City University of New York (CUNY) CUNY Academic Works All Dissertations, Theses, and Capstone Projects Dissertations, Theses, and Capstone Projects 2-2020 Synthesis and Antitumor Activity of Potentially Tumor Targeting Analogues of the Tetrahydrofuran Containing Acetogenins Patricia Gonzalez Periche The Graduate Center, City University of New York How does access to this work benefit ou?y Let us know! More information about this work at: https://academicworks.cuny.edu/gc_etds/3641 Discover additional works at: https://academicworks.cuny.edu This work is made publicly available by the City University of New York (CUNY). Contact: [email protected] SYNTHESIS AND ANTITUMOR ACTIVITY OF POTENTIALLY TUMOR TARGETING ANALOGUES OF THE TETRAHYDROFURAN CONTAINING ACETOGENINS by PATRICIA GONZALEZ PERICHE A dissertation submitted to the Graduate Faculty in Chemistry in partial fulfilment of the requirements for the degree of Doctor of Philosophy, The City University of New York 2020 i © 2020 PATRICIA GONZALEZ PERICHE All rights reserved ii SYNTHESIS AND ANTITUMOR ACTIVITY OF POTENTIALLY TUMOR TARGETING ANALOGUES OF THE TETRAHYDROFURAN CONTAINING ACETOGENINS by Patricia Gonzalez Periche This manuscript has been read and accepted for the Graduate Faculty in Chemistry In satisfaction of the dissertation requirement for the degree of Doctor of Philosophy ________________________ ______________________________________ David R. Mootoo Date Chair of Examining Committee ________________________ ______________________________________ Brian R. Gibney Date Executive office Supervisory Committee: Ryan P. Murelli Akira Kawamura Naga Vara Kishore Pillarsetty THE CITY UNIVERSITY OF NEW YORK iii ABSTRACT Synthesis and antitumor activity of potentially tumor targeting analogues of the tetrahydrofuran containing acetogenins by Patricia Gonzalez Periche Advisor: Professor David R. Mootoo The tetrahydrofuran containing acetogenins (THF-ACGs) are a naturally occurring class of compounds with potent toxicity against a broad range of tumors, including multidrug resistant (MDR) strains. However, the equally high toxicity to normal cells presents a hurdle for their clinical advancement. This study entails synthetic and tumor targeting strategies that are relevant to their therapeutic development. Chapter 1 presents a review of the synthesis and biological activity of the THF-ACGs. Chapter 2 covers a modular approach for the synthesis of libraries of THF-ACGs. This strategy is illustrated in the synthesis of C-10 epimers of 4-deoxyannonomontacin (4-DAN), one of the more potent monotetrahydrofuran acetogenins. Towards the discovery of synthetically more accessible and tumor-selective analogues, Chapter 3 describes the application of this methodology to analogues of 4-DAN where the THF ring is substituted by mannose with different chain lengths and/or the butenolide is substituted by simpler heterocycles. To address the issue of systemic toxicity, Chapter 4 presents tumor-targeting strategies for the THF- ACGs and their mimics. In the ‘chameleon’ approach the THF moiety was replaced with the 3-O- carbamoyl-mannose residue, present in bleomycin, a family of clinically used anti-tumor agents and believed to be responsible for their tumor selectivity. Therefore, the carbamoyl containing mannose residue could confer both drug potency and selectivity. A prodrug strategy in which a naturally occurring THF-ACG or synthetic analogue is conjugated via a cleavable linker to a tumor vector, is also described. The guiding hypothesis is that following uptake by the tumor cell, the prodrug will be degraded to release iv the active cytotoxic agent. As proof of principle, prodrugs comprising a sugar analogue of 4-DAN or the naturally occurring acetogenin annonacin, 2-[3-(1,3-dicarboxypropyl)ureido] pentanedioic acid (DUPA), a highly specific vector for prostate specific membrane antigen (PSMA) in prostate cancer (PCa), and a traceless disulfide-based linker were synthesized. Chapter 5 presents the cytotoxicity data for the new THF-ACGs and structure activity conclusions. Using an MTT assay at both 16 and 48 hours, the cell viability of the THF-ACGs and their mimetics were evaluated against five cancer cell lines: MDA-MD-231 (triple negative breast), MCF-7 (breast), HCT116 (colon), PC-3 (PSMA negative prostate cancer) and LNCaP (PSMA positive prostate). THF containing analogues generally showed a lower IC50 (ca 0.5-5 micromolar) across the panel of cell lines than ones in which the THF is replaced by a mannose. For the analogues with butenolide replacements, the thiophene-2-carboxamide residue was the most active substitute and their derivatives showed comparable or higher activity than their butenolide parents. The cytotoxicity of sugar mimetics with alkyl ether chains of different lengths and positions was also determined. While individual analogues displayed low micromolar potency and selectivity across the five cell lines tested, no clear structure activity trends emerged. An ACG mimetic in which the THF and butenolide segments were replaced with 3-O- carbamoyl-mannose and thiophene-2-carboxamide residues respectively, showed potent activity in the 0.1-10 M range against LNCaP and PC-3. Although not evaluated, the tumor selectivity of this mimetic is particularly interesting given its structural similarity to the bleomycins. The cytotoxicity activity of the DUPA-linked prodrugs derived from a mannose based ACG mimetic and the naturally occurring ACG annonacin was also evaluated. In the 16 h MTT assay against LNCaP (PSMA positive), the two prodrugs were more active than their parent drugs. However, against the PSMA negative cell lines, MDA-MD-231, MCF-7, HCT116 and PC-3 both prodrugs and their parent drugs showed similar activity and were appreciably less active than for LNCaP. The higher toxicity for LNCaP over the PSMA negative cell lines, albeit modest (ca 2:1), supports the notion that this selectivity is connected to DUPA-PSMA binding, and that PSMA is an internalizing receptor. However, in a 48 h MTT assay, while the cytotoxicity of the prodrugs and parent drugs against MDA-MD-231, MCF-7, HCT116 was similar to that observed at 16 h, the activity of the prodrugs (but not the parent drugs), against PC-3 v increased markedly. This unexpected trend for PC-3 does not align with our prodrug hypothesis and may be an indication that these DUPA-linked ACGs have a different mechanism of action than their parent drugs. More robust studies are needed to resolve this issue, as well as to improve prodrug selectivity. vi “Our greatest weakness lies in giving up. The most certain way to succeed is always to try just one more time.” Thomas Edison vii ACKNOWLEDGEMENTS First, I would like to thank my mentor, Professor Mootoo, for his constant support, patience and for helping me grow both as a scientist and as a person. Thank you, Professor. The opportunity that you provided for undergraduate research inspired me to pursue a Ph.D. I would also like to thank my committee members, Professor Akira Kawamura, Professor Ryan Murelli and Professor Naga Vara Kishore Pillarsetty for their support, guidance and stimulating conversations over the years. To the other members of the Mootoo lab, both past and present. Particularly, Dr. Stewart Bachan, Dr. Ahmad Altiti and Dr. Clayton Mattis, Steven Truong, Amanda Ramdular, Dayanni Bhagwandin, Avelyn de los Reyes, I am forever thankful for your help, encouragement and support over the years. Thanks to everyone in the Chemistry Department at Hunter College. In particular, to Dr. Matthew Devany for his help and patience with NMR characterization. To Professor Donna McGregor, thank you for helping me come from Spain and for being a role model as a strong and successful woman in science. Thank you for constant guidance. I would also like to acknowledge the academic and technical staff in the department. I am very grateful to Graduate Center for creating such a supportive work environment and for allowing me the opportunity to earn a Ph.D. In particular, I would like to acknowledge my classmates for their support. To my friends in New York, my family away from home, thank you for the good times, the support and the endless encouragement. To my friends and family back home, thank you for always being there, even with an ocean in between us. To my mom and my sister, thank you for being there every step of the way and for helping me get where I am today. viii ix TABLE OF CONTENTS ABSTRACT.................................................................................................................................................. iv ACKNOWLEDGEMENTS .......................................................................................................................... viii TABLE OF CONTENTS ............................................................................................................................... x LIST OF SYMBOLS AND ABBREVIATIONS ........................................................................................... xiii LIST OF CANCER CELL LINES .............................................................................................................. xvii LIST OF FIGURES .................................................................................................................................. xviii LIST OF SCHEMES .................................................................................................................................... xx LIST OF TABLES .....................................................................................................................................