Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Triazolobenzodiazepines and -triazepines as Protein Interaction Inhibitors Targeting Bromodomains of the BET Family Matthias Alfons Wrobel aus Weiden i.d.OPf. 2013 Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. November 2011 von Herrn Prof. Dr. Franz Bracher betreut. Eidesstattliche Versicherung Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet. München, 09.12.2013 Matthias Wrobel Dissertation eingereicht am 10.12.2013 1. Gutachter: Prof. Dr. Franz Bracher 2. Gutachter: Prof. Dr. Stefan Knapp Mündliche Prüfung am 21.01.2014 This dissertation was carried out in close collaboration with the Structural Genomics Consortium of the University of Oxford. Prof. Dr. Stefan Knapp Dr. Panagis Filippakopoulos Screening via DSF / ITC / Co-crystallization …für Caro …für meine Familie Acknowledgements I would like to thank Prof. Dr. Franz Bracher for giving me the possibility to work on this interesting topic, the helpful advices in meetings, always having a sympathetic ear for all kind of affairs and his support during the time of my PhD Thesis. I would also like to thank all the members of the Board of Examiners. In gratitude for the opportunity to visit his group at the University of Oxford (UK) and his considerable commitment for our collaboration, I would like to thank Prof. Dr. Stefan Knapp. Special thanks goes to Dr. Panagis Filippakopoulos for his supervision during my stays at the SGC and his patience and support in uncountable discussions about co-crystallization, structure optimization and critical appraisal of screening results. I thank the staff of the analytical division under supervision of Dr. Lars Allmendinger, Christoph Müller for assistance in respect of HPLC purity and Martina Stadler for preparation of MTT and ADT assays, Dr. Olaf Morgenstern for providing us a selection of compounds, Dr. Sarah Picaud for carrying out the DSF screenings, performing ITC measurements and preparing plates for co- crystallization as well as Dr. Oleg Fedorov for interesting discussions. Thanks goes to all staff and co-workers of the Bracher group, especially to Sebastian Dittrich for qualified discussions concerning chemistry and to Dr. Marco Keller for his support in the lab and sharing his office with me. Special thanks goes to Dr. André Gehring and Markus Luber for the enjoyable working atmosphere in our lab and the important assistance in practical and theoretical questions. I am exceptionally grateful to my parents Elisabeth and Wolfgang and to my sister Susanne, without their support I would not have made it to this point. With all my heart, I thank my fiancée Caro, for her love, support and encouragement over the last years. Contents CHAPTER I – INTRODUCTION ...... 1 1.1 EPIGENETICS .............................................................................. 1 1.1.1 Background information .................................................................. 1 1.1.2 From DNA to chromosomes ............................................................ 2 1.1.3 Epigenetic marks .............................................................................. 4 1.1.3.1 DNA methylation .......................................................................... 5 1.1.3.2 Histone modifications ................................................................... 6 1.2 BROMODOMAINS ......................................................................... 9 1.2.1 An epigenetic recognition module .................................................. 9 1.2.2 The BET family ................................................................................ 11 1.3 BENZODIAZEPINE PHARMACEUTICALS – AN IMPORTANT SUBSTANCE CLASS OF DRUGS .............................. 12 CHAPTER II – PROJECT & STRATEGY OF SYNTHESIS ............. 15 2.1 AIM OF THIS WORK .................................................................... 15 2.2 STRATEGY OF SYNTHESIS .......................................................... 18 2.2.1 Synthesis development for 6-ethyltriazolobenzodiazepines ...... 18 2.2.2 Synthesis of triazolobenzotriazepines .......................................... 19 2.2.2.1 4-(2-Hydroxyethyl)triazolobenzotriazepine and derivatives ........ 20 2.2.2.2 4-Benzyltriazolobenzotriazepine ................................................ 21 2.2.2.3 4-Substituted 8-chloro-6-phenyl-4H-triazolobenzotriazepine ..... 22 2.2.2.4 4H-Triazolobenzotriazepines with modified core scaffold .......... 24 2.2.2.5 Further optimization of the annulated benzo ring system ........... 25 Contents CHAPTER III – METHODS FOR STRUCTURE OPTIMIZATION .......... 29 3.1 DIFFERENTIAL SCANNING FLUORIMETRY ..................................... 30 3.1.1 The principle of measurement ....................................................... 30 3.1.2 Measurement and analysis ............................................................ 31 3.1.3 Thermodynamic view on DSF: entropy vs. enthalpy ................... 33 3.2 ISOTHERMAL TITRATION CALORIMETRY ....................................... 36 3.3 CO-CRYSTALLIZATION OF PROTEINS WITH LIGANDS ...................... 38 CHAPTER IV – SYNTHESIS, RESULTS & DISCUSSION ................ 41 4.1 TRIAZOLOBENZODIAZEPINES: THE FIRST CLASS OF TARGET COMPOUNDS .................................. 42 4.1.1 Synthesis of the benzodiazepinone core scaffold ....................... 42 4.1.2 Preparation of 6-ethyltriazolobenzodiazepines ............................ 44 4.1.3 Benzotriazepines provided by the University of Greifswald ....... 46 4.1.4 Screening of the benzodiazepines and -triazepines .................... 47 4.1.5 Discussion of the benzodiazepine screening results .................. 50 4.2 FIRST OPTIMIZATION OF TRIAZOLOBENZOTRIAZEPINES .................. 54 4.2.1 Synthesis and derivatization of 4-(2-hydroxyethyl)triazolo- benzotriazepine ............................................................................... 54 4.2.2 Interesting aspects of NMR analysis of special carbamates ...... 58 4.2.3 Synthesis of 4-benzyltriazolobenzotriazepine.............................. 60 4.2.4 Screening of 4-(2-hydroxyethyl)triazolobenzotriazepine, its derivatives and the 4-benzyl analogon .................................... 61 4.2.5 Discussion of temperature shift results of the first triazolobenzotriazepine series ....................................................... 62 Contents 4.3 8-CHLORO-6-PHENYLTRIAZOLOBENZOTRIAZEPINES WITH AROMATIC SIDE CHAINS IN POSITION 4 ......................................... 65 4.3.1 Preparation of 4H-triazolobenzotriazepine 11 .............................. 65 4.3.1.1 Approach starting from synthesized 4-benzyltriazolobenzotriazepine 10 ............................................ 66 4.3.1.2 Synthesis of compound 11 using 4-methoxybenzyl as protecting group .................................................................... 66 4.3.2 Derivatization of 4H-triazolobenzotriazepine 11 with various substituted 4-benzyl residues .......................................... 69 4.3.2.1 Benzylation via Mitsunobu reaction ............................................ 69 4.3.2.2 Benzylation via N-alkylation of compound 11 using benzyl bromides or chlorides ...................................................... 70 4.3.3 Preparation of 1,4-substituted 1,2,3-triazole compounds ........... 72 4.3.4 Screening and co-crystallization results of synthesized triazolobenzotriazepines with 4-benzyl and 4-(triazol-4-yl)methyl moieties ....................................................... 76 4.3.5 Explanation and discussion of recently screened triazolobenzotriazepines ................................................................ 78 4.4 4-BENZYL SERIES WITH DIFFERENT SUBSTITUTED SCAFFOLDS ....... 81 4.4.1 Synthesis of a "nitro" and a "dichloro" substituted 4H-triazolobenzotriazepine ............................................................ 81 4.4.2 The nitro series: Derivatization of 1-methyl-8-nitro- 6-phenyl-4H-triazolobenzotriazepine 14 ....................................... 83 4.4.3 Preparation of the dichloro series by N-alkylation of 8-chloro- 6-(2-chlorophenyl)-1-methyl-4H-triazolobenzotriazepine 15 ....... 84 4.4.4 Isotope effects in mass spectra obtained from multi-halide substituted compounds ................................................................. 86 4.4.5 Temperature shift results and discussion of the nitro and the dichloro series .................................................................. 89 4.4.6 Lead structure optimization by comparison of the three benzyl series ................................................................................... 91 Contents 4.5 MODIFICATION OF 6-(2-CHLOROPHENYL)-4-(3-METHOXYBENZYL)- TRIAZOLOBENZOTRIAZEPINE IN POSITIONS 8 AND 9 ...................... 95 4.5.1 Preparation of 2-aminobenzophenones ........................................ 95 4.5.1.1 Two step synthesis using photochemistry .................................. 95 4.5.1.2 Synthesis by "ortho selective acylation" of aniline derivatives .... 97 4.5.2 Preparation of (3-methoxybenzyl)hydrazine hydrochloride 86 ... 98 4.5.3 Synthesis of 6-(2-chlorophenyl)-4-(3-methoxybenzyl)- triazolobenzotriazepines ................................................................ 99 4.5.4 Screening results and discussion of novel triazolobenzotriazepines .............................................................. 101 4.6 ADDITIONAL PERFORMED BIOASSAYS ......................................