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Developing Small Molecule Inhibitors of ALK2: a Serine/Threonine Kinase Implicated in Diffuse Intrinsic Pontine Glioma

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Developing Small Molecule Inhibitors of ALK2: a Serine/Threonine Kinase Implicated in Diffuse Intrinsic Pontine Glioma Developing Small Molecule Inhibitors of ALK2: a Serine/Threonine Kinase Implicated in Diffuse Intrinsic Pontine Glioma by Deeba Ensan A thesis submitted in conformity with the requirements for the degree of Master of Science Pharmacology and Toxicology University of Toronto © Copyright by Deeba Ensan 2020 Developing Small Molecule Inhibitors of ALK2: a Serine/Threonine Kinase Implicated in Diffuse Intrinsic Pontine Glioma Deeba Ensan Master of Science Pharmacology and Toxicology University of Toronto 2020 Abstract Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric cancer for which no effective chemotherapeutic drugs exist. Analysis of the genomic landscape of this disease has led to the identification of the serine/threonine kinase ALK2 as a potential target for therapeutic intervention. In this work, we developed two separate series of potent type I inhibitors of ALK2 based on the previously reported inhibitor LDN-214117. The first structure-activity relationship (SAR) study focuses on improving the selectivity, permeability and pharmacokinetic profile of M4K2149, a benzamide analogue with reduced off-target affinity for the hERG potassium channel. The second part of this thesis highlights the efforts made to develop a conformationally constrained inhibitor that was rigidified into the biologically active configuration of M4K2009, the lead compound of this project. Future studies will assess the permeability of select compounds in a Caco-2 permeability assay and their PK profiles in vivo. ii Acknowledgments Completing my Master’s degree at the Ontario Institute for Cancer Research (OICR) was truly an eye-opening experience. The beginning of my training was most certainly taxing. However, the support I received from the Drug Discovery team facilitated my adaptation to a new environment. As time went on, I discovered that I possessed competencies and capabilities of which I was previously unaware. There were many individuals at the institute who helped me to realize my potential. To them, I am much obliged. I would like to primarily thank Dr. Carlos A. Zepeda-Velázquez for being my mentor for the past two years. Carlos was instrumental in bringing my research project to fruition. Drug discovery and design is quite a niche field, incorporating elements of biochemistry, physiology, pharmacology and organic chemistry. It is a subject that is not taught in university. Therefore, Carlos ensuring that I was able to fully grasp its core principles was no small feat. From setting aside time to answer my inquiries to helping me troubleshoot reactions and develop synthetic methods, Carlos was able to instill in me the importance of scientific reasoning, critical thinking and independence. I am grateful to Dr. David Smil for his guidance throughout the ALK2 project and for actively promoting the exchange of ideas related to drug discovery and research. I also acknowledge Babu Joseph, Dr. Iain D. G. Watson, Brian J. Wilson, Dr. Methvin B. Isaac, Dr. Taira Kiyota, Dr. Shiva Kalhor-Monfared and Julie Owen for their invaluable assistance. Lastly, I wish to express my deepest gratitude to Dr. Rima Al-awar for giving me the opportunity to complete my graduate studies in the Drug Discovery Program at the OICR. Rima created an environment conducive to student learning and always took the time to ensure that I was making progress throughout my degree. The work presented herein could not have been done without the continuous support of my parents, sister and friends. They were always there when I needed them most. iii Table of Contents Acknowledgments.......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables ................................................................................................................................. vi List of Figures ............................................................................................................................... vii List of Abbreviations ................................................................................................................... viii Chapter 1 Introduction .....................................................................................................................1 Introduction .................................................................................................................................1 1.1 Diffuse Intrinsic Pontine Glioma .........................................................................................1 1.1.1 Pathology and Prognosis ..........................................................................................1 1.1.2 Current Standard of Care and Prospective Therapies ..............................................1 1.1.3 Epigenetic Drug Targets ..........................................................................................3 1.1.4 Activin receptor-like kinase 2 (ALK2) ....................................................................5 1.2 Kinase inhibitors ..................................................................................................................7 1.2.1 Targeting Kinases: Strengths and Limitations .........................................................7 1.2.2 Brain Penetrant Kinase Inhibitors ............................................................................9 1.3 Study Objective ..................................................................................................................11 Chapter 2 Structure Activity Relationship of M4K2149 ...............................................................15 Structure Activity Relationship of M4K2149 ...........................................................................15 2.1 Chemical Syntheses ...........................................................................................................15 2.2 Results and Discussion ......................................................................................................22 2.2.1 Binding Mode of M4K2149 with ALK2 ...............................................................22 2.2.2 Optimizing Potency and Selectivity.......................................................................23 2.2.3 Caco-2 Studies .......................................................................................................29 iv 2.2.4 Pharmacokinetic Studies and Assessment of Off-target Activity ..........................32 2.2.5 Exploring Alternative Ortho Substituents .............................................................34 Chapter 3 Structure Activity Relationship of Conformationally Constrained Derivatives of M4K2009 ..................................................................................................................................38 Structure Activity Relationship of Conformationally Constrained Derivatives of M4K2009 ..................................................................................................................................38 3.1 Chemical Syntheses ...........................................................................................................38 3.2 Results and Discussion ......................................................................................................41 3.2.1 Binding Mode of M4K2009 with ALK2 ...............................................................41 3.2.2 Optimizing Potency and Selectivity.......................................................................43 Chapter 4 Conclusions and Future Studies ....................................................................................48 Conclusions and Future Studies ................................................................................................48 Chapter 5 Synthetic Procedures and Compound Characterizations ..............................................50 Synthetic Procedures and Compound Characterizations ..........................................................50 5.1 Chemical Syntheses ...........................................................................................................50 5.2 NMR Spectra .....................................................................................................................85 References ....................................................................................................................................119 Appendices ...................................................................................................................................131 Copyright Acknowledgements.....................................................................................................151 v List of Tables Table 1: Target values of lead ALK2 inhibitor 13 Table 2: Inhibitory and off-target activities of 14a-b, 7a-b and 8a 25 Table 3: Inhibitory and off-target activities of 8b-c, 18a-b, 20a-e and 31b 26 Table 4: Inhibitory and off-target activities of 2-fluoro-6-methoxybenzamide and 2,6- dimethoxybenzamide analogues, 26a-f 30 Table 5: In vitro permeability and oral in vivo PK studies of 2-fluoro-6-methoxybenzamide analogues 33 Table 6: Inhibitory activity of benzamide analogues against WT and DIPG-linked mutant forms of ALK2 34 Table 7: Inhibitory and off-target activities of 31a, 31c-f and 36 36 Table 8: Caco-2 permeability of 31a and 31e-f 37 Table 9: Inhibitory and off-target activities of M4K2009 and
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