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