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Small‐Molecule Inhibitors of Dna Polymerase Function

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Small‐Molecule Inhibitors of Dna Polymerase Function SMALL‐MOLECULE INHIBITORS OF DNA POLYMERASE FUNCTION DISSERTATION zur Erlangung des Akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Tobias Strittmatter aus Küssaberg‐Kadelburg an der Universität Konstanz Mathematisch‐Naturwissenschaftliche Sektion Fachbereich Chemie 2014 Tag der mündlichen Prüfung: 05. Dezember 2014 Prüfungsvorsitz: Prof. Dr. Gerhard Müller 1. Referent: Prof. Dr. Andreas Marx 2. Referent: Prof. Dr. Thomas U. Mayer Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-274434 „Meinen Eltern” Danksagung Die vorliegende Arbeit entstand in der Zeit von November 2009 bis August 2014 in der Arbeitsgruppe von Prof. Dr. Andreas Marx am Lehrstuhl für Organische und Zelluläre Chemie am Fachbereich Chemie der Universität Konstanz. Nach diesem Zeitraum geprägt von intensiver Forschungsarbeit liegt nun meine Dissertation vor und ein weiteres Kapitel meiner beruflichen Karriere kann nun erfolgreich abgeschlossen werden. Aus diesem Grund ist es jetzt auch an der Zeit, mich bei all denen zu bedanken, die mich während dieser spannenden Zeit begleitet, unterstützt und gefördert haben. Meinem Doktorvater Prof. Dr. Andreas Marx danke ich ganz herzlich für die Überlassung der sehr interessanten und interdisziplinären Themenstellung, sowie für das in mich gesetzte Vertrauen, welches mir viel Raum für die selbstständige Bearbeitung und kreative Gestaltung des Themas erlaubte. Ihm, wie auch den weiteren Mitgliedern meines „Thesis Committee“, Prof. Dr. Thomas Mayer und Herrn Prof. Dr. Michael Berthold, danke ich zudem für die anregenden wissenschaftlichen Diskussionen und die geleisteten Hilfestellungen. Prof. Dr. Thomas Mayer danke ich außerdem für die Übernahme des Zweitgutachtens und Herrn Prof. Dr. Gerhard Müller für die Übernahme des Prüfungsvorsitzes. Natürlich möchte ich mich auch bei allen früheren und jetzigen Freunden und Kollegen der Arbeitsgruppe Marx und der Graduiertenschule Chemische Biologie für die unvergessliche Zeit, die tolle Arbeitsatmosphäre und die Hilfsbereitschaft bedanken. Besonderer Dank gilt hier Dr. Norman Hardt, Magdalena Grzywa, Matthias Drum und Dr. Nina Blatter für die sehr gute, langjährige Zusammenarbeit und die tolle Laboratmosphäre. Zudem danke ich Dr. Karl‐ Heinz Jung für die interessanten wissenschaftlichen Fachgespräche. Weiterhin danke ich ganz herzlich meinen sehr talentierten Bachelorstudenten Annika Hantusch, Moritz Pott, Joos Aschenbrenner und Melina Hoffmann, sowie allen Mitarbeiterpraktikanten und wissenschaftlichen Hilfskräften für ihre Leistung, ihr Engagement und ihr Interesse. Bei Dr. Thomas Huhn, Dr. Timo Immel und Malin Bein bedanke ich mich für die Durchführung der ersten Zytotoxizitätsmessungen. Bei Herrn Prof. Dr. Thomas Brunner und Anette Brockmann möchte ich mich besonders für die großartige Hilfe bei komplexen zellbiologischen Fragestellungen und die fruchtbare Zusammenarbeit bei der Durchführung der Zellassays bedanken. Meinen Lektoren Dr. Norman Hardt, Karin Reichardt, Matthias Drum und Joachim Braun danke ich für die Durchsicht meiner schriftlichen Arbeit. Meinen Freunden, meiner Familie und ganz besonders meinen Eltern danke ich für die liebevolle und bedingungslose Unterstützung in allen Lebenslagen. Publikationen Teile dieser Arbeit sind veröffentlicht in: T. Strittmatter, B. Bareth, T. A. Immel, T. Huhn, T. U. Mayer & A. Marx ”Small Molecule Inhibitors of Human DNA Polymerase λ” ACS Chem. Biol. 2011, 6 (4), 314 – 319 T. Strittmatter, J. Aschenbrenner, N. Hardt & A. Marx ”Synthesis of 4′‐C‐alkylated‐5‐iodo‐2′‐deoxypyrimidine nucleosides” ARKIVOC 2013, (ii) Issue in Honor of Prof. Richard R. Schmidt, 46 – 59 T. Strittmatter, A. Brockmann, M. Pott, A. Hantusch, T. Brunner & A. Marx ”Expanding the Scope of Human DNA Polymerase λ and β Inhibitors” ACS Chem. Biol. 2014, 9 (1), 282–290. Weitere Publikationen: M. Catarinella, T. Grüner, T. Strittmatter, A. Marx & T. U. Mayer ”BTB‐1: A Small Molecule Inhibitor of the Mitotic Motor Protein Kif18A” Angew. Chem. Int. Ed. 2009, 48, 9072 – 9076 Angew. Chem. 2009, 121, 9236 – 9240 B. Reichmann, M. Drexler, B. Weibert, N. Szesni, T. Strittmatter & H. Fischer ”Amino‐substituted Butatrienes: Unusual η1Ligands Formed by an Unusual Reaction” Organometallics 2011, 30 (5), 1215 – 1223 O. B. Gutiérrez Acosta, N. Hardt, S. M. Hacker, T. Strittmatter, B. Schink & A. Marx ”TPP stimulates acetone activation by D. biacutus as monitored by a fluorogenic ATP analogue” ACS Chem. Biol. 2014, 9 (6), 1263 – 1266. A. Brockmann, T. Strittmatter, S. May, A. Marx & T. Brunner ”Structure‐function relationship of thiazolide‐induced apoptosis in colorectal tumor cells” ACS Chem. Biol. 2014, 9 (7), 1520 – 1527. J. Braun, M. M. Möckel, T. Strittmatter, A. Marx, U. Groth & T. U. Mayer ”Synthesis and Biological Evaluation of Optimized Inhibitors of the Mitotic Kinesin Kif18A” ACS Chem. Biol. (DOI: 10.1021/cb500789h), in press. Table of contents Table of Contents Introduction................................................................................................... 3 1) Chemical genetics................................................................................................ 3 2) DNA polymerases ................................................................................................ 5 2.1) General ................................................................................................................5 2.2) DNA polymerases and the polymerisation reaction.................................................6 2.3) DNA polymerase λ and β .......................................................................................8 2.4) Herpes virus DNA polymerase..............................................................................11 3) DNA polymerases as drug targets ....................................................................... 12 3.1) General ..............................................................................................................12 3.2) Screening methods for DNA polymerase inhibitors ...............................................14 Concepts and objectives................................................................................20 Results and discussions .................................................................................22 4) Small‐molecule inhibitors of human pol λ and pol β............................................. 22 4.1) Introduction .......................................................................................................22 4.2) Biochemical evaluation of the 1st small‐molecule generation.................................24 4.2.1) Conclusion .......................................................................................................29 4.3) Establishment of a 2nd generation compound library of potential active molecules against pol λ and β...............................................................................................29 4.3.1) Design of the 2nd small‐molecule generation SM12, 29‐58 ..................................29 4.3.2) Synthesis of 2nd small‐molecule generation SM12, 29‐58 ....................................31 4.3.3) Conclusion .......................................................................................................34 4.4) Biochemical evaluation 2nd small‐molecule generation..........................................35 4.4.1) Evaluation of the 2nd small‐molecule generation ................................................35 4.4.2) Side‐by‐side comparison of SM1 and SM49 with reported inhibitors...................38 4.4.3) Discussion and structure‐activity relationships...................................................39 4.4.4) Conclusion .......................................................................................................41 4.5) Cellular investigations of the 1st and 2nd small‐molecule generation .......................42 4.5.1) Introduction.....................................................................................................42 4.5.2) Cell viability measurements of potential rhodanine probes ................................43 4.5.3) Co‐treatment experiments with genotoxic agents and probes SM1 or SM49........44 4.5.4) Conclusion .......................................................................................................47 5) Synthesis of 4’‐C‐alkylated‐5‐iodo‐2’‐deoxypyrimidine nucleosides as potential antiviral drugs and synthetic building blocks ........................................................ 49 5.1) Introduction .......................................................................................................49 5.2) Synthesis of 4‐C‐modified carbohydrate building blocks ........................................52 5.3) Synthesis of 4’‐C‐alkylated‐pyrimidine nucleosides ...............................................54 5.4) Synthesis of 4’‐C‐alkylated‐5‐iodo‐2’‐deoxypyrimidine nucleosides........................54 1 Table of contents 5.5) Evaluation of the synthons ‐ exemplified by 3’,5’‐di‐O‐acetyl‐2’‐deoxy‐5‐iodo‐4’‐C‐ propyluridine N12c in a Sonagashira test‐reaction .................................................56 5.6) Discussion and Conclusion...................................................................................57 Conclusion ....................................................................................................59 Zusammenfassung ........................................................................................65 Materials and methods
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