PART 1. SYNTHESIS OF N-15 LABELED (R)-DEUTERIOGLYCINE PART 2. SYNTHESES OF CARBON-LINKED ANALOGS OF RETINOID GLYCOSIDE CONJUGATES DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Joel R. Walker * * * * * The Ohio State Univeristy 2003 Dissertation Committee: Approved by Robert W. Curley, Jr., Ph.D., Adviser Werner Tjarks, Ph.D. ________________________ Adviser Karl A. Werbovetz, Ph.D. College of Pharmacy ABSTRACT (R)-Glycine-d-15N has been used to permit assignments of the prochiral α-protons of glycine residues in the FK-506 binding protein. A key and low yielding step in the synthetic route to (R)-glycine-d-15N occurred in the ruthenium tetraoxide-mediated degradation of N-t-BOC-p-methoxybenzyl amine to the N-t-BOC-glycine after both 2H and 15N are incorporated. In order to improve this step, investigation of the oxidation reaction conditions along with various aromatic ring carboxylate precursors were undertaken. It was found that using ruthenium chloride, periodic acid as the stoichiometric re-oxidant, and N-(p-methoxyphenylmethylamine)-2,2,2-trichloroethyl carbamate were the optimal conditions and substrate. This improvement was paramount for the applicability of this route for large scale production of labeled glycine that could be used in other biological applications. The retinoic acid analog N-(4-hydroxyphenyl)retinamide (4-HPR) is an effective chemopreventative and chemotherapeutic for numerous types of cancer. In vivo, 4-HPR is metabolized to 4-HPR-O-glucuronide (4-HPROG), which has been shown to be more effective than the parent molecule in rat mammary tumor models. To investigate whether 4-HPROG was an active agent, the carbon linked analog (4-HPRCG) was synthesized ii and subsequently found to be a more effective chemopreventative than 4-HPROG or 4- HPR. In the original synthesis of 4-HPRCG, the route to a key C-glycoside was lengthy and inefficient. In order to investigate 4-HPRCG as a chemotherapeutic, the synthesis was redesigned and significantly improved by access to a key C-benzyl-glucuronide intermediate through employment of a Suzuki coupling reaction between an exoanomeric methylene sugar and an aryl bromide. Subsequently, 4-HPRCG was tested in an animal model and shown to possess effective chemotherapeutic actions. In vivo, potential cleavage of the amide bond of 4-HPR would liberate retinoic acid, which may explain some of its side effects. This same cleavage may occur with 4- HPRCG, thus the fully carbon linked analog of 4-HPROG (4-HBRCG) was proposed and synthesized. The synthetic route for 4-HBRCG focused on the production of a key C- benzylbromide-glucuronide obtained through the employment of Suzuki coupling chemistry. Once the benzylbromide was obtained, a key alkylation of a retinal Umpolung derivative yielded the carbon skeleton for 4-HBRCG. Subsequent biological testing will reveal the actions and potency of 4-HBRCG. iii Dedicated to Mom, Dad, and Aphayphone iv ACKNOWLEDGMENTS For all the guidance, training, assistance, and answered questions, I would like to thank my advisor, Dr. Robert W. Curley, Jr. From your instruction, I will take with me patience, critical thinking, ethics, and a little bit of musical talent. Also, I would like to thank the rest of the Medicinal Chemistry faculty for their teaching and experience, including John Fowble and Charles Cottrell. I would also like to show appreciation for Joan Dandrea and Kathy Brooks for their help with administrative matters. I am grateful to my classmates, Dave, James, and Tanit, for all your brainstorming during our coursework. Often times, it seemed I would not have made it through without your assistance. I would like to thank Young for his lightning speed with fresh chemistry ideas and on the basketball court. Also, I would like to recognize former and present students in the department for enriching both my academic and social lives. I would like to acknowledge my fellow dungeon dwellers, Derek, Kevin, and Serena, for their help in numerous matters. From physical labor to mental exercises to bouncing off the walls, we managed to sneak in lots of fun. Kevin, thanks for all the sound advice and the standards, “Is it in the literature?” and “What about a model reaction?” I would especially like to recognize Derek for being detail oriented, logical, and always available for assistance in and out of the lab. Also thanks for being the comic v relief of the group and an associate member of Benzene. Without your solid rhythm section, Benzene would have been especially horrible. I am grateful that we became good friends over the infinite amount of time we were locked up together. Let’s just say, I’ll never forget your birthday. Finally, I am eternally grateful for the support given to me from my wife, Aphayphone. Between the long hours studying, the long hours in the lab, and the occasional basketball game, you probably were tired of wondering, “When is he coming home?” Without your sacrifices, this goal would not have been reached. Also, I would like to thank my Dad for his unwavering encouragement to persevere until the problems were solved. Furthermore, I would like to thank the rest of my family, my wife’s family, and all my friends for asking every month or so, “When are you graduating?” vi VITA May 11, 1972……………………….Born – Muncie, Indiana 1995....……………………………...B.S. Chemistry, University of Nevada, Las Vegas 1994-1997…………………………..Associate Scientist, Lockheed Environmental Systems and Technologies Company Las Vegas, Nevada 1997-2003…………………………..Teaching and Research Assistant, The Ohio State University PUBLICATIONS Steinberg, S.; Walker, J. R. Colorimetric Analysis of Benzene for Use in Environmental Screening. Chemosphere 1995, 31, 3771-3781. Walker, J. R.; Curley, R. W., Jr. Improved Synthesis of (R)-Glycine-d-15N. Tetrahedron 2001, 57, 6695-6701. Walker, J. R.; Alshafie, G.; Abou-Issa, H.; Curley, R. W., Jr. An Improved Synthesis of the C-Linked Glucuronide of N-(4-Hydroxyphenyl)retinamide. Bioorg. Med. Chem. Lett. 2002, 12, 2447-2450. vii FIELDS OF STUDY Major Field: Pharmacy Specialization: Medicinal Chemistry viii TABLE OF CONTENTS Page Dedication………………………………………………………………………………...iv Acknowledgments…………………………………………………………………………v Vita……………………………………………………………………………………....vii List of Figures…………………………………………………………………………...xiii List of Schemes……………………………………………………………………….....xvi List of Spectra………………………………………………………………………….xviii List of Tables…………………………………………………………………………....xix List of Abbreviations…………………………………………………………………….xx Part 1 – Synthesis of N-15 Labeled (R)-Deuterioglycine Chapter 1 – Stereospecific Synthesis of Doubly Labeled (R)-2H,15N-Glycine 1.1 Introduction……………………………………………………………………......2 1.1.1 NMR Theory………………………………………………………………2 ix 1.1.2 Uses of Labeled Amino Acids in Enzymology…………………………....4 1.1.3 Uses of Labeled Amino Acids in Protein NMR…………………………..5 1.1.4 Uses of Labeled Chiral Glycine…………………………………………...8 1.2 Synthesis of Chiral Glycines……………………………………………………..11 1.3 Synthesis of Doubly Labeled Glycine…………………………………………...12 1.4 Conclusions………………………………………………………………………27 Part 2 – Syntheses of Carbon-linked Analogs of Retinoid Glycoside Conjugates Chapter 2 – Introduction for Retinoids 2.1 Discovery…………………………………………………………………….......30 2.2 Structures…………………………………………………………………….......31 2.3 Natural and Synthetic Sources of Vitamin A………………………………….....33 2.4 Absorption, Storage, and Metabolism……………………………………….......35 2.5 Biological Effects of Retinoids………………………………………………......39 2.5.1 Vision………………………………………………………………….....39 2.5.2 Growth and Differentiation……………………………………………....42 2.5.3 Embryonic Development…………………………………………….......43 2.5.4 Immune System……………………………………………………….....44 2.6 Cellular Mechanisms of Retinoids…………………………………………….....45 2.7 Therapeutic Vitamin A and Retinoids……………………………………….......48 2.8 Toxicity of Retinoids………………………………………………………….....52 2.9 Synthetic Retinoids……………………………………………………………....53 2.9.1 Receptor Dependent Retinoids………………………………………......53 2.9.2 Receptor Independent Retinoids……………………………………........56 2.10 N-(4-Hydroxyphenyl)retinamide………………………………………………...58 2.11 N-(4-Hydroxyphenyl)retinamide-O-glucuronide………………………………...60 x Chapter 3 – Synthesis of the C-Linked Analog of 4-HPR-O-glucuronide 3.1 Rationale…………………………………………………………………………64 3.2 C-Glycosides……………………………………………………………………..65 3.3 Previous Studies with 4-HPR-C-glucuronide……………………………………69 3.4 Redesign of the Synthetic Route to 4-HPRCG…………………………………..74 3.4.1 Carbon Monoxide Insertion Route………………………………….........74 3.4.2 Suzuki Coupling Route…………………………………………………..79 3.5 Synthesis of 4-HPRCG………………………………………………………......79 3.6 Biological Evaluation of 4-HPRCG………………………………………….......92 3.7 Conclusions……………………………………………………………………....96 Chapter 4 – Synthesis of the Fully C-Linked Analog of 4-HPR-O-glucuronide 4.1 Rationale……………………………………………………………………........97 4.2 Retinoid C-Glycosides……………………………………………………….....100 4.3 Synthesis of 4-HBRCG………………………………………………………....100 4.4 Synthesis of 4-HBRC-glucoside……………………………………………......114 4.5 Biological Evaluation of 4-HBRCG…………………………………………....114 4.6 Efforts toward the Synthesis of Retinoyl-β-C-glucuronide…………………….117 4.7 Conclusions……………………………………………………………………..120 Chapter 5 – Summary and Conclusions 5.1 Part 1……………………………………………………………………………122 5.2 Part 2……………………………………………………………………………125 Chapter 6 – Experimental Section 6.1 General Methods………………………………………………………………..131 6.2 Glycine Synthesis…………………………………………………………….....132 xi 6.3 4-HPRCG