P Zhang er al/ European Joumal of Medicinal Chemistry 61(2013)95-10 washed as before. Finally, 100 HL of a solution containing 0.03% References H202 and 2 mg/mL O-phenylenediamine in 0.1 mM citrate buffer. [1 P. Cohen, D. Yellowlees, A. A. Donella-Deana, B.A. Hemmings, ture until color emerged. The reaction was terminated by the ddition of 50 HL of 2 M H2S04, and the plate was read using a multi 11(2010)539-551 well spectrophotometer(VERSA maxTM, Molecular Devices, Sun- 14A. Sudher Babu, V.N. Balaji, Expert Opin Ther. Targets yale, CA, USA)at 490 nm. The inhibition rate(%)was calculated [51 D. Alonso, A Martinez, Glycogen Synthase Kinase 3(GSK-3)and Its Inhibitors, using the following equation: [1-(A490A490 control )x 100% howley Sons N.USA2006,pp.307-331 [6] A Martinez, C. Gil. Alzheimers Dis.2011(2011)280502. 图ma 28(2008)773-796. 4.3. Molecular modeling per, G K Smith, K Schneider, J Med. Chem. nan, A. Licht-Murava, S. Pietrokovski, M. Eisenstein, Biochim. The gSK-3B protein-ligand complex crystal structure(PDB IPYX)was chosen as the template for docking analysis, which was [101 A. Martinez, M. Alonso, A Castro, C. Perez, F Moreno, J. Med. Chem. 4. 2002)1292-1299 6.9 with the MMFF94 force field until a 0.01 kcal/mol gradient was reached For enzyme preparation, the hydrogen atoms were added, [13] D.L. Perez, S. Conde, C. Perez, C. Gil, D. Simon, L Gelpi, F Luque, A Martinez, Bioorg. Med. Chem. 17(2009)6914-6925 and all of ligands, metal, water, and cocrystallized phosphates were [14] D 1. Perez, V Palomo, C.P. rez, C. Gil, P D Dans, F.J. Luque onde, A martinez removed. The binding as defined as a sphere of 10 A radius Aed.chem54(2011)4042-4056. around the residue Arg 209. Docking calculations were performed [16jM. Hamann, D. Alonso, E. Martin-Aparicio, A Fuertes, Ml Perez-Puerto, docking complex were extracted for conformational cluster analysis Prod. 70 2007 139,-1405 varro, M. del Mlonte-Millan, M. Medina. J. Nat. in order to find the docking results with the highest probabili [17 JC Lancelot, B. Letois, C. Saturnono, P. De Caprariis, M. Robba, Org. Prep. [18] A Levai, H Duddeck, Pharmazie 38(1983)827-828. S.F. Wnuk, S.M. Chowdhury, P L. Garcia Jr, M.L. Robins, ] Org Chem. 67(2002) 6-1819 [20] A. Baki, A. Bielik, L Molnar, G Szendrei, G M. Keseru, Assay Drug Dev. TechnoL e ully acknowledge the financial support by speci [211Gold.Version5.0,TheCambridgeCrystallographicDataCentrehttp://www. Research Fund for the Doctoral Program of Higher Education of China(Grant No. 20070246089), the Shanghai Committee of [ V Palomo, L Soteras, Dd Perez, C Perez, C. Gill, NE Campillo, A Martinez. Science and Technology of China(Grant No. 10ZR1401800), and ThanksareduetoProfessorJianzhongLu(departMentofPhar-/24/nccelrYs.com National Drug Innovative Program(Grant No. 2009ZX09301-011) Mouelhi, M. Bellassoued, Synth. Commun. 31(2001)1007-1011 assoued, N. Lensen, M. Bakasse, S. Mouelhi, J. Org. Chem. 63(1998 E.R. Spitzmiller, C.F. Turk, Med Chem. 6 (1963)544-546 Nesmeyanov, LI. Zakharkin, R Kh Freidlina, Dokl. Akad. Nauk SSSR 111 Medica) for assistance with the bioassay and biological evaluation Appendix A. Supplementary data hardt, S Dove, M. Zabel, S Elz, R Seifert, A Buschauer, ] Med Chem. (2008)7193-7204. Supplementary data related to this article can be found at [30] YWa http://dx.doinorg/10.1016j.ejmech.2012.09.021. [31] Sybyl Version 6.9, Triops Inc, St. Louis, MO.washed as before. Finally, 100 mL of a solution containing 0.03% H2O2 and 2 mg/mL o-phenylenediamine in 0.1 mM citrate buffer, pH 5.5, was added and samples were incubated at room tempera￾ture until color emerged. The reaction was terminated by the addition of 50 mL of 2 M H2SO4, and the plate was read using a multi well spectrophotometer (VERSA max, Molecular Devices, Sun￾nyvale, CA, USA) at 490 nm. The inhibition rate (%) was calculated using the following equation: [1
(A490/A490 control)]  100%. 4.3. Molecular modeling The GSK-3b proteineligand complex crystal structure (PDB ID: 1PYX) was chosen as the template for docking analysis, which was proceeded using GOLD 5.0 software. For ligand preparation, the structure of compound 6v was generated and minimized using Sybyl 6.9 with the MMFF94 force field until a 0.01 kcal/mol gradient was reached. For enzyme preparation, the hydrogen atoms were added, and all of ligands, metal, water, and cocrystallized phosphates were removed. The binding site was defined as a sphere of 10 A radius around the residue Arg 209. Docking calculations were performed with other parameters set in their default values. Top five scored docking complex were extracted for conformational cluster analysis in order to find the docking results with the highest probability. Acknowledgments We gratefully acknowledge the financial support by Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20070246089), the Shanghai Committee of Science and Technology of China (Grant No.10ZR1401800), and National Drug Innovative Program (Grant No. 2009ZX09301-011). Thanks are due to Professor Jianzhong Lu (Department of Phar￾maceutical Analysis, School of Pharmacy, Fudan University) and to Professor Meiyu Geng (Division of Antitumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica) for assistance with the bioassay and biological evaluation. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2012.09.021. References [1] P. Cohen, D. Yellowlees, A. Aitken, A. Donella-Deana, B.A. Hemmings, P.J. Parker, Eur. J. Biochem. 124 (1982) 21e35. [2] J.R. Woodgett, Sci. STKE 100 (2001) 12. [3] E.M. Hur, F.Q. Zhou, Nat. Rev. Neurosci. 11 (2010) 539e551. [4] A. Kannoji, S. Phukan, V. Sudher Babu, V.N. Balaji, Expert Opin. Ther. Targets 12 (2008) 1443e1455. [5] D. Alonso, A. Martinez, Glycogen Synthase Kinase 3 (GSK-3) and Its Inhibitors, JohnWiley & Sons, Hoboken, NJ, USA, 2006, pp. 307e331. [6] A. Martinez, C. Gil, D.I. Perez, Int. J. Alzheimer’s Dis. 2011 (2011) 280502. [7] A. Martinez, Med. Res. Rev. 28 (2008) 773e796. [8] P. Bamborough, D. Drewry, G. Harper, G.K. Smith, K. Schneider, J. Med. Chem. 51 (2008) 7898e7914. [9] H. Eldar-Finkelman, A. Licht-Murava, S. Pietrokovski, M. Eisenstein, Biochim. Biophys. Acta 1804 (2010) 598e603. [10] A. Martinez, M. Alonso, A. Castro, C. Pérez, F.J. Moreno, J. Med. Chem. 45 (2002) 1292e1299. [11] T. del Ser, Alzheimer’s Dementia 6 (2010) S147. [12] S. Conde, D.I. Pérez, A. Martínez, C. Perez, F.J. Moreno, J. Med. Chem. 46 (2003) 4631e4633. [13] D.I. Perez, S. Conde, C. Pérez, C. Gil, D. Simon, F. Wandosell, F.J. Moreno, J.L. Gelpí, F.J. Luque, A. Martínez, Bioorg. Med. Chem. 17 (2009) 6914e6925. [14] D.I. Perez, V. Palomo, C.P. rez, C. Gil, P.D. Dans, F.J. Luque, S. Conde, A. Martinez, J. Med. Chem. 54 (2011) 4042e4056. [15] H. Eldar-Finkelman, M. Eisenstein, Curr. Pharm. Des. 15 (2009) 2463e2470. [16] M. Hamann, D. Alonso, E. Martín-Aparicio, A. Fuertes, M.J. Pérez-Puerto, A. Castro, S. Morales, M.L. Navarro, M. del Monte-Millán, M. Medina, J. Nat. Prod. 70 (2007) 1397e1405. [17] J.C. Lancelot, B. Letois, C. Saturnono, P. De Caprariis, M. Robba, Org. Prep. Proced. Int. 24 (1992) 204e208. [18] A. Levai, H. Duddeck, Pharmazie 38 (1983) 827e828. [19] S.F. Wnuk, S.M. Chowdhury, P.I. Garcia Jr., M.J. Robins, J. Org. Chem. 67 (2002) 1816e1819. [20] A. Baki, A. Bielik, L. Molnar, G. Szendrei, G.M. Keseru, Assay Drug Dev. Technol. 5 (2007) 75e83. [21] GOLD, Version 5.0, The Cambridge Crystallographic Data Centre, http://www. ccdc.cam.ac.uk. [22] V. Palomo, I. Soteras, D.I. Perez, C. Perez, C. Gil, N.E. Campillo, A. Martinez, J. Med. Chem. 54 (2011) 8461e8470. [23] Discovery Studio, Version 3.0, Accelrys Software Inc., http://www. accelrys.com. [24] N. Lensen, S. Mouelhi, M. Bellassoued, Synth. Commun. 31 (2001) 1007e1011. [25] M. Bellassoued, N. Lensen, M. Bakasse, S. Mouelhi, J. Org. Chem. 63 (1998) 8785e8789. [26] J. Krapcho, E.R. Spitzmiller, C.F. Turk, J. Med. Chem. 6 (1963) 544e546. [27] A.N. Nesmeyanov, L.I. Zakharkin, R. Kh Freidlina, Dokl. Akad. Nauk SSSR 111 (1956) 114e116. [28] A. Lebedev, A. Lebedeva, V. Sheludyakov, E. Kovaleva, O. Ustinova, I. Kozhevnikov, Russ. J. Gen. Chem. 75 (2005) 1113e1124. [29] P. Ghorai, A. Kraus, M. Keller, C. Gotte, P. Igel, E. Schneider, D. Schnell, G.n. Bernhardt, S. Dove, M. Zabel, S. Elz, R. Seifert, A. Buschauer, J. Med. Chem. 51 (2008) 7193e7204. [30] Y. Wang, J. Ai, Y. Chen, L. Wang, G. Liu, M. Geng, A. Zhang, J. Med. Chem. 54 (2011) 2127e2142. [31] Sybyl Version 6.9, Triops Inc, St. Louis, MO. P. Zhang et al. / European Journal of Medicinal Chemistry 61 (2013) 95e103 103