alanthamine and galanthamine-galanthaminium salts. Bioorg. Med. hem.Le.2000,10,637-639 10 cells/well (200 ul) into 96-well plates and allowed to adhere (11) Carlier, P. R. Du, D. M.: Han, Y. F. Liu, J Perola, E; W and grow. When cells reached the required confluence, they were L. D ; Pang. Y. P. Dimerization of an inactive fragment of huperzine laced into serum-free medium and treated with the synthesized A produce a drug with twice the potency of the natural product Chem,lnt.Ed.2000,39,1775-1777 compounds 5h and 5i. Twenty-four hours later the surv ( 12)Feng, S; Wang. Z; He, X; Zheng, S: Xia, Y. Jiang, H; Tang, X: as determined by MTT assay. Briefly, after incubat Bai, D. Bis-huperzine B: highly potent ar L of MTT(5 mg/mL: Sigma, St Louis, MO)at 37 erase inhibitors. J Med. Chem. 2005. 48. 655-657 living cells containing MTT formazon crystals were (13)Munoz-Torrero, D: Camps, P. Dimeric and hybrid anti-Alzheime in 200 uL of dimethyl sulfoxide(DMsO, Sigma). The absor- drug candidates. Curr. Med. Chem. 2006. 13. 399-422 ance of each well was measured using a microculture plate (14)Harel, M. Schalk, L; Ehret-Sabatier, L; Bouet, F. Goeldner. M. HirtI reader with a test wavelength of 570 nm and a reference C ; Axelsen, P H. Silman, L; Sussman, J. L. Quatemary ligand binding Proc. Natl. Acad. Sci. U.S.A. 1993. 90. 9031-9035 Acknowledgment. We thank the National Natural Science (15)Li, W: Hao, J; Tang, Y; Chen, Y; Qiu, Z Comparative studies of Foundation of China(Grants 30472088, 30772553, and cophore. Acta. Pharmacol Sin. 2005, 26, 334-338 30371731), the Program of Shanghai Subject Chief Scientist (16)Ennis, C. Haroun, F. and Lattimer, N. Can the effects of meptazinol Grant 06XD14011), and the Major Basic Research Project of aceyl guinea-pig isolated ileum be explained by inhibition of Shanghai Municipal Science and Technology Commission (17) Chen, Y Studies on the Synthesis, Resolution and Optical Isomers (Grant 07DJ14005) for financial support. We also gratefully Meptazinol. Ph. D. Dissertation, Fudan University, Shanghai, P. R thank Dr Manuela Bartolini(University of Bologna, Italy) and China. 2004 Dr. Margarita Dinamarca(Pontificia Universidad Catolica, ( 18)Xie, Q: Tang, Yun ; Li, w: Wang, X; Qiu, Z Investigation of the Chile) for their valuable suggestions in the experiments on binding mode of (-)-meptazinol and bis-meptazinol derivatives on acetylcholinesterase using a molecular docking method. J Mol Model. AChE-induced AB aggregation. 2006.12,390-397 (19) Savin Note Added after ASAP Publication. This manuscript was Chiasserini. L: Pellerano. C: Novellino. E: Mckissic. D: Saxena released ASAP on March 12, 2008 with errors in the Experimental and Acknowledgment Sections. The correct version posted recognition sites. Rational design of novel, selective, and highly potent March 15. 2008 cholinesterase inhibitors. Med. Chem. 2003. 46.1-4 (20) Bartolini, M: Bertucci, C. Cavrini, V: Andrisano, V. 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Mec c Mol. Biol. 1997, 267.o of a genetic algorithm for flexible docking ao. Chem. 1998. 6, 1835-1850.(b)Guillou, C: Mary, A Renko, D Z Dong, C. Z; Heymans, F; Chen, H. Z Pharma Gras, E: Thal, C. Potent acetylcholinesterase inhibitors: des PMS777, a new AChE inhibitor with PAF antago ynthesis and structure-activity relationships of alkylene linked bis- J. Neuropsychopharmacol. 2007, 10, 21-2100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C. Cells were plated at 5 × 104 cells/well (200 µl) into 96-well plates and allowed to adhere and grow. When cells reached the required confluence, they were placed into serum-free medium and treated with the synthesized compounds 5h and 5i. Twenty-four hours later the survival of cells was determined by MTT assay. Briefly, after incubation with 20 µL of MTT (5 mg/mL; Sigma, St. Louis, MO) at 37 °C for 3 h, living cells containing MTT formazon crystals were solubilized in 200 µL of dimethyl sulfoxide (DMSO, Sigma). The absor￾bance of each well was measured using a microculture plate reader with a test wavelength of 570 nm and a reference wavelength of 655 nm. Acknowledgment. We thank the National Natural Science Foundation of China (Grants 30472088, 30772553, and 30371731), the Program of Shanghai Subject Chief Scientist (Grant 06XD14011), and the Major Basic Research Project of Shanghai Municipal Science and Technology Commission (Grant 07DJ14005) for financial support. We also gratefully thank Dr. Manuela Bartolini (University of Bologna, Italy) and Dr. Margarita Dinamarca (Pontificia Universidad Católica, Chile) for their valuable suggestions in the experiments on AChE-induced A
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