Duquesne University Duquesne Scholarship Collection Electronic Theses and Dissertations 2013 Classical Antifolates: Synthesis of 5-Substituted, 6-Substituted and 7-Substituted Pyrrolo[2,3-d]Pyrimidines as Targeted Anticancer Therapies Yiqiang Wang Follow this and additional works at: https://dsc.duq.edu/etd Recommended Citation Wang, Y. (2013). Classical Antifolates: Synthesis of 5-Substituted, 6-Substituted and 7-Substituted Pyrrolo[2,3-d]Pyrimidines as Targeted Anticancer Therapies (Doctoral dissertation, Duquesne University). Retrieved from https://dsc.duq.edu/etd/1335 This Immediate Access is brought to you for free and open access by Duquesne Scholarship Collection. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Duquesne Scholarship Collection. For more information, please contact [email protected]. CLASSICAL ANTIFOLATES: SYNTHESIS OF 5-SUBSTITUTED, 6-SUBSTITUTED AND 7-SUBSTITUTED PYRROLO[2,3- d]PYRIMIDINES AS TARGETED ANTICANCER THERAPIES A Dissertation Submitted to the Graduate School of Pharmaceutical Sciences Duquesne University In partial fulfillment of the requirements for the degree of Doctor of Philosophy By Yiqiang Wang May 2013 Copyright by Yiqiang Wang 2013 Name: Yiqiang Wang Dissertation: CLASSICAL ANTIFOLATES: SYNTHESIS OF 5-SUBSTITUTED, 6-SUBSTITUTED AND 7-SUBSTITUTED PYRROLO[2,3-d]PYRIMIDINES AS TARGETED ANTICANCER THERAPIES Degree: Doctor of Philosophy Date Feb 06, 2013 APPROVED & ACCEPTED Aleem Gangjee, Ph.D. (Committee Chair) Professor of Medicinal Chemistry Mylan School of Pharmacy Distinguished Professor Graduate School of Pharmaceutical Sciences Duquesne University, Pittsburgh, Pennsylvania APPROVED Marc W. Harrold, Ph.D. (Committee Member) Professor of Medicinal Chemistry Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, Pennsylvania APPROVED Patrick T. Flaherty, Ph.D. (Committee Member) Associate Professor of Medicinal Chemistry Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, Pennsylvania APPROVED David J. Lapinsky, Ph.D. (Committee Member) Assistant Professor of Medicinal Chemistry Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, Pennsylvania APPROVED Lawrence H. Block, Ph.D. (Committee Member) Professor emeritus of Pharmaceutics Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, Pennsylvania APPROVED Douglas J. Bricker, Ph.D. Dean of Mylan School of Pharmacy, Professor of Pharmacology-Toxicology Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, Pennsylvania iii ABSTRACT CLASSICAL ANTIFOLATES: SYNTHESIS OF 5-SUBSTITUTED, 6-SUBSTITUTED AND 7-SUBSTITUTED PYRROLO[2,3- d]PYRIMIDINES AS TARGETED ANTICANCER THERAPIES By Yiqiang Wang May 2013 Dissertation supervised by Professor Aleem Gangjee, Ph.D. This dissertation comprises an introduction, background and current research progress in the area of classical antifolates as the targeted anticancer therapies. Mammalian cells have a sophisticated folate uptake and retention system. Transport into the cell is usually facilitated by one or more of three folate transporters: reduced folate carrier (RFC), the membrane folate receptor (FR), and the proton coupled folate transporter (PCFT). The expression of RFC in both normal and tumor cells presents a potential obstacle to antitumor selectivity. Thus, it is of interest to design targeted antifolates that are substrates for transporters other than RFC to restrict drug transport into normal tissues selectivity. This rationale provides the foundation to develop targeted agents that can be selectively transported into tumors by FRs and PCFT over RFC, with potent intracellular targets inhibition. iv At the same time, it has been of interest not only to design potent antifolates against specific enzymes such as dihydrofolate reductase (DHFR), thymidylate synthase (TS), glycinamide- ribonucleotide formyl transferase (GARFTase) and amino-imidazole- carboxamide-ribonuleotide formyl transferase (AICARFTase) but also to design and synthesize single agents that have potent multiple inhibitory activity against these enzymes. This strategy is particularly promising in anticancer chemotherapy against the multiple drug resistant cancers. Such a single agent could act at more than one active site and provide “combination chemotherapy” benefits. In this study, twelve series of classical 5-, 6- and 7-substituted pyrrolo[2,3- d]pyrimidines were designed and synthesized. Extensive structure modifications of the pyrrolo[2,3-d] pyrimidine scaffold were investigated to determine selective transport via FR or/and PCFT and tumor targeted antifolates with GARFTase or multiple folate metabolizing enzyme inhibition. The design strategies employed include: variation of the side chain substitution position (5-,6- and 7-substituted); variation of the side chain length (n=1-6); isosteric replacement of the 1,4-disubstituted phenyl ring with 1,2- and 1,3- disubstituted phenyl ring and 2,5- disubstituted thiophenyl ring; replacement the L-glutamate with variation at the α and γ carboxylic acids. As a part of this study, a total of one hundred and fifty six new compounds (including new intermediates) were synthesized and separated. Of these, twelve series consisting of forty two classical antifolate final compounds were submitted for biological evaluation. In addition, bulk synthesis of some potent final compounds (2, 2.0 g; 161, 500 mg; 175, 1.0 g; 166, 500 mg; 194, 500 mg) was carried out to facilitate in vivo evaluation. v During the synthesis of the target compounds, several synthetic improvements were achieved successfully including: 1. α-Bromo ketones instead of α-chloro ketones were synthesized to react with 2,4- diamino-6-hydroxypyrimidine to selectively afford pyrrolo[2,3-d]pyrimidines without side product furo[2,3-d]pyrimidines. 2. Instead of using the reported reaction condition to get 72% yield in the hydrogenation of 235, a 10% Pd/C, 5 h condition was employed to get a complete transformation (100% yield of 236) without any partial reduction. The troublesome separation of 236 was avoided. More importantly, a new Heck coupling of the thiophene iodide 301 and allyl alcohols to synthesize aldehydes in one step was discovered. The reaction condition is mild (45 oC) with a good yield (65%) and the labile ester group of 301 is tolerated at this condition. In addition, the reaction is fast (2 h) and easy to handle (no argon protection needed). Due to its potential use in analog synthesis of clinically used antifolates such as RTX and PMX, this mild conditioned and easy to handle Heck coupling reaction is highly attractive. During this study, the SAR of folate transporters (RFC, FR and PCFT) and GARFTase inhibitors were extensively explored. The 6-substituted straight chain compound 166 (n=7) was extremely potent against KB tumor cells (IC50=1.3 nM, about 80-fold more potent than clinically used PMX) without any RFC activity. The intracellular enzyme target of 166 was subsequently identified as GARFTase. The 5- substituted phenyl compound 175 (n=4) showed AICARFTase as the primary target with potent KB tumor cell inhibition (IC50=7.9 nM, about 8-fold more potent than PMX) and vi also indirectly activated AMPK cell signaling pathway via ZMP accumulation which transmits an inhibitory signal to the mTOR complex leading to tumor cell apoptosis. Both of these compounds were selected for animal study to determine the antitumor activity against human tumor xenograft in mice. Due to their potent antitumor activities, these two compounds serve as leads for future structural optimization. vii DEDICATION Dedicated To My Family For Their Love And Support viii ACKNOWLEDGEMENT I would like to thank all of those who contributed to this work. In particular I am most grateful to my supervisor, Professor Dr. Aleem Gangjee, for his guidance and support which made this dissertation possible. I am indebted to him not just for his scientific guidance but also for his encouragement, day after day, financial support and his friendship. I would like to thank my dissertation committee: Drs. Marc W. Harrold, Patrick T. Flaherty, David J. Lapinsky, Lawrence H. Block and Aleem Gangjee for their advice and support. I wish to thank Dr. Larry H. Matherly at Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine and Dr. Roy L. Kisliuk at Tufts University School of Medicine for evaluating all the compounds for enzyme inhibitory activity and antitumor activity. I wish to express my sincere appreciation to Nancy Hosni and Jackie Farrer in the office of pharmacy school for their help and assistance. I would like to give thanks to all my colleagues in the Graduate School of Pharmaceutical Sciences for their help and stay with me at Duquesne University. I would like to thank the Graduate School of Pharmaceutical Sciences for the financial support. Finally, I would like to express my love and gratitude to my beloved family for their understanding & endless love, through the duration of my studies. ix TABLE OF CONTENTS Page Abstract .......................................................................................................................... iv Dedication .................................................................................................................... viii Acknowledgement .......................................................................................................... ix List of Tables ................................................................................................................