INVESTIGATIONS ON AURONES AS MODULATORS OF ABCG2 SIM HONG MAY (B. Sc. (Pharmacy) (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgements First and foremost, I would like to express my heartfelt gratitude to my supervisor Assoc. Prof. Go Mei Lin for her unwavering support and encouragement throughout the whole course of my research. This piece of thesis work would not be possible without her advice and insights into the research topic. I would also like to thank Dr Rachel Ee Pui Lai for her advice in my research and the offer of cell lines (MDA-MB-231/V and R) as well as the opportunity to carry out biological assays (Western Blotting) in her laboratory in the Department of Pharmacy. Special thanks to my senior, Dr Lee Chong Yew for the contribution of his aurones to my research work as well as the constant help in bench work and the encouragement rendered especially in the early years of my graduate studies. Many thanks to the past and present members of the Medicinal Chemistry Laboratory at the Department of Pharmacy for their camaraderie and sharing of knowledge and research experiences: Dr. Liu Xiao Ling, Dr. Suresh Kumar Gorla, Dr Zhang Wei, Dr Leow Jolene, Mr Yeo Wee Kiang, Ms Nguyen Thi Hanh Thuy, Mr Wee Xi Kai, Mr Pondy Murgappan Ramanujulu, Dr Yang Tian Ming, Ms Tan Kheng Lin and Ms Chen Xiao. Appreciation also goes out to the past research assistants and final year students: Ms Tee Hui Wearn, Ms Audrey Chan Xie Wah, and Ms Loh Ker Yun for their help in my project. I would also like to thank Dr Han Yi, Dr Tian Quan, Ms Leow Pay Chin and Ms Ong Zhan Yuin from Dr Rachel Ee’s laboratory for their advice and assistance in biological work. Appreciation goes to Mdm Oh Tang Booy, Ms. Ng Sek Eng and the rest of the technical staff at the Department of Pharmacy. Research is made easier with their help in the purchasing of chemicals/biologicals and trouble-shooting of machines. I would like to acknowledge the financial support for my graduate studies from the National University of Singapore Research Scholarship. ii Last but not least, I would like to thank my parents and family for their constant support, understanding and concern throughout the course of my graduate studies. iii Conferences and Publications 1) Hong-May Sim, Pui-Lai Rachel Ee and Mei-Lin Go, 4, 6-Dimethoxyaurones as Dual Modulators of P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), European School of Medicinal Chemistry (ESMEC), 6th to 11th July 2008, Urbino, Italy. 2) Hong-May Sim and Mei-Lin Go, Structurally Modified Aurones as Inhibitors of the ATP Binding Cassette Transport Protein ABCG2 (Breast Cancer Resistance Protein), 10th Asia Pacific Rim Universities Doctoral Students Conference (APRU DSC), 6th to 10th July 2009, Kyoto, Japan. 3) Hong-May Sim and Mei-Lin Go, Structurally Modified Aurones: Inhibitors of the ATP Binding Cassette Transport Proteins Breast Cancer Resistance Protein (ABCG2) and P- glycoprotein (ABCB1), 7th International Symposium for Chinese Medicinal Chemists (ISCMC), 1st to 5th Feb 2010, Kaoshiung, Taiwan. 4) Sim. H.M., Lee, C.Y., Ee, P.L.R., Go, M.L., Dimethoxyaurones: Potent inhibitors of ABCG2 (breast cancer resistance protein) European Journal of Pharmaceutical Sciences, 35, 2008, 293-306. Manuscripts in preparation: 1) Sim. H.M., Lee, C.Y., Yeo, W.K., Loh, K.Y., Ee, P.L.R., Go, M.L., Structurally modified aurones as modulators of ABCG2: Structure-activity relationships and pharmacophore modeling. 2) Sim. H.M., Wu. C.P., Ee, P.L.R., Ambudkar. S., Go, M.L., In vitro and in vivo evaluations of structurally modified aurones as modulators of ABCG2 iv Table of Contents Acknowledgements………………………………………………………………………………...ii Conferences and Publications………………………………………………………………..........iv Table of contents…...........................................................................................................................v Summary……………………………………………………………………………………..........vi List of Tables…………………………………………………………………………………….viii List of Figures……………………………………………………………………………………...x List of Schemes…………………………………………………………………………………...xv List of Abbreviations…………………………………………………………………………….xvi Chapter 1: Introduction……………………………………………………………………..1 1.1. Multidrug resistance (MDR) and ATP-Binding Cassette (ABC) transporters……………1 1.2. Structural features of ABCB1 and ABCG2………………………………………………2 1.3. Functions of ABCB1 and ABCG2………………………………………………………..4 1.4. Substrates and Inhibitors of ABCB1 and ABCG2………………………………………..6 1.5. Flavonoids as MDR reversal agents……………………………………………………..12 1.6. Aurones as modulators of ABC transporter proteins …………………………………...16 1.7. Statement of Purpose…………………………………………………………………….18 Chapter 2: Design and Synthesis of Target Compounds…………………………………21 2.1. Introduction…………………………………………………………………………21 2.2. Rationale of design………………………………………………………………….22 2.2.1. Series 1-5……………………………………………………………………………23 2.2.2. Series 6-12…………………………………………………………………………..29 2.3. Chemical considerations.............................................................................................37 2.3.1. Aurones (Series 1, 2, 5) and Dehydroaurones (Series 8)…………………………....37 2.3.2. Indanones (Series 6)………………………………………………………………...42 v 2.3.3. Aza-aurones (Series 7)………………………………………………………………43 2.3.4. Chalcones (Series 11)……………………………………………………………….47 2.3.5. Flavones (Series 12)………………………………………………………………...47 2.4. Assignment of configurations……………………………………………………….49 2.4.1. Aurones (Series 1, 2, 5) and dehydroaurones (Series 8)……………………………49 2.4.2. Indanones (Series 6)………………………………………………………………...51 2.4.3. 4, 6-Dimethoxy aza-aurones (Series 7)…………………………………………......52 2.4.4. 2′-Hydroxy, 4’6’-dimethoxy chalcones (Series 11)………………………………...55 2.5. Experimental methods………………………………………………………………57 2.5.1. General details……………………………………………………………………....57 2.5.2. General procedure for the synthesis of series 1, 2, 6 and 8 (4, 6-dimethoxy-aurones, 4, 5, 6-trimethoxy-aurones, unsubstituted (Ring A) aurones and 4, 5, 6-trimethoxy dehydro aurone)……………………………………………………………………………………….58 2.5.2.1. 4, 6-Dimethoxy-aurones…………………………………………………………….58 2.5.2.1.1. 3, 5-Dimethoxyphenoxyacetic acid (A-1)…………………………………………58 2.5.2.1.2. 4, 6-Dimethoxybenzofuran-3(2H)-one (A-3)……………………………………...59 2.5.2.2. 4, 5, 6-Trimethoxy-aurones………………………………………………………….59 2.5.2.2.1. 3, 4, 5-Trimethoxyphenoxyacetic acid (A-2)……………………………………...59 2.5.2.2.2. 4, 5, 6-Dimethoxybenzofuran-3(2H)-one (A-4)…………………………………...59 2.5.2.3 General procedure for the synthesis of 4, 6-dimethoxyaurones and 4, 5, 6- trimethoxyaurones (Compounds 1-1 to 1-28, 2-1 to 2-10)…………………………………..60 2.5.2.4 Procedure for synthesis of (Z)-2-(cyclohexylmethylene)-4, 5, 6- trimethoxybenzofuran-3(2H)-one (2-11)…………………………………………………….61 2.5.2.5 General procedure for the synthesis of unsubstituted aurones (Compounds 5-1 to 5- 8).…………………………………………………………………………………………….61 vi 2.5.2.6 General procedure for the synthesis of 4, 5, 6-trimethoxydehydroaurone (Compound 8-3)……………………………………………………………………………...62 2.5.3 General procedure for the synthesis of 4, 6-dimethoxy-indanones (Compounds 6-1 to 6- 2)……………………………………………………………………………………………..62 2.5.3.1. 3, 5-Dimethoxypropanoic acid (I-1)………………………………………………...62 2.5.3.2. 5, 7-Dimethoxy-2, 3-dihydro-1H-inden-1-one (I-2)………………………………...63 2.5.3.3. Condensation of I-2 with substituted benzaldehydes………………………………..64 2.5.4 General procedure for the synthesis of 4, 6-dimethoxy-aza-aurones (Compounds 7-1 to 7-8)…………………………………………………………………………………………...64 2.5.4.1. 2-Amino-4,6-dimethoxy-α-chloroacetophenone (AZ-1)……………………………64 2.5.4.2. 1-Acetyl-4,6-dimethoxy-2,3-dihydro-1H-indole-3-one (AZ-2)……………………..65 2.5.4.3. Condensation of AZ-2 with substituted benzaldehydes……………………………..66 2.5.5. General procedure for the synthesis of 2′-hydroxy-4′, 6′-dimethoxychalcones (Compounds 11-1 to 11-5)…………………………………………………………………...66 2.5.6. General procedure for the synthesis of 5, 7-dimethoxy flavones (Compounds 12-1 to 12-5)………………………………………………………………………………………….67 2.5.7. Protection and deprotection of phenolic hydroxyl groups on 3-hydroxybenzaldehyde, 4-hydroxybenzaldehyde, 3, 4-dihydroxybenzaldehyde, 3, 5-dihydroxybenzaldehyde, 3- hydroxy-4-methoxybenzaldehyde and 4-hydroxy-3-methoxybenzaldehyde………………..67 2.5.8. X-ray crystallography of compounds 1-2 and 6-20…………………………………..68 2.5.9 High pressure liquid chromatography (HPLC) analyses………………………………68 2.6 Summary…………………………………………………………………………………69 Chapter 3: Effects of aurones and related compounds on the efflux of pheophorbide a (PhA) by ABCG2 over-expressing human breast cancer (MDA-MB-231/R) cells and calcein-AM (CAM) by ABCB1 over-expressing Mardin-Darby canine kidney (MDCK) cells………………………………………………………………………………………….70 3.1. Introduction……………………………………………………………………………..70 3.2. Experimental…………………………………………………………………………….70 3.2.1. Cell lines and materials for biological assay…………………………………………..70 vii 3.2.2. Procedure for western blot analysis…………………………………………………...71 3.2.3. Procedure for PhA accumulation assay………………………………………………..71 3.2.4. Procedure for calcein-AM (CAM) accumulation assay……………………………….73 3.2.5. Statistical Analysis…………………………………………………………………….75 3. 3. Results…………………………………………………………………………………..76 3.3.1. Determination of EC50 from PhA assay……………………………………………….76 3.3.2. Determination of EC50 from CAM assay……………………………………………...78 3.3.3. Effect of structural modifications on efflux of PhA by ABCG2 over-expressing human breast cancer (MDA-MB-231/R) cells……………………………………………………….81 3.3.3.1. Series 1-5 compounds with mono-substituted