Design, Synthesis, and Evaluation ofIndolebutylamines as a Novel Class of SelectiveDopamine D3 Receptor Ligands

Peng Du1,†, Lili Xu1,†, Jiye Huang2,†, Kunqian Yu2,Rui Zhao3, Bo Gao3, Hualiang Jiang2, WeiliZhao1, Xuechu Zhen3 and Wei Fu1,*

1Department of Medicinal Chemistry & Key Laboratory ofSmart Drug Delivery, Ministry of Education & PLA, Schoolof Pharmacy, Fudan University, Shanghai, 201203, China2Drug Discovery and Design Center, State Key Laboratoryof Drug Research, Shanghai Institute of MateriaMedica,Chinese Academy of Sciences, Shanghai, 201203, China3Department of Pharmacology, College of PharmaceuticalSciences, 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 selectiveligands for the dopamine 3 receptor. The most potentcompound 11q binds to dopamine 3 receptor with a Ki

value of 124 nM and displays excellent selectivity overthe dopamine 1 receptor and dopamine 2 receptor.Investigation based on structural information indicatesthat site S182 located in extracellular loop 2 mayaccount for high selectivity of compounds. Interactionmodels of the dopamine 3 receptor-11q complex andstructure-activity relationships were discussed by inte-grating all available experimental and computationaldata 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 andaccepted for publication 26 April 2013

Since the discovery by Sokoloff et al. in 1990 (1), dopamine3 receptor (D3R) has been proved to be a promising thera-peutic target for drug discovery. Dopamine 3 receptorantagonist was shown to play a key role in the treatment ofschizophrenia (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 dopaminereceptor subtypes (D2, D3, and D4). For instance, hD3Rand hD2R share 78% sequence homology within theseven transmembrane domains and 94% sequencehomology within the active site (10,11). To date, most ofD3R selective ligands are 4-Phenylpiperazines and theirclose analogs (12,13). Considering the importance of theD3R 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 potentand selective D3R ligands. Using a strategy that com-bines synthetic chemistry, binding assays, and a set ofcomputational approach (integrating active site mapping,pharmacophore-based virtual screening, and automatedmolecular docking), we designed a series of IBA deriva-tives as a new type of highly selective D3R antagonists.Furthermore, the molecular determinants critical to thebinding specificity and selectivity of D3R were identifiedand the structure-activity relationships (SAR) was investi-gated.

Methods and Materials

Structure-based pharmacophore model generationDopamine 3 receptor was obtained from the Protein DataBank (PDB ID: 3PBL) (11). The GRID22 program (14) wasemployed to map the active sites of the optimized X-raystructure of D3R with five types of chemical probes, thatis, negative ionizable (COO�), positive ionizable (N1+),hydrogen-bond acceptor (O), hydrogen-bond donor (N1),and hydrophobic probes (DRY). For each of the fiveprobes used in the grid calculations, grid points weresuperimposed to identify clusters of positions. The mem-bers of each identified clusters were combined into onepharmacophore feature, and the centers of each pharma-cophore features were set at the geometric centers of themembers in each clusters (15). Finally, a four-feature phar-macophore model was generated.

Virtual screeningThe obtained pharmacophore model was used to screenthe Asinex GOLD and Maybridge collection databasewhich 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

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Research Article

match our pharmacophore model. For each molecule inthe database, a maximum of 250 conformations with anenergy threshold of 20 kcal/mol were generated usingFAST algorithm. Only compounds with a fit value greaterthan three were retained. Then Lipinski’s Rule of Five wasapplied to reject non-drug-like compounds. The hitsobtained were overlaid on the active site of the D3R andthose creating steric clashes were discarded. GoldScorewas used to rank the hits. The interaction analyses incombination with scoring function was used to guide thefinal selection.

Molecular dockingMolecular docking was carried out using GOLD 5.0.1(16).The binding site was defined to include all residues withina 15.0 �A radius of the conserved D3.32Cc carbon atom. Ahydrogen-bond constraint was set between the protonatednitrogen atom (N1) of ligand and D3.32 side chain. Tenconformations were produced for each ligand, and Gold-Score was used as scoring function. Other parameterswere set as standard default. High-scoring complexeswere inspected visually to select the most reasonable solu-tion.

Biological evaluation

Binding assaysAll the synthesized new compounds were subjected tocompetitive binding assays for the human dopamine (D1,D2, and D3) receptors, using membrane preparationobtained from HEK293 cells stably transfected respectivereceptor. [3H] SCH23390 (D1) and [3H]-Spiperone (D2and D3) were used as standard radioligands. The per-centage displacement of radioligand and Ki values ofthese compounds is reported in Table 1. Duplicatedtubes 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 [3H]SCH23390 (for D1R), or [3H]Spiperone (for D2R and D3R) in a final volume of 200 lLbinding buffer containing 50 mM Tris, 4 mM MgCl2, pH7.4. Non-specific binding was determined by parallelincubations with either 10 lM SCH23390 for D1 or Spip-erone for D2, D3 dopamine receptors, respectively. TheIC50 and Ki values were calculated by non-linear regres-sion (PRISM; Graphpad, San Diego, CA, USA) using asigmoidal function.

[35S]GTPcS binding assaysThe [35S]GTPcS binding assay was performed at 30 °C for30 min with 10 lg of membrane protein in a final volumeof 100 lL with various concentration of the compound.The antagonism effects of the compounds were tested inthe 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 guanosinetriphosphate. The reaction was initiated by adding of [35S]GTPcS (final concentration of 0.1 nM). Non-specific bindingwas measured in the presence of 100 lM 5′-guanylimid-odiphosphate (Gpp(NH)p).

Experimental Section

Chemicals and solvents were purchased and used withoutfurther purification. 1H and 13C NMR spectra wererecorded on a Bruker AMX-400 instrument. The chemicalshifts were referenced to the solvent peak, namelyd = 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 spectrawere given with an electric ionization (ESI) produced byHP5973 N analytical mass spectrometer. All tested com-pounds had a minimal purity of 95% assessed by HPLCmethod (Schemes 1 and 2).

General procedures for the preparation ofcompounds 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 addedto a solution of N-cyclohexylaniline (3.23 g, 18.43 mmol)and Et3N (1.86 g, 18.43 mmol) in anhydrous CH2Cl2 at0 °C under N2 atmosphere and then stirred at room tem-perature for 5 h. The reaction was diluted with CH2Cl2 andwashed with brine, and the organic layer was dried overNa2SO4, 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 � 2512 496.0 4 1 3.73 3.29 69.0 2203 � 913 364.5 3 2 2.93 3.30 59.1 2814 � 35Spiperone 0.48 � 0.1

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mixture was refluxed for 8 h, and the reaction mixture wasevaporated to dryness. The residue was dissolved inEtOAc (100 mL), washed with H2O (50 mL), dried, andevaporated to obtain the crude product, which was puri-fied by flash chromatography (CH2Cl2/MeOH, 40:1) toafford tert-butyl 4-(2-(cyclohexyl(phenyl)amino)-2- oxoethyl)piperazine-1-carboxylate 9 as a yellow liquid (4.58 g, yield95.4%), (iii) To a solution of compound 9 (3.0 g,8.68 mmol) in CH2Cl2 (15 mL) was added trifluoroaceticacid (15 mL). The mixture was stirred at room temperaturefor 12 h, then concentrated, washed with PE and Et2Oseparately 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 for12 h, then the reaction mixture was evaporated to dry-ness, water added, and the mixture was extracted withCH2Cl2 (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). 1H NMR (400 MHz, CDCl3) d8.11 (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-MSm/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%). 1H 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%). 1H 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%). 1H 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) d168.72, 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-MSm/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%). 1H 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%). 1H 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]+.

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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%). 1H 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/z507.2 [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%). 1H 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%). 1H 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) d169.18, 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/z569.2 [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%). 1H 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/z459.4 [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%). 1H 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%). 1H 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%). 1H 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%). 1H 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]+.

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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%). 1H 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/z503.4 [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%). 1H 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%). 1H 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/z521.4 [M + H]+.

Results and Discussion

Pharmacophore-based virtual screeningThe obtained pharmacophore model was shown inFigure 1A. As a result of our virtual screening protocol, 16compounds were selected to purchase and submitted topharmacological experiments (Tables S1 and S2). To ourdelight, three of them revealed moderate D3R activities.Their chemical structures and corresponding bindingassays 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 thepharmacophore model quite well (Figure S1) and repre-sents a novel class of D3R ligands. It was identified tobind hD3R with 2161 nM affinity and was thus chosen asthe lead compound for further optimization.

Rational design and structure-activityrelationshipsThe structural analysis and the ligand–receptor interactionelucidated by molecular docking were investigated toguide the structure modification and optimization of com-pound 11a. Compound 11a is characterized by an indolehead, a linear alkyl linker and the N-phenylacetamide tailconnected to a piperazine moiety. To rationalize the designof the derivatives, the structural model of the complexD3R-11a was constructed by combining molecular dock-ing and all available experimental data (Figure 2A). Threeimportant interactions were identified in the D3R-11amodel: the conserved salt bridge interaction between theprotonated nitrogen atom (N1) of 11a and the carboxylategroup of D3.32; the cation-p contact between the proton-ated nitrogen atom and F6.51; and the hydrogen bondformed by the oxygen atom of carbonyl group in 11a andY7.35 in D3R. It indicates that the piperazine ring and thecarbonyl group are critical to the activity, as these indis-pensable interactions determined the binding orientation ofthe 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 theextracellular segments of TM-III, -VII) (18). The hollowspace was found in the OBS and SBP (Figure 2A), sug-gesting that 11a could be optimized by appending largergroups 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 toimprove the potency of this series of ligands (Table 2).

As our lead compound 11a already carries an aromatichead and a bulky tail, the length of the linker was first con-sidered to be incremented to fill the hollow space in theactive site and 11p was obtained, providing a delightfulimprovement in the binding affinity (Ki = 636 nM). To verifyour predicted binding mode, the effect of the length of thelinker (n = 2–4) on affinity was further examined. Indeed,the affinity of 11o with a 4-carbon linker is superior tothose of 11m with 2-carbon and/or 11k with 3-carbonlinkers. As predicted, it proved that the longer linker could

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allow the molecule to extend to the hollow space in theOBS and SBP, thus the affinity was increased. In addition,the affinity of 11a with larger cyclohexyl group on the tailwas higher than those of 11b, 11c, and 11d with smallergroups. It further confirms that the tight binding in OBS

and SBP by introducing proper functional groups to themolecules could improve the affinity to D3R.

Compounds 11f, 11h, and 11g were next prepared toevaluate 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 hydrophobicelements (Hydro-1 and Hydro-2) were shown in cyan spheres, the positive ionizable element POS in red, and a hydrogen-bond-acceptorelement (HBA) in green. Three exclusion volumes (Excl-1, -2, -3) are shown in black spheres, corresponding to the positions of conservedresidues 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)

NR3O

NN

NH

R1n

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 � 2511b 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 � 11511f 3 Cl H Cyclohexyl 37.84% 54.25% 292 � 1111g 3 Cl 2-OMe Cyclohexyl 18.04% 42.93% 950 � 8111h 3 Br H Cyclohexyl 42.04% 20.13% 2136 � 911i 3 Br 2-OMe Cyclohexyl 19.29% 24.57% 1513 � 12111j 3 H H Cyclohexyl 26.57% 34.68% 73.23%11k 3 H 2-OMe Cyclohexyl 28.32% 58.69% 1497 � 811l 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 � 211p 4 F H Cyclohexyl 3.58% 18.76% 635 � 511q 4 F 2-OMe Cyclohexyl 8.65% 35.01% 123 � 3SCH23390 1.69 � 0.12 –a –Spiperone – 1.08 � 0.09 0.48 � 0.07

aThe activity was not determined. All values are means of three separate experiments. The values of Ki were given only when the inhibitionrate of compound is >90%.

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the indole head on binding affinity. The results indicatedthat all halogenated derivatives displayed improved bindingaffinity, and the chlorinated compound performs betterthan the fluorinated or brominated one (11f > 11a > 11h)when the length of linker is 3 (n = 3). In addition, whenn = 4, the binding affinity of 11p with a 5-fluorine on indoleis superior to 11n with a hydrogen, demonstrating theimportant role played by halogens in D3R ligand design.

We noted that the o-methoxy group in the phenyl ring ofthe 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 theaffinity to D3R. Close examination indicated that the o-methoxy substituent in the phenyl ring played an optimalrole when a 4-carbon linker was present. Among thesemolecules, 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 activityat D3R, and the calculated antagonistic potencies (IC50) of11q were 828 nM for the D3R (Table 3).

Molecular docking study was promptly conducted toinvestigate the binding mode of 11q with D3R to revealthe role played by the o-methoxy group and the structuralfeatures responsible for the increased affinity (Figure 2B).Consistent with the binding mode of D3R with the lead11a (without the o-methoxy group), the salt bridge formedwith D3.32, the cation-p interaction with F6.51, and thehydrogen 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 indolehead to forms p–p interaction with F6.52 in a parallel dis-placed manner. The interactions illustrated by our modelare 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 intothe D3R-11q model to find the intrinsic factor that givesthis novel series of compounds a unique pharmacological

activity toward D3R. The D3R-11q model shows that theinteractions formed by the protonated nitrogen of pipera-zine and the negatively charged D3.32, and the amidemoiety 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 hydrophobicresidues V3.33, V5.39, W6.48, F6.51, F6.52, H6.55(Figure 3B). In the meantime, the N-phenylacetamide tailextends to the SBP and participates in the hydrophobicinteractions with I183 in ECL2 and V2.61, L2.64, F3.28,V3.29, Y7.43 (Figure 3B). Our predicted binding mode isconsistent with the site-directed mutagenesis studies,which indicated that the mutation of F6.51 and V2.61caused significant reduction in binding affinities of D3Rligands (22,23). Thus, the hydrophobic interactions in theOBS and SBP, and the hydrogen-bond interactionsformed with the key residues D3.32 and Y7.35 mostlycontribute to the activity to D3R.

It is worth noting that the designed series of compoundshave high selectivity to D3R. As D2R and D3R share highsequence identity in the active site, we sought to identifythe structural determinants for such a high selectivity overthe 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). AsD1R and D3R share less sequence identity, the non-con-served residues at positions 7.35, 7.43, and 6.55 in D1Rmake collision with 11q, hence, it is not difficult to under-stand that this series of compounds display no activity forhD1R. Whereas in the case of D2R and D3R, which havealmost identical residues in the binding pocket, what con-tributes to their selectivity? Only three of 22 residues arenot 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 theD3R

Compound EC50 (nM) IC50 (nM)

11q –a 827.63Haloperidol – 248.02Quinpirole 25.673 –

a[35S]GTPcS binding activity could not be detected.

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D2R (Figure 3B), namely V6.56I, S184A, and S182I. Theside chains of both V6.56I and S184A stretch outwardfrom the binding pocket, indicating that the two sites maycontribute little to the selectivity. S182I, however, is facingthe binding pocket and might be the key factor for theselectivity. We noted that I182 in D2R acts as a cap (Fig-ure S2a) and plugs up the entrance of ligand. It probablyprevents this series of compounds from entering the activesites, 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), thesmall-size S182 allows the smooth entrance of designedcompounds and forms favorable hydrophobic interactionwith them. Hence, we conclude that S182 in ECL2 inD3R might be the key residue that determines the high

selectivity of this series of D3R ligands over D2R. Heinrichet al., reported a series of indolebutylamines (IBAs) andphenylpiperazine derivatives showing high activities against5-HT1AR and D2R (24,25). Our lead compound 11a

obtained from virtual screening bears N-phenylacetamidetail, and it displays different pharmacological profile fromphenylpiperazine derivatives and shows high selectivitytoward D3R instead. The introduction of amide groupcauses the lost of D2R activity.

Conclusions

In summary, a new type of compounds with the corestructure of IBA and N-phenylacetamide were identified ashigh selective D3R ligands over D1R and D2R by combin-ing a series of computational approaches, the syntheticchemistry, and binding assays. Compound 11q displaysrelatively high binding affinity (Ki = 124 nM), and it wasevaluated as D3R antagonist by [35S]GTPcS binding assay.The results indicated that a fluorine substituted indolehead, 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 bindingmode of 11q with D3R constructed by molecular dockingnot only elucidated SARs in detail, but also recognized themolecular determinants critical for D3R affinity as well asselectivity. The carboxylate group of D3.32 formed con-served salt bridge interaction with the protonated nitrogenatom (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. Andtogether 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 entranceto D3R and was found to be the molecular structuraldeterminant for the selectivity of D3R over D2R. As thesethree residues have not obtained much attention in thepreviously reported studies on selective D3R ligands, thisstudy may give a hint in the design of novel selective D3Rligands. Further optimization of this series of compoundsis ongoing.

A

B

Figure 3: (A) The sequence alignment in the binding pocket ofD1R, D2R, and D3R; (B) Predicted binding mode of compound11q (yellow sticks) with D3R (blue cartoon), and D2R (graycartoon) is superimposed to D3R.

R1

NHNH2

NH

R1OH

NH

R1 Bra b

NH

F Br

NH

F

OH

NH

F

Brfc,d,e

3a: R1=H3b: R1=F3c: R1=Cl3d: 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.

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Acknowledgment

This work was supported by the National Natural ScienceFoundation of China (No. 81172919, 81130023,30825042) and grants from the National High TechnologyResearch and Development Program of China (863 Pro-gram) (No. 2012AA020301), the State Key Program ofBasic 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 alsoappreciated.

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HN R3

a

NR3Cl

O

b

N R3NOBocN

cN R3N

OHN

NR3O

NN

NH

R1 nd

7 8 9

10 11a-11q

R2

R2

R2

R2

R2

Scheme 2: Syntheses of compounds 11a–q. Reagents and conditions: (a) chloroacetylchloride, TEA, DCM; (b) Boc-piperazine, K2CO3,CH3CN; (c) TFA/DCM; (d) 3a–3d, 6b, K2CO3, CH3CN.

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Note

aInc., A. S.; Release 3.5 ed.; San Diego: Accelrys SoftwareInc.: 2012.

Supporting Information

Additional Supporting Information may be found in theonline 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 compoundsfor dopamine D1, D2, and D3 receptors.

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