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Parallel synthesis of small molecule compound libraries : isoform selective Rho-Kinase inhibitors Mohamed, Safwat 2015

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  PARALLEL SYNTHESIS OF SMALL MOLECULE COMPOUND LIBRARIES: ISOFORM SELECTIVE RHO-KINASE INHIBITORS   by  Safwat Mohamed B.Pharm., Assiut University, Egypt 2000 M.Sc., Assiut University, Egypt 2006  A THESIS SUBMITTED IN PARTIAL FUFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY   in The Faculty of Graduate and Postdoctoral Studies (Pharmaceutical Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2015   © Safwat Mohamed, 2015   ii  Abstract  The objective of this thesis was to use parallel synthesis to build small molecule libraries of novel compounds with potential as isoform selective type II rho-kinase (ROCK1/2) inhibitors. Considerable effort is ongoing to identify isoform selective ROCK inhibitors, as hyperactivity of the ROCK is implicated in cardiovascular diseases, cancer, diabetes and many other diseases that affect millions of Canadians, and billions of people worldwide.  In the absence the extensive structural details of the type II kinase inhibitor binding mode, we have used an empirical approach to the design of type II ROCK inhibitors, based on the conception that such molecules correspond to elongated structures with H-bonding functional elements in their central portion, a motif at one end that mimics the adenine ring in ATP, and a hydrophobic moiety at the other end of the molecule that will interact with an allosteric pocket in the ATP binding region of the kinase. Based on these very general structural requirements, eleven different representative libraries of novel compounds (not described in CAS) were designed and synthesized. In the initial series of compounds, a 2-pyridinone motif was employed as the hinge binding element, and the central portion corresponded to carboxamide substituted  oxazoline, oxadiazole, or 2-aminothiazole  system, joined through the amide bond to a set of structurally diverse aromatic, heteroaromatic and benzylamine subunits, corresponding to the diversity elements. The preliminary assay results demonstrated that, overall, these compounds were weak and non-selective ROCK1/2 inhibitors when compared to H-1152 as a positive control at 10 μM concentration. However, further structural modification revealed the interest in using an indazole motif as the hinge binder in conjunction with the 2-aminothiazole carboxamide linker. Indeed, more potent activities were observed in the single point assay for a significant portion of the libraries of molecules built around these structural components. Further evaluation of 18 active compounds in a 10-point assay, by Invitrogen, to determine IC50’s revealed that indazole-based inhibitors are active at low micromolar concentrations (1-10 µM), but do not display any significant isoform selectivity.   iii  Preface  This dissertation is submitted for the degree of Doctor of Philosophy at the University of British Columbia. The research described in this dissertation was performed under the supervision of Professor David Grierson in the Department of Pharmaceutical Sciences, University of British Columbia, between January 2009 and January 2015. With guidance and supervision from Dr. Grierson, I was responsible for the planning, designing, and conducting all of the chemistry experiments, data analysis, and interpretation of the results. The biological assays were conducted in Dr. Kathleen MacLeod laboratory by Dr. Guorong Lin. To the best of my knowledge, this work is original except where references are made to previous work. The cited figures in my thesis are used with permission from applicable sources. This work was financia1ly supported by the Egyptian government in the first two years (Jan 2009-Jan 2011) in the form of scholarship (Egyptian Channel System).       iv  Table of Contents  Abstract ......................................................................................................................................... ii Preface ......................................................................................................................................... iii Table of Contents ......................................................................................................................... iv List of Tables ............................................................................................................................. viii List of Figures ............................................................................................................................... x List of Symbols and Abbreviations .......................................................................................... xiv Acknowledgements ..................................................................................................................... xx Dedication ................................................................................................................................... xxi Chapter 1: Introduction ............................................................................................................... 1 1.1  Drug Discovery and Compound Libraries ......................................................................... 1 1.2 Pharmaceutical Industry In-House Libraries and HTS ...................................................... 2 1.3 Combinatorial Chemistry - Parallel Synthesis and Structural Diversity ............................ 3 1.3.1 Parallel Synthesis: Appendage Diversity Strategy ................................................. 4 1.3.2 Parallel Synthesis: Privileged Scaffolds for Library Design .................................. 5 1.4 Thesis Project: Development of Isoform Selective Rho-Kinase Inhibitors ....................... 9 1.5 Protein Kinases ................................................................................................................. 10 1.5.1 Rho-associated, Coiled-Coil Containing, Protein Kinase (ROCK) ...................... 11 1.5.2 ROCK Isoform Homology, Functional Roles, and Tissue Distribution .............. 12 1.5.3 ROCK and Cardiovascular Diseases .................................................................... 14 1.5.4 ROCK and Diabetes ............................................................................................. 15 1.5.5 ROCK and Cancer ................................................................................................ 16 1.6 Design of Kinase Inhibitors .............................................................................................. 17 v  1.6.1 ROCK Structure and Catalytic Mechanism ......................................................... 17 1.6.2 Type 1 Kinase Inhibitors ...................................................................................... 19 1.6.3 Type II Kinase Inhibitors ...................................................................................... 21 1.6.4 Other Kinase Inhibitors ........................................................................................ 24 1.7 Current Status of ROCK Inhibitor Development ............................................................. 25 1.7.1 First Generation ROCK Inhibitors ....................................................................... 25 1.7.2 Second Generation Isoquinoline-Based ROCK Inhibitors ................................... 27 1.7.3 Pyridine-Based ROCK Inhibitors  ........................................................................ 28 1.7.4 Azaindole-Based ROCK Inhibitors  ..................................................................... 29 1.7.5 Indazole-Based ROCK Inhibitors  ........................................................................ 30 1.7.6 Pyrazole-Based ROCK Inhibitors  ....................................................................... 32 1.7.7 Aminofurazan-Based ROCK Inhibitors  .............................................................. 33 1.7.8 Others ................................................................................................................... 34 1.8 Prospective ....................................................................................................................... 34 1.9 Scope of My Work ........................................................................................................... 34 Chapter 2:  Objectives and Evolution of my Thesis Project .................................................. 35 2.1 Starting Point for a Rho-Kinase (ROCK) Inhibitor Library ............................................. 35 Chapter 3: Pyridinone Dihydroxazole/Oxadiazole-Based Compounds ................................ 41 3.1 Introduction ...................................................................................................................... 41 3.2 Synthesis of Pyridinone-Oxazole Carboxamides 22a-f and 23a-f ................................... 42 3.2.1 Synthesis of the 2-Pyridinone-5-Carboxylic Acid Building Blocks 7 and 8 ........ 42 3.2.2 Synthesis of the Serine Carboxamide Building Blocks 15a-f .............................. 45 3.2.3 Synthesis of Target Compounds 22a-f and 23a-f ................................................. 47 3.3 Synthesis of Pyridinone-Oxadiazole Carboxamide Based Series 34a-ah ........................ 49 vi  3.3.1 Optimization of Peptide Coupling Conditions for the Synthesis of 34i-ah ......... 54 3.4 Synthesis of Isomeric Pyridinone-Oxadiazole Based Derivatives 41a-g ......................... 56 3.5 Synthesis of Modified Pyridinone Oxadiazole-Based Compounds 45a-f ........................ 58 Chapter 4: Structural Elongation of the Linker Component ................................................. 60 4.1 Introduction ...................................................................................................................... 60 4.2 Synthesis of Pyridinone-Piperazine-Based Compounds 50a-d ........................................ 61 4.3 Synthesis of Pyridinone Aminothiazoles-Based Compounds 65a-p and 66o,p............... 62 Chapter 5: Indazole/Pyridine-Based ROCK Inhibitors: Introduction of Diversity  Elements on  the Left Side of the Molecules .......................................................... 67 5.1 Introduction ...................................................................................................................... 67 5.2 Preparation of N-Aryl Aminothiazole Ester Intermediates 72a-j ..................................... 68 5.2.1 Ethyl 2-Bromothiazole-4-Carboxylate as a Common Intermediate to Prepare N-Aryl Aminothiazole 72a-j .................................................................................... 68 5.2.2 Hantszch Approach to Prepare N-Aryl Aminothiazole Esters 72f-j .................... 72 5.3 Preparation of Compounds 76a-j and 77a-j ..................................................................... 73 Chapter 6: Indazole-Based ROCK1/ROCK2 Inhibitors ........................................................ 75 6.1 Introduction ...................................................................................................................... 75 6.2 Synthesis of 2-(Indazole-5-yl)aminothiazoles-4-Carboxamide Derivatives 80a-q ......... 76 6.3 Synthesis of 2-(Indazole-5-yl)aminothiazoles-5-Carboxamide Derivatives 85a-o .......... 78 6.4 Synthesis of 2-(Indazole-4-yl)aminothiazoles-4-Carboxamide Derivatives 93a-x .......... 79 Chapter 7: In Vitro Biological Assays and Results .................................................................. 82 7.1 Enzyme-Linked Immunosorbent Assay (ELISA) ............................................................ 83 7.1.1 Cyclex® ROCK Assay ......................................................................................... 83 7.1.2 Millipore® ROCK Assay ..................................................................................... 86 vii  7.2 Rho-Kinase (ROCK) Activity Assay using FRET Technique ......................................... 96 7.3 Factors that may Contribute to the Differences in Biological Results Between the ELISA-Based and FRET-Based ROCK Activity Assay Methods ................................... 99 7.3.1 ATP Concentration ............................................................................................... 99 7.3.2 Enzyme Concentration ....................................................................................... 100 7.3.3 Buffer Composition ............................................................................................ 101 7.3.4 Detection Method  .............................................................................................. 102 7.3.5 Screening Concentrations ................................................................................... 102 7.4 Enzyme-Linked Immunosorbent-Based Assay Protocol ................................................ 103 7.5 FRET-Based Assay Protocol .......................................................................................... 104 Chapter 8: Experimental ......................................................................................................... 105 8.1 Materials and Methods ................................................................................................... 105 8.2 Experimental Details ...................................................................................................... 106 Conclusions and Future Directions ......................................................................................... 268 Bibliography .............................................................................................................................. 270     viii  List of Tables  Table 1: Selection of peptide coupling agent for 13a-f synthesis. ............................................... 46 Table 2: Optimization of peptide coupling agents for 34i-ah synthesis. ...................................... 54 Table 3: Optimization of base for 34i-ah synthesis. .................................................................... 55 Table 4: SNAr of bromothiazole under different base conditions. ................................................ 70 Table 5: Buchwald-Hartwig cross coupling trials to prepare 72f. ................................................ 72 Table 6: Inhibitory activity of pyridinone oxazoline carboxamide-based compounds 20a-f and 21a-f against ROCK2 ................................................................................................... 84 Table 7: Inhibitory activity of pyridinone oxadiazole carboxamide-based compounds 34a-ah against ROCK2 ............................................................................................................. 85 Table 8: Inhibitory activity of pyridinone oxadiazole carboxamide-based compounds 34a-ah against ROCK1/2 .......................................................................................................... 86 Table 9: Inhibitory activity of isomeric pyridinone oxadiazole carboxamide-based compounds 41a-g against ROCK1/2 ................................................................................................ 88 Table 10: Inhibitory activity of pyridinone piperazine compounds 50a-d against ROCK1/2.  ... 89 Table 11: Inhibitory activity of pyridinone aminothiazole carboxamide-based compounds 65a-p and 66o,p against ROCK1/2 ......................................................................................... 90 Table 12: Inhibitory activity of aminothiazole carboxamide-based compounds 76a-j and 77a-j against ROCK1/2 .......................................................................................................... 91 Table 13: Inhibitory activity of 2-(indazole-5-yl)aminothiazoles-4-carboxamide derivatives 80a-q against ROCK1/2 ............................................................................................... 93 Table 14: Inhibitory activity of 2-(indazole-5-yl)aminothiazoles-5-carboxamide derivatives 85a-o against ROCK1/2 ................................................................................................ 95 ix  Table 15: IC50 values of compounds tested in Invitrogen ............................................................ 98 Table 16: NMR data of compounds 13a-f .................................................................................. 113 Table 17: NMR data of compounds 14a-f .................................................................................. 116 Table 18: NMR data of compounds 15a-f .................................................................................. 118 Table 19: NMR data of compounds 16a-f .................................................................................. 122 Table 20: NMR data of compounds 17a-f .................................................................................. 125 Table 21: NMR data of compounds 19a-f .................................................................................. 131 Table 22: NMR data of compounds 20a-f .................................................................................. 135 Table 23: NMR data of compounds 21a-f .................................................................................. 138 Table 24: NMR data of compounds 34a-ih ................................................................................ 157 Table 25: NMR data of compounds 41a-g ................................................................................. 169 Table 26: NMR data of 45a-f ..................................................................................................... 177 Table 27: NMR data of compounds 65a-p ................................................................................. 194 Table 28: NMR data of compounds 76a-j: ................................................................................. 215 Table 29: NMR data of compounds 77a-j .................................................................................. 220 Table 30: NMR data of compounds 80a-q ................................................................................. 229 Table 31: NMR data of compounds 85a-o ................................................................................. 245 Table 32: NMR data of compounds 93a-x ................................................................................. 263          x  List of Figures  Figure 1: Drugs identified in high-throughput screening. .............................................................. 2 Figure 2: Ugi reaction for the generation of amide bond ............................................................... 3 Figure 3: Development of inhibitors of HIV-1 alternative splicing ............................................... 5 Figure 4: Bezodiazepine as a privileged scaffold ........................................................................... 6 Figure 5: Chromone as a privileged scaffold ................................................................................. 6 Figure 6: 1,4-Dihydropyridine as a privileged scaffold ................................................................. 7 Figure 7: Quinoline as a privileged scaffold .................................................................................. 7 Figure 8: 2-Aminothiazole as a privileged scaffold ....................................................................... 8 Figure 9: Applications of purine ring-based chemical libraries ..................................................... 8 Figure 10: Phosphorylation of protein substrate by kinases ......................................................... 10 Figure 11: a) Human protein kinases classification, b) Graphical representation of human kinome tree ................................................................................................................. 10 Figure 12: Regulation of Rho-G protein activity ......................................................................... 11 Figure 13: A schematic of the molecular structure of Rho-kinase isoforms ................................ 12 Figure 14: ROCK substrates ......................................................................................................... 13 Figure 15: Structures of the approved ROCK inhibitor fasudil, and hydroxyfasudil .................. 14 Figure 16: General kinase domain architecture ............................................................................ 17 Figure 17: Organization of the kinase active site, ATP binding and catalytic sites ..................... 18 Figure 18: The various conformations of the activation loop ...................................................... 19 Figure 19: a) ATP binding site, b) Topological view of ATP binding site .................................. 20 Figure 20: a) Type I inhibitor, PD166326, b) Topological representation of PD166326 in the ABL1 .......................................................................................................................... 20 xi  Figure 21: Examples of type 1 inhibitors, a) Erlotinib in EGFR, b) Sunitinib in FGFR1, c) Dasatinib in c-Abl ....................................................................................................... 21 Figure 22: a) Imatinib in complex with c-Abl,  b) Topological representation of imatinib in the c-Abl ........................................................................................................................... 22 Figure 23: Closed activation loop conformation accompanied by increased size of back “Hyp1” pocket .......................................................................................................................... 22 Figure 24: Examples of type II inhibitors .................................................................................... 23 Figure 25: a) Topological representation of sorafenib in B-Raf, b) Doramapimod in p38 MAP, c) Fuoro-pyrimidine derivative in VEGFR2 .............................................................. 24 Figure 26: Schematic representation of kinase inhibitors Type III and Type IV binding modes 25 Figure 27: Molecular structures of four Rho-kinase inhibitors .................................................... 26 Figure 28: a) Binding interactions of fasudil within ROCK-ATP site, b) Topological representation of fasudil in ROCK c) Binding interactions of hydroxyfasudil within ROCK-ATP site, d) Topological representation of hydroxyfasudil in ROCK ........... 27 Figure 29: Isoquioline-based ROCK inhibitors ............................................................................ 28 Figure 30: Pyridine-based ROCK inhibitors ................................................................................ 29 Figure 31: Schematic presentation of RKI-1447 XXV interactions with the ROCK1-ATP site . 29 Figure 32: Azaindole-based ROCK inhibitors ............................................................................. 30 Figure 33: Indazole-based ROCK inhibitors ................................................................................ 30 Figure 34: a) Schematic presentation of the key interactions between XVIII and ROCK1; b) XVIII bound to ROCK1 DFG-in conformation c) Imatinib bound to Bcr-Abl ......... 31 Figure 35: Superposition of XXXI on XXXVI ............................................................................ 32 Figure 36: Pyrazole-based ROCK inhibitors ................................................................................ 32 Figure 37: Compounds XXXVII and XXXIX docked into ROCK2 ATP-binding   site ............ 33 xii  Figure 38: Aminofurazan-based ROCK inhibitors ...................................................................... 34 Figure 39: Designing of type II kinase inhibitors by attaching type I head to a type II tail ......... 36 Figure 40: The binding options of 2-pyridinone-based scaffold .................................................. 37 Figure 41: Initial choice of the core motif .................................................................................... 37 Figure 42: Overall strategy of the thesis ...................................................................................... 38 Figure 43: Type II-like rho-kinase inhibitors ............................................................................... 40 Figure 44: Summary of synthesis of compounds 22a-f and 23a-f ............................................... 41 Figure 45: Synthesis of 4-alkoxy-6-oxo-1,6-dihydropyridine-3-carboxylic acids ....................... 42 Figure 46: Tautomerism of 2-pyridinone and its effect on alkylation reaction ............................ 43 Figure 47: Mitsunobu reaction mechanism .................................................................................. 44 Figure 48: Boc deprotection mechanism ...................................................................................... 45 Figure 49: Synthesis of serine carboxamide ................................................................................. 46 Figure 50: Mechanism of BOP-Cl in amide bond formation ....................................................... 47 Figure 51: Side reaction from TFA cleavage of O,N-ketal .......................................................... 47 Figure 52: Synthesis of pyridinone oxazole-based compounds ................................................... 48 Figure 53: General method for 1,2,4-oxadiazole synthesis .......................................................... 50 Figure 54: Synthesis of pyridinone oxadiazole carboxamide-based compounds ......................... 51 Figure 55: Dehydration of primary amides by CyCl .................................................................... 52 Figure 56: Trimethylaluminium-mediated amide formation ........................................................ 53 Figure 57: Isomeric modification of 6-pyridinone-based compounds ......................................... 57 Figure 58: Difference between pyridinones 34a-ah, and 41a-g in their orientation within the hinge residue ............................................................................................................... 57 Figure 59: Synthesis of isomeric pyridinone oxadiazole carboxamide-based compounds .......... 58 Figure 60: Linker shortening of pyridinone oxadiazole-based compounds ................................. 58 xiii  Figure 61: Modified pyridinone oxadiazoles 45a-f ...................................................................... 59 Figure 62: Pyridinone piperazine and pyridinone aminothiazole-based compounds ................... 60 Figure 63: Synthesis of pyridinone piperazine-based compounds ............................................... 61 Figure 64: Reported 2-aminothiazole-based kinase inhibitors ..................................................... 62 Figure 65: Synthesis of pyridinone aminothiazole-based compounds ......................................... 63 Figure 66: Plausible binding interactions for 66o, and 66p ......................................................... 65 Figure 67: Aminothiazole amide-based compound library .......................................................... 67 Figure 68: Binding interactions for LI with ROCK ..................................................................... 68 Figure 69: Building compounds 76a-j and 77a-j based on 2-bromothiazole ester 70 ................. 68 Figure 70: SNAr reaction of bromothiazole intermediate 70 with (hetero)arylamines 71a-j ....... 69 Figure 71: SNAr reaction of 2-bromothiazole with (hetero)arylamines in acidic medium .......... 70 Figure 72: Amination of 2-bromothiazole using Goldberg reaction ............................................ 71 Figure 73: Amination of 2-bromothiazole using Buchwald-Hartwig reaction ............................ 71 Figure 74: Hantszch synthesis of thiazoles 72f-j ......................................................................... 72 Figure 75: Suggested mechanism for Boc cleavage in basic medium.......................................... 73 Figure 76: Aminothiazole carboxamide-based compounds 76a-j and 77a-j ............................... 74 Figure 77: Conception of the three isomeric indazole-aminothiazole carboxamide families ...... 75 Figure 78: Synthesis of indazole-5-aminothiazole-4-carboxamide derivatives 80a-q ................ 76 Figure 79: Proposed mechanism for the reduction of the nitro group using hydrazine ............... 77 Figure 80: Synthesis of indazole-5-aminothiazole-5-carboxamide derivatives 85a-o ................. 78 Figure 81: Synthesis of indazole-4-aminothiazole-4-carboxamide derivatives 93a-x ................. 80 Figure 82: Effect of kinase concentration on assay signal ......................................................... 101    xiv  List of Symbols and Abbreviations  ABL1 Abelson murine leukemia viral oncogene homolog 1 ACN Acetonitrile ADP Adenosine diphosphate AGC Protein kinases A, G, and C AIDS Acquired immunodeficiency syndrome AMPA α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor AKT  Protein kinase B Ar Aryl ATP Adenosine triphosphate Bcr-Abl Breakpoint cluster region- Abelson murine leukemia virus Boc tert-Butoxy carbonyl BOP-Cl Bis-(2-oxo 3-oxazolidinyl)phosphonic chloride Braf  B-Raf proto-oncogene, serine/threonine kinase br s Broad singlet   13C Carbon NMR oC Degrees Celsius calcd. Calculated CAS  Chemical Abstracts Service CCK  Cholecystokinin Cdc42 Cell division control gene CDK Cyclin-dependent kinase CML Chronic myeloid leukemia CNS Central nervous system CRD Cysteine Rich Domain CyCl Cyanuric chloride xv  d Doublet dd Doublet of doublets ddd Doublet of doublet of doublets DAPK Death-associated protein kinase DAST Bis(2-methoxyethyl) amino-sulfur trifluoride DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DCM Dichloromethane DFG Asp-Phe-Gly DHA Diheteroaryl amides DHP Dihydropyridine DIAD Diisopropyl azodicarboxylate DIEA Diisopropylethylamine DMF Dimethylformamide DMF-DMA N,N-Dimethylformamide dimethoxyacetal DMAP 4-(Dimethylamino)pyridine DMSO-d6 Deuterated dimethylsulfoxide DOS Diversity-oriented synthesis EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide EDCF Endothelium-derived constricting factors EDRF Endothelium-derived relaxing factors EGFR Epidermal growth factor receptor ELISA Enzyme-linked immunosorbent assay eNOS Endothelial nitric oxide synthase equiv. equivalents ESI Electrospray ionization et al.     Et alii, and others EtOAc Ethyl acetate xvi  EtOH Ethanol FDA US Food and Drug Administration FGFR  Fibroblast growth factor receptor FP Front pocket FRET  Fluorescence resonance energy transfer g Gram(s) GAPs GTPase-activating proteins GDP Guanosine diphosphate GDI Guanine nucleotide dissociation inhibitor GEF Guanine nucleotide exchange factor GTP Guanosine triphosphate GTPases Guanosine triphosphatase GSK Glycogen synthase kinase h Hour(s) 1H Proton NMR HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate  HBTU O-Benzotriazole-N,N,N`,N`-tetramethyl-uronium-hexafluorophosphate HCV Hepatitis C virus HIV Human immunodeficiency virus infection HOAt 1-Hydroxy-7-aza-benzotriazole HOBT Hydoxybenzotriazole HRMS High resolution mass spectrometry Hyp Hydrophobic pocket HTS High-throughput screening Hz Hertz IC50 Inhibitory concentration 50 xvii  IP3K Inositol 1,4,5-trisphosphate 3-kinase J Coupling constant Km Michaelis constant L Liter LIMK LIM kinase LTCC L-Type Calcium Channel µ Micro µL Microliter μM Micromole M Molar m Multiplet MAPK Mitogen-activated protein kinases MBS Myosin binding subunit MCR Multi-component reaction Me Methyl MeOH Methanol mg Milligram MHz Mega hertz min minutes mL Milliliter MLCK Ca2+-calmodulin-dependent myosin light chain MLCP Myosin light chain phosphatase mmol Millimole mol Mole MS Mass spectrometry MSK  Mitogen and stress activated protein kinase MW Microwave irradiation xviii  MYPT  Myosin phosphatase target subunit m/z Mass to charge ratio NMR Nuclear magnetic resonance  NO Nitric oxide P38 P38 mitogen-activated protein kinases P450 Cytochromes P450 PDE Phosphodiesterase Ph Phenyl PG Protecting group PKA Protein kinase A PKC Protein kinase C PH Pleckstrin Homology ppm Parts per million PyBOP Benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate q Quartet Rac1 Ras-related C3 botulinum toxin substrate 1 RBD Rho Binding Domain Rho Small G protein Rho ROCK Rho-Associated, coiled-coil containing protein Kinase rt Room temperature s Singlet SAR Structural activity relationship SI Selectivity index SNAr Nucleophilic aromatic substitution SR Serine/arginine-rich proteins Src Proto-oncogene tyrosine-protein kinase SRSF1 Serine/arginine-rich splicing factor 1 xix  STZ  Streptozotocin Syk Spleen tyrosine kinase t Triplet t Tert TBAF Tetrabutylammonium fluoride TBS tert-Butyldimethylsilyl TCDI  1,1'-Thiocarbonyldiimidazole Tec  Tyrosine-protein kinase Tec TFA Triflouoacetic acid THF Tetrahydrofuran TLC Thin layer chromatography Ts Tosyl pTsOH p-Toluenesulfonic acid UBC The University of British Columbia VEGFR Vascular endothelial growth factor receptor     xx  Acknowledgments   First and foremost, I would like to express my gratitude to Professor David Grierson, my supervisor, for accepting me as a graduate student in his laboratory, for his constant support and encouragement over the years. Also thank, Dr. Grierson, for giving me the opportunity to be the first doctoral student to graduate from his laboratory at UBC, and most importantly for his patience with me. I am really proud to have worked under his supervision because he taught me invaluable lessons which equipped me with the required basics to be able to start my scientific journey. I hope I will continue to live up to his expectations. Words are inadequate to thank all members of Dr. Grierson lab, past and present, for their friendship and support. I will never forget our insightful discussions every Thursday morning in our lab meetings. I would not have been able to go forward without their help and constructive critiques. Second, I would like to thank our collaborator Professor Kathleen MacLeod for her willingness to run the biological testing in her lab, and Dr. Guorong Lin, for running the biological assays and troubleshooting when nothing appeared to be working as it should. A special thanks to the members of my committee, including my chair Professor Wayne Riggs, for their helpful suggestions and constructive comments throughout the years of my thesis. In particular, I would like to thank Professor Glenn Sammis for teaching me advanced organic chemistry topics in his interesting course Chemistry 566.  I would like to thank the Egyptian government, the Mission office, and El-Minia University for funding my research in the first two years. I am thankful to Dr. Markus Heller from CDRD for his assistance in running NMR samples, and Andras Szeitz for his assistance with mass spectroscopy. I would also like to extend my thank-you’s to the administrative office of UBC Pharmaceutical Sciences. Also, I would like to thank Jamal Kurtu for his continuous support. I would like to forward my appreciation to all of my friends, past and present, especially Hesham Soliman who helped me a lot during my journey. Finally, I will close out by thanking my wife for her endless support and encouragement. I am eternally grateful to her for her sacrifices that helped to make my dream come true.     xxi             This thesis is dedicated to my mother, my wife, and my two sons (Muhammad and Omar) for their endless support and prayers 1  Chapter 1  Introduction   1.1  Drug Discovery and Compound Libraries The development of rapid readout in vitro and cellular assay protocols for biological targets of therapeutic interest (e.g. enzyme, receptor, nucleic acids) and their integration into robotic platform technologies for the high-throughput screening (HTS) of large sets of molecules heralded the beginning of a new paradigm in drug discovery.1,2 Prior to this point, biologically active compounds were almost exclusively discovered using assays designed to test one molecule, or small groups of molecules at a time.  The subsequent optimization of an active “hit” to produce a “lead” compound reTuired the synthesis of analoJues of the primary hit.3-5 This process, referred to as a structure-activity relationship (SAR) study, involved making small modifications in the hit structure and evaluation of how these changes impact activity. In this way, making a series of incremental changes in the hit structure helps, on the one hand, to determine what modifications enhance or diminish activity/target affinity, and, on the other hand, it provides crucial information concerning the composition of the active molecule binding site. Although the SAR strategy has proven hugely successful for the development of a large number of therapeutics, in the modern context this iterative activity driven approach to drug discovery is too time consuming and incompatible with the advent of HTS. To respond to the technological challenge HTS represented, industry countered by systematically reevaluating in-house libraries against new therapeutic targets. Further, both industrial and academic laboratories engaged in the development of combinatorial chemistry and parallel synthesis techniques, whereby a sequence of reactions and/or functionalized (privileged) scaffolds could be used to create new and structurally diverse chemical entities imbued with drug-like properties. Small molecule libraries can be used as a chemical-biology tool to explore biology space.6 Indeed, it was recognized, that traditional drug development programs created molecules around a specific objective, and therefore explored only a tiny part of the supposedly almost infinite number of variations in the nature of interactions that could exist between a protein and small molecules. The underlying tenant in the compound library approach is that testing diversity libraries against all new, and established, targets of therapeutic interest would reveal 2  new and unimagined molecules displaying biological activity. Importantly, these discoveries serve as the starting point for new drug development programs. In essence, this approach corresponds to the synthetic chemists complement to what nature has achieved through the assembly of complex natural products, which has fueled the development over the past century of a myriad of drugs (antibiotics, anticancer agents, etc.).  The fundamental paradigm shift that has occurred following efforts to explore the utility of compound libraries in drug discovery is that, in contrast to the traditional SAR approach, diversity driven small molecule library synthesis generally precedes biological evaluation, rather than being guided by it.   1.2 Pharmaceutical Industry In-House Libraries and HTS Essentially all pharmaceutical companies have “in-house libraries”, which have been built up over decades from directed (SAR driven) medicinal chemistry efforts around particular pharmacophores. Indeed, the process of taking a hit molecule through the drug development pipeline to identify a clinical candidate level generally involves the synthesis and screening of 500 to !1000 molecules. As only one molecule finds application in each case, the other “failed” molecules comprise the in-house library.7 These libraries, or a selection from these libraries, are systematically retested using HTS against new, or other established therapeutic targets. In this way screening series of phenylaminopyrimidines and anilinoquinazolines ultimately led to the development of kinase inhibitors imatinib I (Bcr-Abl inhibitor) and gefitinib II (EGFR inhibitor), respectively (Figure 1).8,9 Similarly, HTS was at the origin of discovery of many other drugs and drug candidates such as eltrombopag III (thrombopoietin receptor agonist) and BMS-858 IV (HCV inhibitor in clinical trials) (Figure 1).9,10     Figure 1: Drugs identified in high-throughput screening.   Interestingly, marketed drugs are often included in new screening programs, as even marketed druJs display “off-tarJet effects” that may be of therapeutic interest.11 The advantage in finding 3  a new application for an existing drug is that their in vivo properties are already optimized (low molecular weight molecules (<500 Da), ability to cross biological membranes, and good solubility in water and dimethyl sulphoxide, etc.12 Indeed, most drug candidates (up to 90%) fail to reach the market due to problems encountered during in vivo evaluation phase.  A potential disadvantage of in house libraries is that they comprise large numbers of analogues of specific hit structures, and although the number of molecules in the library may be large, they are limited in structural diversity, i.e. they explore only a small part of chemical-biology space.  1.3 Combinatorial Chemistry - Parallel Synthesis and Structural Diversity Under the pressure of HTS, many innovative advances have been made to construct libraries and to optimize their diversity. In concept, “combinatorial library synthesis” refers to the production of mixtures of compounds, obtained by reacting sets of reacting partners (building blocks) with each other in the same reaction vessel.13 For example, the reaction of a set of amines with a set of carboxylic acids in the presence of a peptide coupling reagent will, in principle, result in the formation of a mixture of all possible amide products. This amide bond forming reaction can also be an integral part of different multi-component reaction (MCR) processes (ex. Ugi reactions) (Figure 2).14   Figure 2: Ugi reaction for the generation of amide bond. The interest in the combinatorial chemistry strategy is that, even in a single operation, a very diverse set of reaction components can be engaged, and the result is the formation of a complex and equally diverse set of product molecules that are tested together.15 However, a drawback to the combinatorial approach is the necessity, when biological activity is found, to separate the library constituents from each other in order to identify the active component. Although ingenious strategies have been developed to deconvolute compound libraries, this process is time consuming relative to the alternative parallel synthesis approach to small molecule library construction.16 In the parallel synthesis version of combinatorial chemistry all combinations of the reactions are carried out simultaneously (i.e. in parallel), but importantly, they are carried out in separate reaction vessels. Thus, in contrast to the combinatorial chemistry, each reaction vessel will contain a single predetermined product. Parallel synthesis can be significantly 4  accelerated using robotic technology, and when optimally planned, product purification is simplified, and in some cases unnecessary.17 Today, the combinatorial chemistry approach has been largely surpassed by the use of parallel synthesis strategies to construct small molecule libraries of “druJ-like” molecules in a minimum timeline.  In its multiple applications, parallel synthesis has been used to identify new ligands that bind and modulate the function of known families of proteins, such as kinases, G-protein coupled receptors, proteases, and ion channels.18 Importantly, it finds its full potential in the construction of libraries of molecules for testing against protein targets of therapeutic interest for which there is little or no structural information available. Further, structural comparison of active and no-active molecules in a compound library provides a preliminary glimpse at SAR.19   Looking at combinatorial chemistry-parallel synthesis more closely, one sees that large collections of molecules having diverse structure and functionality can be prepared from simple starting materials.20 In these libraries structural complexity/diversity can be efficiently generated in four ways: a) skeleton (scaffold) diversity by incorporation of different core structures in the library, b) functional group diversity by varying functional groups, c) appendage diversity (or building-block diversity) through variation in structural elements around a common skeleton or functional group, d) stereochemical diversity through variation in the orientation of the elements that interact with the macromolecules.21-23 Grouping combinatorial chemistry-parallel synthesis toJether under the term “diversity-oriented synthesis” '2S, one sees that '2S is a powerful and efficient tool for generating libraries of complex and diverse molecules.21,22 As practiced in our laboratory, parallel synthesis is used to generate libraries of new chemical entities using primarily the appendage diversity and scaffold diversity approaches.  1.3.1 Parallel Synthesis: Appendage Diversity Strategy  As an example of the appendage diversity strategy, efforts are being directed in our laboratory to define an alternative to the anti-HIV agent IDC16 V (Figure 3). This tetracyclic indole compound was the first small molecule identified that inhibits the exonic splicing enhancer activity of the human spliceosome SR protein factor SRSF1.24 It was discovered through random screening of the Institute Curie in house library of 6700 molecules, which was initially synthesized as anti-tumor agents.24 Our current objective is to construct ring-opened, non-toxic and more synthetically accessible “mimics” of I'C1. 7his included the parallel synthesis of a library of 240 diheteroarylamide compounds VI. In this program, the expectation was that 5  compound VII, containing the right side pyridine ring appendage (ring D in IDC16) would be active (see superposition in Figure 3). To our surprise, this molecules was totally inactive. However, the four related molecules VIIIa-d, possessing a common benzoisosthiazole appendage in their structure, displayed potent anti-HIV activity.  These latter molecules differ significantly in structure from IDC16, and, in fact, in a traditional SAR study, we would never have envisaged their synthesis.25    Figure 3: Development of inhibitors of HIV-1 alternative splicing. 1.3.2 Parallel Synthesis: Privileged Scaffolds for Library Design Chemical library construction is often based on the chemistry of a suitably functionalized (activated) “privileJed scaffold”. 7his approach, first coined by (vans in the late 10s, refers to the idea of using a molecular framework or substructure that has the ability to serve as a ligand for multiple, diverse classes of targets.26 The benzodiazepines as anxiolytic drugs were discovered in the 1950s by Hoffman-LaRoche Company. In 1994, Elman and colleagues constructed a library of structurally diverse 1, 4-benzodiazapine-based compounds as antagonists of the cholecystokinin (CCK) receptor A (for treatment of gastrointestinal cancer). The 1,4-benzodiazapine core was further used for a wide array of therapeutic targets (Figure 4).26,27  6   Figure 4: Benzodiazepine as a privileged scaffold. The chromone scaffold has also been used as a privileged scaffold. Indeed, decorating the chromone scaffold leads to a variety of therapeutic agents used to treat different disorders (Figure 5).26,28-31  Figure 5: Chromone as a privileged scaffold. 7  The 1,4-dihydropyridine (1,4-DHP) has been used as a scaffold to construct L-type Calcium Channel (LTCC) blockers for treatment for hypertension. However, structural modifications involving introduction of diverse functionality on the 1,4-DHP scaffold has resulted in the identification of many pharmacological probes for other pathologies (Figure 6).26,32-36  Figure 6: 1,4-Dihydropyridine as a privileged scaffold. Quinoline-containing compounds are widely used as "parental" compounds to synthesize molecules with diverse biological application, including antimalarial, antibacterial, antiarrhythmic, and irritable bowel syndrome treatment (Figure 7).26,37-40  Figure 7: Quinoline as a privileged scaffold. 8  The 2-aminothiazole motif (fused or non-fused) appears as an integral feature in many compounds of therapeutic interest and marketed drugs. As indicated in Figure 8, 2-aminothiazole-based compounds showed broad spectrum of biological activities.41-46   Figure 8: 2-Aminothiazole as a privileged scaffold. Finally, libraries of purine containing compounds have been screened against a wide variety of biological targets and they resulted in the development of many therapeutic agents. A review by Legraverend and Grierson illustrates the many different applications that arise through changes in the functionality on the purine ring (Figure 9).47  Figure 9: Applications of purine ring-based chemical libraries.  9  There are many other examples in the literature describing the development of compound libraries based on privileged scaffold structures.  1.4 Thesis Project: Development of Isoform Selective Rho-kinase Inhibitors In our laboratory the parallel synthesis approach is being used to progressively create a library of “druJ-like” small molecules25 that will be screened to discover biologically active compounds targeting a wide range of systems of therapeutic interest. This is being achieved, using as a starting point the synthesis of targeted libraries for several projects that are aimed at developing molecules as inhibitors of protein-protein interactions and inhibitors of specific classes of enzymes.  A particularity of each of these projects is the absence of precise structural knowledge of the nature of the interactions between our compounds and the protein target that we could use to guide the design of new inhibitors. The parallel synthesis strategy is thus well adapted to our objectives. From a fundamental research perspective, the exploration of new therapeutic targets, and the development of new ligands against existing targets implicated in important pathologies, places our effort at the forefront in new areas of biomedical research.  As outlined in detail in Chapter 2, the objective of my thesis research is to use a combination of the appendage and scaffold-based parallel synthesis strategies to create a library of selective inhibitors of the two isoforms of Rho-kinase (ROCK1 and ROCK2). The precise role of these two kinase isoforms in cancer and diabetic cardiovascular disease is currently being unfolded. Consequently, there is considerable interest in the pharmaceutical sector to secure isoform selective ROCK1/2 inhibitors in order to determine the pathophysiological role of each isoform, and to validate ROCK as a therapeutic target. The development of kinase inhibitor-based drugs has undergone an exponential growth with the arrival of 23 new, marketed drugs during the past ten years.48 These drugs are categorized as being either type I or II kinase inhibitors on the basis of their mode of binding in the ATP binding site. Interestingly, although the structural basis for the design of type I kinase inhibitors is very solidly established, there is still an incomplete picture of how to design type II inhibitors, which stabilize the activation loop closed conformation.49 In fact, despite considerable effort, no type II  rho-kinase inhibitors have been described to date. My research project focused on the use of diversity driven parallel synthesis to explore different structural options to identify isoform selective type 1 and type II ROCK inhibitors. Before entering into the foundations of my research and the results obtained, a brief introduction to 10  kinases and Type 1/2 kinase inhibitors will be given, along with a summary of the biology of our kinase targets, ROCK1/2.   1.5 Protein Kinases Protein kinases comprise one of the largest families of phosphotransferase enzymes that catalyze the transfer of the terminal, or -phosphate moiety of adenosine triphosphate (ATP) to a serine, threonine (serine/threonine kinases), or tyrosine residue (tyrosine kinase) located on a substrate protein (Figure 10).50  Figure 10: Phosphorylation of protein substrate by kinases. The human genome has about 518 protein kinase genes, and they represent about 2% of all human genes.49 Protein kinases are classified into eight major groups according to their sequence similarity in catalytic domain (Figure 11).49   Figure 11: a) Human protein kinases classification, b) Graphical representation of the human kinome tree.49 11  Protein kinases are essential for controlling and modulating many diverse cellular regulatory functions, including signal transduction, gene expression, cell differentiation, and apoptosis.51-54 Although these enzymes are essential to normal physiology, aberrant expression of protein kinases is associated with major pathologies, including cancers (e.g. EGFR, VEGFR, ABL, CDKs, Braf, c-Kit, AKT), diabetes mellitus and cardiovascular disorders (e.g. PKA, PKC, and ROCK), inflammation (e.g. Src, Tec, and Syk), central nervous system (CNS) diseases (e.g. GSK3, DAPK1, and MAPK), and many others.51-54   1.5.1 Rho-associated, Coiled-Coil Containing, Protein Kinase (ROCK) Rho-associated, coiled-coil containing protein kinase (ROCK) is a kinase enzyme belonging to the AGC family of serine/threonine protein kinases with a molecular mass of ~160 kDa. Rho-kinase is activated by upstream G-protein, as a part of cell signaling, called RhoA. Rho G proteins belong to the Ras superfamily of monomeric GTPases and it contains 20 members of ubiquitously expressed protein in eukaryotes, including RhoA, RhoB, RhoC, Rac1 and Cdc42.  Rho G proteins regulate various cellular aspects such as cell shape, cell motility, and cell proliferation. Rho acts as molecular “on-off” switches that cycle between inactive GDP-bound form and active GTP-bound form interacting with different downstream effectors to trigger a series of cellular effects. The activity of Rho is regulated by three regulatory factors (Figure 12), including guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs). GEFs act as a positive modulator and activate Rho proteins by catalyzing the exchange of GDP for GTP while GAPs stimulate the intrinsic GTP hydrolysis activity and inactivate Rho. GDIs suppress the transformation between Rho-GDP and Rho-GTP forms and block the spontaneous activation (Figure 10). Rho-GDP is present in the cytoplasm while Rho-GTP is predominantly bound to the cell membrane. 55-57  Figure 12: Regulation of Rho-G protein activity.55  12  In the active form, GTP-bound, Rho GTPases activate many downstream effectors. One of the first and best-characterized downstream effectors of the small GTP-binding protein RhoA which is Rho-kinase (ROCK).  1.5.2  ROCK Isoform Homology, Functional Roles, and Tissue Distribution Identified in 1996, Rho-kinase has two isoforms known as ROCK-1 and ROCK-2 or Rho-kinase ȕ and α, respectively (Figure 13).58 The two isoforms are encoded by two different genes. The ROCK-1 gene is present in chromosome 18 and encodes a 1354 amino acid protein, while the ROCK-2 gene is present in chromosome 2 and encodes a 1388 amino acid protein. ROCK-1 and -2 exhibit an overall 65% homology in amino acid sequence and the two isoforms are almost identical in their kinase domains (92% sequence identity).59   Figure 13: A schematic of the molecular structure of Rho-kinase isoforms.58 Activation of ROCK by GTP-bound RhoA leads to phosphorylation of different substrates modulating a wide range of important cellular functions such as vascular smooth muscle contraction, cell adhesion, cell migration, actin cytoskeleton organization, cytokinesis, stress fiber formation, and gene expression.58,60 In fact, due to the high degree of structural similarity, ROCK1 and ROCK2 are believed to share more than 30 downstream substrates including myosin light chain (MLC), LIM kinases (LIMK), ezrin, radixin, moesin, and adducin (Figure 14).58 ROCK plays a crucial role in regulation of vascular smooth muscle contraction through phosphorylation of the regulatory myosin light chain MLC.60 Smooth muscle contraction is controlled by the phosphorylation of MLC, which is regulated by the opposing activities of MLC kinase (MLCK) and MLC phosphatase (MLCP). MLCP dephosphorylates MLC, leading to vascular smooth muscle relaxation. A rise in concentration of free Ca2+in the cytosol causes activation of Ca2+-calmodulin-dependent myosin light chain kinase (MLCK) and in turn phosphorylation of MLC and smooth muscle contraction. However, MLC phosphorylation and smooth muscle contraction can be induced in the absence of significant increases in Ca2+ (Ca2+ -independent smooth muscle contraction or calcium sensitization). This is accomplished through 13  phosphorylation of the regulatory myosin binding subunit (MBS) of MLCP by ROCK leading to the inhibition of MLCP activity and an increase in the level of phosphorylated MLC.60   Figure 14: ROCK substrates.58 Although the two ROCK isoforms are ubiquitously expressed in most tissues; they have been found to possess differential tissue localization. ROCK1 mRNA was found highly expressed in lung, kidney, liver and spleen; whereas ROCK2 mRNA was found preferentially expressed in the heart, brain and skeletal muscles.61 It was initially assumed that the two ROCK isoforms would be functionally redundant. However, recent studies have shown that they often have distinct non-redundant physiological roles.62 Genetic deletion of one ROCK isoform does not exhibit a compensatory up-regulation of the other isoform expression.62 Genetic deletion, knockout, of ROCK1 (ROCK1-1-) in mice resulted in reducing cardiac fibrosis in chronic high blood pressure models, inhibition of cell detachment induced by doxorubicin treatment, and insulin resistance.59,62,63 Whereas ROCK2-knockout (ROCK2-1-) resulted in intrauterine growth retardation, increased fetal death, and impaired motor function.64,65 Additionally, Both ROCK1 and ROCK2 knockdown resulted in eyelid open at birth (EOB) phenotype, omphalocele phenotype.66 The roles of ROCK in many diseases have been studied extensively with the aim to find out a new treatment. In the scope of my thesis, I will focus on the role of ROCK in cardiovascular diseases (collaboration with Dr. Kath MacLeod), diabetes, and cancer.   14  1.5.3 ROCK and Cardiovascular Diseases RhoA/ROCK-pathway abnormalities  are a central feature of the molecular pathology of arterial hypertension and it is regarded as a promising strategy for the treatment of hypertension.67 ROCKs influence arterial smooth muscle contraction through inhibition of MLCP activity and thus increased activity of the Rho/Rho-kinase pathway plays a central role in arterial hypercontraction.68 Rho/Rho-kinase pathway might be also involved in the angiotensin II-induced hypertension since ROCKs are considered one of the downstream effectors for angiotensin II.69 Dysregulated cell proliferation of vascular smooth muscle cells can be considered as a one feature of hypertension.  ROCKs are involved in the regulation of SMC proliferation so ROCKs might also modulate vascular hypertension via increasing cell proliferation.70 The non-selective ROCK inhibitor, fasudil IX, (Figure 15) has been shown to lower blood pressure in hypertensive rats versus normotensive rats.71 Rho-kinase pathway plays a crucial role in the pathogenesis of coronary vasospasm which in turn favors the development of variant angina, myocardial infarction, and sudden death.72 Accumulating evidence indicated that the expression of ROCKs has increased in the arteriosclerotic coronary lesions.73 It was demonstrated that the injection of fasudil IX and hydroxyfasudil X (Figure 15) in the coronary arteries suppress the coronary vasospasm and decrease ischemia/reperfusion (I/R) injury.74,75   Figure 15: Structure of the approved ROCK inhibitor fasudil IX, and the experimental drug hydroxyfasudil (keto form) X. The Rho/Rho-kinase-mediated pathway also plays an important role in the pathogenesis of cardiac hypertrophy and heart failure via phosphorylating troponin and inhibiting tension generation in cardiac myocytes.76 Recent evidence indicated that Rho-kinase abnormalities are substantially involved in the pathogenesis of fatal pulmonary hypertension through multiple mechanisms including vascular smooth muscles hypercontraction, pulmonary cell proliferation leading to vascular remodeling, and inÀammatory cell miJration.77 Long-term inhibition of Rho-kinase with fasudil caused a marked improvement of the pulmonary hypertension.78 15  1.5.4 ROCK and Diabetes Recently, a great deal of attention has been focused on Rho/Rho-kinase pathway in diabetes-related research as a new strategy in the management of diabetic complications. The studies have demonstrated a strong correlation between the overexpression/hyperactivity of Rho-kinase and the development or progression of diabetic complications.79 The possibility of using Rho-kinase inhibitors for treatment of diabetic neuropathy, sexual dysfunction, cardiomyopathy and other diabetic complications has been suggested.80 In this context, ongoing research in Dr. Kath MacLeod’s laboratory highlighted the role of RhoA/ROCK in the ventricular dysfunction that is associated with diabetic cardiomyopathy.81 Lin and coworkers found that the activation of the RhoA/ROCK signaling pathway plays a crucial role in the development of diabetic cardiomyopathy, and inhibition of ROCK acutely improved the contractile dysfunction in a rat model of diabetic cardiomyopathy.82  Erectile dysfunction (ED), a prevalent complication of diabetes, is associated with an impairment of the relaxation of corporal smooth muscle. It was reported that RhoA/ROCK pathway plays an important role in the erectile dysfunction in the STZ-diabetic rat penis. 83 The mechanism involved the increase in MYPT1phosphorylation and the down-regulation of eNOS expression/activity that both inhibit the corporal smooth muscle relaxation. ROCK and eNOS proteins were found colocalized in the endothelium of the corpus cavernosum in the STZ-diabetic rats. The erectile responsiveness in diabetic animals was significantly improved by intracavernous injection of Y-27632 that caused an increase in the maximal intracavernosal pressure without affecting mean arterial pressure.84 Diabetic vascular diseases such as neuropathy, retinopathy and nephropathy are initiated by endothelial dysfunction.85 The endothelial dysfunction results from an imbalance between the endothelium-derived relaxing factors (EDRF) such as nitric oxide (NO) and endothelium-derived constricting factors (EDCF) such thromboxane A2, and angiotensin II.85 RhoA/Rho-kinase activation has siJni¿cant effects on the development of diabetic vascular diseases. 52C. has been implicated in the down-regulation of nitric oxide (NO) synthesis via reducing the endothelial nitric oxide synthase (eNOS) mRNA stability and consequently decreasing expression of the (eNOS).86 Rho Kinase Inhibitors were found to ameliorate impairment of diabetic endothelial dysfunction in kidney vasculature.87 Insulin resistance and obesity play an important role in the development of type II diabetes. RhoA/Rho-kinase pathway has recently been found to modulate insulin signaling through 16  phosphorylation of serine residue of insulin receptor substrate-1 (IRS-1).88 Selective deletion of ROCK2 improved insulin sensitivity, suggesting that ROCK2 inhibition may be a novel approach for treatment of diabetes.81 1.5.5  ROCK and Cancer Mounting evidence has suggested that the Rho/ROCK overexpression has a distinct contribution in a number of human cancers. ROCK2 has been found over-expressed in colon, bladder, hepatocellular, and lung tumors.89-91 Furthermore, elevated levels of ROCKI were reported in hormone-refractory prostate cancer.92 Aberrant expression of ROCKs plays an important role in the cell migration and metastasis of the tumor cells via regulating assembly/disassembly of actin-cytoskeleton.93 Moreover, ROCK isoforms mediate vascular endothelial growth factor-A (VEGF)-driven angiogenesis suggesting that the overexpression of ROCK protein plays an important role in the formation of new blood vessels for tumor tissues.94 Because of this, Rho-kinase might be a potential therapeutic target for cancer therapies in the future and ROCK inhibitors may be a potential treatment of the disease via delaying tumor progression or preventing tumor migration. Y27632, has been reported to reduce human breast cancer cell migration, proliferation and metastasis to human bone.95 Moreover, Y27632 was reported to inhibit the Rho-mediated activation of actomyosin and the dissemination of MM1 hepatoma cells.91 In Addition, fasudil and hydroxyfasudil, as Rho-kinase inhibitors, were reported to inhibit tumor cell migration and tumor progression in human and rat tumor models.96  The crucial role of Rho-kinase in the pathogenesis of various diseases strongly motivates the discovery of ROCK inhibitor. ROCK is considered as a promising drug target for the search of cure for these diseases. Designing Rho-kinase inhibitors require understanding of the structure of the kinase domain (catalytic site), the binding mode of ATP, and the factors that govern the specific binding of the inhibitor to the catalytic site.97   17  1.6 Design of Kinase Inhibitors  A fundamental step toward designing kinase inhibitors is to understand the composition and geometry of the ATP binding site, the nature of the interactions between ATP and the kinase, and the effect of the activation loop status on the organization of the kinase active site. 1.6.1 ROCK Structure and Catalytic Mechanism Kinases have the same general structure and they are highly conserved in the ATP binding-catalytic sites. The typical protein kinase domain is composed of two lobes (Figure 16): a smaller N-terminal lobe containing a twisted five-stranded anti-parallel ȕ-sheet and the C-helix and a larJely α-helical C-terminal lobe. ATP binds in the cleft between the N- and C-terminal lobes, such that the adenine ring forms crucial donor-acceptor hydrogen bonds to the backbone amide NH and CO components in the short segment of amino acids, referred to as “hinJe residues”, which connect the N- and C-lobes (Figures 16, 17). The ribose ring in ATP binds into a sugar pocket and the triphosphate motif in ATP binds in a hydrophilic channel, which corresponds to the catalytic site.105,106  Other important features of the ATP binding-catalytic site include the glycine-rich loop, which allows ATP to approach the peptide backbone closely, the activation loop, which (as discussed below) controls entry of ATP and the protein substrate, and the phosphate binding site, where the catalytic reaction occurs (Figure 16).98-100     Figure 16: General kinase domain architecture and residue numbering for the residues of ATP binding site in ROCK1 and ROCK2.100   Activation loop Hinge ATCatalytic site N-terminal domain C-terminal domain 18   Figure 17: Organization of the kinase active site, ATP binding and catalytic site.  In normally functioning cells, kinase activity is regulated via a number of different mechanisms. The activation loop is a crucial regulatory element as its movement defines the organization of the catalytic site as well as access of ATP and the protein substrate to their respective binding sites. At the N-terminus of the activation loop is a conserved DFG (Asp-Phe-Gly) sequence, which contributes to positioning the triphosphate group in ATP for phosphate transfer. There are two extreme conformations of the activation loop: open and closed (Figure 18). When the activation loop adopts the open or “in” conformation, i.e. the DFG active conformation, the catalytic site is correctly structured and substrate/ATP can bind, resulting in catalysis. In the “DFG-in” conformation, the aspartic acid ' residue points toward the terminal phosphate group of bound ATP and helps to coordinate a catalytically important Mg2+ ion. Further, the phenylalanine (F) in the DFG motif points away from the ATP binding sites to the back cleft.101-104  Alternatively, when the activation loop adopts the “closed” or “out” conformation (DFG inactive conformation) the catalytic site is disorganized and substrates and ATP cannot access the active site. In the “DFG-out” conformation, the phenylalanine rotates away from the nearby αC heli[ and proMects into the A7P pocket, thereby blockinJ the accessibility of ATP and protein substrate to their binding sites (Figure 18). 102    19   Figure 18: The various conformations of the activation loop.104   1.6.2 Type 1 Kinase Inhibitors The vast majority of kinase inhibitors developed to date (>10,000) are type 1 inhibitors. Type I kinase inhibitors are ATP competitive inhibitors, in that they compete with ATP for occupation of the ATP binding site in the activation loop open (active) conformation.105 Like ATP, a key feature in type I inhibitors is the presence of a subunit that forms one to three H-bonds to the hinge region residues. However, with few exceptions type 1 kinase inhibitors are not designed such that structural elements project into the sugar and phosphate binding regions. The reason for this is that the ATP binding site and adjoining catalytic site are highly conserved between kinases. Indeed, due to the only minor differences between these sites in kinases, it has proven to not be possible (to date) to develop kinase selective inhibitors that exploit uniquely these binding regions.106 Fortunately, it has proven possible to develop type 1 kinase inhibitors displaying a good to high level of selectivity between kinase by designing molecules that project structural elements into two hydrophobic pockets (hydrophobic region I and II), which touch the ATP site, but are not involved in ATP binding.105,106 Figures 19a and b illustrate the position of these two additional or supplementary hydrophobic binding pockets, relative to the binding regions for ATP. In the 2D representation in Figure 19a, the pocket located in the interior of the ATP site is designated as Hyp I and the other pocket, which connects with the solvent (H2O) exposed surface of the enzyme is referred to as Hyc II. In the more topological representation in Figure 19b, the regions corresponding to Hyp I are BP1 and BP2 and the regions making up Hyc II on the front cleft are E0, which serves as the entrance of the ligand and E1,which serves as entrance for solvent-exposed surface.105 These two pockets have different size, shape, and composition between kinases. The gatekeeper 20  residue represented by the “.” motif is a further selectivity feature, located at the entrance to the Hyp1 pocket. Its size/steric bulk, determines to what extent motifs in the inhibitor structure can access this pocket. In general, the gatekeeper corresponds to alanine/threonine (small) or to phenyl alanine/leucine/methionine (bulky) residues. In ROCK1/2 the gatekeeper is a Met residue at positions 153/169, respectively.98,105  Figure 19: a) ATP binding sites divided into adenine-binding region, ribose-binding region, and phosphate-binding region, hydrophobic pockets I and II around ATP binding site, b) Topological view of ATP binding site in activation loop open conformation.105,106   Figure 20 illustrates the structural elements in kinase inhibitor PD166326 (ABL1 inhibitor) that bind to the hinge and interact with the selectivity pockets Hyp I and Hyc II.103,105    Figure 20:  a) Type I inhibitor, PD166326 XI, exploiting hydrophobic pockets I and II of ABL1. b) Topological representation of PD166326 XI in the ABL1, the aminopyrimidine fragment occupies the adenine pocket, thiomethyl group extends towards E0, dichlorophenyl inserted into back pocket BP-II.103,105  Type 1 inhibitors can accommodate different heterocyclic rings that occupy the purine binding site and serves as a scaffold for appending side chains that interact/bind in the adjacent 21  hydrophobic regions I and II.103 Examples of the diversity of functionality and structural motifs that enable the design of kinase selective kinase inhibitors is illustrated in Figure 21.105   Figure 21: Examples of type 1 inhibitors, a) Erlotinib XII in EGFR, quinazoline ring occupies the adenine pocket, the ether functionalities extends towards E0 and E1, and the acetylene moiety is extended toward BP-1, b) Sunitinib XIII in FGFR1, indolinone ring bound to the adenine region and the 3o amine extends towards  E1, c) Dasatinib XIV in c-Abl, the aminothiazole moiety binds in the adenine pocket, the hydroxyethyl piperidine extends toward E1, and the substituted phenyl inserted into back pocket BP-II.105 1.6.3 Type II Kinase Inhibitors The Type II kinase inhibition mode was initially revealed from the structure of imatinib (Gleevec®) I in complex with Bcr-Abl.107 Imatinib is a non-ATP competitive inhibitor that binds to the inactive, activation loop closed, form of the enzyme. Schematically, the interactions involved are shown in Figures 22a and b. The process whereby imatinib ultimately binds to the kinase involves interaction of the drug with the kinase at some point in the equilibrium between the activation loop open (active) and closed (inactive) conformations, such that it captures and stabilizes the inactive conformation (thereby shifting the equilibrium to the inactive state).108 22   Figure 22: a) Imatinib (type II inhibitor) I in complex with c-Abl,103  b) Topological representation of imatinib I in the c-Abl, the 2-aminopyrimidine and the attached pyridine ring occupies the adenine pocket, the substituted benzamide group binds to the back cleft BP-I and DFG pockets. The piperazine moiety occupies the back pocket BP-IV.103,105 Important is that the shape adopted by the activation loop in the closed conformation differs considerably to the open state. This movement occludes binding of ATP and the protein substrate, and effectively destroys the organization of the catalytic site. Further, it most freTuently results in a siJnificant shift on the position of the ')* seTuence to the “')*-out” position (see Figure 18 above). Indeed, the DFG motif will move up 10 angstrom during the closing of the activation loop, and this is accompanied by a significant increase in the volume of the back pocket (BPI-IV) such that an allosteric site exists beyond the DFG and Lys-Glu residues. The resulting increase in the volume of the PB-I or BP-IV pockets provides the room necessary for large molecules such as imatinib to bind to the kinase.104 This movement can be pictorially represented in Figure 23.   Figure 23: Closed activation loop conformation accompanied by increased size of back “Hyp1” pocket and its involvement in the new allosteric zone. 23  It has been suggested that, as the ATP binding region has a unique structure for each kinase when the activation loop is closed, type II kinase inhibitors can naturally “select” between kinases.51,104 However, more recent data points to the possibility that this phenomenon may not be general.109 The combination of a hinge binding component, additional functionality that H-bonds to the DFG sequence and/or the Lys-Glu pair and a third component that interacts with the newly exposed hydrophobic region beyond the DFG and Lys/Glu motifs are characteristics found in the currently known Type II kinase inhibitors.105 In other terms, Type II inhibitors are typically elongated-type structures that contain a head motif designed to interact with the hinge residue, an amide/urea linker functions in the middle of the molecule that harbor hydrogen-bond donor/acceptor groups essential to form H-bonds to the DFG sequence, and a tail (terminal) aromatic ring designed to occupy the allosteric site in the reorganized/restructured ATP binding region (Figure 24).109 The positioning of these structural elements in the type II binding site are illustrated in the topology diagrams  in Figure 25.105   Figure 24: Examples of type II inhibitors: The hinge-binding motif is indicated in red, the linker motif in gray, amide/urea function in blue, and the terminal hydrophobic in green.   24       Figure 25: a) Topological representation of sorafenib XV in B-Raf, the pyrdinyl-carboxamide moiety occupies the adenine pocket, the phenyl urea fragment binds to the back cleft BP-III and DFG-out pockets. The terminal substituted phenyl moiety inserted into the back pocket BP-IV,105 b) Doramapimod XVI in p38 MAP, and c) Fuoro-pyrimidine derivative XVII in VEGFR2.105   The number of Type II kinase inhibitors reaching the market is increasing, as they display good to excellent selectivity profiles relative to the target kinase. Further, they possess low association rate constant (kon) and a profoundly lower dissociation rate constant (koff) resulting in a long acting and extended kinase inhibition. The reason for the low koff is assumed to be due to the higher lipophilicity and extra hydrogen bonding of these molecules.110  1.6.4 Other Kinase Inhibitors Additional efforts have been exerted to develop alternative modalities for inhibiting kinases. These efforts paved the way to develop more selective inhibitors, Type III, IV, and V, that can target regions outside the highly conserved ATP-binding. Unfortunately, the rational design of these type of inhibitors is challenged by our incomplete understanding of these binding options and a lack of screening methods.111   Type III kinase inhibitors are small molecules, which act as non-ATP site binders (ATP-noncompetitive). They exclusively bind to an allosteric pocket adjacent to the catalytic domain of the kinase and negatively modulate kinase activity (Figure 26).109,111  Type IV kinase inhibitors also bind to a site apart from the ATP binding pocket, allosteric site (Figure 26). Upon binding to the allosteric site, they induce conformational changes of the 25  active site of the enzyme that make the kinase inactive. The exact location of the allosteric site is not known and may be anywhere on the kinase except for the pocket exploited by type III.109,111 Type V kinase inhibitors refers to bi-substrate, bivalent, inhibitors that target both the ATP and the substrate binding regions concurrently.111  Figure 26: Schematic representation of kinase inhibitors binding modes highlighting type III                       and type IV binding modes.111  1.7 Current Status of ROCK Inhibitor Development Significant research effort worldwide has been directed towards the discovery of potent and selective ROCK inhibitors. However, as mentioned earlier, due to the high sequence identity between ROCK isoforms in their kinase domain (92%), the development of ROCK-isoform-selective inhibitors has been, and continues to be a challenging task.  1.7.1 First Generation ROCK Inhibitors Currently, the only ROCK inhibitor clinically approved is fasudil IX (HA-1077)  (Figure 15), which has been used safely in Japan since 1995 for the treatment of cerebral vasospasm after subarachnoid hemorrhage (SAH). Although, fasudil is a more potent Type I inhibitor of ROCK (IC50= 1.2 and 0.82 µM for ROCK1 and ROCK2, respectively) relative to related AGC family kinases, it is a non-selective ROCK kinase inhibitor drug.71,112 The half-life of fasudil hydrochloride was found very short, less than 15 minutes. However, the long lasting effect has been attributed to its active hydroxylated metabolite, hydroxyfasudil X, rather than fasudil itself. Hydroxyfasudil X (Figure 15) has been prepared and proved to be more potent and selective toward ROCK1/2 relative to other kinases than fasudil.113 Further optimization of fasudil led to the discovery of the dimethylfasudil XVIII, H-1152P, (Figure 27), which was found to be more 26  potent than fasudil as Rho-kinase inhibitor but still retains inhibitory activity against other kinases.114 In 1997 Uehata and coworkers discovered a monocyclic pyridine-based compound, Y-27632 XIX (Figure 27), as slightly more potent (Ki of 220 nM) and more selective for Rho-kinase inhibitor than fasudil.60,71 Fasudil, hydroxyfasudil, dimethylfasudil (H-1152P), and Y-27632 are widely used as a molecular probes to evaluate the function of Rho-kinases in various diseases. They are further used to study the structure of the enzyme binding site.99    Figure 27: Molecular structures of four Rho-kinase inhibitors. Fasudil (IX) and hydroxyfasudil (keto form, X) are also presented in Figure 15.  Crystallographic data for fasudil IX, and hydroxyfasudil X in complex with ROCK (Figure 28) reveal that the isoquinoline ring occupies the adenine binding region of ATP and forms typical two hydrogen bonds with the hinge region of the enzyme. In fasudil, the isoquinoline nitrogen and C1-H form H-bonds with Met156/172(NH) and Glu154/170(C=O) hinge residues, respectively. Interestingly, the isoTuinolone motif in hydro[yfasudil is oriented in a “flipped” orientation relative to fasudil, in order to enable both the isoquinolone NH and C=O motifs to form H-bonds to the main chain of the hinge Glu154/170(C=O) and Met156/172(NH) residues.98,99,115  The weak isoform selectivity observed for these four Type I ROCK1/2 inhibitors is a consequence of the fact that the remaining polar functionality is projected into the sugar (R) or phosphate (P) binding regions (which are the same for all kinases), rather than into one of the two hydrophobic selectivity pockets (HypI or HycII).99 27                 Figure 28: a) Binding interactions of fasudil IX within ROCK-ATP site, b) Topological representation of fasudil IX in ROCK c) Binding interactions of hydroxyfasudil X within ROCK-ATP site, d) Topological representation of hydroxyfasudil X in ROCK.99 Over the past decade, many second generation Rho-kinase inhibitors have been identified, belonging to various chemical classes.  1.7.2 Second Generation Isoquinoline-Based Inhibitors Building on the isoquinoline scaffold found in fasudil, Iwakubo and coworkers showed that the R-enantiomer of compound XX is highly active in a cell-free kinase assay.116 Ray et al. used a fragment-based NMR screening strategy to develop compound XXI as ROCK1 inhibitor.117 Wu and coworkers showed that compound XXII displayed 10-fold improvement in potency over fasudil and Y-27632.118 In another study, the conjugate of 5-isoquinolinsulfonylamide and oligo-D-arginine (ARC-3002 XXIII) was reported to possess high affinity toward ROCK2 (ROCK1 not tested) with subnanomolar dissociation constant (Kd= 20 pM) 119 (Figure 29). 28   Figure 29: Isoquioline-based ROCK inhibitors. 1.7.3 Pyridine-Based Compounds The Y-27632 XIX analogue Wf536 XXIV was reported by Nakajima to inhibit metastasis in animal model systems120. Patel et al described the pyridine-thiazole-urea RKI-1447 XXV as a potent ROCK1 and ROCK2 inhibitor (Figure 30) with siJni¿cant anti-invasive and antitumor activities in breast cancer.121 As revealed from X-ray diffraction studies (Figure 31), the pyridine head group of XXV resides in the adenine pocket of the ATP-binding site of ROCK1 forming one key hydrogen bond between the N of the ring and the NH of M156.  The urea system forms two H-bond interactions with the backbone NH/C=O of Lys105 and Asp216 in the DFG motif, respectively.121 It is important to note that, despite the presence of both a urea functionality and a terminal non-polar aromatic motif in this elongated structure, it binds in ROCK1 as a type-I inhibitor rather than via the type-2 mode.121   Boland et al. developed ROCK inhibitor XXVI with excellent intraocular pressure lowering effect when applied topically to ocular normotensive NZW rabbits (ROCK2 IC50 = 2.5 nM). Further development of this compound series by Amakem resulted in the discovery of AMA0076 (undisclosed structure), which is currently in clinical trials for the treatment of glaucoma.122 Pfizer discovered PF-4950834 XXVII as a selective Rho-kinase inhibitor with anti-inflammatory properties (ROCK1/2 (IC50= 8-33 nM)).123 Finally, Li et al. showed that AS 1892802 XXVIII decreases both inflammatory and non-inflammatory pain in rat models. Moreover it was found to be slightly more selective for ROCK2 (IC50 = 100 nM) than ROCK1 (IC50 = 1690 nM) (SI = 17).124  29   Figure 30: Pyridine-based ROCK inhibitors.  Figure 31: Schematic presentation of RKI-1447 XXV interactions with the ROCK1-ATP site.121 1.7.4 Azaindole-Based Compounds  Efforts to optimize Y-27632 XIX by substituting the pyridine ring with a pyrrolo-pyridine, azaindole, ring system led to the development of Y39983 XXIX (ROCK1/2 IC50= 2.5-5.1 nM) and XXX (ROCK1/2 IC50= 0.6-1.1 nM) (Figure 32) as a more potent Rho-kinase inhibitors. Currently, Y39983 XXIX was submitted to phase II clinical trials as a treatment for ocular hypertension or glaucoma. 125,126  Molecular modeling studies (Figure 32) indicated that the replacement of the pyridine ring in Y-27632 XIX by an azaindole motif, as in XXX, gave rise to an extra hydrogen bond interaction in the hinge region between the azaindole N1-H and carbonyl O from Glu154. The insertion of a secondary amino group between the central difluorobenzene and terminal pyrimidine ring also 30  resulted in the second extra hydrogen bond interaction with the side chain carbonyl O of Asp160.127,128    Figure 32: Azaindole-based ROCK inhibitors and a molecular modeling image of XXX docked into ROCK1.127 1.7.5 Indazole-Based Compounds  Indazole-based compounds GSK429286A XXXI and DW 1865 XXXII have displayed good potency against ROCK1 (IC50= 14 and 250 nM respectively).129,130 SR-1459 XXXIII and SR-899 XXXIV displayed low nanomolar binding affinity against ROCK2 (IC50= 13, 100 nM respectively).131 Compound XXXV, discovered by a fragment-based strategy, also shows moderate potency for ROCK (IC50= 650-670 nM range).124 Most importantly, KD025 (formerly SLx-2119) XXXVI has proven to be a novel and potent ROCK2 inhibitor (IC50= 105 nM), with a 200-fold selectivity (SI) over ROCK1 (IC50= 24000 nM)132 (Figure 33).    Figure 33: Indazole-based ROCK inhibitors. 31  Analysis of the data for binding of XXXV into the ATP-binding site of ROCK1 (Figure 34) revealed that, like the pyridine-thiazole-urea compound XXV, it binds as a type-I inhibitor (DFG-in conformation).49,124 Indeed, although the urea motif captures a key H-bonding interaction with the Asp residue in the DFG sequence, the terminal phenyl ring interacts with the front pocket (Hyp1 or E1) of the ATP binding site. The results for XXXV and XXV suggests that simply incorporating an amide/urea and a terminal hydrophobic aromatic component behind the hinge scaffold may not be a sufficient condition for the design of a type II ROCK inhibitor.   Figure 34: a) Schematic presentation of the key interactions between XXXV and ROCK1; b) structural analysis indicates that XXXV binds to ROCK1 in the DFG-in conformation and the terminal aromatic ring resides in the front pocket (FP2, also known as E1)49, c) Imatinib binding in Bcr-Abl in the DFG-out conformation showing a detailed description of the hydrophobic pockets around ATP binding site.49,124 It is noteworthy to mention that the variation in the positioning of the motifs on the middle portion of the molecules XXXI to XXXVI seems acceptable, i.e. compatible with activity. In this context, we find it compelling to imagine, from the superposition of compound XXXI on XXXVI (KD025) (Figure 35), that the origin of the ROCK1/2 selectivity for compound XXXVI resides in the presence of the added substituted aromatic motif that is inside the blue box. Whether or not compound XXXVI is a type II ROCK inhibitor remains to be determined.   32   Figure 35: Superposition of XXXI (blue) on XXXVI (red), note close structural identify,except for aryl motif in black box. 1.7.6 Pyrazole-Based Compounds SR-3677 XXXVII is one of several pyrazole-based inhibitors that have been identified from a high-throughput screen as a selective ROCK2 (ROCK2/ROCK1= 20-fold) (Figure 36).133 Compound XXXVIII was identified by Fang and coworkers as ROCK inhibitor and has been used as a starting point for the development of compound XXXIX as a potent ROCK2 inhibitor (IC50 = 7 nM).134 Other compounds possessing a pendant side chain have been developed. Of these compounds, XL-XLIV with remarkable binding affinity against ROCK2 isoform (IC50 §1 nM).135-137    Figure 36: Pyrazole-based ROCK inhibitors.  33  Compounds XXXVII and XXXIX were docked into the ATP-binding site of the ROCK2 and the enzyme±ligand complex showed that they all bind as Type-I inhibitors (Figure 37). Beyond the interactions of the pyrazole motif with the hinge residues, a third hydrogen bond is formed in the phosphate binding region between the carboxamide C=O and the side chain NH of Lys121. A fourth H-bonding interaction was found between the protonated tertiary amine of the dimethylaminoethyl group and the side chain carbonyl group of Asp176. This interaction is probably the key binding element that contributes for the higher selectivity of these compounds for ROCK over other kinases. Hydrophobic interaction was found between the terminal aromatic ring and a hydrophobic pocket under the P-loop. It is believed that this hydrophobic interaction is the dominant factor that contributes to the high potency of the these compounds.133,134    Figure 37: Structure of compounds A) XXXVII docked into ROCK2 ATP-binding site, B) XXXIX docked into ROCK2 ATP-binding site.133,134  1.7.7 Aminofurazan-Based Compounds  GlaxoSmithKline described two potent compounds: GSK269962A XLVI and SB-772077-B XLVII based upon earlier work on compound XLV, a MSK-1 inhibitor.138 Adding extra functionalities/substituents to the prototype molecule XLV resulted in loss of affinity for MSK-1, AGC family kinase member, and the development of high affinity for ROCK isoforms. GSK269962A XLVI and SB-772077-B XLVII have high potency (IC50= 1.6 to 6 nM) and good selectivity for human ROCK1/2 relative to other protein kinases138-140 (Figure 38).  34   Figure 38: Aminofurazan-based ROCK inhibitors. 1.7.8 Others Continued medicinal chemistry efforts to develop Rho-kinase inhibitors have resulted in the discovery of numerous other scaffolds that are noteworthy in their ability to inhibit ROCK1/2. These include benzoxaborole141, benzimidazole142, phenyl urea143, and 3-nitropyridine144. Other potent and non-isoform selective ROCK inhibitors are undergoing clinical evaluation, but their structures are not disclosed. 1.8 Prospective The growing evidence of the implication of ROCK isoforms in the pathogenesis of many diseases and knowing that these two isoforms are functionally non-redundant drives the need to synthesize drug-like molecules that are isoform selective, and avoid off-target binding. In view of the high degree (92%) of sequence identity for the kinase domains in ROCK1 and ROCK2, the design of isoform selective inhibitors is going to be challenging.  Further, the available data indicates that the currently known ROCK inhibitors bind at the ATP-binding site in the Type I binding mode.60 At present KD025 remains the only Rho-kinase inhibitor to exhibit significant ROCK2 versus ROCK1 selectivity (200-fold). The structural origin of this selectivity remains obscure.  1.9 Scope of My Work  The work presented in this dissertation aims to develop isoform selective ROCK inhibitors inhibiting ROCK by type II binding mode that can specifically inhibit ROCK isoforms. To accomplish this goal, we have developed the synthesis of 11 different small molecule libraries as outlined in Chapter 2.    35  Chapter 2 Objectives and Evolution of my Thesis Project 2.1 Starting Point for a Rho-kinase (ROCK) Inhibitor Library  The ultimate aim of my research is to generate a small molecule library based on different scaffolds and appendages that will be tested against different targets of therapeutic interest. In the scope defined for this thesis, the discussion is centralized around one target, ROCK. The development of rho-kinase inhibitors has been an active field of research over the past 10 years, and will continue to be, due to the escalating evidence of their implication in many pathophysiological disorders. As described in chapter one, the ATP binding sites in ROCK1/2 are 92% identical, which makes the goal of development of isoform-selective inhibitors difficult to achieve. Despite this obstacle, based on experiments using the non-selective inhibitor Y-27632, there is a noted difference between the two sites in their affinity (Km) for ATP binding (Km values for ATP were estimated to be 0.15 µM and 0.25 µM for ROCK1 and ROCK2, respectively).145 These data suggest that it may, in fact, be possible to design isoform selective type II ROCK inhibitors. Importantly, this small, but significant difference in ATP binding site geometry/composition may accentuate differences in the activation loop closed conformations of the two enzymes, which may further permit the design of type II Rho-kinase inhibitors that select between the two ROCK isoforms. At the outset of my work, structural data were available for fasudil, hydroxyfasudil, and Y-27632 only in complex with ROCK1 in the activation loop open conformation.98,99 This enabled us to analyze the binding of these type I kinase inhibitors in the ATP binding site of ROCK. In contrast, no corresponding structural information was available that could provide insight as to the geometry of the activation loop closed conformation of ROCK. Consequently, it was not possible to enJaJe a proJram to “rationally desiJn” type II isoform selective 52C. inhibitors using structural data. For this reason we leaned heavily toward the use of parallel synthesis to study a wide range of structures as potential type II rho kinase inhibitors. Targeting the ROCK activation loop in a closed conformation was a pioneering idea from the research perspective.  In the conception of our parallel synthesis approach we were guided by the generalized observation that known type II kinase inhibitors (Chapter 1, Figure 24) typically have a hinge 36  binding moiety at one end, which forms at least one H-bonding interaction with the hinge residues, an internal, or central, segment containing an amide or urea motif, which H-bonds to either the DFG activation loop subunit or the C-loop Glu residue, and at the other end a hydrophobic aryl/heteroaryl subunit, which interacts with the allosteric pocket created by the DFG-out flip. In other words, type II inhibitors are elongated molecules, which are just too large (complicated), to fit into the hydrophobic pocket in the ATP binding site in the type I model. This point is illustrated (Figure 39) by one tactic that has been developed to transform type I kinase inhibitors to type II inhibitors by simply adding a amide/urea and hydrophobic subunits onto the HYP1 binding pocket motif.106     Figure 39: Designing of type II kinase inhibitors by attaching type I head (black) to a type II tail (red).106 The crucial choice at the beginning of my project was the selection of the hinge binder motif (head part) that is amenable to more structural modifications and optimization. Inspired by the presence of the cyclic amide function in hydroxyfasudil X and several other kinase inhibitors,146 and considering synthetic accessibility of diversely functionalized 2-pyridinone systems,147 the 2-pyridinone motif was initially chosen as the hinge binding motif. The 2-pyridinone system has the possibility to bind to the hinge region via two hydrogen bond interactions between the carbonyl oxygen/NH of the ring and the amide NH of Met156/ the C=O of Glu154 respectively  or between the carbonyl oxygen/NH of the ring and NH/C=O of Glu154, respectively (Figure 40). Further, in our initial work the decision was taken to connect the spacer to the 2-pyridinone ring via the C-5.  37   Figure 40: The binding options of 2-pyridinone-based scaffold..  Considering the easy accessibility of the requisite 2-pyridinone-5-carboxylic acid derivatives, the synthesis of a series of diheteroaryl amides 'HA’s XLVIII was envisaged (Figure 41).   Figure 41: Initial choice of the core motif. Although the preparation of a diverse library of DHA compounds XLVIII could easily be prepared by peptide-type coupling reactions, it was anticipated that such compounds would not be sufficiently elongated for them to act as type II kinase inhibitors. An alternative choice for the spacer-DFG binding element, which would provide a more elongated structure, is the acyl urea motif found in XLIX. However, N-acyl ureas are metabolically unstable. For this reason, priority was given, as presented in Figure 42, to incorporate a stable amide bioisostere, such as an oxazole and oxadiazole, in the linker segment.148 This idea is explored in chapter 3 of my thesis. Indeed, in this chapter, we described our effort to synthesize the oxazoles 22/23a-f, and the synthesis of oxadiazoles 34a-ah. Note that right side of the molecules will be varied so as to create a diverse library of analogues that will permit us to explore means to optimize binding to the allosteric site in the type II binding mode. This work is extended to include the isomeric oxadiazole compounds 41a-g, and the oxadiazoles 45a-f lacking the central amide motif. These compounds were prepared in order to determine whether such compounds could in fact function as type I ROCK inhibitors.  38   Figure 42: Overall strategy of the thesis.     39  In chapter 4 we described different structural modifications to the linker part of the pyridinone based scaffold, which were studied to evaluate the optimal length of the linker. The strategy adopted for these modifications included: 1) incorporating six-membered saturated ring (piperazine) and hydrogen-bond acceptor (C=O), 2) incorporating five-membered hateroaromatic ring (thiazole) and hydrogen-bond donor (NH).  In chapter 5 we described further structural modification that were prompted by the biological results obtained in chapter 4. A different strategy was ultimately employed by changing the lefts side of our scaffold by introducing divergent (hetero)aromatic rings as hinge binder motifs while having either an indazole or a pyridine motif on the right side. In chapter 6 we described structural optimization to improve upon the established scaffold, indazole-aminothiazole based scaffold, to increase potency and selectivity based on biological results obtained in chapter 5. The design of these analogs acted to closely mimic type-II inhibitors. In Chapter 7, we discussed the biological results obtained for a selection of the molecules that have been synthesized. It is important to note, that during the course of these studies, a relatively large number of publications have appeared exploring the potential the different ROCK inhibitor classes described in the introductory chapter (Sections 1.7.1 to 1.7.8). Many of these molecules, illustrated in Figure 43, have structural features that satisfy the criteria established for Type II kinase inhibitor binding.  However, the structural data and docking studies for complexes of these molecule types with ROCK indicate that they all interact via either the type I inhibitor model (DFG-in), or an intermediate state between the DFG-in and DFG-out, (DFG-out like conformation), where the inhibitors interact with the FP I and/or II pockets, and not the inner pocket as found for essentially all other type II kinase inhibitors (Figure 34c, Chapter 1).49 As surprising as these results are, they underscore the importance and interest of studies, such as ours, to further explore different structural types in an effort to identify type II ROCK Inhibitors. Indeed, the structural features of the activation loop closed conformation of ROCK appear to differ significantly from many of the other kinases investigated. Although there is significant progress made in the development of ROCK inhibitors such as KD025 XXXVI (Chapter 1), ROCK-isoform-selective inhibitors have not been optimized yet in terms of structural features and binding mode. . 40   Figure 43: Type II-like rho-kinase inhibitors; hinge binding motif is indicated in red, the linker motif in black, amide/urea function in blue, and the terminal hydrophobic in green.        41  Chapter 3  Pyridinone-Dihydroxazole/Oxadiazole-Based Compounds 3.1 Introduction As briefly mentioned in the previous chapter, the first objective fixed was to construct a library of compounds where the separation between the hinge binding 2-pyridinone component and the diverse set of aryl and heteroaryl motifs incorporated on the other extremity is larger than that found in the simple diheteroaryl amide (DHA) analogues XLVIII (Figure 44). Such molecules can be built by introducing an additional amide motif into the central portion of XLVIII. However, it was anticipated that the resulting acylurea-type compounds XLIX would be unstable, relative to molecules where an oxazole ring is used as a stable bioisostere of one of the amide functions. Our initial goal was to synthesize a library of 2-pyridinone-oxazole carboxamides 22a-f/23a-f as potential ROCK inhibitors.   Figure 44: Summary of synthesis of compounds 22a-f and 23a-f.    42  3.2 Synthesis of Pyridinone-Oxazole Carboxamides 22a-f and 23a-f  Based upon the method developed by Pattenden,149 Wipf,150 and others151,152 to construct substituted oxazoles from l- or d-serine, the synthesis of our target library of compounds 22a-f/23a-f can be divided into 4 stages (Figure 44): i) synthesis of the 2-pyridinone-5 carboxylic acids 7 and 8,  ii) synthesis of the O-protected serine carboxamide intermediates 15a-f iii) condensation of 7 and 8 with 15a-f, iv) dehydrocyclization of intermediates 18a-f/19a-f followed by oxidation to get the desired oxazole products 22a-f/23a-f.   Note, a key feature in this synthesis is to form the crucial amide bond that introduces the diversity element prior to formation of the oxazole ring. It was anticipated that the acid function in the simple serine derivative 12 would be more reactive than a carboxyl function attached to C-4 of an oxazole. 3.2.1 Synthesis of the 2-Pyridinone-5-Carboxylic Acid Building Blocks 7 and 8 The pyridinone-5-carboxylic acid derivative 3 that we initially chose to use bears an additional OH substituent at C-4, which can be exploited in SAR to improve binding to the ROCK enzyme and/or to improve solubility and other physico-chemical properties. Compounds 7 and 8 were obtained in 5 steps from diethyl 1,3-acetonedicarboxylate 1 (Figure 45).153   Figure 45: Synthesis of 4-alkoxy-6-oxo-1,6-dihydropyridine-3-carboxylic acids.  43  In the first step, diester 1 was reacted with triethyl orthoformate in acetic anhydride to give intermediate 2.  Without isolation, this intermediate was reacted with ammonia to give the expected pyridinone product 3 in 68% yield through a 1,4-addition ± cyclization process.It was hoped that, in the next step, it would be possible to carry out a selective  O-benzylation/methylation of the C-4 OH group in 3 to give compounds 6a and b directly.  However, this proved to be challenging, as the lactam NH and lactim OH in the two tautomeric forms of 3 are also reactive. Although X-ray crystallography shows that the 2-pyridinone form 3a is the largely predominant tautomer in the solid state,154 it is well known that the desired reaction of the C-4 phenolic OH in 3 can be accompanied by alkylation of at either the NH (lactam tautomer) and OH (lactim tautomer) centers (Figure 46).155    Figure 46: Tautomerism of 2-pyridinone ring system and its effect on alkylation reaction. Indeed, attempts to selectively O-methylate/benzylate the C-4 OH in 3 using Mitsunobu conditions resulted in the formation of a mixture of N1-, O2-, and O4-alkylated products (Figure 46), which could not be separated by flash chromatography.155 To circumvent this problem, the N1-Boc protected pyridinone 4 was prepared by reacting 3 with di-tert-butyl dicarbonate Boc2O in THF, using sodium hydride as a catalyst, (68% yield). The chemoselective N-protection over O-protection results from the greater stability of (N-Boc) carbamate versus the O-Boc carbonate in basic medium.156  Having protected the lactam nitrogen, the O-alkyaltion of the C-4 OH in 4 was attempted under different basic conditions (NaH, cesium and potassium carbonate) in THF. Surprisingly, under the same base conditions used to put the Boc group on nitrogen it was deprotected, and multiple products were formed. A plausible explanation for these results is that the THF was not completely anhydrous, and that adventitious water acted as a nucleophile, which effected hydrolysis of the amide bond. In fact, N-Boc protected nitrogen heterocycles are known to be susceptible to base promoted amide hydrolysis, due to the stability of the N-anion that is produced as a leaving group in such reactions.157  In light of these results, attention was turned to using the  Mitsunobu reaction for the aryl-alkyl ether formation step, as base is not included in 44  the protocol.158 The Mitsunobu reaction was carried out using benzyl alcohol/methyl alcohol in combination of triphenylphosphine and diisopropyl azodicarboxylate (DIAD) in THF at room temperature. The required O4-alkylated products 5a,b were isolated in 51% and 47% yield, respectively. The moderate yields in these reactions was a consequence of difficulties encountered during separation of the products from hydrazine and triphenylphosphine oxide by-products. Formation of the O4-alkylation products was confirmed by the presence of deshielded protons of O-CH2, of 5a, and O-CH3, of 5b at 5.23 and 3.51 ppm, respectively in the 1H NMR spectrum.  Mechanistically, the Mitsunobu reaction involves: 1) formation of protonated betaine from the reaction between triphenyl phosphine and DEAD, 2) the activation of an alcoholic hydroxyl group by the protonated betaine to form the oxyphosphonium intermediate, and 3) oxyphosphonium attack by the pyridinone anion, nucleophile, in SN2 mechanism with carbon-oxygen bond cleavage and formation of the final ether (Figure 47). One of the main considerations for the Mitsunobu reaction is the pKa of nucleophile. The acidic proton in the nucleophile must have a pKa less than 15 for the reaction to be successful.158 In our case, 4-hydroxypyridine is sufficiently acidic (Pka= 11.12) for its participation in the Mitsunobu reaction.159   Figure 47: Mitsunobu reaction mechanism. 45  Subsequent deprotection of the N-Boc group in 5a,b was achieved by treatment with trifluoroacetic acid in dichloromethane to give the ester 6a,b in 90 and 84% yields, respectively. In this deprotection step (Figure 48) the t-butyl ester group undergoes cleavage in acidic conditions forming carbon dioxide and isobutylene by-products.   Figure 48: Boc deprotection mechanism. In the last step, saponification of ester 6a,b using LiOH in THF:MeOH:H2O (2:2:1) at room temperature  provided the desired acids 7 and 8 in high yield (88% and 84%, respectively).  The structures of compounds 3-8 were confirmed by 1H NMR and mass spectroscopy. For all compounds there are two characteristic peaks in the proton NMR: one peak around 5.8-5.6 ppm for the C-3 H and the other peak at 8.0-7.8 ppm for the C-6 H.  The different chemical shifts for these two protons is a function of their chemical environment. The C- proton is located on a ȕ-carbon to the exocyclic carboxylate ester or acid which is an electron withdrawing groups while C- proton is located on a ȕ-carbon to the exocyclic hydroxyl or alkoxy groups which are electron donating groups. 3.2.2 Synthesis of the Serine Carboxamide Building Blocks 15a-f The approach adopted for the synthesis of the serine carboxamide components 15a-f is illustrated in Figure 49. The first step involved N-Boc protection of L-serine methyl ester 9 (HCl salt) through reaction with Boc2O in the presence of triethylamine to give 10 in 90% yield.160  Subsequent conversion of 10 to the fully O/N-protected ketal o[azolidine or *arner’s ester) 11 was acheived using DMP in the presence of BF3-Et2O as catalyst.161 The ester 11 was then saponified to the corresponding acid 12 using aqueous lithium hydroxide in MeOH at room temperature for 12 hours. Acid 12 was then reacted with the library amines under BOP-Cl/Et3N conditions to produce amides 13a-f in 51-75% yield. The 1H NMR spectra for compounds 13a-f showed that the two protons on CH2 of the serine residue are not equivalent: one multiplet siJnal for one proton appeared at į .50-4.13 ppm and the other multiplet appeared at į .0-3.85 ppm.   46   Figure 49: Synthesis of serine carboxamide.   Exploratory experiments were then carried out using aniline as a representative amine component in order to find optimal conditions for the peptide-type coupling reaction that introduce the diversity elements. The results, listed in Table 1, showed that the best (isolated) yield was obtained using BOP-Cl (Table 1, entry 2) so BOP-Cl was applied in the formation of derivatives 13b-f.  Entry Coupling agent Equiv. Isolated yield (%)  1  BOP  1.2  48%  2  BOP-Cl  1.2  78%  3  PyBOP  1.2  57%  4  HBTU  1.2  28%  Conditions: acid (1 eq), amine (1 eq), Et3N (3 eq) in THF at room temperature for 12h Table 1: Selection of peptide coupling agent for 13a-f synthesis. 47  BOP-Cl activation of carboxylic acids (Figure 50) depends on base-catalyzed formation of a mixed anhydride, which is subsequently attacked by the amine forming the amide.162   Figure 50: Mechanism of BOP-Cl in amide bond formation. Cleavage of both the O,N-ketal and the Boc group in the following step was initially effected using TFA/CH2Cl2 (1:1). However, competing trifluoroacetylation of the liberated amine functionality was observed (Figure 51). Even though cleavage of the trifluoroacetyl group could be readily affected by rendering the reaction medium basic prior to work-up, the use of TFA was abandoned in favor of the use of HCl. Indeed, treatment of compounds 13a-f with methanolic hydrogen chloride at RT for 3 h afforded the β-hydroxy amines 14a-f as hydrochloride salts in good yields (71-84%).   Figure 51: Side reaction from TFA cleavage of O,N-ketal. In the subsequent step, the liberated alcohol function in 14a-f was O-TBS protected, so as to avoid any competing ester formation during the EDC mediated amide coupling reaction. In the experiment, reaction of alcohol 14a-f with tert-butyldimethylsilyl chloride (TBS) in acetonitrile in the presence of imidazole afforded the ȕ-silylated hydroxy amines 15a-f in 66-75% isolated yields.  3.2.3 Synthesis of Target Compounds 22a-f and 23a-f The synthetic pathway to access compounds 22/23a-f (Figure 52) began with formation of amides 16a-f and 17a-f. In cases where racemization is not a concern, one of the most popular ways to form an amide bond is to react an acid chloride with the requisite amine in the presence of a base to trap the HCl generated during the reaction.163 In our case, formation of the acid chlorides of 7 and 8 through reaction with thionyl chloride was tricky. The reaction was sluggish 48  and resulted in the formation of many by-products, as shown by TLC. However, the reaction of acids 7 and 8 with amines 15a-f in the presence of EDC/HOBt/DIEA afforded the desired amides 16a-f and 17a-f in moderate to good yields (42-67%).   Figure 52: Synthesis of pyridinone oxazole-based compounds. A problem encountered with this reaction was that considerable quantities of product were lost during the aqueous extraction process. In an attempt to overcome this situation, the concentrated reaction mixture was taken up in a small volume of DCM and applied directly to  a silica-gel column. However, even using this strategy purification was difficult, resulting in loss of valuable material.    Intermediates 16a-f and 17a-f were initially desilyated using TBAF in THF to give the ȕ-hydroxy amides 18a-f and 19a-f. However, again, the aqueous extraction during work up to remove excess TBAF and by-products from TBAF was not ideal because the product had significant water solubility. Efforts to remove excess TBAF with an alternative work-up method using sulfonic acid resin and calcium carbonate also proved unsatisfacory. Ultimately, these problems were circumvented by treating compounds 16 and 17 with methanolic hydrogen chloride, as work-up consisted in simple evaporation of the methanol solvent. The crude product was taken on to the ring closure step without purification.  A variety of reaJents and conditions have been developed that effect cyclodehydration of ȕ-hydroxy amides 18a-f and 19a-f to oxazolines 20a-f and 21a-f, including the use of 49  diethylaminosulfur trifluoride (DAST) and Burgess reagent (methyl N-(triethylammoniumsulfonyl)carbamate).164,165 In our hands, the use of DAST at low temperature in the presence of base gave consistently better results.164 Compounds 20a-f and 21a-f were obtained in 16-46% isolated yields (see experimental section, Chapter 8).  Different conditions have also been developed for the oxidation of oxazolines to the corresponding oxazoles.166-168 However, it was recognized that this transformation can be problematic. Indeed, efforts to dehydrogenate oxazolines 20a-f and 21a-f using using BrCCl3/ DBU, MnO2 or NBS were all unsuccessful. In all cases, formation of multiple products was observed when the reaction was carried out at room temperature, or on mild heating, or at higher temperature (above 80°C).  The structures for the oxazoline derivatives 20a-f and 21a-f were confirmed by NMR (Chapter 8, Table 22, and 23) and mass spectroscopy.  In the 1H NMR spectra, the disappearance of amide N-H and the O-H protons was noted, and there was a downfield shift in the oxazoline CH (C-4) and CH2 (C-5) protons. All 12 of the pyridinone-oxazolines 20a-f and 21a-f correspond to novel compounds (not described in CAS). As considerable difficulties were encountered in the conversion of the oxazolines to oxazoles 22a-f and 23a-f, the dihydrooxazole derivatives 20a-f and 21a-f were instead evaluated as potential ROCK2 inhibitors using the in vitro ELISA assay developed in collaboration with Dr. .ath Mac/eod’s Jroup. The 12 compounds were tested at a single point concentration (10 µM) using Y27632 as a positive control. Disappointingly, eight compounds (20a, 20b, 20c, 20e, 21a, 21b, 21e, and 21f) were only found active (Chapter 7, Table 6) and they exhibited very weak, up to 20%, inhibitory activity against ROCK2 compared to the positive control.  As the synthetic route to the desired oxazoles was long and tedious (purification problems at essentially every step) and it did not prove possible to effect the final oxidation step, we decided abandon further work in this direction. 3.3 Synthesis of Pyridinone-Oxadiazole Carboxamide based series 34a-ah In view of the difficulty encountered to oxidize the oxazoline intermediates 20/21a-f to the target oxazoles 22/23a-f, we turned our attention toward the use of the 1,2,4-oxadiazole ring as an alternative and more readily accessible amide bioisostere. The 1,2,4-oxadiazole motif is found in a number of clinically used drugs, as well as in many molecules currently in late stage clinical trials.169-171 This amide/ester bioisostere has the advantage of higher metabolic and 50  hydrolytic stability.172 Due to the significance of 1,2,4-oxadiazoles in drug development, numerous approaches have been described in literature for the synthesis of this ring system. One of the common methods used involves the condensation of amidoximes with carboxylic acid derivatives (acid chlorides, nitriles, esters, or other activated acid derivatives) (Figure 53).173-175  Figure 53: General method for 1,2,4-oxadiazole synthesis. We have used this approach to synthesize of a series of 34 novel pyridinone-1,2,4-oxadiazoles based compounds 34a-ah (not described in CAS), incorporating the N-substituted amide function at C-5 of the oxadiazole ring (Figure 54). The synthetic pathway to access compounds 34a-ah (Figure 54) began with the CDI-mediated conversion of the commercially available acid 24 to amide 26. In this process, the in situ generated N-acylimidazole 25 intermediate was treated with ammonium hydroxide at room temperature to afford the primary amide 26 in 77% yield.  Examination of the 1H-NMR spectrum of the amide product 26 showed the appearance of new broad sinJlet at į1. ppm corresponding to NH2. The peaks corresponding to H-2, H-4, and H-5 in the proton spectrum were observed at 7.9-8.0 ppm, 7.8-7.7 ppm, and 6.4-6.3 ppm respectively. Dehydration of amide 26 using cyanuric chloride (CyCl) in DMF at 40 oC subsequently afforded nitrile 27. The advantage of using these conditions was that it required only one third of equivalent of cyanuric chloride for the reaction to go to completion, and the cyanuric acid by-product was easy to remove by aqueous extraction.  51   Figure 54: Synthesis of pyridinone oxadiazole carboxamide-based compounds. 52  The mechanism for the cyanuric chloride-mediated dehydration of amide 26 is depicted in Figure 55. In the first step, the hydroxyl group of DMF is activated by a nucleophilic aromatic substitution SNAr to form the reactive species, a Vilsmeier adduct. The next step involves the dehydration of the amide by the in-situ generated Vilsmeier adduct to produce the nitrile function.176 In the 1H-NMR of nitrile 27 a downfield shift for the H-2 signal occurred relative to the same proton in the spectrum of amide 26. This was attributed to the more electron-withdrawing effect of nitrile group.   Figure 55: Dehydration of primary amides by CyCl. Nitrile 27 was converted to amidoxime 28 (an amino oxime) upon reaction with hydroxylamine hydrochloride in the presence of sodium bicarbonate as a base in an ethanol/water mixture (4:1). This involved nucleophilic attack on the nitrile carbon atom by the nitrogen atom of hydroxylamine.177 One of the challenges encountered during work±up procedures for the amidoxime 28 was its high polarity, which made the purification step difficult. Efforts to condense amidoxime 28 with the acid chloride of monoethyloxalate 30, in the presence of triethylamine as a catalyst, to give 32 using different solvent and temperature conditions resulted in the formation of a complex mixture of products (as seen by TLC). This is presumably because there is, once again, competing acylation of NH of lactam and OH of lactim tautomer. However, this situation was effectively circumvented using new conditions we developed, based upon the condensation of the O-silylated amidoxime 29 with the acyl fluoride 31. Acid fluorides have the advantage of being more stable toward oxygen nucleophiles, such as water or methanol, than the corresponding acid chlorides due to the nature of the C-F bond.163 In addition, acid fluorides react more efficiently with amines than do acid chlorides.163 Further, reaction of acid fluoride with silylated hydroxyl group would avoid base catalysis since the reaction is driven forward by the formation of strong Si-F bond.178 The silylated amidoxime 29 53  was obtained by silylation of the hydroxyl group of 28 using tert-butyldimethylsilyl chloride in DMF in the presence of imidazole as a base, while the acid fluoride 31 was prepared by heating the acid chloride of monoethyloxalate 30 with sodium fluoride in high-boiling polar aprotic solvent, sulfolane, and then distilling off the product.179 Reaction of the silylated amidoxime 29 with acid fluoride 31 at room temperature in presence of catalytic amount of TBAF afforded O-acylated amidoxime 32. TBAF act as a catalyst initiating the desilylation of oxygen to generate a reactive oxygen anion that attacks the acid fluoride carbonyl group. The fluoride ion released in this step engages in the O-desilyaltion process. After solvent change from THF to dioxane, the acyclic intermediate 32 cyclized to ester 33 by heating at 120 oC for 20 minutes under microwave conditions (61% yield).  This involved the intramolecular nucleophilic attack of the amine nitrogen on the carbonyl carbon followed by dehydration and aromatization. The 1H NMR spectrum of compound 33 confirmed the formation of the product (absence of signal of the starting material 29 at 6.05 (s, 2H, NH2) and the appearance of chemical shift changes of the protons at C-’, and C-’relative to the startinJ material 29. These chemical changes are assumed to be due to the electron-withdrawing effect of the oxadiazole.  Ester 33 was converted directly to amides 34a-h by reaction with the requisite amines in presence of trimethylaluminium as a catalyst at 110 oC for 24h (23-41% yields). The mechanism of ester aminolysis is illustrated in Figure 56.180,181 This synthetic strategy was used to synthesize the eight novel compounds 34a-h (not described in CAS). The structure of these target compounds were confirmed by 1H-NMR (Chapter 8, Table 24) and high resolution mass spectroscopy.   Figure 56: Trimethylaluminium-mediated amide formation. However, as the amide bond forming conditions were harsh and the yields were not good, we opted to use classical peptide coupling reagents to synthesize the remaining target compounds 34i to 34ah. In preparation for this operation, ester 33 was hydrolyzed (NaOH, EtOHaq, heat, 12 h) to acid 35.     54  3.3.1 Optimization of Peptide Coupling Conditions for the Synthesis of 34i-ah  To find the optimal conditions for the peptide-coupling step, the reaction of the acid 35 with aniline with different coupling reagents was studied (Table 2)    Entry Coupling agent Equiv. Isolated yield (%)  1  EDC  1.2  18%  2  BOP-Cl  1.2  20%  3  PyBOP  1.2  15%  4  EDC/HOBT  1.2/1.2  48%  5  EDC/HOAt  1.2/1.2  58%  6  HOAt/HATU  1.2/1.2  51%  Conditions: Acid (1 eq), amine (1 eq), DIEA (3 eq) in DMF at room temperature for 24h Table 2: Optimization of peptide coupling agents for 34i-ah synthesis. The results showed that reactions using EDC, BOP-Cl, and PyBOP did not go to completion, as the product yields were low up to 0ௗ and most of the starting material was recovered. However, using the coupling agent pairs EDC/HOBT, EDC/HOAt, and HOAt/HATU resulted in greater conversion of the starting material to the product, and the yields were effectively doubled (48-58%). The best yield for the coupling product was observed with EDC/HOAt mixture (Table 2, entry 5).  Having established using HOAt/EDC as coupling mixture, we then explored the optimization of the base used during coupling. We performed a series of experiments (Table 3) using different equivalents of non-nucleophilic HuniJ’s base DIEA together with of EDC/HOAt (1.2 eq) coupling agents in DMF at room temperature for 24h. The base screen showed that DIEA in 2.5 equivalents (Table 3, entry 2) gave the best conversion so it was selected as the optimal base condition for the coupling reaction.     55  Entry Hunig’s base Equiv. Isolated yield (%)  1  DIEA  1.2  35%  2  DIEA  2.5  56%  3  DIEA  3  56%  Table 3: Optimization of base for 34i-ah synthesis. The reaction of the acid 35 (1 eq) with aniline (1 eq) in presence of EDC/HOAt (1.2 eq) and DIEA (2.5 eq) in DMF was monitored, by TLC, at different temperatures (rt, 50 oC, and 80 oC).  At high temperatures, the reaction failed to provide the full conversion and the yields separated were less than that at room temperature (56%, 18%, and 7% yields obtained at rt, 50 oC, and 80 oC respectively). Furthermore, Microwave-assisted peptide coupling was tried at 100 oC but it was very sluggish and led to the formation of several byproducts. The peptide coupling reaction of acid 35 with aniline was monitored by TLC over 48h and no change happened in TLC after the first 24h so it was chosen as the optimal time for the reaction.   DMF was also chosen as a solvent for the peptide coupling reaction because of the high polarity of the starting acid which made it insoluble in various solvents like DCM, THF, and ACN.  Having established conditions for the coupling reaction, acid 35 was reacted with the set of 26 amines to give pyridinone-oxadiazole carbocamides 34i-ah. Although the preparation of these amides worked well in most cases, the observed variation in yield from 18%, for the worst cases 34v, to 56%, for the best case 34ad, was found to be dependent on the nucleophilicity of the amine. Amines bearing electron-donating substituents, such as amine components in 34ad, generally behave better than the ones bearing electron-withdrawing substituents, such as amine component in 34v. It is also a consequence of the limited solubility of amides 34i-ah in typical organic solvents (DCM, EtOAc, etc), and DMSO, which made it difficult to purify them by flash chromatography. The reason for this insolubility is presumably due to the complimentary hydrogen bond donors and acceptors inside the molecule, which results in strong solute-solute interactions and self-assembly.   The structures of the target compounds 34a-ah, issuing from both amide bond forming techniques, were confirmed by 1H-NMR (Chapter 8, Table 24) and high resolution mass spectroscopy. As expected, the 1H NMR spectra for compounds 34a-ah showed that only minor 56  chemical shift changes occur for the signature peaks for the C-’, and C-’ protons relative to the starting pyridin-6-one-3-carboxylic acid 24. These chemical shift changes are assumed to reflect the electron-withdrawing effect of the oxadiazole ring as well as the polarity of the (hetero)aromatic substituent on the oxadiazole ring. The diverse amine components, used to construct the library of molecules 34a-ah, were chosen amongst different aryl, heteroaryl, benzyl and cycloalkyamines, such that we could identify motifs that productively occupy the type 2 inhibitor allosteric site in the activation loop closed conformation in ROCK1/2. However, due to limited budget resources, only 10 compounds (34a, 34f, 34j, 34l, 34n, 34r, 34z, 34aa, 34ac, 34af) were initially submitted for screening in the ROCK2 inhibitory assay developed by Dr. Kath Mac/eod’s Jroup see chapter 7).  The compounds were tested at a single point concentration (10 µM) using Y-27632 as a positive control. Among the tested compounds, eight compounds (34a, 34f, 34j, 34l, 34r, 34aa, 34ac, 34af) showed a weak Rho-kinase inhibitory activity (Chapter 7, Table 7). After optimizing the test conditions by 'r. Mac/eod’s Jroup,  compounds 34a-c, 34e-g, 34j-n, 34p-r, 34u,  34w-ac, 34ae, and 34af) were tested against ROCK1 and ROCK2 at a single point concentration (10 µM) using H-1152 as a positive control. The results (Chapter 7, Table 8) showed improvement on ROCK inhibitory activity compared to oxazoline compounds 20/21a-f. Additionally, all of active compounds inhibited ROCK1 in preference to ROCK2. In particular, compounds 34g (71% inhibition), 34q (55% inhibition), 34af (62% inhibition), and 34ag (58% inhibition). In summary, pyridinone-oxadiazole based scaffold can serve as starting point for further structural optimization to improve inhibitory activity and isoform selectivity. 3.4 Synthesis of Isomeric Pyridinone-Oxadiazole Based Derivatives 41a-g At this juncture, the decision was made to evaluate whether modifying the geometry of the pyridinone-oxadiazole carboxamide structure, by changing the point of attachment of the oxadiazole ring to the pyridinone scaffold from C-5 to C-3, would enhance binding affinity to ROCK1/2. A series of seven isomeric pyridinone-based derivatives 41a-g were prepared, where the amine components were chosen amongst those conferring activity to compounds 34a-ah (Figure 57).  57   Figure 57: Isomeric modification of 6-pyridinone-based compounds.  In principle, this modification would enable us to explore an alternate arrangement of H-bond donor/acceptor interactions with the hinge residues and whether such molecules more readily access the inner type 1 and type 2 inhibitor selectivity pockets in the ATP binding region (Figure 58).    Figure 58: Difference between pyridinones 34a-ah, and 41a-g in their orientation within the hinge residue.  The synthesis of compounds 41 a-g was accomplished by an alternative and more convergent route to that used for compounds 34a-ah, which involves assembly of the oxadiazole ring through condensation of the amidoxime intermediate 38 with the requisite oxalate amide derivatives 40a-g (Figure 59).182  This four-step approach started with the transformation of commercially available 2-chloronicotinonitrile 36 to 2-hydroxylnicotinonitrile 37 in excellent yield (96%) via heating in glacial acetic acid at 125oC for five hours. The derived pyridinone 37 was then transformed into amidoxime 38 by reaction with hydroxylamine hydrochloride and sodium bicarbonate for six hours in refluxing aqueous ethanol. The amidoxime 38 was isolated with a good yield (71%) and was characterized by 1H NMR (Chapter 8, Table 25) and mass spectrometry. The 2-(arylamino)-2-oxoacetates esters 40a-g, obtained by reacting the set of amines with ethyl chlorooxoacetate 39 in the presence of triethylamine, were coupled with amidoxime 38 in the presence of sodium hydride to provide an intermediate O-acylamidoxime, which was heated at 60 oC in DMF to furnish the target pyridinone-oxadiazoles carboxamide derivatives 41a-g in yields ranging from 23-44%. 58   Figure 59: Synthesis of isomeric pyridinone oxadiazole carboxamide-based compounds.  The synthesized compounds were screened in vitro against ROCK1 and ROCK2 at a single point concentration (10 µM) using H-1152 as a positive control. To our disappointment, attachment of the oxadiazole ring to the pyridinone scaffold at C-3 did not improve affinity for ROCK. The results (Chapter 7, Table 9) indicated that six compounds, 41a-e, and 41g, exhibited weak inhibitory activity (less than 40% inhibition), compared to the positive control, and weak selectivity against ROCK isoforms.  3.5 Synthesis of Modified Pyridinone Oxadiazole-Based Compounds 45a-f To complete our study of the pyridinone oxadiazole-based compounds, a series of six compounds  45a-f were prepared to probe the effect of omitting the carboxamide component of the linker. In principle, this should provide us with molecules acting as type I ROCK inhibitors (Figure 60).   Figure 60: Linker shortening of pyridinone oxadiazole-based compounds.  59  The synthesis of compounds 45a-f was achieved by condensing two key building blocks: the silylated amidoxime 28 and the acid fluorides 43a-f as depicted in Figure 61. The aroyl fluorides 43a-f were prepared by reacting the corresponding benzoic acid derivatives 42a-f with cyanuric fluoride in DCM at room temperature. Acylation of the silylated amidoxime 28 with the acid fluoride at room temperature in the presence of catalytic amount of TBAF afforded O-acyalted amidoxime 44a-f. This acyclic intermediate 44a-f was heated under microwave conditions at 100 0C for 20 minutes, to accelerate the ring closure reaction rate. The cyclodehydration afforded the target compounds 45a-f in yields ranging from 25-63%.  Figure 61: Modified pyridinone oxadiazoles 45a-f.  The derived tricyclic compounds 45a-f were assayed for their potential as Rho-kinase inhibitor. Disappointingly, no activity was observed. This suggests that the carboxamide component in the more elongated pyridinone-oxadiazole is beneficial for activity. Although these results obtained for the oxazolines and oxadiazoles compounds were very preliminary (primitive), they did encourage us to move forward in our goal to design and identify isoform selective ROCK inhibitors molecules possessing the essential structural characteristics typical of Type II kinase inhibitors.   60  Chapter 4  Structural Elongation of the Linker Component   4.1 Introduction Progressing forward with our goal to identify type II Rho-kinase inhibitors, two modifications to the central linker portion in the pyridinone compounds 34a-ah were chosen for evaluation of their influence on isoform selective ROCK inhibition. The first option (Figure 62) was to replace the oxadiazole ring by an N-acylpiperazine motif as found in 50a-d, and the second was to replace the oxadiazole ring by a 2-aminothiazole motif, as found in molecules with general formula 65a-p/66o,p. Both of these modifications provide novel structure types, which are more elongated relative to the pyridinone-oxadiazoles 34a-ah. Further, an additional H-bonding option is created in each molecule-type, and both series of molecules are easily adaptable to parallel synthesis of compound libraries.  Note, that, once again, it is the right side component of the molecules that corresponds to the diversity element.   Figure 62: Pyridinone piperazine and pyridinone aminothiazole-based compounds.   61  4.2 Synthesis of Pyridinone-Piperazine-Based Compounds 50a-d  The rationale behind choosing to incorporate a piperazine ring in compounds 50a-d derives from literature reports on rho-kinase inhibitors having this substructure (compounds I, XIV, XXI, XXIII, XXIV, Chapter 1).105,117,131 Indeed, docking studies on this class of inhibitors showed that cycloalkylamines (piperidine and piperazine), fit into and fully occupy the F region of the ligand-binding pocket of the Rho kinase. Additionally, the presence of the piperazine ring was determined to improve water solubility and oral bioavailability.131 We initially envisioned the synthesis of a trial set of four compounds 50a-d using commercially available aryl isocyanates 47-a-d, which differ in electron density in the aromatic ring (Figure 63). Reaction of these isocyanates with mono Boc-protected piperazine 46 in DCM at room temperature afforded the urea derivatives 48a-d in good yield (71-88%). Subsequent, N-Boc deprotection of 48a-d under acidic conditions (TFA/CH2Cl2) gave the N-substituted phenylpiperazine-1-carboxamides 49a-d. The target compounds 50a-d were obtained in 38-61% overall yield by coupling acid 23 with amines 49a-d using HOAt/EDC peptide coupling reagents in DMF at room temperature for 12 hours.   Figure 63: Synthesis of pyridinone piperazine-based compounds. The preliminary biological results (Chapter 7, Table 10) showed that the substitution of oxadiazole substructure of 34a-ah with carbonyl-piperazine in 50a-d resulted in diminished inhibitory activity.  62  4.3 Synthesis of Pyridinone Aminothiazoles-Based Compounds 65a-p and 66o,p The 2-aminothiazole motif is frequently encountered in biologically active compounds, including inhibitors of different kinases such as dasatinib (c-Abl kinase inhibitor), BMS-605541 (VEGFR-2 kinase inhibitor), and masatinib (c-kit kinase inhibitor) (Figure 64).105,183,184  Note, that relative to the oxadiazole carboxamides studied in chapter 3 , the longer C-S bond in the thiazole ring was anticipated to alter the geometry and directional orientation of the carboxamide substituents coming off this ring. It was further hoped that this oxadiazole to thiazole modification, plus the added conformational flexibility resulting from incorporation of an NH group between the pyridinone and thiazole rings, would enhance affinity for binding to ROCK. The secondary amino group between the pyridinone head and thiazole ring also provides the opportunity for supplementary H-bonding.127,128    Figure 64: Reported 2-aminothiazole-based kinase inhibitors. The synthetic route employed to obtain the target pyridinone-aminothiazole-carboxamide compounds 65a-p is illustrated in Figure 65. This six-step reaction sequence, based on the Hantzsch synthesis of 2-aminothiazoles from thioureas and bromopyruvate derivatives,185 began with reduction of the 5-nitro group in 51 at atmospheric pressure using 10% palladium on carbon as catalyst, to form the corresponding 5-aminopyridinone 53 in 68% yield. These conditions proved vastly superior to the alternate option where nitro compound 51 was heated with a mixture of iron and aqueous hydrochloric acid (Fe-HCl) in methanol. In this instance, the reduction was sluggish and the expected compound was obtained in only 18% yield. The 1H NMR spectrum for compound 53 showed the appearance of broad singlet signal at 4.25-4.15 ppm for the -NH2- protons.  The next step involved the construction of the thiourea motif. Initially, we attempted to prepare thiourea 57 directly by reacting amine 53 with 1,1’-thiocarbonyldiimidazole (TCDI) followed by quenching with 25% ammonia solution. However, the reaction proceeded in very poor yield (<10%). We also tried another single-step reaction to prepare thiourea 57, involving reaction of 63  amine 53 with ammonium thiocyanate in the presence of concentrated hydrochloric acid. To our surprise, the reaction failed to give any product, and the starting material was recovered. However, the reaction of amine 53 with the benzoylisothiocyante in acetonitrile at room temperature for 6 hours afforded the disubstituted thiourea derivative 55 in good yield (75%).186 The 1H NMR spectrum for this product revealed the appearance of broad singlets at į 1.01 ppm and į 11.-11.64 ppm, attributed to the N-H protons of the thiourea, and a multiplet siJnal at į 8.01±7.49 ppm for the phenyl ring protons.    Figure 65: Synthesis of pyridinone aminothiazole-based compounds. 64  Treatment of N-acylthiourea 55 with sodium hydroxide in methanol at 70 oC readily cleaved the benzoyl group to give the monosubstituted thiourea 57 in good yield (70%). The mechanism for this reaction involves the attack of the hydroxide ion on the electrophilic benzoyl carbonyl group of the N-acylthiourea, forming a tetrahedral intermediate which quickly expels benzoic acid.187 The structure of the hydrolysis product was confirmed by the absence of peaks for the phenyl group in the 1H-NMR spectrum, and the appearance of a broad sinJlet siJnal at į .1-6.89 ppm for the NH2 group. With thiourea 57 in hand, it was converted to the aminothiazole 61 by reaction with ethyl bromopyruvate in hot EtOH (Hantzsch reaction). This ring forming process is initiated by alkylation of the thiourea sulfur (present in its enethiol form) to form a S-alkyl intermediate that cyclizes through nucleophilic attack of the NH at the carbonyl carbon. Elimination of water from the derived thiazoline intermediate 59 then occurs, completing formation of the 2-aminothiazole-4-carboxylate system ring.185,188 The 1H-NMR spectrum of the product revealed the disappearance of NH2 signal  of thiourea moiety, and the appearance new signals for the C-5 proton of the 1,3-thiazole ring and ethyl group of carboxylate ester.  Ester 61 was subsequently hydrolyzed to the corresponding carboxylic acid 63 (71%) by heating in aqueous sodium hydroxide for 12 hours. Formation of the free acid was confirmed by 1H-NMR (broad singlet signal for the carboxylic proton at 12.65-12.07 ppm, which disappeared by D2O exchange). The last step involved building the library of amide derivatives 65a-o (13-56% yields) by reacting the set of 15 amines with acid 61 under peptide coupling conditions (HOAt/EDC/DIEA in DMF). Note that, once again reaction work-up and chromatographic purification was complicated by the poor solubility of the amide products in common organic solvents. Note also, that the variation in reaction yields reflected the differences in nucleophilicity of the nitrogen atom in the amines partners (good yields were observed with ʌ-rich (hetero)aromatic amines and vice versa). Compounds 65a-p were screened against the two ROCK isoforms. Interestingly, it was observed that the most active analogues were compounds 65g (ROCK1 % inhibition= 81% and ROCK2 % inhibition= 63%) and 65o (ROCK1 % inhibition= 91% and ROCK2 % inhibition=  72%) (Chapter 7, Table 11), having 3-aminopyridine and 5-aminoindazole, respectively, as the amine coupling component. Indeed, these results posed the fascinating question as to whether it 65  was the 2-pyridinone motif or the benzopyrazole (indazole) or pyridine motifs that H-bond to the hinge residues.  To distinguish between these possibilities for compound 65o, the corresponding analogues 65p, 66o, and 66p, were prepared, where either, or both, the pyridine NH and the indazole N-2 nitrogen are N-methylated.  It was envisaged that N-methylation would disrupt H-bonding to the hinge C=O, and render the molecules ineffective as ROCK inhibitors. Compound 65p was prepared by coupling carboxylic acid intermediate 63 with N2-methyl aminoindazole. To access compounds 66o,p, 1-methyl 5-nitropyridin-2-one 52 was prepared from 51 (Figure 65) and carried through the steps to give the N-methylated carboxylic acid intermediate 64. It was, in turn, reacted with indazole and N2-methyl indazole to give the target compounds 66o,p. Compounds 66o,p were evaluated against ROCK1 and ROCK2 at a single point concentration (10 µM). According to our initial view that the 2-pyridinone motif binds to the hinge residues, it was anticipated that compounds 66o, and 66p would be inactive. However, activity was observed for both compounds and for the N(2)-methylindazole analogue 65p (Chapter 7, Table 11). That activity was observed for the dimethylated compound 66p, was particularly surprising. However, pazopanib L an N2-methylated indazole-based kinase inhibitor, (Figure 66) is a potent VEGFR tyrosine kinase inhibitor, marketed for treatment of renal cell carcinoma. The drug was found bound to the hinge region of the enzyme via a one hydrogen bond interaction with N1-H (Figure 66).189 Taking into consideration that N-methylation of the indazole ring may not influence activity, these results suggest that binding of compounds 65o,p and 66o/p in ATP site of ROCK most likely involves the indazole ring. These result further suggest that central 2-amino-4-carboxythiazole motif that links the two terminal rings can be oriented in either direction. In a similar way, it may be the pyridine nitrogen in compound 64g that preferentially binds to the hinge residue NH.   Figure 66: Plausible binding interactions for 66o, and 66p.189 66  Based on these observations, it was decided to explore (Chapter 5) the activity of a series of indazole compounds 76a-j, wherein the indazole is kept on the right side and diversity is introduced on the left side of the molecule by replacing the pyridinone component found in 65a-p by different aryl and heteroraryl subunits.  As the 3-pyridine motif also gave good activity, the corresponding compounds 77a-j were also included in this study. Further, to complete the investigation of ROCK inhibitors based on indazole hinge binder, we also decided to replace the pyridinone as the hinge binder on the left side by indazole, and to introduce diversity on the right side, as in compounds 80a-q, 85a-o, and 93a-x (Chapter 6).   67  Chapter 5  Indazole/Pyridine-based ROCK Inhibitors: Introduction of Diversity Elements on the Left Side of the Molecules  5.1 Introduction The biological data for the N-methylated pyridinone-indazole compounds, 65p and 66o,p, and pyridinone-pyridine based compound 65g, suggested that these molecules may display a stronger affinity for ROCK1/2 when it is the indazole and pyridine subunits, rather than the 2-pyridinone motif, that bind H-bond to the hinge residues. To test this idea, the synthesis of a library of compounds 76a-j and 77a-j (Figure 67) was undertaken, in which the left side of the molecule corresponds to different aromatic/hetero-aromatic motifs and the right side is either an indazole 76a-j or a pyridine hinge binding motif 77a-j.  Figure 67: aminothiazole amide-based compound library. The indazole ring has been successfully used as a building block for the development of ROCK inhibitors (Chapter 1, section 1.7.5). The docking model of LI with ROCK (Figure 68) showed that the indazole ring resides in the hinge region and forms two hydrogen bond interactions between the N1-H/N2 of the pyrazole ring and the backbone C=O group of Glu165/the amide NH of Met167 respectively.190 Additionally, inspired by the binding interactions of compound  XXV (Chapter 1), we can assume that the 3-pyridine-based compounds we propose can similarily bind via H- bond interactions between the N of the pyridine ring and the NH of Met156.  68   Figure 68: Binding interactions for LI with ROCK as determined by homology modeling.190  5.2 Preparation of N-Aryl Aminothiazole Ester Intermediates 72a-j  5.2.1 Ethyl 2-Bromothiazole-4-Carboxylate as a Common Intermediate to Prepare N-Aryl Aminothiazole 72a-j In the previous chapters, the strategy followed was to introduce the diversity element into the molecule in the last step, through reaction of a suitable activated acid intermediate with a series of aryl and heteroarylamines. To access compounds 76a-j and 77a-j an alternate approach was envisaged in which the diverse set of amines is reacted in the first step with ethyl 2-bromothiazole carboxylate, and the two selected hinge binder components are then introduced in an amide bond forming reaction, involving the C-4 acid function in this pivotal thiazole intermediate (Figure 69).     Figure 69: Building compounds 76a-j and 77a-j based on 2-bromothiazole ester 70. 69  The required 2-bromo substituted thiazole intermediate 70 was conveniently prepared in 56% overall yield Figure 70), by condensation of thiourea 67 with ethyl bromopyruvate 68, and reaction of the derived 2-aminothiazole intermediate 69 (obtained in 89% yield) with isoamyl nitrite and copper(II) bromide in acetonitrile at 80°C (Sandmeyer reaction).  Figure 70: SNAr reaction of bromothiazole intermediate 70 with (hetero)arylamines 71a-j. For the next step two different strategies were studied for N-arylation of the thiazole ring at C-2: displacement of the bromo substituent in 70 via an SNAr reaction,191 and transition metal (Cu(II)192,193 and Pd(0)194,195) catalyzed coupling processes.  According to the literature, formation of the N-arylated thiazoles via SNAr reactions is considered challenging, due to low nucleophilicity of aniline and other heterocyclic amines.196 To explore this process, a study of the influence of base on the conversion of 70 to compound 72a (Table 5) was undertaken using 2-aminopyridine 64a as a model heterocyclic amine (THF, 60°C, 5 hours). As can be seen, the yields were comparable using either weak or stronger base, but the best result was obtained using sodium hydride as a strong base (Table 4, entry 4).   70  Entry Base Solvent Yield (%) 1 K2CO3 THF 41 2 Cs2CO3 DMF 48 3 t-BuONa DMF 51 4 NaH THF 54  Table 4: SNAr of bromothiazole under different base conditions. In this reaction, the idea was to quantitatively convert 3-aminopyridine to the corresponding amide anion, such that the nucleopilicity of the exocyclic amino group would be significantly enhanced.191 Using these conditions it proved possible to prepare compounds 65a-d. However, the reaction failed for the other amines 64e-j studied.  Looking at this substitution reaction from the opposite viewpoint, an alternate way to compensate for poor reactivity of substituted anilines and heteroaromatic amines amines was to activate of thiazole ring toward SNAr reaction via its protonation under acid conditions.196 In the experiment, reaction of 2-bromothiazole 70 with amines 71e-j was studied in the presence of pTsOH (Figure 71).196 These conditions worked for the preparation of target compound 72e, but were unsuccessful for the other aryl and heteroarylamines.   Figure 71: SNAr reaction of 2-bromothiazole with (hetero)arylamines in acidic medium.  71  As the SNAr reaction approach proved to not be readily generalizable to all the (hetero)aromatic amines studied, attention was directed to perfecting copper(II) catalysed C-N cross coupling conditions (Goldberg reaction) for the preparation of  the N-aryl thiazoles 72f-j (Figure 72).193 . Indeed, the Goldberg reaction has been used to couple a wide range of (hetero)aromatic amines with (hetero)aryl halides.192 Based on this, bromothiazole derivative 70 was reacted with 3-aminopyridine 71f using a catalytic amount (5 mol%) of copper iodide and N,N’-dimethylethylamine as the chelating diamine ligand.  However, even in experiments where the quantity of ligand is varied considerably (10-50 mol%), product formation was not observed, and the starting material was almost totally recovered.   Figure 72: Amination of 2-bromothiazole using Goldberg reaction. In view of these results, we tried to use another approach for C-N cross coupling based on Buchwald-Hartwig N-arylation of 2-bromothiazole 70 (Figure 73).194,195 The reaction was initially studied for 3-amnopyridine 71f using Pd2(dba)3 as a catalyst, Xantphos as a ligand,  t-BuONa as a base in dioxane at 110 oC for 24h (nitrogen atmosphere).   Figure 73: Amination of 2-bromothiazole using Buchwald-Hartwig reaction. Under these conditions, the product 72f was obtained in very low (<10%) yield. In order to increase the yield, we ran the reaction in presence of different ligands and different bases as summarized in Table 5. However, the yield remained inferior to 10%. Indeed, 5-membered ring heteroaryl halides containing multiple heteroatoms were found recalcitrant in Pd-catalyzed C±N cross-coupling reactions.197 72  Entry Pd source Ligand Base Solvent Yield (%) 1 Pd2(dba)3 Xantphos t-BuONa DMF 3% 2 Pd2(dba)3  XPhos t-BuONa DMF 5% 3 Pd2(dba)3 XPhos Cs2CO3 DMF 8% 3 Pd2(dba)3 Xantphos Cs2CO3 DMF 6%  Table 5: Buchwald-Hartwig cross coupling trials to prepare 72f. As for the SNAr reactions, the origin of the disappointingly poor yields observed for the copper and palladium catalyzed cross-coupling reactions is likely rooted in the low reactivity of both reacting components. We thus decided not to pursue any further use of 2-bromothiazole 70 as a pivotal substructure for the introduction of both sides of the product molecules.  5.2.2 Hantszch Approach to Prepare N-Aryl Aminothiazole Esters 72f-j The more linear approach based on the Hantszch method for the construction of the thiazole ring, enabled the synthesis of the remaining N-aryl aminothiazole ester intermeidates 72f-j required for the preparation our compound library (Figure 74).   Figure 74: Hantszch synthesis of thiazoles 72f-j.   In a similar fashion to the synthetic pathway shown in Figure 65, The protocol for the synthesis of compounds 72f-j (Figure 74) started with reacting heteroaryl amines 71f-j (the protected  N-73  Boc-71g/h were used) with benzoylisothiocyanate in acetonirile at room temperature to form the N,N’-disubstituted  thiourea 73f-j. Deacylation of disubstituted thiourea 73f-j with lithium hydroxide in methanol afforded the key N-substituted thiourea 74f-j intermediates. Not suprizingly (see earlier) the Boc protecting group on N-Boc-71g/h was cleaved during the deacylation step. The Boc cleavage in basic medium could happen through the mechanism illustrated in Figure 75.198  Figure 75: suggested mechanism for Boc cleavage in basic medium. The cyclocondenastion of thiourea 74f-j with ethyl bromopyruvate lead to the formation of aminothiazole carboxylate ester 72f-j in yields ranging from 51%-73.  5.3 Preparation of Compounds 76a-j and 77a-j  The final stage in the synthesis of the library of compounds 76 and 77 was to attach the 3-pyridyl and 5-indazolyl hinge binder components to acids 75 via an amide bond forming reaction, as illustrated in Figure 76.  The acid precursors 75a-j were prepared by hydrolysis of esters 72a-e (obtained from the SNAR approach) and 72f-j (obtained from Hantszch approach) using aqueous lithium hydroxide in methanol for 12 hours (44-76% yields). It should be noted that the separation of two acid derivatives, 75g and 75j was troublesome. Unlike the others, these two compounds did not precipitate out of the solution after acidification, and required purification by silica column chromatography (dry adsorbed onto the column). Coupling acids 75a-j with 5-aminoindazole and 3-aminopyridine was carried out using HOAt/EDC (DMF at rt for 18 hours) to give a library of 20 novel compounds (not described in CAS) 76a-j and 77a-j, respectively in in moderate yields (38-%54%). The structures of these compounds were confirmed by 1H-NMR and high resolution mass spectroscopy as described in the experimental section, Tables 28,29.  74   Figure 76: Aminothiazole carboxamide-based compounds 76a-j and 77a-j.  These compounds were evaluated against ROCK1 and ROCK2 using the ELISA technique in Dr. MacLeod’s lab. The compounds were tested at a single point concentration (10 µM) using H1152 as a positive control. Seven compounds showed inhibitory activity against ROCK isoforms (Chapter 7, Table 12). Out of these seven active compounds, four compounds (76f, 76g, 76h, and 76i) had high inhibitory activity against ROCK1 (92%, 95%, 105%, and 95%, respectively compared to the positive control) and they all have in common the 5-aminoindazole ring on the right side of the molecule. These results highlight the significance of this ring in our future plans (Chapter 6).     75  Chapter 6  Indazole-Based ROCK1/ROCK2 Inhibitors  6.1 Introduction The results described in Chapters 4 and 5 for the aminothiazole compounds 65o and 76h (Figure 77), which contain an indazole motif as the diversity component on the right and/or left side of their structure, encouraged us to look further into the importance of the indazole ring in our effort to develop isoform selective ROCK inhibitors. For that reason, we undertook the synthesis and evaluation of three sets of isomeric indazole-aminothiazole carboxamide derivatives 80a-q, 85a-o, and 93a-x. All three incorporate the diversity elements on the right side of the molecule, but they differ in either the positioning of the carboxamide substituent on the thiazole ring or the point of attachment of the aminothiazole unit onto the left side indazole nucleus.  Although only a single orientation is represented for these molecules, it is clearly apparent from the structures that they will be oriented in the ATP site of rho-kinase in distinctly different ways.  In particular, it was hoped that the geometry of one of these systems would be optimal such that right side motif would interrogate the allosteric pocket accessible to type II kinase inhibitors.   Figure 77: Conception of the three isomeric indazole-aminothiazole carboxamide families of potential type II rho kinase inhibitors: compounds 80a-q, 85a-o, and 93a-x.  76  6.2 Synthesis of 2-(Indazole-5-yl)aminothiazoles-4-Carboxamide Derivatives 80a-q The reaction sequence that was elaborated to access compounds 80a-q (Figure 78) required in the first steps that we protect the indazole NH in 5-nitroindazole 78 with a Boc group ( Boc2O, Et3N, RT; 86%) to give 79, and reduce the nitro group to give N(1)-Boc-5-aminoindazole 71h (see chapter 5 for first use of this intermediate).   Figure 78: Synthesis of indazole-5-aminothiazole-4-carboxamide derivatives 80a-q. Reduction of the nitro compound 79 using hydrazine hydrate in the presence of palladium on carbon as a catalyst afforded 71h in high yield (81%). The appearance of -NH2 protons in 1H NMR spectrum, as well as the shift in the peaks for the aromatic C-H’s confirmed the formation of the amine. The mechanism for this reduction (Figure 79) involves nucleophilic attack of hydrazine on the nitro group nitrogen atom to form 1-hydroxy-1-aryltriazane 1-oxide. This intermediate loses hydroxide anion, which then abstracts a proton from the terminal NH2. This is followed by the release diazene and formation of nitroso intermediate. This nitroso intermediate 77  is then attacked by a second hydrazine molecule forming 1-hydroxy-1-aryltriazane, which rearranges to form the aromatic amine product, water, and nitrogen.199   Figure 79: Proposed mechanism for the reduction of the nitro group using hydrazine Reaction of amine derivative 71h with benzoylisothiocyanate in acetone at room temperature gave N-benzoyl-N′-indazolylthiourea 73h.  Deacylation of this N,N’-disubstituted thiourea with LiOH resulted in both debenzoylation and N-BOC deprotection, affording the monosubstituted thiourea 74h.198 Compound 74h was then converted to the aminothiazole 72h by S-alkylation of the thiourea function with ethyl bromopyruvate followed by cyclization (Hantzsch approach). The ester 72h was hydrolyzed by stirring with LiOH in aqueous methanol at room temperature for 12h. This provided the pivotal acid intermediate 75h in good yield (70%). In the library forming step acid 75h was reacted with a set of 17 amines under peptide coupling conditions (HOAt-EDC and DIEA in DMF, RT, 18h) to give compounds 80a-q (not described in CAS) in yields ranging from 22% to 61%. The structures of the library compounds 80a-q were confirmed by 1H-NMR (Chapter 8, Table 30) and high resolution mass spectroscopy.1H-NMR showed the appearance of new singlet signal belonging to carboxamide NH as well as the appearance of new set aromatic protons in the aromatic region for the diversity elements on the right side of the molecule. These compounds were submitted as their HCl salts for in vitro testing against ROCK1 and 52C. in 'r. Mac/eod’s lab usinJ the ELISA technique. The compounds were tested at a single point concentration (10 µM) using H1152 as a positive control. It was satisfying that most of the synthesized compounds showed a marked improvement in inhibitory activity over pyridinone-based compounds, and a number of these tested compounds were also superior to positive control H1152 (Chapter 7, Table 13).  Additionally, compounds 80b and 80f-j were submitted for testing in Invitrogen using fluorescence resonance energy transfer (FRET) technique. These compounds were tested at 10-78  point concentrations and the IC50 for the tested compound was determined (Chapter 7, Table 15).   6.3 Synthesis of 2-(Indazole-5-yl)aminothiazole-5-Carboxamide Derivatives 85a-o The alternate strategy to access the isomeric C-5 carboxamide substituted 2-aminothiazole system in compounds 85a-o is outlined in Figure 80.    Figure 80: Synthesis of indazole-5-aminothiazole-5-carboxamide derivatives 85a-o. Indeed, a novel and convergent two-step synthetic route was developed that permitted us to avoid the tedious peptide coupling step with amines. Treatment of the available N1-(indazol-5-yl) thiourea 74h with N,N-dimethylformamide dimethoxyacetal (DMF-DMA) 81 in ethanol at reflux for 3h afforded the key intermediate N,N’-disubstituted thiourea 82 in excellent yield (91%). In the second step, reaction of 82 with the chloroacetamide derivatives 83a-o (prepared by reacting set of amines with chloroacetyl chloride in the presence of Et3N as a base) in 79  methanol at 80 oC for 6 h and furnished the 15 novel compounds 85a-o in overall yields ranging from 29 - 49%. In this sequence, the disubtituted thiourea 82 provided the two ring carbon atoms (C-2 and C-4) and the heteroatoms (S and N) of the resultant thiazole ring, while the remaining carbon atom (C-5) was supplied by the methylene group in the chloroacetamide derivatives 83a-o. Mechanistically, the reaction is believed to involve S-alkylation of thiourea 82 forming intermediate 84a followed by nucleophilic attack of the active methylene group on the in situ generated iminium ion.  This latter step forms the 5-membered ring intermediate 84b, which spontaneously loses Me2NH to give afford the target aminothiazoles 85a-o.   All of these compounds 85a-o were tested In-vitro against ROCK1 and ROCK2 at a single point concentration (10 µM), using H1152 as a positive control. The results revealed that improved inhibitory activity was achieved by altering the substitution pattern on the thiazole ring and all of the synthesized compounds inhibited ROCK1 more than ROCK2 (Chapter 7, Table 14). Subsequently, compounds 85a, 85d, 85i and 85n were submitted for testing in Invitrogen using FRET technique. These compounds were tested at 10-point concentrations and the IC50 for the tested compound was determined (Chapter 7, Table 15).  6.4 Synthesis of 2-(Indazole-4-yl)aminothiazoles-4-Carboxamide Derivatives 93a-x In our continuing effort to optimize the activity and selectivity of the synthesized compounds against ROCK isoforms, we turned our attention to shifting the position of the aminothiazole motif on the indazole ring from C-5 to C-4 (Figure 77). The desired indazole-aminothiazole-caboxamide based compounds 93a-x were synthesized following the route outlined in Figure 81. Although the 4-nitroindazole 87 is commercially available, an economically practical synthesis was achieved via diazaotization (aqueous sodium nitrite under acidic conditions at 0 oC) of 2-methyl-3-nitroaniline 86 followed by intraomolecular cyclization to affords 87 in high yield (89%).200 The strategy followed for the synthesis of 80a-q was then followed to access the target molecules. Reduction of the nitro group in 87 using hydrazine hydrate and Pd/C (cat) afforded 4-aminoindazole intermediate 88. The N-benzoyl-N’-indazolyl thiourea intermediate 89 was then obtained by reacting amine 88 with benzoylisothiocyanate in acetone at room temperature for 2 hours. Hydrolysis of thiourea derivative 89 by heating with lithium hydroxide afforded the monosubstituted derivative 90 in good yield (77%). The reaction of indazole amine 88 with ammonium thiocyanate in acidic medium was also explored as an alternative route to prepare N-(indazol-4-yl) thiourea 90, but it failed to give the desired product (the starting amine 80  was recovered). Similarily, reaction of amine 88 with 1,1’-thiocarbonyldiimidazole (TCDI) followed by quenching with 25% ammonia solution to form 90 failed.    Figure 81: Synthesis of indazole-4-aminothiazole-4-carboxamide derivatives 93a-x. Reaction of  thiourea derivative 90 with ethyl bromopyruvate proceeded as wanted to give thiazole ester 91 in 72% yield.  This ester was saponified to acid 92 by stirring with LiOH in aqueous methanol at room temperature for 12h. Acid 92 was subsequently reacted with the 81  requisite aryl, heteroaryl and benzylamines to give the 24 membered library novel indazole-aminothiazole carboxamides 93a-x (not described in CAS) using standard peptide coupling conditions HOAt/ EDC/ DIEA. The structures of the analogues 93a-x were confirmed by 1H-NMR spectroscopy (Chapter 8, Table 32) and high resolution mass spectrometry (Chapter 8, Table ).  These compounds were submitted for in vitro testing against ROCK1 and ROCK2 at single-point concentration (10 µM) using H1152 as positive control. To our disappointment, this structural modification resulted in complete loss of inhibitory activity against ROCK isoforms. As none of the 93a-x analogues reached the potency range of congeners 80a-q, this suggest that position-4 was not the ideal for improving activity and selectivity.    82  Chapter 7  In Vitro Biological Assays and Results  As described in Chapters 2 to 6, a library synthesis approach has been used to generate molecule-types that incorporate the qualitative characteristics of type II kinase inhibitors. This has been achieved by varying the left (hinge binder) and right side of the molecules, as well as the nature of the linker element. Selected members from each subset of molecules were evaluated for their activity/isoform selectivity using in vitro assays developed in Dr. Kath Mac/eod’s laboratory. SubseTuent and independent evaluation of isoform selectivity for the more active compounds was subsequently carried out using the industry standard ROCK1/2 assay developed by InvitrogenTM Life Technologies. The results of these preliminary screens informed us whether a given sub-library of compounds exhibited activity/isoform selectivity. This in turn motivated the study of further iterative modifications to the active skeleton that were summarized in Figure 42, Chapter 2. It must be emphasized that the objective in the library strategy we adopted during this research was to identify structural types that display activity, rather than to establish structure-activity relationships (SAR) within a given set of compounds.  In fact, only in the next phase of our project will effort be more specifically directed to optimizing the activity/isoform selectivity for the most active compounds.   The evaluation of the in vitro inhibitory activity and selectivity of the ROCK inhibitor libraries described in Chapters 3 to 6 was initially carried out in 'r. Mac/eod’s laboratory usinJ an Enzyme-Linked Immunosorbent Assay (ELISA) technique. The initial testing focused on ROCK2 activity, as a consequence of the interest of the MacLeod group to have in hand isoform selective ROCK2 inhibitors as probes to study diabetic cardiovascular disease (see Chapter 1). These tests were carried out using the kinase assay kit developed by Cyclex to measure ROCK2 inhibition activity. As the project advanced, an alternative kit supplied by Millipore was employed, as it permitted us to measure both ROCK1 and ROCK2 inhibition by our molecules.    83  7.1 Enzyme-Linked Immunosorbent Assay (ELISA)  This biochemical method was used for Cyclex® and Millipore® ROCK assay protocols. The method is based on using plates precoated with recombinant ROCK substrate (MYPT1) which contains a Thr696 residue as a phosphorylation site for ROCK1 and ROCK2. MYPT1 Thr696 phosphorylation is detected by a primary antibody that specifically detects only phosphorylated MYPT1. Subsequently, a horseradish peroxidase (HRP)-conjugated secondary antibody is added followed by the chromogenic substrate tetramethylbenzidine (TMB) which yields a blue color upon conversion to the diimine derivative by HRP. A stop solution is added to change the color to yellow which is read at 450 nm and is proportional to the amount of MYPT1 Thr696 phosphorylation (an index of ROCK activity).202,203 Compounds were tested in duplicate at single-point concentration 10 μM to determine the  inhibition against ROCK2, for the Cyclex assay, and against ROCK1 and ROCK2 isoforms, for the Millipore® assay. 7.1.1 Cyclex® ROCK Assay  7.1.1.1 Screening of Pyridinone Oxazoline Carboxamide-Based Compounds 20a-f and 21a-f  Pyridinone-oxazoliine carboxamide derivatives 20a-f and 21a-f were tested in vitro for their ROCK2 inhibitory activity. The compounds were tested at single-point concentration 10 μM using Y-27632 as a positive control. Only eight compounds exhibited weak ROCK2 inhibitory activity as illustrated in Table 6. At this testing concentration, compound 21e exhibited the highest inhibitory activity (20% inhibition).   84  Table 6: Inhibitory activity of pyridinone oxazoline carboxamide-based compounds 20a-f and 21a-f against ROCK2  Compound R  % Inhibition for ROCK2 (at 10 μM)  Y-27632     98 20a CH2Ph   4 20b CH2Ph   18 20c CH2Ph   9 20e CH2Ph   5 21a CH3   10 21b CH3   8 21e CH3   20 21f CH3  3   7.1.1.2 Screening of Pyridinone Oxadiazole Carboxamide-Based Compounds 34a-ah We tested ten compounds (34a, 34f, 34j, 34l, 34n, 34r, 34z, 34aa, 34ac, 34af) from 34a-ah compounds against ROCK2 using Y-27632 as a positive control. As illustrated in Table 7, these results showed that the replacement of the oxazoline ring by an oxadiazole ring slightly improved ROCK2 inhibitory activity. Eight compounds had weak inhibitory activity against 85  ROCK2 compared to the positive control (Table 7). Compounds 34j, 34ac, and 34af exhibited the highest inhibitory activity against ROCK2 compared to the other derivatives.  Table 7: Inhibitory activity of pyridinone oxadiazole carboxamide-based compounds 34a-ah against ROCK2  Compound  ROCK2 % Inhibition Y-27632  101  34a  2 34f  13 34j  48 34l  16 34r  3 34aa  28 34ac  41 34af  38  As we progressed along, an alternative kit supplied by Millipore was employed that permitted us to measure both ROCK1 and ROCK2 inhibition by our molecules. The conditions were optimized so the inhibition curves, generated by 'r. Mac/eod’s Jroup, of H-1152 for ROCK1 and ROCK2 correlated with the literature. 86  7.1.2 Millipore® ROCK Assay In this assay, compounds were tested against ROCK1/2 at a single-point concentration (10 μM using H-1152 as a positive control.  7.1.2.1 Screening of Pyridinone Oxadiazole Carboxamide-Based Compounds 34a-ah Twenty-four compounds (34a-c, 34e-g, 34j-n, 34p-r, 34u, 34w-ac, 34ae, and 34af) were retested against ROCK1 and ROCK2 using H-1152 as a positive control. Out of these derivatives, fifteen compounds were found active (Table 8). The results showed that the replacement of oxazoline ring by oxadiazole ring had a slight improvement on ROCK inhibitory activity. None of the tested compounds had a strong inhibitory activity toward ROCK isoforms compared to the positive control H-1152. As shown in Table 9, compounds 34g, 34q, 34u, 34af, and 34ag exhibited the highest inhibitory activity against ROCK1. It should be noted that compounds 34g, 34g, 34ag had a bicyclic system on the right side, tail segment while 34q and 34u share phenyl ring bearing electron-donating on the tail segment.  As for the selectivity between ROCK1 and ROCK2, all of the active compounds inhibited ROCK1 in preference to ROCK2. In particular, compounds 34g, and 34af with a bicyclic system on the tail segment displayed 9- to 12-fold more activity against ROCK1 than ROCK2. On the other hand, compound 34ag bearing 1,4-benzodioxan-6-yl motif at the right side inhibited ROCK1 (58% inhibition) without any effect on ROCK2.  Table 8: Inhibitory activity of pyridinone oxadiazole carboxamide-based compounds 34a-ah against ROCK1/2   87  Compound  ROCK1 % Inhibition ROCK2 %Inhibition H-1152  96 101 34a  43 12 34e  29 10 34f  44 9 34g  71 6 34j  39 10 34l  19 8 34n  39 13 34q  55 7 34u  46 37 34x  33 22 34aa  41 17 34ac  38 4 34ae  22 10 34af  62 7 34ag  58 0.0 88  7.1.2.2 Screening of Isomeric Pyridinone Oxadiazole Carboxamide-Based Derivatives 41a-g Trials to improve inhibitory activity by synthesizing isomeric pyridinone-based derivatives 41a-g did not succeed in improving either activity or selectivity. The compounds were tested at single-point concentration 10 μM usinJ H-1152 as a positive control. Six compounds showed activity as illustrated in Table 9. At this testing concentration, compound 41d exhibited the highest inhibitory activity (37% inhibition) compared to the positive control.  Table 9: Inhibitory activity of isomeric pyridinone oxadiazole carboxamide-based compounds 41a-g against ROCK1/2  Compound  ROCK1 % Inhibition ROCK2 % Inhibition H-1152  93 98  41a  28  2 41b  31 11  41c  35  14 41d  37  17  41e  13  17 41g  19  8  89  7.1.2.3 Screening of Pyridinone Piperazine-Based Compounds 50a-d Derivatives of pyridinone piperazine-based compounds 50a-d have been tested for inhibitory activity against ROCK1 and ROCK2 at 10 μM concentration using H-1152 as a positive control. The results of these four derivatives, as demonstrated in Table 10, showed that substitution of oxadiazole substructure of 34a-ah with carbonyl-piperazine in 50a-d resulted in diminished inhibitory activity. Table 10: Inhibitory activity of pyridinone piperazine-based compounds 50a-d against ROCK2   Compound  ROCK1 % Inhibition ROCK2 % Inhibition H-1152  93 98 45a  13 17 45b  11 3 45c  32 12 45d  31 11  7.1.2.4 Screening of Pyridinone Aminothiazole Carboxamide-Based Compounds 65a-p and 66o,p Aminothiazole was chosen as a substitute fragment for the oxadiazole substructure in 34a-ah to afford compounds 65a-p, and 66o,p. These analogues were tested at 10 μM concentration against ROCK1 and ROCK2. Only eight compounds were found active as shown in Table 11. The inhibitory activity of compounds 65o and 66o against ROCK1 was found very close to the positive control H-1152 at this testing concentration. Compounds 65c, 65g, 65p, and 66p moderately inhibited both isoforms at the previously mentioned concentration. Substitutions on 90  the ring nitrogen of the pyrdinone or indazole ring by methyl group, as found in 65p and 66o,p, were well tolerated.  As for the selectivity between ROCK1 and ROCK2, most of the active inhibited ROCK1 more than ROCK2, less than 2-fold more activity against ROCK1 than ROCK2. In summary, this modification had a positive impact on ROCK inhibitory activity whereas it did not improve selectivity.  Table 11: Inhibitory activity of pyridinone aminothiazole carboxamide-based compounds 65a-p and 66o,p against ROCK1/2.  Compound  ROCK1 % Inhibition ROCK2 % Inhibition H-1152  96 101 65a  32 35 65c  58 51 65g  81 63 65j  41 40 65o  91 72 65p  56 51 66o  95 91 66p  68 54  91  7.1.2.5 Screening of Aminothiazole Carboxamide-Based Compounds 76a-j and 77a-j We next turned our attention to change the left side of the molecule by using different aromatic/hetero-aromatic motifs while keeping the 3-pyridyl, of 65g, or 5-indazolyl moieties, of 65o, on the right side. Results showed that active compounds, Table 12, maintained the weak isoform selectivity but showed an increase in ROCK inhibitory activity. As shown in Table 12, seven compounds were found active at 10 μM concentration. At this testing concentration, compound 76h, 77h exhibited inhibitory activity toward ROCK1 greater than the positive control H-1152. Analogues 76f, 76i, 77f, and 77i potentially inhibited ROCK1 more than ROCK2. Compound 76g had almost the same inhibitory activity against the two isoforms. Although these structural modifications improved inhibitory activity toward ROCK isoforms as compared with the previous series, they decreased the isoform selectivity. Most of the compounds inhibited ROCK1 more than ROCK2, less than 2-fold. Out of the six active compounds, four compounds had an indazole ring on the left side of the molecule. Therefore, we hypothesized the importance of this ring as a hinge binder motif.  Table 12: Inhibitory activity of aminothiazole carboxamide-based compounds 76a-j and 77a-j against ROCK1/2   Compound  ROCK1 % Inhibition ROCK2 % Inhibition H-1152  96 101 76f  92 75 76g  95 93 92  Compound  ROCK1 % Inhibition ROCK2 % Inhibition 76h  105 85 76i  95 68 77f  91 56 77h  102 70 77i  71 28  7.1.2.6 Screening of 2-(Indazole-5-yl)aminothiazole-4-Carboxamide Derivatives 80a-q To examine the importance of the indazole ring as a hinge binder motif, we prepared compounds 80a-q and evaluated their ROCK-inhibitory activity. As shown in Table 13, fifteen compounds were tested and all of the tested compounds exhibited remarkable inhibitory activity toward ROCK1. Seven compounds 80b, 80c, 80d, 80h, 80j, 80n, and 80q exhibited inhibitory activity toward ROCK1 greater than the positive control H-1152 at 10 μM concentrations. Some analogues, 80d, 80i, 80o, 80p, and 80q, exhibited weak inhibitory activity against ROCK2. As for the isoform selectivity, these modifications improved isoform selectivity. Compounds 80a-q inhibited ROCK1 more than (3- to 11-fold)  ROCK2 compared to the previous series. However, the selectivity did not significantly increase to the extent that we can claim that we have good isoform selectivity. In summary, compounds 80a-q with a new hinge binder motif, indazole, displayed a remarkable improvement in the inhibitory activity toward ROCK1 whereas a little effect against ROCK2 was exhibited. This suggested that the indazole ring is a good choice for hinge binding but we still need more structural optimization. 93  Table 13: Inhibitory activity of 2-(indazole-5-yl)aminothiazole-4-carboxamide derivatives 80a-q against ROCK1/2   Compound  ROCK1 % Inhibition ROCK2 % Inhibition H-1152  94 99 80a  70 13 80b  104 19 80c  110 11 80d  102 24 80e  75 7 80g  91 22 80h  100 15 80i  87 24 80j  109 12 80k  72 17 80l  90 29 94  Compound  ROCK1 % Inhibition ROCK2 % Inhibition 80n  100 15 80o  95 23 80p  103 49 80q  116 25  7.1.2.7 Screening of 2-(Indazole-5-yl)aminothiazole-5-Carboxamide Derivatives 85a-o Having demonstrated the importance of the indazole ring as a hinge binder motif, we considered it interesting to continue and expand this investigation by synthesizing indazole-aminothiazole carboxamide derivatives 85a-o having the carboxamide motif at C-5 of the thiazole ring. Improved inhibitory activity was achieved by altering the substitution pattern on the thiazole ring. As displayed in Table 14, all of the synthesized compounds inhibited ROCK1 more than ROCK2. Compounds 85h-j, and 85l-o having phenyl ring substituted with electron-donating groups, methoxy and methylthio, exhibited high inhibitory activity against ROCK1 superior to the positive control H-1152 at 10 μM concentration. )urthermore, the unsubstituted phenyl and the 4-fluoro substituted analogues 85a, 85g, respectively displayed high inhibitory activity toward ROCK1. For the isoform selectivity, the results showed that compounds 85a-o inhibited ROCK1 (2- to 7-fold) at the testing concentration which was lower than the previous analogues 80a-q.  In summary, analogues having carboxamide derivatives at 5-position of thiazole ring, 85a-o, displayed higher inhibitory activity but less selectivity than those having carboxamide derivatives at 4-position, 80a-q.   95  Table 14: Inhibitory activity of 2-(indazole-5-yl)aminothiazole-5-carboxamide derivatives 85a-o against ROCK1/2   Compound  ROCK1 % Inhibition ROCK2 % Inhibition H-1152  94 99 85a  104 48 85b  90 31 85c  94 18 85d  85 15 85e  103 14 85f  98 14 85g  112 31 85h  104 40 85i  102 20 85j  109 20 96  Compound  ROCK1 % Inhibition ROCK2 % Inhibition 85k  89 18 85l  109 44 85m  104 27 85n  106 36 85o  112 41  7.1.2.8 Screening of 2-(Indazole-4-yl)aminothiazole-4-Carboxamide Derivatives 93a-x Continuing our efforts to develop potent and selective ROCK inhibitors, we next investigated repositioning amionthiazole-carboxamide substructure from 5- to 4-position of indazole ring to form 93a-x. This structural modification showed a significant reduction in inhibition of ROCK isoforms and almost of compounds were inactive. It may be speculated that the diminished activity displayed by 93a-x is due partly to unfavorable orientation of the linker and tail segments with respect to their binding sites. 7.2 Rho-Kinase (ROCK) Activity Assay using FRET Technique Some compounds were submitted for reevaluation in the industry standard assay developed by  Invitrogen Life Technologies to ensure the reliability of the results and to determine the IC50. These compounds were tested using FRET-based assay =’-LYTE®. 7he =’-LYTE® technology is a homogenous biochemical assay based on fluorescence resonance energy transfer (FRET). It is designed to utilize the differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. The assay uses peptide substrate that is labeled with two fluorophores (coumarin donor and fluorescein acceptor) one at each end and in close proximity to one another (typically 10-100 Å). These two fluorophores make up a FRET pair.204 In the 97  initial reaction referred to as the kinase reaction, the .inase transfers the Ȗ-phosphate of ATP to serine/threonine residue in the synthetic peptide substrate. In the secondary reaction (the development Reaction), a site-specific protease recognizes and cleaves non-phosphorylated peptides.204 The phosphorylated substrate exhibits suppressed cleavage by the development reagent. The uncleaved phosphorylated peptides maintain FRET whereas cleavage disrupts FRET emission, non-radioactive energy transfer, between the coumarin donor and fluorescein acceptor fluorophores on the peptide. After excitation of the donor fluorophore at 400 nm, The FRET signal is calculated as the ratio between coumarin (i.e., donor ) emission at 445-460 nm to fluorescein (i.e., acceptor) emission at 520-535nm.204,205  Emission Ratio = Coumarin Emission (445 nm)/ Fluorescein Emission (520 nm) Eighteen compounds were tested usinJ =’-LYTE® ROCK assay kit (Table 15). The compounds were tested in duplicate at 10-point concentrations to obtain a dose-response curve. In the retest campaign, compound activity was calculated as percentage inhibition in relation to the positive control, staurosporine. The IC50 values for the tested compounds were determined by the company using fitted curves with GraphPad Prism 5 software.  As shown in Table 15, the major finding from these results was that nine compounds, 76e, 76g, 76h, 76i, 80b, 80h, 80i, 80q, 85i, inhibited ROCK1 more than ROCK2. Structural analysis of these compounds showed that all have indazole ring at one side of the molecules, indazole-based compounds. The other side of these molecules was found to contain either a bicyclic system (as in case of 76g, 76h, 76i, 80h, and 80q) with hydrogen bond donor and/or acceptor or a monocyclic ring bearing electron-donating groups (as in 80b, and 80i).  On the other hand, compounds, 80j, 85a, 85n, had more ROCK2 inhibitory activity in InvitroJen’s test whereas they were more active aJainst 52C.1 in Millipore’s test.  Among the tested compounds, compound 76i, with 6-methoxy pyridine-6-ylat the right side of the molecule, displayed the highest ROCK1 potency (IC50  1.1 μM whereas compound 85n had higher affinity for ROCK2 (IC50  .1 μM. Indazole-based compounds 80f, 80k, and 85d with  indanyl, benzothiazolyl, and benzodioxolyl moieties, respectively exhibited the weakest ROCK inhibitory activity with IC50 !10 μM. Changing the substitution pattern of the linker segment in compound 93d diminished activity. With regard to the isoform selectivity, the tested compounds displayed weak isoform selectivity. Analogue 80h showed the highest selectivity against ROC.1 §.5-fold). On the other hand, 98  analogues 80g, 85n, and 85n, inhibited ROCK2 (3-folds) more than ROCK1. Actually, these results differed from the ones obtained from Millipore’s test  Table 15: IC50 values of compounds tested in Invitrogen  Compound ROCK1 IC50 (μM) ROCK2 IC50 (μM) Staurosporin 0.43 0.42 34af >10 >10 76e 4.6 7.8 76g 6.4 >10 76h 1.7 2.5 76i 1.1 2.6 80b 8.6 >10 80f >10 >10 80g >10 3.3 80h 3.7 11 80i 1.3 2.7 80j 7.7 4.6 80k >10 >10 80q 4.8 >10 85a 9.3 3.5 85d >10 >10 85i 3.4 7 85n 7.4 2.1 93d >100 >100    99  7.3 Factors that may Contribute to the Differences in Biological Results Between the ELISA-Based and FRET-Based ROCK Activity Assay Methods There are several factors to be considered during ROCK assay design that might affect the results. Herein, I will discuss the major factors that might lead to the differences between the ELISA-based (EMD Millipore) and FRET-based (Z'-LYTE®, Invitrogen) ROCK activity assay results. 7.3.1 ATP Concentration Optimizing ATP concentration is a key factor in developing a ROCK biochemical assay. Too low ATP concentration will limit enzyme activity whereas high ATP concentration will saturate the enzyme and reduce the chance of inhibitor binding. In order to achieve good linearity between signal and kinase activity, an optimal ATP concentration must be used. There are three different approaches for choosing ATP concentration for the activity assay. The first option relies on using a standard fixed ATP concentration, usually between 10 and 100 µM, for all different protein kinase assays. This option has the advantage of making the experimental procedure easier especially if several protein kinases are involved. However, the disadvantage is that it cannot be used to sort the potency of a given inhibitor between different kinases; in other words, the binding affinity of the inhibitor is not a good measure of the inhibitor selectivity.  According to Cheng±Prusoff equation, the IC50, concentration of inhibitor that results in 50% inhibition of maximal enzyme activity, of an ATP-competitive inhibitor is linearly related to ATP concentration.206  Equation of Cheng±Prusoff: Ki= IC50/(1 + [S]/Km) Where the Ki is the inhibition constant that describes the binding affinity between the inhibitor and the enzyme, Km is Michaelis±Menten constant that describes the affinity between the ATP and the enzyme and [S] is a fixed substrate concentration. Here is an example to demonstrate the effect of using a fixed ATP concentration on inhibitor selectivity studies. Consider two hypothetical kinases, kinase 1 with KmATP 10 μM and kinase  with KmATP 1.5 μM. An inhibitor ; has a .i of 0.01μM for kinase 1 and 0.00 μM for kinase 2. The IC50 for ; measured at an A7P concentration of 10 μM for kinase 1 would be larJer than that for kinase  whereas at A7P concentration of 0 μM, the IC50 measured for kinase 1 would be smaller than that for kinase 2. Thus, inhibitor selectivity ranking between different kinases using assays with a fixed ATP concentration may be misleading.207 100  The second option is to use an ATP concentration that mimics the intracellular ATP levels. This option is complicated by the fact that little information about the exact cellular ATP concentrations is known. Moreover, ATP concentrations continuously fluctuate, may differ from one cellular compartment to another, from one cell type to another, and between diseased and normal cells which makes the determination of cellular level of ATP challenging.207 The third option for choosing the ATP concentration is to use ATP at a concentration that equals its Km value, the concentration that allows half maximal reaction velocity, for the investigated kinase. ATP Km concentration for ROCK2 was determined to be 39.5 µM. At ATP Km, the IC50 equals twice the Ki value and represents a good measure of affinity between the inhibitor and the tested kinase. Thus, the selectivity of a given inhibitor against several different kinases can be ranked on the basis of its intrinsic affinity (potency). 207,208 In summary, working exactly at ATP Km concentration is preferred when more than one kinase is tested and will ensure that all types of inhibition are detected. 7.3.2 Enzyme Concentration A very important aspect of ROCK assay design is the enzyme concentration used. There is a strong correlation between the reaction velocity and enzyme concentration. Too low enzyme concentrations may lead to weak signal and increased error. At optimal kinase concentrations, the assay signal is strong enough to give reproducible data and linearity can be achieved between assay signal and kinase activity. The best approach is to use an enzyme concentration well within the linear region of the curve. At high kinase concentrations, accumulation of phosphorylated substrate after the given reaction time and deviation from linearity between kinase activity and assay signal will be observed (Figure 82, nonlinear region). As the concentration of enzyme increases more, depletion of the substrate and the conversion of all ATP into ADP will result in a plateau (insensitive) region (Figure 82). In this region, higher kinase concentration cannot increase the reaction velocity further.207,209, 210        101              Figure 82: Effect of kinase concentration on assay signal.207 7.3.3 Buffer Composition Typically, in vitro biochemical assays are conducted at physiological pH in an attempt to mimic the intracellular environment of the native enzyme. Finding a buffer is an important step in designing a robust and sensitive kinase assay. Buffer components have a major influence on determining the lowest possible kinase concentration that can be used and the maximal enzyme capacity that can be obtained. Compatible assay buffers guarantee the lowest IC50 value that can be measured for an inhibitor.207 It is important to mention that the IC50 depends on enzyme concentration in a way that the assay wall equals half the kinase concentration used in the assay. The IC50 can never get lower than assay wall even if the tested compound binds tightly to the enzyme. For example, the lowest IC50 value that can be measured for an inhibitor in an assay that uses 20 nM kinase is 10 nM even if the real IC50 of the inhibitor is less.207,209,211  A previous study investigated the influence of different physiological buffers (MES, MOPS, HEPES and Tris) used in kinase assays on enzyme inhibitor screening assays 207. Buffers were tested at 20 mM, pH=7.5 in the presence of 10 mM MgCl2, 0.01% Triton X-100, and 1 mM DTT. The study found that inhibition of ROCK2 was sensitive to the composition of the assay buffer. MOPS had the least effect on enzyme activity and was recommended to maximize the catalytic activity of ROCK2 in activity assays 207. The buffer composition provided with ROCK Kinase activity Assay signal region of low assay signal linear region insensitive region 102  ELISA assay kit by EMD Millipore is different from that of the Z'-LYTE® kit by Invitrogen. Thus, this may be a reason for the difference in results between the two kits.  To optimize the addition of phospahatase inhibitors to ROCK2 assay, it was found that sodium orthovanadate and ȕ-glycerophosphate decrease enzyme activity. On the basis of these results, sodium orthovanadate and ȕ-glycerophosphate should be avoided.207 7.3.4 Detection Method Various assay detection methods have been used in ROCK activity assays. Herein, I will discuss the differences between ELISA and FRET as methods used for our compound libraries assay.  ELISA is one of the earliest antibody dependent assays used to detect phosphorylated peptides from kinase reaction. This assay offers the advantages of being relatively fast and simple. The main drawback is that it has limited use for Ser/Thr protein kinases because of the possibility of cross-reactivity between the secondary antibody and many substrate sequences. As well, these assays require many washing steps.212 FRET-based assay is a non-antibody based format with the advantages of minimizing the absolute and relative errors. With this in mind, the FRET technology is more attractive than ELISA for ROCK assays. 7.3.5 Screening Concentrations Selection of inhibitor concentrations is an key factor in kinase inhibition assays. Screening inhibitors using a single concentration, as was done using the ELISA activity assay cannot be used to determine the IC50. Thus, the generated data in the form of % inhibition could not be used in inhibitor profiling.209 In the other hand, screening the inhibitor at several different concentrations (10 inhibitor concentrations) as was done with the FRET-based assay enables the generation of a dose response curve for each test compound.209 The IC50, can be determined from the curve and used to profile the potency and selectivity of each test compound. Thus, screening strategies for reporting IC50 require testing the inhibitor at several different concentrations.  In conclusion, the differences in inhibitory potencies of our compounds between EMD Millipore and InvitroJen’s assays is likely due to different assay conditions, substrate concentration, ATP concentration, and buffer components. The preliminary results obtained can form the basis for developing more potent and selective ROCK inhibitors using ligand-based drug design approach.      103   7.4 Enzyme-Linked Immunosorbent-Based Assay Protocol  1- Active, N-terminal His6-tagged recombinant human ROCK-II residues 11-552 and active, N-terminal His6-tagged, recombinant, human ROCK-I amino acids 17-535 can be used be used directly in the assay or further diluted with kinase buffer. Each sample were assayed in duplicate. Initiate the kinase reaction by addinJ 50 μ/ of kinase buffer 50 μM A7P, 15mM MgCl2, 0mM M2PS, pH ., 5mM ȕ-glycerol phosphate, 5mM EGTA, 1mM sodium orthovanadate, 1mM dithiothreitol).  2- Add μ/ of the diluted solution of ROCK positive control (H-1152) or the different test compounds 10μM.  3- Recombinant ROCK1 or ROCK2 (1 mUnit/µL) was added 4- The mixture was incubated for 30 minutes at 30ºC with medium agitation on an orbital shaker.  5- The mixture was discarded and the wells were washed with wash buffer (0.05% tween 20 in 5% TBS) 2-3 times. 6- After washinJ and adeTuate blottinJ, the precoated wells were incubated with a 100 μ/ of anti-phospho-MYPT1 (Thr696) antibody for 1 hour at room temperature with medium agitation.  7- The unbound antibody was discarded and the wells were washed 2-3 times. 8- 7he wells were incubated with a 100 μ/ of H5P-conjugated secondary antibody for 1 hour at room temperature with continuous agitation, after which the unbound antibody was discarded and wells were washed 2-3 times. 9- 100 µL of TMB/E substrate were added to each well and the plate was placed in the dark for not more than 15 minutes for blue color development with continuous monitoring. 10- The reaction was terminated by adding 100 μ/ of stop solution 1M sulphuric acid to each well and the yellow color generated was measured at 450 nm.    104  7.5 FRET-Based Assay Protocol Active, N-terminal GST-tagged recombinant human ROCK1(1-535) and ROCK2(1-552) enzymes (Invitrogen, cat. PR7028A and PV3759, respectively) were employed in this study.  The procedure was as follows: 1. In each well, .5 μ/ of ; test compound solution in 1 'MS2, 5 μ/ of ; Peptide.inase mi[ture and .5 μ/ of ; A7P Solution were mi[ed and shaken 0 seconds on a plate shaker. 2. The kinase reaction mixture was then incubated at room temperature for 60 minutes.  3. At the end of the kinase reaction, 5 μ/ of development reaJent solution for proteolytic cleavage of non-phosphorylated peptides) were added and the mixture was shaken for 30 seconds on a plate shaker and then incubated at room temperature for another 60 minutes.  4. Fluorescence developed was read at 445-460 nm (coumarin emission) and 520-535 nm (fluorescein emission) using a fluorescence plate reader.  Test compounds were used at 10 different concentrations and a dose-response curve was constructed for each compound.  Z-Lyte® Kinase Assay Ser/Thr 07 and 13 peptides (Invitrogen, cat. PV3180 and PV3793) were used as substrates for ROCK1 and ROCK2, respectively, in accordance with the manufacturer's instructions. Final peptide concentration in the reaction mixture was 2 μM for both peptides.   Final concentrations of ROCK1 and ROCK2 in the kinase reaction was 378 ng/mL (3.8573 nM) and 116 ng/mL (1.2775 nM), respectively.  Final ATP concentration in the reaction mixture was 5 μM for 52C.1 and 50 μM for ROCK2. This was based on a A7P .m of .1 μM for 52C.1 and .5 μM for 52C..   The reaction buffer was composed of 50 mM HEPES buffer, pH 7.5, 10 mM MgCl2, 1 mM EGTA, and 0.01% Brij-35.   Staurosporine was used as a positive control. IC50 of staurosporine for ROCK1 was 4.26 nM and for ROCK2 was 4.15 nM using the substrates mentioned above.  Coumarin/fluorescein emission ratio is determined and % phosphorylation of substrate is calculated.  105  Chapter 8  Experimental 8.1 Materials and methods All reactions were performed in flame-dried or oven-dried glassware unless otherwise mentioned. DMSO-d6, CDCl3, Acetonitrile (anhydrous), and all chemicals were purchased from commercial sources and used as received. THF was pre-dried by refluxing over sodium benzophenone ketyl under nitrogen atmosphere and distilling prior to use. Dimethylformamide (DMF) was distilled under reduced pressure over molecular sieves and degassed prior to use. Dichloromethane (DCM) was pre-dried over CaH2 and distilled prior to use Thin layer chromatography (TLC) was done using precoated silica gel 60 F254 plates. Flash chromatography was performed using Silica Flash P60 silica gel (230-400 mesh). Biotage Isolera Spektra One flash chromatography was also used in the purification. Proton nuclear magnetic resonance (1H NMR) spectra and Carbon nuclear magnetic resonance (13C NMR) were measured using a Bruker AV-400 spectrometer (400MHz for 1H and 100MHz for 13C, respectively). Chemical shifts are reported in parts per million (ppm) and are referenced relative to the center line of deuterated dimethylsulfoxide DMSO-d6 (2.5 ppm 1H NMR; 39.51 ppm 13C NMR), and chloroform-d (7.27 ppm 1H NMR; 77.0 ppm 13C NMR). All spectra were performed at room temperature. The splitting patterns (multiplicities) were represented as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of doublets, td = triplet of doublets, and ddd = doublet of doublet of doublets. High resolution mass spectra were measured with Thermo Scientific Q Exactive Orbitrap High Resolution Mass Spectrometer and low resolution mass spectra were recorded using AB Sciex QTRAP 5500-Agilent 1290. Microwave experiments were performed on Biotage Initiator microwave reactor.    106    8.2 Experimental Details  Ethyl 4-hydroxy-6-oxo-1,6-dihydropyridine-3-carboxylate 3:  According to reference 213, a mixture of diethyl 1,3-acetonedicarboxylate 1 (25 ml, 138mmol), acetic anhydride (26.05 ml, 276mmol) and triethyl orthoformate (22.9 ml, 138mmol) was heated at 130°C for 1 hr. The reaction mixture was then cooled to room temperature and the solution was concentrated in vacuo to remove excess acetic anhydride.  The resulting oily residue was cooled to 0ºC and 30% ammonia (45 ml) was added, followed by water (100 ml).  The reaction mixture was allowed to stand in an ice bath for 1h, and the white precipitate that formed was isolated by filtration, washed with water. The filtrate was acidified to pH 4.0 using 1N HCl and cooled to 0ºC. The additional precipitated material filtered off, washed with water and combined with the previously isolated solid. This yellow solid was crystallized from MeOH: H2O (1:1) to give 3 (17.18 g, 68%) as a yellowish solid. 1H-NMR (400 MHz, DMSO -d6): 11.78 (1H, br s, OH), 10.74 (1H, br s, N1-H), 8.02 (1H, br s, H-2), 5.61 (1H, s, H-5), 4.27 (2H, q, J=7.1Hz, CH2), 1.29 (3H, t, J=7.1Hz, CH3). MS ESI (m/z): 182 [M-H]-.  1-(tert-Butyl) 3-ethyl 4-hydroxy-6-oxopyridine-1,3(6H)-dicarboxylate 4:  To a suspension of NaH (60% in a mineral oil, 5.35 g, 134 mmol) in dry THF (100 mL), was added compound 3 (6.13 g, 33.5 mmol). The mixture was stirred at 50 °C for 1 h. After cooling to room temperature, a solution of di-Tert-Butyl dicarbonate (29.2 g, 134 mmol) in dry THF (50 mL) was added and the mixture was stirred at 50 °C for 2 h. After cooling, the mixture was quenched slowly with saturated aqueous NH4Cl (500 mL) and extracted with EtOAc. The combined organic fractions were dried over Na2SO4 and the solvent was removed in vacuo. The oily residue was purified by flash column chromatography on silica gel (gradient elution, 0-5% MeOH in CH2Cl2) to give 4 (6.64 g, 68%) as a white solid.  107  1H-NMR (400 MHz, DMSO -d6):  12.33 (1H, br s, OH), 8.10 (1H, s, H-2), 6.20 (1H, s, H-5), 4.18 (2H, q, J=7.1Hz, CH2), 1.46 (9H, s, C(CH3)3), 1.25 (3H, t, J=7.1Hz, CH3). MS ESI (m/z): 282 [M-H]-.  1-(tert-Butyl) 3-ethyl 4-(benzyloxy)-6-oxopyridine-1,3(6H)-dicarboxylate 5a:   A mixture of N-Boc pyridinone 4 (4.6 g, 16.3 mmol), benzyl alcohol (2.7 g, 25 mmol) and PPh3 (6.82g, 25 mmol) in dry THF (80mL) was stirred under N2 until complete dissolution. The solution was cooled to 0 oC and DIAD (5.12 mL, 26 mmol) was added dropwise. After stirring the mixture at room temperature for 18 hours, it was diluted with water (100-150 ml) and extracted with EtOAc. The combined organic phase was dried over sodium sulfate (Na2SO4), filtered and then concentrated in vacuo. The crude material was purified by falsh column chromatography on silica gel (gradient elution, 0-3% MeOH in CH2Cl2) to give 5a as white semisolid (3.1 g, 51%). 1H-NMR (400 MHz, DMSO -d6):  8.70 (1H, s, H-2), 7.39-7.33 (2H, dd, J=6.3Hz, J=7.9Hz, H-’,5’), 7.31-7.27 (3H, m, H-’,’,’), 6.34 (1H, s, H-5), 5.23 (2H, s, OCH2Ph), 4.20 (2H, q, J=7.1Hz, CH2), 1.46 (9H, s, C(CH3)3), 1.25 (3H, t, J=7.1Hz, CH3). MS ESI (m/z): 372 [M-H]-.  1-(tert-Butyl) 3-ethyl 4-methoxy-6-oxopyridine-1,3(6H)-dicarboxylate 5b:  Prepared according to the procedure for 5a, N-Boc pyridinone 4 (4.6 g, 16.3 mmol) was reacted with methanol (0.8 g, 25 mmol mmol). The crude product was purified by flash column chromatography on silica gel (gradient elution, 1-4% MeOH in CH2Cl2) to give 5b as white semisolid (2.28 g, 47%). 1H-NMR (400 MHz, DMSO -d6):  8.58 (1H, s, H-2), 6.27 (1H, s, H-5), 4.20 (2H, q, J=7.1Hz, CH2), 3.51 (3H, s, OCH3), 1.46 (9H, s, C(CH3)3), 1.26 (3H, t, J=7.1Hz, CH3). MS ESI (m/z): 296 [M-H]-.    108  Ethyl 4-(benzyloxy)-6-oxo-1,6-dihydropyridine-3-carboxylate 6a:   To a solution of 5a (2.5 g, 6.7 mmol) in dry CH2Cl2 (12 mL) was added TFA (12 mL). The solution was stirred at room temperature for 3 h. The reaction mixture was evaporated in vacuo and the resulting residue was dissolved in EtOAc, and washed with saturated aqueous NaHCO3. The organic layer was separated and the aqueous layer was extracted with EtOAc. The combined extracts were dried over Na2SO4 and the solvent was removed in vacuo. The residue was purified by flash column chromatography on silica gel (gradient elution, 1-5% MeOH in CH2Cl2) to give 6a as a white solid (1.65g, 90%).  1H-NMR (400 MHz, DMSO -d6):  8.53 (1H, s, H-2), 7.37-7.31 (2H, dd, J=6.4Hz, J=7.9Hz, H-’,5’), 7.30-7.24 (3H, m, H-’,’,’), 5.67 (1H, s, H-5), 5.14 (2H, s, OCH2Ph), 4.03 (2H, q, J=7.1Hz, CH2), 1.17 (3H, t, J=7.1Hz, CH3). MS ESI (m/z): 272 [M-H]-.  Ethyl 4-methoxy-6-oxo-1,6-dihydropyridine-3-carboxylate 6b:  Prepared according to the procedure for 6a, compound 5b (2.0 g, 6.7 mmol) was reacted with TFA (10 mL) in in dry CH2Cl2 (10 mL). The crude product was purified by flash column chromatography on silica gel (gradient elution, 1-5% MeOH in CH2Cl2) to give 6b as a white solid (1.11g, 84%).  1H-NMR (400 MHz, DMSO -d6):  10.72 (1H, br s, N1-H), 8.48 (1H, s, H-2), 5.68 (1H, s, H-5), 4.28 (2H, q, J=7.1Hz, CH2), 3.43 (3H, s, OCH3), 1.30 (3H, t, J=7.1Hz, CH3). MS ESI (m/z): 196 [M-H]-.  4-(Benzyloxy)-6-oxo-1,6-dihydropyridine-3-carboxylic acid 7:  109  LiOH (0.40 g, 16.74 mmol) in water (5 ml) was added to a solution of ethyl 4-(benzyloxy)-6-oxo-1,6-dihydropyridine-3-carboxylate 6a (1.52 g, 5.58 mmol) in THF:MeOH:H2O 2:2:1 (15ml), and the mixture was stirred at ambient temperature for 12 h. The reaction mixture was then concentrated in vacuo and the resulting residue was dissolved in water, and the pH was adjusted to pH 4.0 by careful addition of 1N HCl. Upon cooloing in an ice bath, the precipitate formed was isolated by filtration, washed with water and dried in vacuo to afford the desired compounds 7 as an off white solid (1.20g, 88%).  1H-NMR (400 MHz, DMSO -d6):  12.45 (2H, br s, N1-H, COOH), 8.56 (1H, s, H-2), 7.38-7.31 (2H, dd, J=6.4Hz, J=7.9Hz, H-’,5’), 7.30-7.24 (3H, m, H-’,’,’), 5.67 (1H, s, H-5), 5.14 (2H, s, OCH2Ph). MS ESI (m/z): 244 [M-H]-.  4-Methoxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid 8:   Following the procedure for 7, compound 6b (1.1 g, 5.58 mmol) was hydrolyzed to give 8 as an off white solid (0.80 g, 84%).  1H-NMR (400 MHz, DMSO -d6):  12.34 (2H, br s, N1-H, COOH), 8.49 (1H, s, H-2), 5.64 (1H, s, H-5), 3.42 (3H, s, OCH3). MS ESI (m/z): 168 [M-H]-.  N-(tert-Butoxycarbonyl)-L-serine, methyl ester 10:   According to reference 214, l-serine methyl ester hydrochloride 9 (15.25 g, 98 mmol) was dissolved in dry CH2Cl2 (250 mL).  Et3N (28.5 mL, 196 mmol) and di-tert-Butyl dicarbonate (25.5 g, 117.5 mmol) were added, and the reaction mixture was stirred at room temperature for 24 h under nitrogen.  The mixture was diluted with water and extracted with EtOAc. The combined organic layer was washed with saturated NaHCO3 (250 mL) and brine (250 mL), dried over Na2SO4 and concentrated in vacuo to afford N-Boc-L-serine methyl ester 10 as colourless oil (19.28 g, 90%) which was used in the next step without further purification.     110  2,2-Dimethyl-oxazolidine-3,4-dicarboxylic acid 3-tert-butyl ester 4-methyl ester 11:   According to reference 214, the crude product 10 (16.24 g, 74.08 mmol) was dissolved in acetone (250mL) and 2,2 dimethoxypropane DMP (80 mL). BF3·Et2O (0.5 mL) was added as a catalyst. The resulting mixture was stirred at room temperature for 3 h or till reaction completion (monitored by TLC). The solvent was removed in vacuo, the residual oil is dissolved in CH2Cl2 (250 mL) and washed with saturated aqueous NaHCO3, brine (100 mL), The organic phase was dried over Na2SO4 and the solvent evaporated in vacuo to give compound 11 as a pale yellow oil (16.90g, 88%), which was used in the next step without further purification.   3-(tert-Butoxycarbonyl)-2,2-dimethyloxazolidine-4-carboxylic acid 12:   According to reference 214, to an ice cooled solution of methyl ester 11 (13.68 g, 52.77 mmol) in MeOH (200 mL) was added LiOH (3.79 g, 158.31 mmol) in water (15 mL) with stirring. The mixture was allowed to warm to room temperature and stirred for 12h. When the reaction was complete (TLC monitoring) the solvent was removed in vacuo, and the resulting residue was dissolved in water (200 mL) and neutralized with 10% citric acid. The solution was extracted with EtOAc, dried over Na2SO4, and concentrated to give the acid 12 as a yellow semisolid (11.13 g, 86%), which was used for the next step without further purification. A small amount of the product was purified for 1H-NMR and mass analysis by flash column chromatography on silica gel (gradient elution, 2-5% EtOAc in hexane) 1H-NMR (400 MHz, CDCl 3): į .1 1H, br s, C22H, .5±4.40 (1H, m, CH-N), 4.22±4.12 (2H, m, CH2-O), 1.67-1.51 (15H, m, 5 CH3). MS ESI (m/z): 244 [M-H]-.  tert-Butyl 2,2-dimethyl-4-(phenyl carbamoyl)oxazolidine-3-carboxylate 13a:   111  3-(Tert-butoxycarbonyl)-2,2-dimethyloxazolidine-4-carboxylic acid 12 (2.45 g, 10 mmol) and aniline (0.93 g, 10 mmol) in dry THF (25 mL) was treated with BOP-Cl (3.04 g, 12 mmol) and Et3N (4.17 mL, 30 mmol). The resulting solution was stirred at ambient temperature for 12 h. The mixture was poured in water and extracted with EtOAc. The organic layer was dried over Na2SO4, filtered and then concentrated in vacuo. The crude material was purified by flash column chromatography on silica gel (gradient elution, 0-2% MeOH in CH2Cl2) to give the product 13a as an off white solid (2.4 g, 75%). 1H-NMR (400 MHz, DMSO -d6): see Table 16. MS ESI (m/z): 319 [M-H]-.  tert-Butyl 4-((4-methoxyphenyl)carbamoyl)-2,2-dimethyloxazolidine-3-carboxylate 13b:  According to procedure for 13a, compound 12 (2.45 g, 10 mmol) was reacted with p-anisidine (1.23 g, 10 mmol). The crude product was purified by flash column chromatography on silica gel (gradient elution, 0-4% MeOH in CH2Cl2) to afford 13b as an off white solid (2.5 g, 71%).  1H-NMR (400 MHz, DMSO -d6): see Table 16 MS ESI (m/z): 351 [M+H]+.  tert-Butyl 2,2-dimethyl-4-(4-(4-trifluoromethylphenyl)carbamoyl)oxazolidine-3-carboxylate 13c:  According to procedure for 13a, compound 12 (2.45 g, 10 mmol) was reacted with 4-trifluromethylaniline (1.62 g, 10 mmol). The crude product was purified by flash column chromatography on silica gel (gradient elution, 0-2% MeOH in CH2Cl2) to afford 13c as an off white solid (1.99 g, 51%).  1H-NMR (400 MHz, DMSO -d6): see Table 16. MS ESI (m/z): 387 [M-H]-.    112  tert-Butyl 2,2-dimethyl-4-(4-(4-chlorophenyl)carbamoyl)oxazolidine-3-carboxylate 13d:  According to procedure for 13a, compound 12 (2.45 g, 10 mmol) was reacted with 4-chloroaniline (1.27 g, 10 mmol). The crude product was purified by flash column chromatography on silica gel (gradient elution, 0-2% MeOH in CH2Cl2) to afford 13d as a pale yellow solid (1.89 g, 53%).  1H-NMR (400 MHz, DMSO -d6): see Table 16. MS ESI (m/z): 353 [M-H]-.  tert-Butyl 2,2-dimethyl-4-(pyridine-3-yl carbamoyl)oxazolidine-3-carboxylate 13e:  According to procedure for 13a, compound 12 (2.0 g, 8.16 mmol) was reacted with 3-aminopyridine (0.77 g, 8.16 mmol), dry THF (20 mL), BOP-Cl (2.48 g, 9.78 mmol) and Et3N (3.4 mL, 24.46 mmol). The crude product was purified by flash column chromatography on silica gel (gradient elution, 0-5% MeOH in CH2Cl2) to give the desired product 13e as a white powder (1.68 g, 64%). 1H-NMR (400 MHz, DMSO -d6): see Table 16. MS ESI (m/z): 320 [M-H]-.  tert-Butyl 2,2-dimethyl-4-(thiazol-2-yl carbamoyl)oxazolidine-3-carboxylate 13f:  According to procedure for 13a, compound 12 (1.5 g, 6.12 mmol) was reacted with 2-aminothiazole (0.61 g, 6.12 mmol), dry THF (15 mL), BOP-Cl (1.86 g, 7.33 mmol) and Et3N (2.55 mL, 18.35 mmol). The crude product was purified by flash column chromatography on silica gel (gradient elution, 0-3% MeOH in CH2Cl2) to give the desired product 13f as a light yellow powder (1.36g, 68%).  1H-NMR (400 MHz, DMSO-d6): see Table 16. MS ESI (m/z): 328 [M+H]+.  113  Table 16: NMR data of compounds 13a-f    Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  13a   10.07 (1H, d, J=14.1Hz, N-H), 4.48-4.37 (1H, m, CH-N), 4.24-4.13 (1H, m, one of CH2-O), 3.98-3.85 (1H, m, one of CH2-O), 1.63-1.22 (15H, m, 5 CH3) 7.58 (2H, d, J=7.9Hz, H-’,’ , 7.31 (2H, t, J=7.9Hz, H-’,5’, .05 (1H, t, J=7.9Hz, H-’)  13b   10.13 (1H, d, J=14.1Hz, N-H), 4.45-4.35 (1H, m, CH-N), 4.25-4.13 (1H, m, one of CH2-O), 3.99-3.86 (1H, m, one of CH2-O), 1.65-1.20 (15H, m, 5 CH3) 7.51 (2H, d, J=9.1Hz, H-’,’, . (2H, d, J=9.1Hz, H-’,5’, 3.71 (3H, s, OCH3)  13c   10.47 (1H, d, J=14.6Hz, N-H), 4.51-4.39 (1H, m, CH-N), 4.25-4.16 (1H, m, one of CH2-O), 4.01-3.90 (1H, m, one of CH2-O), 1.67-1.22 (15H, m, 5 CH3) 7.81 (2H, dd, J=8.4Hz, H-’,5’ , 7.7 (2H, dd, J=8.4Hz, H-’,’  13d   10.22 (1H, d, J=14.6Hz, N-H), 4.45-4.35 (1H, m, CH-N), 4.22-4.14 (1H, m, one of CH2-O), 3.97-3.87 (1H, m, one of CH2-O), 1.62-1.19 (15H, m, 5 CH3) 7.61 (2H, dd, J=8.4Hz, H-’,5’ , 7.38 (2H, dd, J=8.4Hz, H-’,’  13e   10.32 (1H, d, J=13.9Hz, N-H), 4.50-4.38 (1H, m, CH-N), 4.26-4.14 (1H, m, one of CH2-O), 4.02-3.88 (1H, m, one of CH2-O), 1.67-1.17 (15H, m, 5 CH3). 8.72 (1H, s, H-’, . 1H, d, J=4.5Hz, H-’, .05 1H, d, J=8.2Hz, H-’, . 1H, dd, J=4.5Hz, J=8.2Hz, H-5’ 114  Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  13f   12.31 (1H, d, J=30.1Hz, N-H), 4.61-4.52 (1H, m, CH-N), 4.24-4.13 (1H, m, one of CH2-O), 4.01-3.88 (1H, m, one of CH2-O), 1.64-1.18 (15H, m, 5 CH3). 7.48 (1H, d, J=4.0Hz, H-’, . (1H, d, J=4.0Hz, H-5’  2-Amino-3-hydroxy-N-phenylpropanamide hydrochloride 14a:  To 13a (2 g, 6.24 mmol) in MeOH (12 mL) was added methanolic HCl (5 mL, 3M), and the reaction was stirred for 3h at room temperature. When the reaction was complete (TLC monitoring) the solvent was removed in vacuo. The resulting residue was then washed with ethyl ether and collected by filtration to the give the product 14a as a white powder (1.1 g, 81%). 1H-NMR (400 MHz, DMSO -d6): see Table 17. MS ESI (m/z): 217 [M+H]+.  2-Amino-3-hydroxy-N-(4-methoxyphenyl)propanamide hydrochloride 14b:   According to the procedure for 14a, oxazolidine 13b (2.5 g, 7.13 mmol) was converted to 14b, isolated as a white powder (1.48 g, 84%). 1H-NMR (400 MHz, DMSO -d6): see Table 17. MS ESI (m/z): 211 [M+H]+.  2-Amino-3-hydroxy-N-(4-(trifluoromethyl)phenyl)propanamide hydrochloride 14c:   115  According to the procedure for 14a, oxazolidine 13c (2.0 g, 5.15 mmol) was converted to 14c, isolated as a white powder (1.07 g, 73%). 1H-NMR (400 MHz, DMSO -d6): see Table 17. MS ESI (m/z): 249 [M+H]+ .  2-Amino-N-(4-chlorophenyl)-3-hydroxypropanamide hydrochloride 14d:  According to the procedure for 14a, oxazolidine 13d (2.0 g, 5.64 mmol) was converted to 14d, isolated as a white powder (1.0 g, 71%). 1H-NMR (400 MHz, DMSO -d6): see Table 17. MS ESI (m/z): 252 [M+H]+.  2-Amino-3-hydroxy-N-(pyridin-3-yl)propanamide hydrochloride 13e:  According to the procedure for 14a, oxazolidine 13e (2.5 g, 7.78 mmol) was converted to 14e, isolated as a white powder (1.34 g, 79%). 1H-NMR (400 MHz, DMSO -d6): see Table 17. MS ESI (m/z): 218 [M+H]+.  2-Amino-3-hydroxy-N-(thiazol-2-yl)propanamide hydrochloride 14f:  According to the procedure for 14a, oxazolidine 13f (2.0 g, 6.11 mmol) was converted to 14f, isolated as a white powder (1.05 g, 77%). 1H-NMR (400 MHz, DMSO -d6): see Table 17. MS ESI (m/z): 224 [M+H]+.    116  Table 17: NMR data of compounds 14a-f   Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  14a   10.45 (1H, s, CONH), 8.22 (2H, s, NH2), 5.60 (1H, t, J=4.8Hz, OH), 3.99 (1H, t, J=4.8Hz, CH-N), 3.84 (2H, t, J=4.8Hz, CH2-O) 7.60 (2H, d, J=7.7Hz, H-’,’, .5 (2H, t, J=7.7Hz, H-’,5’, .11 1H, t, J=7.7Hz, H-’  14b   10.52 (1H, s, CONH), 8.26 (2H, s, NH2), 5.55 (1H, t, J=5.0Hz, OH), 3.98 (1H, t, J=5.0Hz, CH-N), 3.83 (2H, t, J=5.0Hz, CH2-O)  7.54 (2H, d, J=9.1Hz, H-’,’, . (2H, d, J=9.1Hz, H-’,5’, . H, s, OCH3)  14c   11.06 (1H, s, CONH), 8.30 (2H, s, NH2), 5.60 (1H, t, J=4.5Hz, OH), 4.10 (1H, t, J=4.5Hz, CH-N), 3.95-3.83 (2H, m, CH2-O) 7.88 (2H, d, J=8.5Hz, H-’,’, .5 (2H, d, J=8.5Hz, H-’,5’  14d   11.06 (1H, s, CONH), 8.35 (2H, s, NH2), 5.60 (1H, s, OH), 4.08 (1H, s, CH-N), 3.96-3.83 (2H, m, CH2-O) 7.88 (2H, d, J=8.9Hz, H-’,’, .5 (2H, d, J=8.9Hz, H-’,5’  14e   10.73 (1H, s, CONH), 8.27 (2H, s, NH2), 5.59 (1H, s, OH), 4.09-4.0 (1H, m, CH-N), 3.92-3.82 (2H, m, CH2-O) 8.81 (1H, d, J=2.4Hz, H-’, . (1H, dd, J=1.4Hz, J=4.8Hz, H-’, 8.06 (1H, ddd, J=1.4Hz, J=2.4Hz, J=8.5Hz, H-’, .5 1H, dd, J=4.8Hz, J=8.5Hz, H-5’  14f   12.68 (1H, s, CONH), 8.56 (2H, s, NH2), 4.20-4.09 (1H, m, CH-N), 3.99-3.84 (2H, m, CH2-O). 7.53 (1H, d, J=3.6Hz, H-’, . (1H, d, J=3.6Hz, H-5’  117  2-Amino-3-((tert-butyldimethylsilyl)oxy)-N-phenylpropanamide 15a:  To a solution of 14a (1.08 g, 5 mmol) in acetonitrile (12 mL) was added imidazole (0.85 g, 12.5 mmol). The mixture was stirred for 30 minutes before the addition of tert-butylchlorodimethylsilane (1.13 g, 7.5 mmol). The mixture was stirred at room temperature for 12h., then concentrated in vacuo. The oily residue was purified by flash column chromatography on silica gel (gradient elution, 0-6% MeOH in CH2Cl2) to give the desired product 15a as a semi-solid (73%). 1H-NMR (400 MHz, DMSO -d6): see Table 18. MS ESI (m/z): 295 [M+H]+.  2-Amino-3-((tert-butyldimethylsilyl)oxy)-N-(4-methoxyphenyl)propanamide 15b:  According to the procedure for 15a, aminoalcohol 14b (1.23 g, 5 mmol) was converted to 15b, isolated as a semi-solid (1.22 g, 75%). 1H-NMR (400 MHz, DMSO -d6): see Table 18. MS ESI (m/z): 325 [M+H]+.  2-Amino-3-((tert-butyldimethylsilyl)oxy)-N-(4-(trifluoromethyl)phenyl)propanamide 15c:  According to the procedure for 15a, aminoalcohol 14c (1.42 g, 5 mmol) was converted to 15b, isolated as a semi-solid (1.2 g, 66%). 1H-NMR (400 MHz, DMSO -d6): see Table 18 . MS ESI (m/z): 363 [M+H]+. 2-Amino-3-((tert-butyldimethylsilyl)oxy)-N-(4-chlorophenyl)propanamide 15d:  118  According to the procedure for 15a, aminoalcohol 14d (1.25 g, 5 mmol) was converted to 15d, isolated as a semi-solid (1.09 g, 66%). 1H-NMR (400 MHz, DMSO -d6): see Table 18.  2-Amino-3-((tert-butyldimethylsilyl)oxy)-N-(pyridin-3-yl)propanamide 15e:  According to the procedure for 15a, aminoalcohol 14e (1.09 g, 5 mmol) was converted to 15e, isolated as a white powder (1.05 g, 71%). 1H-NMR (400 MHz, DMSO -d6): see Table 18. MS ESI (m/z): 296 [M+H]+.  2-Amino-3-((tert-butyldimethylsilyl)oxy)-N-(thiazol-2-yl)propanamide 15f:  According to the procedure for 15a, aminoalcohol 14f (1.12 g, 5 mmol) was converted to 15f, isolated as a white powder (1.03 g, 68%). 1H-NMR (400 MHz, DMSO -d6): see Table 18.  MS ESI (m/z): 302 [M+H]+.   Table 18: NMR data of compounds 15a-f    Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  15a   9.84 (1H, s, CONH), 3.77-3.64 (2H, m, CH2-O), 3.41(1H, t, J=5.5Hz, CH-N), 0.81 (9H, s, SiC(CH3)3), -0.01 (6H, s, SiCH3CH3) 7.63 (2H, d, J=7.6Hz, H-’,’, . (2H, t, J=7.6Hz, H-’,5’, .0 1H, t, J=7.6Hz, H-’ 119  Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  15b   9.66 (1H, s, CONH), 3.74-3.63 (5H, m, CH2-O, OCH3), 3.37(1H, t, J=5.2Hz, CH-N), 0.82 (9H, s, SiC(CH3)3), -0.01 (6H, s, SiCH3CH3) 7.53 (2H, d, J=8.9Hz, H-’,’, . (2H, d, J=8.9Hz, H-’,5’, 3.74-3.63 (5H, m, OCH3)  15c   9.66 (1H, s, CONH), 3.80-3.63 (2H, m, CH2-O), 3.37(1H, t, J=5.4Hz, CH-N), 0.8 (9H, s, SiC(CH3)3), -0.01 (6H, s, SiCH3CH3) 7.86 (2H, d, J=8.4Hz, H-’,’, .5 (2H, d, J=8.4Hz, H-’,5’  15d   9.96 (1H, s, CONH), 3.78-3.62 (2H, m, CH2-O), 3.4 (1H, t, J=5.2Hz, CH-N), 0.81 (9H, s, SiC(CH3)3), -0.01 (6H, s, SiCH3CH3) 7.68 (2H, d, J=8.7Hz, H-’,’, .5 (2H, d, J=8.7Hz, H-’,5’  15e   3.92-3.74 (2H, m, CH2-O), 3.68 (1H, t, J=5.3Hz, CH-N), 0.81 (9H, s, SiC(CH3)3), -0.01 (6H, s, SiCH3CH3). 8.79 (1H, d, J=2.5Hz, H-’, . (1H, dd, J=1.4Hz, J=4.7Hz, H-’, 8.07 (1H, ddd, J=1.4Hz, J=2.5Hz, J=8.5Hz, H-’, . 1H, dd, J=4.7Hz, J=8.5Hz, H-5’  15f   5.24 (2H, br s, NH2), 3.79-3.64 (2H, m, CH2-O), 3.68 (1H, dd, J=4.9 Hz, J= 6.2Hz, CH-N), 0.78 (9H, s, SiC(CH3)3), -0.03 (6H, s, SiCH3CH3). 7.46 (1H, d, J=3.7Hz, H-’, .1 (1H, d, J=3.7Hz, H-5’   4-(Benzyloxy)-N-(3-((tert-butyldimethylsilyl)oxy)-1-oxo-1-(phenylamino)propan-2-yl)-6-oxo-1,6-dihydropyridine-3-carboxamide 16a:  To a solution of 4-benzyloxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid 7 (0.12 g, 0.5 mmol) and 2-amino-3-hydroxy-N-phenyl propanamide 15a (0.15 g, 0.5  mmol) in DMF (5 120  ml) was added HOBt (0.08 g, 0.6 mmol), EDC (0.12 g, 0.6 mmol), and DIEA (0.19 g, 1.5 mmol) at room temperature. The mixture was stirred at ambient temperature for 12 h. The solvent was evaporated under vacuum. The resulting oily residue was flash column chromatographed on silica gel (gradient elution, 3-10% MeOH in CH2Cl2) to give 16a (0.17g, 67%) as a colorless oil. 1H-NMR (400 MHz, DMSO -d6): see Table 19. MS ESI (m/z): 520 [M-H]-.  4-(Benzyloxy)-N-(3-((tert-butyldimethylsilyl)oxy)-1-((4-methoxyphenyl)amino)-1-oxopropan-2-yl)-6-oxo-1,6-dihydropyridine-3-carboxamide 16b:    According to the procedure for 16a, amine 15b (0.16 g, 0.5 mmol) was converted to 16b, isolated as a as a colorless oil (0.17 g, 63%). 1H-NM R (400 MHz, DMSO-d6): see Table 19. MS ESI (m/z): 550 [M-H]-.  4-(Benzyloxy)-N-(3-((tert-butyldimethylsilyl)oxy)-1-oxo-1-((4(trifluoromethyl)phenyl)amino) propan-2-yl)-6-oxo-1,6-dihydropyridine-3-carboxamide 16c:  According to the procedure for 16a, amine 15c (0.18 g, 0.5 mmol) was converted to 16c, isolated as a colorless oil (0.14 g, 49%). 1H-NMR (400 MHz, DMSO -d6): see Table 19. MS ESI (m/z): 588 [M-H]-.    121  4-(Benzyloxy)-N-(3-((tert-butyldimethylsilyl)oxy)-1-((4-chlorophenyl)amino)-1-oxopropan-2-yl)-6-oxo-1,6-dihydropyridine-3-carboxamide 16d:  According to the procedure for 16a, amine 15d (0.16 g, 0.5 mmol) was converted to 16d, isolated as a colorless oil (0.15 g, 53%). 1H-NMR (400 MHz, D MSO-d6): see Table 19. MS ESI (m/z): 555 [M-H]-.   4-(Benzyloxy)-N-(3-((tert-butyldimethylsilyl)oxy)-1-oxo-1-(pyridin-3-ylamino)propan-2-yl)-6-oxo-1,6-dihydropyridine-3-carboxamide 16e:  According to the procedure for 16a, amine 15e (0.15 g, 0.5 mmol) was converted to 16e, isolated as a colorless oil (0.15 g, 58%). 1H-NMR (400 MHz, DMSO -d6): see Table 19. MS ESI (m/z): 521 [M-H]-.  4-(Benzyloxy)-N-(3-((tert-butyldimethylsilyl)oxy)-1-oxo-1-(thiazol-2-ylamino)propan-2-yl)-6-oxo-1,6-dihydropyridine-3-carboxamide 16f:  According to the procedure for 16a, amine 15f (0.15 g, 0.5 mmol) was converted to 16f, isolated as a colorless oil (0.17 g, 65%). 1H-NMR (400 MHz, DMSO -d6): see Table 19. MS ESI (m/z): 527 [M-H]-. 122  Table 19: NMR data of compounds 16a-f    Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  16a   10.13 (1H, s, CONH), 8.61 (1H, s, H-2), 7.41-7.22 (8H, m , Ar-H,  CONH), 5.21 (1H, s, H-5), 5.18 (2H, s, CH2Ph), 4.83-4.70 (1H, m, CH-N), 4.06-3.87 (2H, m, CH2-O), 0.61 (9H, s, SiC(CH3)3), -0.12 (6H, s, SiCH3CH3). 7.58 (2H,d, J=7.2Hz H-’,’, 7.41-7.22 (8H, m, H-’,5’, .05 1H, t, J=7.2Hz, H-’  16b   12.31 (1H, s, N1-H), 9.97 (1H, s, CONH), 8.64 (1H, s, CONH), 8.46 (1H, s, H-2), 7.37-7.22 (5H, m , Ar-H), 5.75 (1H, s, H-5), 5.13 (2H, s, CH2Ph), 4.74-4.66 (1H, m, CH-N), 3.96-3.78 (2H, m, CH2-O), 0.81 (9H, s, SiC(CH3)3), -0.02 (6H, s, SiCH3CH3). 7.49 (2H, d, J=8.8Hz, H-’,’, . (2H, d, J=8.8Hz, H-’,5’, .1 H, s, OCH3)   16c   12.35 (1H, s, N1-H), 10.49 (1H, s, CONH), 8.65 (1H, s, CONH), 8.47 (1H, s, H-2), 7.37-7.22 (5H, m , Ar-H), 5.75 (1H, s, H-5), 5.13 (2H, s, CH2Ph), 4.74-4.66 (1H, m, CH-N), 3.96-3.78 (2H, m, CH2-O), 0.81 (9H, s, SiC(CH3)3), -0.02 (6H, s, SiCH3CH3). 7.81 (2H, d, J=8.8Hz, H-’,5’, . (2H, d, J=8.8Hz, H-’,’   16d   12.37 (1H, s, N1-H), 10.26 (1H, s, CONH), 8.67 (1H, s, CONH), 8.44 (1H, s, H-2), 7.35-7.21 (5H, m , Ar-H), 5.75 (1H, s, H-5), 5.13 (2H, s, CH2Ph), 4.77-4.67 (1H, m, CH-N), 3.99-3.79 (2H, m, 7.62 (2H, d, J=8.9Hz, H-’,’, . (2H, d, J=8.9Hz, H-’,5’  123  Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm CH2-O), 0.79 (9H, s, SiC(CH3)3), -0.03 (6H, s, SiCH3CH3).  16e   12.36 (1H, s, N1-H), 10.36 (1H, s, CONH), 8.63 (1H, d, J=7.2Hz, CONH), 8.48 (1H, s, H-2), 7.38-7.23 (6H, m , Ar-H), 5.75 (1H, s, H-5), 5.14 (2H, s, CH2Ph), 4.80-4.70 (1H, m, CH-N), 4.03-3.83 (2H, m, CH2-O), 0.81 (9H, s, SiC(CH3)3), -0.03 (6H, s, SiCH3CH3). 8.75 (1H, d, J=2.5Hz, H-’, 8.27 (1H, dd, J=1.4Hz, J=4.7Hz, H-’, 8.02 (1H, ddd, J=1.4Hz, J=2.5Hz, J=8.5Hz, H-’, 7.38-7.23 (6H, m , H-5’  16f   12.35 (1H, s, N1-H), 8.73 (1H, s, CONH), 8.43 (1H, s, H-2), 7.36-7.21 (7H, m , Ar-H, CONH), 5.75 (1H, s, H-5), 5.13 (2H, s, CH2Ph), 4.84-4.77 (1H, m, CH-N), 4.07-3.85 (2H, m, CH2-O), 0.78 (9H, s, SiC(CH3)3), -0.04 (6H, s, SiCH3CH3). 7.47 (1H, d, J=4.2Hz, H-’, .-7.21 (7H, m , H-5’  N-(3-((tert-Butyldimethylsilyl)oxy)-1-oxo-1-(phenylamino)propan-2-yl)-4-methoxy-6-oxo-1,6-dihydropyridine-3-carboxamide 17a:  To a solution of 4-methoxy-6-oxo-1,6-dihydropyridine-3-carboxylic acid 8 (0.07 g, 0.4 mmol) and 2-amino-3-hydroxy-N-phenyl propanamide 15a (0.12 g, 0.4  mmol) in DMF (5 ml) was added HOBt (0.06 g, 0.48 mmol), EDC (0.1 g, 0.48 mmol), and DIEA (0.15 g, 1.2 mmol) at room temperature. The mixture was stirred at ambient temperature for 12 h, and concentrated in vacuo. The resulting oily residue was flash column chromatographed on silica gel (gradient elution, 4-12% MeOH in CH2Cl2) to give 17a as a white powder (0.11 g, 61%). 1H-NMR (400 MHz, DMSO -d6): see Table 20. 124  MS ESI (m/z): 444 [M-H]-. N-(3-((tert-Butyldimethylsilyl)oxy)-1-((4-methoxyphenyl)amino)-1-oxopropan-2-yl)-4-methoxy-6-oxo-1,6-dihydropyridine-3-carboxamide 17b:  According to the procedure for 17a, amine 15b (0.13 g, 0.4 mmol) was reacted with acid 8 (0.07 g, 0.4 mmol) to give 17b, isolated as a white powder (0.1 g, 53%) after flash column chromatography on silica gel (gradient elution, 6-15% MeOH in CH2Cl2). 1H-NMR (400 MH z, DMSO-d6): see Table 20. MS ESI (m/z): 474 [M-H]-.  N-(3-((tert-Butyldimethylsilyl)oxy)-1-oxo-1-((4-(trifluoromethyl)phenyl)amino)propan-2-yl)-4-methoxy-6-oxo-1,6-dihydropyridine-3-carboxamide 17c:  According to the procedure for 17a, amine 15c (0.18 g, 0.5 mmol) was reacted with acid 8 (0.09 g, 0.5 mmol) to give  17c, isolated as a white powder (0.11 g, 43%) after flash column chromatography on silica gel (gradient elution, 2-11% MeOH in CH2Cl2). 1H-NMR (400 MHz, DMSO -d6): see Table 20. MS ESI (m/z): 512 [M-H]-.  N-(3-((tert-Butyldimethylsilyl)oxy)-1-((4-chlorophenyl)amino)-1-oxopropan-2-yl)-4-methoxy-6-oxo-1,6-dihydropyridine-3-carboxamide 17d:  According to the procedure for 17a, amine 15d (0.16 g, 0.5 mmol) was reacted with acid 8 (0.09 g, 0.5 mmol) to give 17d, isolated as a yellowish powder (0.099 g, 41%) after flash column chromatography on silica gel (gradient elution, 2-11% MeOH in CH2Cl2).  1H-NMR (400 MHz, DMSO -d6): see Table 20. MS ESI (m/z): 479 [M-H]-. 125  N-(3-((tert-Butyldimethylsilyl)oxy)-1-oxo-1-(pyridin-3-ylamino)propan-2-yl)-4-methoxy-6-oxo-1,6-dihydropyridine-3-carboxamide 17e:  According to the procedure for 17a, amine 15e (0.12 g, 0.4 mmol) was reacted with acid 8 (0.07 g, 0.4 mmol)  to give 17e, isolated as a white powder (0.1 g, 55%) after flash column chromatography on silica gel (gradient elution, 4-15% MeOH in CH2Cl2).  1H-NMR (400 MHz, DMSO -d6): see Table 20. MS ESI (m/z): 445 [M-H]-.  N-(3-((tert-Butyldimethylsilyl)oxy)-1-oxo-1-(thiazol-2-ylamino)propan-2-yl)-4-methoxy-6-oxo-1,6-dihydropyridine-3-carboxamide 17f:  According to the procedure for 17a, amine 15f (0.12 g, 0.4 mmol) was reacted with acid 8 (0.07 g, 0.4 mmol) to give 17f, isolated as a yellowish powder (0.09 g, 57%) after flash column chromatography on silica gel (gradient elution, 3-12% MeOH in CH2Cl2).  1H-NMR (400 MHz, DMSO -d6): see Table 20. MS ESI (m/z): 451 [M-H]-.  Table 20: NMR data of compounds 17a-f  Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppms R 1H-NMR (400 MHz, DMSO-d6)  ppm  17a   12.23 (1H, s, N1-H), 10.13 (1H, s, CONH), 8.60 (1H, s, CONH), 8.41 (1H, s, H-2), 5.69 (1H, s, H-5), 4.81-4.71 (1H, m, CH-N), 3.99-3.81 (2H, m, CH2-O), 3.42 (3H, s, OCH3), 0.81 (9H, s, SiC(CH3)3), -0.01 (6H, s, SiCH3CH3). 7.59 (2H, d, J=8.2Hz H-’,’, 7.34-7.27 (2H, m, H-’,5’, .05 1H, t, J=8.2Hz, H-’ 126  Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppms R 1H-NMR (400 MHz, DMSO-d6)  ppm  17b   12.19 (1H, s, N1-H), 9.99 (1H, s, CONH), 8.58 (1H, s, CONH), 8.41 (1H, s, H-2), 5.69 (1H, s, H-5), 4.76-4.67 (1H, m, CH-N), 3.97-3.80 (2H, m, CH2-O), 3.41 (3H, s, OCH3), 0.81 (9H, s, SiC(CH3)3), -0.01 (6H, s, SiCH3CH3). 7.50 (2H, d, J=8.7Hz, H-’,’, . (2H, d, J=8.7Hz, H-’,5’, . H, s, OCH3)   17c   12.22 (1H, s, N1-H), 10.51 (1H, s, CONH), 8.61 (1H, s, CONH), 8.41 (1H, s, H-2), 5.70 (1H, s, H-5), 4.81-4.73 (1H, m, CH-N), 3.97-3.80 (2H, m, CH2-O), 3.42 (3H, s, OCH3), 0.80 (9H, s, SiC(CH3)3), -0.02 (6H, s, SiCH3CH3). 7.81 (2H, d, J=8.7Hz, H-’,5’, . (2H, d, J=8.7Hz, H-’,’   17d   12.22 (1H, s, N1-H), 10.28 (1H, s, CONH), 8.60 (1H, s, CONH), 8.40 (1H, s, H-2), 5.69 (1H, s, H-5), 4.80-4.69 (1H, m, CH-N), 3.99-3.79 (2H, m, CH2-O), 3.41 (3H, s, OCH3), 0.80 (9H, s, SiC(CH3)3), -0.02 (6H, s, SiCH3CH3). 7.63 (2H, d, J=8.8Hz, H-’,’, . (2H, d, J=8.8Hz, H-’,5’   17e   12.29 (1H, s, N1-H), 10.40 (1H, s, CONH), 8.65 (1H, s, CONH), 8.41 (1H, s, H-2), 5.71 (1H, s, H-5), 4.81-4.72 (1H, m, CH-N), 4.03-3.83 (2H, m, CH2-O), 3.41 (3H, s, OCH3), 0.80 (9H, s, SiC(CH3)3), -0.02 (6H, s, SiCH3CH3). 8.76 (1H, d, J=2.4Hz, H-’, 8.27 (1H, dd, J=1.4Hz, J=4.6Hz, H-’, 8.03 (1H, ddd, J=1.4Hz, J=2.4Hz, J=8.5Hz, H-’, 7.34 (1H, dd, J=4.6Hz, J=8.5Hz, H-5’)  17f   12.36 (1H, s, N1-H), 12.18 (1H, s, CONH), 8.62 (1H, br s, CONH), 8.39 (1H, s, H-2), 5.71 (1H, s, H-5), 4.90-4.78 (1H, m, CH-N), 4.11-3.85 (2H, m, CH2-O), 3.41 (3H, s, OCH3), 0.79 (9H, s, SiC(CH3)3), -0.04 (6H, d, J=11.9Hz, SiCH3CH3). 7.48 (1H, d, J=3.9Hz, H-’, . (1H, d, J=3.9Hz, H-5’    127  4-(Benzyloxy)-N-(3-hydroxy-1-oxo-1-(phenylamino)propan-2-yl)-6-oxo-1,6-dihydropyridine-3-carboxamide 18a:  To 16a (0.21g, 0.4 mmol) in methanol (5 mL) was added methanolic HCl (1 mL, 3M), and the reaction was stirred for 5h (complete by TLC analysis), then concentrate in vacuo. The residue was washed with ethyl ether to give 18a as a white solid (0.13 g, 82%), which was used without further purification.  MS ESI (m/z): 406 [M-H]-.  4-(Benzyloxy)-N-(3-hydroxy-1-((4-methoxyphenyl)amino)-1-oxopropan-2-yl)-6-oxo-1,6-dihydropyridine-3-carboxamide 18b:  According to the procedure for 18a, intermediate 16b (0.22 g, 0.4 mmol) was converted to 18b (white solid; 0.14 g, 79%). MS ESI (m/z): 436 [M-H]-.  4-(Benzyloxy)-N-(3-hydroxy-1-oxo-1-((4-(trifluoromethyl)phenyl)amino)propan-2-yl)-6-oxo-1,6-dihydropyridine-3-carboxamide 18c:  According to the procedure for 18a, intermediate 16c (0.21 g, 0.36 mmol) was converted to 18c (yellowish solid; 0.12 g, 72%). MS ESI (m/z): 474 [M-H]-.  128  4-(Benzyloxy)-N-(1-((4-chlorophenyl)amino)-3-hydroxy-1-oxopropan-2-yl)-6-oxo-1,6-dihydropyridine-3-carboxamide 18d:  According to the procedure for 18a, intermediate 16d (0.2 g, 0.36 mmol) was converted to 18d (yellowish solid; 0.11 g, 68%). MS ESI (m/z): 440 [M-H]-.  4-(Benzyloxy)-N-(3-hydroxy-1-oxo-1-(pyridin-3-ylamino)propan-2-yl)-6-oxo-1,6-dihydropyridine-3-carboxamide 18e:  According to the procedure for 18a, intermediate 16e (0.21 g, 0.4 mmol) was converted to 18e (white solid; 0.11 g, 69%). MS ESI (m/z): 407 [M-H]-.  4-(Benzyloxy)-N-(3-hydroxy-1-oxo-1-(thiazol-2-ylamino)propan-2-yl)-6-oxo-1,6-dihydropyridine-3-carboxamide 18f:   According to the procedure for 18a, intermediate 16f (0.18 g, 0.34 mmol) was converted to 18c (white solid; 0.08 g, 56%). MS ESI (m/z): 413 [M-H]-.    129  N-(3-Hydroxy-1-oxo-1-(phenylamino)propan-2-yl)-4-methoxy-6-oxo-1,6-dihydropyridine-3-carboxamide 19a:  To 17a (0.14 g, 0.32 mmol) in methanol (4 mL) was added methanolic HCl (1 mL, 2M) and the reaction was stirred for 3h. After completion of the reaction (TLC monitoring), the precipitated compound was collected, washed with methanol, and dried in vacuo to give product 19a as a white solid (0.9 g, 84%).  1H-NMR (400 MHz, DMSO -d6): see Table 21. MS ESI (m/z): 330 [M-H]-.  N-(3-Hydroxy-1-((4-methoxyphenyl)amino)-1-oxopropan-2-yl)-4-methoxy-6-oxo-1,6-dihydropyridine-3-carboxamide 19b;  According to the procedure for 19a, intermediate 17b (0.14 g, 0.30 mmol) was converted to 19b (white solid; 0.09 g, 80%). 1H-NMR (400 MHz, DMSO -d6): see Table 21. MS ESI (m/z): 360 [M-H]-.  N-(3-Hydroxy-1-oxo-1-((4-(trifluoromethyl)phenyl)amino)propan-2-yl)-4-methoxy-6-oxo-1,6-dihydropyridine-3-carboxamide 19c:  According to the procedure for 19a, intermediate 17c (0.15 g, 0.30 mmol) was converted to 19c (white solid; 0.09 g, 74%). 1H-NMR (400 MHz, DMSO -d6): see Table 21. MS ESI (m/z): 398 [M-H]-.  130  N-(1-((4-Chlorophenyl)amino)-3-hydroxy-1-oxopropan-2-yl)-4-methoxy-6-oxo-1,6-dihydropyridine-3-carboxamide 19d:  According to the procedure for 19a, intermediate 17d (0.14 g, 0.30 mmol) was converted to 19d (white solid; 0.08 g, 71%). 1H-NMR (400 MHz, DMSO -d6): see Table 21. MS ESI (m/z): 364 [M-H]-.  N-(3-Hydroxy-1-oxo-1-(pyridin-3-ylamino)propan-2-yl)-4-methoxy-6-oxo-1,6-dihydropyridine-3-carboxamide 19e:   According to the procedure for 19a, intermediate 17e (0.13 g, 0.30 mmol) was converted to 19c (white solid; 0.08 g, 85%). 1H-NMR (400 MHz, DMSO -d6): see Table 21. MS ESI (m/z): 331 [M-H]-.  N-(3-Hydroxy-1-oxo-1-(thiazol-2-ylamino)propan-2-yl)-4-methoxy-6-oxo-1,6-dihydropyridine-3-carboxamide 19f:  According to the procedure for 19a, intermediate 17f (0.14 g, 0.31 mmol) was converted to 19f (white solid; 0.07 g, 69%). 1H-NMR (400 MHz, DMSO -d6): see Table 21. MS ESI (m/z): 337 [M-H]-.     131  Table 21: NMR data of compounds 19a-f    Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  19a   10.13 (1H, s, CONH), 8.63 (1H, d, J=7.4Hz, CONH), 8.49 (1H, s, H-2), 5.70 (1H, s, H-5), 5.37 (1H, br s, OH), 4.70-4.62 (1H, m, CH-N), 3.82-3.69 (2H, m, CH2-O), 3.43 (3H, s, OCH3). 7.62 (2H, d, J=8.4Hz H-’,’, 7.34-7.27 (2H, m, H-’,5’, .05 1H, t, J=7.4Hz, H-’  19b   12.24 (1H, s, N1-H), 9.96 (1H, s, CONH), 8.58 (1H, d, J=7.3Hz, CONH), 8.46 (1H, s, H-2), 5.67 (1H, s, H-5), 4.66-4.59 (1H, m, CH-N), 3.78-3.68 (5H, m, CH2-O), 3.42 (3H, s, OCH3). 7.52 (2H, d, J=8.6Hz, H-’,’, . (2H, d, J=8.6Hz, H-’,5’, .78-3.68 (5H, m, OCH3)   19c   10.55 (1H, s, CONH), 8.68 (1H, d, J=7.4Hz, CONH), 8.54 (1H, s, H-2), 5.67 (1H, s, H-5), 4.71-4.62 (1H, m, CH-N), 3.89-3.70 (2H, m, CH2-O), 3.43 (3H, s, OCH3). 7.85 (2H, d, J=8.5Hz, H-’,5’, . (2H, d, J=8.5Hz, H-’,’   19d   12.70 (1H, s, N1-H), 10.24 (1H, s, CONH), 8.81 (1H, s, CONH), 8.42 (1H, s, H-2), 5.64 (1H, s, H-5), 5.20 (1H, br s, OH), 4.70-4.59 (1H, m, CH-N), 3.84-3.66 (2H, m, CH2-O), 3.40 (3H, s, OCH3). 7.65 (2H, d, J=8.7Hz, H-’,’, . (2H, d, J=8.7Hz, H-’,5’)   19e   11.33 (1H, s, CONH), 8.76 (1H, d, J=7.4Hz, CONH), 8.65-8.55 (3H, m, H-2), 5.76 (1H, s, H-5), 5.20 (1H, br s, OH), 4.72-4.65 (1H, m, CH-N), 3.97-3.73 (2H, m, CH2-O), 3.42 (3H, s, OCH3). 9.25 (1H, d, J=2.5Hz, H-’, 8.65-8.55 (3H, m, H-’,’, .00 (1H, dd, J=4.6Hz, J=8.5Hz, H-5’) 132  Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  19f   12.31 (1H, s, N1-H), 10.24 (1H, s, CONH), 8.71 (1H, d, J=7.4Hz,  CONH), 8.52 (1H, s, H-2), 5.76 (1H, s, H-5), 4.77-4.70 (1H, m, CH-N), 3.89-3.72 (2H, m, CH2-O), 3.43 (3H, s, OCH3). 7.48 (1H, d, J=3.9Hz, H-’, . (1H, d, J=3.9Hz, H-5’  2-(4-Benzyloxy-6-oxo-1,6-dihydropyridin-3-yl)-N-phenyl-4,5-dihydrooxazole-4-carboxamide 20a:  To a solution of 18a (0.04 g, 0.1 mmol) in DMF (3 mL) at íƒC dry ice-acetone), was slowly added bis(2-methoxyethyl) amino-sulfur trifluoride (DAST) (0.016 g, 0.12 mmol) under nitrogen.  The reaction was stirred at íƒC for 1h. Anhydrous K2CO3, (0.04 g, 0.28 mmol,) was added and the reaction was allowed to warm to room temperature and stirred for 5h. Once the reaction has completed (monitored by TLC) the reaction mixture was quenched by addition of 10% aqueous NaHCO3 solution and extracted with EtOAc. The combined organic layers were dried over Na2SO4, and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (gradient elution, 2-8% MeOH in CH2Cl2) to give the product 20a as a white solid (0.018 g, 46%). 1H-NMR (400 MHz, DMSO -d6): see Table 22. MS ESI (m/z): 388 [M-H]-.  2-(4-(Benzyloxy)-6-oxo-1,6-dihydropyridin-3-yl)-N-(4-methoxyphenyl)-4,5-dihydrooxazole-4-carboxamide 20b:  133  According to the procedure for 20a, intermediate 18b (0.05 g, 0.12 mmol) was reacted with DAST to give 20b as a white solid (0.020 g, 41%) after silica flash chromatography (gradient elution, 3-12% MeOH in CH2Cl2). 1H-NMR (400 MHz, DMSO -d6): see Table 22. MS ESI (m/z): 418 [M-H]-, HRMS: m/z calculated for C23H21N3O5-: 418.14084, found: 418.14127. 2-(4-(Benzyloxy)-6-oxo-1,6-dihydropyridin-3-yl)-N-(4-(trifluoromethyl)phenyl)-4,5-dihydrooxazole-4-carboxamide 20c:  According to the procedure for 20a, intermediate 18c (0.05 g, 0.12 mmol) was reacted with DAST to give 20c as a pale yellow solid (0.014 g, 32%) after silica flash chromatography (gradient elution, 2-8% MeOH in CH2Cl2). 1H-NMR (400 MHz, DMSO -d6): see Table 22. MS ESI (m/z): 456 [M-H]-. 2-(4-(Benzyloxy)-6-oxo-1,6-dihydropyridin-3-yl)-N-(4-chlorophenyl)-4,5-dihydrooxazole-4-carboxamide 20d:  According to the procedure for 20a, intermediate 18d (0.044 g, 0.10 mmol) was reacted with DAST to give 20d as a white solid (0.012 g, 29%) after silica flash chromatography (gradient elution, 3-8% MeOH in CH2Cl2). 1H-NMR (400 MHz, DMSO -d6): see Table 22. MS ESI (m/z): 422 [M-H]-.    134  2-(4-(Benzyloxy)-6-oxo-1,6-dihydropyridin-3-yl)-N-(pyridin-3-yl)-4,5-dihydrooxazole-4-carboxamide 20e:  According to the procedure for 20a, intermediate 18e (0.041 g, 0.10 mmol) was reacted with DAST to give 20e as a white solid (0.014 g, 35%) after silica flash chromatography (gradient elution, 5-12% MeOH in CH2Cl2). 1H-NMR (400 MHz, DMSO -d6): see Table 22. MS ESI (m/z): 389 [M-H]-, HRMS: m/z calculated for C21H18N4O4-: 389.12553, found: 389.12592.  2-(4-(Benzyloxy)-6-oxo-1,6-dihydropyridin-3-yl)-N-(thiazol-2-yl)-4,5-dihydrooxazole-4-carboxamide 20f:  According to the procedure for 20a, intermediate 18f (0.054 g, 0.13 mmol) was reacted with DAST to give 20f as a white solid (0.014 g, 28%) after silica flash chromatography (gradient elution, 3-12% MeOH in CH2Cl2). 1H-NMR (400 MHz, DMSO -d6): see Table 22. MS ESI (m/z): 395 [M-H]-, HRMS: m/z calculated for C19H16N4O4S-: 395.08195, found: 395.08197.  Table 22: NMR data of compounds 20a-f   135  Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  20a   11.83 (1H, s, N1’-H), 10.31 (1H, s, CONH), 8.47 (1H, s, H-’, 7.37-7.21 (5H, m , Ar-H), 5.76 (1H, s, H-5’), 5.16 (2H, s, CH2Ph), 5.04 (1H, dd, J=7.2Hz, 9.9Hz,  H-4), 4.68-4.59 (2H, m, H-5). 7.61 (2H, d, J=8.4Hz H-’,’, 7.33-7.26 (2H, m, H-’,5’, 7.04 (1H, t, J=7.4Hz, H-’  20b   12.00 (1H, s, N1’-H), 10.20 (1H, s, CONH), 8.48 (1H, s, H-’, 7.36-7.23 (5H, m , Ar-H), 5.78 (1H, s, H-5’), 5.16 (2H, s, CH2Ph), 5.01 (1H, dd, J=7.2Hz, 9.9Hz,  H-4), 4.68-4.59 (2H, m, H-5). 7.53 (2H, d, J=8.7Hz, H-”,”), 6.90 (2H, d, J=8.7Hz, H-”,5”), 3.72 (3H, s, OCH3)   20c   12.02 (1H, s, N1’-H), 10.68 (1H, s, CONH), 8.45 (1H, s, H-’, .0-7.22 (5H, m , Ar-H), 5.77 (1H, s, H-5’), 5.16 (2H, s, CH2Ph), 5.08 (1H, dd, J=7.1Hz, 9.9Hz,  H-4), 4.68-4.59 (2H, m, H-5). 7.86 (2H, d, J=8.6Hz, H-”,5”, 7.71 (2H, d, J=8.6Hz, H-”,”   20d   11.87 (1H, s, N1’-H), 10.49 (1H, s, CONH), 8.45 (1H, s, H-’, .-7.22 (5H, m , Ar-H), 5.76 (1H, s, H-5’), 5.16 (2H, s, CH2Ph), 5.04 (1H, dd, J=7.1Hz, 9.9Hz,  H-4), 4.68-4.59 (2H, m, H-5). 7.67 (2H, d, J=8.6Hz, H-”,”, 7.39 (2H, d, J=8.6Hz, H-”,5”   20e   10.55 (1H, s, CONH), 8.49 (1H, s, H-’, 7.40-7.25 (6H, m, Ar-H), 5.78 (1H, s, H-5’), 5.16 (2H, s, CH2Ph), 5.08 (1H, dd, J=6.9Hz, 9.9Hz, H-4), 4.72-4.60 (2H, m, H-5). 8.80 (1H, d, J=2.5Hz, H-”, 8.30 (1H, dd, J=1.4Hz, J=4.6Hz, H-”), 8.06 (1H, ddd, J=1.4Hz, J=2.5Hz, J=8.5Hz, H-”),    7.40-7.25 (6H, m , H-5”)  20f   11.71 (1H, s, N1’-H), 8.47 (1H, s, H-’, .-7.21 (6H, m, Ar-H), 5.76 (1H, s, H-5’), 5.16 (2H, s, CH2Ph), 5.04 (1H, dd, J=7.2Hz, 9.9Hz, H-4), 4.75-4.50 (2H, m, H-5). 7.53 (1H, d, J=3.8Hz, H-”, 7.28 (1H, d, J=3.8Hz, H-5”    136  2-(4-Methoxy-6-oxo-1,6-dihydropyridin-3-yl)-N-phenyl-4,5-dihydrooxazole-4-carboxamide 21a:  To 19a (0.05 g, 0.15 mmol) in DMF (4 mL) at íƒC dry ice-acetone), was slowly added bis(2-methoxyethyl) amino-sulfur trifluoride DAST (0.024 g, 0.18 mmol) under nitrogen.  The reaction was stirred at íƒC for 1h. Anhydrous K2CO3, (0.04 g, 0.28 mmol,) was then added, and the reaction was allowed to warm to room temperature and stirred for 5h. Once the reaction has completed (monitored by TLC) the reaction mixture was quenched by addition of 10% aqueous NaHCO3 solution and extracted with EtOAc. The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The resulting residue was purified by flash column chromatography on silica gel (gradient elution, 4-10% MeOH in CH2Cl2) to give the desired product 21a as a white solid (0.018 g, 39%). 1H-NMR (400 MHz, DMSO -d6): see Table 23. MS ESI (m/z): 312 [M-H]-, HRMS: m/z calculated for C16H15N3O4-: 312.09898, found: 312.09903.  2-(4-Methoxy-6-oxo-1,6-dihydropyridin-3-yl)-N-(4-methoxyphenyl)-4,5-dihydrooxazole-4-carboxamide 21b:  According to the procedure for 21a, intermediate 19b (0.05 g, 0.14 mmol) was reacted with DAST to give 21b as a white solid (0.015 g, 31%) after silica flash chromatography (gradient elution, 2-12% MeOH in EtOAc). 1H-NMR (400 MHz, DMSO -d6): see Table 23. MS ESI (m/z): 342 [M-H]-, HRMS: m/z calculated for C17H17N3O5-: 342.10954, found: 342.10956.    137  2-(4-Methoxy-6-oxo-1,6-dihydropyridin-3-yl)-N-(4-(trifluoromethyl)phenyl)-4,5-dihydrooxazole-4-carboxamide 21c:  According to the procedure for 20a, intermediate 19c (0.052 g, 0.13 mmol) was reacted with DAST to give 21c as a white solid (0.009 g, 18%) after silica flash chromatography (gradient elution, 2-10% MeOH in CH2Cl2). 1H-NMR (400 MHz, DMSO -d6): see Table 23. MS ESI (m/z): 380 [M-H]-, HRMS: m/z calculated for C17H14N3O4F3-: 380.08636, found: 380.08658.  N-(4-Chlorophenyl)-2-(4-methoxy-6-oxo-1,6-dihydropyridin-3-yl)-4,5-dihydrooxazole-4-carboxamide 21d:  According to the procedure for 20a, intermediate 19d (0.047 g, 0.13 mmol) was reacted with DAST to give 21d as a white solid (0.009 g, 22%) after silica flash chromatography (gradient elution, 3-12% MeOH in CH2Cl2). 1H-NMR (400 MHz, DMSO -d6): see Table 23. MS ESI (m/z): 346 [M-H]-, HRMS: m/z calculated for C16H14N3O4Cl-: 346.06001, found: 346.06003.  2-(4-Methoxy-6-oxo-1,6-dihydropyridin-3-yl)-N-(pyridin-3-yl)-4,5-dihydrooxazole-4-carboxamide 21e:  According to the procedure for 20a, intermediate 19e (0.05 g, 0.15 mmol) was reacted with DAST to give 21e as an off white solid (0.01 g, 22%) after silica flash chromatography (gradient elution, 3-12% MeOH in EtOAc). 1H-NMR (400 MHz, DMSO -d6): see Table 23. 138  MS ESI (m/z): 313 [M-H]-, HRMS: m/z calculated for C15H14N4O4-: 313.09423, found: 313.09445.  2-(4-Methoxy-6-oxo-1,6-dihydropyridin-3-yl)-N-(thiazol-2-yl)-4,5-dihydrooxazole-4-carboxamide 21f:  According to the procedure for 20a, intermediate 19f (0.05 g, 0.15 mmol) was reacted with DAST to give 21f as a white solid (0.008 g, 16%) after silica flash chromatography (gradient elution, 3-12% MeOH in CH2Cl2). 1H-NMR (400 MHz, DMSO -d6): see Table 23. MS ESI (m/z): 319 [M-H]-, HRMS: m/z calculated for C13H12N4O4S-: 319.05065, found: 319.05084.  Table 23: NMR data of compounds 21a-f  Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  21a   11.94 (1H, s, N1’-H), 10.36 (1H, s, CONH), 8.38 (1H, s, H-2’), 5.73 (1H, s, H-5’), 5.05 (1H, dd, J=7.0Hz, 10.0Hz, H-4), 4.74-4.59 (2H, m, H-5), 3.44 (3H, s, OCH3). 7.62 (2H, dd, J=8.6Hz, 1.2Hz, H-”,”), 7.37-7.28 (2H, m, H-”,5”), 7.12-7.03 (1H, m, H-4”)  21b   11.84 (1H, s, N1’-H), 10.41 (1H, s, CONH), 8.38 (1H, s, H-’, 5. 1H, s, H-5’, 5.05 1H, dd, - .0Hz, 10.0Hz, H-4), 4.73-4.59 (2H, m, H-5), 3.44 (3H, s, OCH3). 7.52 (2H, d, J=8.7Hz, H-”,”, 6.89 (2H, d, J=8.7Hz, H-”,5”, 3.73 (3H, s, OCH3)  139  Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  21c   11.89 (1H, s, N1’-H), 10.69 (1H, s, CONH), 8.37 (1H, s, H-’, 5.73 (1H, s, H-5’), 5.09 (1H, dd, J=7.0Hz, 10.0Hz, H-4), 4.75-4.59 (2H, m, H-5), 3.44 (3H, s, OCH3). 7.86 (2H, d, J=8.6Hz, H-”,5”, 7.71 (2H, d, J=8.6Hz, H-”,”)   21d   11.89 (1H, s, N1’-H), 10.48 (1H, s, CONH), 8.37 (1H, s, H-’, 5.73 (1H, s, H-5’), 5.05 (1H, dd J=7.0Hz, 10.0Hz, H-4), 4.72-4.59 (2H, m, H-5), 3.44 (3H, s, OCH3). 7.67 (2H, d, J=8.6Hz, H-”,”), 7.39 (2H, d, J=8.6Hz, H-”,5”)   21e   10.57 (1H, s, CONH), 8.38 (1H, s, H-’, 5.74 (1H, s, H-5’), 5.09 (1H, dd J=7.0Hz, 10.0Hz, H-4), 4.72-4.60 (2H, m, H-5), 3.44 (3H, s, OCH3). 8.80 (1H, d, J=2.6Hz, H-2”), 8.30 (1H, dd, J=1.4Hz, J=4.6Hz, H-”), 8.07 (1H, ddd, J=1.4Hz, J=2.6Hz, J=8.5Hz, H-”), 7.38 (1H, dd, J=4.6Hz, J=8.5Hz, H-5”)  21f   12.68 (1H, s, N1’-H), 8.42 (1H, s, H-’, 5.75 (1H, s, H-5’), 5.22 (1H, dd J=7.0Hz, 10.0Hz, H-4), 4.80-4.63 (2H, m, H-5), 3.52 (3H, s, OCH3). 7.53 (1H, d, J=3.8Hz, H-4”), 7.28 (1H, d, J=3.8Hz, H-5”)  6-Oxo-1,6-dihydropyridine-3-carboxamide 26:  To a solution of 6-oxo-1,6-dihydropyridine-3-carboxylic acid 24 (13.91 g, 100 mmol) in dry DMF (120 mL), 1,1­carbonyldiimidazole (17.84 g, 110 mmol) was added portionwise at room temperature. The mixture was heated to 60 oC until bubbling ceased, then cooled to room temperature. Concentrated 28% ammonium hydroxide (60 mL) was added and the mixture stirred at 0 oC for 2h. The precipitate that formed was isolated by filteration, washed with water and dried under vacuum at 50 oC to give 26 as a white solid (10.64 g, 77%). 1H-NMR (400 MHz, DMSO -d6):  į 1. H, s, 11-H, CONH2), 7.98 (1H, d, J=2.6Hz, H-2), 7.78 (1H, dd, J=2.6Hz, J=9.6Hz, H-4), 6.34 (1H, d, J=9.6Hz, H-5). MS ESI (m/z): 137 [M-H]-. 140  6-Oxo-1,6-dihydropyridine-3-carbonitrile 27:   To a solution of 6-oxo-1,6-dihydropyridine-3-carboxamide 26 (10 g, 72.4 mmol) in DMF (100 mL) was added cyanuric chloride (6.68 g, 36.2 mmol) portionwise at room temperature. Then the mixture was stirred at 40 oC for 2h, and the suspension was then poured into ice water (60 mL) and stirred at 0 oC for 2h. The precipitate that formed was isolated by filtration, washed with water and dried under vacuum at 50 oC to give 27 as a pale yellow solid (5.91 g, 68%). 1H-NMR (400 MHz, DMSO -d6): į 12.43 (1H, s, N1-H), 8.25 (1H, d, J=2.5Hz, H-2), 7.65 (1H, dd, J=2.5Hz, J=9.6Hz, H-4), 6.41 (1H, d, J=9.6Hz, H-5). MS ESI (m/z): 119 [M-H]-.  N'-Hydroxy-6-oxo-1,6-dihydropyridine-3-carboximidamide 28:   To a hot solution of 6-oxo-1,6-dihydropyridine-3-carbonitrile 27 (7 g, 58.33 mmol) in EtOH (100 mL), was added a solution of hydroxylamine hydrochloride (8.1 g, 116.66 mmol) and NaHCO3 (9.8 g, 116.66 mmol) in water (80 mL). The reaction mixture was heated to reflux for 6h and then concentrate in vacuo. The resulting residue was dry loaded onto silica gel and flash column chromatographed (gradient elution, 10-30% MeOH in EtOAc) to give 28 as a white solid (6.96 g, 78%). 1H-NMR (400 MHz, DMSO -d6):   11.71 (1H, s, N1-H), 9.50 (1H, s, OH), 7.72 (1H, dd, J=2.3Hz, J=9.6Hz, H-4), 7.65 (1H, d, J=2.3Hz, H-2), 6.31 (1H, d, J=9.6Hz, H-5), 5.72 (2H, br s, NH2). MS ESI (m/z): 154 [M+H]+.  N'-((tert-Butyldimethylsilyl)oxy)-6-oxo-1,6-dihydropyridine-3-carboximid-amide 29:   141  Amidoxime 28 (6.5 g, 42.44 mmol) was dissolved in DMF (100 mL) and imidazole (4.29 g, 63.66 mmol) was added. The mixure was cooled to 0oC and tert-butyldimethylsilyl chloride (8.06 g, 55.55 mmol) was added. The reaction mixture was stirred for 12h at ambient temperature, and concentrated in vacuo. The resulting crude oil was purified by flash column chromatography on silica gel (gradient elution, 2-8% MeOH in CH2Cl2)  to afford 29 as a white solid (7.5 g, 66%). 1H-NMR (400 MHz, DMSO -d6):  11.80 (1H, s, N1-H), 7.75-7.65 (2H, m, H-2,4), 6.34 (1H, d, J=9.6Hz, H-5), 6.05 (2H, br s, NH2), 0.92 (9H, s, SiC(CH3)3), 0.12 (6H, s, SiCH3CH3). MS ESI (m/z): 266 [M-H]-.  Ethyl fluorooxoacetate 31:  A mixture of ethyl chloroxoacetate 30 (12.2 g, 89.4 mmol) and dry sodium fluoride (3.8g, 89.4 mmol) in sulfolane (80 mL) was refluxed at 130 oC for 4h. The reaction mixture was distilled under reduced pressure to give compound 31 as colorless oil (5.8g, 54%).  Ethyl 3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxylate 33:  To the O-silylated amidoxime 29 (1.03g, 3.85 mmol) solution in dry THF (12 mL), was added ethyl fluoroxoacetate 31 (0.45g, 3.85 mmol) and a catalytic amount of TBAF (0.2 µL, 0.21 mmol). The reaction mixture was stirred overnight at ambient temperature, and the solvent was then removed in vacuo. The residue was dissolved in dioxane (5 mL) in a microwave vial and irradiated for 20 minutes at 120 oC. The solvent was evaporated and the residue was silica flash column chromatographed (gradient elution, 2-8% MeOH in CH2Cl2) to give compound 33 as white solid (0.38 g, 61%). 1H-NMR (400 MHz, DMSO -d6): 12.18 (1H, s, N1’-H), 8.09 (1H, d, J=2.6Hz, H-’, . (1H, dd, J=2.6Hz, J=9.6Hz, H-’, .5 1H, d, - .Hz, H-5’, .5 H, T, - .1Hz, CH2), 1.36 (3H, t, J=7.1Hz, CH3).  142  MS ESI (m/z): 236 [M+H]+.  3-(6-Oxo-1,6-dihydropyridin-3-yl)-N-phenyl-1,2,4-oxadiazole-5-carboxamide 34a:  Under nitrogen atmosphere, trimethylaluminium (2 M solution in heptane; 0.63 ml, 1.27mmol) was added to a stirred solution of aniline (0.12 g, 1.27 mmol) in dioxane (4 ml) at ambient temperature. The mixture was stirred at room temperature for 2 h. A solution of ester 33 (0.08 g, 0.32 mmol) in dioxane (3 mL) was then added. The mixture was heated to 110 oC for 24h. The mixture was cooled to room temperature and quenched with water (2 mL). After 15 minutes stirring, Na2SO4 was added and stirring was continued for another 15 minutes. The mixture was then filtered and the cake was washed with dioxane. The filtrate was concentrated in vacuo and the residue was silica flash column chromatographed (gradient elution, 3-10% MeOH in CH2Cl2) to give 34a as a white solid (0.027 g, 30%).  1H-NMR (400 MHz, DMSO -d6): see Table 24. MS ESI (m/z): 281 [M-H]-, HRMS: m/z calculated for C14H10N4O3-: 281.06801, found: 281.06805.  N-(4-Methoxyphenyl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34b:  Following the procedure described for 34a, ester 33 (0.08 g, 0.32 mmol) was condensed with p-anisidine (0.16 g, 1.27 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-12 % MeOH in CH2Cl2) to give 34b as an off white solid (0.028 g, 29%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. 13C NM R (100MHz, DMSO-d6): 169.5, 166.2, 162.6, 157.0, 151.4, 145.3, 140.9, 138.1, 130.7, 122.9, 121.5, 117.7, 114.4, 104.7, 55.7. MS ESI (m/z): 311 [M-H]-, HRMS: m/z calculated for C15H12N4O4-: 311.07858, found: 311.07874. 143  N-(2,3-Dimethylphenyl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34c:  Following the procedure described for 34a, ester 33 (0.08 g, 0.32 mmol) was condensed with 2,3-dimethylaniline (0.15 g, 1.27 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-10 % MeOH in CH2Cl2) to give 34c as an off white solid (0.037 g, 38%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. MS ESI (m/z): 309 [M-H]-, HRMS: m/z calculated for C16H14N4O3-: 309.09931, found: 309.09949.  3-(6-Oxo-1,6-dihydropyridin-3-yl)-N-(p-tolyl)-1,2,4-oxadiazole-5-carboxamide 34d:  Following the procedure described for 34a, ester 33 (0.08 g, 0.32 mmol) was condensed with p-toludine (0.16 g, 1.28 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-12 % MeOH in CH2Cl2) to give 34d as a white solid (0.025 g, 26%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. 13C NMR (100MHz, DMSO -d6): 166.2, 159.2, 151.9, 149.9, 144.7, 142.4, 140.2, 135.3, 129.8, 121.3, 116.8, 106.5, 105.7, 103.8, 21.0. MS ESI (m/z): 295 [M-H]-, HRMS: m/z calculated for C15H12N4O3-: 295.08366, found: 295.08368.  N-Benzyl-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34e:  144  Following the procedure described for 34a, ester 33 (0.08 g, 0.32 mmol) was condensed with benzylamine (0.14 g, 1.28 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-8 % MeOH in CH2Cl2) to give 34e as a white solid (0.039 g, 41%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. 13C NMR (100MHz, DMSO -d6): 169.3, 166.1, 162.5, 153.4, 138.5, 138.1, 137.6, 128.8, 128.0, 127.6, 122.9, 121.4, 104.6, 43.1. MS ESI (m/z): 295 [M-H]-, HRMS: m/z calculated for C15H12N4O3-: 295.08366, found: 295.08380.  N-(4-Methoxybenzyl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34f:  Following the procedure described for 34a, ester 33 (0.08 g, 0.32 mmol) was condensed with 4-methoxybenzylamine (0.17 g, 1.28 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-10 % MeOH in CH2Cl2) to give 34f as a white solid (0.04 g, 39%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. 13C NMR (100MHz, DMSO -d6): 169.4, 166.1, 162.5, 158.9, 153.3, 138.1, 137.5, 130.5, 129.5, 122.8, 121.4, 114.2, 104.7, 55.5, 42.6. MS ESI (m/z): 325 [M-H]-, HRMS: m/z calculated for C16H14N4O4-: 325.09423, found: 325.09415.  N-(2,3-Dihydro-1H-inden-5-yl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34g:  145  Following the procedure described for 34a, ester 33 (0.08 g, 0.32 mmol) was condensed with 4-methoxybenzylamine (0.17 g, 1.28 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-6 % MeOH in CH2Cl2) to give 34g as a white solid (0.03 g, 30%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. MS ESI (m/z): 321 [M-H]-, HRMS: m/z calculated for C17H14N4O3-: 321.09931, found: 321.09930.  3-(6-Oxo-1,6-dihydropyridin-3-yl)-N-(pyridin-2-ylmethyl)-1,2,4-oxadiazole-5-carboxamide 34h:  Following the procedure described for 34a, ester 33 (0.08 g, 0.32 mmol) was condensed with 2-picolyamine (0.14 g, 1.28 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-6 % MeOH in CH2Cl2) to give 34h as white solid (0.02 g, 23%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. 13C NMR (100MHz, DMSO -d6): 169.2, 166.1, 162.5, 157.4, 153.6, 149.4, 138.1, 137.5, 137.3, 122.9, 121.7, 121.5, 104.7, 45.0. MS ESI (m/z): 296 [M-H]-, HRMS: m/z calculated for C14H11N5O3-: 296.07891, found: 296.07895. 3-(6-Oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxylic acid 35:  NaOH (2M; 16 mL) was added to a stirred suspension of 33 (2.5g, 10.62 mmol) in ethanol (50ml), and the mixture was heated at 90 oC for 12 h. The reaction mixture was then concentrated in vacuo, and the residue was dissolved in water and acidified to pH 4.0 by careful addition of 1N HCl. After cooling, the precipitate was isolated by filtration and dried in vacuo to afford the desired compound 35 as a white powder (1.6 g, 73%). The product was taken forward to the next step without further purification. 146  1H-NMR (400 MHz, DMSO -d6):  11.51 (2H, s, N1’-H, COOH), 8.25 (1H, d, J=2.6Hz, H-’, .5 1H, dd, - .Hz, - .Hz, H-’, .1 1H, d, - .Hz, H-5’. MS ESI (m/z): 206 [M-H]-.  N-(3-Chlorophenyl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34i:   To a solution of 3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxylic acid 35 (0.07 g, 0.33 mmol) in DMF (5 mL) was added HOAt (0.055 g, 0.4 mmol), EDC (0.076 g, 0.4 mmol) and DIEA (0.11 g, 0.83 mmol) . After stirring for 30 minutes, 3-chloroaniline (0.04 g, 0.33 mmol) was added and stirring was continued at room temperature for 24h. The solvent was removed, and the concentrate was azeotroped twice with CH2Cl2. The residue was silica flash column chromatographed (gradient elution, 3-10% MeOH in CH2Cl2) to afford 34i as a white solid (0.027 g, 26%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. MS ESI (m/z): 315 [M-H]-, HRMS: m/z calculated for C14H9ClN4O3-: 315.02904, found: 315.02921.   N-(3-Chloro-2-methoxyphenyl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34j:  Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 3-chloro-o-anisidine (0.052 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-12 % MeOH in CH2Cl2) to give 34j as a white solid (0.032 g, 28%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. MS ESI (m/z): 345 [M-H]-, HRMS: m/z calculated for C15H11ClN4O4-: 345.03961, found: 345.03989.  147  N-(2-Chloro-4-methylphenyl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34k:  Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 2-chloro-4-methylaniline (0.047 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-9 % MeOH in CH2Cl2) to give 34k as pale yellow solid (0.03 g, 28%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. 13C NMR (100MHz, DMSO -d6): 169.0, 166.3, 162.5, 152.1, 139.0, 138.1, 137.7, 130.8, 130.4, 129.2, 128.9, 127.9, 121.5, 104.5, 20.8. MS ESI (m/z): 329 [M-H]-, HRMS: m/z calculated for C15H11N4O3Cl-: 329.04469, found: 329.04529.  N-(4-Chlorophenyl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34l:  Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 4-chloroaniline (0.04 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-10 % MeOH in CH2Cl2) to give 34l as a pale green coloured solid (0.022 g, 21%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. MS ESI (m/z): 315 [M-H]-, HRMS: m/z calculated for C14H9N4O3Cl-: 315.02904, found: 315.02911.  3-(6-Oxo-1,6-dihydropyridin-3-yl)-N-(4-(trifluoromethyl)phenyl)-1,2,4-oxadiazole-5-carboxamide 34m:  148  Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 4-(trifluoromethyl)aniline (0.053 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-10 % MeOH in CH2Cl2) to give 34m as an off white solid (0.022 g, 21%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. MS ESI (m/z): 349 [M-H]-, HRMS: m/z calculated for C15H9N4O3F3-: 349.0554, found: 349.05542.  N-(4-Cyanophenyl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34n:  Following the procedure described for 34i, acid 35 (0.09 g, 0.43 mmol) was condensed with 4-aminobenzonitrile (0.051 g, 0.43 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-10 % MeOH in CH2Cl2) to give 34n as an off white solid (0.025 g, 19%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. MS ESI (m/z): 306 [M-H]-, HRMS: m/z calculated for C15H9N5O3-: 306.06326, found: 306.06326.  N-(2,6-dichlorophenyl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34o:  Following the procedure described for 34i, acid 35 (0.09 g, 0.43 mmol) was condensed with 2,6-dichloroaniline (0.07 g, 0.43 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-8 % MeOH in CH2Cl2) to give 34o as a yellowish white solid (0.035 g, 23%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. MS ESI (m/z): 348 [M-H]-, HRMS: m/z calculated for C14H8N4O3Cl2-: 348.99007, found: 348.99011.  149  N-(3,5-Dimethoxyphenyl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34p:  Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 3,5-dimethoxyaniline (0.05 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 3-15 % MeOH in CH2Cl2) to give 34p as an off white solid (0.047 g, 42%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. MS ESI (m/z): 341 [M-H]-, HRMS: m/z calculated for C16H14N4O5-: 341.08914, found: 341.08917.  N-(3,4-Dimethoxyphenyl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34q:  Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 3,4-dimethoxyaniline (0.05 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-14 % MeOH in CH2Cl2) to give 34q as an off white solid (0.05 g, 45%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. MS ESI (m/z): 341 [M-H]-, HRMS: m/z calculated for C16H14N4O5-: 341.08914, found: 341.08939.  N-(3,5-Difluorophenyl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34r:  Following the procedure described for 34i, acid 35 (0.09 g, 0.43 mmol) was condensed with 3,5-difluoroaniline (0.055 g, 0.43 mmol). The crude product mixture was separated by flash 150  column chromatography on silica gel (gradient elution, 2-10 % MeOH in CH2Cl2) to give to give 34r as a white solid (0.04 g, 30%). 1H-NMR (400 MHz , DMSO-d6): see Table 24. MS ESI (m/z): 317 [M-H]-, HRMS: m/z calculated for C14H8F2N4O3-: 317.04917, found: 317.04916.  N-(3,5-Dichlorophenyl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34s:  Following the procedure described for 34i, acid 35 (0.09 g, 0.43 mmol) was condensed with 3,5-dichloroaniline (0.07 g, 0.43 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-10 % MeOH in CH2Cl2) to give 34s as a pale yellow solid (0.035 g, 23%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. 13C NMR (100MHz, DMSO -d6): 169.0, 166.3, 162.6, 152.3, 145.3, 140.9, 138.1, 137.7, 134.6, 124.8, 121.5, 119.6, 117.7, 104.6. MS ESI (m/z): 348 [M-H]-, HRMS: m/z calculated for C14H8N4O3Cl2-: 348.99007, found: 348.99048. N-(4-(Methylthio)phenyl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34t:  Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 4-(methylthio)aniline (0.046 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-12 % MeOH in CH2Cl2) to give 34t as pale red solid (0.055 g, 51%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. 13C NMR (100MHz, DMSO -d6): 169.3, 166.2, 162.6, 151.6, 140.8, 138.1, 137.8, 135.0, 129.2, 127.0, 122.0, 121.5, 104.7, 15.5. 151  MS ESI (m/z): 327 [M-H]-, HRMS: m/z calculated for C15H12N4O3S-: 327.05573, found: 327.05573.  N-(2-(Methylthio)phenyl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34u:  Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 2-(methylthio)aniline (0.046 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-10 % MeOH in CH2Cl2) to give 34u as a white solid (0.051 g, 48%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. 13C NMR (100MHz, DMSO -d6): 169.1, 166.2, 162.6, 152.0, 138.1, 137.7, 134.6, 134.1, 128.2, 126.4, 122.5, 121.5, 104.5, 16.0.   MS ESI (m/z): 327 [M-H]-, HRMS: m/z calculated for C15H12N4O3S-: 327.05573, found: 327.05591.  N-(2-Bromophenyl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34v:  Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 2-bromoaniline (0.057 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-6 % MeOH in CH2Cl2) to give 34v as a white solid (0.021 g, 18%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. 13C NMR (100MHz, DMSO -d6): 169.1, 166.3, 162.6, 152.1, 138.2, 137.8, 135.1, 133.4, 129.3, 128.9, 128.4, 121.5, 120.2, 104.6. MS ESI (m/z): 358 [M-H]-, HRMS: m/z calculated for C14H9N4O3Br-: 358.97853, found: 358.97894. 152  N-([1,1'-Biphenyl]-2-yl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34w:  Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 2-aminobiphenyl (0.057 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-8 % MeOH in CH2Cl2) to give 34w as a white solid (0.057 g, 48%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. MS ESI (m/z): 357 [M-H]-, HRMS: m/z calculated for C20H14N4O3-: 357.09931, found: 357.09982. 3-(6-Oxo-1,6-dihydropyridin-3-yl)-N-(3-phenoxyphenyl)-1,2,4-oxadiazole-5-carboxamide 34x:  Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 3-phenoxyaniline (0.06 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-10 % MeOH in CH2Cl2) to give  34x as a white solid (0.057 g, 46%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. 13C NMR (100MHz, DMSO -d6): 169.2, 166.2, 162.5, 157.6, 156.6, 151.8, 139.3, 138.1, 137.7, 130.8, 130.6, 124.3, 122.7, 121.5, 119.6, 116.0, 115.5, 111.1, 104.6. MS ESI (m/z): 373 [M-H]-, HRMS: m/z calculated for C20H14N4O4-: 373.09423, found: 373.09427.  N-(Naphthalen-2-yl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34y:  153   Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 2-naphthylamine (0.047 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-6 % MeOH in CH2Cl2) to give 34y as an off white solid (0.05 g, 50%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. MS ESI (m/z): 331 [M-H]-, HRMS: m/z calculated for C18H12N4O3-: 331.08366, found: 331.08368.  3-(6-Oxo-1,6-dihydropyridin-3-yl)-N-(thiazol-2-yl)-1,2,4-oxadiazole-5-carboxamide 34z:  Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 2-aminothiazole (0.033 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-10 % MeOH in CH2Cl2) to give 34z as a pale yellow solid (0.04 g, 42%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. MS ESI (m/z): 288 [M-H]-, HRMS: m/z calculated for C11H7N5O3S-: 288.01968, found: 288.01968.  3-(6-Oxo-1,6-dihydropyridin-3-yl)-N-(pyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34aa:  Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 3-aminopyridine (0.031 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-15 % MeOH in CH2Cl2) to give 34aa as an off white solid (0.036 g, 38%). 1H-NMR (400  MHz, DMSO-d6): see Table 24. MS ESI (m/z): 282 [M-H]-, HRMS: m/z calculated for C13H9N5O3-: 282.06326, found: 282.06323.  154  3-(6-Oxo-1,6-dihydropyridin-3-yl)-N-(pyridin-4-yl)-1,2,4-oxadiazole-5-carboxamide 34ab:  Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 4-aminopyridine (0.031 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-15 % MeOH in CH2Cl2) to give 34ab as a white solid (0.017 g, 18%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. MS ESI (m/z): 282 [M-H]-, HRMS: m/z calculated for C13H9N5O3-: 282.06326, found: 282.06348.  3-(6-Oxo-1,6-dihydropyridin-3-yl)-N-(quinolin-6-yl)-1,2,4-oxadiazole-5-carboxamide 34ac:   Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 6-aminoquinoline (0.048 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-15 % MeOH in CH2Cl2) to give 34ac as a white solid (0.023 g, 21%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. 13C NMR (100MHz, DMSO -d6): 169.4, 166.3, 162.6, 153.1, 151.3, 148.5, 138.1, 137.7, 132.7, 132.5, 129.5, 128.8, 124.8, 124.6, 121.9, 121.5, 104.7. MS ESI (m/z): 332 [M-H]-, HRMS: m/z calculated for C17H11N5O3-: 332.07891, found: 332.07904.  N-Cyclohexyl-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34ad:  Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with cyclohexylamine (0.033 g, 0.33 mmol). The crude product mixture was separated by flash 155  column chromatography on silica gel (gradient elution, 2-5 % MeOH in CH2Cl2) to give 34ad as a white solid (0.054 g, 56%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. MS ESI (m/z): 287 [M-H]-, HRMS: m/z calculated for C14H16N4O3-: 287.11496, found: 287.11523.  3-(6-Oxo-1,6-dihydropyridin-3-yl)-5-(5-(morpholine-1-carbonyl)-1,2,4-oxadiazole 34ae:   Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with morpholine (0.029 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-8 % MeOH in CH2Cl2) to give 34ae as a white solid (0.027 g, 30%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. MS ESI (m/z): 275 [M-H]-, HRMS: m/z calculated for C12H12N4O4-: 275.07858, found: 275.07858.  N-(Benzo[d][1,3]dioxol-5-yl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34af:  Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 3,4-(methylenedioxy)aniline (0.045 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-12 % MeOH in CH2Cl2) to give 34af as a white solid (0.058 g, 54%). 1H-NMR (400 MHz, DMSO -d6): see Table 24. 13C NMR (100MHz, DMSO -d6): 169.4, 166.2, 162.6, 151.4, 147.6, 144.9, 138.1, 137.7, 131.9, 121.4, 114.7, 108.6, 104.7, 103.2, 101.8. MS ESI (m/z): 325 [M-H]-, HRMS: m/z calculated for C15H10N4O5-: 325.05784, found: 325.05774. 156  N-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34ag:  Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 1,4-benzodioxane-6-amine (0.05 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-12 % MeOH in CH2Cl2) to give 34ag as a yellow solid (0.057 g, 51%). 1H-NMR (400 MHz, DMSO -d6): see Table 24.  MS ESI (m/z): 337 [M-H]-, HRMS: m/z calculated for C16H10N4O5-: 337.05784, found: 339.07330.  N-(1H-Indazol-5-yl)-3-(6-oxo-1,6-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 34ah:  Following the procedure described for 34i, acid 35 (0.07 g, 0.33 mmol) was condensed with 5-aminoindazole (0.044 g, 0.33 mmol). The crude product mixture was separated by flash column chromatography on silica gel (gradient elution, 2-15 % MeOH in CH2Cl2) to give 34ah as an off white solid (0.015 g, 14%).  1H-NMR (400 MHz, DMSO -d6): see Table 24. MS ESI (m/z): 321 [M-H]-, HRMS: m/z calculated for C15H10N6O3-: 321.07416, found: 321.07413.  Table 24: NMR of compounds 34a-ah    157   Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  34a   12.22 (1H, s, N1’-H), 11.22 (1H, s, CONH), 8.16 (1H, d, J=2.6Hz, H-’), 7.98 (1H, dd, J=2.6Hz, J=9.6Hz, H-’), 6.55 (1H, d, J=9.6Hz, H-5’ 7.81 (2H,d, J=7.6Hz H-”,” . (2H,  t, J=7.6Hz, H-”,5”, .0 1H, t, J=7.6Hz, H-”  34b   12.22 (1H, s, N1’-H), 11.12 (1H, s, CONH), 8.15 (1H, d, J=2.5Hz, H-’), 7.98 (1H, dd, J=2.5Hz, J=9.6Hz, H-’), 6.54 (1H, d, J=9.6Hz, H-5’  7.73 (2H, d, J=9.0Hz, H-”,”, . (2H, d, J=9.0Hz, H-”,5”, . H, s, OCH3)   34c   12.24 (1H, s, N1’-H), 10.93 (1H, s, CONH), 8.15 (1H, d, J=2.3Hz, H-’), 7.98 (1H, dd, J=2.3Hz, J=9.4Hz, H-’), 6.54 (1H, d, J=9.4Hz, H-5’  7.21-7.11(3H, m, H-”,5”,”  34d   12.24 (1H, s, N1’-H), 11.16 (1H, s, CONH), 8.15 (1H, d, J=2.5Hz, H-’), 7.98 (1H, dd, J=2.5Hz, J=9.6Hz, H-’), 6.54 (1H, d, J=9.6Hz, H-5’  7.69 (2H, d, J=9.0Hz, H-”,”, .1 (2H, d, J=9.0Hz, H-3”,5”, .0 H, s, CH3)  34e   12.21 (1H, s, N1’-H), 9.98 (1H, t, J=6.4Hz, CO-NH), 8.08 (1H, d, J=2.5Hz, H-’), 7.94 (1H, dd, J=2.5Hz, J=9.6Hz, H-’), 6.52 (1H, d, J=9.6Hz, H-5’  7.35 (4H, d, J=4.4Hz, H-”,”,5”,”, 7.31-7.24 (1H, m, H-”, . 1H, d, J=6.4Hz, CH2)   34f   12.19 (1H, s, N1’-H), 9.88 (1H, t, J=6.4Hz, CO-NH), 8.07 (1H, d, J=2.5Hz, H-’), 7.93 (1H, dd, J=2.5Hz, J=9.6Hz, H-’), 6.52 (1H, d, J=9.6Hz, H-5’ 7.27 (2H, d, J=8.7Hz, H-”,”, .0 (2H, d, J=8.7Hz, H-”,5”, .0 H, d, J=6.3Hz, CH2), 3.73 (3H, s, OCH3) 158  Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm   34g   12.22 (1H, s, N1’-H), 10.87 (1H, s, CONH), 8.15 (1H, d, J=2.4Hz, H-’), 7.98 (1H, dd, J=2.4Hz, J=9.6Hz, H-’), 6.54 (1H, d, J=9.6Hz, H-5’  7.26 (1H, dd, J=2.9Hz, J=6.1Hz, H-5”, .-7.13 (2H, m,  H-”,”, . (2H, t, J=7.4Hz, benzylic CH2), 2.84 (2H, t, J=7.4Hz, benzylic CH2), 2.01 (2H, p, J=7.4Hz, CH2)   34h   12.19 (1H, s, N1’-H), 9.93 (1H, t, J=6.4Hz, CO-NH), 8.09 (1H, d, J=2.5Hz, H-’), 7.95 (1H, dd, J=2.5Hz, J=9.6Hz, H-’), 6.53 (1H, d, J=9.6Hz, H-5’  8.53 (1H, dd, J=1.7Hz, J=4.8Hz, H-”, . 1H, dt, - 1.Hz, - .Hz, H-”, .0 1H, d, - .Hz, H-” , 7.32-7.26 (1H, m, H-5”, 4.59 (2H, d, J=5.9Hz, CH2)   34i   12.22 (1H, s, N1’-H), 11.39 (1H, s, CONH), 8.16 (1H, d, J=2.4Hz, H-’), 7.98 (1H, dd, J=2.4Hz, J=9.5Hz, H-’), 6.54 (1H, d, J=9.5Hz, H-5’  7.96 (1H, d, J=2.1Hz, H-”, . (1H, ddd, J=0.8Hz, J=2.1Hz,  J=8.2Hz, H-”, .5 1H, t, - .1Hz, H-5”, . 1H, ddd, - 0.Hz, J=2.1Hz, J=8.1Hz, H-”   34j   12.22 (1H, s, N1’-H), 10.55 (1H, s, CONH), 8.15 (1H, d, J=2.4Hz, H-’), 7.98 (1H, dd, J=2.4Hz, J=9.5Hz, H-’), 6.55 (1H, d, J=9.5Hz, H-5’  7.79 (1H, dd, J=1.5Hz, J=8.1Hz, H-”, . 1H, dd, - 1.5Hz, - .1Hz, H-”, . 1H, t, - .1Hz, H-5”, 3.82 (3H, s, OCH3)  34k   12.21 (1H, s, N1’-H), 10.91 (1H, s, CONH), 8.14 (1H, d, J=2.6Hz, H-’), 7.97 (1H, dd, J=2.6Hz, J=9.5Hz, H-’), 6.55 (1H, d, J=9.5Hz, H-5’ 7.52 (1H, d, J=8.1Hz, H-”, . (1H, d, J=1.1Hz, H-”, . 1H, dd, J=1.1Hz, J=8.1Hz, H-5”, . H, s, CH3)  34l   12.25 (1H, s, N1’-H), 11.37 (1H, s, CONH), 8.16 (1H, d, J=2.6Hz, H-’), 7.98 (1H, dd, J=2.6Hz, J=9.5Hz, H-’), 6.54 (1H, d, J=9.5Hz, H-5’  7.86 (2H, d, J=8.9Hz, H-”,”, . (2H, d, J=8.9Hz, H-”,5”  159  Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  34m   12.21 (1H, s, N1’-H), 11.56 (1H, s, CONH), 8.16 (1H, d, J=2.6Hz, H-’), 7.98 (1H, dd, J=2.6Hz, J=9.5Hz, H-’), 6.55 (1H, d, J=9.5Hz, H-5’ 8.06 (2H, d, J=9.0Hz, H-”,”, .0 (2H, d, J=9.0Hz, H-”,5”   34n   12.23 (1H, s, N1’-H), 11.57 (1H, s, CONH), 8.16 (1H, d, J=2.5Hz, H-’), 7.98 (1H, dd, J=2.5Hz, J=9.5Hz, H-’), 6.54 (1H, d, J=9.5Hz, H-5’  8.04 (2H, d, J=9.0Hz, H-”,”, . (2H, d, J=9.0Hz, H-”,5”    34o   12.23 (1H, s, N1’-H), 11.49 (1H, s, CONH), 8.14 (1H, d, J=2.6Hz, H-’), 7.98 (1H, dd, J=2.6Hz, J=9.5Hz, H-’), 6.55 (1H, d, J=9.5Hz, H-5’  7.64 (2H, d, J=8.1Hz,  H-”,5” , . (1H, t, J=8.1Hz,  H-”    34p   12.27 (1H, s, N1’-H), 11.10 (1H, s, CONH), 8.15 (1H, d, J=2.5Hz, H-’), 7.97 (1H, dd, J=2.5Hz, J=9.6Hz, H-’), 6.54 (1H, d, J=9.6Hz, H-5’ 7.11 (2H, d, J=2.2Hz, H-”,”, . (1H, t, J=2.2Hz, H-”, . H, s,  OCH3)   34q   12.23 (1H, s, N1’-H), 11.06 (1H, s, CONH), 8.15 (1H, d, J=2.5Hz, H-’), 7.98 (1H, dd, J=2.5Hz, J=9.6Hz, H-’), 6.54 (1H, d, J=9.6Hz, H-5’  7.48 (1H, d, J=2.3Hz, H-”, . (1H, dd, J=2.3Hz, J=8.8Hz, H-”, 6.98 (1H, d, J=8.8Hz, H-5”, . (6H, s, 2 OCH3)    34r   12.27 (1H, s, N1’-H), 11.10 (1H, s, CONH), 8.15 (1H, d, J=2.5Hz, H-’), 7.97 (1H, dd, J=2.5Hz, J=9.6Hz, H-’), 6.54 (1H, d, J=9.6Hz, H-5’  7.62 (2H, d, J=2.2Hz, H-”, .0 (2H, d, J=2.2Hz, H-”, .10 1H, t, J=2.2Hz, H-”.   34s   12.24 (1H, s, N1’-H), 11.52 (1H, s, CONH), 8.15 (1H, d, J=2.4Hz, H-’), 7.98 (1H, dd, J=2.4Hz, J=9.5Hz, H-’), 6.55 (1H, d, J=9.5Hz, H-5’  7.92 (2H, d, J=1.8Hz, H-”, ”, . (1H, t, J=1.8Hz, H-” 160  Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm   34t   11.75 (2H, d, N1’-H,  CONH), 8.15 (1H, d, J=2.3Hz, H-’), 7.98 (1H, dd, J=2.3Hz, J=9.5Hz, H-’), 6.54 (1H, d, J=9.5Hz, H-5’  7.78 (2H, d, J=8.7Hz, H-”,”, .1 (2H, d, J=8.7Hz, H-”,5”, . H, s, SCH3)    34u   12.22 (1H, s, N1’-H), 10.78 (1H, s, CONH), 8.13 (1H, d, J=2.5Hz, H-’), 7.97 (1H, dd, J=2.5Hz, J=9.6Hz, H-’), 6.55 (1H, d, J=9.6Hz, H-5’  7.56 (1H, dd, J=1.4Hz, J=7.9Hz, H-” , . 1H, dd, - 1.Hz, - .Hz, H-”, . 1H, dt, - 1.Hz, J=7.6Hz, H-5”, . 1H, dt, J=1.4Hz, J=7.6Hz, H-”, .51H, s, SCH3)    34v   12.23 (1H, s, N1’-H), 10.95 (1H, s, CONH), 8.14 (1H, d, J=2.6Hz, H-’), 7.98 (1H, dd, J=2.6Hz, J=9.5Hz, H-’), 6.55 (1H, d, J=9.5Hz, H-5’  7.77 (1H, dd, J=1.1Hz, J=8.1Hz, H-”, .5 1H, dd, - 1.Hz, - .Hz, H-”, . 1H, dt, - 1.1Hz, J=7.8Hz, H-5”, .0 1H, dt, J=1.2Hz, J=7.8Hz, H-”    34w   12.20 (1H, s, N1’-H), 10.65 (1H, s, CONH), 8.01 (1H, d, J=2.6Hz, H-’), 7.90 (1H, dd, J=2.4Hz, J=9.4Hz, H-’), 6.52 (1H, d, J=9.6Hz, H-5’  7.68 (1H, d, J=7.6Hz), 7.51-7.34 (8H, m, Ar-H)   34x   12.22 (1H, s, N1’-H), 11.27 (1H, s, CONH), 8.14 (1H, d, J=2.4Hz, H-’), 7.96 (1H, dd, J=2.4Hz, J=9.4Hz, H-’), 6.53 (1H, d, J=9.4Hz, H-5’  7.63 (1H, ddd, J=0.9Hz, J=2.7Hz, J=8.1Hz, Ar-H) 7.53 (1H, t, J=2.7Hz, H-”, .-7.37 (3H, m, Ar-H), 7.22-7.14 (1H, m, Ar-H), 7.10-7.03 (2H, m, Ar-H), 6.89-6.84 (1H, ddd, J=0.9Hz, J=2.7Hz, J=8.1Hz, Ar-H)   34y  12.27 (1H, s, N1’-H), 11.37 (1H, s, CONH), 8.19 (1H, d, J=2.6Hz, H-’), 8.08-7.96* (3H, m,  H-’), 6.57 (1H, d, J=9.6Hz, H-5’ 8.08-7.96* (3H, m, Ar-H), 7.95 (1H, d, J=7.5Hz, Ar-H), 7.68-7.55 (4H, m, Ar-H) 161  Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm   34z   12.70 (2H, s, N1’-H, CONH), 8.25 (1H, d, J=2.6Hz, H-’), 7.65 (1H, dd, J=2.6Hz, J=9.6Hz, H-’), 6.41 (1H, d, J=9.6Hz, H-5’ 7.53 (1H, d, J=4.2Hz, H-”, . (1H, d, J=4.2Hz, H-5”   34aa   12.00 (1H, s, N1’-H), 8.15 (1H, d, J=2.5Hz, H-’), 7.98 (1H, dd, J=2.5Hz, J=9.6Hz, H-’), 6.54 (1H, d, J=9.6Hz, H-5’  8.92 (1H, d, J=2.3Hz, H-”, .5 (1H, dd, J=1.4Hz, J=4.7Hz, H-”, 8.19 (1H, ddd, J=1.4Hz, J=2.3Hz, J=8.2Hz, H-”, . 1H, dd, J=4.7Hz, J=8.2Hz, H-5”   34ab   12.21 (1H, s, N1’-H), 11.53 (1H, s, CONH), 8.15 (1H, d, J=2.5Hz, H-’), 7.98 (1H, dd, J=2.5Hz, J=9.6Hz, H-’), 6.54 (1H, d, J=9.6Hz, H-5’ 8.56 (2H, d, J=6.3Hz, H-”,”, . (2H, d, J=6.3Hz, H-”,5”    34ac   12.23 (1H, s, N1’-H), 11.49 (1H, s, CONH), 8.19 (1H, d, J=2.4Hz, H-’, 8.08-7.97* (m, 2H, H-’, .5 (1H, d, J=9.6Hz, H-5’  8.96 (1H, dd, J=1.5Hz,  J=4.2Hz, H-” ), 8.49 (1H, dd, J=1.5Hz, J=8.6Hz, H-”), 8.08-7.97* (2H, m, H-”), 7.84 (1H, t, J=7.3Hz, H-”), 7.71 (1H, dd, J=1.0Hz, J=7.3Hz, H-”), 7.59 (1H, dd, J=4.2Hz, J=8.6Hz, H-”)   34ad   12.21 (1H, s, N1’-H), 9.24 (1H, d, J=8.4Hz, CO-NH), 8.09 (1H, d, J=2.5Hz, H-’), 7.93 (1H, dd, J=2.5Hz, J=9.6Hz, H-’), 6.51 (1H, d, J=9.6Hz, H-5’ 1.90-1.02 (10H, m, H-”,”,”,5”,”, .81-3.67 (1H, m, H-1”   34ae   12.00 (1H, s, N1’-H), 8.08 (1H, d, J=2.5Hz, H-’), 7.93 (1H, dd, J=2.5Hz, J=9.6Hz, H-’), 6.52 (1H, d, J=9.6Hz, H-5’ 3.80-3.75 (2H, m, CH2), 3.69 (4H, s, 2 CH2), 3.67-3.62 (2H, m, CH2)  162  Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm   34af   12.21 (1H, s, N1’-H), 11.17 (1H, s, CONH), 8.14 (1H, d, J=2.4Hz, H-’), 7.97 (1H, dd, J=2.4Hz, J=9.4Hz, H-’), 6.54 (1H, d, J=9.4Hz, H-5’  7.42 (1H, d, J=2.1Hz, H-”, .0 (1H, dd, J=2.1Hz, J=8.4Hz, H-”, 6.95 (1H, d, J=8.4Hz, H-”, .0 (2H, s, OCH2O)    34ag   12.22 (1H, s, N1’-H), 11.07 (1H, s, CONH), 8.15 (1H, d, J=2.5Hz, H-’), 7.97 (1H, dd, J=2.5Hz, J=9.6Hz, H-’), 6.54 (1H, d, J=9.6Hz, H-5’  7.40 (1H, d, J=2.5Hz H-5”, . (1H, dd, J=2.5Hz, J=8.8Hz, H-”, 6.88 (1H, d, J=8.8Hz, H-”, . (4H, d, J=1.0Hz, 2 OCH2)   34ah   12.26 (1H, br s, N1’-H), 11.28 (1H, br s, CONH), 8.17 (1H, d, J=2.4Hz, H-’), 7.99 (1H, dd, J=2.4Hz, J=9.4Hz, H-’), 6.55 (1H, d, J=9.4Hz, H-5’  8.29 (1H, d, J=1.7Hz, H-”,  .1 (1H, s, H-”, .0 1H, dd, - 1.Hz, J=8.8Hz, H-”, .5 1H, d, J=8.8Hz, H-”  2-Oxo-1,2-dihydropyridine-3-carbonitrile 37:  According to reference 215, a solution of 2-chloronicotinonitrile 36 (5.00 g, 36.1mmol) in AcOH (100 mL) and H2O (10 mL) was heated at 125 oC for 5 h. The cooled suspension was then poured onto ice. The solid was collected, washed with water and dried under vacuum to give 37 (4.16 g, 96%) which was used in the next step without further purification. 1H-NMR (400 MHz, DMSO -d6): ppm 12.56 (1H, br s, N1-H), 8.14 (1H, dd, J=1.7Hz, J=6.8Hz, H-4), 7.78 (1H, dd, J=1.7Hz, J=6.8Hz, H-6), 6.34 (1H, t, J=6.8Hz, H-5). MS ESI (m/z): 119 [M-H]-.  N'-Hydroxy-2-oxo-1,2-dihydropyridine-3-carboximidamide 38:  163  To a hot solution of 2-oxo-1,2-dihydropyridine-3-carbonitrile 37 (3.50 g, 29.17 mmol) in EtOH (50 mL), was added a solution of hydroxylamine hydrochloride (4.05 g, 58.33 mmol) and NaHCO3 (4.9 g, 58.33 mmol) in water (40 mL). The reaction mixture was heated at reflux for 6h. The solvent was then removed in vacuo and the resulting residue was dry-loaded silica gel flash column chromatographed (10-20% MeOH in EtOAc) to give the 38 as a white solid (6.33 g, 71%). 1H-NMR (400 MHz, DMSO -d6):  ppm 12.07 (1H, br s, N1-H), 9.48 (1H, s, OH), 7.94 (1H, dd, J=1.4Hz, J=6.8Hz, H-4), 7.50 (1H, dd, J=1.4Hz, J=6.8Hz, H-6), 6.30 (3H, t, J=7.8Hz, H-5, NH2). MS ESI (m/z): 154 [M+H]+.  Ethyl 2-((2-(methylthio)phenyl)amino)-2-oxoacetate 40a:  Ethyl chlorooxoacetate 39 (0.5 mL, 4.48 mmol) was added dropwise to a mixture of 2-methylthioaniline (0.62 g, 4.48 mmol) and Et3N (0.75 mL, 5.37 mmol) in CH2Cl2 (8 mL) at 0 oC. The reaction mixture was allowed to warm slowly to room temperature and stirred for 12 h. The mixture was then filtered to remove the ammonium salts, and the filtrate was washed with water. The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The crude oil was flash column chromatographed on silica gel (gradient elution, 5-10% EtOAc in hexane) to afford 40a as an off white solid (0.95 g, 89%). 1H-NMR (400 MHz, DMSO -d6):   ppm 10.22 (1H, br s, N-H), 7.66-7.61 (1H, m, H-3’), 7.49-7.43 (1H, m, H-6’), 7.30-7.24 (2H, m, H-4’, H-5’), 4.32 (2H, q, J=7.1Hz, CH2), 2.43 (3H, s, SCH3), 1.32 (3H, t, J=7.1Hz, CH3). MS ESI (m/z): 240 [M+H]+.  Ethyl 2-((2,3-dihydro-1H-inden-4-yl)amino)-2-oxoacetate 40b:  Indan-4-amine (0.6 g, 4.48 mmol) was reacted with ethyl chlorooxoacetate 39 (0.5 mL, 4.48 mmol) according to the procedure for 40a. The crude product was flash column 164  chromatographed on silica gel (gradient elution, 3-10% EtOAc in hexane) to afford 40b as a brown solid (0.87 g, 83%).  1H-NMR (400 MHz, DMSO -d6):   ppm 10.28 (1H, br s, N-H), 7.26 (1H, dd, J=2.9Hz, J=6.1Hz, H-5’), 7.18-7.09 (2H, m,  H-’,’), 4.30 (2H, q, J=7.1Hz, ethyl CH2), 2.92 (2H, t, J=7.4Hz, benzylic-H), 2.84 (2H, t, J=7.4Hz, benzylic-H), 2.01 (2H, p, J=7.4Hz, CH2(2’)), 1.31 (3H, t, J=7.1Hz, CH3).  MS ESI (m/z): 234 [M+H]+.  Ethyl 2-((3,4-dimethoxyphenyl)amino)-2-oxoacetate 40c:  3,4-Dimethoxyaniline (0.69 g, 4.48 mmol) was reacted with ethyl chlorooxoacetate 39 (0.5 mL, 4.48 mmol) according to the procedure for 40a. The crude product was flash column chromatographed on silica gel (gradient elution, 3-15% EtOAc in hexane) to afford 40c as a white solid (1.03 g, 91%).  1H-NMR (400 MHz, DMSO -d6):   ppm 10.61 (1H, br s, N-H), 7.40 (1H, d, J=2.2Hz, H-’), 7.33 (1H, dd, J=2.2Hz, J=8.8Hz, H-’), 6.93 (1H, d, J=8.8Hz, H-5’), 4.29 (2H, q, J=7.1Hz, CH2), 3.73 (6H, d, J=2.7Hz, 2 OCH3), 1.31 (3H, t, J=7.1Hz, CH3). MS ESI (m/z): 253 [M+H]+.  Ethyl 2-((3-chloro-2-methoxyphenyl)amino)-2-oxoacetate 40d:  3-Choloro-4-methoxyaniline (0.71 g, 4.48 mmol) was reacted with ethyl chlorooxoacetate 39 (0.5 mL, 4.48 mmol) according to the procedure for 40a. The crude product was flash column chromatographed on silica gel (gradient elution, 3-12% EtOAc in hexane) to afford 40d as a yellowish white solid (0.82 g, 71%).  1H-NMR (400 MHz, DMSO -d6):   ppm 10.01 (1H, br s, N-H), 7.87 (1H, dd, J=1.6Hz, J=8.1Hz, H-’), 7.34 (1H, dd, J=1.6Hz, J=8.1Hz, H-’), 7.19 (1H, t, J=8.1Hz, H-5’), 4.32 (2H, q, J=7.3Hz, CH2), 3.82 (3H, s, OCH3), 1.31 (3H, t, J=7.3Hz, CH3).  MS ESI (m/z): 258 [M+H]+.    165  Ethyl 2-oxo-2-((3-phenoxyphenyl)amino)acetate 40e:  3-Phenoxyaniline (0.83 g, 4.48 mmol) was reacted with ethyl chlorooxoacetate 39 (0.5 mL, 4.48 mmol) according to the procedure for 40a. The crude product was flash column chromatographed on silica gel (gradient elution, 3-12% EtOAc in hexane) to afford 40e as off white solid (1.0 g, 78%).  1H-NMR (400 MHz, DMSO -d6):   ppm 10.84 (1H, br s, N-H), 7.53 (1H, ddd, J=0.9Hz, J=2.6Hz, J=8.1Hz, Ar-H) 7.47 (1H, t, J=2.6Hz, H-’), 7.44-7.32 (3H, m, Ar-H), 7.20-7.13 (1H, m, Ar-H), 7.07-7.00 (2H, m, Ar-H), 6.83-6.73 (1H, ddd, J=0.9Hz, J=2.6Hz, J=8.1Hz, Ar-H), 4.29 (2H, q, J=7.2Hz, CH2), 3.82 (3H, s, OCH3), 1.30 (3H, t, J=7.2Hz, CH3).  Ethyl 2-oxo-2-(p-tolylamino)acetate 40f:   4-Methylaniline (0.48 g, 4.48 mmol) was reacted with ethyl chlorooxoacetate 39 (0.5 mL, 4.48 mmol) according to the procedure for 40a. The crude product was flash column chromatographed on silica gel (gradient elution, 3-12% EtOAc in hexane) to afford 40f as a white solid (0.84 g, 90%).  1H-NMR (400 MHz, DMSO -d6):   ppm 10.67 (1H, br s, N-H), 7.61 (2H, d, J=8.9Hz, H-’,’), 7.16 (2H, d, J=8.9Hz, H-’,5’), 4.29 (2H, q, J=7.2Hz, CH2), 3.82 (3H, s, OCH3), 2.27 (3H, s, CH3), 1.30 (3H, t, J=7.2Hz, ethyl CH3).  MS ESI (m/z): 208 [M+H]+.  Ethyl 2-(benzo[d][1,3]dioxol-5-ylamino)-2-oxoacetate 40g:  3,4-(Methylenedioxy)aniline (0.61 g, 4.48 mmol) was reacted with ethyl chlorooxoacetate 39 (0.5 mL, 4.48 mmol) according to the procedure for 40a. The crude product was flash 166  column chromatographed on silica gel (gradient elution, 3-15% EtOAc in hexane) to afford 40g as a white solid (0.98 g, 92%).  1H-NMR (400 MHz, DMSO -d6):   ppm 10.68 (1H, br s, N-H), 7.36 (1H, d, J=2.3Hz, H-’), 7.22 (1H, dd, J=2.3Hz, J=8.9Hz, H-’), 6.90 (1H, d, J=8.9Hz, H-’), 6.01 (2H, s, OCH2O), 4.29 (2H, q, J=7.1Hz, CH2), 1.30 (3H, t, J=7.1Hz, CH3). MS ESI (m/z): 238 [M+H]+.   N-(2-(Methylthio)phenyl)-3-(2-oxo-1,2-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 41a:  Sodium hydride 60% suspension in oil (200 mg, 5.0 mmol) was added to a stirred solution of ethyl 2-((2-(methylthio)phenyl)amino)-2-oxoacetate 40a (0.31 g, 1.31mmol) and amidoxime 38 (0.2g, 1.31mmol) in dry DMF (6 mL) at 0 oC under nitrogen. After stirring for 2 hours at room temperature, then the reaction mixture was stirred at 60 oC for 12 hours. The solvent was removed in vacuo and the crude oil silica flash column chromatographed (gradient elution, 3-10% MeOH in CH2Cl2) to afford 41a as a white solid (0.18 g, 43%). 1H-NMR (400 MHz, DMSO -d6): see Table 25. 13C NMR (100MHz, DMSO -d6): 169.1, 166.4, 162.6, 152.0, 138.2, 137.7, 134.6, 134.3, 128.2, 126.7, 122.5, 121.1, 104.8, 16.1.   MS ESI (m/z): 327 [M-H]-, HRMS: m/z calculated for C15H12N4O3S-: 327.05573, found: 327.05597.  N-(2,3-Dihydro-1H-inden-4-yl)-3-(2-oxo-1,2-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 41b:  Following the procedure for 41a, ethyl 2-((2,3-dihydro-1H-inden-4-yl)amino)-2-oxoacetate 40b (0.31 g, 1.31 mmol) was reacted with amidoxime 38 (0.2g, 1.31mmol). The crude 167  product was silica flash column chromatographed (gradient elution, 3-8% MeOH in CH2Cl2) to afford 41b as a white solid (0.17 g, 40%). 1H-NMR (400 MHz, DMSO -d6): see Table 25. MS ESI (m/z): 321 [M-H]-, HRMS: m/z calculated for C17H14N4O3-: 321.09931, found: 321.09930.  N-(3,4-Dimethoxyphenyl)-3-(2-oxo-1,2-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 41c:  Following the procedure for 41a, ethyl 2-((3,4-dimethoxyphenyl)amino)-2-oxoacetate 40c (0.33 g, 1.31 mmol) was reacted with amidoxime 38 (0.2g, 1.31mmol). The crude product was silica flash column chromatographed (gradient elution, 3-15% MeOH in CH2Cl2) to afford 41c as white solid (0.16 g, 36%). 1H-NMR (400 MHz, DMSO -d6): see Table 25. MS ESI (m/z): 341 [M-H]-.  N-(3-Chloro-2-methoxyphenyl)-3-(2-oxo-1,2-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 41d:  Following the procedure for 41a, ethyl 2-((3-chloro-2-methoxyphenyl)amino)-2-oxoacetate 40d (0.34 g, 1.31 mmol) was reacted with amidoxime 38 (0.2g, 1.31mmol). The crude product was silica flash column chromatographed (gradient elution, 3-10% MeOH in CH2Cl2) to afford 41d as a white solid (0.1 g, 23%). 1H-NMR (400 MHz, DMSO -d6): see Table 25. MS ESI (m/z): 345 [M-H]-, HRMS: m/z calculated for C15H11ClN4O4-: 345.03961, found: 345.03992.   168  3-(2-Oxo-1,2-dihydropyridin-3-yl)-N-(3-phenoxyphenyl)-1,2,4-oxadiazole-5-carboxamide 41e:  Following the procedure for 41a, ethyl 2-oxo-2-((3-phenoxyphenyl)amino)acetate 40e (0.37 g, 1.31 mmol) was reacted with amidoxime 38 (0.2g, 1.31mmol). The crude product was silica flash column chromatographed (gradient elution, 3-10% MeOH in CH2Cl2) to afford 41e as an off white solid (0.19 g, 37%). 1H-NMR (400 MHz, DMSO -d6): see Table 25. 13C NMR (100MHz, DMSO -d6): 168.1, 166.2, 159.2, 157.6, 156.6, 152.1, 144.3, 140.2, 139.3, 130.8, 130.6, 124.3, 122.7, 121.2, 119.6, 116.7, 116.0, 115.5, 111, 105.3. MS ESI (m/z): 373 [M-H]-, HRMS: m/z calculated for C20H14N4O4-: 373.09423, found: 373.09406.  3-(2-Oxo-1,2-dihydropyridin-3-yl)-N-(p-tolyl)-1,2,4-oxadiazole-5-carboxamide 41f:  Following the procedure for 41a, ethyl 2-oxo-2-(p-tolylamino)acetate 40f (0.27 g, 1.31 mmol) was reacted with amidoxime 38 (0.2g, 1.31mmol). The crude product was silica flash column chromatographed (gradient elution, 3-10% MeOH in CH2Cl2) to afford 41f as an off white solid (0.16 g, 41%). 1H-NMR (400 MHz, DMSO -d6): see Table 25. 13C NMR (100MHz, DMSO -d6): 166.1, 159.1, 151.9, 149.9, 144.9, 142.4, 140.2, 134.3, 129.6, 121.3, 116.8, 106.5, 104.7, 101.8, 21.2. MS ESI (m/z): 295 [M-H]-, HRMS: m/z calculated for C15H12N4O3-: 295.08366, found: 295.08368.   169  N-(Benzo[d][1,3]dioxol-5-yl)-3-(2-oxo-1,2-dihydropyridin-3-yl)-1,2,4-oxadiazole-5-carboxamide 41g:  Following the procedure for 41a, ethyl 2-(benzo[d][1,3]dioxol-5-ylamino)-2-oxoacetate 40g (0.31 g, 1.31 mmol) was reacted with amidoxime 38 (0.2g, 1.31mmol). The crude product was silica flash column chromatographed (gradient elution, 3-10% MeOH in CH2Cl2) to afford41g as an off white solid (0.19 g, 44%). 1H-NMR (400 MHz, DMSO -d6): see Table 25. MS ESI (m/z): 325 [M-H]-, HRMS: m/z calculated for C15H10N4O5-: 325.05784, found: 325.05817.  Table 25: NMR data of compounds 41a-g  Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  41a   12.32 (1H, s, N1’-H), 10.76 (1H, s, CONH), 8.30 (1H, dd, J=2.1Hz, J=7.1Hz, H-’, . 1H, dd, J=2.1Hz, J=6.5Hz, H-’, .5 1H, t, J=6.8Hz, H-5’  7.58 (1H, dd, J=1.4Hz, J=7.9Hz, H-” ), 7.47 (1H, dd, J=1.4Hz, J=7.9Hz, H-”, .5 1H, dt, - 1.Hz, - .Hz, H-5”, . 1H, dt, - 1.Hz, J=7.6Hz, H-”, . H, s, SCH3)   41b   12.38 (1H, s, N1’-H), 10.86 (1H, s, CONH), 8.31 (1H, dd, J=2.2Hz, J=7.1Hz, H-’, .1 1H, dd, J=2.2Hz, J=6.5Hz, H-’, . 1H, t, J=6.8Hz, H-5’  7.28 (1H, dd, J=2.9Hz, J=6.1Hz, H-5”, .1-7.22 (2H, m,  H-”,”, . (2H, t, J=7.4Hz, benzylic-H), 2.85 (2H, t, J=7.4Hz, benzylic-H), 2.01 (2H, p, J=7.4Hz, CH2)  170  Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  41c    12.31 (1H, s, N1’-H), 11.08 (1H, s, CONH), 8.33 (1H, dd, J=2.1Hz, J=7.2Hz, H-’, .1 1H, dd, J=2.1Hz, J=6.4Hz, H-’, . 1H, t, J=6.8Hz, H-5’  7.48 (1H, d, J=2.3Hz, H-”, . 1H, dd, J=2.3Hz, J=8.8Hz, H-”, 6.98 (1H, d, J=8.8Hz, H-5”, . H, s,  OCH3)   41d   12.35 (1H, s, N1’-H), 10.54 (1H, s, CONH), 8.33 (1H, dd, J=2.1Hz, J=7.2Hz, H-’, . 1H, dd, J=2.1Hz, J=6.4Hz, H-’, . 1H, t, J=6.8Hz, H-5’ 7.82 (1H, dd, J=1.6Hz, J=8.0Hz, H-”, . 1H, dd, - 1.Hz, - .0Hz, H-”, . 1H, t, - .0Hz, H-5”, 3.82 (3H, s, OCH3)   41e   12.33 (1H, s, N1’-H), 11.30 (1H, s, CONH), 8.32 (1H, dd, J=2.2Hz, J=7.1Hz, H-’, . 1H, dd, J=2.2Hz, J=6.5Hz, H-’, . 1H, t, J=6.8Hz, H-5’ 7.63 (1H, ddd, J=0.9Hz, J=2.6Hz, J=8.1Hz, Ar-H) 7.53 (1H, t, J=2.6Hz, H-”, .47-7.37 (3H, m, Ar-H), 7.22-7.15 (1H, m, Ar-H), 7.13-7.02 (2H, m, Ar-H), 6.89-6.84 (1H, ddd, J=0.9Hz, J=2.6Hz, J=8.1Hz, Ar-H)  41f   12.34 (1H, s, N1’-H), 11.18 (1H, s, CONH), 8.33 (1H, dd, J=2.2Hz, J=7.1Hz, H-’, . 1H, dd, J=2.2Hz, J=6.5Hz, H-’, .5 1H, t, J=6.8Hz, H-5’  7.70 (2H, d, J=9.0Hz, H-”,”, .1 (2H, d, J=9.0Hz, H-”,5”, .0 (3H, s, CH3)  41g   12.34 (1H, s, N1’-H), 11.17 (1H, s, CONH), 8.32 (1H, dd, J=2.1Hz, J=7.2Hz, H-’, . 1H, dd, J=2.1Hz, J=6.4Hz, H-’, . 1H, t, J=6.8Hz, H-5’ 7.43 (1H, d, J=2.2Hz, H-”, .0 1H, dd, J=2.2Hz, J=8.3Hz, H-”, .5 (1H, d, J=8.3Hz, H-”, .0 H, s, OCH2O)   Benzoyl fluoride derivatives 43a:  Cyanuric fluoride (0.3 mL, 5 mmol) was added dropwise to a mixture of benzoic acid 42a (0.41 g, 3.33 mmol) and pyridine (0.81 mL, 10 mmol) in CH2Cl2 (8 mL) at 0 oC. The 171  reaction mixture was allowed to warm slowly to room temperature and stirred for 12 h. After completion of the reaction (monitored by TLC) the mixture was poured onto water and extracted with CH2Cl2. The combined organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The residue was silica flash column chromatographed (gradient elution, 2-6% EtOAc in hexane) to afford benzoyl fluoride 43a as a colourless oil (0.29 g, 71%). 19F-NMR  (400 MHz, CDCl3): ppm.    4-Methoxybenzoyl fluoride 43b:  Prepared according to the procedure followed in 43a, 4-methoxybenzoic acid 42b (0.51 g, 3.34 mmol) was reacted with cyanuric fluoride (0.3 mL, 5 mmol). The crude product was silica flash column chromatographed (gradient elution, 2-8% EtOAc in hexane) to afford 43b as white semisolid (0.37 g, 73%).  2,4-Dimethoxybenzoyl fluoride 43c:  Prepared according to the procedure followed in 43a 2,4-dimethoxybenzoic acid 42c (0.61 g, 3.34 mmol) was reacted with cyanuric fluoride (0.3 mL, 5 mmol). The crude product was silica flash column chromatographed (gradient elution, 2-10% EtOAc in hexane) to afford 43c as white powder (0.46 g, 75%). 4-Chlorobenzoyl fluoride 43d:  Prepared according to the procedure followed in 43a, 4-chlorobenzoic acid 42d (0.52 g, 3.33 mmol) was reacted with cyanuric fluoride (0.3 mL, 5 mmol). The crude product was silica flash column chromatographed (gradient elution, 2-6% EtOAc in hexane) to afford 43d as a colourless oil (0.34 g, 64%).   172  4-Methylbenzoyl fluoride 43e:  Prepared according to the procedure followed in 43a using p-toluic acid 42e (0.45 g, 3.33 mmol). The crude product was purified by flash column chromatography on silica gel (gradient elution, 2-5% EtOAc in hexane) to afford 43e as a colourless oil (0.33 g, 73%).  4-(Trifluoromethyl)benzoyl fluoride 43f:   Prepared according to the procedure followed in 43a, 4-(trifluoromethyl)benzoic acid 42f (0.63 g, 3.33 mmol) was reacted with cyanuric fluoride (0.3 mL, 5 mmol). The crude product was purified by flash column chromatography on silica gel (gradient elution, 2-5% EtOAc in hexane) to afford 43f as a colourless oil (0.38 g, 59%). N'-(Benzoyloxy)-6-oxo-1,6-dihydropyridine-3-carboximidamide 44a:  To a stirred solution` of amidoxime 28 (0.25g, 1.64 mmol) and benzoyl fluoride 43a (0.2 g, 1.64 mmol) in dry THF (6 mL) was added a catalytic amount of TBAF (15 µL). The reaction mixture was stirred at ambient temperature for 4h. After removal of the THF in vacuo, the residue was silica flash column chromatographed (gradient elution, 3-8% MeOH in CH2Cl2) to afford 44a as a white solid (0.33 g, 79%). 1H-NMR (400 MHz, DMSO -d6):  11.96 (1H, s, N-H), 8.18-8.13 (2H, m, H-’,’, . (1H, d, J=2.4Hz, H-2), 7.79 (1H, dd, J=2.4Hz, J=9.6Hz, H-4), 7.69-7.63 (1H, m, H-’, .5-7.49 (2H,  m, H-’,5’,  . H, br s, NH2), 6.39 (1H, d, J=9.6Hz, H-5). MS ESI (m/z): 256 [M-H]-.      173  N'-((4-Methoxybenzoyl)oxy)-6-oxo-1,6-dihydropyridine-3-carboximidamide 44b:  According to the procedure for 44a, 4-methoxybenzoyl fluoride 43b (0.25 g, 1.64 mmol) was reacted with amidoxime 28 (0.25g, 1.64 mmol). The crude product was silica flash column chromatographed (gradient elution, 3-10% MeOH in CH2Cl2) to afford 44b as an off white solid (0.34 g, 73%).  1H-NMR (400 MHz, DMSO -d6):  11.94 (1H, s, N-H), 8.11 (2H, d, J=9.0Hz, H-’,’, 7.86 (1H, d, J=2.3Hz, H-2), 7.78 (1H, dd, J=2.3Hz, J=9.5Hz, H-4), 7.04 (2H,  d, J=9.0Hz,  H-’,5’,  .0 (2H, br s, NH2), 6.38 (1H, d, J=9.5Hz, H-5), 3.85 (3H, s, OCH3). MS ESI (m/z): 285 [M-H]-.  N'-((2,4-Dimethoxybenzoyl)oxy)-6-oxo-1,6-dihydropyridine-3-carboximidamide 44c:  According to the procedure for 44a, 2,4-dimethoxybenzoyl fluoride 43c (0.30 g, 1.64 mmol) was reacted with amidoxime 28 (0.25g, 1.64 mmol). The crude product was silica flash column chromatographed (gradient elution, 3-10% MeOH in CH2Cl2) to afford 44c as an off white solid (0.37 g, 71%). 1H-NMR (400 MHz, DMSO -d6):  11.92 (1H, s, N-H), 7.86 (1H, d, J=2.3Hz, H-2), 7.77 (1H, dd, J=2.3Hz, J=9.5Hz, H-4), 7.46 (1H, d, J=8.1Hz, H-”, . 1H, d, - .Hz, H-”, 6.64 (1H, dd, J=2.2Hz, J=8.1Hz, H-5”, 6.68 (2H, br s, NH2), 6.39 (1H, d, J=9.5Hz, H-5), 3.87 (3H, s, OCH3), 3.76 (3H, s, OCH3). MS ESI (m/z): 316 [M-H]-.  N'-((4-Chlorobenzoyl)oxy)-6-oxo-1,6-dihydropyridine-3-carboximidamide 44d:  174  According to the procedure for 44a, 4-chlorobenzoyl fluoride 43d (0.26 g, 1.64 mmol) was reacted with amidoxime 28 (0.25g, 1.64 mmol). The crude product was silica flash column chromatographed (gradient elution, 2-8% MeOH in CH2Cl2) to afford 44d as an off white solid (0.37 g, 79%). 1H-NMR (400 MHz, DMSO -d6):  11.98 (1H, s, N-H), 8.18 (2H, d, J=8.9Hz, H-’,’, 7.87 (1H, d, J=2.4Hz, H-2), 7.77 (1H, dd, J=2.4Hz, J=9.5Hz, H-4), 7.60 (2H,  d, J=8.9Hz,  H-’,5’,  .9 (2H, br s, NH2), 6.39 (1H, d, J=9.5Hz, H-5). MS ESI (m/z): 290 [M-H]-.  N'-((4-Methylbenzoyl)oxy)-6-oxo-1,6-dihydropyridine-3-carboximidamide 44e:  According to the procedure for 44a, 4-methylbenzoyl fluoride 43e (0.23 g, 1.66 mmol) was reacted with amidoxime 28 (0.25g, 1.66 mmol). The crude product was silica flash column chromatographed (gradient elution, 2-6% MeOH in CH2Cl2) to afford 44e as a pale yellow solid (0.30 g, 69%). 1H-NMR (400 MHz, DMSO -d6):  11.95 (1H, s, N-H), 8.05 (2H, d, J=8.5Hz, H-’,’, 7.87 (1H, d, J=2.4Hz, H-2), 7.78 (1H, dd, J=2.4Hz, J=9.6Hz, H-4), 7.33 (2H,  d, J=8.5Hz,  H-’,5’,  .1 (2H, br s, NH2), 6.39 (1H, d, J=9.6Hz, H-5), 2.39 (3H, s, CH3). MS ESI (m/z): 270 [M-H]-.   6-Oxo-N'-((4-(trifluoromethyl)benzoyl)oxy)-1,6-dihydropyridine-3-carboximidamide 44f:  According to the procedure for 44a, 4-(trifluoromethyl)benzoyl fluoride 43f (0.32 g, 1.66 mmol) was reacted with amidoxime 28 (0.25g, 1.66 mmol).  The crude product was silica flash column chromatographed (gradient elution, 2-8% MeOH in CH2Cl2) to afford 44f as an off white solid (0.40 g, 75%). 175  1H-NMR (400 MHz, DMSO -d6):  12.03 (1H, s, N-H), 8.36 (2H, d, J=8.3Hz, H-’,’, 7.93-7.86 (3H, m, H-2,’,5’, .8 (1H, dd, J=2.4Hz, J=9.5Hz, H-4), 6.95 (2H, br s, NH2), 6.40 (1H, d, J=9.5Hz, H-5). MS ESI (m/z): 324 [M-H]-. 5-(5-Phenyl-1,2,4-oxadiazol-3-yl)pyridin-2(1H)-one 45a:   N'-(Benzoyloxy)-6-oxo-1,6-dihydropyridine-3-carboximidamide 44a (0.13 g, 0.5 mmol) was dissolved in dioxane (4 mL) and irradiated for 20 minutes at 100 oC under microwave conditions. The solvent was then evaporated and the residue was silica flash column chromatographed (gradient elution, 2-10% MeOH in CH2Cl2) to give compound 45a as a white solid (0.07 g, 58%). 1H-NMR (400 MHz, DMSO -d6): see Table 26. MS ESI (m/z): 238 [M-H]-, HRMS: m/z calculated for C13H9N3O2-: 238.06220, found: 238.0621. 5-(5-(4-Methoxyphenyl)-1,2,4-oxadiazol-3-yl)pyridin-2(1H)-one 45b:  Following the procedure for 45a, compound 44b (0.14 g, 0.5 mmol) was converted to 45b, obtained as a white solid (0.08 g, 61%) after silica flash column chromatography (gradient elution, 2-10% MeOH in CH2Cl2). 1H-NMR (400 MHz, DMSO -d6): see Table 26. MS ESI (m/z): 268 [M-H]-.    176  5-(5-(2,4-Dimethoxyphenyl)-1,2,4-oxadiazol-3-yl)pyridin-2(1H)-one 45c:  Following the procedure for 45a, compound 44c (0.16 g, 0.5 mmol) was converted to 45c, obtained as a white solid (0.08 g, 53%) after silica flash column chromatography (gradient elution, 2-12% MeOH in CH2Cl2).  1H-NMR (400 MHz, DMSO -d6): see Table 26. MS ESI (m/z): 298 [M-H]-.  5-(5-(4-Chlorophenyl)-1,2,4-oxadiazol-3-yl)pyridin-2(1H)-one 45d:  Following the procedure for 45a, compound 44d (0.15 g, 0.51 mmol) was converted to 45d, obtained as an off white solid (0.05 g, 38%) after silica flash column (gradient elution, 2-6% MeOH in CH2Cl2). 1H-NMR (400 MHz, DMSO -d6): see Table 26. MS ESI (m/z): 272 [M-H]-, HRMS: m/z calculated for C13H8ClN3O2-: 272.02323, found: 272.02344.  5-(5-(p-Tolyl)-1,2,4-oxadiazol-3-yl)pyridin-2(1H)-one 45e:  177  Following the procedure for 45a, compound 44e (0.14 g, 0.51 mmol) was converted to 45e, obtained as a white solid (0.08 g, 63%) after silica flash column chromatography (gradient elution, 2-6% MeOH in CH2Cl2). 1H-NMR (400 MH z, DMSO-d6): see Table 26. MS ESI (m/z): 252 [M-H]-, HRMS: m/z calculated for C14H11N3O2-: 252.07785, found: 252.07779.  5-(5-(4-(Trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)pyridin-2(1H)-one 45f:  Following the procedure for 45a, compound 44f (0.16 g, 0.5 mmol) was converted to 45f, obtained as a white solid (0.04 g, 25%) after silica flash column (gradient elution, 2-6% MeOH in CH2Cl2) to afford 45f. 1H-NMR (400 MHz, DMSO -d6): see Table 26. MS ESI (m/z): 306 [M-H]-.  Table 26: NMR data of 45a-f   Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  45a   12.16 (1H, s, N-H), 8.12 (1H, d, J=2.4Hz, H-6), 7.97 (1H, dd, J=2.4Hz, J=9.6Hz, H-4), 6.53 (1H, d, J=9.6Hz, H-3)  8.16 (2H, m, H-”,”, 7.77-7.71 (1H, m, H-”, 7.69-7.63 (2H,  m, H-”,5”  178  Entry   Scaffold  1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  45b   12.14 (1H, s, N-H), 8.15-8.06* (3H, m,  H-6), 7.95 (1H, dd, J=2.5Hz, J=9.6Hz, H-4), 6.52 (1H, d, J=9.6Hz, H-3) 8.15-8.06* (3H, m, H-”,”, 7.19 (2H, d, J=9.0Hz, H-”,5”, .88 (3H, s, OCH3)   45c    8.03-7.98 (1H, m, H-6), 7.58-7.53 (1H, m, H-4), 7.10-7.04 (1H, m, H-3)  7.50 (1H, d, J=8.1Hz, H-”, 6.75 (1H, d, J=2.2Hz, H-”, 6.70 (1H, dd, J=2.2Hz, J=8.1Hz, H-5”, 3.87 (3H, s, OCH3), 3.76 (3H, s, OCH3)   45e   12.13 (1H, s, N-H), 8.10 (1H, d, J=2.2Hz, H-6), 7.96 (1H, dd, J=2.2Hz, J=9.5Hz, H-4), 6.52 (1H, d, J=9.5Hz, H-3) 8.05 (2H, d, J=8.7Hz, H-”,”, .46 (2H, d, J=8.7Hz, H-”,5”, .42 (3H, s, CH3)  45f   12.19 (1H, s, N-H), 8.15 (1H, d, J=2.0Hz, H-6), 7.99 (1H, dd, J=2.0Hz, J=9.2Hz, H-4), 6.54 (1H, d, J=9.2Hz, H-3) 8.37 (2H, d, J=8.4Hz, H-”,”, .0 (2H, d, J=8.4Hz, H-”,5”   tert-Butyl 4-((3-methoxyphenyl)carbamoyl)piperazine-1-carboxylate 48a:  3-Methoxyphenyl isocayante 47a (0.26 g, 1.75 mmol) was added dropwise to a solution of N-Boc-piperazine 46 (0.33 g, 1.75 mmol) in CH2Cl2 (5 mL) under N2. The reaction mixture was stirred at ambient temperature for 6 h. After completion of the reaction (monitored by TLC) the mixture was poured onto water and extracted with CH2Cl2. The combined organic layer was dried over Na2SO4, filtered and concentrated in vacuo. The residue was silica flash column chromatographed (gradient elution, 5-15% EtOAc in hexane) to afford 48a as colourless oil (0.51 g, 88%). 1H-NMR (400 MHz, DMSO -d6):  8.53 (1H, br s, CONH), 7.14 (1H, t, J=2.4Hz, H-’, 7.11 (1H, d, J=8.2Hz, H-5’), 7.03 (1H, ddd, J=0.8Hz, J=2.4Hz, J=8.2Hz, H-’), 6.51 (1H, 179  ddd, J=0.8Hz, J=2.4Hz, J=8.2Hz, H-’), 3.70 (3H, s, OCH3), 3.45±3.32 (m, 8H, NCH2), 1.42 (9H, s, C(CH3)3). MS ESI (m/z): 334 [M-H]-.  tert-Butyl 4-((3-bromophenyl)carbamoyl)piperazine-1-carboxylate 48b:  Prepared according to the procedure for 48a, 3-bromophenyl isocyanate 47b (0.35 g, 1.75 mmol) was reacted with N-Boc-piperazine 46 (0.33 g, 1.75 mmol). The residue was silica flash column chromatographed (gradient elution, 5-10% EtOAc in hexane) to afford 48b as colourless oil (0.48 g, 71%). 1H-NMR (400 MHz, DMSO -d6):  8.73 (1H, br s, CONH), 7.78 (1H, t, J=2.1Hz, H-’, 7.43 (1H, ddd, J=0.8Hz, J=2.1Hz, J=8.1Hz, H-’), 7.19 (1H, t, J=8.1Hz, H-5’), 7.10 (1H, ddd, J=0.8Hz, J=2.1Hz, J=8.1Hz, H-’), 3.46±3.33 (m, 8H, NCH2), 1.42 (9H, s, C(CH3)3). MS ESI (m/z): 407 [M++2]+.  tert-Butyl 4-((3-chlorophenyl)carbamoyl)piperazine-1-carboxylate 48c:  Prepared according to the procedure for 48a, 3-chlorophenyl isocyanate 47c (0.27 g, 1.75 mmol) was reacted with N-Boc-piperazine 46 (0.33 g, 1.75 mmol).  The residue was silica flash column chromatographed (gradient elution, 5-10% EtOAc in hexane) to afford 48c as colourless oil (0.43 g, 72%). 1H-NMR (400 MHz, DMSO -d6):  8.74 (1H, br s, CONH), 7.64 (1H, t, J=2.1Hz, H-’, 7.38 (1H, ddd, J=0.8Hz, J=2.1Hz, J=8.1Hz, H-’), 7.25 (1H, t, J=8.1Hz, H-5’), 6.97 (1H, ddd, J=0.8Hz, J=2.1Hz, J=8.1Hz, H-’), 3.46±3.32 (m, 8H, NCH2), 1.42 (9H, s, C(CH3)3). tert-Butyl 4-((2,4-dimethoxyphenyl)carbamoyl)piperazine-1-carboxylate 48d:  180  Prepared according to the procedure for 48a, 2,4-dimethoxyphenyl isocyanate 47d (0.31 g, 1.75 mmol) was reacted with N-Boc-piperazine 46 (0.33 g, 1.75 mmol).  The residue was silica flash column chromatographed (gradient elution, 5-15% EtOAc in hexane) to afford 48d as colourless oil (0.49 g, 77%). 1H-NMR (400 MHz, DMSO -d6):  7.61 (1H, br s, CONH), 7.32 (1H, d, J=8.8Hz, H-’, 6.57 (1H, d, J=2.7Hz, H-’), 6.44 (1H, d, J=2.7Hz, J=8.8Hz, H-5’), 3.76 (3H, s, OCH3), 3.73 (3H, s, OCH3), 3.41±3.32 (m, 8H, NCH2), 1.42 (9H, s, C(CH3)3).  N-(3-Methoxyphenyl)piperazine-1-carboxamide 49a:  To a solution of 48a (0.4 g, 1.2 mmol) in dry CH2Cl2 (3 mL) was added TFA (3 mL). The solution was stirred at room temperature for 3 h. The mixture was evaporated in vacuo and the residue was dissolved in EtOAc, and washed with saturated aqueous NaHCO3. The organic layer was separated and the aqueous layer was extracted with EtOAc. The combined EtOAc extracts were dried over Na2SO4 and concentrated in vacuo. The residue was silica flash column chromatographed (gradient elution, 2-6% MeOH in CH2Cl2) to give 49a as a colourless semi-solid (0.2 g, 72%). 1H-NMR (400 MHz, DMSO -d6):  8.88 (1H, br s, NH), 8.70 (1H, br s, NH), 7.15 (1H, d, J=7.8Hz, H-5’), 7.12 (1H, t, J=2.2Hz, H-’, 7.03 (1H, ddd, J=0.8Hz, J=2.2Hz, J=7.8Hz, H-6’), 6.54 (1H, ddd, J=0.8Hz, J=2.2Hz, J=7.8Hz, H-’), 3.70 (3H, s, OCH3), 3.67±3.61 (m, 4H, NCH2), 3.18±3.10 (m, 4H, NCH2). MS ESI (m/z): 236 [M+H]+.  N-(3-Bromophenyl)piperazine-1-carboxamide 49b:  Prepared according to the procedure for 49a, compound 48b (0.42 g, 1.1 mmol) was reacted with TFA. The residue was silica flash column chromatographed (gradient elution, 2-4% MeOH in CH2Cl2) to give 49b as a colourless semi-solid (0.21 g, 68%). 181  1H-NMR (400 MHz, DMSO -d6):  8.97 (1H, br s, NH), 8.91 (1H, br s, NH), 7.77 (1H, t, J=2.1Hz, H-’, 7.43 (1H, ddd, J=0.8Hz, J=2.1Hz, J=8.1Hz, H-’), 7.21 (1H, t, J=8.1Hz, H-5’), 7.13 (1H, ddd, J=0.8Hz, J=2.1Hz, J=8.1Hz, H-’), 3.70±3.60 (m, 4H, NCH2), 3.20±3.09 (m, 4H, NCH2). MS ESI (m/z): 286 [M+2]+.  N-(3-Chlorophenyl)piperazine-1-carboxamide 49c:  Prepared according to the procedure for 49a, compound 48c (0.41 g, 1.2 mmol) was reacted with TFA. The residue was silica flash column chromatographed (gradient elution, 2-4% MeOH in CH2Cl2) to give 49c as a colourless semi-solid (0.19 g, 65%). 1H-NMR (400 MHz, DMSO -d6):  9.02 (1H, br s, NH), 8.94 (1H, br s, NH), 7.63 (1H, t, J=2.0Hz, H-’, 7.38 (1H, ddd, J=0.8Hz, J=2.0Hz, J=7.8Hz, H-’), 7.27 (1H, t, J=7.8Hz, H-5’), 7.13 (1H, ddd, J=0.8Hz, J=2.0Hz, J=7.8Hz, H-’), 3.70±3.60 (m, 4H, NCH2), 3.20±3.09 (m, 4H, NCH2). MS ESI (m/z): 240 [M+H]+.  N-(2,4-Dimethoxyphenyl)piperazine-1-carboxamide 49d:  Prepared according to the procedure for 49a, compound 48d (0.4 g, 1.1 mmol) was reacted with TFA. The residue was silica flash column chromatographed (gradient elution, 2-6% MeOH in CH2Cl2) to give 49d as a colourless semi-solid (0.21 g, 73%). 1H-NMR (400 MHz, DMSO -d6):  8.87 (1H, br s, NH), 7.85 (1H, br s, NH), 7.27 (1H, d, J=8.8Hz, H-’, 6.58 (1H, d, J=2.7Hz, H-’), 6.46 (1H, d, J=2.7Hz, J=8.8Hz, H-5’), 3.76 (3H, s, OCH3), 3.74 (3H, s, OCH3), 3.64±3.50 (m, 4H, NCH2), 3.15±3.07 (m, 4H, NCH2). MS ESI (m/z): 266 [M+H]+.   182  N-(3-Methoxyphenyl)-4-(6-oxo-1,6-dihydropyridine-3-carbonyl)piperazine-1-carboxamide 50a:  To a solution of 6-oxo-1,6-dihydropyridine-3-carboxylic acid 23 (0.1 g, 0.72 mmol) and N-(3-methoxyphenyl)piperazine-1-carboxamide 49a (0.17 g, 0.72 mmol) in DMF (5 mL) was added HOAt (0.12 g, 0.86 mmol), EDC (0.17 g, 0.86 mmol) and DIEA (0.19 g, 1.44 mmol). The mixture was stirred at room temperature for 12h. The solvent was evaporated in vacuo and azeotroped twice with CH2Cl2. The residue was silica flash chromatographed (gradient elution, 5-10% MeOH in CH2Cl2) to afford 50a as a colourless semisolid (0.14 g, 54%). 1H-NMR (400 MHz, DMSO -d6): 11.88 (1H, br s, N1’-H), 8.66 (1H, br s, NH), 7.62 (1H, d, J=2.4Hz, H-’, .51 1H, dd, -=2.4Hz, J=9.4Hz, H-’, .11 (1H, t, J=2.2Hz, H-”, .05 (1H, ddd, J=0.8Hz, J=2.2Hz, J=7.8Hz, H-”, .5 1H, ddd, - 0.Hz, - .Hz, - .Hz, H-”, .5 1H, d, - .Hz, H-5’, .74 (3H, s, OCH3), 3.58±3.44 (m, 8H, NCH2). MS ESI (m/z): 357 [M+H]+.  N-(3-Bromophenyl)-4-(6-oxo-1,6-dihydropyridine-3-carbonyl)piperazine-1-carboxamide 50b:  Prepared according to the procedure for 50a, compound 49b (0.2 g, 0.72 mmol) was reacted with acid 23 (0.1 g, 0.72 mmol). The residue was silica flash column chromatographed (gradient elution, 2-6% MeOH in CH2Cl2) to afford 50b as a colourless semisolid (0.12 g, 41%). 1H-NMR (400 MHz, DMSO -d6):  11.88 (1H, br s, N1’-H), 8.79 (1H, s, NH), 7.78 (1H, t, J=2.1Hz, H-”, 7.61 (1H, d, J=2.4Hz, H-’, 7.52 (1H, dd, J=2.4Hz, J=9.5Hz, H-’), 7.44 (1H, ddd, J=0.8Hz, J=2.1Hz, J=8.1Hz, H-”), 7.20 (1H, t, J=8.1Hz, H-5”), 7.11 (1H, ddd, J=0.8Hz, J=2.1Hz, J=8.1Hz, H-”), 6.36 (1H, d, J=9.5Hz, H-5’, 3.58±3.45 (m, 8H, NCH2). MS ESI (m/z): 407 [M+2] +.  183  N-(3-Chlorophenyl)-4-(6-oxo-1,6-dihydropyridine-3-carbonyl)piperazine-1-carboxamide 50c:  Prepared according to the procedure for 50a, compound 50c (0.17 g, 0.72 mmol) was reacted with acid 23 (0.1 g, 0.72 mmol). The residue was silica flash column chromatographed (gradient elution, 2-6% MeOH in CH2Cl2) to afford 50c as a colorless semisolid (0.1 g, 38%). 1H-NMR (400 MHz, DMSO -d6):  11.88 (1H, br s, N1’-H), 8.78 (1H, br s, NH), 7.64 (1H, t, J=2.2Hz, H-”, 7.61 (1H, d, J=2.4Hz, H-’, 7.52 (1H, dd, J=2.4Hz, J=9.4Hz, H-’), 7.39 (1H, ddd, J=0.8Hz, J=2.2Hz, J=8.4Hz, H-”), 7.26 (1H, t, J=8.4Hz, H-5”), 6.98 (1H, ddd, J=0.8Hz, J=2.2Hz, J=8.4Hz, H-”), 6.36 (1H, d, J=9.4Hz, H-5’, 3.60±3.45 (m, 8H, NCH2). MS ESI (m/z): 361 [M+H]+.  N-(2,4-Dimethoxyphenyl)-4-(6-oxo-1,6-dihydropyridine-3-carbonyl)piperazine-1-carboxamide 50d:  Prepared according to the procedure followed in 50a compound 50d (0.19 g, 0.72 mmol) was reacted with acid 23 (0.1 g, 0.72 mmol). The residue was silica flash column chromatographed (gradient elution, 2-10% MeOH in CH2Cl2) to afford 50d as a white semisolid (0.17 g, 61%). 1H-NMR (400 MHz, DMSO -d6):  11.89 (1H, br s, N1’-H), 8.65 (1H, br s, NH), 7.60 (1H, d, J=2.4Hz, H-’, 7.52 (1H, dd, J=2.4Hz, J=9.4Hz, H-’), 7.27 (1H, d, J=8.8Hz, H-6"), 6.57 (1H, d, J=2.6Hz, H-”), 6.45 (1H, dd, J=2.6Hz, J=8.8Hz, H-5”), 6.36 (1H, d, J=9.4Hz, H-5’, 3.77 (3H, s, OCH3), 3.71 (3H, s, OCH3), 3.57±3.41 (m, 8H, NCH2). MS ESI (m/z): 387 [M+H]+. 1-Methyl-5-nitropyridin-2(1H)-one 52:  184  To a solution of 5-nitropyridin-2(1H)-one 51 (2.0 g, 14.28 mmol) in DMF (25 mL) was added NaH (0.74 g, 18.56 mmol) at 0 oC, and the suspension was stirred at room temperature for 1h. CH3I (2.2 g, 15.71 mmol) was then added dropwise at 0 oC, and the mixture was allowed to warm to room temperature and stirred for 6h. Upon reaction completion (monitored by TLC) the solvent was evaporated in vacuo. The residue was dissolved in EtOAc and washed with saturated NaHCO3 and water. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was silica flash column chromatographed (gradient elution, 0-4% MeOH in CH2Cl2) to afford 52 as a pale yellow solid (1.41 g, 64%).  1H-NMR (400 MHz, DMSO -d6):  9.20 (1H, d, J=2.9Hz, H-6), 8.13 (1H, dd, J=2.9Hz, J=9.4Hz, H-4), 6.47 (1H, d, J=9.4Hz, H-3), 3.55 (3H, s, CH3). MS ESI (m/z): 153 [M-H]-.  5-Aminopyridin-2(1H)-one 53:  A mixture of 51 (2.0 g, 14.28 mmol) and Pd/C (500mg, 10% w/w) in ethanol (50 mL) was degassed and purged with N2. The mixture was then hydrogenated at 1 atmosphere pressure for 12 h at room temperature. The mixture was then filtered through a small pad of celite, and concentrated.  The residue was silica flash column chromatographed (gradient elution, 10-20% MeOH in CH2Cl2) to give 53 as a white solid (1.07 g, 68%). 1H-NMR (400 MHz, DMSO -d6): 10.55 (1H, s, N1-H), 7.03 (1H, dd, J=2.9Hz, J=9.4Hz, H-4), 6.74 (1H, d, J=2.9Hz, H-6), 6.21 (1H, d, J=9.4Hz, H-3), 4.25 (2H, s, NH2). MS ESI (m/z): 111 [M+H]+.  1-Methyl-5-nitropyridin-2(1H)-one 54:  Prepared following the procedure for 53, compound 52 (2.2 g, 14.28 mmol) was hydrogenated. The crude mixture was silica flash column chromatographed (gradient elution, 5-10% MeOH in CH2Cl2) to afford 54 as a white solid (1.3 g, 74%). 1H-NMR (400 MHz, DMSO -d6):  7.04 (1H, dd, J=2.9Hz, J=9.4Hz, H-4), 6.38 (1H, d, J=2.9Hz, H-6), 6.23 (1H, d, J=9.4Hz, H-3), 4.31 (2H, s, NH2), 3.32 (3H, s, CH3). MS ESI (m/z): 125 [M+H]+. 185  1-Benzoyl-3-(6-oxo-1,6-dihydropyridin-3-yl) thiourea 55   To a solution of 53 (1.0 g, 9.08 mmol) in acetonitrile (12 mL) was added benzoyl isothiocyanate (1.34 mL, 9.97 mmol). The mixture was stirred at ambient temperature for 6h. After completion of reaction (TLC monitoring) the precipitate was filtered, washed with acetonitrile, and dried to afford the product 55 as a brick red solid (1.86 g, 75%). 1H-NMR (400 MHz, DMSO -d6):  12.01 (1H, br s, NH), 11.64 (2H, br s, NH), 8.01-7.92 (2H, m, H-2”,6”), 7.70-7.62 (2H, m, H-4’, ”), 7.58-7.49 (3H, m, H-2’,3”,5”), 6.36 (1H, d, J=9.4Hz, H-5’). MS ESI (m/z): 272 [M-H]-.  1-Benzoyl-3-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl) thiourea 56:  Prepared according to the procedure for 55, compound 54 (1.13 g, 9.08 mmol) was reacted with benzoyl isothiocyanate (1.34 mL, 9.97 mmol) to afford 56 as a brick red solid (2.04 g, 78%). 1H-NMR (400 MHz, DMSO -d6): 12.01 (1H, br s, NH), 11.69 (1H, br s, NH), 7.99-7.94 (3H, m, H-4’,2”,6”), 7.70-7.63 (1H, m, H-”), 7.58-7.50 (3H, m, H-2’,3”,5”), 6.40 (1H, d, J=9.5Hz, H-5’), 3.43 (3H, s, CH3). MS ESI (m/z): 286 [M-H]-.  1-(6-Oxo-1,6-dihydropyridin-3-yl)thiourea 57:   To a suspension of 55 (1.0 g, 3.66 mmol) in MeOH (10 mL) was added aqueous NaOH (0.1 g in 1 mL H2O) at room temperature. The reaction mixture was heated to 70 oC for 2h. After completion of reaction (TLC monitoring), the precipitated compound was separated and 186  washed on the filter with water and dried under high vacuum to give 57 as a white solid (0.43 g, 70%). The obtained product was taken to the next step without further purification. 1H-NMR (400 MHz, DMSO -d6): 11.47 (1H, br s, NH), 9.09 (1H, br s, NH), 7.67-7.04 (4H, m, H-’,’, NH2), 6.30 (1H, d, J=8.9Hz, H-5’. MS ESI (m/z): 168 [M-H]-.  1-(1-Methyl-6-oxo-1,6-dihydropyridin-3-yl)thiourea 58:  Prepared according to the procedure followed in 57, treatment of compound 56 (1.05 g, 3.66 mmol) with aqueous NaOH afforded 58 as a white solid (0.5 g, 75%). 1H-NMR (400 MHz, DMSO -d6):  9.09 (1H, s, NH), 8.14-6.89 (4H, m, H-’,’, NH2), 6.33 (1H, d, J=8.9Hz, H-5’, 3.38 (3H, s, CH3). MS ESI (m/z): 182 [M-H]-.  Ethyl 2-((6-oxo-1,6-dihydropyridin-3-yl)amino)thiazole-4-carboxylate 61:   To a suspension of 1-(6-oxo-1,6-dihydropyridin-3-yl) thiourea 57 (1.5 g, 8.87 mmol) in EtOH (15 ml), ethyl bromopyruvate  (1.11 mL, 8.87 mmol) was added, and the mixture was heated at 90 oC for 3 h. After cooling to room temperature, the solvent was removed under reduced pressure. The resulting crystalline solid was collected by filtration, washed with cold ethanol and dried to give 61 as an off white solid (1.6 g, 68%). The obtained product was taken forward to the next step without further purification. 1H-NMR (400 MHz, DMS O-d6):  10.26 (1H, br s, NH), 8.30 (1H, d, J=2.3Hz, H-’, 7.75 (1H, s, H-5), 7.62 (1H, dd, J=2.3Hz, J=9.5Hz, H-’, 6.63 (1H, d, J=9.5Hz, H-5’, 4.25 (2H, q, J=7.1Hz, CH2), 1.28 (t, 3H, J=7.1Hz, CH3). MS ESI (m/z): 266 [M+H]+. Ethyl 2-((1-methyl-6-oxo-1,6-dihydropyridin-3-yl)amino)thiazole-4-carboxylate 62:  187  Prepared according to the procedure followed in 61 compound 58 (1.63 g, 8.87 mmol) was converted to tiazole 62, isolated as an off white solid (1.81 g, 73%). 1H-NMR ( 400 MHz, DMSO-d6):  9.91 (1H, br s, NH), 8.21 (1H, d, J=2.4Hz, H-’, 7.70 (1H, s, H-5), 7.46 (1H, dd, J=2.4Hz, J=9.4Hz, H-’, 6.46 (1H, d, J=9.4Hz, H-5’, 4.24 (2H, q, J=7.1Hz, CH2), 3.43 (3H, s, NCH3), 1.28 (t, 3H, J=7.1Hz, CH3). MS ESI (m/z): 279 [M+H]+. 2-((6-Oxo-1,6-dihydropyridin-3-yl)amino)thiazole-4-carboxylic acid 63:   To a stirred suspension of 61 (1.0 g, 3.77 mmol) in ethanol (10ml), sodium hydroxide (0.45g, 11.31 mmol) in water (5 mL) was added. The mixture was heated at 90 oC for 12 h. The mixture was then concentrated in vacuo, and the residue was dissolved in water (10 mL), and the pH was adjusted to PH-4 by careful addition of 1N HCl. The precipitate was isolated by filtration, washed by water then MeOH, and dried in vacuo to afford the compound 63 as a brick red solid (0.63 g, 71%). The obtained product was taken forward to the next step without further purification.      1H-NMR (400 MHz, DMSO -d6):  12.07 (1H, br s, COOH), 9.98 (1H, br s, NH), 8.15 (1H, d, J=2.8Hz, H-’, 7.65 (1H, s, H-5), 7.45 (dd, 1H, J=2.8Hz, J=9.7Hz, H-’, 6.41 (1H, d, J=9.7Hz, H-5’. MS ESI (m/z): 236 [M-H]-, HRMS: m/z calculated for C9H7N3O3S-: 236.01354, found: 236.01328. 2-((1-Methyl-6-oxo-1,6-dihydropyridin-3-yl)amino)thiazole-4-carboxylic acid 64:   Prepared according to the procedure for 63 compound 62 (1.05 g, 3.77 mmol) was hydrolyzed to afford 64 as a brick red solid (0.69 g, 73%). 1H-NMR (400 MHz, DMSO -d6):  12.65 (1H, br s, COOH), 9.83 (1H, br s, NH), 8.19 (1H, d, J=2.5Hz, H-’, 7.64 (1H, s, H-5), 7.45 (dd, 1H, J=2.5Hz, J=9.5Hz, H-’, 6.43 (1H, d, J=9.5Hz, H-5’, 3.43 (3H, s, NCH3). MS ESI (m/z): 250 [M-H]-.  188  2-((6-Oxo-1,6-dihydropyridin-3-yl)amino)-N-phenylthiazole-4-carboxamide 65a:    To a solution of the acid 63 (0.07 g, 0.3 mmol) and aniline (0.044 g, 0.48 mmol) in DMF (5 mL), was added HOAt (0.04 g, 0.32 mmol), EDC (0.06 g, 0.32 mmol) and DIEA (0.12 g, 0.9 mmol). The mixture was stirred at ambient temperature for 24h, and then concentrated in vacuo.  The residue was dry-loaded onto a silica column and column chromatographed (gradient elution, 2-10% MeOH in CH2Cl2) to afford 65a as a pale yellow solid (0.04 g, 44%). 1H-NMR (400 MHz, DMSO -d6): see Table 27. 13C NMR (100MHz, DMSO -d6): 164.5, 160.8, 159.7, 146.3, 138.8, 136.2, 135.0, 129.1, 128.8, 125.6, 124.3, 122.7, 120.8, 120.0, 114.4. MS ESI (m/z): 311 [M-H]-, HRMS: m/z calculated for C15H12N4O2S-: 311.06082, found: 311.06113.  2-((6-Oxo-1,6-dihydropyridin-3-yl)amino)-N-(p-tolyl)thiazole-4-carboxamide 65b:  Following the procedure for 65a, p-toluidine (0.05 g, 0.48 mmol) was reacted with acid 63 (0.07 g, 0.3 mmol). The crude product mxture was separated by silica flash column chromatography (gradient elution, 2-10% MeOH in CH2Cl2) to afford 65b as a beige solid (0.07 g, 46%). 1H-NMR (400 MHz, DMSO -d6): see Table 27.  MS ESI (m/z): 325 [M-H]-, HRMS: m/z calculated for C16H14N4O2S-: 325.07647, found: 325.07657.  N-(4-Methoxyphenyl)-2-((6-oxo-1,6-dihydropyridin-3-yl)amino)thiazole-4-carboxamide 65c:  189  Following the procedure for 65a, p-anisidine (0.06 g, 0.48 mmol) was reacted with acid 63 (0.07 g, 0.3 mmol). The crude product mxture was separated by silica flash column chromatography (gradient elution, 2-12% MeOH in CH2Cl2) to afford 65c as a white solid (0.076 g, 48%). 1H-NMR (400 MHz, DMSO -d6): see Table 27. MS ESI (m/z): 341 [M-H]-, HRMS: m/z calculated for C16H14N4O3S-: 341.07138, found: 341.07138.  N-(2,3-Dimethylphenyl)-2-((6-oxo-1,6-dihydropyridin-3-yl)amino)thiazole-4-carboxamide 65d:  Following the procedure for 65a, 2,3-dimethylaniline (0.06 g, 0.5 mmol) was reacted with acid 63 (0.073 g, 0.31 mmol). The crude product mxture was separated by silica flash column chromatography (gradient elution, 2-10% MeOH in CH2Cl2) to afford 65d as a beige solid (0.095 g, 56%). 1H-NMR (400 MHz, DMSO -d6): see Table 27. MS ESI (m/z): 339 [M-H]-, HRMS: m/z calculated for C17H16N4O2S-: 339.09212, found: 339.09213.  N-(3,5-Dimethoxyphenyl)-2-((6-oxo-1,6-dihydropyridin-3-yl)amino)thiazole-4-carboxamide 65e:  Following the procedure for 65a, 3,5-dimethoxyaniline (0.076 g, 0.5 mmol) was reacted with acid 63 (0.073 g, 0.31 mmol). The crude product mxture was separated by silica flash column chromatography (gradient elution, 4-14% MeOH in CH2Cl2) to afford 65e as a pale beige solid (0.1 g, 55%). 1H-NMR (400 MHz, D MSO-d6): see Table 27. 13C NMR (100MHz, DMSO -d6): 164.5, 160.9, 160.8, 159.7, 146.2, 140.5, 136.2, 132.5, 125.5, 122.6, 120.1, 117.2, 114.5, 99.0, 96.2, 55.6, 54.0.  190  MS ESI (m/z): 371 [M-H]-, HRMS: m/z calculated for C17H16N4O4S-: 371.08195, found: 371.08191.  N-(4-(Dimethylamino)phenyl)-2-((6-oxo-1,6-dihydropyridin-3-yl)amino)thiazole-4-carboxamide 65f:  Following the procedure for 65a, N,N-dimethyl-p-phenylenediamine (0.076 g, 0.5 mmol) was reacted with acid 63 (0.073 g, 0.31 mmol). The crude product mxture was separated by silica flash column chromatography (gradient elution, 2-8% MeOH in CH2Cl2) to afford 65f as dark green solid (0.08 g, 47%). 1H-NMR (400 MHz, DMSO -d6): see Table 27. MS ESI (m/z): 354 [M-H]-, HRMS: m/z calculated for C17H17N5O2S-: 354.10302, found: 354.10336.  2-((6-Oxo-1,6-dihydropyridin-3-yl)amino)-N-(pyridin-3-yl)thiazole-4-carboxamide 65g:  Following the procedure for 65a, 3-aminopyridine (0.044 g, 0.48 mmol) was reacted with acid 63 (0.07 g, 0.3 mmol). The crude product mxture was separated by silica flash column chromatography (gradient elution, 4-15% MeOH in CH2Cl2) to afford 65g as a white solid (0.046 g, 31%). 1H-NMR (400 MHz, DMSO -d6): see Table 27. MS ESI (m/z): 312 [M-H]-, HRMS: m/z calculated for C14H11N5O2S-: 312.05607, found: 312.05637.  N-(6-Methoxypyridin-3-yl)-2-((6-oxo-1,6-dihydropyridin-3-yl)amino)thiazole-4-carboxamide 65h:  Following the procedure for 65a, 5-amino-2-methoxypyridine (0.06 g, 0.48 mmol) was reacted with acid 63 (0.07 g, 0.3 mmol). The crude product mxture was separated by silica 191  flash column chromatography (gradient elution, 4-15% MeOH in CH2Cl2) to afford 65h as pale beige solid (0.05 g, 30%). 1H-NMR (400  MHz, DMSO-d6): see Table 27. 13C NMR (100MHz, DMSO -d6): 164.5, 160.8, 160.5, 159.9, 146.0, 139.7, 136.2, 133.3, 129.8, 125.6, 122.8, 120.0, 114.5, 110.4, 53.9. MS ESI (m/z): 342 [M-H]-, HRMS: m/z calculated for C15H13N5O3S-: 342.06663, found: 342.06683.  2-((6-Oxo-1,6-dihydropyridin-3-yl)amino)-N-(quinolin-6-yl)thiazole-4-carboxamide 65i:  Following the procedure for 65a, 6-aminoquinoline (0.07 g, 0.48 mmol) was reacted with acid 63 (0.07 g, 0.3 mmol). The crude product mxture was separated by silica flash column chromatography (gradient elution, 4-15% MeOH in CH2Cl2) to afford 65j as a pale beige solid (0.03 g, 18%). 1H-NMR (400 MHz, DMSO -d6): see Table 27. MS ESI (m/z): 362 [M-H]-, HRMS: m/z calculated for C18H13N5O2S-: 362.07172, found: 362.07202.  N-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-((6-oxo-1,6-dihydropyridin-3-yl)amino)thiazole-4-carboxamide 65j:  Following the procedure for 65a, 1,4-benzodioxan-6-amine (0.07 g, 0.48 mmol) was reacted with acid 63 (0.07 g, 0.3 mmol). The crude product mxture was separated by silica flash column chromatography (gradient elution, 2-10% MeOH in CH2Cl2) to afford 65j as an off white solid (0.06 g, 35%). 1H-NMR (400 MHz, DMSO -d6): see Table 27. 13C NMR (100MHz, DMSO-d6): 164.4, 160.7, 159.4, 146.4, 143.4, 140.2, 136.2, 132.5, 125.5, 122.6, 120.1, 117.2, 114.0, 109.8, 64.7, 64.4. MS ESI (m/z): 369 [M-H]-, HRMS: m/z calculated for C17H14N4O4S-: 369.06630, found: 369.06662.  192  N-(1H-Indol-5-yl)-2-((6-oxo-1,6-dihydropyridin-3-yl)amino)thiazole-4-carboxamide 65k:  Following the procedure for 65a, 6-aminoindole (0.06 g, 0.48 mmol) was reacted with acid 63 (0.07 g, 0.3 mmol). The crude product mxture was separated by silica flash column chromatography (gradient elution, 4-15% MeOH in CH2Cl2) to afford 65k as a pale brown solid (0.03 g, 19%). 1H-NMR (400 MHz, DMSO -d6): see Table 27. MS ESI (m/z): 350 [M-H]-, HRMS: m/z calculated for C17H13N5O2S-: 350.07172, found: 350.07196.  N-(1H-Benzo[d]imidazol-6-yl)-2-((6-oxo-1,6-dihydropyridin-3-yl)amino)thiazole-4-carboxamide 65l:  Following the procedure for 65a, 6-aminobenzimidazole (0.06 g, 0.48 mmol) was reacted with acid 63 (0.07 g, 0.3 mmol). The crude product mxture was separated by silica flash column chromatography (gradient elution, 4-15% MeOH in CH2Cl2) to afford 65l as a pale pink solid (0.02 g, 13%). 1H-NMR (400 MHz, DMSO -d6): see Table 27. MS ESI (m/z): 351 [M-H]-, HRMS: m/z calculated for C16H12N6O2S-: 351.06697, found: 351.06711.  N-(Benzo[d]thiazol-6-yl)-2-((6-oxo-1,6-dihydropyridin-3-yl)amino)thiazole-4-carboxamide 65m:  Following the procedure for 65a, 6-aminobenzothiazole (0.07 g, 0.48 mmol) was reacted with acid 63 (0.07 g, 0.3 mmol). The crude product mxture was separated by silica flash column chromatography (gradient elution, 2-8% MeOH in CH2Cl2) to afford 65m as an off white solid (0.06 g, 34%). 1H-NMR (400 MHz, DMSO -d6): see Table 27. 193  MS ESI (m/z): 368 [M-H]-, HRMS: m/z calculated for C16H11N5O2S2-: 368.02814, found: 368.02817.  N-Benzyl-2-((6-oxo-1,6-dihydropyridin-3-yl)amino)thiazole-4-carboxamide 65n:  Following the procedure for 65a, benzylamine (0.05 g, 0.48 mmol) was reacted with acid 63 (0.07 g, 0.3 mmol). The crude product mxture was separated by silica flash column chromatography (gradient elution, 2-8% MeOH in CH2Cl2) to afford 65n as a pale green solid (0.087 g, 56%). 1H-NMR (400 MHz, DMSO -d6): see Table 27. MS ESI (m/z): 325 [M-H]-, HRMS: m/z calculated for C16H14N4O2S-: 325.07647, found: 325.07666.  N-(1H-Indazol-5-yl)-2-((6-oxo-1,6-dihydropyridin-3-yl)amino)thiazole-4-carboxamide 65o:  Following the procedure for 65a, 5-aminoindazole (0.06 g, 0.47 mmol) was reacted with acid 63 (0.07 g, 0.3 mmol). The crude product mxture was separated by silica flash column chromatography (gradient elution, 2-14% MeOH in CH2Cl2) to afford 65o as a pale beige solid (0.03 g, 20%). 1H-NMR (400 MHz, DMSO-d6): see Table 27. MS ESI (m/z): 351 [M-H]-, HRMS: m/z calculated for C16H12N6O2S-: 351.06697, found: 351.0672.  N-(2-Methyl-2H-indazol-5-yl)-2-((6-oxo-1,6-dihydropyridin-3-yl)amino)thiazole-4-carboxamide 65p:  Following the procedure for 65a, 2-methyl-2H-indazol-5-amine (0.07 g, 0.48 mmol) was reacted with acid (0.07 g, 0.3 mmol). The crude product mxture was separated by silica flash 194  column chromatography (gradient elution, 2-10% MeOH in CH2Cl2) to afford 65p as a pale green solid (0.046 g, 26%). 1H-NMR (400 MHz, DMSO -d6): see Table 27. MS ESI (m/z): 365 [M-H]-, HRMS: m/z calculated for C17H14N6O2S-: 365.08262, found: 365.08252.  Table 27: NMR data of compounds 65a-p  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  65a   11.37 (1H, br s, N1’-H), 9.92 (1H, br s, C2-NH), 9.68 (1H, br s, CO-NH), 8.10 (1H, d, J=2.9Hz, H-’, 7.57 (1H, s, H-5) 7.55-7.32* (3H,  m, H-’), 6.40 (1H, d, J=9.6Hz, H-5’ 7.78-7.72 (2H, m, H-”,” .55-7.32* (3H,  m, H-”,5”, .11 (1H, t, J=7.4Hz, H-”   65b   11.32 (1H, br s, N1’-H), 9.93 (1H, br s, C2-NH), 9.61 (1H, s, CO-NH), 8.11 (1H, d, J=2.9Hz, H-’, 7.57 (1H, s, H-5), 7.53 (1H, dd, J=2.9Hz, J=9.6Hz, H-’, 6.40 (1H, d, J=9.6Hz, H-5’ 7.63 (2H, d, J=8.2Hz, H-”,”, 7.16 (2H, d, J=8.2Hz, H-”,5”, 2.28 (3H, s, CH3)     65c   11.30 (1H, br s, N1’-H), 9.91 (1H, br s, C2-NH), 9.60 (1H, s, CO-NH), 8.10 (1H, d, J=2.9Hz, H-’), 7.55-7.52 (2H, m, H-5,’ , 6.40 (1H, d, J=9.7Hz, H-5’) 7.65 (2H, d, J=9.1Hz, H-”,”, 6.93 (2H, d, J=9.1Hz, H-”,5”, 3.75 (3H, s, OCH3)  65d   11.34 (1H, br s, N1’-H), 9.97 (1H, br s, C2-NH), 9.41 (1H,br  s, CO-NH), 8.01 (1H, d, J=2.7Hz, H-’), 7.67-7.50* (3H,  m, H-5,’), 6.40 (1H, d, J=9.6Hz) 7.67-7.50* (3H, m, H-” , .11 (1H, t, J=7.7Hz, H-5”, .0 1H, d, J=7.7Hz, H-”, . H, s, CH3), 2.16 (3H, s, CH3)  195  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  65e   11.39 (1H, br s, N1’-H), 9.96 (1H, br s, C2-NH), 9.65 (1H, br s, CO-NH), 8.14 (1H, d, J=2.9Hz, H-’), 7.60 (1H, s, H-5), 7.51 (1H, dd, J=2.9Hz, J=9.6Hz, H-’), 6.41 (1H, d, J=9.6Hz, H-5’) 7.06 (2H, d, J=2.2Hz, H-”,”), 6.29 (1H, t, J=2.2Hz, H-4”), 3.74 (6H, s, 2 OCH3)   65f   11.30 (1H, br s, N1’-H), 9.91 (1H, br s, C2-NH), 9.60 (1H, br s, CO-NH), 8.10 (1H, d, J=2.9Hz, H-’), 7.61-7.45* (4H, m, H-5,’ , 6.40 (1H, d, J=9.7Hz, H-5’) 7.61-7.45* (4H, m, H-”,”, .72 (2H, d, J=9.1Hz, H-”,5”, . (6H, s, 2 CH3)    65g   11.34 (1H, br s, N1’-H), 9.99 (1H, br s, C2-NH), 9.95 (1H, br s, CO-NH), 8.13 (1H, d, J=2.9Hz, H-’, 7.64 (1H, s, H-5), 7.53 (1H, dd, J=2.9Hz, J=9.6Hz, H-’, 6.40 (1H, d, J=9.6Hz, H-5’ 8.91 (1H, d, J=2.3Hz, H-”, . (1H, dd, J=1.4Hz, J=4.7Hz, H-”, 8.20 (1H, ddd, J=1.4Hz, J=2.3Hz, J=8.2Hz, H-”, .0 1H, dd, J=4.7Hz, J=8.2Hz, H-5”   65h   11.34 (1H, br s, N1’-H), 9.90 (1H, br s, C2-NH), 9.83 (1H, br s, CO-NH), 8.11 (1H, d, J=2.9Hz, H-’, 7.59 (1H, s, H-5), 7.55 (1H, dd, J=2.9Hz, J=9.2Hz, H-’, 6.40 (1H, d, J=9.2Hz, H-5’ 8.50 (1H, d, J=2.7Hz, H-”, .05 (1H, dd, J=2.7Hz, J=8.9Hz, H-”, 6.84 (1H, d, J=8.9Hz, H-5”, . (3H, s, OCH3)    65i   11.34 (1H, br s, N1’-H), 10.14 (1H, br s, C2-NH), 10.03 (1H, br s, CO-NH), 8.09 (1H, d, J=2.9Hz, H-’, 7.59 (1H, s, H-5), 7.77-7.70* (2H, m, H-’, 6.43 (1H, d, J=9.2Hz, H-5’  8.68 (1H, dd, J=1.6Hz, J=4.2Hz, H-”, 8.58 (1H, d, J=9.0Hz, H-”,  8.47 (1H, dd, J=1.6Hz, J=8.6Hz, H-”, .0 1H, d, - .0Hz, H-”, 7.77-7.70* (2H, m,  H-5”, 7.46 (1H, dd, J=4.2Hz, J=8.6Hz, H-”   65j   11.32 (1H, br s, N1’-H), 9.91 (1H, br s, C2-NH), 9.55 (1H, br s, CO-NH), 8.10 (1H, d, J=2.9Hz, H-’), 7.67 (1H, s, H-5), 7.52 (1H, dd, J=2.9Hz, J=9.6Hz, H-’), 6.39 (1H, d, J=9.6Hz, H-5’) 7.37 (1H, d, J=2.5Hz H-5”,  .1 (1H, dd, J=2.5Hz, J=8.8Hz, H-”, 6.83 (1H, d, J=8.8Hz, H-”, . (4H, dd, J=5.1Hz, J=8.8Hz, 2 OCH2)  196  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  65k   11.34 (1H, br s, N1’-H), 10.02 (1H, br s, C2-NH), 9.51 (1H, br s, CO-NH), 8.15 (1H, d, J=2.7Hz, H-’), 7.55 (1H, s, H-5), 7.40-7.32* (3H, m, H-’), 6.44-6.38* (2H, m, H-5’) 11.07 (1H, br s, N1”-H) 7.95 (1H, s, H-”, .5 1H, d, - .Hz, H-” ), 7.40-7.32* (3H, m, H-”,” ), 6.44-6.38* (2H, m, H-”   65l   11.33 (1H, br s, N1’-H), 9.95 (1H, br s, C2-NH), 9.71 (1H, s, CO-NH), 8.14 (1H, d, J=2.7Hz, H-’), 7.59 (1H, s, H-5), 7.58-7.50* (2H, m, H-’), 6.41 (1H, d, J=9.2Hz, H-5’) 12.44 (1H, s, N1”-H) 8.18 (1H, s, H-”, .1 1H, s, H-” .5-7.50* (2H, m, H-”, .5 1H, d, J=8.6Hz, H-5”    65m    11.37 (1H, br s, N1’-H), 10.01 (1H, br s, C2-NH), 9.97 (1H, br s, CO-NH), 8.15 (1H, d, J=2.9Hz, H-’), 7.65 (1H, s, H-5), 7.55 (1H, dd, J=2.9Hz, J=9.6Hz, H-’), 6.41 (1H, d, J=9.6Hz, H-5’) 9.31 (1H, s, H-”, . 1H, d, J=2.1Hz, H-”, .0 1H, d, J=8.8Hz, H-”, . (1H, dd, J=2.1Hz, J=8.8Hz, H-5”  65n    11.22 (1H, br s, N1’-H), 9.82 (1H, br s, C2-NH), 8.57 (1H, t, J=6.3Hz, CO-NH), 7.99 (1H, d, J=2.8Hz, H-’), 7.58 (1H, dd, J=2.8Hz, J=9.6Hz, H-’), 7.44 (1H, s, H-5), 6.38 (d, 1H, J=9.6Hz, H-5’)  7.36-7.27 (4H, m, H-”,”,5”,”, 7.26-7.19 (1H, m, H-”, .51 1H, d, J=6.4Hz, CH2)  65o    11.37 (1H, br s, N1’-H), 9.94 (1H, br s, C2-NH), 9.74 (1H, br s, CO-NH), 8.13 (1H, d, J=2.8Hz, H-’), 7.59 (1H, s, H-5), 7.58-7.49* (2H,m, H-’), 6.41 (1H, d, J=9.6Hz, H-5’)  13.03 (1H, s, N1”-H), 8.21 (1H, d, J=1.9Hz, H-”, .0 1H, s, H-”, 7.62 (1H, dd, J=1.9Hz, J=8.9Hz, H-”, .5-7.49* (2H, m, H-”   65p   11.37 (1H, br s, N1’-H), 9.95 (1H, br s, C2-NH), 9.65 (1H, br s, CO-NH), 8.13 (1H, d, J=2.9Hz, H-’), 7.62-7.51* (3H, m, H-5), 7.46 (1H, dd, J=2.0Hz, J=9.6Hz, H-’), 6.42 (1H, d, J=9.6Hz, H-5’) 8.30 (1H, s, H-”, .0 1H, d, J=1.8Hz, H-” .-7.51* (3H, m, H-”, ”, .16 (3H, s, CH3)  197  N-(1H-Indazol-5-yl)-2-((1-methyl-6-oxo-1,6-dihydropyridin-3-yl)amino)thiazole-4-carboxamide 66o:  To a solution of the acid 64 (0.12 g, 0.5 mmol) and the 5-aminoindazole (0.07 g, 0.51 mmol) in DMF (5 mL), was added HOAt (0.074 g, 0.55 mmol), EDC (0.11 g, 0.55 mmol) and DIEA (0.16 g, 1.25 mmol). The mixture was stirred at ambient temperature for 24h. The solvent was removed in vacuo and the residue was silica flash column chromatographed (gradient elution, 2-12% MeOH in CH2Cl2) to afford 66o as a pale green solid (0.03 g, 18%). 1H-NMR (400 MHz, DMSO -d6):  13.03 (1H, s, N1”-H), 9.99 (1H, s, C2-NH), 9.89 (1H, s, CO-NH), 8.58 (1H, d, J=2.9Hz, H-’, . 1H, d, - 1.Hz, H-”, .0 1H, s, H-”, .5 (1H, dd, J=1.8Hz, J=8.9Hz, H-”, .1 1H, s, H-5), 7.43 (1H, dd, J=2.9Hz, J=9.6Hz, H-’, 7.53 (1H, d, J=8.9Hz, H-”, .5 1H, d, - .Hz, H-5’, .5 H, s, 11-CH3). 13C NMR (100MHz, DMSO -d6): 164.1, 160.4, 159.6, 146.5, 140.1, 137.6, 135.4, 134.0, 131.8, 128.9, 123.1, 122.0, 121.1, 114.5, 111.6, 110.5, 37.7. MS ESI (m/z): 365 [M-H]-, HRMS: m/z calculated for C17H14N6O2S-: 365.08262, found: 365.08279.  N-(2-Methyl-2H-indazol-5-yl)-2-((1-methyl-6-oxo-1,6-dihydropyridin-3-yl)amino)thiazole-4-carboxamide 66p:  Prepared according to the procedure for 66o, acid 64 was reacted with 2-methyl-2H-indazol-5-amine (0.073 g, 0.5 mmol). The residue was silica flash column chromatographed (gradient elution, 2-10% MeOH in CH2Cl2) to afford 66p as a dark green solid (0.042 g, 24%). 1H-NMR (400 MHz, DMSO -d6):  9.99 (1H, s, C2-NH), 9.80 (1H, s, CO-NH), 8.58 (1H, d, J=2.9Hz, H-’, .0 1H, s, H-”, . 1H, d, - 1.Hz, H-”, .1 1H, s, H-5), 7.54 (1H, d, J=8.9Hz, H-”, . 1H, dd, - 1.8Hz, J=8.9Hz, H-”, . 1H, dd, - .Hz, - .Hz, H-’, . 1H, d, - .Hz, H-5’, .15 H, s, 1”-CH3), 3.53 (3H, s, N1-CH3). 13C NMR (100MHz, DMSO -d6): 164.1, 160.4, 159.6, 146.5, 146.3, 135.4, 132.1, 129.0, 124.9, 122.4, 121.8, 121.75, 120.0, 117.5, 114.4, 110.1, 37.7. 198  MS ESI (m/z): 379 [M-H]-, HRMS: m/z calculated for C18H16N6O2S-: 379.09827, found: 379.09875.  Ethyl 2-aminothiazole-4-carboxylate 69:   To a suspension of thiourea 67 (5.0 g, 65.69 mmol) in ethanol (30 mL), was slowly added ethyl bromopyruvate 68 (8.25 mL, 65.69 mmol). The mixture was heated to 90 °C for 4 h. After reaction completion (monitored by TLC) the mixture was cooled to room temperature, and concentrated in vacuo. The crystalline solid was collected by filtration, washed with ethanol and dried. The crude product was recrystallized from ethylacetate/hexane to afford 69 (10.07 g, 89%) as an off white solid. 1H-NMR (400 MHz, DMSO -d6): 7.24 (1H, s, H-5), 7.45 (2H, br s, NH2), 4.19 (1H, q, J=7.1Hz, CH2), 1.25 (1H, t, J=7.1Hz, CH3). MS ESI (m/z): 173 [M+H]+.    Ethyl 2-bromothiazole-4-carboxylate 70:  Tert-Butyl nitrite (3.42 mL, 25.8 mmol) was added dropwise to a solution of ethyl 2-aminothiazole-4-carboxylate 69 (3 g, 17.22 mmol) and copper(II)-bromide (5.77 g, 25.86 mmol) in MeCN (75 mL) The mixture was heated at 80 °C for 2 h, then cooled and partitioned between CH2Cl2, 1N HCl, and water. The aqueous layer was further extracted with CH2Cl2 and the combined organic layers were dried over Na2SO4 and concentrated in vacuo The residue was silica flash column chromatographed (gradient elution, 5-15% EtOAc in hexane) to afford 70 as a yellow solid (2.3 g, 56%). 1H-NMR (400 MHz, DMSO -d6): 7.28 (1H, s, H-5), 4.21 (1H, q, J=7.1Hz, CH2), 1.25 (1H, t, J=7.1Hz, CH3). MS ESI (m/z): 238 [M+2]+.   tert-Butyl tert-butyl 5-nitro-1H-indole-1-carboxylate 71g-NO 2 (N 1-Boc):  199  To a stirred solution of 5-nitroindole (5g, 30.65 mmol) in CH2Cl2 (60 mL) were successively added Et3N (5.31 mL, 38.31 mmol), (Boc)2O (8.36 g, 38.31 mmol), and DMAP (0.38 g, 3.1 mmol). The mixture was stirred for 24 h at room temperature before addition of saturated aqueous NH4Cl and extraction with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (gradient elution, 1-3% MeOH in CH2Cl2) to give the product 71g-NO 2 (N 1-Boc) as a yellow solid (7.34g, 91%).  1H-NMR (400 MHz, DMSO-d6): 8.61 (1H, d, J=2.3Hz, H-4), 8.26-8.18 (2H, m, H-6,7), 7.90 (1H, d, J=3.5Hz, H-2), 6.96 (1H, d, J=3.5Hz, H-3), 1.64 (9H, s, C(CH3)3).  tert-Butyl 5-amino-1H-indole-1-carboxylate 71g ( N 1-Boc):   Palladium on carbon (0.8 g, 10% w/w) and NH2NH2.H2O (1.7 mL, 34 mmol) were added to a solution of tert-butyl 5-nitro-1H-indole-1-carboxylate 71g-NO 2 (N 1-Boc) (6.82 g, 26 mmol) in MeOH (70 mL). The mixture was heated under reflux for 3h, then filtered while hot through a bed of celite and washed with hot MeOH. The solvent was evaporated in vacuo and the residue was silica flash column chromatographed (gradient elution, 2-5 % MeOH in CH2Cl2) to afford 71g (N 1-Boc) as a white powder (4.92g, 81%).              1H-NMR (400 MHz, DMSO -d6): 7.70 (1H, d, J=8.6Hz, H-7), 7.46 (1H, d, J=3.7Hz, H-2), 6.70 (1H, d, J=2.3Hz, H-4), 6.61 (1H, dd, J=8.6Hz, J=2.3Hz, H-6), 6.45 (1H, d, J=3.5Hz, H-3),  4.86 (2H, br s, NH2), 1.56 (9H, s, C(CH3)3). MS ESI (m/z): 233 [M+H]+.  tert-Butyl 5-amino-1H-indazole-1-carboxylate 71h (N 1-Boc):    Following the procedure 71g (N 1-Boc), the nitro group in tert-butyl 5-nitro-1H-indazole-1-carboxylate 79 (6.6 g, 25 mmol) was reduced. The crude mixture was silica flash column chromatographed (gradient elution, 2-6% MeOH in CH2Cl2) to afford 71h (N 1-Boc) as an orange solid (4.3g, 74%). 200  1H-NMR (400 MHz, DMSO -d6): 8.11 (1H, s, H-3), 7.75 (1H, d, J=8.9Hz, H-7), 6.91 (1H, dd, J=2.2Hz, J=8.9Hz, H-6), 6.83 (1H, d, J=2.2Hz, H-4), 5.18 (2H, br s, NH2), 1.62 (s, 9H, C(CH3)3). MS ESI (m/z): 234 [M+H]+.  Ethyl 2-(pyridin-2-ylamino)thiazole-4-carboxylate 72a:  To a suspension of NaH (60% in a mineral oil, 0.54 g, 13.4 mmol) in dry THF (10 mL) 2-aminopyridine 71a (0.42 g, 4.47 mmol) was added. The mixture was stirred at 60 °C for 30 minutes. Then ethyl 2-bromothiazole-4-carboxylate 70 (1.05 g, 4.47 mmol) was added and the reaction was stirred at 60 °C for an additional 5h. After cooling, the mixture was quenched slowly with saturated aqueous NH4Cl and extracted with EtOAc. The combined organic extracts were dried over Na2SO4 and concentrated in vacuo. The oily residue was silica flash column chromatographed (gradient elution, 2-10% MeOH in CH2Cl2) to afford 72a as an off white solid (0.34 g, 31%). 1H-NMR (400 MHz, DMSO -d6): 9.64 (1H, br s, NH), 8.36 (1H, dd, 1H, J=1.3Hz, J=4.9Hz, H-’), 8.18 (1H, d, J=8.3Hz, H-’), 7.96 (1H, s, H-5), 7.87 (1H, dt, J=1.5Hz, J=8.3Hz, H-’), 7.19 (ddd, 1H, J=1.5Hz, J=4.9Hz, J=8.3Hz, H-5’), 4.56 (1H, q, J=7.2Hz, CH2), 1.41 (1H, t, J=7.2Hz, CH3). MS ESI (m/z): 250 [M+H]+.   Ethyl 2-((4-cyanophenyl)amino)thiazole-4-carboxylate 72b:   Following the protocol for 72a, the sodium anion of 4-aminobenzonitrile (0.53 g, 4.74 mmol) was reacted with bromothiazole 70 (1.05 g, 4.47 mmol). The crude product was silica flash column chromatographed (gradient elution, 2-10% MeOH in CH2Cl2) to afford 72b as an off white solid (0.34 g, 31%). 201  1H-NMR (400 MHz, DMSO -d6): 10.92 (1H, br s, NH), 7.90 (1H, s, H-5), 7.81 (2H, d, J=9.0Hz, H-’,5’), 7.77 (2H, d, J=9.0Hz, H-’,’, 4.27 (1H, q, J=7.2Hz, CH2), 1.29 (1H, t, J=7.2Hz, CH3).  MS ESI (m/z): 274 [M+H]+.  Ethyl 2-(pyrimidin-2-ylamino)thiazole-4-carboxylate 72c:   Following the protocol for 72a, the sodium anion of 2-aminopyrimidine (0.42 g, 4.47 mmol) was reacted with bromothiazole 70 (1.05 g, 4.47 mmol).  The crude product was silica flash column chromatographed (gradient elution, 2-10% MeOH in CH2Cl2) to afford 72c as an off white solid (0.27 g, 24%). 1H-NMR (400 MHz, DMSO -d6): 1H-NMR (400 MHz, DMSO -d6): 10.64 (1H, br s, NH), 8.64 (2H, d, J=4.9Hz, H-’,’, . 1H, s, H-5), 7.08 (1H, t, J=4.9Hz, H-5’, .5 (1H, q, J=7.2Hz, CH2), 1.41 (1H, t, J=7.2Hz, CH3). MS ESI (m/z): 251 [M+H]+.  Ethyl 2-((4-nitrophenyl)amino)thiazole-4-carboxylate 72d:   Following the protocol for 72a, the sodium anion of 4-nitroaniline (0.62 g, 4.47 mmol) was reacted with bromothiazole 70 (1.05 g, 4.47 mmol).  The crude product was silica flash column chromatographed (gradient elution, 2-8% MeOH in CH2Cl2) to afford 72d as an orange solid (0.27 g, 24%). 1H-NMR (400 MHz, DMSO -d6): 11.02 (1H, br s, NH), 8.21 (2H, d, J=9.0Hz, H-’,5’, 7.92 (1H, s, H-5), 7.82 (2H, d, J=9.0Hz, H-’,’, 4.25 (1H, q, J=7.2Hz, CH2), 1.28 (1H, t, J=7.2Hz, CH3).  MS ESI (m/z): 294 [M+H]+.    202  Ethyl 2-(phenylamino)thiazole-4-carboxylate 72e:  To a solution of aniline (0.51 g, 5.46 mmol) in iPrOH (10 mL) was added ethyl 2-bromothiazole-4-carboxylate 70 (0.86 g, 3.64 mmol) and pTosOH (0.62 g, 3.64 mmol). The mixture was stirred at 90 °C for 48h, then cooled, diluted with EtOAc, and washed with saturated NaHCO3 and water. The organic layer was dried over Na2SO4 and concentrated in vacuo. The oily residue was silica flash column chromatographed (gradient elution, 2-10% MeOH in CH2Cl2) to give 72e (0.4 g, 44%) as a white solid. 1H-NMR (400 MHz, DMSO -d6): 9.84 (1H, br s, NH), 7.90 (1H, s, H-5), 7.71-7.67 (2H, m, H-’,’), 7.31-7.250 (2H, m, H-’,5’), 7.01-6.94 (1H, m, H-’), 4.24 (1H, q, J=7.2Hz, CH2), 1.23 (1H, t, J=7.2Hz, CH3).  MS ESI (m/z): 249 [M+H]+.  1-Benzoyl-3-(pyridin-3-yl) thiourea 73f:  To a solution of 3-aminopyridine 71f (1.7 g, 18 mmol) in acetonitrile (20 mL) was added benzoyl isothiocyanate (2.37 mL, 18 mmol). The mixture was stirred at ambient temperature for 2h, and then concentrated. The residue was silica flash column chromatographed (gradient elution, 2-5% MeOH in CH2Cl2) to afford the product 73f as a pale red solid (3.6 g, 77%). MS ESI (m/z): 256 [M-H]-.  tert-Butyl 5-(3-benzoylthioureido)-1H-indole-1-carboxylate 73g (N 1-Boc):   To a solution of 71g (N 1-Boc) (4.18 g, 18 mmol) in acetonitrile (20 mL) was added benzoyl isothiocyanate (2.37 mL, 18 mmol). The mixture was stirred at ambient temperature for 2h, 203  and then concentrated. The residue was silica flash column chromatographed (gradient elution, 2-7% MeOH in CH2Cl2) to afford 73g (N 1-Boc) as pale red solid (5.05 g, 71%). 1H-NMR (400 MHz, DMSO -d6): 12.66 (1H, br s, NH), 11.59 (1H, br s, NH), 8.08 (1H, d, J=8.6Hz, H-7), 8.05-7.98 (3H, m, H-,’,’, 7.74 (1H, d, J=3.7Hz, H-2), 7.69 (1H, t, J=7.5Hz, H-’), 7.57 (2H, t, J=7.5Hz, H-’,5’), 7.52 (1H, dd, J=8.6Hz, J=2.3Hz, H-6), 6.78 (1H, d, J=3.5Hz, H-3),  1.56 (9H, s, C(CH3)3). MS ESI (m/z): 394 [M-H]-.  tert-Butyl 5-(3-benzoylthioureido)-1H-indazole-1-carboxylate 73h ( N 1-Boc):      To a solution of 71h (N 1-Boc) (4.2 g, 18 mmol) in acetonitrile (20 mL) was added benzoyl isothiocyanate (2.37 mL, 18 mmol). The mixture was stirred at ambient temperature for 2h, and then concentrated. The residue was silica flash column chromatographed (gradient elution, 2-8% MeOH in CH2Cl2) to afford 73h (N 1-Boc) as pale red solid (5.2 g, 73%). 1H-NMR (400 MHz, DMSO -d6): 12.66 (1H, br s, NH), 11.65 (1H, br s, NH), 8.46 (1H, s, H-3), 8.27 (1H, d, J=1.9Hz, H-4), 8.09 (1H, d, J=8.9Hz, H-7), 8.00 (2H, d, J=7.4Hz, H-’,’), 7.76 (1H, dd, J=1.9Hz, J=8.9Hz, H-6), 7.68 (1H, t, J=7.4Hz, H-’), 7.55 (2H, t, J=7.4Hz, H-’,5’), 1.66 (9H, s, C(CH3)3). MS ESI (m/z): 395 [M-H]-.  1-Benzoyl-3-(benzo[d]thiazol-6-yl) thiourea 73i:   To a solution of 71i (2.7 g, 18 mmol) in acetonitrile (20 mL) was added benzoyl isothiocyanate (2.37 mL, 18 mmol). The mixture was stirred at ambient temperature for 2h, 204  and then concentrated. The residue was silica flash column chromatographed (gradient elution, 2-8% MeOH in CH2Cl2) to afford 73i as yellow solid (3.8 g, 68%). 1H-NMR (400 MHz, DMS O-d6): 12.71 (1H, br s, NH), 11.67 (1H, br s, NH), 9.41 (1H, s, H-2), 8.57 (1H, d, J=1.9Hz, H-7), 8.11 (1H, d, J=8.9Hz, H-4), 8.00 (2H, d, J=7.4Hz, H-’,’), 7.74 (1H, dd, J=1.9Hz, J=8.9Hz, H-5), 7.68 (1H, t, J=7.4Hz, H-’), 7.55 (2H, t, J=7.4Hz, H-’,5’). MS ESI (m/z): 312 [M-H]-.  1-Benzoyl-3-(quinolin-6-yl) thiourea 73j:  To a solution of 71j (2.6 g, 18 mmol) in acetonitrile (20 mL) was added benzoyl isothiocyanate (2.37 mL, 18 mmol). The mixture was stirred at ambient temperature for 2h, and then concentrated. The residue was silica flash column chromatographed (gradient elution, 2-10% MeOH in CH2Cl2) to afford 73j as yellow solid (3.5 g, 63%). 1H-NMR (400 MHz, DMSO -d6): 12.83 (1H, br s, NH), 11.72 (1H, br s, NH), 8.92 (1H, dd, J=1.6Hz, J=4.2Hz, H-2), 8.46-8.38 (2H, m, Ar-H), 8.09-7.98 (4H, m, Ar-H), 7.70 (1H, t, J=7.4Hz, H-’), 7.61-7.53 (3H, m, Ar-H). MS ESI (m/z): 306 [M-H]-.    1-(Pyridin-3-yl)thiourea 74f:  To a solution of 73f (3.1 g, 12 mmol) in MeOH (30 mL) at room temperature was added LiOH (0.2 g) in water 2 mL. The reaction mixture was heated up to 75 oC for 1h.  After completion of reaction (TLC monitoring), the precipitate was separated, washed with water and dried in vacuo to give 74f as a white solid (1.26 g, 69%). 1H-NMR (400 MHz, DMSO -d6): 9.97 (1H, br s, NH), 8.94 (1H, d, J=2.3Hz, H-2), 8.35 (1H, dd, J=1.4Hz, J=5.1Hz, H-6), 8.19 (1H, ddd, J=1.4Hz, J=2.3Hz, J=8.4Hz, H-4), 7.54 (1H, d, J=5.1Hz, J=8.4Hz, H-5). MS ESI (m/z): 154 [M+H]+.  205  1-(1H-Indol-5-yl)thiourea 74g:  As described for 74f, intermediate 73g ( N 1-Boc) (4.35 g, 11 mmol) was hydrolyzed to afford 74g as an off white solid (1.5 g, 71%).  1H-NMR (400 MHz, DMSO -d6): 11.1 (1H, br s, NH), 9.49 (1H, br s, NH), 7.43-7.33 (3H, m, H-2,4,7), 6.93 (1H, dd, J=8.6Hz, J=2.3Hz, H-6), 6.44-6.39 (1H, m, H-3). MS ESI (m/z): 192 [M+H]+.  1-(1H-Indazol-5-yl)thiourea 74h:  As described for 74f , intermediate 73h (N 1-Boc) (4.75 g, 12 mmol) was hydrolyzed to afford 74h as an off white solid (1.73 g, 75%).  1H-NMR (400 MHz, DMSO -d6): 13.04 (1H, br s, NH), 9.61 (1H, s, NH), 8.04 (1H, s, H-3), 7.68 (1H, s, H-4), 7.49 (1H, d, J=8.8Hz, H-7), 7.23 (1H, dd, J=1.7Hz, J=8.8Hz, H-6). MS ESI (m/z): 193 [M+H]+.  1-(Benzo[d]thiazol-6-yl)thiourea 74i;  As described for 74f, intermediate 73i (3.76 g, 12 mmol) was hydrolyzed to afford 74i as an off white solid (1.58 g, 63%).  1H-NMR (400 MHz, DMSO -d6): 9.87 (1H, br s, NH), 9.31 (1H, s, H-2), 8.25 (1H, d, J=1.9Hz, H-7), 8.01 (1H, d, J=8.8Hz, H-4), 7.46 (1H, dd, J=1.9Hz, J=8.8Hz, H-5). MS ESI (m/z): 210 [M+H]+.  1-(Quinolin-6-yl)thiourea 74j:  206  As described for 74f, itermediate 63j (3.69 g, 12 mmol) was hydrolyzed to afford 74j as an off white solid (1.32 g, 54%).  1H-NMR (400 MHz, DMSO -d6): 9.97 (1H, br s, NH), 8.81 (1H, dd, J=1.6Hz, J=4.2Hz, H-2), 8.29 (1H, dd, J=1.7Hz, J=8.6Hz, H-4), 8.06 (1H, d, J=2.4Hz, H-5), 7.95 (1H, d, J=9.1Hz, H-8), 7.75 (1H, dd, J=2.4Hz, J=9.1Hz, H-7), 7.49 (1H, dd, J=4.2Hz, J=8.6Hz, H-3). MS ESI (m/z): 204 [M+H]+.  Ethyl 2-(pyridin-3-ylamino)thiazole-4-carboxylate 72f:  To a suspension of 1-(pyridin-3-yl)thiourea 74f (1.07 g, 7 mmol) in ethanol (12 mL), was added ethyl 2-bromopyruvate (0.88 mL, 7 mmol). The reaction was heated at 90 oC for 3h and then allowed to cool to room temperature. The solvent was removed in vacuo, and the residue was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to give ester 72f as an off white solid (1.57 g, 63%). 1H-NMR (400 MHz, DMSO -d6): 10.38 (1H, br s, NH), 9.04 (1H, d, J=2.3Hz, H-2’), 8.37 (1H, dd, J=1.4Hz, J=4.9Hz, H-’), 8.29 (1H, ddd, J=1.4Hz, J=2.3Hz, J=8.3Hz, H-’), 7.83 (1H, s, H-5), 7.61 (1H, d, J=4.9Hz, J=8.3Hz, H-5’), 4.28 (2H, q, J=7.1Hz, CH2), 1.29 (3H, t, J=7.1Hz, CH3). MS ESI (m/z): 248 [M-H]-.  Ethyl 2-((1H-indol-5-yl)amino)thiazole-4-carboxylate 72g:  Prepared according to 72f, compound 74g (1.33 g, 7 mmol) was reacted with ethyl 2-bromopyruvate (0.88 mL, 7 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í8% MeOH in CH2Cl2) to afford ester 72g as pale brown solid (1.43 g, 71%). 1H-NMR (400 MHz, DMSO -d6): 11.04 (1H, br s, N1’-H), 10.08 (1H, br s, C2-NH), 7.81 (1H, d, J=2.1Hz, H-’), 7.62 (1H, s, H-5), 7.36 (1H, d, J=8.5Hz, H-’), 7.33 (1H, t, J=3.1Hz, H-2’), 7.13 (1H, dd, J=2.1Hz, J=8.5Hz, H-’), 6.39 (1H, t, J=3.1Hz, H-’), 4.26 (2H, q, J=7.1Hz, CH2), 1.30 (3H, t, J=7.1Hz, CH3). 207  MS ESI (m/z): 286 [M-H]-.  Ethyl 2-((1H-indazol-5-yl)amino)thiazole-4-carboxylate 72h:   Prepared according to 72f, compound 74h (1.34 g, 7 mmol) was reacted with ethyl 2-bromopyruvate (0.88 mL, 7 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to afford ester 72h as an off white solid (1.23 g, 61%). 1H-NMR (400 MHz, DMSO -d6): 12.98 (1H, br s, N1’-H), 10.33 (1H, br s, C2-NH), 8.18 (1H, d, J=1.9Hz, H-’), 8.02 (1H, s, H-’), 7.72 (1H, s, H-5), 7.52 (1H, d, J=8.9Hz, H-’), 7.37 (1H, dd, J=1.9Hz, J=8.9Hz, H-’), 4.27 (2H, q, J=7.1Hz, CH2), 1.30 (3H, t, J=7.1Hz, CH3). MS ESI (m/z): 287 [M-H]-.  Ethyl 2-(benzo[d]thiazol-6-ylamino)thiazole-4-carboxylate 72i:  Prepared according to 72f, compound 74i (1.46 g, 7 mmol) was reacted with ethyl 2-bromopyruvate (0.88 mL, 7 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í6% MeOH in CH2Cl2) to afford ester 72i as a grrenish white solid (1.56 g, 73%). 1H-NMR (400 MHz, DMSO -d6): 10.70 (1H, br s, C2-NH), 9.22 (1H, s, H-’), 8.62 (1H, d, J=2.2Hz, H-’), 8.03 (1H, d, J=8.6Hz, H-’), 7.83 (1H, s, H-5), 7.60 (1H, dd, J=2.2Hz, J=8.6Hz, H-5’, 4.29 (2H, q, J=7.1Hz, CH2), 1.31 (3H, t, J=7.1Hz, CH3). MS ESI (m/z): 304 [M-H]-.  Ethyl 2-(quinolin-6-ylamino)thiazole-4-carboxylate 72j:  208  Prepared according to 72f, compound 74j (1.42 g, 7 mmol) was reacted with ethyl 2-bromopyruvate (0.88 mL, 7 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to afford ester 72j as a white solid (1.07 g, 51%). 1H-NMR (400 MHz, DMSO -d6): 11.19 (1H, br s, C2-NH), 9.05 (1H, dd, J=1.6Hz, J=4.2Hz, H-’), 8.85 (1H, d, J=8.6Hz, H-’), 8.67 (1H, d, J=2.4Hz, H-5’), 8.20 (1H, d, J=8.8Hz, H-’), 8.10 (1H, dd, J=2.4Hz, J=8.8Hz, H-’, 7.92 (1H, dd, J=4.2Hz, J=8.6Hz, H-’), 7.81 (1H, s, H-5), 4.31 (2H, q, J=7.1Hz, CH2), 1.34 (3H, t, J=7.1Hz, CH3). MS ESI (m/z): 298 [M-H]-.  2-(Pyridin-2-ylamino)thiazole-4-carboxylic acid 75a:  A solution of LiOH (0.29g, 12 mmol) in water (3mL) was added to a stirred suspension of ester 72a (1.0 g, 4 mmol) in MeOH (12ml). The mixture was stirred at room temperature for 12 h, and then concentrated in vacuo. The residue was dissolved in water (10 mL), and the pH was adjusted to 4 by careful addition of 1N HCl. The resulting precipitate was isolated by filtration, washed by water, and dried in vacuo to afford 75a as an off white solid (0.51 g, 58%).  1H-NMR (400 MHz, DMSO -d6): 12.66 (1H, br s, COOH), 11.61 (1H, br s, NH), 8.32 (1H, dd, J=1.5Hz, J=4.9Hz, H-’, .1 1H, d, - .Hz, H-’, . 1H, s, H-5), 7.74 (1H, dt, J=1.5Hz, J=8.3Hz, H-’, .0 ddd, 1H, - 1.5Hz, - .Hz, - .Hz, H-5’. MS ESI (m/z): 220 [M-H]-.  2-((4-Cyanophenyl)amino)thiazole-4-carboxylic acid 75b:  Prepared by the procedure for 75a, ester 72b (1.1 g, 4 mmol) was hydrolyzed to afford 75b as an off white solid (0.6 g, 61%).  1H-NMR (400 MHz, DMSO -d6): 12.95 (1H, br s, COOH), 11.07 (1H, br s, NH), 7.90 (1H, s, H-5), 7.84 (2H, d, J=9.0Hz, H-’,5’), 7.79 (2H, d, J=9.0Hz, H-’,’. MS ESI (m/z): 244 [M-H]-.  209  2-(Pyrimidin-2-ylamino)thiazole-4-carboxylic acid 75c:  Prepared by the procedure for 75a, ester 72c (1.0 g, 4 mmol) was hydrolyzed to afford 75c as an off white solid (0.47 g, 54%).  1H-NMR (400 MHz, DMSO -d6): 11.95 (1H, br s, NH), 8.67 (2H, d, J=4.9Hz, H-’,’), 7.91 (1H, s, H-5), 7.08 (1H, t, J=4.9Hz, H-5’). MS ESI (m/z): 221 [M-H]-.  2-((4-Nitrophenyl)amino)thiazole-4-carboxylic acid 75d:  Prepared by the procedure for 75a, ester 72d (1.17 g, 4 mmol) was hydrolyzed to afford 75d as a yellow solid (0.6 g, 57%).  1H-NMR (400 MHz, DMSO -d6): 13.05 (1H, br s, COOH), 11.17 (1H, br s, NH), 8.26 (2H, d, J=9.1Hz, H-’,5’), 7.95-7.84 (3H, m, H-5,’,’. MS ESI (m/z): 264 [M-H]-.  2-(Phenylamino)thiazole-4-carboxylic acid 75e:  Prepared by the procedure for 75a, ester 72e (1.0 g, 4 mmol) was hydrolyzed to afford 75e as an off white solid (0.72 g, 68%).  1H-NMR (400 MHz, DMSO -d6): 12.68 (1H, br s, COOH), 10.41 (1H, br s, NH), 7.84 (1H, s, H-5), 7.68-7.61 (2H, m, H-’,’), 7.31-7.25 (2H, m, H-’,5’), 6.98-6.94 (1H, m, H-’). MS ESI (m/z): 219 [M-H]-.     2-(Pyridin-3-ylamino)thiazole-4-carboxylic acid 72f:  210  Prepared by the procedure for 75a, ester 72f (1.0 g, 4 mmol) was hydrolyzed to afford 75f as an off white solid (0.58 g, 66%).  1H-NMR (400 MHz, DMSO -d6): 12.92 (1H, br s, COOH), 11.09 (1H, br s, NH), 9.12 (1H, d, J=2.1Hz, H-’), 8-41-8.34 (2H, m, H-’,’, 7.85 (1H, s, H-5), 7.68 (1H, d, J=5.1Hz, J=8.4Hz, H-5’). MS ESI (m/z): 220 [M-H]-. 2-((1H-Indol-5-yl)amino)thiazole-4-carboxylic acid 75g:  Prepared by the procedure for 75a, ester 72g (1.15 g, 4 mmol) was hydrolyzed. Note, the crude product mixture was dry loaded onto a silica column and chromatographed (gradient elution, 10-30% MeOH in EtOAc). Compound 75g was isolated as a yellow solid (0.49 g, 47%). 1H-NMR (400 MHz, DMSO -d6): 12.75 (1H, br s, COOH), 11.02 (1H, br s, N1’-H), 10.03 (1H, br s, C2-NH), 7.87 (1H, d, J=2.1Hz, H-’), 7.57 (1H, s, H-5), 7.35 (1H, d, J=8.5Hz, H-’), 7.32 (1H, t, J=3.1Hz, H-’), 7.16 (1H, dd, J=2.1Hz, J=8.5Hz, H-’), 6.39 (1H, t, J=3.1Hz, H-’. MS ESI (m/z): 258 [M-H]-. 2-((1H-Indazol-5-yl)amino)thiazole-4-carboxylic acid 75h:  Prepared by the procedure for 75a, ester 72h (1.15 g, 4 mmol) was hydrolyzed to afford 75h as a reddish solid (0.73g, 70%).  1H-NMR (400 MHz, DM SO-d6): 12.89 (2H, s, COOH, N1’-H), 10.28 (1H, s, C2-NH), 8.25 (1H, d, J=1.8Hz, H-’), 8.03 (1H, s, H-’), 7.66 (1H,s, H-5), 7.51 (1H, d, J=8.9Hz, H-’), 7.38 (1H, dd, J=1.8Hz, J=8.9Hz, H-’). MS ESI (m/z): 259 [M-H]-, HRMS: m/z calculated for C11H8N4O2S-: 259.02952, found: 259.02951.   211  2-(Benzo[d]thiazol-6-ylamino)thiazole-4-carboxylic acid 75i:  Prepared by the procedure for 75a, ester 72i (1.22 g, 4 mmol) was hydrolyzed to afford 75i as a yellow solid (0.84g, 76%).  1H-NMR (400  MHz, DMSO-d6): 12.77 (1H, br s, COOH), 10.65 (1H, br s, C2-NH), 9.19 (1H, s, H-’), 8.68 (1H, d, J=2.2Hz, H-’), 8.02 (1H, d, J=8.6Hz, H-’), 7.77 (1H, s, H-5), 7.61 (1H, dd, J=2.2Hz, J=8.6Hz, H-5’. MS ESI (m/z): 276 [M-H]-.  2-(Quinolin-6-ylamino)thiazole-4-carboxylic acid 75j:  Prepared by the procedure followed in 75a, ester 72j (1.19 g, 4 mmol) was hydrolyzed. Note, the crude product mixture was dry loaded onto a silica column and flash chromatographed (gradient elution, 10-30% MeOH in EtOAc). Compound 75j was isolated as a yellow solid (0.48g, 44%). 1H-NMR (400 MHz, DMSO -d6): 13.03 (1H, br s, COOH), 10.75 (1H, br s, C2-NH), 8.75 (1H, dd, J=1.6Hz, J=4.2Hz, H-’), 8.40 (1H, d, J=2.4Hz, H-5’), 8.23 (1H, dd, J=1.6Hz, J=8.6Hz, H-’), 7.99 (1H, d, J=8.8Hz, H-’), 7.85 (1H, dd, J=2.4Hz, J=8.8Hz, H-’, 7.81 (1H, s, H-5), 7.48 (1H, dd, J=4.2Hz, J=8.6Hz, H-’). MS ESI (m/z): 270 [M-H]-.  N-(1H-Indazol-5-yl)-2-(pyridin-2-ylamino)thiazole-4-carboxamide 76a:  To a solution of acid 75a (0.11 g, 0.5 mmol) in DMF (5 mL) was added HOAt (0.09 g, 0.55 mmol), EDC (0.11 g, 0.55 mmol) and DIEA (0.22 mL, 1.25 mmol). After stirring for 30 minutes, 5-aminoindazole (0.066 g, 0.5 mmol) was added and stirring was continued at room temperature for 18h. The mixture was then concentrated in vacuo and azeotroped twice with 212  CH2Cl2. The resulting residue was silica flash column chromatographed (gradient elution, 3í15% MeOH in CH2Cl2) to afford 76a as an off white solid (0.045 g, 27%). 1H-NMR (400 MHz, DMSO -d6): see Table 28. MS ESI (m/z): 335 [M-H]-, HRMS: m/z calculated for C16H12N6OS-: 335.07205, found: 335.07251. 2-((4-Cyanophenyl)amino)-N-(1H-indazol-5-yl)thiazole-4-carboxamide 76b:  Prepared according to 76a, acid 75b (0.14 g, 0.5 mmol) was reacted with 5-aminoindazole (0.066 g, 0.5 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to afford 76b as an off white solid (0.1 g, 31%). 1H-NMR (400 MHz, DMSO -d6): see Table 28. MS ESI (m/z): 359 [M-H]-, HRMS: m/z calculated for C18H12N6OS-: 359.07205, found: 359.07230. N-(1H-Indazol-5-yl)-2-(pyrimidin-2-ylamino)thiazole-4-carboxamide 76c:  Prepared according to 76a, acid 75c (0.11 g, 0.5 mmol) was reacted with 5-aminoindazole (0.066 g, 0.5 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í14% MeOH in CH2Cl2) to afford 76c as a white solid (0.04 g, 24%). 1H-NMR (400 MHz, DMSO -d6): see Table 28. MS ESI (m/z): 336 [M-H]-, HRMS: m/z calculated for C15H11N7OS-: 336.06730, found: 336.06778.  N-(1H-Indazol-5-yl)-2-((4-nitrophenyl)amino)thiazole-4-carboxamide 76d:  Prepared according to 76a, acid 75d (0.13 g, 0.5 mmol) was reacted with 5-aminoindazole (0.066 g, 0.5 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to afford 76d as a pale yellow solid (0.04 g, 21%). 1H-NMR (400 MHz, DMSO -d6): see Table 28. 213  MS ESI (m/z): 379 [M-H]-, HRMS: m/z calculated for C17H12N6O3S-: 379.06188, found: 379.06219. N-(1H-Indazol-5-yl)-2-(phenylamino)thiazole-4-carboxamide 76e:  Prepared according to 76a, acid 75e (0.11 g, 0.5 mmol) was reacted with 5-aminoindazole (0.066 g, 0.5 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to afford 76e as an off white solid (0.09g, 54%). 1H-NMR (400 MHz, DMSO -d6): see Table 28. MS ESI (m/z): 334 [M-H]-, HRMS: m/z calculated for C17H13N5OS-: 334.07680, found: 334.07721.  N-(1H-Indazol-5-yl)-2-(pyridin-3-ylamino)thiazole-4-carboxamide 76f:  Prepared according to 76a, acid 75f (0.11 g, 0.5 mmol) was reacted with 5-aminoindazole (0.066 g, 0.5 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í15% MeOH in CH2Cl2) to afford 76f as an off white solid (0.03 g, 18%). 1H-NMR (400 MHz, DMSO -d6): see Table 28. 13C NMR (100MHz, DMSO -d6): 163.3, 159.8, 150.7, 146.5, 142.7, 140.1, 138.0, 135.0, 131.6, 128.9, 124.4, 123.1, 120.7, 115.4, 112.4, 110.5. MS ESI (m/z): 335 [M-H]-, HRMS: m/z calculated for C16H12N6OS-: 335.07205, found: 335.07211.  2-((1H-Indol-5-yl)amino)-N-(1H-indazol-5-yl)thiazole-4-carboxamide 76g:  Prepared according to 76a, acid 75g (0.13 g, 0.5 mmol) was reacted with 5-aminoindazole (0.066 g, 0.5 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í15% MeOH in CH2Cl2) to afford 76g as an orange solid (0.09 g, 48%). 1H-NMR (400 MHz, DMSO -d6): see Table 28. 214  MS ESI (m/z): 373 [M-H]-, HRMS: m/z calculated for C19H14N6OS-: 373.08770, found: 373.08798. 2-((1H-Indazol-5-yl)amino)-N-(1H-indazol-5-yl)thiazole-4-carboxamide 76h:  Prepared according to 76a, acid 75g (0.13 g, 0.5 mmol) was reacted with 5-aminoindazole (0.066 g, 0.5 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í15% MeOH in CH2Cl2) to afford 76h as a brown solid (0.08 g, 43%). 1H-NMR (400 MHz, DMSO -d6): see Table 28. MS ESI (m/z): 374 [M-H]-, HRMS: m/z calculated for C18H13N7OS-: 374.08295, found: 374.08292.  2-(Benzo[d]thiazol-6-ylamino)-N-(1H-indazol-5-yl)thiazole-4-carboxamide 76i:  Prepared according to 76a, acid 75i (0.14 g, 0.5 mmol) was reacted with 5-aminoindazole (0.066 g, 0.5 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to afford 76i as an off white solid (0.1 g, 51%). 1H-NMR (400 MHz, DMSO -d6): see Table 28. 13C NMR (100MHz, DMSO -d6): 163.5, 159.8, 154.3, 148.6, 146.6, 138.9, 137.8, 135.4, 134.0, 131.7, 123.5, 122.7, 117.7, 115.1, 112.5, 110.5, 110.0, 49. MS ESI (m/z): 391 [M-H]-, HRMS: m/z calculated for C18H12N6OS2-: 391.04412, found: 391.04462.  N-(1H-Indazol-5-yl)-2-(quinolin-6-ylamino)thiazole-4-carboxamide 76j:  Prepared according to 76a, acid 75j (0.14 g, 0.5 mmol) was reacted with 5-aminoindazole (0.066 g, 0.5 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í15% MeOH in CH2Cl2) to afford 76j as a pale yellow solid (0.04 g, 21%). 1H-NMR (400 MHz, DMSO -d6): see Table 28. 215  13C NMR (100MHz, DMSO -d6): 163.3, 159.7, 148.6, 146.7, 144.6, 138.8, 137.8, 135.9, 134.0, 131.8, 130.2, 129.5, 123.2, 122.8, 122.5, 122.2, 115.4, 112.2, 112.1, 110.5. MS ESI (m/z): 385 [M-H]-, HRMS: m/z calculated for C20H14N6OS-: 385.08770, found: 385.08795.  Table 28: NMR data of compounds 76a-j:  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6)  ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm   76a   13.03 (1H, br s, N1’-H), 11.52 (1H, br s, C2-NH), 9.75 (1H, br s, CO-NH), 8.27 (1H, d, J=1.7Hz, H-’,  .0 1H, s, H-’, . (1H, s, H-5), 7.60 (1H, dd, J=1.7Hz, J=8.8Hz, H-’, .5 1H, d, - .Hz, H-’ 8.34 (1H, ddd, J=0.8Hz J=1.8Hz, J=4.9Hz, H-”, .5 1H, ddd, J=1.8Hz, J=7.2Hz, J=8.3Hz, H-”, 7.13 (1H, dd, J=1.8Hz, J=8.3Hz, H-”, . 1H, ddd, - 1.Hz, J=4.9Hz, J=7.2Hz, H-5”    76b   13.04 (1H, s, N1’-H), 10.93 (1H, s, C2-NH), 9.90 (1H, s, CO-NH), 8.20 (1H, d, J=1.8Hz, H-’,  .0 1H, s, H-’, .1 1H, s, H-5), 7.65 (1H, dd, J=1.8Hz, J=9.0Hz, H-’, .5 (1H, d, J=9.0Hz, H-’ .5 H,  d, - .Hz, H”,5”, 7.77 (2H,  d, J=7.7Hz, H-”,”    76c   13.04 (1H, br s, N1’-H), 11.94 (1H, br s, C2-NH), 9.80 (1H, s, CO-NH), 8.25 (1H, d, J=1.7Hz, H-’,  .0 1H, s, H-’, . (1H, s, H-5), 7.59 (1H, dd, J=1.7Hz, J=8.8Hz, H-’, .5 1H, d, - .Hz, H-’ 8.68 (2H, d, J=4.9Hz, H-”,”, 7.09 (1H, t, J=4.9Hz, H-5”    76d   13.06 (1H, br s, N1’-H), 11.19 (1H, br s, C2-NH), 9.90 (1H, br s, CO-NH), 8.21 (1H, d, J=1.7Hz, H-’,  .0 1H, s, H-’, . (1H, s, H-5), 7.67 (1H, dd, J=1.7Hz, J=8.6Hz, H-’, .5 1H, d, - .Hz, H-’ .5 H, d, - .Hz, H”,5”, .0 (2H, d, J=8.8Hz, H-”,”  216  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6)  ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm   76e   13.03 (1H, br s, N1’-H), 10.37 (1H, br s, C2-NH), 9.80 (1H, br s, CO-NH), 8.21 (1H, d, J=1.7Hz, H-’,  .0 1H, s, H-’, . (1H, s, H-5), 7.64 (1H, dd, J=1.7Hz, J=8.9Hz, H-’, .5 1H, d, - .Hz, H-’ 7.77-7.72 (2H, m,  H-”,”, 7.38-7.32 (2H, m,   H-”,5”, 7.05-6.95 (1H, m, H-”    76g   13.04 (1H, s, N1’-H), 11.04 (1H, s, C2-NH), 9.77 (1H, s, CO-NH), 8.25 (1H, d, J=1.9Hz, H-’,  .0 1H, s, H-’, . 1H, dd, J=1.9Hz, J=8.5Hz, H-’, .5 1H, s, H-5), 7.54 (1H, d, J=8.5Hz, H-’ 10.04 (1H, s, N1”-H), 7.91 (1H, d, J=2.0Hz, H-”, .5 1H, d, J=8.3Hz, H-”, . 1H, t, J=2.5Hz, H-”, .5 1H, dd, J=2.0Hz, J=8.7Hz, H-”, . 1H, t, J=2.5Hz, H-”   76h   13.05 (1H, s, N1’-H), 10.33 (1H, s, C2-NH), 9.84 (1H, s, CO-NH), 8.26 (1H, d, J=1.9Hz, H-’,  .07 (1H, s, H-’, . 1H, dd, J=1.9Hz, J=8.7Hz, H-’, . 1H, s, H-5), 7.55* (2H, t, J=8.0Hz, H-’ 12.98 (1H, s, N1”-H), 8.38 (1H, d, J=1.8Hz, H-”,  .0 1H, s, H-”, 7.55* (2H, t, J=8.6Hz, H-”, . (1H, dd, J=1.8Hz, J=8.6Hz, H-”   76i   13.05 (1H, s, N1’-H), 10.69 (1H, s, C2-NH), 10.00 (1H, s, CO-NH), 8.25 (1H, d, J=1.8Hz, H-’,  .0 1H, s, H-’, . (1H, s, H-5), 7.68 (1H, dd, J=1.8Hz, J=8.6Hz, H-’, .5 1H, d, - .Hz, H-’ 9.21 (1H, s, H-”, .03(1H, d, J=2.2Hz, H-”,  .01H, d, J=8.8Hz, H-”, .551H, dd, J=2.2Hz, J=8.8Hz, H-5”   76j   13.05 (1H, s, N1’-H), 10.78 (1H, s, C2-NH), 10.00 (1H, s, CO-NH), 8.29 (1H, d, J=1.8Hz, H-’,  .0 1H, s, H-’, . (1H, s, H-5), 7.71 (1H, dd, J=1.8Hz, J=8.6Hz, H-’, .5 1H, d, - .Hz, H-’ 8.82 (1H, dd, J=1.6Hz, J=4.2Hz, H-”, . 1H, d, - .Hz, H-5”, 8.43 (1H, dd, J=1.6Hz, J=8.6Hz, H-”, . 1H, d, - .1Hz, H-”, 7.79 (1H, dd, J=2.3Hz, J=9.1Hz, H-”,  .50 (1H, dd, J=4.2Hz, J=8.6Hz, H-”  2-(Pyridin-2-ylamino)-N-(pyridin-3-yl)thiazole-4-carboxamide 77a:  217  To a solution of acid 75a (0.11 g, 0.5 mmol) in DMF (5 mL) was added HOAt (0.09 g, 0.55 mmol), EDC (0.11 g, 0.55 mmol) and DIEA (0.22 mL, 1.25 mmol). After stirring for 30 minutes, 3-aminopyridine (0.047 g, 0.5 mmol) was added and stirring continued at room temperature for 18h. The mixture was then concentrated in vacuo and azeotroped twice with CH2Cl2. The residue was silica flash column chromatogrpahed (Jradient elution, í10 MeOH in CH2Cl2 to afford 77a as an off white solid (0.046 g, 31%). 1H-NMR (400 MHz, DMSO -d6): see Table 29. MS ESI (m/z): 296 [M-H]-, HRMS: m/z calculated for C14H11N5OS-: 296.06115, found: 296.06119. 2-((4-Cyanophenyl)amino)-N-(pyridin-3-yl)thiazole-4-carboxamide 77b:  Prepared according to 77a, acid 75b (0.12 g, 0.5 mmol) was reacted with 3-aminopyridine (0.047 g, 0.5 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to afford 77b as an off white solid (0.014 g, 12%). 1H-NMR (400 MHz, DMSO -d6): see Table 29. MS ESI (m/z): 320 [M-H]-, HRMS: m/z calculated for C16H11N5OS-: 320.06115, found: 320.06119.  N-(Pyridin-3-yl)-2-(pyrimidin-2-ylamino)thiazole-4-carboxamide 77c:  Prepared according to 77a, acid 75c (0.11 g, 0.5 mmol) was reacted with 3-aminopyridine (0.047 g, 0.5 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í14% MeOH in CH2Cl2) to afford 77c as an off white solid (0.04 g, 28%). 1H-NMR (400 MHz, DMSO -d6): see Table 29. MS ESI (m/z): 297 [M-H]-, HRMS: m/z calculated for C13H10N6OS-: 297.05640, found: 297.05643. 2-((4-Nitrophenyl)amino)-N-(pyridin-3-yl)thiazole-4-carboxamide 77d:  218  Prepared according to 77a, acid 75d (0.13 g, 0.5 mmol) was reacted with 3-aminopyridine (0.047 g, 0.5 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to afford 77d as a yellow solid (0.04 g, 28%). 1H-NMR (400 MHz, DMSO -d6): see Table 29. 13C NMR (100MHz, DMSO -d6): 162.5, 159.9, 151.3, 146.8, 145.9, 143.3, 141.1, 140.1, 135.1, 129.2, 128.7, 126.0, 124.0, 121.0, 117.3. MS ESI (m/z): 340 [M-H]-, HRMS: m/z calculated for C15H11N5O3S-: 340.05098, found: 340.05099.  2-(Phenylamino)-N-(pyridin-3-yl)thiazole-4-carboxamide 77e:  Prepared according to 77a, acid 75e (0.11 g, 0.5 mmol) was reacted with 3-aminopyridine (0.047 g, 0.5 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to afford 77e as a pale yellow solid (0.074 g, 50%). 1H-NMR (400 MHz, DMSO -d6): see Table 29. MS ESI (m/z): 295 [M-H]-, HRMS: m/z calculated for C15H12N4OS-: 295.06591, found: 295.06595. N-(Pyridin-3-yl)-2-(pyridin-3-ylamino)thiazole-4-carboxamide 77f:  Prepared according to 77a, acid 75f (0.11 g, 0.5 mmol) was reacted with 3-aminopyridine (0.047 g, 0.5 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í15% MeOH in CH2Cl2) to afford 77f as a pale pink solid (0.068 g, 46%). 1H-NMR (400 MHz, DMSO -d6): see Table 29. MS ESI (m/z): 296 [M-H]-, HRMS: m/z calculated for C14H11N5OS-: 296.06115, found: 296.06116.  2-((1H-Indol-5-yl)amino)-N-(pyridin-3-yl)thiazole-4-carboxamide 77g:  219  Prepared according to 77a, acid 75g (0.13 g, 0.5 mmol) was reacted with 3-aminopyridine (0.047 g, 0.5 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 77g as a pale yellow solid (0.045 g, 47%). 1H-NMR (400 MHz, DMSO -d6): see Table 29. MS ESI (m/z): 334 [M-H]-, HRMS: m/z calculated for C17H13N5OS-: 334.07680, found: 334.07675.  2-((1H-Indazol-5-yl)amino)-N-(pyridin-3-yl)thiazole-4-carboxamide 77h:  Prepared according to 77a, acid 75h (0.13 g, 0.5 mmol) was reacted with 3-aminopyridine (0.047 g, 0.5 mmol).  The crude mixture was silica flash column chromatographed (gradient elution, 2í16% MeOH in CH2Cl2) to afford 77h as an off white solid (0.079 g, 47%). 1H-NMR (400 MHz, DMSO -d6): see Table 29. MS ESI (m/z): 335 [M-H]-, HRMS: m/z calculated for C16H12N6OS-: 335.07205, found: 335.07211.  2-(Benzo[d]thiazol-6-ylamino)-N-(pyridin-3-yl)thiazole-4-carboxamide 77i:  Prepared according to 77a, acid 75i (0.14 g, 0.5 mmol) was reacted with 3-aminopyridine (0.047 g, 0.5 mmol).  The crude mixture was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to afford 77i as a yellowish green solid (0.1 g, 57%). 1H-NMR (400 MHz, DMSO -d6): see Table 29. MS ESI (m/z): 352 [M-H]-, HRMS: m/z calculated for C16H11N5OS2-: 352.03322, found: 352.03329.  N-(Pyridin-3-yl)-2-(quinolin-6-ylamino)thiazole-4-carboxamide 77j:  220  Prepared according to 77a, acid 75j (0.14 g, 0.5 mmol) was reacted with 3-aminopyridine (0.047 g, 0.5 mmol).  The crude mixture was silica flash column chromatographed (gradient elution, 2í16% MeOH in CH2Cl2) to afford 77j as a yellow solid (0.048 g, 28%). 1H-NMR (400 MHz, DMSO -d6): see Table 29. 13C NMR (100MHz, DMSO -d6): 163.4, 160.1, 148.6, 145.9, 144.6, 143.0, 138.8, 135.9, 134.8, 130.2, 129.4, 128.4, 127.2, 124.0, 122.8, 118.8, 116.2, 112.3. MS ESI (m/z): 346 [M-H]-, HRMS: m/z calculated for C18H13N5OS-: 346.07680, found: 346.07730.  Table 29: NMR data of compounds 77a-j  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  77a   11.51 (1H, br s, C2-NH), 10.00 (1H, s, CO-NH), 8.92 (1H, d, J=2.3Hz, H-’, 8.32 (1H, dd, J=1.5Hz, J=4.8Hz, H-’, 8.13 (1H, ddd, J=1.5Hz, J=2.3Hz, J=8.5Hz, H-’, . 1H, s, H-5),7.40 (1H, dd, J=4.8Hz, J=8.5Hz, H-5’  8.34 (1H, ddd, J=0.8Hz J=1.8Hz, J=4.9Hz, H-”, .5 1H, ddd, J=1.8Hz, J=7.2Hz, J=8.3Hz, H-”, 7.14 (1H, dd, J=1.8Hz, J=8.3Hz, H-”, . 1H, ddd, - 1.Hz, J=4.9Hz, J=7.2Hz, H-5”   77b   10.95 (1H, br s, C2-NH), 10.10 (1H, s, CO-NH), 8.95 (1H, d, J=2.3Hz, H-’, 8.34 (1H, dd, J=1.5Hz, J=4.6Hz, H-’, 8.19 (1H, ddd, J=1.5Hz, J=2.3Hz, J=8.5Hz, H-’, . 1H, s, H-5), 7.42 (1H, dd, J=4.6Hz, J=8.5Hz, H-5’ . H,  d, - .Hz, H”,5”, 7.77 (2H,  d, J=7.7Hz, H-”,”   77c   11.91 (1H, br s, C2-NH), 10.05 (1H, s, CO-NH), 8.92 (1H, s, H-’, . 1H, dd, J=1.3Hz, J=4.0Hz, H-’, .1 1H, dd, J=1.3Hz, J=8.5Hz, H-’, . 1H, s, H-5), 7.40 (1H, dd, J=4.0Hz, J=8.5Hz, H-5’ 8.68 (2H, d, J=4.9Hz, H-”,”, 7.09 (1H, t, J=4.9Hz, H-5”  221  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  77d   11.23 (1H, br s, C2-NH), 10.08 (1H, s, CO-NH), 8.98 (1H, d, J=2.3Hz, H-’, 8.37 (1H, dd, J=1.5Hz, J=4.6Hz, H-’, 8.22 (1H, ddd, J=1.5Hz, J=2.3Hz, J=8.5Hz, H-’, .5 1H, s, H-5),7.45 (1H, dd, J=4.6Hz, J=8.5Hz, H-5’ . H, d, - .Hz, H”,5”, .0 (2H, d, J=8.7Hz, H-”,”   77e   10.39 (1H, br s, C2-NH), 10.00 (1H, s, CO-NH), 8.95 (1H, d, J=2.3Hz, H-’, 8.33 (1H, dd, J=1.5Hz, J=4.7Hz, H-’, 8.20 (1H, ddd, J=1.5Hz, J=2.3Hz, J=8.3Hz, H-’, . 1H, s, H-5), 7.41 (1H, dd, J=4.7Hz, J=8.3Hz, H-5’ 7.77-7.72 (2H, m,  H-”,”, 7.38-7.32 (2H, m,   H-”,5”, 7.05-6.95 (1H, m, H-”   77f   10.63 (1H, br s, C2-NH), 10.09 (1H, s, CO-NH), 8.94 (1H, d, J=2.3Hz, H-’, 8.34 (1H, dd, J=1.4Hz, J=4.7Hz, H-’, 8.18 (1H, ddd, J=1.4Hz, J=2.3Hz, J=8.5Hz, H-’, .0 1H, s, H-5),7.41 (1H, dd, J=4.7Hz, J=8.5Hz, H-5’ 8.81 (1H, d, J=2.3Hz, H-”,  . (1H, ddd, J=1.4Hz, J=2.3Hz, J=8.5Hz, H-”, .1 1H, dd, J=1.4Hz, J=4.7Hz, H-”, . 1H, dd, J=4.7Hz, J=8.5Hz, H-5”    77g   10.70 (1H, br s, C2-NH), 9.77 (1H, s, CO-NH), 8.92 (1H, d, J=2.2Hz, H-’, 8.30 (1H, dd, J=1.5Hz, J=4.7Hz, H-’, .1 (1H, ddd, J=1.5Hz, J=2.2Hz, J=8.5Hz, H-’, . 1H, s, H-5), 7.39 (1H, dd, J=4.7Hz, J=8.5Hz, H-5’ 9.88 (1H, s, N1”-H), 7.30 (1H, d, J=1.9Hz, H-”, . 1H, d, J=8.3Hz, H-”, . 1H, d, J=2.5Hz, H-”, .08 (1H, dd, J=1.9Hz, J=8.7Hz, H-”, . 1H, d, J=2.5Hz, H-”   77h   10.70 (1H, br s, C2-NH), 10.16 (1H, s, CO-NH), 8.99 (1H, d, J=2.2Hz, H-’, 8.35 (1H, dd, J=1.5Hz, J=4.7Hz, H-’, 8.24 (1H, ddd, J=1.5Hz, J=2.2Hz, J=8.5Hz, H-’, .0 1H, s, H-5), 7.44 (1H, dd, J=4.7Hz, J=8.5Hz, H-5’ 12.97 (1H, s, N1”-H), 8.38 (1H, d, J=1.8Hz, H-”,  .0 1H, s, H-”, .55 1H, d, - .Hz, H-”, 7.46 (1H, dd, J=1.8Hz, J=8.6Hz, H-”   77i  10.70 (1H, br s, C2-NH), 10.16 (1H, s, CO-NH), 8.99 (1H, d, J=2.2Hz, H-’, 8.35 (1H, dd, J=1.5Hz, J=4.7Hz, H-’, 8.24 (1H, ddd, J=1.5Hz, J=2.2Hz, J=8.5Hz, H-’, .0 1H, s, H-5), 7.44 9.21 (1H, s, H-”, .01H, d, J=2.1Hz, H-”,  .01H, d, J=8.7Hz, H-”, .51H, dd, J=2.1Hz, J=8.7Hz, H-5” 222  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6) ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  (1H, dd, J=4.7Hz, J=8.5Hz, H-5’   77j   10.83 (1H, br s, C2-NH), 10.19 (1H, s, CO-NH), 9.03 (1H, d, J=2.2Hz, H-’, 8.35 (1H, dd, J=1.5Hz, J=4.7Hz, H-’, 8.27 (1H, ddd, J=1.5Hz, J=2.2Hz, J=8.5Hz, H-’, . 1H, s, H-5), 7.44 (1H, dd, J=4.7Hz, J=8.5Hz, H-5’  8.76 (1H, d, J=2.2Hz, H-5”, . (1H, dd, J=1.6Hz, J=4.2Hz, H-”, 8.41 (1H, dd, J=1.6Hz, J=8.6Hz, H-”, . 1H, d, - .0Hz, H-”, 7.76 (1H, dd, J=2.2Hz, J=9.0Hz, H-7”,  .50 1H, dd, J=4.2Hz, J=8.6Hz, H-”  tert-Butyl 5-nitro-1H-indazole-1-carboxylate 79:   To a stirred solution of 5-nitroindazole 78 (5g, 30.65 mmol) in CH2Cl2 (60 mL) were successively added Et3N (5.31 mL, 38.31 mmol), (Boc)2O (8.36 g, 38.31 mmol), and DMAP (0.38 g, 3.1 mmol). The mixture was stirred for 24 h at room temperature before addition of saturated aqueous NH4Cl and extraction with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (gradient elution, 1-5% MeOH in CH2Cl2) to give the product 79 as an orange solid (6.94g, 86%).  1H-NMR (400 MHz, DMSO -d6): 8.89 (1H, d, J=2.3Hz, H-4), 8.65 (1H, s, H-3), 8.44 (1H, dd, J=2.3Hz, J=9.2Hz, H-6), 8.26 (1H, d, J=9.2Hz, H-7), 1.67 (9H, s, C(CH3)3).  2-((1H-Indazol-5-yl)amino)-N-phenylthiazole-4-carboxamide 80a:  223  To a solution of acid 75h (0.1g, 0.38 mmol) in DMF (5 mL), was added HOAt (0.07 g, 0.42 mmol), EDC (0.081 g, 0.42 mmol) and DIEA (0.2 mL, 1.14 mmol). After stirring for 30 minutes, aniline was added (0.035 g, 0.38 mmol) and stirring was continued at room temperature for 18h. The mixture was then concentrated in vacuo and azeotroped twice with CH2Cl2. The residue was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 80a as an off white solid (0.065 g, 51%). 1H-NMR (400 MHz, DMSO -d6): see Table 30. 13C NMR (100MHz, DMSO -d6): 164.8, 159.6, 145.7, 138.8, 137.0, 134.6, 133.5, 129.1, 124.3, 123.4, 121.0, 120.4, 117.2, 114.1, 111.3, 108.2. MS ESI (m/z): 334 [M-H]-, HRMS: m/z calculated for C17H13N5OS-: 334.07680, found: 334.07721.  2-((1H-Indazol-5-yl)amino)-N-(4-methoxyphenyl)thiazole-4-carboxamide 80b:  Prepared according to 80a, acid 75h (0.1g, 0.38 mmol) was reacted with p-anisidine (0.047 g, 0.38 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í14% MeOH in CH2Cl2) to afford 80b as a white solid (0.076 g, 56%). 1H-NMR (400 MHz, DMSO -d6): see Table 30. MS ESI (m/z): 364 [M-H]-, HRMS: m/z calculated for C18H15N5O2S-: 364.08737, found: 364.08786.  2-((1H-Indazol-5-yl)amino)-N-(3-methoxyphenyl)thiazole-4-carboxamide 80c:   Prepared according to 80a, acid 75h (0.1g, 0.38 mmol) was reacted with m-anisidine (0.047 g, 0.38 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í14% MeOH in CH2Cl2) to afford 80c as an off white solid (0.079 g, 58%). 1H-NMR (400 MHz, DMSO -d6): see Table 30. 224  13C NMR (100MHz, DMSO -d6): 164.8, 159.9, 159.6, 145.5, 140.0, 137.0, 134.6, 133.4, 129.9, 123.4, 120.6, 114.2, 113.2, 111.4, 109.8, 108.4, 106.6, 55.5. MS ESI (m/z): 364 [M-H]-, HRMS: m/z calculated for C18H15N5O2S-: 364.08737, found: 364.08832.  2-((1H-Indazol-5-yl)amino)-N-(3,4-dimethoxyphenyl)thiazole-4-carboxamide 80d:  Prepared according to 80a, acid 75h (0.1g, 0.38 mmol) was reacted with 3,4-dimethoxyaniline (0.058 g, 0.38 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í16% MeOH in CH2Cl2) to afford 80d as a pale yellow solid (0.074 g, 49%). 1H-NMR (400 MHz, DMSO -d6): see Table 30. MS ESI (m/z): 394 [M-H]-, HRMS: m/z calculated for C19H17N5O3S-: 394.09793, found: 394.09818. 2-((1H-Indazol-5-yl)amino)-N-(4-fluorophenyl)thiazole-4-carboxamide 80e:  Prepared according to 80a, acid 75h (0.1g, 0.38 mmol) was reacted with 4-fluoroaniline (0.042 g, 0.38 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 80e as an off white solid (0.07 g, 52%). 1H-NMR (400 MHz, DMSO -d6): see Table 30. 13C NMR (100MHz, DMSO -d6): 160.1, 159.7, 157.7, 145.7, 137.0, 135.3, 134.6, 133.5, 123.4, 123.1, 123.0, 120.4, 115.8, 115.6, 114.3, 111.3, 108.2. MS ESI (m/z): 352 [M-H]-, HRMS: m/z calculated for C17H12N5OSF-: 352.06738, found: 352.06772. 2-((1H-Indazol-5-yl)amino)-N-(2,3-dihydro-1H-inden-4-yl)thiazole-4-carboxamide 80f:  225  Prepared according to 80a, acid 75h (0.1g, 0.38 mmol) was reacted with 4-aminoindan (0.05 g, 0.38 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to afford 80f as a pale yellow solid (0.067 g, 47%). 1H-NMR (400 MHz, DMSO -d6): see Table 30. MS ESI (m/z): 374 [M-H]-, HRMS: m/z calculated for C20H17N5OS-: 374.10810, found: 374.10831.  2-((1H-Indazol-5-yl)amino)-N-(benzo[d][1,3]dioxol-5-yl)thiazole-4-carboxamide 80g:  Prepared according to 80a, acid 75h (0.1g, 0.38 mmol) was reacted with 3,4-(methylenedioxy) aniline (0.052 g, 0.38 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í14% MeOH in CH2Cl2) to afford 80g as an off white solid (0.083 g, 58%). 1H-NMR (400 MHz, DMSO -d6): see Table 30. MS ESI (m/z): 378 [M-H]-, HRMS: m/z calculated for C18H13N5O3S-: 378.06663, found: 378.06705.  2-((1H-Indazol-5-yl)amino)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)thiazole-4-carboxamide 80h:  Prepared according to 80a, acid 75h (0.1g, 0.38 mmol) was reacted with 1,4-benzodioxan-6-amine (0.052 g, 0.38 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í14% MeOH in CH2Cl2) to afford 80h as an off white solid (0.077 g, 52%). 1H-NMR (400 MHz, DMSO -d6): see Table 30. 13C NMR (100MHz, DMSO -d6): 159.4, 151.6, 146.0, 143.3, 140.3, 137.0, 135.1, 133.5, 132.5, 129.3, 121.2, 117.1, 114.4, 113.8, 111.3, 110.1, 108.2, 64.7, 64.4. MS ESI (m/z): 392 [M-H]-, HRMS: m/z calculated for C19H15N5O3S-: 392.08228, found: 392.08270. 226  2-((1H-Indazol-5-yl)amino)-N-(6-methoxypyridin-3-yl)thiazole-4-carboxamide 80i:  Prepared according to 80a, acid 75h (0.1g, 0.38 mmol) was reacted with 5-amino-2-methoxypyridine (0.047 g, 0.38 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í14% MeOH in CH2Cl2) to afford 80i as a pale pink solid (0.067 g, 48%). 1H-NMR (400 MHz, DMSO -d6): see Table 30. 13C NMR (100MHz, DMSO -d6): 164.6, 160.5, 160.0, 146.1, 140.0, 136.9, 134.6, 133.8, 133.6, 129.8, 123.6, 119.9, 114.2, 111.1, 110.4, 108.0, 53.7. MS ESI (m/z): 365 [M-H]-, HRMS: m/z calculated for C17H14N6O2S-: 365.08262, found: 365.08301.  2-((1H-Indazol-5-yl)amino)-N-(1H-indol-5-yl)thiazole-4-carboxamide 80j:  Prepared according to 80a, acid 75h (1 equiv) was reacted with 6-aminoindole (0.05 g, 0.38 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 80j as a pale beige solid (0.055 g, 39%). 1H-NMR (400 MHz, DMSO -d6): see Table 30. 13C NMR (100MHz, DMSO -d6): 164.8, 159.2, 145.8, 137.1, 134.6, 133.6, 133.4, 130.5, 127.9, 126.5, 123.4, 120.6, 116.6, 113.3, 112.7, 111.7, 111.4, 108.4, 101.6. MS ESI (m/z): 373 [M-H]-, HRMS: m/z calculated for C19H14N6OS-: 373.08770, found: 373.08792.  2-((1H-Indazol-5-yl)amino)-N-(benzo[d]thiazol-6-yl)thiazole-4-carboxamide 80k:  227  Prepared according to 80a, acid 75h (0.1g, 0.38 mmol) was reacted with 6-aminobenzothiazole (0.057 g, 0.38 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 80k as an off white solid (0.077 g, 52%). 1H-NMR (400 MHz, DMSO -d6): see Table 30. MS ESI (m/z): 391 [M-H]-, HRMS: m/z calculated for C18H12N6OS2-: 391.04412, found: 391.04434.  2-((1H-Indazol-5-yl)amino)-N-(quinolin-6-yl)thiazole-4-carboxamide 80l:  Prepared according to 80a, acid 75h (0.1g, 0.38 mmol) was reacted with 6-aminoquinoline (0.055 g, 0.38 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í18% MeOH in CH2Cl2) to afford 80l as off whit solid (0.032 g, 22%). 1H-NMR (400 MHz, DMSO -d6): see Table 30. MS ESI (m/z): 385 [M-H]-, HRMS: m/z calculated for C20H14N6OS-: 385.08770, found: 385.08804.  2-((1H-Indazol-5-yl)amino)-N-benzylthiazole-4-carboxamide 80m:  Prepared according to 80a, acid 75h (0.1g, 0.38 mmol) was reacted with benzylamine (0.04 g, 0.38 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to afford 80m as white solid (0.08 g, 61%). 1H-NMR (400 MHz, DMSO -d6): see Table 30. 13C NMR (100MHz, DMSO -d6): 164.4, 161.4, 146.4, 140.2, 136.8, 134.7, 133.7, 128.7, 127.6, 127.2, 123.6, 119.8, 117.3, 113.0, 111.0, 107.6, 42.7. MS ESI (m/z): 348 [M-H]-, HRMS: m/z calculated for C18H15N5OS-: 348.09245, found: 348.09277.  228  2-((1H-Indazol-5-yl)amino)-N-(4-methoxybenzyl)thiazole-4-carboxamide 80n:  Prepared according to 80a, acid 75h (0.1g, 0.38 mmol) was reacted with 4-methoxy benzylamine (0.04 g, 0.38 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 80n as a white solid (0.09 g, 64%). 1H-NMR (400 MHz, DMSO -d6): see Table 30. MS ESI (m/z): 378 [M-H]-, HRMS: m/z calculated for C19H17N5O2S-: 378.10302, found: 378.10309.  2-((1H-Indazol-5-yl)amino)-N-(pyridin-2-ylmethyl)thiazole-4-carboxamide 80o:  Prepared according to 80a, acid 75h (0.1g, 0.38 mmol) was reacted with 2-picolylamine (0.04 g, 0.38 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to afford 80o as an off white solid (0.077 g, 58%). 1H-NMR (400 MHz, DMSO -d6): see Table 30. 13C NMR (100MHz, DMSO -d6): 164.3, 162.0, 154.7, 146.8, 145.2, 141.7, 136.8, 134.7, 133.5, 126.1, 125.9, 123.5, 120.1, 114.3, 111.1, 107.6, 44.7. MS ESI (m/z): 349 [M-H]-, HRMS: m/z calculated for C17H14N6OS-: 349.08770, found: 349.08792.  N-(4-(1H-Imidazol-1-yl)phenyl)-2-((1H-indazol-5-yl)amino)thiazole-4-carboxamide 80p:  Prepared according to 80a, acid 75h (0.1g, 0.38 mmol) was reacted with 4-(1H-Imidazol-1-yl)aniline (0.06 g, 0.38 mmol). The crude mixture was silica flash column chromatographed 229  (gradient elution, 2í10% MeOH in CH2Cl2) to afford 80p as an off white solid (0.06 g, 40%). 1H-NMR (400 MHz, DMSO -d6): see Table 30. MS ESI (m/z): 400 [M-H]-, HRMS: m/z calculated for C20H15N7OS-: 400.09860, found: 400.09882.  2-((1H-Indazol-5-yl)amino)-N-(4-morpholinophenyl)thiazole-4-carboxamide 80q:  Prepared according to 80a, acid 75h (0.1g, 0.38 mmol) was reacted with 4-morpholinoaniline (0.067 g, 0.38 mmol). The crude mixture was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 80q as an off white solid (0.084 g, 54%). 1H-NMR (400 MHz, DMSO -d6): see Table 30. MS ESI (m/z): 419 [M-H]-, HRMS: m/z calculated for C21H20N6O2S-: 419.12957, found: 419.13062. was reduced. The crude mixture was silica flash column chromatographed (gradient elution, 2-8% MeOH in CH2Cl2) to afford 71h (N 1-Boc) as an orange solid (4.3g, 74%). 1H-NMR (400 MHz, DMSO -d6): 8.11 (1H, s, H-3), 7.75 (1H, d, J=8.9Hz, H-7), 6.91 (1H, dd, J=2.2Hz, J=8.9Hz, H-6), 6.83 (1H, d, J=2.2Hz, H-4), 5.18 (2H, br s, NH2), 1.62 (s, 9H, C(CH3)3). MS ESI (m/z): 234 [M+H]+.  Table 30: NMR data of compounds 80a-q  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6)  ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  80a  12.97 (1H, br s, N1’-H), 10.31 (1H, br s, C2-NH), 9.77 (1H, s, CO-NH), 8.33 (1H, d, J=1.7Hz, H-4’, 8.04 (1H, s, H-’, 7.84-7.75 (2H, m, H-”,”, 7.41-7.33 (2H,  m, H-”,5”, 7.17-7.07 (1H, m, H-” 230  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6)  ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  7.63 (1H, s, H-5), 7.53 (1H, d, J=8.9Hz, H-’, 7.44 (1H, dd, J=1.7Hz, J=8.9Hz, H-’   80b   12.96 (1H, br s, N1’-H), 10.29 (1H, br s, C2-NH), 9.68 (1H, br s, CO-NH), 8.34 (1H, d, J=1.8Hz, H-’), 8.03 (1H, s, H-’), 7.59 (1H, s, H-5), 7.52 (1H, d, J=8.8Hz, H-’), 7.43 (1H, dd, J=1.8Hz, J=8.8Hz, H-’) 7.69 (2H, d, J=9.0Hz, H-”,”, .5 (2H, d, J=9.0Hz, H-”,5”, . 3H, s, OCH3)   80c   12.96 (1H, br s, N1’-H), 10.30 (1H, br s, C2-NH), 9.74 (1H, br s, CO-NH), 8.32 (1H, d, J=1.9Hz, H-’), 8.04 (1H, s, H-’), 7.63 (1H, s, H-5), 7.53 (1H, d, J=8.9Hz, H-’), 7.44 (1H, dd, J=1.9Hz, J=8.9Hz, H-’) 7.51 (1H, d, J=1.2Hz, H-”, . 1H, dd, J=1.2Hz, J=7.9Hz, H-”, . 1H, t, J=7.9Hz, H-5”, .1 1H, dd, J=1.2Hz, J=7.9Hz, H-”, . H, s, OCH3)  80d   12.96 (1H, br s, N1’-H), 10.29 (1H, br s, C2-NH), 9.66 (1H, br s, CO-NH), 8.35 (1H, d, J=2.0Hz, H-’), 8.04 (1H, s, H-’), 7.59 (1H, s, H-5), 7.55-7.48* (2H, m, H-’), 7.43 (1H, dd, J=2.0Hz, J=8.9Hz, H-’) 7.55-7.48* (2H, m, H-”, . 1H, dd, J=2.4Hz, J=8.7Hz, H-”, .5 1H, d, J=8.7Hz, H-5”, . H, d, J=11.5Hz, OCH3)   80e    12.96 (1H, br s, N1’-H), 10.30 (1H, br s, C2-NH), 9.88 (1H, br s, CO-NH), 8.36 (1H, d, J=1.7Hz, H-’), 8.03 (1H, s, H-’), 7.63 (1H, s, H-5), 7.52 (1H,d, J=8.9Hz, H-’), 7.44 (1H, dd, J=1.7Hz, J=8.9Hz, H-’) 7.88-.1 H, m, H”,”, .-7.19 (2H, m, H-”,5”   80f   13.00 (1H, br s, N-H1’), 10.43 (1H, br s, C2-NH), 9.42 (1H, br s, CO-NH), 8.44 (1H, d, J=2.0Hz, H-’), 7.97 (1H, s, H-’), 7.65 (1H, s, H-5), 7.53 (1H, d, J=9.0Hz, H-’), 7.38 (1H, dd, J=2.0Hz, J=9.0Hz, H-’)   7.92 (1H, d, J=8.2Hz, H-5” .1 1H, t, J=7.8Hz H-”, .05 1H, d, - .Hz, H-”, . H, td, - .Hz, J=22.3Hz, benzylic CH2), 2.14 (2H, p, J=7.2Hz, CH2)  231  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6)  ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm   80g   12.96 (1H, br s, N1’-H), 10.28 (1H, br s, C2-NH), 9.71 (1H, br s, CO-NH), 8.33 (1H, d, J=2.0Hz, H-’), 8.03 (1H, s, H-’), 7.59 (1H, s, H-5), 7.52 (1H, d, J=8.9Hz, H-’), 7.44 (1H, dd, J=2.0Hz, J=8.9Hz, H-’) 7.47 (1H, d, J=2.1Hz, H-”, . (1H, dd, J=2.1Hz, J=8.4Hz, H-”, . 1H, d, J=8.4Hz, H-”, .0 H, s, OCH2O)   80h   12.96 (1H, br s, N1’-H), 10.28 (1H, br s, C2-NH), 9.64 (1H, br s, CO-NH), 8.32 (1H, d, J=1.8Hz, H-’), 8.03 (1H, s, H-’), 7.58 (1H, s, H-5), 7.52 (1H, d, J=8.9Hz, H-’), 7.45 (1H, dd, J=1.8Hz, J=8.9Hz, H-’) 7.44 (1H, d, J=2.5Hz, H-5”,  .0 1H, dd, J=2.5Hz, J=8.8Hz, H-”, . 1H, d, J=8.8Hz, H-”, . H, dd, J=5.1Hz, J=8.6Hz, 2 OCH2)   80i    12.97 (1H, br s, N1’-H), 10.31 (1H, br s, C2-NH), 9.90 (1H, br s, CO-NH), 8.40 (1H, d, J=1.8Hz, H-’), 8.05 (1H, s, H-’), 7.65 (1H, s, H-5), 7.53 (1H, d, J=8.9Hz, H-’), 7.45 (1H, dd, J=1.8Hz, J=8.9Hz, H-’) 8.57 (1H, d, J=2.7Hz, H-”, .10 1H, dd, J=2.7Hz, J=8.9Hz, H-”, . d, 1H, J=8.9Hz, H-5”, . H, s, OCH3)    80j    12.96 (1H, br s, N1’-H), 11.06 (1H, br s, C2-NH), 9.60 (1H, br s, CO-NH), 8.33 (1H, d, J=1.9Hz, H-’), 8.05 (1H, s, H-’), 7.58 (1H, s, H-5), 7.53 (1H,d, J=9.0Hz, H-’), 7.46 (1H, dd, J=1.9Hz, J=9.0Hz, H-’) 10.32 (1H, s, N1”-H), 7.99 (1H, d, J=1.5Hz, H-”, .-7.41 (2H, m, H-”,”, .5 1H, t, - .5Hz, H-”, 6.43 (1H, t, J=2.5Hz, H-”   80k   12.97 (1H, br s, N-H1’), 10.33 (1H, br s, C2-NH), 10.06 (1H, br s, CO-NH), 8.36 (1H, d, J=1.9Hz, H-’), 8.06 (1H, s, H-’), 7.68 (1H, s, H-5), 7.53 (1H,d, J=9.0Hz, H-’), 7.46 (1H, dd, J=1.9Hz, J=9.0Hz, H-’) 9.32 (1H, s, H-”, . 1H, d, J=2.1Hz, H-”, .0 1H, d, J=8.9Hz, H-”, .0 1H, dd, - .1Hz, - .Hz H-5”  80l   12.98 (1H, br s, N1’-H), 10.34 (1H, br s, C2-NH), 10.13 (1H, br s, CO-NH), 8.38 (1H, d, J=2.0Hz, H-’), 8.07 (1H, s, H-’), 7.70 (1H, s, H-5), 7.56 (1H, d, J=9.0Hz, H-’), 7.47 (1H, dd, J=2.0Hz, 8.74 (1H, dd, J=1.7Hz, J=4.1Hz, H-”, 8.55 (1H, d, J=2.2Hz, H-5”, . 1H, dd, J=1.6Hz, J=8.3Hz, H-”, .10 1H, dd, J=2.2Hz, J=9.1Hz, H-”, .0 1H, d, J=9.1Hz, H-”, .5 1H, dd, 232  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6)  ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm J=8.9Hz, H-’) J=4.1Hz, J=8.3Hz, H-”  80m   12.93 (1H, br s, N1’-H), 10.23 (1H, br s, C2-NH), 8.61 (1H, t, J=6.4Hz, CO-NH), 8.32 (1H, d, J=2.0Hz, H-’), 7.97 (1H,s, H-’), 7.49 (2H, m, H-5, H-’), 7.42 (1H, dd, J=2.0Hz, J=8.9Hz, H-’)  7.34 (4H, d, J=4.4Hz, H-”,”,5”,”, 7.28-7.20 (1H, m, H-”, .51 1H, d, J=6.4Hz, CH2)  80n   12.93 (1H, br s, N1’-H), 10.22 (1H, br s, C2-NH), 8.52 (1H, t, J=6.3Hz, CO-NH), 8.30 (1H, d, J=2.0Hz, H-’), 7.97 (1H, s, H-’), 7.49 (1H, d, J=8.9Hz, H-’), 7.46 (1H, s, H-5), 7.41 (1H, dd, J=2.0Hz, J=8.9Hz, H-’) 7.20 (2H, d, J=8.7Hz, H-”,”, .0 (2H, d, J=8.7Hz, H-”,5”, . H, d, J=6.3Hz, CH2), 3.72 (3H, s, OCH3)   80o   12.95 (1H, br s, N1’-H), 10.28 (1H, br s, C2-NH), 8.73 (1H, t, J=5.9Hz, CO-NH) 8.35 (1H, d, J=1.6Hz, H-’), 7.98 (1H, s, H-’), 7.52-7.47 (2H, m, H-5,’), 7.43 (1H, dd, J=2.0Hz, J=8.9Hz, H-’) 8.55 (1H, dd, J=1.7Hz, J=4.8Hz, H-”, 7.79 (1H, dt, J=2.2Hz, J=7.7Hz, H-”, 7.38-7.34 (1H, m, H-” , .-7.28 (1H, m, H-5”, . H, d, - 5.Hz, CH2)   80p   12.97 (1H, br s, N1’-H), 10.31 (1H, br s, C2-NH), 9.98 (1H, br s, CO-NH), 8.37 (1H, d, J=1.9Hz, H-’), 8.04 (1H, s, H-’), 7.74 (1H, s, H-5), 7.53 (1H, d, J=8.8Hz, H-’), 7.44 (1H, dd, J=1.9Hz, J=8.9Hz, H-’) 8.25 (1H, s, H-”’, . H, d, J=8.9Hz, H-”,5”, .0-7.63 (3H, m, H-”,”, ”’, .1 1H, s, H-5”’  80q   12.96 (1H, br s, N1’-H), 10.29 (1H, br s, C2-NH), 9.60 (1H, br s, CO-NH), 8.33 (1H, d, J=1.9Hz, H-’), 8.03 (1H, s, H-’), 7.57 (1H, s, H-5), 7.52 (1H, d, J=8.8Hz, H-’), 7.43 (1H, dd, J=1.9Hz, J=8.9Hz, H-’) 7.65 (2H, d, J=9.0Hz, H-”,”, . (2H, d, J=9.0Hz, H-”,5”, 3.79-3.69 (4H, m, H-”’,”’, .1-3.03 (4H, m, H-”’,5”’.    233  N'-((1H-indazol-5-yl)carbamothioyl)-N,N-dimethylformimidamide 82:    N,N-Dimethylformamide dimethoxyacetal 81 (2.5 mL, 18.75 mmol) was added to a suspension of indazolyl thiourea 74h (3.0g, 15.61 mmol) in EtOH (25 mL). The mixture was heated under reflux for 3h. The reaction was concentrated in vacuo to remove solvent and excess DMF-DMA. The resulting crude product was purified by flash column chromatography (gradient elution, 1-10% MeOH in CH2Cl2) to afford 81 as a white solid (3.5 g, 91%).  1H-NMR (400 MHz, DMSO -d6): 12.98 (1H, d, J=4.5Hz, NH), 10.49 (1H, d, J=4.5Hz, NH), 8.77 (1H, d, H-3), 8.24-7.85 (2H, m, H-4,7), 7.55 (1H, dd, J=1.7Hz, J=8.8Hz, H-6), 7.47-7.39 (1H, m, N=CH), 3.23-2.95 (6H, m, 2 CH3). MS ESI (m/z): 246 [M-H]-.  2-Chloro-N-phenylacetamide 83a:  Aniline (0.3 g, 3.22 mmol) was dissolved in dry CH2Cl2 (8 mL) and Et3N (0.54 mL, 3.87 mmol) was added. The reaction mixture was cooled to 0 oC, and then 2-chloroacetyl chloride (0.31 mL, 3.87 mmol) was added dropwise. The reaction mixture was warmed to room temperature and stirred for an additional 12 h. The solvent was removed in vacuo and the residue was purified by flash column chromatography (gradient elution, 2-8% EtOAc in hexane) to afford 83a as an off white solid (0.5 g, 91%).  1H-NMR (400 MHz, DMSO -d6): +br s, NH), 7.61-7.55 (2H, m, H-’,’), 7.36-7.30 (2H,  m, H-’,5’), 7.12-7.05 (1H, m, H-’), 4.25 (2H, s, CH2). MS ESI (m/z): 168 [M-H]-.  2-Chloro-N-(3-phenoxyphenyl)acetamide 83b:  234  Prepared according to the procedure followed in 83a using 3-phenoxyaniline (0.6 g, 3.22 mmol). The crude product was purified by flash column chromatography (gradient elution, 2-12% EtOAc in hexane) to afford 83b as an off white solid (0.73 g, 87%). MS ESI (m/z): 260 [M-H]-.  2-Chloro-N-(3-methoxyphenyl)acetamide 83c:  Prepared according to the procedure followed in 83a using m-anisidine (0.4 g, 3.22 mmol). The crude product was purified by flash column chromatography (gradient elution, 3-14% EtOAc in hexane) to afford 83c as an off white solid (0.57 g, 89%). 1H-NMR (400 MHz, DMSO -d6): +br s, NH), 7.27 (1H, t, J=2.1Hz H-’), 7.23 (1H, t, J=8.0Hz H-5’), 7.12 (1H, dd, J=2.1Hz, J=8.0Hz,  H-’), 6.67 (1H, td, J=2.1Hz, J=8.0Hz, H-’), 4.23 (2H, s, CH2), 3.75 (3H, s, OCH3). MS ESI (m/z): 198 [M-H]-.  N-(Benzo[d][1,3]dioxol-5-yl)-2-chloroacetamide 83d:  Prepared according to the procedure followed in 83a using 3,4-(methylenedioxy) aniline (0.44 g, 3.22 mmol). The crude product was purified by flash column chromatography (gradient elution, 3-14% EtOAc in hexane) to afford 83d as an off white solid (0.64 g, 93%). 1H-NMR (400 MHz, DMSO -d6): 10.20 (1H, br s, NH), 7.27 (1H, d, J=2.1Hz, H-’), 6.96 (1H, dd, J=2.1Hz, J=8.4Hz, H-’), 6.87 (1H, d, J=8.4Hz, H-’), 5.98 (2H, s, OCH2O), 4.21 (2H, s, COCH2). MS ESI (m/z): 212 [M-H]-.  2-Chloro-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acetamide 83e:  Prepared according to the procedure followed in 83a using 1,4-benzodioxan-6-amine (0.44 g, 3.22 mmol). The crude product was purified by flash column chromatography (gradient elution, 2-14% EtOAc in hexane) to afford 83e as an off white solid (0.65 g, 89%). 235  1H-NMR (400 MHz, DMSO -d6):  10.11 (1H, br s, NH), 7.21 (1H, d, J=2.1Hz, H-5’), 6.96 (1H, dd, J=2.1Hz, J=8.4Hz, H-7’), 6.80 (1H, d, J=8.4Hz, H-’), 4.24-4.18 (6H, m, 2 OCH2,  COCH2).  MS ESI (m/z): 226 [M-H]-.  2-Chloro-N-(3,4-dimethoxyphenyl)acetamide 83f:  Prepared according to the procedure followed in 83a using 3,4-dimethoxyaniline (0.49 g, 3.22 mmol). The crude product was purified by flash column chromatography (gradient elution, 5-20% EtOAc in hexane) to afford 83f as an off white solid (0.68 g, 92%). 1H-NMR (400 MHz, DMSO -d6):  10.13 (1H, br s, NH), 7.26 (1H, d, J=2.3Hz, H-’), 7.10 (1H, dd, J=2.3Hz, J=8.7Hz, H-’), 6.90 (1H, d, J=8.7Hz, H-5’), 4.21 (2H, s, COCH2), 3.72 (6H, d, J=7.8Hz, 2 OCH3). MS ESI (m/z): 228 [M-H]-.  2-Chloro-N-(4-fluorophenyl)acetamide 83g:  Prepared according to the procedure followed in 83a using 4-fluoroaniline (0.49 g, 3.22 mmol). The crude product was purified by flash column chromatography (gradient elution, 2-12% EtOAc in hexane) to afford 83g as an off white solid (0.5 g, 83%). 1H-NMR (400 MHz, DMSO -d6):  10.31 (1H, br s, NH), 7.66-7.56 (2H, m, H-’,’, .21-7.12 (2H, m, H-’,5’, .5 (2H, s, COCH2). MS ESI (m/z): 186 [M-H]-.   2-Chloro-N-(4-methoxyphenyl)acetamide 83h:  Prepared according to the procedure followed in 83a using p-anisidine (0.4 g, 3.22 mmol). The crude product was purified by flash column chromatography (gradient elution, 4-14% EtOAc in hexane) to afford 83h as an off white solid (0.58 g, 91%). 236  1H-NMR (400 MHz, DMSO -d6):  10.16 (1H, br s, NH), 7.50 (2H, d, J=9.0Hz, H-’,’, 6.90 (2H, d, J=9.0Hz, H-’,5’, .1 (2H, s, COCH2), 3.72 (3H, s, OCH3). MS ESI (m/z): 198 [M-H]-.  2-Chloro-N-(6-methoxypyridin-3-yl)acetamide 83i:  Prepared according to the procedure followed in 83a using 5-amino-2-methoxypyridine (0.4 g, 3.22 mmol). The crude product was purified by flash column chromatography (gradient elution, 5-18% EtOAc in hexane) to afford 83i as a pale pink solid (0.56 g, 87%). 1H-NMR (400 MHz, DMSO -d6):  10.31 (1H, br s, NH), 8.34 (1H, d, J=2.6Hz, H-’), 7.89 (1H, dd, J=2.6Hz, J=8.9Hz, H-’), 6.82 (1H, d, J=8.9Hz, H-5’), 4.25 (2H, s, COCH2), 3.82 (3H, s, OCH3). MS ESI (m/z): 199 [M-H]-.  2-Chloro-N-(4-methoxybenzyl)acetamide 83j:  Prepared according to the procedure followed in 83a using 4-methoxybenzylamine (0.4 g, 3.22 mmol). The crude product was purified by flash column chromatography (gradient elution, 2-12% EtOAc in hexane) to afford 83j as an off white solid (0.65 g, 95%). 1H-NMR (400 MHz, DMSO -d6):  8.64 (1H, t, J=6.1Hz, NH), 7.19 (2H, d, J=8.8Hz, H-’,’), 6.88 (2H, d, J=8.8Hz, H-’,5’), 4.22 (2H, d, J=6.1Hz, NCH2), 4.09 (2H, s, COCH2), 3.72 (3H, s, OCH3). MS ESI (m/z): 212 [M-H]-.  2-Chloro-N-(3,4,5-trimethoxyphenyl)acetamide 83k:  237  Prepared according to the procedure followed in 83a using 3,4,5-trimethoxyaniline (0.59 g, 3.22 mmol). The crude product was purified by flash column chromatography (gradient elution, 5-18% EtOAc in hexane) to afford 83k as an off white solid (0.72 g, 86%). 1H-NMR (400 MHz, DMSO -d6):  10.24 (1H, br s, NH), 6.98 (2H, s, H-’,’), 4.24 (2H, s, COCH2), 3.76 (6H, s, 2 OCH3), 3.63 (3H, s, OCH3). MS ESI (m/z): 258 [M-H]-.  2-Chloro-N-(4-(methylthio)phenyl)acetamide 83l:  Prepared according to the procedure followed in 83a using 4-(methylthio)aniline (0.45 g, 3.22 mmol). The crude product was purified by flash column chromatography (gradient elution, 2-12% EtOAc in hexane) to afford 83l as a pale yellow solid (0.65 g, 94%). 1H-NMR (400 MHz, DMSO -d6):  10.29 (1H, br s, NH), 7.54 (2H, d, J=9.0Hz, H-’,’), 7.24 (2H, d, J=9.0Hz, H-’,5’), 4.23 (2H, s, COCH2), 2.44 (3H, s, SCH3).  2-Chloro-N-(3,4-dimethoxybenzyl)acetamide 83m:  Prepared according to the procedure followed in 83a using 3,4-dimethoxybenzylamine (0.54 g, 3.22 mmol). The crude product was purified by flash column chromatography (gradient elution, 5-15% EtOAc in hexane) to afford 83m as an off white solid (0.69 g, 88%). 1H-NMR (400 MHz, DMSO -d6):  8.63 (1H, t, J=6.1Hz, NH), 6.92-6.82 (2H, m, H-’,5’), 6.78 (1H, dd, J=1.5Hz, J=8.2Hz, H-’), 4.23 (2H, d, J=6.1Hz, NCH2), 4.10 (2H, s, COCH2), 3.73 (6H, d, J=7.8Hz, 2 OCH3). MS ESI (m/z): 242 [M-H]-.  2-Chloro-N-(4-(dimethylamino)phenyl)acetamide 83n:  Prepared according to the procedure followed in 83a using N,N-dimethyl-p-phenylenediamine (0.44 g, 3.22 mmol). The crude product was purified by flash column 238  chromatography (gradient elution, 5-18% EtOAc in hexane) to afford 83n as a pale green solid (0.55 g, 81%). 1H-NMR (400 MHz, DMSO -d6):  7.12 (2H, d, J=9.0Hz, H-’,’), 6.75 (2H, d, J=9.0Hz, H-’,5’), 4.53 (2H, s, COCH2), 2.95 (6H, s, 2 CH3). MS ESI (m/z): 213 [M+H]+.  2-Chloro-N-(4-morpholinophenyl)acetamide 79o:  Prepared according to the procedure followed in 83a using 4-morpholinoaniline (0.57 g, 3.22 mmol). The crude product was purified by flash column chromatography (gradient elution, 4-16% EtOAc in hexane) to afford 83o as a pale yellow solid (0.67 g, 82%). 1H-NMR (400 MHz, DMSO -d6):  10.05 (1H, br s, NH), 7.44 (2H, d, J=9.0Hz, H-’,’), 6.91 (2H, d, J=9.0Hz, H-’,5’), 4.19 (2H, s, COCH2), 3.76-369 (4H, m, H-”,”), 3.08-3.00 (4H, m, H-”,5”). MS ESI (m/z): 253 [M-H]-.  2-((1H-Indazol-5-yl)amino)-N-phenylthiazole-5-carboxamide 85a:   Chloroacetamide derivative 83a (0.068 g, 0.4 mmol) was added to a suspension of disubstituted thiourea derivative 82 (0.1 g, 0.4 mmol) in MeOH (5 mL). The mixture was heated under reflux for 6h. The completion of the reaction was monitored by TLC, and then the solvent was evaporated under vacuum. The resulting residue was silica flash column chromatographed (gradient elution, 3í12% MeOH in CH2Cl2) to afford 85a as an off white solid (0.05 g, 37%). 1H-NMR (400 MHz, DMSO -d6): see Table 31.   239  MS ESI (m/z): 334 [M-H]-, HRMS: m/z calculated for C17H13N5OS-: 334.0768, found: 334.07709.  2-((1H-Indazol-5-yl)amino)-N-(3-phenoxyphenyl)thiazole-5-carboxamide 85b:  Prepared according to 85a, disubstituted thiourea derivative 82 (0.1 g, 0.4 mmol) was reacted with chloroacetamide derivative 83b (0.1 g, 0.4 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 85b as an off white solid (0.068 g, 40%). 1H-NMR (400 MHz, DMSO -d6): see Table 31. MS ESI (m/z): 426 [M-H]-, HRMS: m/z calculated for C23H17N5O2S-: 426.10302, found: 426.10345.  2-((1H-Indazol-5-yl)amino)-N-(3-methoxyphenyl)thiazole-5-carboxamide 85c:  Prepared according to 85a, disubstituted thiourea derivative 82 (0.1 g, 0.4 mmol) was reacted with chloroacetamide derivative 83c (0.08 g, 0.4 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í15% MeOH in CH2Cl2) to afford 85c as an off white solid (0.057 g, 39%). 1H-NMR (400 MHz, DMSO -d6): see Table 31. MS ESI (m/z): 364 [M-H]-, HRMS: m/z calculated for C18H15N5O2S-: 364.08737, found: 364.08783.    240  2-((1H-Indazol-5-yl)amino)-N-(benzo[d][1,3]dioxol-5-yl)thiazole-5-carboxamide 85d:  Prepared according to 85a, disubstituted thiourea derivative 82 (0.1 g, 0.4 mmol) was reacted with chloroacetamide derivative 83d (0.085 g, 0.4 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í15% MeOH in CH2Cl2) to afford 85d as an off white solid (0.065 g, 43%). 1H-NMR (400 MHz, DMSO -d6): see Table 31. MS ESI (m/z): 378 [M-H]-, HRMS: m/z calculated for C18H13N5O3S-: 378.06663, found: 378.06729. 2-((1H-Indazol-5-yl)amino)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)thiazole-5-carboxamide 85e:  Prepared according to 85a, disubstituted thiourea derivative 82 (0.1 g, 0.4 mmol) was reacted with chloroacetamide derivative 83e (0.09 g, 0.4 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í15% MeOH in CH2Cl2) to afford 85e as a pale yellow solid (0.075 g, 48%). 1H-NMR (400 MHz, DMSO -d6): see Table 31. 13C NMR (100MHz, DMSO -d6): 168, 159.4, 143.3, 142.5, 140.0, 137.0, 134.2, 133.8, 132.9, 123.4, 123.2, 120.2, 117.2, 113.8, 111.2, 109.7, 108.4, 64.6, 64.4.  MS ESI (m/z): 392 [M-H]-, HRMS: m/z calculated for C19H15N5O3S-: 392.08228, found: 392.08325.   241  2-((1H-Indazol-5-yl)amino)-N-(3,4-dimethoxyphenyl)thiazole-5-carboxamide 85f:   Prepared according to 85a, disubstituted thiourea derivative 82 (0.1 g, 0.4 mmol) was reacted with chloroacetamide derivative 83f (0.092 g, 0.4 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 5í18% MeOH in CH2Cl2) to afford 85f as a beige solid (0.055 g, 35%). 1H-NMR (400 MHz, DMSO -d6): see Table 31. 13C NMR (100MHz, DMSO -d6):  167.9, 159.4, 148.9, 145.5, 142.4, 138.3, 137.8, 134.3, 133.9, 132.8, 123.5, 121.0, 112.6, 111.9, 111.2, 108.4, 105.8, 56.2, 55.8. MS ESI (m/z): 394 [M-H]-, HRMS: m/z calculated for C19H17N5O3S-: 394.09793, found: 394.09866.  2-((1H-Indazol-5-yl)amino)-N-(4-fluorophenyl)thiazole-5-carboxamide 85g:  Prepared according to 85a, disubstituted thiourea derivative 82 (0.1 g, 0.4 mmol) was reacted with chloroacetamide derivative 83g (0.075 g, 0.4 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 85g as a yellow solid (0.04 g, 29%). 1H-NMR (400 MHz, DMSO -d6): see Table 31. 13C NMR (100MHz, DMSO -d6): 168.2, 159.8, 157.4, 143.0, 135.7, 135.0, 134.1, 133.8, 129.0, 123.4, 122.9, 122.4, 120.8, 120.3, 115.8, 115.6, 111.2, 108.6. MS ESI (m/z): 352 [M-H]-, HRMS: m/z calculated for C17H12N5OSF-: 352.06738, found: 352.06812.    242  2-((1H-Indazol-5-yl)amino)-N-(4-methoxyphenyl)thiazole-5-carboxamide 85h:  Prepared according to 85a, disubstituted thiourea derivative 82 (0.1 g, 0.4 mmol) was reacted with chloroacetamide derivative 83h (0.08 g, 0.4 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 3í15% MeOH in CH2Cl2) to afford 85h as an off white solid (0.067 g, 46%). 1H-NMR (400 MHz, DMSO -d6): see Table 31. MS ESI (m/z): 364 [M-H]-, HRMS: m/z calculated for C18H15N5O2S-: 364.08737, found: 364.08804.  2-((1H-Indazol-5-yl)amino)-N-(6-methoxypyridin-3-yl)thiazole-5-carboxamide 85i:   Prepared according to 85a, disubstituted thiourea derivative 82 (0.1 g, 0.4 mmol) was reacted with chloroacetamide derivative 83i (0.08 g, 0.4 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 3í15% MeOH in CH2Cl2) to afford 85i as a pale brown solid (0.048 g, 33%). 1H-NMR (400 MHz, DMSO -d6): see Table 31. MS ESI (m/z): 365 [M-H]-, HRMS: m/z calculated for C17H14N6O2S-: 365.08262, found: 365.08337.  2-((1H-Indazol-5-yl)amino)-N-(4-methoxybenzyl)thiazole-5-carboxamide 85j:  243  Prepared according to 85a, disubstituted thiourea derivative 82 (0.1 g, 0.4 mmol) was reacted with chloroacetamide derivative 83j (0.085 g, 0.4 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to afford 85j as a yellow solid (0.074 g, 49%). 1H-NMR (400 MHz, DMSO -d6): see Table 31. 13C NMR (100MHz, DMSO -d6): 167.5, 161, 158.7, 141.7, 141.5, 137.0, 134.3, 133.7, 132.0, 129.1, 123.4, 123.0, 120.1, 114.2, 111.1, 108.2, 55.5, 42.3. MS ESI (m/z): 378 [M-H]-, HRMS: m/z calculated for C19H17N5O2S-: 378.10302, found: 378.10342.  2-((1H-Indazol-5-yl)amino)-N-(3,4,5-trimethoxyphenyl)thiazole-5-carboxamide 85k:  Prepared according to 85a, disubstituted thiourea derivative 82 (0.1 g, 0.4 mmol) was reacted with chloroacetamide derivative 83k (0.1 g, 0.4 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 3í18% MeOH in CH2Cl2) to afford 85k as a pale brown solid (0.064 g, 38%). 1H-NMR (400 MHz, DMSO -d6): see Table 31. 13C NMR (100MHz, DMSO -d6): 168.1, 159.6, 153.1, 142.7, 137.1, 135.5, 134.1, 134.0, 133.8, 129.0, 123.4, 123.2, 120.9, 120.3, 111.2, 108.5, 98.2, 60.6, 56.2.   MS ESI (m/z): 424 [M-H]-, HRMS: m/z calculated for C20H19N5O4S-: 424.10850, found: 424.10950.  2-((1H-Indazol-5-yl)amino)-N-(4-(methylthio)phenyl)thiazole-5-carboxamide 85l:   244  Prepared according to 85a, disubstituted thiourea derivative 82 (0.1 g, 0.4 mmol) was reacted with chloroacetamide derivative 83l (0.086 g, 0.4 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to afford 85l as a beige solid (0.07 g, 47%). 1H-NMR (400 MHz, DMSO -d6): see Table 31. MS ESI (m/z): 380 [M-H]-, HRMS: m/z calculated for C18H15N5OS2-: 380.06452, found: 380.06528.  2-((1H-Indazol-5-yl)amino)-N-(3,4-dimethoxybenzyl)thiazole-5-carboxamide 85m:  Prepared according to 85a, disubstituted thiourea derivative 82 (0.1 g, 0.4 mmol) was reacted with chloroacetamide derivative 83m (0.097 g, 0.4 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 3í12% MeOH in CH2Cl2) to afford 85m as a pale green solid (0.08 g, 49%). 1H-NMR (400 MHz, DMSO -d6): see Table 31. MS ESI (m/z): 408 [M-H]-, HRMS: m/z calculated for C20H19N5O3S-: 408.11358, found: 408.11441.  2-((1H-Indazol-5-yl)amino)-N-(4-(dimethylamino)phenyl)thiazole-5-carboxamide 85n:  Prepared according to 85a, disubstituted thiourea derivative 82 (0.1 g, 0.4 mmol) was reacted with chloroacetamide derivative 83n (0.085 g, 0.4 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 3í15% MeOH in CH2Cl2) to afford 85n as a pale green solid (0.047 g, 31%). 1H-NMR (400 MHz, DMSO -d6): see Table 31. 245  MS ESI (m/z): 377 [M-H]-, HRMS: m/z calculated for C19H18N6OS-: 377.11900, found: 377.11948.  2-((1H-Indazol-5-yl)amino)-N-(4-morpholinophenyl)thiazole-5-carboxamide 85o:  Prepared according to the procedure followed in 85a using chloroacetamide derivatives 83o (0.1 g, 0.4 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 3í15% MeOH in CH2Cl2) to afford 85o as an off white solid (0.072 g, 43%). 1H-NMR (400 MHz, DMSO -d6): see Table 31. MS ESI (m/z): 419 [M-H]-, HRMS: m/z calculated for C21H20N6O2S-: 419.12957, found: 419.13022.  Table 31: NMR data of compounds 85a-o  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6)  ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  85a   13.02 (1H, br s, N1’-H), 10.59 (1H, br s, C2-NH), 10.05 (1H, br s, CO-NH), 8.19 (1H, d, J=1.7Hz, H-’, 8.13 (1H, s, H-4), 8.06 (1H, s, H-’, 7.54 (1H, d, J=8.9Hz, H-’, 7.41 (1H, dd, J=1.7Hz, J=8.9Hz, H-’   7.70-7.64 (2H, m, H-”,”, 7.36-7.29 (2H,  m, H-”,5”, 7.11-7.04 (1H, m, H-” 246  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6)  ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  85b   13.02 (1H, br s, N1’-H), 10.63 (1H, br s, C2-NH), 10.02 (1H, br s, CO-NH), 8.19 (1H, d, J=1.9Hz, H-’), 8.14 (1H, s, H-4), 8.05 (1H, s, H-’), 7.53 (1H, d, J=9.1Hz, H-’), 7.42 (1H, dd, J=1.9Hz, J=9.1Hz, H-’)  7.63 (1H, ddd, J=0.9Hz, J=2.6Hz, J=8.1Hz, Ar-H) 7.51 (1H, t, J=2.6Hz, H-”), 7.44-7.33 (3H, m, Ar-H), 7.21-7.12 (1H, m, Ar-H), 7.12-7.05 (2H, m, Ar-H), 6.87-6.79 (1H, ddd, J=0.9Hz, J=2.6Hz, J=8.1Hz, Ar-H)  85c   13.03 (1H, br s, N1’-H), 10.61 (1H, br s, C2-NH), 10.04 (1H, br s, CO-NH), 8.20 (1H, d, J=1.9Hz, H-’), 8.14 (1H, s, H-4), 8.07 (1H, s, H-’), 7.55 (1H, d, J=9.1Hz, H-’), 7.42 (1H, dd, J=1.9Hz, J=9.1Hz, H-’) 7.38 (1H, t, J=2.1Hz H-”, .-7.25 (2H, m, H-5”,”, . 1H, td, J=2.1Hz, J=7.9Hz, H-”, . H, s, OCH3)   85d   13.01 (1H, br s, N1’-H), 10.57 (1H, br s, C2-NH), 9.97 (1H, br s, CO-NH), 8.18 (1H, d, J=2.0Hz, H-’), 8.08 (1H, s, H-4), 8.05 (1H, s, H-’), 7.53 (1H, d, J=8.9Hz, H-’), 7.40 (1H, dd, J=2.0Hz, J=8.9Hz, H-’) 7.33 (1H, d, J=2.1Hz, H-”, .0 (1H, dd, J=2.1Hz, J=8.4Hz, H-”, 6.88 (1H, d, J=8.4Hz, H-”, .01 (2H, s, OCH2O)   85e    13.10 (1H, br s, N1’-H), 11.67 (1H, br s, C2-NH), 11.18 (1H, br s, CO-NH), 8.22 (1H, d, J=1.9Hz, H-’), 8.11 (1H, s, H-’), 8.04 (1H, s, H-4), 7.61-7.48* (3H, m, J=8.9Hz, H-’,’) 7.61-7.48* (3H, m, H-”, .1 (1H, s, H-5”, .0 1H, d, - .5Hz, H-”, .04 (2H, s, OCH2), 3.94 (2H, s, OCH2)  85f    13.01 (1H, br s, N1’-H), 10.55 (1H, br s, C2-NH), 9.94 (1H, br s, CO-NH), 8.19 (1H, d, J=2.0Hz, H-’), 8.09 (1H, s, H-4), 8.05 (1H, s, H-’), 7.53 (1H, d, J=8.9Hz, H-’), 7.41 (1H, dd, J=2.0Hz, J=8.9Hz, H-’) 7.35 (1H, d, J=2.3Hz, H-”, .0 (1H, dd, J=2.3Hz, J=8.7Hz, H-”, 6.92 (1H, d, J=8.7Hz, H-5”, .5 (3H, s, OCH3), 3.73 (3H, s, OCH3)  247  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6)  ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm   85g   13.01 (1H, br s, N1’-H), 10.30 (1H, br s, C2-NH), 10.11 (1H, br s, CO-NH), 8.18 (1H, d, J=1.8Hz, H-’), 8.11 (1H, s, H-4), 8.05 (1H, s, H-’), 7.53 (1H, d, J=9.0Hz, H-’), 7.40 (1H, dd, J=1.8Hz, J=9.0Hz, H-’) 7.72-7.65 (2H, m, H”,”, .-7.13 (2H, m, H-”,5”    85h   13.01 (1H, br s, N1’-H), 10.55 (1H, br s, C2-NH), 9.95 (1H, br s, CO-NH), 8.19 (1H, d, J=1.8Hz, H-’), 8.08 (1H, s, H-4), 8.05 (1H, s, H-’), 7.53 (1H, d, J=9.1Hz, H-’), 7.40 (1H, dd, J=1.8Hz, J=9.1Hz, H-’) 7.56 (2H, d, J=9.0Hz, H-”,”, 6.91 (2H, d, J=9.0Hz, H-”,5”, 3.74 (3H, s, OCH3)    85i    13.01 (1H, br s, N1’-H), 10.60 (1H, br s, C2-NH), 10.11 (1H, br s, CO-NH), 8.19 (1H, d, J=2.1Hz, H-’), 8.09 (1H, s, H-4), 8.05 (1H, s, H-’), 7.54 (1H, d, J=9.1Hz, H-’), 7.40 (1H, dd, J=2.1Hz, J=9.1Hz, H-’) 8.41 (1H, d, J=2.6Hz, H-”, .5 (1H, dd, J=2.6Hz, J=8.9Hz, H-”, 6.83 (1H, d, J=8.9Hz, H-5”, . (3H, s, OCH3)   85j    12.98 (1H, br s, N1’-H), 10.45 (1H, br s, C2-NH), 8.77 (1H, t, J=6.5Hz, CO-NH), 8.18 (1H, d, J=1.8Hz, H-’), 8.03 (1H, s, H-’), 7.90 (1H, s, H-4), 7.51 (1H, d, J=8.9Hz, H-’), 7.38 (1H, dd, J=1.8Hz, J=8.9Hz, H-’) 7.23 (2H, d, J=8.8Hz, H-2”,”, 6.90 (2H, d, J=8.8Hz, H-”,5”, 4.34 (2H, d, J=6.1Hz, CH2), 3.73 (3H, s, OCH3)   85k   13.01 (1H, br s, N1’-H), 10.59 (1H, br s, C2-NH), 9.99 (1H, br s, CO-NH), 8.20 (1H, d, J=2.1Hz, H-’), 8.11 (1H, s, H-4), 8.05 (1H, s, H-’), 7.53 (1H, d, J=9.0Hz, H-’), 7.41 (1H, dd, J=2.1Hz, J=9.0Hz, H-’) 7.10 (2H, s, H-”,”, . H, s,  OCH3), 3.63 (3H, s, OCH3)   85l   13.03 (1H, br s, N1’-H), 10.61 (1H, br s, C2-NH), 10.08 (1H, br s, CO-NH), 8.20 (1H, d, J=1.9Hz, H-’), 8.13 (1H, s, H-4), 8.07 (1H, s, H-’), 7.55 (1H, d, J=9.1Hz, H-’), 7.42 (1H, dd, J=1.9Hz, J=9.1Hz, H-’) 7.65 (2H, d, J=9.0Hz, H-”,”, 7.27 (2H, d, J=9.0Hz, H-”,5”, 2.46 (3H, s, SCH3) 248  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6)  ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  85m   12.98 (1H, br s, N1’-H), 10.45 (1H, br s, C2-NH), 8.75 (1H, t, J=6.4Hz, CO-NH), 8.18 (1H, d, J=1.9Hz, H-’), 8.03 (1H, s, H-’), 7.91 (1H, s, H-4), 7.51 (1H, d, J=8.9Hz, H-’), 7.38 (1H, dd, J=1.9Hz, J=8.9Hz, H-’) 6.92 (1H, d, J=1.5Hz, H-”, .1 (1H, d, J=8.2Hz, H-5”, . 1H, dd, J=1.5Hz, J=8.2Hz, H-”, . (2H, d, J=6.1Hz, CH2), 3.74 (3H, s, OCH3), 3.72 (3H, s, OCH3)   85n   13.00 (1H, br s, N1’-H), 10.52 (1H, br s, C2-NH),  9.82 (1H, br s, CO-NH), 8.20 (1H, d, J=2.0Hz, H-’), 8.08-8.02 (2H, m, H-4, ’), 7.53 (1H, d, J=8.9Hz, H-’), 7.40 (1H, dd, J=2.0Hz, J=8.9Hz, H-’) 7.45 (d, 1H, J=9.1Hz, H-”,”, 6.72 (1H, d, J=9.1Hz, H-”,5”, 2.86 (6H, s, 2 CH3)   85o   13.01 (1H, br s, N1’-H), 10.54 (1H, br s, C2-NH),  9.90 (1H, br s, CO-NH), 8.19 (1H, d, J=1.8Hz, H-’), 8.07 (1H, s, H-4), 8.05 (1H, s, H-’),  7.53 (1H, d, J=8.8Hz, H-’), 7.40 (1H, dd, J=1.8Hz, J=8.8Hz, H-’) 7.51 (2H, d, J=9.0Hz, H-”,”, 6.93 (2H, d, J=9.0Hz, H-”,5”, 3.78-3.70 (4H, m, 2 OCH2), 3.10-3.03 (4H, m, 2 NCH2)  4-Nitroindazole 87:  To a cooled solution of 2-methyl-3-nitroaniline 86 (5.0 g, 32.9 mmol) in glacial acetic acid (75 mL) at 0 °C, was added dropwise a cooled  solution of sodium nitrite (2.72 g, 38.63 mmol) in water (12 mL). The resulting solution was stirred at 0 oC for 1h then allowed to warm to room temperature. Then, the reaction mixture is allowed to stand at room temperature for 3 days. The solution was concentrated under reduced pressure. The residue was diluted with EtOAc and washed thoroughly with saturated aqueous NaHCO3, brine, and water. The organic layer was dried over Na2SO4, filtered, and the solvent was removed in vacuo. The resulting residue was silica flash column chromatographed (gradient elution, 2-10% EtOAc in hexane) to afford 87 as a yellow powder in 89% yield. 1H-NMR (400 MHz, DMSO -d6):  13.91 (1H, br s, N1-H), 8.54 (1H, s, H-3), 8.16 (1H, dd, J=0.5Hz, J=7.7Hz, H-5), 8.09 (1H, d, J=8.3Hz, H-7), 7.61 (1H, t, J=7.9Hz, H-6). 249  4-Amino-1H-indazole 88:  To a solution of 4-nitroindazole 87 (3.26 g, 20 mmol) in MeOH (50 mL), Pd/C (0.5 g, 10% w/w) and NH2NH2.H2O (1.3 mL, 26.15 mmol) were added. The reaction mixture was heated under reflux for 3h. The solution was filtered hot through a bed of Celite then washed with hot MeOH. The solvent was removed under reduced pressure and the resulting residue was purified by flash column chromatography (gradient elution, 1-6% MeOH in CH2Cl2) to afford 88 as a white powder (5.04g, 83%). 1H-NMR (400 MHz, DMSO -d6):  12.59 (1H, br s, N1-H), 8.07 (1H, s, H-3), 6.97 (1H, t, J=7.9Hz, H-6), 6.60 (1H, d, J=8.2Hz, H-7), 6.11 (1H, d, J=7.4Hz, H-5), 5.68 (2H, br s, NH2). MS ESI (m/z): 134 [M+H]+.  N-((1H-Indazol-4-yl)carbamothioyl)benzamide 89:  To a solution of 4-amino-1H-indazole 88 (4 g, 30.04 mmol) in acetone (40 mL) was added benzoyl isothiocyanate (4.43 mL, 30.67 mmol). The reaction mixture was stirred at ambient temperature for 2h. After completion of reaction (TLC monitoring), the precipitate was filtered, washed with acetone, and dried to afford the product 89 as a white reddish crystalline solid (6.68 g, 75%) which was used for the next step without further purification. 1H-NMR (400 MHz, DMSO -d6):  13.30 (1H, br s, NH), 13.14 (1H, br s, NH), 11.73 (1H, br s, NH), 8.08 (1H, s, H-3’), 8.03 (2H, d, J=7.5Hz, H-2,6), 7.96 (1H, d, J=7.0Hz, H-5’), 7.68 (1H, t, J=7.3Hz, H-4), 7.56 (2H, t, J=7.7Hz, H-3,5), 7.47 (1H, d, J=8.3Hz, H-7’), 7.40 (1H, t, J=7.8Hz, H-6’). MS ESI (m/z): 295 [M-H]-.    250  1-(1H-Indazol-4-yl)thiourea 90:  To a suspension of  89 (5g, 16.87 mmol) in MeOH (50 mL) was added LiOH solution (0.13 g, 5.6 mmol) in H2O (2 mL) at room temperature,  a clear solution resulted in 15 minutes. The reaction mixture was heated up to 75 oC for 1h. After completion of reaction (TLC monitoring), the solvent was concentrated and the precipitated compound was separated and washed on the filter with methanol and dried under high vacuum to get the desired product 90 as a white solid (2.5g, 77%). The product was used for the next step without further purification. 1H-NMR (400 MHz, DMSO -d6):  13.12 (1H, br s, NH), 9.88 (1H, br s, NH), 8.02 (1H, s, H-’), 7.31 (5H, m, H-5’,’,’, 1H2).  Ethyl 2-((1H-indazol-4-yl)amino)thiazole-4-carboxylate 91:  To a suspension of thiourea derivative 90 (2.0 g, 10.4 mmol) in ethanol (20 mL) was added ethyl 2-bromopyruvate (1.31 mL, 10.4 mmol). The reaction was heated at 90 oC for 3h and allowed to cool to room temperature. The solvent was evaporated under vacuum, and the resulting crude oil was purified by flash column chromatography (gradient elution, 2-8% MeOH in CH2Cl2) to afford 91 as a white powder (2.06g, 72%). 1H-N MR (400 MHz, DMSO-d6):  13.08 (1H, br s, N1’-H), 10.57 (1H, br s, C2-NH), 8.36 (1H, s, H-’), 7.90 (1H, d, J=7.6Hz, H-’), 7.85 (1H, s, H-5), 7.31 (1H, t, J=8.0Hz, H-6’), 7.15 (1H, d, J=8.3Hz, H-5’), 4.29 (2H, q, J=7.1Hz, CH2), 1.31 (3H, t, J=7.1Hz, CH3). MS ESI (m/z): 287 [M-H]-.   251  2-((1H-Indazol-4-yl)amino)thiazole-4-carboxylic acid 92:  To a stirred suspension of ester 91 (2.0g, 6.94 mmol) in MeOH (20ml), LiOH (0.5g, 20.81 mmol) in water (5mL) was added. The mixture was stirred at room temperature for 12 h. The solvent was evaporated in vacuo. The resulting residue was dissolved in water, and the pH was adjusted to 4 by careful addition of 1N HCl. The resulting precipitate was isolated by filtration, washed by water then MeOH, and dried in vacuo to afford the desired compounds 92 as an off white powder (1.5g, 78%) which was used for the next step without further purification. 1H-NMR (400 MHz, DMSO -d6):  13.05 (2H, br s, N1’-H, COOH), 10.52 (1H, br s, C2-NH), 8.38 (1H, s, H-’), 7.99 (1H, d, J=7.6Hz, H-’), 7.79 (1H, s, H-5), 7.31 (1H, t, J=8.0Hz, H-6’), 7.14 (1H, d, J=8.3Hz, H-5’). MS ESI (m/z): 261 [M+H]+, HRMS: m/z calculated for C11H8N4O2S+: 261.04407, found: 261.04428.  2-((1H-Indazol-4-yl)amino)-N-phenylthiazole-4-carboxamide 93a:  To a solution of acid 92 (0.1g, 0.38 mmol) and aniline (0.035 g, 0.38 mmol) in DMF (6 mL) was added HOAt (0.07 g, 0.42 mmol), EDC (0.081 g, 0.42 mmol) and DIEA (0.2 mL, 1.14 mmol). The reaction mixture was stirred at ambient temperature for 24h. The solvent was evaporated under vacuum and azeotroped twice with CH2Cl2. The resulting residue was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 93a as an off white solid (0.065 g, 51%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. 13C NMR (100MHz, DMSO -d6): 163.6, 159.7, 151.6, 146.2, 141.5, 140.1, 138.8, 133.5, 131.8, 129.3, 127.9, 124.4, 121.2, 115.5, 115.0, 107.3, 104.3. 252  MS ESI (m/z): 334 [M-H]-, HRMS: m/z calculated for C17H13N5OS-: 334.07680, found: 334.07727.  2-((1H-Indazol-4-yl)amino)-N-benzylthiazole-4-carboxamide 93b:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with benzylamine (0.04 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to afford 93b as an off white solid (0.074 g, 56%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. 13C NMR (100MHz, DMSO -d6): 163.4, 161.3, 146.3, 141.4, 140.2, 133.5, 131.8, 128.7, 127.9, 127.6, 127.2, 121.1, 114.8, 114.6, 107.1, 104.0, 42.7. MS ESI (m/z): 348 [M-H]-, HRMS: m/z calculated for C18H15N5OS-: 348.09245, found: 348.09244.  2-((1H-Indazol-4-yl)amino)-N-(pyridin-3-yl)thiazole-4-carboxamide 93c:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 3-aminopyridine (0.035 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í14% MeOH in CH2Cl2) to afford 93c as a creamy white solid (0.062 g, 49%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. 13C NMR (100MHz, DMSO -d6): 163.7, 160.3, 150.6, 145.7, 141.5, 140.0, 135.0, 133.4, 131.8, 128.9, 124.0, 120.7, 116.3, 114.9, 107.4, 104.3. MS ESI (m/z): 335 [M-H]-, HRMS: m/z calculated for C16H12N6OS-: 335.07205, found: 335.07248. 253  2-((1H-Indazol-4-yl)amino)-N-(4-fluorophenyl)thiazole-4-carboxamide 93d:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 4-fluoroaniline (0.042 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 93d as an off white solid (0.067 g, 50%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. 13C NMR (100MHz, DMSO -d6): 163.5, 160.1, 159.8, 157.8, 146.2, 141.4, 135.2, 133.5, 131.8, 127.9, 123.1, 115.8, 115.7, 115.6, 114.9, 107.3, 104.2.  MS ESI (m/z): 352 [M-H]-, HRMS: m/z calculated for C17H12FN5OS-: 352.06738, found: 352.06760.  2-((1H-indazol-4-yl)amino)-N-(benzo[d][1,3]dioxol-5-yl)thiazole-4-carboxamide 93e:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 3,4-(methylenedioxy) aniline (0.052 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í14% MeOH in CH2Cl2) to afford 93e as a green solid (0.08 g, 57%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. 13C NMR (100MHz, DMSO -d6): 163.5, 159.6, 147.5, 146.2, 144.0, 141.4, 133.4, 133, 131.8, 127.9, 115.4, 114.9, 114.2, 108.4, 107.3, 104.2, 103.2, 101.5.  MS ESI (m/z): 378 [M-H]-, HRMS: m/z calculated for C18H13N5O3S-: 378.06663, found: 378.06720.    254  2-((1H-Indazol-4-yl)amino)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)thiazole-4-carboxamide 93f:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 1,4-benzodioxan-6-amine (0.052 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í15% MeOH in CH2Cl2) to afford 93f as a beige solid (0.087 g, 58%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. 13C NMR (100MHz, DMSO -d6): 163.5, 159.5, 146.3, 143.3, 141.4, 140.3, 133.5, 132.4, 131.8, 127.9, 117.1, 115.3, 115.0, 114.4, 110.2, 107.3, 104.2, 64.6, 64.4. MS ESI (m/z): 392 [M-H]-, HRMS: m/z calculated for C19H15N5O3S-: 392.08228, found: 392.08249.  2-((1H-Indazol-4-yl)amino)-N-(3,4-dimethoxyphenyl)thiazole-4-carboxamide 93g:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 3,4-dimethoxyaniline (0.058 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 4í15% MeOH in CH2Cl2) to afford 93g as a green solid (0.078 g, 52%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. 13C NMR (100MHz, DMSO -d6): 163.6, 159.5, 148.9, 146.3, 145.8, 141.5, 133.5, 132.2, 131.9, 128.0, 115.2, 114.9, 113.2, 112.3, 107.3, 106.3, 104.3, 56.1, 49.1. MS ESI (m/z): 394 [M-H]-, HRMS: m/z calculated for C19H17N5O3S-: 394.09793, found: 394.09824.   255  2-((1H-Indazol-4-yl)amino)-N-(p-tolyl)thiazole-4-carboxamide 93h:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with p-toluidine (0.04 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to afford 93h as a beige solid (0.082 g, 62%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. 13C NMR (100MHz, DMSO -d6): 163.5, 159.6, 151.40, 146.30, 141.5, 140.1, 136.3, 135.1, 133.5, 133.4, 131.8, 129.5, 127.8, 121.1, 115.3, 107.3, 104.3, 21.0. MS ESI (m/z): 348 [M-H]-, HRMS: m/z calculated for C18H15N5OS-: 348.09245, found: 348.09283. 2-((1H-Indazol-4-yl)amino)-N-(4-methoxyphenyl)thiazole-4-carboxamide 93i:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with p-anisidine (0.047 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 3í14% MeOH in CH2Cl2) to afford 93i as a beige solid (0.077 g, 56%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. MS ESI (m/z): 364 [M-H]-.   2-((1H-Indazol-4-yl)amino)-N-(2-(methylthio)phenyl)thiazole-4-carboxamide 93j:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 2-(methylthio)aniline (0.053 g, 0.38 mmol). The resulting residue was silica flash column chromatographed 256  (gradient elution, 3í12% MeOH in CH2Cl2) to afford 93j as an off white solid (0.068 g, 47%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. MS ESI (m/z): 380 [M-H]-, HRMS: m/z calculated for C18H15N5OS2-: 380.06452, found: 380.06494.  2-((1H-Indazol-4-yl)amino)-N-(4-morpholinophenyl)thiazole-4-carboxamide 93k:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 4-morpholinoaniline (0.067 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 3í14% MeOH in CH2Cl2) to afford 93k as a pale green solid (0.086 g, 54%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. 13C NMR (100MHz, DMSO -d6):163.50, 159.41, 156.20, 151.40, 146.40, 141.40, 140.10, 135.1, 133.5, 131.90, 129.30, 127.90, 122.90, 121.10, 115.10, 114.90, 114.20, 107.20, 104.2, 55.70, 49.0.  MS ESI (m/z): 419 [M-H]-, HRMS: m/z calculated for C21H20N6O2S-: 419.12957, found: 419.13004. 2-((1H-Indazol-4-yl)amino)-N-(3-methoxyphenyl)thiazole-4-carboxamide 93l:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with g m-anisidine (0.047 g, 0.38 mmol).  The resulting residue was silica flash column chromatographed (gradient elution, 3í14% MeOH in CH2Cl2) to afford 93l as a pale beige solid (0.07 g, 51%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. MS ESI (m/z): 364 [M-H]-, HRMS: m/z calculated for C18H15N5O2S-: 364.08737, found: 364.08786.  257  2-((1H-Indazol-4-yl)amino)-N-(2,3-dihydro-1H-inden-5-yl)thiazole-4-carboxamide 93m:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 5-aminoindan (0.05 g, 0.38 mmol).   The resulting residue was silica flash column chromatographed (gradient elution, 2í10% MeOH in CH2Cl2) to afford 93m as a beige solid (0.07 g, 49%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. MS ESI (m/z): 374 [M-H]-, HRMS: m/z calculated for C20H17N5OS-: 374.1081, found: 374.10815.  2-((1H-Indazol-4-yl)amino)-N-(3-phenoxyphenyl)thiazole-4-carboxamide 93n:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 3-phenoxyaniline (0.07 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 93n as a greenish white solid (0.087 g, 54%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. 13C NMR (100MHz, DMSO -d6):163.6, 159.9, 157.3, 156.96, 151.5, 146.1, 141.43, 133.47, 131.8, 130.5, 130.4, 129.2, 127.7, 124.0, 121.2, 119.2, 115.9, 115.8, 115.0, 114.3, 111.0, 107.3, 104.3. MS ESI (m/z): 426 [M-H]-, HRMS: m/z calculated for C23H17N5O2S-: 426.10302, found: 426.10355.    258  2-((1H-Indazol-4-yl)amino)-N-(4-chlorophenyl)thiazole-4-carboxamide 93o:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 4-chloroaniline (0.048 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 93o as an off white solid (0.053 g, 38%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. 13C NMR (100MHz, DMSO -d6):163.6, 159.9, 151.2, 146.0, 141.5, 140.0, 137.9, 135.1, 133.4, 131.9, 129.2, 127.8, 127.8, 115.9, 114.9, 107.3, 104.2. MS ESI (m/z): 368 [M-H]-, HRMS: m/z calculated for C17H12N5OSCl-: 368.03783, found: 368.03784.  2-((1H-Indazol-4-yl)amino)-N-(3,4,5-trimethoxyphenyl)thiazole-4-carboxamide 93p:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 3,4,5-trimethoxyaniline (0.07 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 4í18% MeOH in CH2Cl2) to afford 93p as a whitish green solid (0.07 g, 44%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. 13C NMR (100MHz, DMSO -d6):163.5, 159.7, 153.1, 146.2, 141.4, 134.9, 134.3, 133.4, 131.8, 127.8, 115.4, 115.0, 107.2, 104.2, 98.8, 60.6, 56.2. MS ESI (m/z): 424 [M-H]-, HRMS: m/z calculated for C20H19N5O4S-: 424.10850, found: 424.10944.    259  2-((1H-Indazol-4-yl)amino)-N-(3-chloro-2-methoxyphenyl)thiazole-4-carboxamide 93q:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 3-choloro-4-methoxyaniline (0.06 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 4í15% MeOH in CH2Cl2) to afford 93q as an off white solid (0.044 g, 29%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. MS ESI (m/z): 398 [M-H]-, HRMS: m/z calculated for C18H14N5O2SCl-: 398.04840, found: 398.04846.  2-((1H-Indazol-4-yl)amino)-N-(3,4-dimethoxybenzyl)thiazole-4-carboxamide 93r:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 3,4-dimethoxybenzylamine (0.063 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 93r as an off white solid solid (0.11 g, 69%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. 13C NMR (100MHz, DMSO -d6):163.4, 161.1, 149.0, 148.2, 146.4, 141.4, 133.5, 132.6, 131.8, 127.8, 119.9, 114.8, 114.4, 112.1, 111.9, 107.0, 104.0, 56.0, 55.9, 42.5. MS ESI (m/z): 408 [M-H]-, HRMS: m/z calculated for C20H19N5O3S-: 408.11358, found: 408.11380.    260  2-((1H-Indazol-4-yl)amino)-N-(4-(trifluoromethyl)benzyl)thiazole-4-carboxamide 93s:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 4-(trifluoromethyl)benzylamine (0.066 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 93s as an off white solid solid (0.066 g, 42%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. 13C NMR (100MHz, DMSO -d6):163.4, 161.5, 146.1, 145.2, 141.4, 133.4, 131.66, 128.6, 128.0, 127.9, 127.7, 126.2, 125.7, 125.6, 114.9, 114.8, 107.1, 104.0, 42.5.  MS ESI (m/z): 416 [M-H]-, HRMS: m/z calculated for C19H14N5OSF3-: 416.07984, found: 416.08020.  2-((1H-Indazol-4-yl)amino)-N-(4-(methylthio)phenyl)thiazole-4-carboxamide 93t:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 4-(methylthio)aniline (0.053 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 93t as an off white solid (0.088 g, 61%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. 13C NMR (100MHz, DMSO -d6):163.5, 159.7, 146.2, 141.5, 136.2, 133.5, 133.1, 131.8, 127.9, 127.2, 121.8, 115.5, 114.9, 107.3, 104.3, 15.8. MS ESI (m/z): 335 [M-H]-, HRMS: m/z calculated for C18H15N5OS2-: 335.07205, found: 335.07248.   261  2-((1H-Indazol-4-yl)amino)-N-(6-methoxypyridin-3-yl)thiazole-4-carboxamide 93u:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 5-amino-2-methoxypyridine (0.047 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 3í18% MeOH in CH2Cl2) to afford 93u as a pink solid (0.088 g, 61%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. 13C NMR (100MHz, DMSO -d6):163.5, 160.6, 160.0, 146.0, 141.4, 140.1, 133.8, 133.4, 131.8, 129.7, 127.8, 115.8, 114.9, 110.4, 107.3, 104.2, 53.7. MS ESI (m/z): 365 [M-H]-, HRMS: m/z calculated for C17H14N6O2S-: 365.08262, found: 365.08301.  2-((1H-Indazol-4-yl)amino)-N-(4-methoxy-3-methylphenyl)thiazole-4-carboxamide 93v:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 4-methoxy-3-methylaniline (0.052 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 93v as a pale brown solid (0.086 g, 60%). 1H-NMR (400 MHz, D MSO-d6): see Table 32. 13C NMR (100MHz, DMSO -d6):163.5, 159.4, 154.3, 146.4, 141.3, 133.5, 131.8, 131.3, 127.9, 125.9, 123.9, 120.0, 115.0, 114.9, 110.6, 107.2, 104.1, 55.4, 16.4. MS ESI (m/z): 378 [M-H]-, HRMS: m/z calculated for C19H17N5O2S-: 378.10302, found: 378.10342.    262  2-((1H-Indazol-4-yl)amino)-N-(4-methoxybenzyl)thiazole-4-carboxamide 93w:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 4-methoxybenzylamine (0.052 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 93w as an off white solid (0.096 g, 67%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. 13C NMR (100MHz, DMSO -d6):163.4, 162.7, 161.1, 158.6, 146.4, 141.4, 133.5, 132.2, 131.7, 129.1, 127.85, 114.8, 114.4, 114.1, 107.0, 103.9, 55.5, 42.1. MS ESI (m/z): 378 [M-H]-, HRMS: m/z calculated for C19H17N5O2S-: 378.10302, found: 378.10330.  2-((1H-Indazol-4-yl)amino)-N-(pyridin-2-ylmethyl)thiazole-4-carboxamide 93x:  Prepared according to 93a, acid 92 (0.1g, 0.38 mmol) was reacted with 2-picolylamine (0.041 g, 0.38 mmol). The resulting residue was silica flash column chromatographed (gradient elution, 2í12% MeOH in CH2Cl2) to afford 93x as an off white solid (0.073 g, 55%). 1H-NMR (400 MHz, DMSO -d6): see Table 32. 13C NMR (100MHz, DMSO -d6):162.9, 160.8, 158.1, 148.9, 145.6, 140.9, 136.8, 133.1, 131.3, 127.4, 122.1, 121.1, 114.4, 114.1, 106.7, 103.6, 44.1. MS ESI (m/z): 349 [M-H]-, HRMS: m/z calculated for C17H14N6OS-: 349.08770, found: 349.08792.  263  Table 32: NMR data of compounds 93a-x   Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6)  ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  93a   13.08 (1H, br s, H-1’, 10.5 1H, br s, C2-NH), 9.78 (1H, br s, CO-NH), 8.38 (1H, s, H-’, .0 1H, d, - .Hz, H-’, .86-7.68* (3H, m, H-5), 7.45-7.30* (3H, m, H-’, .1 1H, d, - .Hz, H-5’  7.86-7.68* (3H, m, H-”,” .45-7.30* (3H,  m, H-”,5”, .1 1H, t, J=7.4Hz, H-”  93b   13.06 (1H, br s, H-1’, 10. 1H, br s, C2-NH), 8.65 (1H, t, J=6.4Hz, CO-NH), 8.37 (1H, s, H-’, .1 1H, d, - .Hz, H-’, 7.62 (1H, s, H-5), 7.37-7.27* (5H, m, H-’), 7.13 (1H, d, J=8.3Hz, H-5’ 7.37-7.27* (5H, m, H-”,”,5”,”, 7.26-7.20 (1H, m, H-”, .51 1H, d, J=6.4Hz, CH2)   93c   13.10 (1H, br s, H-1’, 10.55 1H, br s, C2-NH), 10.07 (1H, br s, CO-NH), 8.40 (1H, s, H-’, .1 1H, d, - 7.8  Hz, H-’, 7.84 (1H, s, H-5), 7.38 (1H, t, J=8.0Hz, H-’, .1 1H, d, - .2Hz, H-5’  8.97 (1H, d, J=2.3Hz, H-”, 8.35 (1H, dd, J=1.4Hz, J=4.7Hz, H-”, 8.23 (1H, ddd, J=1.4Hz, J=2.3Hz, J=8.2Hz, H-”, . 1H, dd, J=4.7Hz, J=8.2Hz, H-5”  93d   13.08 (1H, br s, H-1’, 10.5 1H, br s, C2-NH), 9.89 (1H, br s, CO-NH), 8.38 (1H, s, H-’, .11 1H, d, - .Hz, H-’, . (1H, s, H-5), 7.35 (1H, t, J=8.0Hz, H-’, 7.26-7.13* (3H, m, H-5’  7.87-7.78 H, m, H”,”, .26-7.13* (3H, m, H-”,5”  264  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6)  ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  93e   13.08 (1H, br s, H-1’, 10.51 1H, br s, C2-NH), 9.72 (1H, br s, CO-NH), 8.37 (1H, s, H-’, .0 1H, d, - .Hz, H-7’), 7.73 (1H, s, H-5), 7.35 (1H, t, J=8.0Hz, H-’), 7.16 (1H, d, J=8.3Hz, H-5’) 7.46 (1H, d, J=2.1Hz, H-”, .1 (1H, dd, J=2.1Hz, J=8.4Hz, H-”, 6.91 (1H, d, J=8.4Hz, H-”, .0 (2H, s, OCH2O)   93f   13.09 (1H, br s, H-1’, 10.5 1H, br s, C2-NH), 9.65 (1H, br s, CO-NH), 8.37 (1H, s, H-’), 8.08 (1H, d, J=7.6Hz, H-7’), 7.72 (1H, s, H-5), 7.35 (1H, t, J=7.9Hz, H-’), 7.26-7.10* (2H, m, H-5’ 7.41 (1H, s, H-5”, .-7.10* (2H, m, H-”, . 1H, d, - .5Hz, H-”, . H, s,  2CH2)   93g   13.08 (1H, br s, H-1’, 10.5 1H, br s, C2-NH), 9.66 (1H, br s, CO-NH), 8.38 (1H, s, H-’), 8.08 (1H, d, J=7.6Hz, H-7’), 7.72 (1H, s, H-5), 7.35 (1H, t, J=7.9Hz, H-’), 7.17 (1H, d, J=8.3Hz, H-5’) 7.48 (1H, d, J=2.4Hz, H-”), 7.33 (1H, dd, J=2.4Hz, J=8.7Hz, H-”, 6.95 (1H, d, J=8.7Hz, H-5”, . (3H, s, OCH3), 3.74 (3H, s, OCH3)    93h   13.08 (1H, br s, H-1’, 10.5 1H, br s, C2-NH), 9.70 (1H, br s, CO-NH), 8.37 (1H, s, H-’), 8.08 (1H, d, J=7.5Hz, H-7’), 7.74 (1H, s, H-5), 7.36 (1H, t, J=7.9Hz, H-’), 7.22-7.09* (3H, m, H-5’)  7.66 (2H, d, J=8.0Hz, H-”,”, 7.22-7.09* (3H, m, H-”,5”, . (s, 3H, CH3)    93i   13.09 (1H, br s, H-1’, 10.5 1H, br s, C2-NH), 9.70 (1H, br s, CO-NH), 8.38 (1H, s, H-’), 8.11 (1H, d, J=7.6Hz, H-7’), 7.73 (1H, s, H-5),  7.35 (1H, t, J=8.0Hz, H-’), 7.16 (1H, d, J=8.3Hz, H-5’)  7.68 (2H, d, J=9.0Hz, H-”,”, . (2H, d, J=9.0Hz, H-”,5”, .5 (3H, s, OCH3)    93j   13.12 (1H, br s, H-1’, 10. 1H, br s, C2-NH), 10.27 (1H, br s, CO-NH), 8.44-8.36* (2H, m, H-’), 8.32 (1H, d, J=7.6Hz, H-7’), 7.83 (1H, s, H-5),  7.29 (1H, t, J=8.0Hz, H-’), 7.19 (1H, d, J=8.3Hz, H-5’) 8.44-8.36* (2H, m, H-” , .0 dd, 1H, J=1.4Hz, J=7.6Hz, H-”, . (dt, 1H, J=1.4Hz, J=7.6Hz, H-5”, 7.18 (dt, 1H, J=1.4Hz, J=7.6Hz, H-”, .51H, s, SCH3) 265  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6)  ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  93k   13.08 (1H, br s, H-1’, 10.51 1H, br s, C2-NH), 9.61 (1H, br s, CO-NH), 8.37 (1H, s, H-’), 8.08 (1H, d, J=7.6Hz, H-7’), 7.71 (1H, s, H-5),  7.35 (1H, t, J=7.9Hz, H-’), 7.16 (1H, d, J=8.2Hz, H-5’) 7.63 (2H, d, J=9.1Hz, H-”,”, . (2H, d, J=9.1Hz, H-”,5”, .5 (4H, m, 2 OCH2), 3.08 (4H, m, 2 NCH2)   93l   13.10 (1H, br s, H-1’, 10.55 1H, br s, C2-NH), 9.77 (1H, br s, CO-NH), 8.37 (1H, s, H-’, .0 1H, d, - .Hz, H-’, . (1H, s, H-5), 7.27 (1H, t, J=8.0Hz, H-’, 7.17 (1H, d, J=8.3Hz, H-5’ 7.49 (1H, t, J=2.2Hz, H-”, .41-7.33 (2H, m, H-5”,”, .1 1H, ddd, J=1.8Hz, J=2.2Hz, J=8.2Hz, H-”, . H, s, 2CH3)   93m    13.09 (1H, br s, H-1’, 10.5 1H, br s, C2-NH), 9.66 (1H, br s, CO-NH), 8.38 (1H, s, H-’, .0 1H, d, - .Hz, H-’, . (1H, s, H-5), 7.36 (1H, t, J=7.9Hz, H-’, 7.17 (1H, d, J=8.3Hz, H-5’        7.67 (1H, s, H-”, 7.49 (1H, dd, J=1.8Hz, J=8.1Hz, H-”, 7.20 (1H, d, J=8.1Hz, H-”,  2.85 (4H, td, J=7.4Hz, J=15.0Hz, 2 benzylic CH2), 2.03 (2H, p, J=7.4Hz, H-”   93n   13.10 (1H, br s, H-1’, 10.5 1H, br s, C2-NH), 9.91 (1H, br s, CO-NH), 8.38 (1H, s, H-’, .0 1H, d, - .Hz, H-’, . (1H, s, H-5), 7.46-7.32* (4H, m, H-’, 7.22-7.14* (2H, m, H-5’ 7.63 (1H, t, J=2.1Hz, H-”, 7.56 (1H, dd, J=2.1Hz, J=8.1Hz, H-” 7.46-7.32* (4H, m, Ar-H), 7.22-7.14* (2H, m, Ar-H), ), 7.08 (2H, d, J=7.7Hz, Ar-H), 6.79 (1H, dd, J=2.1Hz, J=8.1Hz, H-”)   93o    13.09 (1H, br s, H-1’, 10.5 1H, br s, C2-NH), 9.97 (1H, br s, CO-NH), 8.38 (1H, s, H-’, .11 1H, d, - .Hz, H-’, . (1H, s, H-5), 7.35 (1H, t, J=8.0Hz, H-’, 7.17 (1H, d, J=8.4Hz, H-5’  7.85 (2H, d, J=8.9Hz, H-”,”, . (2H, d, J=8.9Hz, H-”,5” 266  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6)  ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  93p   13.08 (1H, br s, H-1’, 10.5 1H, br s, C2-NH), 9.70 (1H, br s, CO-NH), 8.37 (1H, s, H-’, .05 1H, d, - .Hz, H-’, . (1H, s, H-5), 7.36 (1H, t, J=7.9Hz, H-’, 7.17 (1H, d, J=8.3Hz, H-5’ 7.23 (2H, s, H-”,”, . H, s, 2 OCH3), 3.65 (3H, s, OCH3)   93q   13.14 (1H, br s, H-1’, 10. 1H, br s, C2-NH), 9.83 (1H, br s, CO-NH), 8.37 (1H, s, H-’), 8.06 (1H, d, J=7.6Hz, H-7’), 7.85 (1H, s, H-5), 7.38 (1H, t, J=7.9Hz, H-’), 7.26-7.18* (3H, m, H-5’)  8.39 (1H, dd, J=1.9Hz, J=7.8Hz, H-”, .-7.18* (3H, m, H-”,5”, 3.95 (3H, s, OCH3)   93r   13.08 (1H, br s, H-1’, 10. 1H, br s, C2-NH), 8.56 (1H, t, J=6.3Hz, CO-NH), 8.37 (1H, s, H-’, .1 1H, d, - .Hz, H-’, 7.62 (1H, s, H-5), 7.30 (1H, t, J=8.0Hz, H-’, .1 1H, d, - .4Hz, H-5’ 6.99 (1H, d, J=1.7Hz, H-”, .1 (1H, d, J=8.2Hz, H-5”, . 1H, dd, J=1.7Hz, J=8.2Hz, H-”, . (2H, d, J=6.2Hz, CH2), 3.73 (6H, d, J=7.8Hz, 2 OCH3)  93s   13.01 (1H, br s, H-1’, 10.5 1H, br s, C2-NH), 8.79 (1H, t, J=6.2Hz, CO-NH), 8.35 (1H, s, H-’), 8.12 (1H, d, J=6.9Hz, H-7’), 7.60 (1H, s, H-5), 7.30 (1H, t, J=8.0Hz, H-’), 7.12 (1H, d, J=8.2Hz, H-5’) 7.70 (2H, d, J=8.2Hz, H-”,5”, .5 (2H, d, J=8.2Hz, H-”,”, .5 (1H, d, J=6.3Hz, CH2)   93t   13.08 (1H, br s, H-1’, 10.5 1H, br s, C2-NH), 9.81 (1H, br s, CO-NH), 8.38 (1H, s, H-’), 8.09 (1H, d, J=7.6Hz, H-7’), 7.78 (1H, s, H-5), 7.35 (1H, t, J=8.0Hz, H-’), 7.17 (1H, d, J=8.3Hz, H-5’)   7.78 (2H, d, J=8.7Hz, H-”,”, . (2H, d, J=8.7Hz, H-”,5”, . (3H, s, SCH3)  93u   13.07 (1H, br s, H-1’, 10.51 1H, br s, C2-NH), 9.89 (1H, br s, CO-NH), 8.38 (1H, s, H-’), 8.15 (1H, d, J=7.5Hz, H-7’), 7.76 (1H, s, H-5), 7.35 (1H, t, J=7.9Hz, H-’), 7.16 (1H, d, J=8.4Hz, H-5’) 8.53 (1H, d, J=1.6Hz, H-”, .05 (1H, dd, J=1.6Hz, J=8.8Hz, H-”, 6.86 (1H, d, J=8.8Hz, H-5”, .5 (3H, s, OCH3)  267  Entry  Scaffold 1H-NMR (400 MHz, DMSO-d6)  ppm R 1H-NMR (400 MHz, DMSO-d6)  ppm  93v   13.09 (1H, br s, H-1’, 10.5 1H, br s, C2-NH), 9.60 (1H, br s, 1H, CO-NH), 8.38 (1H, s, H-’), 8.10 (1H, d, J=7.6Hz, H-7’), 7.71 (1H, s, H-5), 7.36 (1H, t, J=7.8Hz, H-’), 7.16 (1H, d, J=8.2Hz, H-5’) 7.56 (1H, dd, J=2.0Hz, J=8.8Hz, H-”, .5 1H, s, H-”, . 1H, d, J=8.8Hz, H-5”, . H, s, 2CH3), 2.17 (3H, s, CH3)   93w   13.07 (1H, br s, H-1’, 10. 1H, br s, C2-NH), 8.57 (1H, t, J=6.5Hz, CO-NH), 8.38 (1H, s, H-’), 8.11 (1H, d, J=7.4Hz, H-7’), 7.60 (1H, s, H-5), 7.29 (1H, t, J=7.8Hz, H-’), 7.14 (1H, d, J=8.3Hz, H-5’) 7.26 (2H, d, J=8.7Hz, H-”,”, .1 (2H, d, J=8.7Hz, H-”,5”, .5 (2H, d, J=6.2Hz, CH2), 3.74 (3H, s, OCH3)   93x   13.02 (1H, br s, H-1’, 10.51 1H, br s, C2-NH), 8.77 (1H, t, J=5.9Hz, CO-NH), 8.37 (1H, s, H-’), 8.13 (1H, d, J=7.4Hz, H-7’), 7.60 (1H, s, H-5), 7.37-7.25* (3H, m, H-’ ), 7.13 (1H, d, J=8.3Hz, H-5’) 8.55 (1H, m, H-”, . 1H, dt, J=1.8Hz, J=7.7Hz, H-”, .-7.25* (3H, m, H-”,5”, .1 H, d, J=5.9Hz, CH2)   268  Conclusions and Future Directions  During the course of this project, my research has focused on the use of the parallel synthesis approach for the design and synthesis of exploratory libraries of heterocyclic molecules, which have the potential to be isoform selective type II rho-kinase (ROCK1/2) inhibitors. There is considerable interest in both academic and industrial laboratories to identify and develop isoform selective ROCK inhibitors for application in a number of therapeutic areas, and in particular diabetic cardiovascular diseases.  There are several approved drugs (such as Imatinib, Sorafenib, Regorafenib, and Nilotinib) that function as type II kinase inhibitors. However, the design of type II kinase inhibitors is a challenging endeavor, as structural details on kinases in their activation loop closed conformation are lacking. This is particularly true for the two ROCK isoforms that possess 92% identity in their ATP binding domain. Basically, the only guidelines available are that the inhibitor should have an elongated structure (i.e. be too large to bind in type I inhibitor mode), and that it should contain a polar neutral amide/urea function in the middle of its structure, and a non-polar aromatic/heteroaromatic motif at the opposite extremity to the subunit that binds to the hinge residues, such that interaction with an allosteric pocket is favored.  In our project, these structural features were included in the different libraries of molecules we have prepared (Chapters 3-6). Building on the fact that hydroxyfasudil (a metabolite of the approved ROCK inhibitor fasudil) is a potent (but non selective) ROCK inhibitor, a 2-pyridinone-based molecule was initially chosen as the hinge binding component, and our objective was to evaluate the influence of adding different appendages to this scaffold on isoform selective ROCK inhibition. Indeed, in chapters 3 to 6, we successively created libraries of larger and more geometrically modified structures, by altering the nature of the linker element (oxazoline-carboxamides, oxadiazole, oxadiazle-carboxamides, and 4- or 5-carboxamide substituted 2-aminothiazoles) and the nature of the diversity elements. Due to budget limitations, only selected molecules in these libraries were evaluated as ROCK inhibitors, using a single point assay where % inhibition relative to a control molecule (H-1152) was measured. However, even with these relatively primitive means, we were able to identify structural families (libraries) that displayed activity against ROCK. These results encouraged us to move forward, and did guide us in the choice of structural modifications that were described 269  in each chapter of the thesis.  Most pertinent, was the observation that the indazole motif proved to be superior to the 2-pyridinone system as a hinge binding element.  In fact, it was determined that the indazole ring conferred activity when it was positioned on either the left or the right side of the inhibitor. This suggested that considerable options still exist to further modify the linker portion in these types of molecules. To have an accurate picture of the potency and isoform selectivity for the most interesting compounds, 18 selected molecules were tested in a 10-point assay (Invitrogen). It was found that the most potent inhibitors were active in the low micromolar range (IC50), and that none of the compounds exhibited significant isoform selectivity.  These results demonstrated that we could design ROCK inhibitors based only on the general knowledge of what structural elements such molecules should contain in their structure. They also indicated to us the likelihood that more potent molecules based on the indazole hinge binding motif could be identified through further modifications of the linker portion of the molecule. Strategies that merit investigation are the incorporation of appending functionality that seeks out supplementary binding interactions (Figure 35, Chapter 1), and the use of entities (splitters) that send functionality into different zones in the inhibitor binding site (Figure 33, Chapter 1). Of greatest importance to any future continuation of our project to identify isoform selective kinase inhibitors, will be to have in hand a simple and inexpensive assay that provides rapid IC50 data against both ROCK enzymes.  270  Bibliography  1) Michael, S.; Auld, D.; Klumpp, C.; Jadhav, A.; Zheng, W.; Thorne, N.; Austin, C. P.; Inglese, J.; Simeonov, A. A robotic platform for quantitative high-throughput screening. Assay Drug Dev. Technol. 2008, 6, 637- 657. 2) Zang, R.; Li, D.; Tang, I.; Wang, J.; Yang, S. Cell-based assays in high-throughput screening for drug discovery.  Int. J. Biotech. Well. Indus. 2012, 1, 31-51. 3) Hughes, J. P.; Rees, S.; Kalindjian, S. B.; Philpott, K. L. 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