pro- ole of the carboxamide linker for D3 receptor selectiv gram)(No. 2012AA020301), the State Key Program of ity. J Med Chem; 54: 3581-3594 Basic Research of China grant (2009CB918502, 8. Micheli F, Arista L, Bertani B, Braggio S, Capell 2010cB912601,2009CB522000,and2011CB5C4403) A M, Cremonesi S, Di-Fabio R. et al. (2010) Explora and National Drug Innovative Program(No. 2009ZX0930 tion of the amine terminus in a novel series of 1.2.4- 011). Support from Priority Academic Program Develop triazolo-3-yl-azabicyclo(3. 1.0 hexanes as selective ment of Jiangsu Higher Education Institutes(PAPD)is also d D3 appreciated. Chem:53:7129-7139 9. Micheli F, Arista L, Bonanomi G, Blaney FE, Braggio S, Capelli A M., Checchia A. et al.(2010)1, 2, 4-Triaz References olyl azabicyclo(3.1.0]hexanes: a new series of potent and selective dopamine D(3)receptor antagonists. J 1. Sokoloff P. Giros B. Martres M.P. Bouthenet m.l. Med chem;:53:374391 Schwartz J.C. (1990)Molecular cloning and character- 10. Li B, Li W, Du P, Yu K.Q., Fu W.(2012)Molecular ization of a novel dopamine receptor(D3)as a target Insights into the D1R Agonist and D2R/D3R Antagonist for neuroleptics. Nature 347: 146-151 Effects of the Natural Product (-)-Stepholidine: molec. 2. Richtand N M, Woods S.C., Berger S.P., Strakowski ular Modeling and Dynamics Simulations. J Phys S M.(2001)D3 dopamine receptor, behavioral sensitize Chem B:116:81218130 tion, and psychosis. Neurosci Biobehav Rev: 25: 427-443. 11. Chien E.Y., Liu W, Zhao Q, Katritch V, Han GW 3. Joyce J.N., Millan M.J.(2005)Dopamine D3 receptor Hanson mA. shi L. Newman A.h., Javitch JA antagonists as therapeutic agents. Drug Discov Cherezov V, Stevens R C.(2010) Structure of the Today;:10:917-925 human dopamine d3 receptor in complex with a D2 4. Heidbreder C.(2008) Selective antagonism at dopa- D3 selective antagonist. Science; 330: 1091-1095 mine D3 receptors as a target for drug addiction phar- 12. Bettinetti L, Schlotter K, Hubner H, Gmeiner P herapy: a review of preclinical evidence. CNS (2002) Interactive SAR studies: rational discovery of Neurol Disord Drug Targets; 7: 410-421 super-potent and highly selective dopamine D3 recep 5. Micheli F, Heidbreder C(2013)Dopamine D3 receptor tor antagonists and partial agonists. J Med antagonists: a patent review(2007-2012). Expert Opin Chem;45:45944597 Ther Pat;23:363-381 13. Robarge M.J., Husbands S.M., Kieltyka A, Brodbeck 6. Chen J, Collins G.T., Zhang J, Yang C.Y., Levant B R, Thurkauf A, Newman A H.(2001)Design and syn Woods J, Wang S(2008)Design, synthesis, and eval- thesis of [(2, 3-dichlorophenyl)piperazin-1-ylJalkylifluore- uation of potent and selective ligands for the dopamine nylcarboxamides as novel ligands selective for the 3(D3)receptor with a novel in vivo behavioral profile dopamine D3 receptor subtype. J Med Med chem:51:5905-5908 Chem;44:31753186 Chem Biol Drug Des 2013: 82: 326-335

Acknowledgment This work was supported by the National Natural Science Foundation of China (No. 81172919, 81130023, 30825042) and grants from the National High Technology Research and Development Program of China (863 Pro￾gram) (No. 2012AA020301), the State Key Program of Basic Research of China grant (2009CB918502, 2010CB912601, 2009CB522000, and 2011CB5C4403), and National Drug Innovative Program (No. 2009ZX09301- 011). Support from Priority Academic Program Develop￾ment of Jiangsu Higher Education Institutes (PAPD) is also appreciated. References 1. Sokoloff P., Giros B., Martres M.P., Bouthenet M.L., Schwartz J.C. (1990) Molecular cloning and character￾ization of a novel dopamine receptor (D3) as a target for neuroleptics. Nature;347:146–151. 2. Richtand N.M., Woods S.C., Berger S.P., Strakowski S.M. (2001) D3 dopamine receptor, behavioral sensitiza￾tion, and psychosis. Neurosci Biobehav Rev;25:427–443. 3. Joyce J.N., Millan M.J. (2005) Dopamine D3 receptor antagonists as therapeutic agents. Drug Discov Today;10:917–925. 4. Heidbreder C. (2008) Selective antagonism at dopa￾mine D3 receptors as a target for drug addiction phar￾macotherapy: a review of preclinical evidence. CNS Neurol Disord Drug Targets;7:410–421. 5. Micheli F., Heidbreder C. (2013) Dopamine D3 receptor antagonists: a patent review (2007–2012). Expert Opin Ther Pat;23:363–381. 6. Chen J., Collins G.T., Zhang J., Yang C.Y., Levant B., Woods J., Wang S. (2008) Design, synthesis, and eval￾uation of potent and selective ligands for the dopamine 3 (D3) receptor with a novel in vivo behavioral profile. J Med Chem;51:5905–5908. 7. Banala A.K., Levy B.A., Khatri S.S., Furman C.A., Roof R.A., Mishra Y., Griffin S.A., Sibley D.R., Luedtke R.R., Newman A.H. (2011) N-(3-fluoro-4-(4-(2-methoxy or 2,3-dichlorophenyl)piperazine-1-yl)butyl)arylcarboxamid￾es as selective dopamine D3 receptor ligands: critical role of the carboxamide linker for D3 receptor selectiv￾ity. J Med Chem;54:3581–3594. 8. Micheli F., Arista L., Bertani B., Braggio S., Capelli A.M., Cremonesi S., Di-Fabio R. et al. (2010) Explora￾tion of the amine terminus in a novel series of 1,2,4- triazolo-3-yl-azabicyclo[3.1.0]hexanes as selective dopamine D3 receptor antagonists. J Med Chem;53:7129–7139. 9. Micheli F., Arista L., Bonanomi G., Blaney F.E., Braggio S., Capelli A.M., Checchia A. et al. (2010) 1,2,4-Triaz￾olyl azabicyclo[3.1.0]hexanes: a new series of potent and selective dopamine D(3) receptor antagonists. J Med Chem;53:374–391. 10. Li B., Li W., Du P., Yu K.Q., Fu W. (2012) Molecular Insights into the D1R Agonist and D2R/D3R Antagonist Effects of the Natural Product (
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Synthesis, Biological Evaluation, and Molecular Modeling 14. Goodford P J.(1985)A computational procedure for 23 Dorfler M, Tschammer N, Hamperl K, Hubner H, determining energetically favorable binding sites on Gmeiner P.(2008)Novel D3 selective dopaminergic biologically important macromolecules. J Med incorporating enyne units as nonaromatic catechol bio Chem;28:849857 isosteres: synthesis, bioactivity, and mutagenesis stud- 15. Leach C.(1988)GRAN: a computer program for the ies. J Med Chem: 51: 6829-6838 cluster analysis of a repertory grid. Br J Clin Psy- 24. Heinrich T, Bottcher H, Bartoszyk GD ho:27(Pt2):173-174 H E, Seyfried C.A., Van Amsterdam C. co可 16. Jones G, Willett P, Glen R.C., Leach A.R., Taylor R. Indolebutylamines as selective 5-HT(1A agonists (1997)Development and validation of a genetic algo- J Med chem;47:4677-4683 rithm for flexible docking. J Mol Biol: 267: 727-748 25. Heinrich T, Bottcher H, Gericke R, Bartoszyk G D 17. Campos K.R., Woo J.C., Lee S, Tillyer RD.(2004)A Anzali S, Seyfried C A, Greiner H E, Van Amsterdam general synthesis of substituted indoles from cyclic C.(2004)Synthesis and structure-activity relationship enol ethers and enol lactones. Org Lett; 6: 79-82 in a class of indolebutylpiperazines as dual 5-HT(1A) 18. Newman A H, Beuming T, Banala A K, Donthamsetti receptor agonists and serotonin reuptake inhibitors P, Pongetti K, Labounty A, Levy B, Cao J, Michino J Med che;47:46844692. M, Luedtke R. R, Javitch J.A., Shi L(2012) Molecular determinants of selectivity and efficacy at the dopa- mine d3 receptor. J Med Chem; 55: 6689-6699 Note 19 Grundt P, Prevatt K M, Cao J, Taylor M, Floresca CZ. Choi J.K.. Jenkins B G, Luedtke R.R., Newman Inc, A.S.; Release 3.5 ed. San Diego: Accelrys Software A H.(2007) Heterocyclic analogues of N-(4-(4-(2, 3-di nc.2012. chlorophenyl)piperazin-1-yi)butyl)arylcarboxamides with functionalized linking chains as novel dopamine D3 receptor ligands: potential substance abuse therapeu- Supporting Information tic agents. J Med Chem: 50: 4135-4146 20. Newman A H, Grundt P, Cyriac G, Deschamps J. R aylor M, Kumar R, Ho D, Luedtke R.R(2009) N-(4- Additional Supporting Information may be found in the (4-(2, 3-dichloro. or 2-methoxyphenyl)piperazin-1-yo) online version of this article butyl) heterobiarylcarboxamides with functionalized link ing chains as high affinity and enantioselective D3 Figure $1. Compound 11a fitted to the four-feature phar eceptor antagonists. J Med Chem; 52: 2559-2570 21. Ehrich K, Gotz A, Bollinger S, Tschammer N, Bettinetti L, Harterich S, Hubner H, Laniq H, Gmeiner P(2009) Figure $2. Key residues in the binding pocket of D3R (a) Dopamine D2, D3, and D4 selective phenylpiperazines and D2R (b)were shown in spheres as molecular probes to explore the origins of subtype specific receptor binding. J Med Chem; 52: 49234935 Table S1. Summary of the molecular properties, fit values 22 Tschammer N, Elsner J. Goetz A. Ehrlich K, Schus- and gold scores of the selected 16 compounds ter S, Ruberg M, Kuhhorn J, Thompson D, Whistler J, Hubner H, Gmeiner P(2011)Highly potent 5-am- Table S2. Binding affinities of the selected 16 compounds inotetrahydropyrazolopyridines: enantioselective dopa- for dopamine D1, D2, and D3 receptors mine D3 receptor binding, functional selectivity, and analysis of receptor-ligand interactions. J Med Chem:54:2477-2491 Chem Bio/ Drug Des 2013: 82: 326-335

14. Goodford P.J. (1985) A computational procedure for determining energetically favorable binding sites on biologically important macromolecules. J Med Chem;28:849–857. 15. Leach C. (1988) GRAN: a computer program for the cluster analysis of a repertory grid. Br J Clin Psy￾chol;27(Pt 2):173–174. 16. Jones G., Willett P., Glen R.C., Leach A.R., Taylor R. (1997) Development and validation of a genetic algo￾rithm for flexible docking. J Mol Biol;267:727–748. 17. Campos K.R., Woo J.C., Lee S., Tillyer R.D. (2004) A general synthesis of substituted indoles from cyclic enol ethers and enol lactones. Org Lett;6:79–82. 18. Newman A.H., Beuming T., Banala A.K., Donthamsetti P., Pongetti K., Labounty A., Levy B., Cao J., Michino M., Luedtke R.R., Javitch J.A., Shi L. (2012) Molecular determinants of selectivity and efficacy at the dopa￾mine d3 receptor. J Med Chem;55:6689–6699. 19. Grundt P., Prevatt K.M., Cao J., Taylor M., Floresca C.Z., Choi J.K., Jenkins B.G., Luedtke R.R., Newman A.H. (2007) Heterocyclic analogues of N-(4-(4-(2,3-di￾chlorophenyl)piperazin-1-yl)butyl)arylcarboxamides with functionalized linking chains as novel dopamine D3 receptor ligands: potential substance abuse therapeu￾tic agents. J Med Chem;50:4135–4146. 20. Newman A.H., Grundt P., Cyriac G., Deschamps J.R., Taylor M., Kumar R., Ho D., Luedtke R.R. (2009) N-(4- (4-(2,3-dichloro- or 2-methoxyphenyl)piperazin-1-yl) butyl)heterobiarylcarboxamides with functionalized link￾ing chains as high affinity and enantioselective D3 receptor antagonists. J Med Chem;52:2559–2570. 21. Ehrlich K., Gotz A., Bollinger S., Tschammer N., Bettinetti L., Harterich S., Hubner H., Laniq H., Gmeiner P. (2009) Dopamine D2, D3, and D4 selective phenylpiperazines as molecular probes to explore the origins of subtype specific receptor binding. J Med Chem;52:4923–4935. 22. Tschammer N., Elsner J., Goetz A., Ehrlich K., Schus￾ter S., Ruberg M., Kuhhorn J., Thompson D., Whistler J., Hubner H., Gmeiner P. (2011) Highly potent 5-am￾inotetrahydropyrazolopyridines: enantioselective dopa￾mine D3 receptor binding, functional selectivity, and analysis of receptor-ligand interactions. J Med Chem;54:2477–2491. 23. Dorfler M., Tschammer N., Hamperl K., Hubner H., Gmeiner P. (2008) Novel D3 selective dopaminergics incorporating enyne units as nonaromatic catechol bio￾isosteres: synthesis, bioactivity, and mutagenesis stud￾ies. J Med Chem;51:6829–6838. 24. Heinrich T., Bottcher H., Bartoszyk G.D., Greiner H.E., Seyfried C.A., Van Amsterdam C. (2004) Indolebutylamines as selective 5-HT(1A) agonists. J Med Chem;47:4677–4683. 25. Heinrich T., Bottcher H., Gericke R., Bartoszyk G.D., Anzali S., Seyfried C.A., Greiner H.E., Van Amsterdam C. (2004) Synthesis and structure–activity relationship in a class of indolebutylpiperazines as dual 5-HT(1A) receptor agonists and serotonin reuptake inhibitors. J Med Chem;47:4684–4692. Note a Inc., A. S.; Release 3.5 ed.; San Diego: Accelrys Software Inc.: 2012. Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Compound 11a fitted to the four-feature phar￾macophore model. Figure S2. Key residues in the binding pocket of D3R (a) and D2R (b) were shown in spheres. Table S1. Summary of the molecular properties, fit values, and gold scores of the selected 16 compounds. Table S2. Binding affinities of the selected 16 compounds for dopamine D1, D2, and D3 receptors. Chem Biol Drug Des 2013; 82: 326–335 335 Synthesis, Biological Evaluation, and Molecular Modeling