University of Tennessee Health Science Center UTHSC Digital Commons Theses and Dissertations (ETD) College of Graduate Health Sciences 5-2008 Flavonoids and Related Compounds as Nucleoside Transporter Inhibitors Surekha Ravaji Pimple University of Tennessee Health Science Center Follow this and additional works at: https://dc.uthsc.edu/dissertations Part of the Medicinal and Pharmaceutical Chemistry Commons, and the Pharmaceutics and Drug Design Commons Recommended Citation Pimple, Surekha Ravaji , "Flavonoids and Related Compounds as Nucleoside Transporter Inhibitors" (2008). Theses and Dissertations (ETD). Paper 200. http://dx.doi.org/10.21007/etd.cghs.2008.0245. This Thesis is brought to you for free and open access by the College of Graduate Health Sciences at UTHSC Digital Commons. It has been accepted for inclusion in Theses and Dissertations (ETD) by an authorized administrator of UTHSC Digital Commons. For more information, please contact [email protected]. Flavonoids and Related Compounds as Nucleoside Transporter Inhibitors Document Type Thesis Degree Name Master of Science (MS) Program Pharmaceutical Sciences Research Advisor John K. Buolamwini, Ph.D. Committee Peter K. Bridson, Ph.D. Issac O. Donkor, Ph.D. Duane D. Miller, Ph.D. DOI 10.21007/etd.cghs.2008.0245 This thesis is available at UTHSC Digital Commons: https://dc.uthsc.edu/dissertations/200 FLAVONOIDS AND RELATED COMPOUNDS AS NUCLEOSIDE TRANSPORTER INHIBITORS A Dissertation Presented for The Graduate Studies Council The University of Tennessee Health Science Center In Partial Fulfillment Of the Requirements for the Degree Master of Science From The University of Tennessee By Surekha Ravaji Pimple May 2008 Copyright © 2008 by Surekha Ravaji Pimple All rights reserved ii Dedication This dissertation is dedicated to my husband, Dr. Yogeshwar Gulabrao Bachhav iii Acknowledgements I would like to thank my major advisor, Dr. John K. Buolamwini for his constant help, guidance and support during the course of this work. In the past two and a half years the encouragement and training that I have received from Dr. Buolamwini has helped develop my independent thinking and skills as a researcher. I am also very grateful to my other committee members, Dr. Peter K. Bridson, Dr. Isaac O. Donkor, and Dr. Duane D. Miller for their valuable suggestions. I extend my sincere thanks to the following past and present members of our laboratory: Dr. Shantaram Kamath, Dr. Shivaputra Patil, Dr. Chunmei Wang, Dr. Amol Gupte, Dr. Zhengxiang Zhu, Dr. Wenwei Lin, JaWanda Grant, Horrick Sharma, and Peihong Guan for all the help and support that they have given me during the tenure of this work. I would like to thank my colleagues at work and my friends, Himabindu, Sucharita, Bharati, Renuka and Varun, at the University of Tennessee for their support and cooperation. I would also like to thank my husband Dr. Yogeshwar Bachhav for the encouragement and support that he has always given. Finally, I would like to express my sincere gratitude to my parents, parent in-laws, and siblings who have always been a constant source of inspiration and support. iv Abstract Mammalian nucleoside transporters can be classified into two main categories, namely, equilibrative nucleoside transporters (ENTs) and concentrative nucleoside transporters (CNTs). ENTs are ubiquitous, and mediate sodium-independent bi- directional facilitated diffusion nucleoside transport processes. CNTs on the other hand, are secondary active unidirectional transporters that are sodium-dependent. Both the equilibrative and the concentrative nucleoside transporters have several family members which are ENT1 to ENT4 and CNT1 to CNT6. Over the past two decades, important advances in the understanding of nucleoside transporter functions have been made. Identification and molecular cloning of the ENT and CNT families from mammals and protozoan parasites have provided much information about the structure, function, regulation, and tissue and cellular localization. Structure–function analyses of various nucleoside transporter chimeras and mutants have revealed important elements involved in substrate and inhibitor recognition and binding. However, the mechanisms that regulate nucleoside transporters in various tissues and cell types are just beginning to be understood. Because of the ability of these transporters to handle nucleoside analogues used in the treatment of patients with cancer and viral diseases, ongoing research should allow the design of more specifically targeted new compounds or improvements to existing drugs. New drugs are welcome not only in the treatment of cancer and viral diseases, but also in cardiovascular disorders and parasitic infections. Due to the absence of crystal structures and limited information regarding the active sites of nucleoside transporters, the designing of novel inhibitors is confined to ligand-based methods. In an effort to search for novel classes of inhibitors other than the existing ones, a series of 95 different flavone and flavone-like compounds was screened against concentrative nucleoside transporters (CNT 1, 2 and 3) and equilibrative nucleoside transporters (ENT 1 and 2). The results obtained in the form of IC50 values were further utilized to perform quantitative structure–activity relationship studies which indeed helped to understand the effects of different functionalities in the inhibition of nucleoside transporters. The validated 3D-QSAR models were used for design and activity prediction of new compounds. Pharmacophore hypotheses were also generated for hCNT3 using the PHASE pharmacophore mapping program to establish structural criteria for inhibitor design, and for database searching to find new hit molecules. Additionally, fifteen compounds were selected based on SAR and screened for equilibrative nucleoside transporter inhibition for validation of QSAR models. One novel compound, XI was designed with reduced complexity in further attempts to identify the ENT pharmacophore. But the synthetic route followed to prepare compound XI, resulted in the synthesis of compounds XII and XIII, which were evaluated as a mixture and exhibited substantial inhibitory activity against hENT1, but had no significant effect on hENT2 or hCNT3. This work has identified a novel class of CNT and ENT nucleoside transporter inhibitors and delineated structural determinants of potency and transporter subtype selectivity. v Table of Contents Chapter 1: Human Nucleoside Transporters . 1 1.1 Nucleoside Transporters . 1 1.2 Concentrative Nucleoside Transporters . 5 1.2.1 Molecular Characteristics . 5 1.2.2 Tissue Distribution . 7 1.2.3 Transport of Physiologic Nucleosides and Nucleoside Analogs . 7 1.2.4 Molecular Determinants of Concentrative Nucleoside Transporter Substrates . 8 1.2.5 Concentrative Nucleoside Transporter Inhibitors . 9 1.2.5.1 Phloridzin . 9 1.2.5.2 5′-Position Modified Nucleoside Derivatives of Adenosine 9 1.2.5.3 Ribofuranoside Compounds with Benzimidazole Moiety . 10 1.2.5.4 8-Position Modified Purine Nucleoside Derivatives . 10 1.3 Equilibrative Nucleoside Transporters . 11 1.3.1 Molecular Characteristics . 11 1.3.2 Tissue Distribution . 14 1.3.3 Transport of Physiologic Nucleosides and Nucleoside Analogs . 14 1.3.4 Equilibrative Nucleoside Transporter Inhibitors . 16 1.4 Applications of Nucleoside Transporter Inhibitors . 16 1.4.1 Antimetabolite Potentiation . 16 1.4.1.1 Cancer Chemotherapy . 16 1.4.1.2 Viral Infections . 17 1.4.2 Adenosine Potentiation . 17 1.4.2.1 Cardioprotection . 17 1.4.2.2 Ischemic Preconditioning and Heart Transplantation . 17 1.4.2.3 Neuroprotection and Stroke . 18 1.4.2.4 Anti-inflammatory Effects . 18 1.4.3 Host Tissue Protection . 18 1.4.3.1 Protection Against Nucleoside Anticancer Drugs Toxicity . 18 1.4.3.2 Antiprotozoal Chemotherapy . 19 Chapter 2: Research Objectives . 20 Chapter 3: Biological Evaluation and 3D-QSAR Studies on Flavone Analogs for Human Concentrative Nucleoside Transporter Inhibition . 22 3.1 Introduction . 22 3.2 Uridine Uptake Assay . 22 3.3 IC50 Curves of Active Flavones . 22 3.4 Materials and Methods . 30 3.5 Molecular Modeling . 30 3.6 CoMFA and CoMSIA Models . 31 3.7 Results of 3D-QSAR Analyses . 34 vi 3.8 PLS Contour Maps . 38 3.8.1 PLS Contour Interpretation for CNT1 CoMFA . 38 3.8.2 PLS Contour Interpretation for CNT1 CoMSIA . 38 3.8.3 PLS Contour Interpretation for CNT2 CoMFA . 42 3.8.4 PLS Contour Interpretation for CNT2 CoMSIA . 42 3.8.5 PLS Contour Interpretation for CNT3 CoMFA . 42 3.8.6 PLS Contour Interpretation for CNT3 CoMSIA . 42 3.9 Activity Prediction of In silico Designed Compounds Using 3D-QSAR Models for hCNT3. 43 3.10 Discussion . 48 3.11 Conclusion . 49 3.12 Experimental Section: [3H]-Uridine Uptake Assay . 49 Chapter 4: Biological Evaluation and 3D-QSAR Studies on Flavones Analogs for Human Equilibrative Nucleoside Transporter Inhibition . 50 4.1 Introduction . 50 4.2 Biological Evaluation: Uridine Uptake Assay . 50 4.3 3D-QSAR Analyses . 59 4.3.1 Materials and Methods . 59 4.3.2 Molecular Modeling . 59 4.3.3 CoMFA and CoMSIA Models . 59 4.3.4 Results of 3D-QSAR Analyses . 61 4.3.5 PLS Contour Maps . 64 4.3.5.1 PLS Contour Interpretation for ENT1 CoMFA . 72 4.3.5.2 PLS Contour Interpretation for ENT1 CoMSIA . 72 4.3.5.3 PLS Contour Interpretation for ENT2 CoMFA . 72 4.3.5.4 PLS Contour Interpretation for ENT2 CoMSIA . 73 4.4 Activity Prediction of In silico Designed Compounds Using 3D-QSAR Models for hENT. 73 4.5 Evaluation of Related Analogs . 77 4.6 Synthesis and Testing of a New Lead as an Equilibrative Nucleoside Transporter Specific Inhibitor . 77 4.7 Discussion . 77 4.8 Conclusion . 85 4.9 Experimental Section: Chemistry . 85 4.9.1 Reaction Procedure . 86 4.9.2 Mixture of Compounds XII and XIII . 86 Chapter 5: Pharmacophore Mapping of Flavone Derivatives for hCNT3 Inhibition . 87 5.1 Introduction . 87 5.2 Materials and Methods . 87 5.2.1 Preparation of Ligands . 90 5.2.2 Creation of Pharmacophoric Sites . 90 5.2.3 Finding a Common Pharmacophore . 90 vii 5.2.4 Scoring Hypotheses . 90 5.2.5 Building a QSAR Model .