A Thesis Entitled The Attempted Synthesis of Carba-Nicotinic Acid Mononucleotide Methyl Ester using the Zincke Reaction By Peng Zhao Submitted to the Graduate Faculty as partial fulfillment of the requirement for The Master of Science Degree in Medicinal Chemistry James T. Slama, Ph.D., Committee Chair L.M. Viranga Tillekeratne, D.Phil., Committee Member Isaac T. Schiefer, Ph.D., Committee Member Amanda Bryant-Friedrich, Ph.D. rer. Nat., Dean College of Graduate Studies The University of Toledo May 2017 Copyright 2017, Peng Zhao This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An abstract of The Attempted Synthesis of Carba-Nicotinic-Acid Mononucleotide Methyl Ester using the Zincke Reation Peng Zhao Submitted to the Graduate Faculty as partial fulfillment of the requirement for The Master of Science Degree in Medicinal Chemistry The University of Toledo May 2017 According to the retrosynthetic analysis, the carba-nicotinic acid mononucleotide methyl ester can be synthesized from a nucleoside made using a primary amine and a Zincke salt by the Zincke reaction. The primary amine which was the cyclopentane analog of 1-aminoribose was synthesized following the procedure described by Cermak and Vince in 1981 starting from the Vince lactam. The pyridinium part was synthesized as a Zincke salt. However carba-nicotinic acid mononucleotide methyl ester cannot be synthesized using this procedure due to the unanticipated high reactivity of the nicotinic acid esters with primary amine during the Zincke reaction. We found that pyridinium-3-carboxylic esters reacted rapidly with amines resulting in the formation of amids. A series of Zincke reaction were performed to study the Zincke reaction. Even the most sterically hindered protecting group tert-butyl ester could not prevent the formation of nicotinamide. iii Acknowledgments I am deeply grateful to my venerable advisor Dr. James T. Slama, for his valuable academic guidance, continued encouragement and considerate understanding. Without his enlightening instruction, impressive kindness and patience, I could not have completed my researches and my degree. His knowledge and guidance enlightens me not only in this period of study but also in my future study and my future life. I also would like to thank him for his help and support in many other ways during my study at Toledo. I would also like to thank all my teachers and all professors in the College of Pharmacy and Pharmaceutical Sciences who have helped me to develop the fundamental and essential academic competence. Last but not least, I'd like to thank my families and all my friends for their encouragement and support. Peng Zhao The University of Toledo May 2017 iv Table of Contents Abstract …………………….………………………………………………………...iii Acknowledgments.………………………………………………………………….iv Table of Contents……………………………………………………………………v List of Figures .....……………….……………………………………………….vi List of Abbreviations………………….…………………………………………….vii I. Introduction….……………..…………….………………………………………….1 II. Results and discussion………………….…………………………………………10 III. Experimental section……………..………………………………………………16 References…………………..………..……………………………………………29 Appendices: 1H-NMR and 13C-NMR spectra of compounds in Experimental Section………………………………………………………………………...…...…32 v List of Figures Figure 1. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP).…...…………..1 Figure 2. Nicotinamide Adenine Dinucleotide Phosphate (NADP)…..……...…...…..2 Figure 3. Synthesis of NAADP analogs using the enzyme catalyzed pyridine base-exchange reaction…………….………….……….….…...……….…..5 Figure 4. Preparation of NAADP and NAADP derivatives using NAD kinase and cyclic ADP-ribose synthetase……………………………….….......……....6 Figure 5. Carba-Nicotinc Acid Adenine Dinucleotide Phosphate (NAADP)…......…..6 Figure 6. Retrosynthetic analysis of carba-nicotinic acid mononucleotide methyl ester...............................................................................................................7 Figure 7. Zincke reaction……...……………………….................…..….……..……..8 Figure 8. Propose synthesis of 6 from the Vince lactam (8)..........................................8 Figure 9. Propose thesis of 4 by Zincke reaction followed by a selective phosphorylation………………………………………………….…...…….9 Figure 10. Zincke salt synthesis……...…………………………………...………….11 Figure 11. Zincke reaction using reagents 6 and 7…………………….….…...…….12 Figure 12. Zincke reaction to prove the activity of nicotinate……….……..………..13 Figure 13. Total synthesis of 17…………...………………………….….…………..16 Figure 14. The study of the Zincke reaction………………………..………………..17 vi List of Abbreviations cADPR…………Cyclic adenosine diphosphate ribose DI water………..Deionized water DMAP………….Dimethylamino pyridine DMF……………N,N-dimethylformamide DMP……………2,2-Dimethoxypropane DMSO………….Dimethyl sulfoxide ER……………...Endoplasmic reticulum IP3……………...Inositol 1,4,5-trisphosphate NAADP………...Nicotinic acid adenine dinucleotide phosphate NADP…………..Nicotinamide adenine dinucleotide phosphate NMN……………Nicotinamide mononucleotide NMO……………N-Methylmorpholine N-oxide OsO4……………Osmium tetroxide PTSA…………...p-Toluene sulfonic acid monohydrate SR………………Sarcoplasmic reticulum TFA…………….Trifuoroacetic acid THF…………….Tetrahydrofuran TLC…………….Thin-layer chromatography vii Chapter One Introduction Nicotinic acid adenine dinucleotide phosphate (NAADP, 1) (Figure 1) is an intracellular second messenger for calcium ion release. NAADP was first identified as a compound that has a potent intracellular calcium ion mobilizing effect in sea urchin eggs (Lee & Aarhus, 1995), and later NAADP was shown to have the same effect in mammals (Cancela et al., 1999). 3 nicotinic acid mononucleotide half adenosine-2′,5′-diphosphate half 2′ 1 Figure 1. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP, 1) There are three major calcium ion mobilizing messengers (Bootman, et al., 2002). They are inositol 1,4,5-trisphosphate (IP3), cyclic adenosine diphosphate ribose (cADPR) and NAADP. IP3 was discovered 1983 and it was shown to be capable of releasing calcium ion from non-mitochondrial stores in pancreatic acinar cells (Streb, et al., 1983). IP3 mediates calcium ion release by binding to IP3 receptor on a ligand-gated calcium channel on endoplasmic reticulum (ER) which releases calcium 1 ion into the cytoplasm (Barrett, et al., 2009). Studies in sea urchin eggs showed that the pyridine nucleotides, NAD and NADP (2) could mediate calcium ion release from Figure 2. Nicotinamide Adenine Dinucleotide Phosphate (NADP, 2) membranous stores which are different from IP3 (Clapper, et al., 1987). Further studies showed that the effect was caused by the NAD metabolite: cyclic adenosine diphosphate ribose (cADPR) (Lee, et al., 1989) and the NADP metabolite nicotinic acid dinucleotide phosphate (NAADP) (Lee & Aarhus, 1995). IP3 and cADPR were shown to act on the two know ER calcium ion release channels, IP3Rs (IP3 receptors) and RyRs (ryanodine receptors) (Galione, et al., 1991) respectively. NAADP was initially isolated and identified as a contaminant in NADP, and was shown to release calcium ion from intracellular vesicles from sea urchin eggs (Clapper, et al., 1987). The difference between NAADP and NADP is that the nicotinamide base of the NADP is replaced by nicotinic acid in NAADP. This slight difference in molecular structure is essential for calcium ion releasing activity and 2 produces a high degree of discrimination in biological activities. NAADP was found to release calcium ion by a pharmacologically distinct mechanism from different subcellular fractions of egg homogenate, but the precise target for NAADP is unknown. Among these three major calcium mobilizing messengers, NAADP is the most potent, active at picomolar or low-nanomolar concentrations (Lee, 1997), and releasing calcium ion from reserve granules in the sea urchin egg and from lysosome like acidic vesicles in mammalian cells. NAADP mobilizes calcium ion from acidic stores. NAADP induces alkalinization of acidic stores in sea urchin eggs (Morgan & Galione, 2007). The concentration response relationship for NAADP mediated calcium ion release from mammalian cells is a bell shaped (Pitt & Funnell, 2010), indicating that the receptor might possess high affinity stimulatory and low affinity inhibitory binding sites. NAADP could therefore inactivate its response at a high concentrations. The carboxyl group at 3-position of the pyridine ring, the amino group of the adenine ring and the 2′-phosphate are all important for the biological activity of NAADP (Lee, 1997). The first important site is the 2′-phosphate. Lee (1997) claimed that the removal of the 2′-phosphate resulted in the loss of agonist activity of NAADP. Attaching a caging group to the 2′-phosphate similarly produces an inactive analog that can regenerate NAADP on irradiation with ultra violet light and cause a large calcium ion concentration change in many cells which provides additional evidence that the NAADP is a messenger for calcium ion mobilization. Therefore, the 2′-phosphate is essential for receptor recognition. However, the 2′-phosphate could be modified without completely losing its activity. If the 2′-phosphate was changed to 3′-phosphate or cyclically linked to both positions as a 2′,3′-cyclic phosphate the molecule will retain activity with a lower potency (Lee and Aarhus, 1997). The 3 second important site is the 3-carboxylate on pyridine ring. Billington et al. (2005) suggested that the high selectivity