Antitubercular Benzothiazinones: Synthesis, Activity, Properties and SAR Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät I – Biowissenschaften der Martin-Luther-Universität Halle-Wittenberg vorgelegt von Andrea Ines Rudolph geboren am 27.01.1983 in Karl-Marx-Stadt Datum der Verteidigung: 04.06.2014, Halle (Saale) Gutachter: Prof. Dr. Peter Imming Prof. Dr. Martin Schlitzer Dr. Ute Möllmann Prof. Dr. Andrea Sinz I CONTENT Content ..................................................................................................................................... I Abbreviations ............................................................................................................................ VII List of figures ............................................................................................................................. XI List of tables ..............................................................................................................................XV Abstract .................................................................................................................................XVII 1 Tuberculosis and antitubercular drug development ............................................. 1 1.1 Tuberculosis ................................................................................................................. 1 1.2 Mycobacterium tuberculosis ....................................................................................... 1 1.3 Mycobacterial cell envelope ....................................................................................... 3 1.4 Antibiotic treatment of tuberculosis ........................................................................... 4 1.5 The drug pipeline ......................................................................................................... 5 1.5.1 Benzothiazinones ........................................................................................................ 7 1.5.2 Fluoroquinolones ....................................................................................................... 11 1.6 Objective of thesis ..................................................................................................... 13 2 Syntheses .......................................................................................................... 15 2.1 Synthetic pathways to benzothiazinones .................................................................. 15 2.1.1 Method A – the classic pathway ............................................................................... 16 2.1.2 Method B – dithiocarbamate pathway ..................................................................... 17 2.1.3 Method C – alkylxanthogenate pathway .................................................................. 17 2.1.4 Method D – alkylsulfanyl BTZ pathway ..................................................................... 17 2.1.5 Method E – a new pathway: thiourea pathway ........................................................ 17 2.1.6 Evaluation of the synthetic routes ............................................................................ 19 2.1.7 Unfamiliar NMR spectra ............................................................................................ 22 2.2 Novel BTZ derivatives ................................................................................................ 24 2.2.1 Unsubstituted arene moiety ..................................................................................... 25 2.2.2 Shifting the nitro group ............................................................................................. 26 2.2.3 Varying substituents at the arene ............................................................................. 27 2.2.4 Substituents at position 2 of the heterocycle ........................................................... 33 2.2.5 2,3-Dihydro-5H-imidazo[2,1-b][1,3]benzothiazin-5-one derivatives ........................ 45 2.3 Syntheses of benzoxazinones .................................................................................... 47 2.3.1 Adaption of method E ............................................................................................... 47 2.3.2 Adaption of the classic pathway method A .............................................................. 48 2.4 Dual action molecules - thiochromenones ............................................................... 50 2.4.1 Essential pharmacophores of fluoroquinolones and benzothiazinones ................... 50 2.4.2 Synthetic approaches to 3-carboxyl-thiochromen-4-ones ....................................... 51 3 Biological Evaluation ......................................................................................... 55 3.1 Agar diffusion test ..................................................................................................... 55 3.2 Minimal inhibitory concentration ............................................................................. 59 II Content 3.3 In vivo activity: ultra-fast murine model .................................................................. 62 3.4 Cytotoxic and antiproliferative effects ..................................................................... 66 4 Pharmacokinetic Evaluation .............................................................................. 69 4.1 Calculated Lipinski rule-of-five parameters .............................................................. 69 4.2 Solubility .................................................................................................................... 72 4.2.1 Methods of solubility determination ........................................................................ 72 4.2.2 Calculated solubility of selected BTZs and BOZs....................................................... 73 4.2.3 Solubility determination via the shake-flask method ............................................... 74 4.3 Microsomal stability .................................................................................................. 79 5 Co-Crystallization with DprE1 ............................................................................ 83 5.1 Crystal structure of BOZ IR 95 with DprE1 ................................................................ 85 6 Conclusion and suggestions for further BTZ development .................................. 87 7 Experimental Section ........................................................................................ 95 7.1 Chemicals and materials ........................................................................................... 95 7.2 Instrumental settings and analyses .......................................................................... 95 7.3 Pharmacokinetic evaluation methods ...................................................................... 97 7.3.1 Solubility determination ........................................................................................... 97 7.3.2 Calculated Lipinski rule-of-five.................................................................................. 98 7.3.3 Microsomal stability .................................................................................................. 98 7.4 Biological evaluation methods .................................................................................. 99 7.4.1 Agar diffusion assay .................................................................................................. 99 7.4.2 MIC determination .................................................................................................. 100 7.4.3 Antiproliferative and cytotoxicity assays ................................................................ 101 7.4.4 Co-Crystallization experimental methods .............................................................. 102 7.5 Syntheses ................................................................................................................ 105 7.5.1 2-chloro-3-nitro-5-(trifluoromethyl)benzoic acid (IR 05) ....................................... 106 7.5.2 N-[(2-chlorophenyl)carbonyl]piperidine-1-carboimidothioic acid (IR 12) .............. 106 7.5.3 1-([2-chloro-3-nitro-5-(trifluoromethyl)phenyl]carbonyl)piperidine (IR 13) .......... 107 7.5.4 2-(piperidin-1-yl)-4H-1,3-benzothiazin-4-one (IR 16) ............................................. 108 7.5.5 sodium (piperidin-1-yl)carbothioylsulfanide (IR 17) ............................................... 109 7.5.6 2-chloro-3-nitro-5-(trifluoromethyl)benzamide (IR 18) ......................................... 110 7.5.7 8-nitro-2-(piperidin-1-yl)-6-(trifluoromethyl)-4H-1,3-benzothiazin-4-one (IR 20)np ................................................................................................................... 110 7.5.8 2-chloro-4,5-difluoro-3-nitrobenzoic acid (IR 29) ................................................... 112 7.5.9 2-chloro-4,5-difluoro-3-nitrobenzamide (IR 32)n ................................................... 112 7.5.10 2,4-dichloro-5-fluoro-3-nitrobenzamide (IR 39)n ................................................... 113 7.5.11 1-(cyclohexylmethyl)piperazine (IR 40) .................................................................. 114 7.5.12 sodium (ethoxymethanethioyl)sulfanide (IR 42) .................................................... 114 7.5.13 imidazolidine-2-thione (IR 45) ...............................................................................