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Benzisothiazole based anti-viral agents : new chemistry revealed during structure-activity relationship… Lin, Zheng Sonia 2020

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BENZISOTHIAZOLE BASED ANTI-VIRAL AGENTS: NEW CHEMISTRY REVEALED DURING STRUCTURE-ACTIVITY RELATIONSHIP STUDIES  by  Zheng Sonia Lin  B.Sc., The University of British Columbia, 2009 M.Sc., The University of Western Ontario, 2012  A THESIS SUBMITTED IN PARTIAL FULFILLMENT 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 2020 © Zheng Sonia Lin, 2020 ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled:  Benzisothiazole based anti-viral agents: new chemistry revealed during structure-activity relationship studies  submitted by Zheng Sonia Lin in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmaceutical Sciences  Examining Committee: Dr. David Grierson, Faculty of Pharmaceutical Sciences Supervisor  Dr. Adam Frankel, Faculty of Pharmaceutical Sciences Supervisory Committee Member  Dr. Chris Orvig, Department of Chemistry University Examiner Dr. Urs Häfeli, Faculty of Pharmaceutical Sciences University Examiner   iii  Abstract My thesis concerns a study of the fundamental chemistry and reactivity of the benzisothiazole-based antiviral agent “1C8” developed in our laboratory.  Initially identified as an anti-HIV agent that blocks HIV replication through perturbation of HIV pre-mRNA alternative splicing, 1C8 also displays activity against adenovirus, influenza, herpes (HSV-1) and hepatitis B infections, by affecting the function of the SR-protein splicing factor SRSF10.  However, as there are currently no structural data available for SRSF10, and no detailed knowledge concerning its interactions with other factors in the spliceosome machinery at the molecular level, the optimization of the biological activity of 1C8 could only be achieved through classical Structure-Activity Relationship (SAR) studies (iterative modifications of its structure coupled to biological testing at each step).  Chapter 2 describes the SAR studies on 1C8 involving modulation of functional groups in its 1,2-benzisothiazole-amide substructure:  Reduction of the potentially bioactivatable nitro group, oxidation of the benzisothiazole sulfur atom, inversion of the central amide function and its replacement by an aminoether motif.  A more extensive modification of 1C8 involved the extrusion of the benzisothiazole sulfur atom and incorporation of the amide function and the nitrogen of the isothiazole ring into a conformationally rigid pyrimidine ring. The absence of anti-HIV activity for any of the 1C8 analogs prepared pointed to the importance of the nitro group and to the correct orientation of the amide function in its structure for activity.    In Chapter 3, the objective was to prepare a pyridopyrazolone benzisothiazole-type constrained amide analog of 1C8, which mimics a conformation imposed by a crucial H-bonding interaction between the 4-pyridinone C=O and the amide N-H. Pivotal to this effort was the preparation of a 3-hydrazino-5-nitro-1,2-benzisothiazole intermediate through reactions of 3-OMe- and 3-Cl-5-nitrobenzisothiazoles with hydrazine.  These reactions resulted in deep-seated rearrangements of the benzisothiazole ring system leading to the formation of (Z)-methyl-2-amino-5-nitrobenzohydrazonate and 3,3'-thiobis-5-nitro-2,1-benzisothiazole, respectively. Support for the proposed mechanisms for the formation of these products was obtained from Density Function Theory calculations. Further investigations using phenylhydrazine revealed the production of aniline from the N-N cleavage of phenylhydrazine.  The mechanism for this transformation requires further study.    iv  Lay Summary  Structure-activity relationship (SAR) studies relate the impact of the structure of a molecule on its biological activity.  SAR is one of the key aspects of drug discovery from primary screening to “lead” optimization.  We performed SAR studies on the benzisothiazole-containing anti-HIV molecule 1C8 (previously discovered in the Grierson Lab) by synthesizing a series of compounds containing modifications on different components of 1C8 to probe interactions with the putative protein target and to investigate the effect of possible 1C8 conformations on its biological activity.  In the process of these SAR studies, we observed unprecedented rearrangements of nitro-substituted benzisothiazole on reactions with hydrazine.  Further studies of the reactions between phenylhydrazine and non-nitro substituted benzisothiazole, we found that the mode of reactivity depends on the nature of the substituent at C-3 on the benzisothiazole ring.    v  Preface The work described in the thesis were conducted by the author under the supervision and guidance of Professor David Grierson in the Faculty of Pharmaceutical Sciences, the University of British Columbia.  I was responsible for planning, designing, and conducting all the chemistry experiments and interpreting the results.  Professor Grierson has also put in huge efforts in editing this thesis.  This was a valuable learning experience for me.  In Chapter 2, the biological tests were performed by Dr. Peter Cheung at the BC Centre for Excellence for HIV/AIDS at St. Paul’s Hospital.    In Chapter 3, Pr. Pierre Kennepohl and Ms. Xing Tong from the Department of Chemistry at UBC carried out the calculations of the energetics for the transformations from the nitro-benzisothiazoles to the rearranged products 126 and 82 using Density Function Theory (Sections 3.3.1.2 – 3.3.1.3).  Figures 71, 73-75, 78, 80, and 81 were constructed by Pr. Kennepohl and Ms Tong, who have also helped with editing these two sections of the thesis.  Single crystal X-ray diffraction experiments were carried out by Dr. Brian Patrick from the Department of Chemistry at UBC.  vi  Table of Contents  Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Tables .............................................................................................................................. viii List of Figures ............................................................................................................................... ix List of Abbreviations ................................................................................................................ xvii Acknowledgements .................................................................................................................... xxi Dedication ................................................................................................................................. xxiii Chapter 1: Introduction ............................................................................................................... 1 1.1 Drug discovery and medicinal chemistry ................................................................... 1 1.2 IDC 16 and the discovery of 1C8 ............................................................................... 2 1.3 HIV and AIDS ............................................................................................................ 4 1.4 Splicing and alternative splicing ................................................................................. 6 1.5 Serine-Arginine Rich (SR) proteins and alternative splicing ..................................... 7 1.6 HIV and alternative splicing ....................................................................................... 7 1.7 Anti-HIV activity of 1C8 ............................................................................................ 8 1.8 Impact of 1C8 on cellular genes and HIV pre-mRNA splicing .................................. 9 1.9 1C8:  The benzisothiazole motif, a focus for SAR studies ....................................... 10 1.10 Chemistry of benzisothiazole .................................................................................... 10 1.10.1 1,2-Benzisothiazoles ......................................................................................... 10 1.10.2 SNAr-type nucleophilic substitution reaction at C-3 versus formation of benzonitrile products through nucleophile induced cleavage of the S-N bond in benzisothiazoles. ............................................................................................................... 11 1.10.3 Synthetic methods of 1,2-benzisothiazoles ....................................................... 17 1.10.4 Preparation of 3-substituted benzisothiazole’s (3-Cl, sulfonate, OMe, and NH2): Starting material in synthesis ............................................................................................ 25 1.11 My Research Project: Objectives .............................................................................. 27 Chapter 2: SAR studies on 1C8 through functional group modulation ................................ 30 vii  2.1 Introduction ............................................................................................................... 30 2.2 (A)     Inverse amide synthesis and isomeric nitro group placement ........................ 31 2.3 (B) Conversion of -NO2 to -NH2:  Preparation of 1C8 analogue 58 ........................ 37 2.4 (C) Bioactivation of isothiazole:  Benzisoxazole-based inverse amide analogue of 1C8 39 2.5 (D) Efforts to prepare the diheteroaryl aminoether 81:  Synthesis of the sulfur bridged 5-nitro-2,1-benzisothiazole dimer 82. ................................................................................... 45 2.6 (E) Scaffold Deconstruction and Morphing Approach to SAR ................................ 49 2.6.1 Syntheses of 4,6-diheteroaryl pyrimidine-based analogues of 1C8...................... 52 Chapter 3: Conformational Constraint of the Central Amide Linker in 1C8 Through Its Incorporation into a Pyrazolone Motif - The Discovery of New Reactivity of the Benzisothiazole Ring System ...................................................................................................... 60 3.1 Introduction ............................................................................................................... 60 3.2 Approach 1:  Synthesis of Pyrazolopyridinone 44 Via Cu(I) Mediated N-Arylation  of 3-Chloro-5-nitro-1,2-benzisothiazole 42 .............................................................................. 62 3.3 Approach 2: Synthesis of Pyrazolopyridinone 44 Via Cyclization of Hydrazine Intermediate 117 ................................................................................................................... 64 3.3.1 Approach “2a” to Intermediate 117: Attempted Preparation of 3-hydrazino-5-nitrobenzisothiazole 45 ..................................................................................................... 65 3.3.2 Conclusions ........................................................................................................... 84 3.4 Approach 2b(1):  The projected synthesis of key intermediate 117.  Literature/Model studies involving the reaction 3-substituted-5-nitrobenzisothiazoles 42 and 119 with phenylhydrazine .................................................................................................................... 85 3.5 Approach 2b(2) The projected synthesis of key intermediate 117.  Model studies involving the reaction between non-nitro substituted 3-methoxy/triflate-1,2-benzisothiazoles and phenylhydrazine ............................................................................................................. 92 Chapter 4: Conclusions ............................................................................................................ 107 Experiments ................................................................................................................................110 Bbliography ................................................................................................................................141 Appendices ..................................................................................................................................150  viii  List of Tables  Table 1.  Various anti-viral activities of 1C8. ................................................................................ 9 Table 2.  Nucleophilic substitution reactions of 3-Chloro-1,2-benzisothiazole with different nucleophiles.32 ............................................................................................................................... 12 Table 3.  4,6-Diheteroaryl pyrimidines prepared using a 3 component one-pot strategy under microwave conditions. R1 = H, CN. ............................................................................................. 59 Table 5.  Cu coupling reactions of pyridopyrazolone 115. .......................................................... 63 Table 6.  Conditions tried for the reaction between 3-chloro-5-nitro-1,2-benzisothiazole and phenylhydrazine. ........................................................................................................................... 87  ix  List of Figures  Figure 1.  The drug discovery pipeline and the role of medicinal chemistry in this process. ........ 1 Figure 2.  Foundation scheme of the project.  (a) IDC16 was discovered from a library screening in an extension to J. Tazi’s work on staurosporine derivative NB-506.3  (b) The diversity driven compound library synthesis approach used in the Grierson lab to identify IDC16 mimics.  Four compounds were found to be anti-HIV active.4 .............................................................................. 3 Figure 3.  The HIV replication cycle.9 (Image adapted from reference 9) .................................... 5 Figure 4.  mRNA splicing and alternative splicing.13 (Image adapted from reference 13) ........... 6 Figure 5.  Organization of HIV-1 genome and different mRNA splicing products.25 (Figure adapted from reference 25) ............................................................................................................. 8 Figure 6.  Structure of 1C8 and selected other benzisothiazole-containing compounds that show biological activities.4, 29-31 ............................................................................................................. 10 Figure 7.  Nomenclature of benzisothiazoles. (Note:  The junction carbon atoms 8 and 9 are labeled as 3a and 7a in some literature reports)32-33 .................................................................................. 11 Figure 8.  Nucleophilic aromatic substitution of 2-chloropyridine.35 .......................................... 12 Figure 9.  Synthesis of Lurasidone precursor 3-piperizinyl-1,2-benzisothiazole 3 from 3-Cl and 3-OTf-benzisothiazole.36-37 .............................................................................................................. 13 Figure 10.  Two alternate/competing pathways to the 3-C reactivity of 1,2-benzisothiazole.38 .. 13 Figure 11.  Formation of 8 and 9 by reacting 3-chloro-1,2-benzisothiazole 1 with NaCN in aqueous acetone.39 ....................................................................................................................................... 14 Figure 12.  Formation of o-(n-butylthio)benzonitrile 10 by treating 3-chlorobenzisothiazole 1 with n-BuLi in DMF at -70°C.41 ........................................................................................................... 15 Figure 13.  Ring-opening of benzisothiazole by sodium methoxide in methanol resulting in the formation of the thiolate anion 11.42 ............................................................................................. 15 Figure 14.  Isomerization of 3-amino-1,2-benzisothiazole 12 to 12’ in the presence of HCl.44 .. 16 Figure 15.  Proposed reaction pathway when 3-chloro-1,2-benzisothiazole 1 reacts with excess piperazine to form 3-piperizinyl-1,2-benzisothiazole 3.45 ............................................................ 17 Figure 16. Two major ring closure strategies for the synthesis of 1,2-benzisothiazole.38 ........... 17 Figure 17.  Synthesis of 5-nitro-1,2-benzisothiazole 17 via a sulfenyl halide intermediate.46 .... 18 x  Figure 18.  Synthesis of 3-aminobenzisothiazoles 18 with electron withdrawing groups at C-7.47....................................................................................................................................................... 18 Figure 19.  Generation of 1,2-benzisothiazole from 2-(aminomethyl)benzenethiol 19 in the presence of I2 and KI.47 ................................................................................................................. 19 Figure 20.  Mechanism for the formation of 3-chloro-1,2-benzisothiazole 1 from disulfide 7 in the presence of chlorine.48 .................................................................................................................. 19 Figure 21.  Proposed mechanism for the formation of 3-(N-pyrrolidinyl)-1,2-benzisothiazole 24 in the reaction of Mg amides with 2,2’-dithiobiz(benzonitrile) 9.49 ............................................. 20 Figure 22.  Preparation of 1,2-benzisothiazol-3-ones 29, 30 and 32 from (a) 2,2-dithiobis-benzamide upon treatment with Br2,50 (b) cyclization of benzyl sulfide upon treatment with sulfuryl chloride,51 and (c) intramolecular N-S bond formation using Selectfluor.52 ................... 22 Figure 23.  Synthesis of 1,2-benzisothiazoles from 2-(t-butylthio or methylthio) benzaldehyde oximes.47, 53-60 ................................................................................................................................ 23 Figure 24.  Synthesis of 1,2-benzisothiazoles from o-haloarylamidines and elemental sulfur via N–S/C–S bond formation.61 .......................................................................................................... 24 Figure 25.  Reaction between benzyne and 1,2,5-thiadiazoles to form 1,2-benzisothiazoles.62-63....................................................................................................................................................... 24 Figure 26.  Formation of 3-substituted-1,2-benzisothiazoles using Koabayashi precursor.64 ..... 25 Figure 27.  Synthesis of 3-chlorobenzisothiazole 1 using POCl3/DMF.65-66 ............................... 25 Figure 28.  Preparation of 3-piperizinylbenzisothiazole 3 from the 3-sulfonate substituted benzisothiazole 40 (R = CH3, Ph).  Note that 3-OTf-benzisothiazole 2 was prepared by the reaction of 37 with triflic anhydride (Figure 9).37, 67 .................................................................................. 26 Figure 29.  Synthesis of 3-methoxybenzisothiazole 41 from 3-chlorobenzisothiazole 1.68 ........ 26 Figure 30.  Preparation of various 3-amino-1,2-benzisothiazoles under amination conditions.69 27 Figure 31.  Compounds prepared during the SAR studies on 1C8. ............................................. 28 Figure 32.  Strategy for the preparation of the constrained 1C8 analog: pyrazolopydridine 44. 29 Figure 33.  SAR modifications on 1C8........................................................................................ 30 Figure 34.  1C8 and 4-, 5-, and 7-NO2 substituted inverse amides 46a (4-NO2), 46b (5-NO2) and 46c (7-NO2). .................................................................................................................................. 31 Figure 35.  Synthesis of 3-amino-1-methyl-4-pyridinone.70 ........................................................ 32 Figure 36.   Synthesis of nitrobenzisothiazole-3-acetic acids 56a, 56b, and 56c.71-74 ................. 33 xi  Figure 37.  Amide coupling via in situ formation of acyl fluorides 57a-c using DeoxoFluor as the fluorinating agent.  Compound 46a was isolated as an admixture with compounds 46b/46c...... 35 Figure 38.  Conformational analysis of (a) 1C8 and (b) its inverse amide 46b.  The bond of rotation is highlighted in yellow.  Shown on the left are the conformations of both compounds with the C=O of amide and pyridinone in the same direction.  Shown on the right are the conformations of both compounds with C=O in opposite directions.  The corresponding energy levels are indicated by the blue arrow. The amide bonds for the more stable conformers of 1C8 and 46b are circled in white. ............................................................................................................................................. 36 Figure 39.  One- and two-electron reduction of nitroaromatics.80 ............................................... 38 Figure 40.   Reduction of -NO2 to -NH2 in 1C8:  Formation of compound 58. .......................... 38 Figure 41.  Effect of 58 and 1C8 on HIV infected CEM-GXR cells. .......................................... 39 Figure 42.  (a) Bioactivation of thiazole resulting in formation of glutathione conjugate. (b) oxazole (60) and pyrazole (61) structural analogs of 59 (one structure with X = O, NH) ........... 40 Figure 43.  Benzisoselenazol-3(2H)-ones and their corresponding diselenides.  R=H, Me, Et, n-Pr, n-Bu, Ph, Bn.82 ........................................................................................................................ 41 Figure 44.  Synthesis of 1C8 benzisoxazole analogue 69.83 ........................................................ 42 Figure 45.  Saccharin and an example of pseudosaccharin.......................................................... 42 Figure 46.  Attempt to prepare sulfone analogue of 1C8 via mCPBA oxidation. ....................... 43 Figure 47.  Synthesis of a sulfone analogue of 1C8 via amide coupling. .................................... 44 Figure 48.  Attempted synthesis of diarylamino ether 81. Preparation of the rearrangement product 82 (confirmed X-ray diffraction structure).88................................................................................ 46 Figure 49.  Proposed synthetic route for the preparation of desired product 81 via non-NO2 substituted 3-chlorobenzisothiazole 1. .......................................................................................... 47 Figure 50.  Products 83 and 84, formed in the reaction between pyridinone amine 49 and 3-chloro-1,2-benzisothiazole 1. ................................................................................................................... 48 Figure 51.  1H NMR (CDCl3) of 83 (top spectrum) and 84 (bottom spectrum).  Note the lack of C-H signal and the presence of –NH2 signal in 84. ...................................................................... 48 Figure 52.  Nitration of diheteroaryl aminoether 83 gave product 85 which contains -NO2 at C-7 position. ......................................................................................................................................... 49 Figure 53.  Literature examples of scaffold deconstruction and reconstruction.89-91 ................... 50 Figure 54.  Structures of 4,6-diheteroarylpyrimidine-based analogues (92) of 1C8. .................. 51 xii  Figure 55.  The target 4,6-diheteroaryl pyrimidine compound 96 can be made by reacting amidine with (a) 1,3-diketone 94 formed from Claisen condensation between 93 and 74, or (b) α, β-unsaturated ketone 98 formed from Aldol condensation between 93 and 97. .............................. 52 Figure 56.  Synthesis of aldehydes 100 and 97, and the 1,5-diketone products generated from their reactions with acetophenone in the presence of NaOH in EtOH. ................................................. 53 Figure 57.  The Michael acceptor ability of these pyridine-containing azachalcones is activated by the metal ion’s coordination to the N atom of the pyridine.93 ...................................................... 54 Figure 58.  DBU mediated Aldol condensation between 100 and nitroacetophenone gave 104 (obtained as a mixture with the starting aldehyde 100). ............................................................... 55 Figure 59.  DBU-mediated synthesis of azachalcones from 97 and acetophenones 93 and 101.  Yields of 106 and 107 could not be determined as they have the same Rf value as the starting aldehyde. ....................................................................................................................................... 56 Figure 60.  Biginelli type three-component one-pot synthesis of 4,6-diheteroaryl pyrimidines. 58 Figure 60.  (a) Construction of 4,6-diheteroarylpyrimidine analogs of 1C8, which capture the 1C8 conformation where the carbonyls of the amide and the 4-pyridinone are in the same direction; (b) “Constrained” pyrazolopyridine-based analogs of 1C8, which capture a key H-bond interaction between the amide NH and the 4-pyridinone C=O. ...................................................................... 60 Figure 61.  Two studied strategies for the synthesis of pyridopyrazolone 1,2-benzisothiazole.   The corresponding starting materials for each approach are provided. ............................................... 61 Figure 62.  Proposed synthetic pathway for preparation of pyrazolopyridinone 44. ................... 64 Figure 63.  Condensation of 4-chloronicotinic acid methyl ester 116 and 5-nitro-3-hydrazino-1,2-benzisothiazole 45.97 ..................................................................................................................... 65 Figure 64.  Literature procedure for the preparation of 3-hydrazino-1,2-benzisothiazole 125.98 66 Figure 65. Reaction of the non-NO2-substituted 3-methoxy-1,2-benzisothiazole 41 with excess hydrazine hydrate according to known literature procedure: formation of disulfide 9. ............... 67 Figure 66. Formation of the rearrangement product 126 from the reaction of 3-methoxy-5-nitro-1,2-benzisothiazole 119 with excess hydrazine hydrate. .............................................................. 68 Figure 67.  Summary of the rearrangement products formed in the reactions of 3-substituted 5-nitro-1,2-benzisothiazoles 42 and 119 with hydrazine hydrate. ................................................... 69 Figure 68.  Reactivity of benzisothiazole with a nucleophile.  X = Cl, -OCH3. .......................... 70 Figure 69.  Proposed mechanism for the formation of rearrangement product 126.102 ............... 71 xiii  Figure 70.  Proposed mechanism for the formation of rearrangement product 82. ..................... 73 Figure 71.  Electrostatic potential map of 119 and 42 showing relative potential on the two different reactants. Electrostatic potentials were generated with Gaussian09 using the M062X theory and Def2TZVP basis set and then visualised with WebMO. While bright blue and bright red represent -0.15V and 0.15V, respectively, the change in electrostatic potential depicted by change in hue as depicted by the scale is approximate for all electrostatic potential maps visualised with WebMO. ............................................................................................................................... 75 Figure 72.  Three proposed pathways where hydrazine attacks the S atom of 119. The colored arrows represent thermodynamic change in the first step for each pathway. Red represents an endergonic process while green signifies an exergonic step. The Gibbs free energy change is indicated between brackets (unit: kcal/mol). ................................................................................ 76 Figure 73.  Reaction coordinate diagram of the first step in P1and P2 in 119 system. The pink are represent an approximation of where the transition state in the P2 pathway lies in.  The total Gibbs free energy of all reactants was set as baseline (0 kcal/mol). Each plateau represents the total Gibbs free energy of all existing compounds (i.e. reactants, transition state TS, and products) at each stage. ............................................................................................................................................. 77 Figure 74.  Proposed pathway PC. The colored arrows represent thermodynamic change in each pathway. Red represents an endergonic process while green means an exergonic step. Every point (pt) of the linewidth stands for 5 kcal/mol. ................................................................................... 77 Figure 75.  Reaction coordinate diagram of P1, P2 and PC in the 119 reaction system. The total Gibbs free energy of all reactants was set as baseline (0 kcal/mol). Each plateau represents the total Gibbs free energy of all existing compounds at each stage. ......................................................... 78 Figure 76.  Revised pathway for the conversion of 3-methoxy-5-nitrobenzisothiazole 119 to the observed hydrazonate product 126. The coloured arrows represent thermodynamic change in the first step for each pathway. Green represents an exergonic step. Every point (pt) of the linewidth stands for 5 kcal/mol. .................................................................................................................... 79 Figure 77.  Four proposed pathways showing reaction between hydrazine and 42.  Pathways P1, P2, and P3 show proposed pathway when hydrazine attacks S atom.  PC shows the proposed pathway when hydrazine attacks at C-3 giving 45.  The colored arrows represent thermodynamic change in the first step for each pathway. Red represents an endergonic process while green means xiv  an exergonic step. Every point (pt) of the linewidth stands for 5 kcal/mol. The Gibbs free energy change is labeled in the bracket (unit: kcal/mol). ......................................................................... 80 Figure 78.  Reaction coordinate diagram of the first step in P1 and PC in 42 system. The total Gibbs free energy of all reactants was set as baseline (0 kcal/mol). Each plateau represents the total Gibbs free energy of all existing compounds (i.e. reactants, transition state TS) at each stage. .. 81 Figure 79.  Reaction of intermediates 132 and 45 with hydrazine. Formation of amidrazone 133/Meisenheimer complex 134 as the common products issuing from both reactions. Note the calculated energy for the conversion of 45 to 134 (+10.68 kcal/mol). ......................................... 82 Figure 80.  Reaction coordinate diagram of P2 and PC in 42 system. The total Gibbs free energy of all reactants was set as baseline (0 kcal/mol). Each plateau represents the total Gibbs free energy of all existing compounds at each stage.  Both pathways start from 42, and structures involved in each step of transformations are shown. ....................................................................................... 83 Figure 81. Predicted pathway PC to compound 82. The colored arrows represent thermodynamic change in the first step for each pathway. Red represents an endergonic process while green means an exergonic step. Every point (pt) of the linewidth stands for 5 kcal/mol. The reagents above the arrow are reactants in the step, while the reagents below represent products formed in the step. To better compare the energy of each compound, two states of hydrazine were used (NHNH2- and NH2NH2). ...................................................................................................................................... 84 Figure 82.  (i) Approach 2b for the preparation of the key intermediate 117 from 3-substituted-5-nitrobenzisothiazoles 42 and 119.  (ii) Illustrated also is a literature precedent for this transformation (147  148), and (iii) our successful conversion of benzisothiazole 41 to 3-phenylhydrazinobenzisothiazole 149. ........................................................................................... 85 Figure 83.  Rearranged product 151 from the reaction between phenylhydrazine and 5-nitro-3-methoxy-1,2-benzisothiazole 119. ................................................................................................ 86 Figure 84.  The reaction between 3-chloro-5-nitrobenzisothiazole 42 with 1.5 eq. phenylhydrazine in the presence of DBU in DMF: Possible product structures 150 and 152a-b. .......................... 87 Figure 85. (a) 1H NMR and (b) 13C NMR of proposed structure 152.  (c) HSQC of compound obtained from the reaction between 42 and 1.5 eq. phenylhydrazine in the presence of DBU in DMF.  (d) quaternary 13C chemical shifts of 149, 82, 1- and 2-methylbenzopyrazole.  NMR solvent is CDCl3.33..................................................................................................................................... 90 xv  Figure 86.  Proposed mechanism for the formation of 152c and 152e in the reaction between 42 and 1.5 eq. of phenylhydrazine in DMF under the presence of DBU at room temperature. ........ 92 Figure 87.  Reaction between phenylhydrazine and 3-methoxy-1,2-benzisothiazole 41. ........... 93 Figure 88.  Synthesis of 149 using 3-OTf-benzisothiazole 2 as the starting material. ................ 94 Figure 89.  (a)1H NMR (DMSO-d6) of samples taken at different time points during the reaction between 41 and 5.0 eq. phenylhydrazine in MeOH (reflux).  Spectra of 149, aniline, and phenylhydrazine are shown on the top for purpose of comparison.  (b) The expanded aromatic region on 1H NMR of the 73 hr sample and the target compound 149.  Peaks for product 149 are marked by *, and the signal for aniline is marked indicated by an orange arrow. ....................... 95 Figure 90.  1H NMR (DMSO-d6) of samples taken at different time points during the reaction between 3-OTf-1,2-benzisothiazole 2 and 5.0 eq. phenylhydrazine in MeOH (reflux).  The formation of the disulfide species 9 at 2 hr 50 min is indicated by the black box.  1H NMR of 9, 149, phenylhydrazine, and aniline are shown at the top for comparison. Note the formation of aniline started at 2 hr 50 min......................................................................................................... 96 Figure 91.  19F NMR (DMSO-d6) of samples taken at different time points during the reaction between 3-OTf-1,2-benzisothiazole 2 and 5.0 eq. phenylhydrazine in MeOH (reflux).  Note that the signal at -72.34 ppm from the starting triflate 2 disappeared completely at 2 hr 50 min of the reaction, indicating all the starting material has been consumed.  The signal at -77.76 ppm corresponds to the triflate anion. ................................................................................................... 97 Figure 92.  Proposed reaction pathway for the conversion of 3-OTf benzisothiazole 2 to compound 149, via the intermediacy of disulfide 9. ....................................................................................... 98 Figure 93.  Proposed pathway for the formation of 3-(2-cyanophenylthio)-1,2-benzisothiazole 157 from the reaction between disulfide 9 and excess phenylhydrazine. ............................................ 99 Figure 94.  Pathway for the formation of 3-piperizinyl-1,2-benzisothiazole from disulfide 9 proposed by Walinsky.45 ............................................................................................................. 100 Figure 95.  Proposed preparation of 3-phenylhydrazino-1,2-benzisothiazole 149 through the thiosulfonate 161 pathway. ......................................................................................................... 101 Figure 96.  (a) Mono-oxidation of disulfide 9. (b) Attempt to generate thiosulfonate from benzenesulfonyl chloride and 2-mercaptobenzonitrile. (c) Attempt to generate thiosulfonate 161 from benzenesulfonyl fluoride and silylated 2-mercaptobenzonitrile 164. The dimerization to form xvi  9 was preferred, and no thiosulfonate formation was observed even upon heating the reaction at reflux. .......................................................................................................................................... 102 Figure 97.  1H NMR (DMSO-d6) for q-NMR of aniline (using ethylene carbonate as the internal standard) in reactions between phenylhydrazine and (a) 41; (b) 2.  Spectra of aniline and phenylhydrazine are shown for the purpose of comparison. ...................................................... 105  xvii  List of Abbreviations °C degree Celsius µ micro µM micromolar ‡ transition state Å Angstrom AcOH acetic acid AIDS acquired immunodeficiency Syndrome  Ar aryl ART antiviral therapy ADME absorption, distribution, metabolism, and excretion Bcl B-cell lymphoma BCLAF1 Bcl-2-associated transcription factor 1 BIT benzisothiazole Bu butyl CC50 cytotoxic concentration d day(s) DAST  diethylaminosulfur trifluoride DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DCE dichloroethane DCM dichloromethane Deoxo-fluor  bis(2-methoxyethyl)aminosulfur trifluoride DFT density functional theory DIBAL diisobutylaluminum hydride DIEA N,N-diisopropylethylamine xviii  DMF N,N-dimethylformamide DMSO dimethylsulfoxide DNA deoxyribonucleic acid ds double stranded e- electron EC ethylene carbonate eq equivalent(s) equiv. equivalent(s) ESE exon splicing enhancer ESI electrospray ionization ESP electrostatic potential Et ethyl EtOH ethanol g grams GFP green fluorescent protein HIV human immunodeficiency virus hr hour HRMS high-resolution mass spectrometry hrs hours HSV Herpes simplex Hz Hertz IC50 half maximal inhibitory concentration J coupling constant kcal kilocalories LDA lithium diisopropylamide xix  M metal m/z mass over charge ratio mCPBA  meta-chloroperbenzoic acid MDCK Madin-Darby Canine Kidney cells Me methyl MeCN acetonitrile MeOH methanol mg milligrams MHz megahertz min minute(s) mol mole(s) mRNA messenger RNA MW microwave NMR nuclear magnetic resonance NTR nitroreductase Nu nucleophile OEt  ethoxide OMe methoxide OTf triflate Ph phenyl PPA polyphosphoric acid ppm parts per million Pr propyl py pyridine q-NMR quantitative NMR xx  r.t. room temperature RNA ribonucleic acid RRM RNA recognition motifs RT reverse transcriptase SAR  structure-activity relationship Selectfluor 1-Fluoro-4-methyl-1,4-diazoniabicyclo[2.2.2]octanebis(tetrafluoroborate) SNAr nucleophilic aromatic substitution SR protein serine-arginine rich protein SRSF1 Serine and arginine rich splicing factor 1 SRSF10 Serine and arginine rich splicing factor 10 T. temperature TBAF tetrabutylammonium fluoride t-Bu tert-butyl Tf2O triflic anhydride TFFH tetramethylfluoroformamidinium hexafluorophosphate THF tetrahydrofuran TLC thin layer chromatography TMS trimethylsilyl TOPO I  Topoisomerase 1 TS transition state ΔG change in Gibbs free energy ρGS(r) density distribution of the ground state  Ψ single particle wave function   xxi  Acknowledgements First and foremost, I wish to express my deepest appreciation to my supervisor, Dr. David Grierson for his supervision and support for my projects. I am thankful to the guidance, insightful and creative thoughts, suggestions, patience, and encouragement he has given me throughout my Ph.D studies.  In particular, I am grateful that he has given me (someone without a traditional organic chemistry background) the chance to join his research group and to develop skills required for an organic chemist.  Furthermore, I appreciated the many open-minded discussions concerning the mechanisms for the “strange” results from my experiments.     I wish to express my gratitude to my committee members, Drs. Adam Frankel, Shy-dar Li, and Jennifer Love, Peter Soja (chair), Tara Klassen (former committee member), and Stelvio Bandiera (former committee chair) for  their guidance and providing me feedback throughout this journey.  I would also like to thank my committee, the University examiner, and the external examiner for reading my thesis and participating in the examination process. I want to extend my gratitude to Ms. Xing Tong and Dr. Pierre Kennepohl (Chemistry) for carrying out the computational calculations on the mechanism of the formation of the rearranged products in Chapter 3 and helping with editing this section of the thesis.  We would not have been able to gain the depth of insights about the reaction pathways of nitro-substituted 1,2-benzisothiazoles without your help.  I would like to thank Dr. Peter Cheung from the BC Centre for Excellence in HIV/AIDS, for his help with testing the compounds for anti-HIV activities. Also, I want to express my thanks to Dr. Brian Patrick (Chemistry) for the single crystal X-ray diffraction experiments.  I wish to thank Andreas Seitz and Dickson Lai for their help with mass spectrometry. I would like to sincerely thank Dr. Thomas Chang and Ms. Shirley Wong from the Office of Graduate and Postdoctoral Studies in the Faculty for the financial support during my studies.  I would like to thank the support and accompaniment from the previous members of the Grierson lab:  Drs. Ronan Gealageas, Alice Devineau, Safwat Rabea, Ying Gong, Ana Koperniku, and Maryam Zamiri.  I want to thank all my friends for their support throughout these years, Jialin, Ying Wang, and Alice.  Special thanks to Crystal, the person who introduced me to the various personality tests and gaming apps.  Discussions wouldn’t have been so amazing without your vast internal (mind) framework.  I want to thank my teacher Karime Kuri, who introduced me to the beautiful Raqs Sharqi and provided me with support, encouragement, and understanding during this journey.  To xxii  my ATS teacher Tonje Olson, thank you for all the classes and showing the amazing strength in your character.  To Cristina, I look forward to the many hikes in the future.  Finally, I would like to thank my dear and beloved parents, who have always been standing by my side no matter what I choose and encouraging me to pursue what I believe in. xxiii  Dedication           To my parents and the owl family at Beach Grove Park1  Chapter 1: Introduction 1.1 Drug discovery and medicinal chemistry Drug discovery is a process in which new medical/therapeutic candidates are identified through input from translational interactions with researchers in medicine, pharmacology, chemistry, and molecular biology.  Consequently, medicinal chemistry is an interdisciplinary field that is concerned with the discovery and design of new therapeutic agents.  One of the key aspects of medicinal chemistry in drug discovery is to bring molecules down the drug discovery pipeline (hit to lead) (Figure 1).  The “lead” compound is usually derived from a series of structural variants of the initial “hit”.  In practice, medicinal chemistry relates the structure of a molecule to its potential to be a drug.  In other terms, chemistry designs and creates molecules, and it is the input from biological sciences which gives that molecule its value.1    Figure 1.  The drug discovery pipeline and the role of medicinal chemistry in this process.  In both academia and the pharmaceutical industry, compound libraries derived from in-house molecule collections, and from combinatorial and parallel synthesis are screened and tested against both established and new therapeutic targets.  When the specific target structure is known, computational chemistry and molecular modeling can be used to identify the binding site 2  interaction.  If the target structure is unknown, synthesizing and screening molecules containing different structures is essentially the only method, whereby researchers can gain valuable information about the site of interaction in the target.2  The primary objective of my thesis has been to use Structure-Activity Relationship (SAR) studies to optimize the anti-HIV activity of the “hit” compound 1C8, which was discovered in our laboratory through screening of a diversity driven compound library for molecules that block HIV replication via perturbation of the host cell mediated alternative splicing of HIV pre-mRNAs. In this process, new aspects of the chemistry of the “benzisothiazole” subunit in 1C8 have been revealed.  1.2 IDC 16 and the discovery of 1C8 In an extension of work by J. Tazi (Universite de Montpellier II, Fr), which identified the staurosporine derivative NB-506 (Figure 2a) as an inhibitor of SR (serine-arginine rich) protein phosphorylation by the kinase activity of topoisomerase I, the random screening of the Institut Curie compound library of 4600 compounds founded by D. Grierson/C. Monneret and co-workers, revealed that a series of tetracyclic indole compounds related to the natural product ellipticine blocked spliceosome assembly (see section 1-4). Using the intrinsic fluorescence of these active molecules, as a marker in binding affinity experiments, it was found that increasing concentrations of SRSF1, but not TOPO I/kinase, induced 80% quenching of fluorescence. To determine the effectiveness of these indole derivatives in vitro, experiments were designed using HIV-1 pre-mRNA as a model substrate regulated by alternative splicing.  One molecule in particular, IDC16, completely prevented HIV-1 replication at sub-micromolar concentrations (performed on HeLa cell line transfected with pΔPSP plasmid containing HIV-1 pro-viral genome deleted between nucleotides 1511 and 4550).  Preliminary results further indicated that IDC16 inhibited HIV replication through blocking the function of SRSF1.3    3   Figure 2.  Foundation scheme of the project.  (a) IDC16 was discovered from a library screening in an extension to J. Tazi’s work on staurosporine derivative NB-506.3  (b) The diversity driven compound library synthesis approach used in the Grierson lab to identify IDC16 mimics.  Four compounds were found to be anti-HIV active.4  Although an important discovery, IDC16 displays cytotoxicity at concentrations greater than 10 micromolar (IC50), excluding its use in anti-HIV therapy. Indeed, due to their planar polycyclic structure, pyridocarbazole (ellipticine) derivatives, such as IDC16, are known DNA 4  intercalators.5-6 To counter this problem, research in our laboratory was directed toward the design, synthesis and evaluation of IDC16 mimics as alternative splicing inhibitors that block HIV replication. The central concept explored was to identify novel compounds lacking the B- and C-rings of IDC16, which would be more conformationally mobile and display no affinity for DNA. The approach taken was to generate a library of compounds where the A- and D-ring of IDC16 were replaced by different functionalized aromatic and heteroaromatic motifs, and where these subunits are joined by an appropriate linker (Figure 2b). By screening a library of 240 potential IDC16 mimics four diheteroarylamide compounds containing a common benzisothiazole (BIT) ring in their structure, 2D3, 1E5, 3C2, and 1C8, showed anti-HIV-1 activity.4 The most active compound, 1C8, displayed activity (IC50 < 1 micromolar) against HIV mutant strains that are resistant to all current anti-HIV drugs used in ART (combination) therapy. Further, it’s activity to toxicity profile, i.e. selectivity index SI = CC50/EC50) of  >100 demonstrated a clear improvement in terms of safety over IDC16.4    1.3 HIV and AIDS Currently, more than 37.8 million people around the world are living with HIV/AIDS, and statistics from 2018 show that there are approximately 770 000 AIDS related deaths per year versus 1.7 million new HIV infections.7  As a treatment, the antiretroviral therapy (ART) strategy, where drug combinations are used, has proven highly effective at lowering the viral load to levels that are undetectable by current testing techniques.  This helps to maintain the patients’ immune system and to decrease the likelihood of opportunistic infections that could lead to death.  As a consequence, HIV/AIDS is treated as a “chronic infection” in developed countries, and aggressive measures are now being taken to bring ART to the developing world, where the majority of HIV infected people reside.8 The human immunodeficiency virus (HIV), a lentivirus with an RNA genome, is the causative agent for the acquired immunodeficiency Syndrome (AIDS). The term HIV/AIDS is now used to describe the range of pathologies from the initial viral infection to late stage symptoms.  The life cycle of the HIV-1 virus is illustrated in Figure 3.9  Primary infection results from the binding of HIV to the surface gp120 protein of host CD4+ T-cells, with entry of the viral particle into the cell.  A ds-DNA copy of the viral ss-RNA is then produced through reverse transcription, orchestrated by the viral polymerase, reverse transcriptase (RT).  The viral RNA is degraded in the process, and 5  the ds-DNA enters the nucleus of the cell and is integrated in the host DNA by the viral enzyme integrase. The newly integrated DNA now serves as the template for the production of the viral m-RNA via transcription, which serves both as a new copy of the viral genome and as the precursor via translation to key HIV proteins involved in the assembly of new virions.  In this process, the newly formed components accumulate and move toward the cell membrane.  The HIV-protease then cleaves the viral Gag and Gag-pol polyproteins, creating the mature protein components of the infectious virus.  The HIV-induced cell lysis leads to a decrease in CD4 cell counts of the host and therefore the destruction of the host's immune system.10    Figure 3.  The HIV replication cycle.9 (Image adapted from reference 9)   The current drugs used in ART target and block viral entry (entry and fusion inhibitors) or the function of the three crucial viral proteins: reverse transcriptase (nucleoside and non-nucleoside RT inhibitors), HIV-protease, and HIV-integrase. ART uses one or more HIV drugs from several of these drug classes in order to circumvent the problem of resistance that rapidly emerges to each drug (as is evidenced in monotherapy).  Although ART can effectively suppress the chance of the virus escape in the infected individual by using a personalized cocktail of drugs, ART is not a cure.  Furthermore, the long-term use of ART is limited by issues of drug compliance, side effects, and 6  ultimately drug resistance.  To overcome these problems, new drugs, especially those that act through an unexploited mechanism of action, need to be discovered.11 Our research has focused on development of novel molecules that perturb HIV mRNA processing, and, in particular, molecules like 1C8 that block the alternative slicing of HIV pre-mRNA transcripts. Preliminary evidence suggests that targeting this host cell process in HIV infected cells results in minimal or no toxicity to normal cells.12   1.4 Splicing and alternative splicing  Figure 4.  mRNA splicing and alternative splicing.13 (Image adapted from reference 13)  During transcription, RNA is synthesized in the 5' to 3' direction along a template DNA strand.  In eukaryotes, the pre-RNA synthesized during transcriptions contains both coding (exons) and non-coding regions (introns). Splicing is the process whereby the introns of the pre-mRNA are excised and the exons are joined together to give the matured m-RNA that is involved in translation. RNA splicing occurs in the nucleus, and is orchestrated by the spliceosome, a multicomponent “machine” consisting of specialized RNAs and more than 150 proteins.  Splicing can occur in a sequential linear fashion to give a mRNA that codes for a single protein, or it can occur in a non-linear fashion such that the exons are joined in different orders to give a set of different mRNAs, many of which code for a different (often distinct) proteins (Figure 4). This process is called alternative splicing.14 Splicing and alternative splicing are nuclear events that often occur as transcription is advancing. 7   1.5 Serine-Arginine Rich (SR) proteins and alternative splicing SR proteins are intrinsically disordered proteins that play important roles in alternative splicing.  There are 12 SR proteins in human cells.  Each SR protein contains repeating units of arginine (R) and serine (S) and are about 50-300 amino acids in length.  They contain one RS-binding domain at the C-terminus, which is rich in arginine/serine and engages in and regulates protein-protein interactions between different splicing factors.  At any one-time multiple serine residues in the SR-domain are phosphorylated.  Depending upon the task that the SR protein is engaged in, the pattern of phosphorylated serine residues can vary. In the N-terminus of SR proteins one or two RNA recognition motifs (RRM) mediate its interactions with mRNA through binding to exon splicing enhancer (ESE) sequences.15  SR-proteins are very important in selecting splice sites for alternative splicing in that they recognize intron and exon enhancers and silencers (cis-acting elements).16  However, due to their conformational mobility, sparingly little information is available concerning both their structure/conformations and the mechanisms whereby they accomplish their tasks.  1.6 HIV and alternative splicing HIV-1 replicates itself by integrating the reverse transcribed viral ds-DNA into the host's DNA.  Once this happens, the viral DNA is transcribed to form new viral mRNA, and subsequently viral proteins that translocate to the cell surface to assemble into new immature virus particles.  Budding occurs so the new viruses are released from the cell.   The HIV-1 genome is about 9kb in length.  The viral transcript will code for its genomic RNA and the mRNA of the viral Gag and Gag-pol proteins.  The Gag (gene-specific antigen) protein codes for the core structural protein of the retrovirus, while the pol protein is the reverse transcriptase, which is an enzyme that allows the transcription of the viral RNA into DNA.  Gag-pol is a fusion protein, the production of which is dependent on the readthrough of the stop codon at the gag-pol junction.17-20  The successful production of new infectious viruses depends on the balanced expression of seven other viral proteins: Tat, Rev, Nef, Vif, Vpr, Vpu, and Env.  In order to achieve this, alternative splicing or intron retention of the primary transcript and nuclear export of the unspliced transcript must be regulated.17, 21-22 After the viral RNA is reverse-transcribed and integrated into 8  the host's DNA, the cellular RNA Polymerase II transcribes the provirus, producing the polycistronic pre-mRNA, which can result in the production of more than 40 different mRNAs from alternative splicing.21, 23-24   Figure 5.  Organization of HIV-1 genome and different mRNA splicing products.25 (Figure adapted from reference 25)   1.7 Anti-HIV activity of 1C8 Based on biological tests, 1C8 shows antiviral activity in T-cell-based assay against wild-type HIV-1 and multiple drug-resistant strains of HIV-1: 1C8 shows IC50's of 0.45 µM and 0.94 µM when tested against HIV-1IIIB (subtype A, X4-tropic) and HIV-1 97USSN54 (subtype A, R5-tropic), respectively. Unlike IDC16, which targets SRSF1, Chabot and coworkers showed that 1C8 targets SRSF10 function.  SRSF10 has been identified as a splicing repressor in heat shock.26  More recently, it has been found that SRSF10 is the key regulator in Bcl-2-associated transcription 9  factor 1 (BCLAF1) pre-mRNA splicing and is associated with oncogenic activities in human colon cancer cells.27   Using Bcl-x as the reporter gene, they found that 1C8 affects the splicing activity of SRSF10 by promoting its dephosphorylation and increasing its interaction with hTra2, which is a regulator in HIV-1 splicing.  Unlike IDC16, 1C8 has no impact on the splicing activity of SRSF1 and SRSF9.28   In more recent tests (not yet published), it was found that 1C8 also acts as an inhibitor of SR protein kinases. Further it has been found that 1C8 is also a potent inhibitor of adenovirus, herpes simplex 1, influenza and Hepatitis B (confidential) replication (Table 1).  The mechanistic details at the molecular level responsible for these biological activities are not fully understood at this point.  Table 1.  Various anti-viral activities of 1C8. Compound Virus HIV-1 (PBMCs) Adenovirus (A549) HSV-1 (HeLa) Influenza (MDCK) 1C8 IC50 = 0.625 µM IC50 = 1 µM IC50 = 1 µm IC50 = 3 µM CC50 > 10 µM CC50 > 10 µM CC50 > 10 µm CC50 > 10 µM  1.8 Impact of 1C8 on cellular genes and HIV pre-mRNA splicing In HeLa cell lines that are stably expressing a modified HIV provirus, addition of 1C8 leads to decrease in Gag (p24), Env, and Rev proteins.  From qRT-PCR, a decrease in all HIV-1 mRNA (unspliced, singly spliced, and multiply spliced RNAs) is observed when 1C8 is present.  In situ hybridization shows that HIV-1 genomic RNA is reduced and confined to transcription sites inside the nucleus.  This is consistent with the loss of Rev, which is responsible for the transport of unspliced mRNA from the nucleus to the cytoplasm.  In endpoint RT-PCR assay experiments, both Tat1 and Nef1 mRNA decrease in the presence of 1C8.  Furthermore, 1C8 affects transcription even in the presence of Tat (an HIV protein that aids in the transcription of the singly spliced and unspliced viral mRNA), but house-keeping cellular genes are not affected.  At 1 µM of 1C8, only expression of HIV RNAs is affected.  This is the first time where SRSF10 has been found to affect HIV alternative splicing.4, 28    10  1.9 1C8:  The benzisothiazole motif, a focus for SAR studies A key motif in the structure of all four active IDC16 mimics is the 5-nitro-1,2-benzisothiazole ring.  Compounds containing a benzisothiazole motif have been found to display antiproliferative, antimicrobial, anti-inflammatory and antibacterial activities.29-30  Most recently, the atypical antipsychotic drug, Lurasidone, which incorporates a benzisothiazole motif was approved in the USA in 2010 (Figure 6).31   Figure 6.  Structure of 1C8 and selected other benzisothiazole-containing compounds that show biological activities.4, 29-31  Consequently, the development of efficient methods for the facile construction and functionalization of the benzisothiazole system has attracted considerable interest. In particular, as developed in more detail further on in this chapter (Sections 1.11.2 and 1.11.3), and in Chapters 2 and 3, my research project to carry out SAR studies on 1C8 has been focused on the manipulation and modifications of the benzisothiazole component in this molecule. As a preamble to this work, we present pertinent background information concerning the chemistry of this ring system.  1.10 Chemistry of benzisothiazole 1.10.1 1,2-Benzisothiazoles The benzisothiazoles (BIT) ring system corresponds to a benzene ring fused to an isothiazole motif.  As shown in Figure 7, there are two isomeric forms of benzisothiazole, depending on the relative positions of S and N in the heterocycle: the 1,2- or 2,1-benzisothiazole isomers.  Among them, the 1,2-benzisothiazone dioxide, saccharin, which is used as an artificial sweetener (300-400 times sweeter than sucrose) has been the most extensively studied.32   11   SN1234567NS12345671,2-benzisothiazole 2,1-benzisothiazoleSNHOOOsaccharin(1,2-benzisothiazol-3(2H)-one 1,1-dioxide)8989 Figure 7.  Nomenclature of benzisothiazoles. (Note:  The junction carbon atoms 8 and 9 are labeled as 3a and 7a in some literature reports)32-33   Interestingly, although there has been considerable effort directed toward the development of biologically active 1,2-benzisothiazole, an analysis of the literature showed that structure-activity studies (SAR), undertaken during the development of most 1,2-benzisothiazole-based compounds, have been surprisingly narrow in scope.29   An underlying reason for this, as painfully revealed during my PhD thesis studies, is that there are multiple facets to the reactivity of the benzisothiazole ring system, and controlling the different reactivity modes is not straight-forward.  It is the consequence of this “awakening”, that my PhD project, initially focused on the SAR studies of 1C8, progressively evolved toward an investigation of several unexplored aspects of the reactivity of the 1,2-benzisothiazole system.  Prior to presenting the objectives of my thesis a brief overview of the preparation and reactivity of the benzisothiazole ring system will be provided.  Indeed, understanding the chemistry of benzisothiazoles is crucial to any future SAR studies on 1C8.    1.10.2 SNAr-type nucleophilic substitution reaction at C-3 versus formation of benzonitrile products through nucleophile induced cleavage of the S-N bond in benzisothiazoles.  In an analogous way to the ability of other heterocyclic systems to undergo nucleophilic substitution via a SNAr mechanism, a logical process for the preparation of 3-substituted benzisothiazoles would be to react a benzisothiazole intermediate incorporating a suitable leaving group adjacent to the nitrogen atom at C-3 with a nucleophile. As illustrated in Figure 8 for 3-chloropyridine, SNAr-type “ipso” substitution reactions are generally considered to proceed in a two-step manner, involving initial formation of a Meisenheimer complex and subsequent rearomatization to form the C-2 substituted pyridine compound with Cl acting as the leaving group, or, as more recent work suggests they can occur via a concerted mechanism.34   12  N ClNuN ClNuN NuMeisenheimer complex  Figure 8.  Nucleophilic aromatic substitution of 2-chloropyridine.35  Applied to benzisothiazole, the 3-Cl function in 3-chloro-1,2-benzisothiazoles can be displaced through reaction with typically “hard” nucleophiles such as amines (ammonia, primary-tertiary amines) and alkoxides, and by reaction with certain “softer” enolates to give 3-substituted 1,2-benzisothiazoles (Table 2).32  Table 2.  Nucleophilic substitution reactions of 3-Chloro-1,2-benzisothiazole with different nucleophiles.32 SNClR1Nu-SNNuR1  R1 Reagent Nu Yield(%) 4-Cl NH3, H2NCHO, 130-140°C NH2 90 4-Cl NaOMe, MeOH, 60°C, 5h OMe 75 4,5-Cl2 NaOMe, MeOH, 60°C, 5h OMe 75 H NaOEt, EtOH OEt 92 H CH2(CO2Et)2 CH(CO2Et)2 46 H CH2(CN)CO2Et CH(CN)CO2Et 70  Indeed, 3-piperazinyl-1,2-benzisothiazole, one of the major building blocks of the antipsychotic drug Lurasidone was prepared in this way (Figure 9).36-37   13   Figure 9.  Synthesis of Lurasidone precursor 3-piperizinyl-1,2-benzisothiazole 3 from 3-Cl and 3-OTf-benzisothiazole.36-37  However, the positioning of the N and S atoms adjacent to each other in the five-membered ring of benzisothiazole adds to the complexity of the chemistry of this system. Indeed, two alternate/competing modes of reactivity of 3-substituted benzisothiazole’s (ex. X = Cl) with nucleophiles have been identified, which often, in fact, dominate (Figure 10).  In the first of these pathways (pathway (a)), SN2 reaction at the benzisothiazole sulfur atom occurs, and in the second (b) the C-3 X atom/group (depending on its nature) ends up being a “leaving group” in a completely different manner.  In both mechanisms a driving force is N-S bond cleavage, which generates the cyano group found in the benzonitrile products. These reactions may occur in a concerted manner or, formally, as indicated in the Figure 10, through evolution of species 4 and 6.32, 38   Figure 10.  Two alternate/competing pathways to the 3-C reactivity of 1,2-benzisothiazole.38    In the first of these reaction modes (a), “soft” (and some more hard) nucleophilic reagents such as sodium cyanide, thiols, and certain tertiary amine bases react at the sulfur atom in benzisothiazole with opening of the five-membered isothiazole ring to produces an imidate intermediate 4 (in neutral or anionic form), which can “spontaneously” (concomitantly) eliminate X- to reveal the cyano group in benzonitrile 5.38   For example, in the reaction of 3-chloro-1,2-14  benzisothiazole 1 with NaCN in acetone, studied by Clarke et al.  in the early 70’s, (Figure 11), formation of the 3-cyano substituted SNAr product was not observed. In these conditions the dominant reaction product was the pathway “a” compound, 2-thiocyanatobenzonitrile 8 (62%).39    Figure 11.  Formation of 8 and 9 by reacting 3-chloro-1,2-benzisothiazole 1 with NaCN in aqueous acetone.39    Important also was the formation of the more minor by-product, disulfide 9 (22%). At first site, the presence of 9 in the reaction mixture was not surprizing, as 2,2’- cyano substituted disulfides are frequently formed as a by-product in the SN2 reaction of nucleophiles at sulfur in benzisothiazoles. As shown in Figure 10, this can result from cleavage of the S-Nuc bond in 5 to give 7 and dimerization through aerial oxidation or, as indicated in Figure 11, from reaction of 4 with another molecule of 3-chloro-1,2-benzisothiazole 1. Indeed, Watanabe et al. showed that reaction of 3-chloro-1,2-benzisothiazole 1 with different substituted thiophenols resulted in the evolution of HCl gas and the formation of o-cyanophenyl disulfides 33-87% yields.40    However, in the specific example of the reaction of 3-chloro-1,2-benzisothiazole 1 with cyanide ion, the thiocyanate group in the ring opened product 8 is stable. The production of the disulfide dimer 9 in this reaction could, therefore, not originate from further transformation of compound 8. To account for its formation the alternate reaction mode illustrated in pathway b of Figure 10 (and the bottom half of Figure 11) was put forward. In this mechanism, Carrington and coworkers proposed that the attacking cyanide nucleophile reacts with the chlorine atom in the SNClCNSSNClClCNCNSCNCNSSNC17NaCN, acetonePathway aPathway b8 (62%)9 (22%)15  intended leaving group in 1 to produce the N-S bond cleavage ring opened thiolate product 7 (either in a stepwise process via the imidate anion 4 (Figure 10) or via a direct/concerted process).39  Implicit in this mechanism is the idea that benzisothiazole itself can act as the leaving group as CNCl is formed in Pathway b, undergoing transformation to sulfide 7 and disulfide 9 as the reaction proceeds.  Interestingly, this latter reaction mode (pathway b) explains the otherwise unexpected formation of compound 10 (90%) when 3-chloro-1,2-benzisothiazole 1 was reacted with the hard nucleophile n-BuLi in DMF (Figure 12).41  Figure 12.  Formation of o-(n-butylthio)benzonitrile 10 by treating 3-chlorobenzisothiazole 1 with n-BuLi in DMF at -70°C.41    In this case it is proposed that the initially formed cyano sulfide intermediate reacts with the BuCl produced, (or more likely with the residual BuBr present in the solution of the alkyl lithium reagent used (the n-BuLi was prepared from BuBr by the Li halogen exchange method).  Interesting also was the observed formation of the ring opened sulfide 11 through an E2-like reaction of benzisothiazole itself (i.e. no C-3 substituent) with sodium methoxide in methanol (Figure 13).42  This reactivity is analogous to the Kemp elimination in benzisoxazole.43  Figure 13.  Ring-opening of benzisothiazole by sodium methoxide in methanol resulting in the formation of the thiolate anion 11.42  SNHOMeCNSNaNaOMe, MeOH1116   The ease with which S-N bond cleavage can occur is further illustrated by the observed equilibration of 3-aminomethyl substituted benzisothiazole 12 (Figure 14). This process is facilitated by the presence of HCl, which supplies a proton that can protonate the imine nitrogen,  and a nucleophile (Cl-), which on reaction with the sulfur centre generates an activated -SCl species for re-closure of the ring to give 12’.44  SNNHHClSNNHHClNHNHS Cl SNNH-H+12 12' Figure 14.  Isomerization of 3-amino-1,2-benzisothiazole 12 to 12’ in the presence of HCl.44  To summarize on the reactivity of the benzisothiazole system, it may appear attractive to predict whether a nucleophile will react via a desired SNAr type substitution at C-3 or via the mechanisms leading to S-N bond cleavage on the basis of the hard/soft character of the attacking nucleophile. However, as discussed in Chapter 3, we, and others, have found, that there are numerous exceptions to the rule. What is more, recent studies by Walinsky et al., connected to the synthesis of lurasidone, adds a further layer of complexity to the reactivity of benzisothiazoles.45 It was found that when 3-chloro-1,2-benzisothiazole 1 was reacted with piperazine in THF at 65 °C the ring opened compound 13 was formed in high yield, rather than the expected SNAr product (Figure 15). However, on continued heating at higher temperature (165 °C) it was apparent that the piperazine nucleophile reacts with the cyano group in 13 to give the presumed amidine intermediate 14, which undergoes ring closure to give the 3-piperazinyl substituted benzisothiazole compound 3, i.e. the product of direct SNAr substitution at C-3.   On the basis of these observations, it is legitimate to suggest that for certain amines (and perhaps other nucleophiles) the direct SNAr substitution at C-3 may not be occurring at all. Note also that the cyclization of intermediate 14 is reminiscent of several routes developed to construct the five-membered ring in benzisothiazole through thiol activation (see next section).     17   Figure 15.  Proposed reaction pathway when 3-chloro-1,2-benzisothiazole 1 reacts with excess piperazine to form 3-piperizinyl-1,2-benzisothiazole 3.45  Globally, one sees that the reactivity of the benzisothiazole ring system is governed by the electronic/orbital interactions between the S and N atoms (as well as substituents on C-4 to C-7  on benzisothiazole), which impacts the N-S bond strength, and bond length. The system is thus inherently susceptible to the reactions that open the heterocycle and reveal the cyano motif.  Indeed, one can look at the five-membered isothiazole ring in benzisothiazole as a camouflaged nitrile group.   1.10.3 Synthetic methods of 1,2-benzisothiazoles A number of different approaches have been developed for the construction of 1,2-benzisothiazoles. In view of the focus given to the reactivity of the central N-S bond, attention will be directed to the two ring closure strategies highlighted in Figure 16. These ring forming reactions are distinguished by the initial absence (strategy a) or presence (strategy b) of the ring nitrogen atom in the penultimate intermediates.38  CSNSNSNC                             a                                                                          b Figure 16. Two major ring closure strategies for the synthesis of 1,2-benzisothiazole.38     SNXX = Cl1NucpiperazineTHF 65°C NCSNNHNHHN 13SNNHNPip14SNNHN318  Strategy “a”:  The reaction of the benzaldehyde derivative 15 bearing an activated ortho sulfenyl halide substituent with ammonia was shown to give the 5-nitro-1,2-benzisothiazole 16 in 56% yield (Figure 17). 46 CHOSBrconc. NH3SNO2NO2NSO2N NHHBr-HBr16 (56%)alcohol15  Figure 17.  Synthesis of 5-nitro-1,2-benzisothiazole 17 via a sulfenyl halide intermediate.46  In a more recent example, various 7-substituted 3-amino-1,2-benzisothiazoles 18 were prepared by cyclization of in situ generated aminosulfanylarenes 17 bearing a cyano group at the ortho position (Figure 18).47   CNCl(i) NaSH, HMPT120 °C, 24 hr(ii) 3% NaOH CNSSNNH2R17R = Cl, NO22. NH2Cl-5 to 10°C, 1.5 hrSH NaRRCSNH2RNSNHNHRTautomerize18  Figure 18.  Synthesis of 3-aminobenzisothiazoles 18 with electron withdrawing groups at C-7.47  Strategy “b”:  One of the early approaches to construction of the 1,2-benzisothiazole system from precursors already incorporating the future five-membered ring nitrogen involved the oxidative cyclization of 2-(aminomethyl)benzenethiols 19 in the presence of iodine or bromine (Figure 19).  The disulfide 22 is a possible intermediate in the process, but the cyclization step most likely involves intramolecular attack of the amine nitrogen on the initially formed sulfenyl iodide motif 19  in 20.  Subsequent oxidation of benzisothiazole intermediate 22 gives the 1,2-benzisothiazole product.47 KI + I2 KI3NH2S HOHNH2SIIINH2S-HI SSNH2H2NSN89%I19 2021 22oxidationK+ Figure 19.  Generation of 1,2-benzisothiazole from 2-(aminomethyl)benzenethiol 19 in the presence of I2 and KI.47  Diaryl disulfides have been used as starting materials for 1,2-benzisothiazole when a halogen is present as the oxidant (Figure 20).  In the following example, 3-chloro-1,2-benzisothiazole 1 was prepared in 36% yield from the disulfide 9 via the formation of the intermediate sulfenyl chloride 23.48  Figure 20.  Mechanism for the formation of 3-chloro-1,2-benzisothiazole 1 from disulfide 7 in the presence of chlorine.48   Interestingly, Nakamura et al. from the Sumimoto Pharmaceuticals Research Centre (Company that made Lurasidone) has reported a study that also used the diaryldisulfide 9, in this case, as a starting material for the preparation of different 1,2-benzisothiazole derivatives 24, substituted at C-3 by a secondary amine function (84-96% yields) (Figure 21).49  In the first step of their procedure (illustrated using pyrrolidine Mg amide) 2,2’-dithiobiz(benzonitrile) 9 reacts with one equivalent of the Mg amide at the cyano group to give the target product 3-(N-20  pyrrolidinyl)-1,2-benzisothiazole 24 and the Mg salt 25 of 2-sulfanylbenzonitrile.  In the next step, a second equivalent of the Mg amide reacts with 25 to form the 2-sulfanylbenzamidine dianion 26.  This dianion reacts with added pyrrolidine in the reaction mixture to both generate the Mg 2-sulfanylbenzamidine 27 and refurbish the pyrrolidine Mg amide.  Addition of CuCl2 to the cooled reaction mixture then effects ring oxidative ring closure to provide the second molecule of product 24.49  CNSSNCNMgBr1st equiv.SNN+ CNSMgBrNMgBr2nd equiv.SMgBrNMgBrNNMgBrNHSMgBrNHNCuCl2SNN92424 25 2627  Figure 21.  Proposed mechanism for the formation of 3-(N-pyrrolidinyl)-1,2-benzisothiazole 24 in the reaction of Mg amides with 2,2’-dithiobiz(benzonitrile) 9.49    A key feature in the Nakamura chemistry, which provides a background for results obtained in Chapter 3, is the idea that the addition of a nucleophile to the cyano group will generate an imidate species that can react with an “activated” sulfur intermediate to form the benzisothiazole ring. This idea follows from earlier strategies in which amides and their equivalents were used to access synthetically valuable 1,2-benzisothiazol-3-one-based compounds.  For instance, the reaction of 2,2-dithiobis-benzamide 28 with Br2 generates a thienyl bromide intermediate, which cyclizes to form the 1,2-benzisothiazol-3-one 29 (Figure 22a).50  Similarly, the 2-benzithio-4,6-dinitrobenzamide with SO2Cl2, results in cleavage of the S-Bn bond and formation a sulfenyl 21  chloride intermediate that cyclizes to give  4,6-dinitro-1,2-benzisothiazol-3-one 30 (Figure 22b).51  More recently, electrophilic fluorinating reagents such as Selectfluor have been used to convert thiobenzamide 31 to the N-alkylated 1,2-benzisothiazol-3-one 32 (Figure 22c).52  In this reaction, the fluorinating agent acts as a catalyst, or a mediator through the interaction between the F atom and S, resulting in the formation of the cyclic fluorosulfonium salt (N-S bond formation).  Depending on the pathway, the final product is formed either through the loss of Me (to form R3N-Me) or the regeneration of the catalyst.  22   Figure 22.  Preparation of 1,2-benzisothiazol-3-ones 29, 30 and 32 from (a) 2,2-dithiobis-benzamide upon treatment with Br2,50 (b) cyclization of benzyl sulfide upon treatment with sulfuryl chloride,51 and (c) intramolecular N-S bond formation using Selectfluor.52  23  Another strategy  to prepare 3-substituted (H, alkyl, Ph) -1,2-benzisothiazole, in which it is  the sulfur that reacts as a nucleophile in the ring forming step is manifested in the reaction of oxime derivatives 33  with activating agents such as Ac2O and PPA (Figure 23).47, 53-60     Figure 23.  Synthesis of 1,2-benzisothiazoles from 2-(t-butylthio or methylthio) benzaldehyde oximes.47, 53-60  Recently, Deng and Xiao have reported a metal free procedure to synthesize 3-arylamino-1,2-benzisothiazole compounds 36 from amidines and elemental sulfur (Figure 24).61  The first step involves the attack of the imine nitrogen on one of the S atoms of the S8 ring, forming the N-S bond in 34.  Loss of S7 and deprotonation resulting in the formation of the thio-oxime anion 35.  The final step of the reaction is an intramolecular nucleophilic substitution on the C atom bearing the Cl by the sulfur.  24   Figure 24.  Synthesis of 1,2-benzisothiazoles from o-haloarylamidines and elemental sulfur via N–S/C–S bond formation.61  Finally, of the many other strategies developed to construct the benzisothiazole system, one of the most elegant is the [4 + 2] cycloaddition or Kobayashi benzyne approach,62 where the S-N bond is derives from a 1,2,5-thiadiazole precursor (Figure 25).  Depending on the nature of R and R’, the yield of the final product 1,2- benzisothiazole can range from 25-75%.63   NSNR' R'+S NN R'R'R'CNSNR'R' = H, Me, CN, Cl, OEt  Figure 25.  Reaction between benzyne and 1,2,5-thiadiazoles to form 1,2-benzisothiazoles.62-63  In this manner, Chen and Wallis (ref) developed a “dissymmetric” version of this aryne-based route to synthesize substituted benzisothiazoles (Figure 26).64  In this case, the production of 3-amino-substituted-1,2-benzisothiazoles could be achieved using dissymmetric thiadiazoles and 2-(trimethylsilyl)aryl triflates as the starting materials and CsF as the fluoride source.  This approach 25  also allowed the preparation of 3-amino-1,2-benzisothiazoles, which was not reported in earlier studies (the key starting material for lurasidone).  Figure 26.  Formation of 3-substituted-1,2-benzisothiazoles using Koabayashi precursor.64  1.10.4 Preparation of 3-substituted benzisothiazole’s (3-Cl, sulfonate, OMe, and NH2): Starting material in synthesis  3-Chloro-benzisothiazole 1 and its derivatives/congeners is an important precursor to 3-amino and 3-OMe-1,2-benzisothiazole.  A classical synthetic strategy to access this starting material is to react 1,2-benzisothiazol-3-one 37 with POCl3/DMF (Figure 27).65-66  In these acidic conditions 37 tautomerizes to its 3-hydroxy-1,2-benzisothiazole form, which reacts with the in situ generated Vilsmeier reagent) to give 38.   Nucleophilic substitution by Cl- at C-3 of 39 gives the final product 3-chlorobenzisothiazole 1.  Figure 27.  Synthesis of 3-chlorobenzisothiazole 1 using POCl3/DMF.65-66   In a similar way, the reaction of 1,2-benzisothiazol-3-one 37 with sulfonyl chlorides provides a simple way to prepare the reactive 3-sulfonate derivatives of 1,2-benzisothiazole 40 26  (Figure 28).37, 67 In fact, this compound was used to prepare 3-piperizino-1,2-benzisothiazole 3, a precursor to the approved antipsychotic agent Lurasidone.  SNHOSNO SRO O NHHN(3.5 eq.)60°C, EtOH SNNHNSRO OCl(1.0 eq.)Et3N (1.0 eq.)DCM, 0°C3 hr37 40 3 Figure 28.  Preparation of 3-piperizinylbenzisothiazole 3 from the 3-sulfonate substituted benzisothiazole 40 (R = CH3, Ph).  Note that 3-OTf-benzisothiazole 2 was prepared by the reaction of 37 with triflic anhydride (Figure 9).37, 67  3-Methoxy-1,2-benzisothiazole 41 can be readily prepared from 3-chlorobenzisothiazole 1 through reaction with NaOMe/MeOH at reflux (Figure 29).68  Note, there are no literature indications that 3-chlorobenzisothiazole undergoes ring opening under these reaction conditions.  Based on HSAB theory, this reaction proceeds through in the SNAr manner at C-3 to give the desired product.    SNClSNOMeNaOMe (1.1 eq.)MeOH, reflux 1 hr75%1 41  Figure 29.  Synthesis of 3-methoxybenzisothiazole 41 from 3-chlorobenzisothiazole 1.68  3-Aminobenzisothiazole’s are important starting materials for the preparation of 3-acylamino substituted benzisothiazole type compounds. In fact, 5-nitro-3-aminobenzisothiazole 43 served as the starting material for the synthesis of our anti-HIV agent 1C8.   Their preparation is achieved by heating the requisite 3-chlorobenzisothiazoles, such as 5-nitro-3-chlorobenzispthiazole 42, in saturated ammonia/formamide at elevated temperature for 2 hours (Figure 30).69 Note that this is the original literature report on how to prepare this compound. It is entirely possible that today, commercially available 3-amino-5-nitro-1,2-benzisothiazole is prepared using milder conditions (instead of 140-150 °C).   27   Figure 30.  Preparation of various 3-amino-1,2-benzisothiazoles under amination conditions.69   1.11 My Research Project: Objectives  Following the discovery of the anti-HIV (anti-viral) agent 1C8 in our laboratories, the objective for my thesis research project was to prepare and study different variants (analogs or mimics) of this molecule to determine their influence on anti-HIV activity versus toxicity (Selectivity Index) and target binding. Currently, the evidence suggests that 1C8 blocks HIV pre-mRNA alternative splicing by interacting either directly with its putative target SRSF10 (serine arginine rich protein 10) or indirectly with an enzyme that affects the phosphorylation/dephosphorylation of this protein.  At the outset of this project, and still today, we have no detailed structural knowledge concerning the nature of the interactions between 1C8 and its target nor, in fact, do we have a definitive idea of the target’s identity and the mechanism whereby 1C8 exerts its biological activity. “In light of this darkness” we have chosen to currently limit our SAR studies on 1C8 to considerations of how the three-dimensional shape/preferred conformations of this molecule are important to initial “drug-target” recognition. Indeed, the more effective is the recognition between a small molecule drug/inhibitor/probe the greater the probability for strong binding and associated potency.2   Objective 1 (Chapter 2):  SAR on 1C8 through modulation of functional groups and modification (remorphing) of its 1,2-benzisothiazole-amide substructure   Chapter 2 describes the preparation of compounds designed to probe putative target binding of 1C8 and the influence of structural changes on the preferred conformations of 1C8 on activity.  Simple functional group modifications that have been explored include, reduction of the potentially bioactivatable nitro group, oxidation of the benzisothiazole sulfur atom, inversion of the central amide motif and its replacement. The consequence on activity of the more deep-seated 28  modification of the structure of 1C8 wherein the amide function and the nitrogen of the isothiazole ring in 1C8 are integrated “morphed” into a conformationally rigid pyrimidine ring will also be described. These changes are illustrated in Figure 31. Note that biological tests on all new compounds were performed in collaboration with Dr. Peter Cheung from the BC Centre for Excellence in HIV/AIDS.     Figure 31.  Compounds prepared during the SAR studies on 1C8.      Objective 2 (Chapter 3):  Modification of the conformational mobility of the central amide in 1C8 through its incorporation into a pyrazolone motif - The discovery of new chemistry/reactivity of the benzisothiazole ring system.  29  In Chapter 3 the objective was to prepare a constrained amide analog of 1C8, which captures a crucial H-bonding interaction that is believed to be important to 1C8’s 3-dimensional conformational structure (Figure 32). The plan was to incorporate this structural element into a pyrazolone ring, as found in the pyridopyrazolone compound 44.  Pivotal to this effort was the preparation of the 3-hydrazino-5-nitro-1,2-benzisothiazole intermediate 45. However, experiments destined to provide us with this important material revealed unexpected aspects of the reactivity of the benzisothiazole system, which were studied in detail. Interpreting these results required establishing mechanisms for several deep-seated rearrangements that the benzisothiazole ring system underwent.    Figure 32.  Strategy for the preparation of the constrained 1C8 analog: pyrazolopydridine 44.   Support for our mechanistic interpretations was obtained through a much appreciated collaboration with Pr. Pierre Kennepohl and Ms. Xing Tong, a PhD thesis student in his laboratory. These researchers carried out valuable predictions of the energetics for these transformations using Density Function Theory calculations.  30  Chapter 2: SAR studies on 1C8 through functional group modulation  2.1  Introduction As discussed in Chapter 1 of this thesis, 1C8 perturbs the function of the splicing factor SRSF10 in HIV-1 pre-mRNA alternative splicing. Published data indicates that 1C8 may inhibit dephosphorylation of SRSF10 by phosphatases, and more recent (unpublished) data suggests that it also/alternatively inhibits SR protein phosphorylation by SR protein kinases.28 In both situations, 1C8 interferes with substrate (SRSF10) – phosphatase/kinase (protein-protein) interactions.  Indeed, addition and removal of phosphates from the many serine residues in SRSF10 determines the multiple cellular processes it regulates.  Unfortunately, as SRSF10 is an intrinsically disordered protein, there is a lack of a useful structural understanding as to how this conformationally mobile protein undergoes selective phosphorylation/dephosphorylation at important serine residues. In the absence of structural information, we have adopted several classical SAR strategies, involving both structural modulation of the 1C8 structure and investigation of rationally designed alternative structures, to gain an understanding of the nature of binding interactions it engages. It is anticipated that in this way, we may also optimize the anti-HIV activity of 1C8 congeners.   Figure 33.  SAR modifications on 1C8.  This chapter discusses five different modifications of the structure of 1C8 (Figure 33):  31  (A) Inversion of the amide linker in conjunction with varying the position of the nitro group in the benzisothiazole motif.  (B) Reduction of the nitro group in 1C8 (C) Replacement of the benzisothiazole sulfur by an oxygen atom and by a sulfone motif (D) Replacement of the amide function by an aminoether motif (excission of the amide C=O) (E) Replacement of the amide motif and the five-membered isothiazole ring in the benzisothiazole component by a pyrimidine ring.  2.2 (A)     Inverse amide synthesis and isomeric nitro group placement The initial goal of my project was to synthesize compound 46b, the analogue of 1C8 wherein the amide function has been inversed, i.e. oriented in the opposite sense (Figure 34). It was expected that this tactic would provide information concerning the acceptor/donor H-bonding interactions the amide bond in 1C8 may engage in upon binding to its putative target. We were also cognisant of the possibility that the inversion of the amide bond analogue could also result in an altered preferred conformation in compound 1C8. This latter effect may impact recognition by the putative target.  Figure 34.  1C8 and 4-, 5-, and 7-NO2 substituted inverse amides 46a (4-NO2), 46b (5-NO2) and 46c (7-NO2).  To obtain the 3-amino-4-pyridinone 49 needed for amide coupling reaction to give inverse amides 46a-c, the pyridine nitrogen in 3-nitro-4-pyridinone (3-nitro-4-hydroxypyridine) (47) was methylated, and the nitro group in 48 was reduced by Pd catalyzed hydrogenation (at 1 atm H2(g)). Compound 49 was obtained as an orange waxy solid in 38% overall yield (Figure 35).70    32   Figure 35.  Synthesis of 3-amino-1-methyl-4-pyridinone.70 Of the different methods available for construction of the carboxylic acid component needed to prepare compound 46b, we adopted the method involving formation of the isatin intermediate 53 and its rearrangement to 3-carboxybenzisothiazole 54 (Figure 36).71-72 An important consideration in this choice was also the fact that, in addition to formation of the target compound 56b, nitration of intermediate 55 would give rise to the two additional isomeric nitro-substituted compounds 56a and 56c. In this way we could study the influence on anti-HIV activity of both the inverse amide bond and the positioning of the nitro substituent in 1C8.  To obtain compound 53, thiophenol (50) was reacted with oxalyl chloride (51), and the in situ generated monothioester 52, was reacted with AlCl3 to affect the intended ring closure (intramolecular Freidel-Crafts acylation). Isatin 53 was converted to 3-amido-benzisothiazole, 54 in 46% yield through reaction with ammonia and 30% hydrogen peroxide. Under these conditions part of the added ammonia was oxidized to hydroxylamine, which reacted with 53 to give oxime (a). The remaining ammonia reacted at the thiocarbonyl carbon centre in (a) with ring opening of the five-membered ring, leading to sequential formation of b, c and d).  Subsequent reaction of the sulfide anion in (e) with the oxime nitrogen, (g) led to formation of the amide compound 54.  In the remaining two steps, amide hydrolysis of 54 in the presence of base afforded carboxylic acid 55.  Nitration of 55 produced the target compounds 5- and 7-nitrobenzisothiazole-3-carboxylic acids 56b and 56c (in a 1: 2 ratio by 1H NMR), along with a minor amount of the 4-nitro isomer 56a.73-74  All three isomers have the same Rf value on TLC but could be totally/partly separated by recrystallization.  Indeed, separation and isolation of the 5- and 7-nitrobenzisothiazole-3-carboxylic acids 56b and 56c was achieved by multiple recrystallizations from EtOAc/MeCN. However, the 4-NO2 isomer 56a could not be obtained in its pure form, and 56a was carried forward as a mixture with the 5- and 7-NO2 isomers in the final amide bond forming step.   33   Figure 36.   Synthesis of nitrobenzisothiazole-3-acetic acids 56a, 56b, and 56c.71-74  To prepare the inverse amides 46a-c from the two components 49 and 56a-c initial efforts were focused on the use of the acid chloride method. However, extensive decomposition occurred when 56a-c were reacted with SOCl2 and only low product yields were observed using the less SHClClOOether, 4 hr reflux SOClOAlCl3, DCM0 °C to reflux 30 min.SOOH2O2, NH4OHroom T16 hrSNONH2(i) NaOH, MeOH16 hr(ii) HCl, pH~2SNOOHHNO3/H2SO4< 10 °C, 3 hrSNOOHO2NSNOOHNO2+SNOOH+NO245%46%85%SNOHOSNOHONH3SNOHNH3SNOHONH2SNO NH2OHO- H+SNONH2OH505152 53a b cd e f54 5556b 56c56a34  aggressive reagent, oxalyl chloride.  Indeed, much of the crude mixture remained on the baseline on TLC and the identities of this mixture could not be determined.  In the light of these results, effort was directed to using the acid fluoride derivatives 56a-c as the activated acid component in the coupling reaction. Acid fluorides are more stable toward hydrolysis and less reactive than acid chlorides, but remain reactive to amines.75   Consequently, in many cases acid fluorides can be isolated, purified, and stored.76  However, attempts to prepare the acid fluorides of compound 56a-c by reacting them with tetramethylfluoroformamidinium hexafluorophosphate (TFFH) and CsF failed.77 Indeed, the acyl fluoride of our nitrobenzisothiazole-3-carboxylic acids (57a-c) proved to be extremely air and moisture sensitive.  In further attempts to generate the desired acyl fluorides in situ using TFFH, 19F NMR showed disappearance of the acyl fluoride signal (26.5 ppm) after 45 minutes of reaction time, due to its decomposition in the reaction medium.   We then turned to the use of bis(2-methoxyethyl)aminosulfur trifluoride (DeoxoFluor) to generate the acid fluoride in situ.78-79  The acid was stirred in DMF with DeoxoFluor overnight, and the amine was added to the reaction mixture directly without the isolation of acyl fluoride (Figure 37).  In keeping with the results using TFFH, the reaction of acid fluorides of 57a-c with 49 provided the final compounds 46a-c in low, but isolatable yields (under 5%).  Note also, that compound 46a was generated as a mixture, as carboxylic acid 56a was not used as a pure starting material.  The anti-HIV screening of these 1C8 inverse amide compounds was carried out by Dr. Peter Cheung, using a reporter CEM-GXR cell-based assay.  At the start of the experiment (day 0), these reporter CEM-GXR cells were infected by coculture with 1% HIV-1NL4-3 infected (GFP positive) cells.  The reporter CEM-GXR cells are an immortalized CD4+ T-lymphocytes that express GFP when the Tat-dependent promoter is activated upon HIV-1 infection.  The compound required for testing was dissolved in DMSO, and then it was added to the culture in the microplate well immediately after the inoculation by infected cells. The final concentration of the tested compound was fixed at 2 μM, and the final DMSO concentration was <0.1%. The percent of infected cells in the culture was measured by flow cytometry on day 3. The three inverse amides did not show any anti-HIV activities.  The results suggested that the amide function in 1C8 is crucial for its biological activity, and that in the inverse amides, this activity could not be recovered by changing the position of the nitro substituent.  35   Figure 37.  Amide coupling via in situ formation of acyl fluorides 57a-c using DeoxoFluor as the fluorinating agent.  Compound 46a was isolated as an admixture with compounds 46b/46c.  Since the absence of activity may be the consequence of a change in the overall shape of compounds 46a-c relative to 1C8 (and therefore how the molecules occupy 3-dimensional space has been changed), a study of the preferred conformation of compound 1C8 was undertaken by molecular modeling. We used ChemDraw 3D to predict the lowest energy structure of 1C8 and its inverse amide using dihedral angle calculations. The desired bond was selected, and a conformational analysis on this bond using GAMESS (General Atomic and Molecular Electronic Structure System) minimizations. A dihedral angle chart was then generated with the conformational energy (kcal/mol) as a function of angle (degrees).  Figure 38a shows the dihedral angle conformational analysis of 1C8 while Figure 38b show the that of the inverse amide 46b.   36   Figure 38.  Conformational analysis of (a) 1C8 and (b) its inverse amide 46b.  The bond of rotation is highlighted in yellow.  Shown on the left are the conformations of both compounds with the C=O of amide and pyridinone in the same direction.  Shown on the right are the conformations of both compounds with C=O in opposite directions.  The corresponding energy levels are indicated by the blue arrow. The amide bonds for the more stable conformers of 1C8 and 46b are circled in white.    In Figure 38, on the left side in both (a) and (b) are the conformations having the C=O of the amide and pyridinone aligned in the same direction.  On the right side in both (a) and (b) are 37  the conformations with the C=O in different directions.  Note that in both 1C8 and 46b, the conformer containing the C=O in the same direction are always higher in energy, and the conformers containing the C=O pointing in different directions are lower in energy.  For 1C8, the conformer containing C=O in the same directions is about 5 kcal/mol higher in energy compared to the other conformer (second lowest in energy level).  On the other hand, for 46b, the conformer containing the C=O aligned in the same direction is very high in energy (global maximum), most likely due to the steric hindrances and electrostatic repulsions (seen from its 3D structure). The lower energy conformer of 46b, on the other hand, has the C=O in different direction (minimized dipole).  Looking at the most stable conformers of both 1C8 and 46b, compared to 1C8, the inverse amide lacks the six-membered ring hydrogen bonding interaction between the amide hydrogen and the oxygen of the pyridinone C=O.  Further, because of this change in the amide bond orientation, 46b has the amide C=O pointing directly downward, whereas in 1C8, the C=O of the amide bond is pointed slightly at an angle toward the side of the benzisothiazole ring (amide bonds circled in white).  The change in the C=O orientation may contribute to the loss of H-bonding donor and acceptor interaction that exists between 1C8 and its putative target, which contributes to the anti-HIV effect of 1C8.)  2.3 (B) Conversion of -NO2 to -NH2:  Preparation of 1C8 analogue 58 In biological systems, nitro groups, and in particular aromatic NO2 groups, can undergo bioactivation reactions that are catalyzed by redox enzymes (Figure 39).80  This process can occur through either a one or two electron oxidation/reduction mechanisms.  In 2e- reductions, the nitro groups are reduced by type I nitroreductase (NTR) to amines via nitroso and hydroxylamine intermediates. The hydroxylamine intermediate is known to be responsible for methemoglobinemia.  Furthermore, the esterified hydroxylamine, nitro radical anion, and nitroso species formed in the two pathways can have mutagenic and carcinogenic activities.  The 1e- reduction of nitro groups (catalyzed by type II NTR) a nitro radical anion.  Under aerobic conditions, this radical anion can be reoxidized back to the nitro group, releasing a reactive superoxide anion species.  The species that formed during this redox recycling of the nitro radical anion can also have carcinogenic effect on the cell (damage to DNA).   38   Figure 39.  One- and two-electron reduction of nitroaromatics.80 To further improve on the safety (Selectivity Index. CC50/EC50) of 1C8, the nitro group in 1C8 was reduced to the corresponding amine using SnCl2 in concentrated HCl (Figure 40).  Compound 58 was obtained as a white solid in 69% yield.  Disappointingly, however, the anti-HIV assay revealed that, compared 58 was significantly less active than 1C8 (Figure 41).   Figure 40.   Reduction of -NO2 to -NH2 in 1C8:  Formation of compound 58.               NOOHN NSO2NSnCl2/HClroom T.7 daysNOOHN NSH2N1C8 58HCl69%39   Figure 41.  Effect of 58 and 1C8 on HIV infected CEM-GXR cells.  2.4 (C) Bioactivation of isothiazole:  Benzisoxazole-based inverse amide analogue of 1C8 Bioactivation of isothiazole compounds involves the oxidation of the sulfur atom in the molecule by cytochrome enzyme P450.  In the example below, Teferra et al. showed that the sulfur atom in compound 59 was oxidized via a P450 enzyme, leaving a ring carbon susceptible to nucleophilic attack by glutathione, resulting in the formation of the glutathione conjugate (Figure 42a).81  They subsequently prepared the isoxazole and pyrazole analogs 60 and 61 of the drug, which have oxygen and nitrogen, respectively, in place of sulfur (Figure 42b).81 These analogs weren’t susceptible to bioactivation and proved to be less toxic without affecting the drug’s efficacy.  40   Figure 42.  (a) Bioactivation of thiazole resulting in formation of glutathione conjugate. (b) oxazole (60) and pyrazole (61) structural analogs of 59 (one structure with X = O, NH)  Of further interest, in the same way that the S atom in 1,2-benzisothiazole is prone to nucleophilic attack, resulting in the scission of the N-S bond, a similar situation also occurs for its selenium analogue (S replaced by Se).  In one study, it was found that ring-opened diselenides would lose antiviral activity (against EMCV Encephalomyocarditis virus) compared to the corresponding benzisoselenazol-3(2H)-ones (Figure 43).82 To address the issues of bioactivation 41  and ring-opening of the benzisothiazole ring, we decided to make the benzisoxazole inverse amide of 1C8 and the sulfone analogue of 1C8.   SeNORNHRSeSeORHNObenzisoselenazol3(2H)-onesbenzisoselenazol3(2H)-ones—diselenides  Figure 43.  Benzisoselenazol-3(2H)-ones and their corresponding diselenides.  R=H, Me, Et, n-Pr, n-Bu, Ph, Bn.82  Accessing the carboxylic acid, 3-carboxybenzisoxazole (67) required for the preparation of the benzisoxazole inverse amide 69 (Figure 44).  2-nitrophenylacetic acid (62) was esterified to give ethyl ester 63, isolated as a light-yellow solid in 85% yield.  Compound 63 was in turn converted to oxime 64 through reaction with isoamyl nitrite and sodium ethoxide.  Recrystallization from dichloromethane gave pure 64 as pale-yellow crystals (32%).  The intramolecular cyclization of 64 was performed by treating it with NaH (1.0 eq.) in DMF at 130 °C for 8 hours.  The crude product mixture was silica column chromatographed using hexane: ethyl acetate (100: 0 to 92: 8) as the eluent.  The product containing fractions were combined and concentrated to afford the target compound as a white solid in 49% yield.  Compound 65 was then treated with HNO3/H2SO4 at 0°C for 2.5 hr.  Unlike the benzisothiazole 55, compound 65 was selectively nitrated at the 5-position, giving compound 66 as the only observed product.  Compound 67 was subsequently obtained by hydrolysis of 66 under acidic (70% aqueous H2SO4).83  The amide coupling between 67 and the amine 49 was performed by generating the acyl fluoride (68) in situ using DeoxoFluor (Figure 44).  Attempts to isolate the acyl fluoride 68 failed; the acyl fluoride reverted back to its acid form upon exposure to air.  Therefore, the acyl fluoride of 68 could only be formed and used in situ.  The overall yield of the amide 69 (yellow solid) formation reaction was 8.4%.  Like the inverse amide of 1C8, compound 69 did not show any anti-HIV activity. 42   Figure 44.  Synthesis of 1C8 benzisoxazole analogue 69.83  The other strategy envisaged to suppress possible cleavage of the N-S bond in vivo was to oxidize the benzisothiazole sulfur atom to the sulfone level (ex. 3-choloropseudosaccarin; Figure 45).    Saccharin itself is an artificial sweetener, and consequently is both water soluble and biologically safe.32    Figure 45.  Saccharin and an example of pseudosaccharin The most direct way to make this sulfone analogue 70 is to react 1C8 with the oxidizing agent such as  meta-chloroperoxybenzoic acid (mCPBA) (Figure 46).  However, despite our NO2ROONONaOEt, EtOHrt to 60 °C, 2hr32%NO2OEtONOHNaH, DMF130 °C, 8 hrONOOEtHNO3/H2SO40 °C, 2.5 hrONOOEtO2N(1) 70% H2SO480 °C, 4 hr(2) ice (30 minutes), filtration85%ONOOHO2N49%77%DeoxoFluor (1.1 equiv.)DMF, 0 °C to room T.2.5 hrONO2NOFONO2NONHNONONH2(1.2 equiv)DMF, overnight8%62 (R = OH)63 (R = OEt)64 6566 67684969SNHOOO SNClOOsaccharin 3-Cl-pseudosaccharin43  repeated effort, the reaction did not proceed even after 24 hours. None of the starting 1C8 was oxidized, and the starting material was recovered.     NO HNONSO2NmCPBANO HNONSO2NOO1C8 70  Figure 46.  Attempt to prepare sulfone analogue of 1C8 via mCPBA oxidation.  An alternative way to synthesize the 1C8 sulfone analogue 70 was to form the amide bond between the acid of the 4-pyridinone (75) and 5-nitro-3-amino-1,2-benzisothiazole-1,1-dioxide (79a) (Figure 47).  For this approach, 4-chloronicotinic acid (72) was prepared from 4-chloropyridine (71) by ortho-lithiation followed by reaction with CO2(g).84  Reaction of compound 72 in methanolic HCl afforded the methyl ester of 4-methoxy-3-pyridinecarboxylic acid (73) as a white solid in 71% yield.85  The subsequent reaction of compound 73 with 0.4 equivalences of MeI in dry MeCN (120 °C for 1.25 hr using microwave heating) gave the N-methyl 4-pyridinone 74 as a white solid in quantitative yield. This transformation involved initial N-methylation of 73 to give a quaternary methiodide salt, followed by iodide anion induced SN2 displacement of the -OMe methyl group. In this step, the pyridinone C=O is liberated and MeI is regenerated, paving the way for further of N-alkylation of remaining starting material (73). Compound 74 was treated with LiOH (3.0 eq.) in THF: MeOH: H2O (2:2:1) at room temperature overnight to give 75 as a white solid in 72% yield.   44   Figure 47.  Synthesis of a sulfone analogue of 1C8 via amide coupling.   45  To complete the synthesis of the target compound, 5-nitro-3-aminobenzisothiazole-1,1-dioxide (79), we initially adapted a procedure described in a patent, used for the preparation of 7-bromo-3-aminobenzoisothiazole-1,1-dioxide.86  This involved using the Sandmeyer reaction to convert the -NH2 in 77 to a sulfonyl chloride group in 78, which would then cyclized to give the target amine.  However, this procedure failed when we attempted it with the nitro- or CF3-containing starting material 77a and 77b.  No reaction occurred in both of these cases; the starting materials being recovered in-tact.  To circumvent this problem, commercially available 3-aminobenzisothiazole-1,1-dioxide 79c (no substitution at the 5-position) was reacted either in its free amino form, or as its N-TMS derivative, with the acyl fluoride 76 using the technology developed in our laboratory.77  Note, the silylated amine would be more reactive than the free amine, as its reaction with the fluoride ion forms a “hypervalent” silicon species, which decomposes to produce volatile TMSF and an amine anion intermediate that is higher in energy and more nucleophilic).87 In this reaction, a white precipitate was formed, suggesting product formation (80).  However, this material proved to be insoluble in a range of solvents, including DMSO and H2O.  Based on the efficiency of the acyl fluoride – TMS amine coupling protocol our laboratory developed, we were confident that the desired sulfone (dioxide) product 80 had been formed. However, since the identity of this material could not be determined and it was totally insoluble, we abandoned the idea of building the 1C8 sulfone analogue.  2.5 (D) Efforts to prepare the diheteroaryl aminoether 81:  Synthesis of the sulfur bridged 5-nitro-2,1-benzisothiazole dimer 82.  Another strategy to examine the importance of the amide bond and to increase the solubility of 1C8 in water was to make the diheteroaryl aminoether 81, the analogue of 1C8 lacking the amide bond.   In principle, this compound can be accessed by reacting amine 49 with 5-nitro-3-chlorobenzisothiazole 42.  However, it was anticipated that the reactivity of the benzisothiazole system may complicate things. Indeed, this procedure did not give the desire product. By column chromatography at 85:15 (hexane: ethyl acetate), a bright yellow fraction was eluded.  The concentrated fraction was a coloured crystalline compound (15-33% isolated yield) that could not be formally identified by 1H NMR. Furthermore, after the yellow compound was eluded, changing the solvent system to 7 DCM: 3 MeOH recovered about 20-25% of unreacted 49.  Amazingly, the 46  structure of this molecule, determined by single crystal X-ray diffraction corresponded to the rearrangement product 82 (Figure 48). Note that compared to the 1,2-benzsothiazole starting material 42, the sulfur and the nitrogen had switched places in 82, and that two of the newly formed 2,1-benzisothiazole subunits were connected via a bridging sulfur atom.  The formation of this sulfur bridged 5-nitro- 2,1-benzisothiazole dimer, along with other rearrangements concerning the chemistry of benzisothiazoles, will be discussed in Chapter 3.   Figure 48.  Attempted synthesis of diarylamino ether 81. Preparation of the rearrangement product 82 (confirmed X-ray diffraction structure).88  As also alluded to in the next chapter, the formation of the rearrangement product 82 was a consequence of the presence of the NO2 group in the starting benzisothiazole 42. To circumvent this problem, we looked into preparing the target aminoether 81 by first preparing the non-NO2 substituted diheteraryl aminoether 83, followed by its nitration (Figure 49). SNO2NClNONH2ACN, MW 125 °C, 2.5 hr NONHSN NO2NSO2NSS NNO2Desired product was not obtained Observed product (15-33% isolated yield)494281 82SNO2NNH2(i) CuCl2, isoamyl nitriteACN, 65 °C, 1 hr(ii) 20% HCl(iii) NaHCO3, pH 839%4347  SNClNONH2MeCN, MW 125 °C, 2.5 hr NONHSNnitrationNONHSN NO249183 81  Figure 49.  Proposed synthetic route for the preparation of desired product 81 via non-NO2 substituted 3-chlorobenzisothiazole 1.  Interestingly, the reaction between 3-chlorobenzisothiazole 1 and the pyrdinone amine 49 generated two products:  the desired diheteroaryl aminoether 83 (minor product) and, on the basis of its 1H NMR spectrum, compound  84 (major product), in which the benzisothiazole  motif is connected to C-2 of the pyridinone ring (Figure 50).  The 1H NMR spectra (NMR solvent = CDCl3) of 83 and 84 are shown in Figure 51.  Note that 1H signals in 84 are more upfield compared to 83.  Also, compared to the top spectrum (83), the bottom spectrum (84) shows a lack of C-H signal and the presence of -NH2 peak.  Both products were obtained as white solids following silica column chromatography.  Compound 83 was purified using 95 DCM/ 5 MeOH.  After 83 has been eluded, the more polar, C-H activated product 84, was isolated by gradually increasing the polarity of the column eluent to 100% MeOH.  The formation of the two different products can be explained by the dual reactivity of 49.  In the first case, the lone pair electrons of the amine attacks C-3 of benzisothiazole with displacement of Cl- to give 83.  In the second case, the pyridinone component behaves as an enamine, with the nucleophilic beta carbon reacting with the electrophilic C-3 of benzisothiazole to give 84.  Note that the reaction only took place in the presence of cat. Pd/C and Celite (diatomite, SiO2).  No reaction occurred without Pd/C or Celite.   48   SNClNONH2MeCN, MW 125 °C, 2.5 hr NONHSN(1.2 eq)NONH2SN+minor (target compound)major(1.0 eq.)(always <20%)(isolated yield 2.5 to 3x minor product)14983 84 Figure 50.  Products 83 and 84, formed in the reaction between pyridinone amine 49 and 3-chloro-1,2-benzisothiazole 1.  Figure 51.  1H NMR (CDCl3) of 83 (top spectrum) and 84 (bottom spectrum).  Note the lack of C-H signal and the presence of –NH2 signal in 84.  To complete the synthesis of the NO2 substituted diheteroaryl aminoether 81, compound 83 was reacted under classical nitration conditions with HNO3/H2SO4 (Figure 52).  Surprisingly, this reaction gave rise to 7-NO2 substituted diheteroaryl aminoether 85 exclusively.  No nitration at the C-5 of benzisothiazole was observed.  Evaluation of the anti-HIV activity of compound 85 is pending.  49  SNHN NO HNO3, H2SO40 °C, 2 hrSNHN NONO283 8522.1% Figure 52.  Nitration of diheteroaryl aminoether 83 gave product 85 which contains -NO2 at C-7 position.  2.6 (E) Scaffold Deconstruction and Morphing Approach to SAR Deconstructing and re-growing the framework of a hit compound has demonstrated its effectiveness in the analysis of molecular scaffolds in drug discovery.  Such strategies allow for one to replace the core motif of a molecule while retaining/amplifying important functional interactions.  This can lead to improvement in binding, pharmacokinetics, solubility, as well as the accessibility of various structural types through synthesis.  Examples where this tactic has been employed are shown in Figure 53.  In the work on Bruton’s Tyrosine Kinase (BTK) inhibitor by Lou and coworkers, the imidazole ring and the amide in CGG11746 were both replaced by 2-pyridinone motifs.  The resulting new molecule, RN486, showed improved binding affinity to BTK and HWB potency (Figure 53a).89  In Figure 53b, a series of bicyclic nitroimidazoles 86 with various R1, R2, and R3 groups were designed and prepared by linking the amide and imidazole nitrogen of the nitroimidazoxazines (molecule on the left).  The derived compounds 87, containing the bicyclic system were non-cytotoxic against mammalian cells and active against G. lamblia, T.b. brucei, and M. tuberculosis.90  In Figure 53c scaffold morphing was also used to mimic the hydrogen bonding interaction (dashed rectangular box) in the thiophene carboxamide urea-based molecule 88 through creation of a ring.  This led to the identification of the bicyclic checkpoint kinase 1 (CHK1) inhibitors: 7-carboxamide thienopyridines and 7-carboxamide indoles (compounds 89, 90, and 91).  It was also revealed that the potency of the compound relied heavily on the intramolecular noncovalent sulfur-oxygen interaction (dashed ellipse), which allowed the alignment of the hinge-binding carboxamide group to the thienopyridine core in a coplanar manner.  In this series, compound 90 showed increased potency (IC50 = 0.006 µM) and reduced hERG (human Ether-à-go-go-related gene) liability (>31.6 µM).91  50  HNNOONNNNHONHNNHNONNHO OFCG11746 RN486disconnectioncyclization(a)(b)NNHHNOR1O2NNNNO2NOR3R2R1cyclization(c)NHHNH2N ONHOHN SHNH2N ONHNSHNH2N OHNN SHNH2N ONHCHK1 IC50:  0.03  µMhERG IC50: 12 µMCHK1 IC50:  0.005  µMhERG IC50: >31.6 µMCHK1 IC50:  0.006  µMhERG IC50: >31.6 µMCHK1 IC50:  2.6  µMhERG IC50: >31.6 µM88 89 9091nitroimidazole framework novel nitroimidazopyrazinones86 87 Figure 53.  Literature examples of scaffold deconstruction and reconstruction.89-91   As discussed in the following section, in our SAR study of 1C8, we have employed the strategy of scaffold deconstruction and regrowth to examine the influence on anti-HIV activity of eliminating the central amide bond in 1C8, breaking open the isothiazole ring and extruding the sulfur atom in a manner such that these elements are incorporated into a pyrimidine ring as in 92 (Figure 54). The advantage to the development of this type of 1C8 analogue is that it would 51  simplify SAR studies by rendering more available compounds where “R” is varied on the phenyl ring. As will become more evident in Chapter 3, generating the corresponding series of benzisothiazole-based 1C8 analogues compounds bearing “R” group(s) on the benzisothiazole “benzo” ring would be particularly challenging. Note that the alignment of the amide C=O and the benzisothiazole C=N is conserved in compounds 92.  Note also, that pyrimidine 92 captures preferentially the “structure” of the 1C8 conformer wherein the pyridinone and amide C=O groups are aligned/pointing in the same direction, and not the alternate, and potentially somewhat lower energy conformer (1C8’) where these carbonyls are opposed (see Figure 38).  Figure 54.  Structures of 4,6-diheteroarylpyrimidine-based analogues (92) of 1C8. 52   2.6.1 Syntheses of 4,6-diheteroaryl pyrimidine-based analogues of 1C8 The initially explored route to prepare the 4,6-diheteroaryl pyrimidine compound 96 implicated the cyclization of the 1,3-dicarbonyl intermediate 94 (formed by condensation of 4-pyridinone ester 74 and 3-nitroacetophenone 93 (Figure 55 route (a)) through reaction with amidine 95.  However, this Claisen condensation reaction, using NaH in anhydrous DMF at various temperatures (0 °C, room temperature, at reflux) produced unidentifiable sticky black tars, which changed colour upon solubilization in various NMR solvents analysis.  In fact, Claisen condensations involving 3-nitroaryl substituted 1,3-diketone derivatives of 4-methoxy pyridine or 4-pyridinones have not been described in the literature.    Figure 55.  The target 4,6-diheteroaryl pyrimidine compound 96 can be made by reacting amidine with (a) 1,3-diketone 94 formed from Claisen condensation between 93 and 74, or (b) α, β-unsaturated ketone 98 formed from Aldol condensation between 93 and 97.    53   Since we were not able to prepare 1,3-diketone 94 after multiple attempts under various reaction conditions, and could not obtain useful information from the 1H NMR spectra concerning what went wrong, we then considered preparing the α, β-unsaturated ketone 98 as the key intermediate toward the synthesis of the target compound 96 (Figure 55 route (b)).    Figure 56.  Synthesis of aldehydes 100 and 97, and the 1,5-diketone products generated from their reactions with acetophenone in the presence of NaOH in EtOH.   The key starting material for preparation of intermediate 98 was 4-pyridinone aldehyde 97.  The synthesis of 97 is described in Figure 56.  In the first step, ester 73 was reduced to the corresponding alcohol 99 (light-yellow solid) using DIBAL.92  Alcohol 99 was then oxidized in the presence of manganese dioxide to give 4-methoxy-3-pyridinecarboxaldehyde 100 as a light-54  yellow solid (86%).  Reaction of aldehyde 100 with a sub stoichiometric amount of MeI under microwave condition gave 97 as a white solid in 60% yield.   In the key Aldol condensation step between 3-nitroacetophenone and 93 to access the key intermediate 96, NaOH in EtOH conditions were employed. Unfortunately, the reaction resulted in polymerization and only gave black tar (unable to obtain any useful information from 1H NMR; the dissolved component also kept changing colour in the NMR tube).  We thought that this could be due to the presence of -NO2 groups.  To test this idea, we then tried the Aldol condensation between 100/97 and acetophenone (101) (overnight reaction).  In both these cases, the 1, 5-diketone products (102 and 103) were isolated in 50% and 51% yield, respectively.  No α, β-unsaturated product was observed/isolated.  Nonetheless, the formation of the 1, 5-diketone product means that the α, β-unsaturated ketone must have been formed!  (and subsequently reacted with another molecule of acetophenone via Michael addition).   NOd++MM = Na, KO Figure 57.  The Michael acceptor ability of these pyridine-containing azachalcones is activated by the metal ion’s coordination to the N atom of the pyridine.93  Indeed, the formation of 1, 5-diketones resulting from the Aldol condensation between a pyridinecarboxaldehyde and acetophenone in the presence of inorganic bases has been mentioned by Down et al.93  In their work, the metal ion from the inorganic bases was suggested to chelate to the N atom of the pyridine, enhancing the Michael acceptor property of the azachalcone (Figure 57).  To circumvent this problem, excess aldehyde and organic bases such as DBU was used in the reaction, in order to generate the desired α, β-unsaturated ketones.  Using the conditions outlined in their paper, we first attempted the aldol condensation between 100 (2.0 eq.) and nitroacetophenone (1.0 eq.). In this way formation of the Michael adducts was and no longer observed.  Indeed, the reaction gave the expected azachalcone (104), which was eluded out from the column (95% DCM: 5% MeOH) at the same time as the starting aldehyde 100.  However, 55  prolonged reaction time resulted in formation of black tars and the loss of product (spot diminishing on TLC).  Note that in this case, we also did not observe any β-hydroxyketone.  We then applied the same strategy to the aldol condensation between 97 and acetophenone and nitroacetophenone.  When compound 97 was reacted with acetophenone in the presence of DBU, α, β-unsaturated ketone (colourless wax) (105) (32%) and β-hydroxyketone (106) were formed.  However, when NO2 is present in the starting ketone, we only obtained 9% of the target α, β-unsaturated ketone (98) in the form of a pale-yellow solid.  In the reaction mixture, we also observed the presence of β-hydroxyketone (107).  The yield of the β-hydroxyketones (106 and 107) in both of these reactions could not be determined, as they eluded out at the same time as the starting aldehyde (which was in excess).  The 1H NMR spectrum contain both the starting materials and the β-hydroxyketones.  The hydrogen bonding between the hydrogen of the β-hydroxyl and the oxygen of the pyridinone is likely to be the reason contributing to the presence of the β-hydroxyketone at the end of the reaction.  Note that in the reaction involving 3-nitroacetophenone, the reaction was stopped after two hours due to the formation of side products (dark red oily materials).  Figure 58.  DBU mediated Aldol condensation between 100 and nitroacetophenone gave 104 (obtained as a mixture with the starting aldehyde 100). 56   Figure 59.  DBU-mediated synthesis of azachalcones from 97 and acetophenones 93 and 101.  Yields of 106 and 107 could not be determined as they have the same Rf value as the starting aldehyde.  To improve the efficiency of the reactions for synthesizing the target compound 94, the method we used to make 4,6-diheteroaryl pyrimidine compounds involved a 3-component (4-pyridinone amine, aryl ketone, and guanidine) one-pot synthesis.  Such Biginelli-type reactions have been used to gain access to a variety of 2-amino-5-alkoxycarbonyl-3,4-dihydropyrimidines.94-96  To synthesize the 4,6-diheteroaryl pyrimidines of our interest, we adopted the strategy developed by Val and coworkers for the synthesis of 2-aminopyrimidine-5-carbonitriles from α-cyanoketones, carboxaldehydes, and guanidine.96  The pathway for the formation of 4,6-diheteroaryl pyrimidines is outlined in Figure 60.  The first step involves the attack of guanidine on the carbonyl of the aldehyde in 97 to form 109, which gives the iminium intermediate 110 upon the loss of a water molecule.  Intermediate 110 acts as an electrophile for the nucleophilic addition of the enolate of the aryl ketone to form 112. In the next step, NH2 attacks and the ketone carbonyl of 112, to give the intramolecular cyclized product 113.  Upon loss of water, intermediate 113 gives 114, which can tautomerize to 114’, a 1,4-dihydropyrimidine.  As Val and coworkers pointed out in their NOHOOONO2DBU, ACNroom T., overnightDBU, ACNroom T., 2 hrsNO ONO ONO2NO OO+NO ONO2O+NOHO(33%)(10%)9797105 10698 107HH10193(2.0 eq.)(2.0 eq.)(1.0 eq.)(1.0 eq.)57  study, intermediate 114’ subsequently underwent aromatization (in the presence of base in this case) to afford the desired 4,6-diheteroaryl pyrimidines.   Using Val’s procedures, six pyrimidine analogs of 1C8, containing different Ar groups on the right hand side of the pyrimidine ring, were synthesized under microwave conditions ( Table 3).  Note that we have attempted the three-component synthesis by replacing guanidine with amidine.  However, the reaction only gave black tars and we were not able to isolate any identifiable product by column chromatography.  Furthermore, the final compounds containing NO2 were obtained in lower yield (similar to what have been observed previously).  Anti-HIV assays, performed by Dr. Peter Cheung, showed that none of these 4, 6-diheteroaryl pyrimidine compounds showed anti-HIV activities. These results suggested that: - The presence of the sulfur atom in the benzisothiazole ring is important - The orientation of the C=O of the amide relative to the pyridinone C=O plays a crucial role in the anti-HIV activity of 1C8 At this point, we directed our attention to the preparation of pyridinopyrazolone 5-nitro-1, 2-benzisothiazoles (see next Chapter).  58   Figure 60.  Biginelli type three-component one-pot synthesis of 4,6-diheteroaryl pyrimidines.  NOHO97H2N NH2NHNONOHHNHNH2NONH NHNH2ArOArONONHNHAr OHNH-H2ONONHNHNHArNONNNH2Araromatization in the presence of baseNONNHNH2Artautomerize109112 113 114114'4,6-diheteroaryl pyrimidines-H2ONONHNHNH2110Na2CO3(3.0 eq.)92108 11159  Table 3.  4,6-Diheteroaryl pyrimidines prepared using a 3 component one-pot strategy under microwave conditions. R1 = H, CN.   60  Chapter 3: Conformational Constraint of the Central Amide Linker in 1C8 Through Its Incorporation into a Pyrazolone Motif - The Discovery of New Reactivity of the Benzisothiazole Ring System  3.1 Introduction  Figure 60.  (a) Construction of 4,6-diheteroarylpyrimidine analogs of 1C8, which capture the 1C8 conformation where the carbonyls of the amide and the 4-pyridinone are in the same direction; (b) “Constrained” pyrazolopyridine-based analogs of 1C8, which capture a key H-bond interaction between the amide NH and the 4-pyridinone C=O.    In the previous chapter, a Structure-Activity-Relationship (SAR) study was carried out on 1C8 to explore the influence or contribution of one of the low energy rotational conformations of 1C8 on target binding/activity (Figure 60a). This involved building a series of analogs, in which the central amide bond and the five-membered isothiazole ring was captured (remorphed) in the form of a 4,6-diheteroarylpyrimidine ring system.  None of the molecules in this series showed anti-HIV activity, suggesting that it may be the other lower energy conformer of 1C8 that plays a crucial role in receptor/target binding/recognition.  In order to study this conformational hypothesis (Figure 60b), where a hydrogen bond exists between the N-H atom in the amide bond and the oxygen atom of the 4-pyridionne in 1C8, the plan was to synthesize structurally constrained molecules that mimic this H-bonded structure (i.e. we wanted to freeze the shape of 1C8).   As 61  presented in this chapter, the idea was to integrate the amide bond into a five-membered pyrazolone ring system, as found in the pyridopyrazolone benzisothiazole compound 44 (Figure 61). During our attempts to synthesize this novel bis-heterocyclic molecule we discovered several novel and unforeseen transformations of the benzisothiazole ring system.  Figure 61.  Two studied strategies for the synthesis of pyridopyrazolone 1,2-benzisothiazole.   The corresponding starting materials for each approach are provided.    We envisaged that the pyridopyrazolone 1,2-benzisothiazole 44 could be accessed via the two different approaches (1 and 2) shown in Figure 61.  In the first (direct) approach transition metal mediated (Cu(I)) coupling conditions would be used to condense pyridopyrazolone 115 and 62  3-chloro-5-nitrobenzisothiazole (42).  The second (indirect) approach would involve cyclization of the hydrazine intermediate 117 followed by selective N-methylation of the pyridine nitrogen. It was anticipated that intermediate 117 could be “readily’ prepared by either: a) reacting 4-chloronicotinic acid methyl ester 116 and 3-hydrazino-5-nitro-benzisothiazle 45, or b) SNAr reaction between 4-hydrazino-nicotinic acid methyl ester 118 and 3-methoxy-5-nitrobenzisothiazole 119 (or 3-chloro-5-nitrobenzisothiazole 42).  3.2 Approach 1:  Synthesis of Pyrazolopyridinone 44 Via Cu(I) Mediated N-Arylation  of 3-Chloro-5-nitro-1,2-benzisothiazole 42  The more direct approach for the synthesis of the target compound 44 via a Cu(I) catalyzed N-arylation of N-methylpyridopyrazolone (115) with 3-chloro-5-nitro-1,2-benzisothiazole (42) has the advantage that it can also be used to attach a variety of other aryl/heteroaryl motifs onto the pyridopyrazolone nitrogen. In this way, Dr. Stoyan Karagiozov in our lab had previously studied the Cu(I) coupling of pyridopyrazolone 115 with a series of eight Ar-Xs and HetAr-Xs (Table 5).    To extend this methodology to the preparation of the target pyridopyrazolone benzisothiazole 44 required an efficient means to prepare the starting pyrazolone 115 (Figure 62).  This was achieved by reacting 4-chloronicotinic acid (72) with oxalyl chloride and subsequently reacting the in situ generated acid chloride 121 with hydrazine hydrate.  Hydrazine was added to the stirred solution at room temperature, and the resulting reaction mixture was refluxed for four hours. The crude mixture was concentrated and column chromatographed (90 EtOAc - 10 MeOH to 55 EtOAc - 45 MeOH) to give 122 as a yellow solid in 55% yield.   In a subsequent “one pot” process, compound 122 was reacted with NaH (2.0 eq.) in refluxing THF (4 hours), and then with added methyl iodide at room temperature (24 hours). The reaction mixture was concentrated and silica column chromatographed (90 EtOAc/10 MeOH to 45 EtOAc/55 MeOH) to give 115 as a yellow solid in 48% yield.  Separately, 3-chloro-5-nitro-benzisothiazole 42 was prepared from 3-amino-5-nitrobenzisothiazole (43) using the Sandmeyer reaction (procedure for the preparation of 42 has been described in Chapter 2).   63  Table 5.  Cu coupling reactions of pyridopyrazolone 115.    NNNHONNNO110 °C, 24hrRR-X115 120  a for entry (f): contains the starting compound.   With the required intermediates in hand, the N-arylation reaction to obtain 115 was attempted. Unfortunately, under the conditions optimized by Dr. Karagiosov extensive decomposition of the benzisothiazole component occurred. Indeed, the reaction produced a dark paste that showed a string of spots on TLC, none of which could be isolated.   Entry Exp. Amide (equiv) Ligand (mol%) CuI (mol%) DMF (ml) K3PO4 (equiv) X R Yield (%) a AVK107 1.2 20 10 1.0 2 I  43 b AVK121 1.2 100 50 1.5 4 I N  42 c AVK125 1.2 100 50 1.5 2 Br N  33 d AVK131 1.2 100 50 1.8 2 I O 86 e AVK136 1.34 100 50 2.0 2 Br NO 50 f AVK137 1.2 100 50 1.8 2 Br SN 65a g AVK140 1.7 90 45 1.8 2 Br NH  61 h AVK145 1.2 100 50 1.5 2 B N 65 64   Figure 62.  Proposed synthetic pathway for preparation of pyrazolopyridinone 44.  3.3 Approach 2: Synthesis of Pyrazolopyridinone 44 Via Cyclization of Hydrazine Intermediate 117 As the N-functionalization of pyridopyrazolone 115 failed under the Cu coupling reaction conditions, attention was directed to the alternate strategy that would allow us to construct the pyrazolone ring system through cyclization of hydrazine compound 117 (Figure 61). Of the two routes envisaged (a and b) to access this intermediate, attention was initially focused on strategy a. In this scenario, 4-chloronicotinic acid methyl ester 116 is condensed 3-hydrazino derivative 45 of 5-nitrobenzisothiazole. Note that this strategy has been extensively used by others for the synthesis of pyrazolo-quinolone type systems from different arylhydrazines (Figure 63).97   NClCOOH(1) oxalyl chloride, DMF, DCM (0 °C to room temperature)(2) hydrazine THF (reflux 4 hrs)NNHHNO(i) NaH, THFreflux 4 hrs(ii) MeI, room temperature overnightNNHNONaNO2, CuCl2ACN (65 °C 1 hr)39%SNO2NClCuI, DMF,NH HNK3PO4, MW 120 °CNNNONSO2NSNO2NNH2NClClO+55%47%72 120121114 42434465   Figure 63.  Condensation of 4-chloronicotinic acid methyl ester 116 and 5-nitro-3-hydrazino-1,2-benzisothiazole 45.97    3.3.1 Approach “2a” to Intermediate 117: Attempted Preparation of 3-hydrazino-5-nitrobenzisothiazole 45 The key requirement for the success of strategy 2a was the preparation of 3-hydrazino-5-nitro-1,2-benzisothiazole intermediate 45.  In principle, this was to be a straight-forward transformation, involving a direct displacement (SNAr reaction) of a suitable leaving group X in 5-nitro-1,2-benzisothiazole 119 (X = 3-OMe) or 42 (X =Cl).  To date, literature reports indicate that 3-chlorobenzisothiazoles undergo normal substitution at C-3 with ethoxide and amines. However, as we have seen in the introductory chapter (Figure 15), the preparation of 3-piperazinyl substituted benzisothiazole 3 from 3-chloro-1,2-benzisothiazole can be a complicated process 66  involving ring-opening of the isothiazole as the initial step. There is no literature precedence for ring-opening reactions between 3-methoxy benzisothiazoles and amines.45   Taking these comments into consideration, we were intrigued that there are only two literature reports concerning the reaction of 3-substituted benzisothiazole with hydrazine as the amine nucleophile. Indeed, in the brief report by Mangia et al, 3-hydrazino-1,2-benzisothizole 125 was obtained by heating 3-methoxy-1,2-benzisothiazole 41 with hydrazine hydrate (6.0 eq.) in methanol at 60 °C for 30 minutes (Figure 64).98 The expected product was isolated in 36% yield by recrystallization from MeOH. Subsequently, Sawhney et al. used this non-substituted 3-hydrazino-1,2-benzisothiazole as precursor to prepare a wide range of molecules displaying anti-inflammatory activity.99  Unfortunately, the presumed large scale preparation of 125  was not described by the authors, and indeed (private communication with the lead author), the preparation of 125 was poorly reproducible, due to problems associated with its stability in the reaction medium.  SNCl NaOMe (1.1 eq.), MeOHreflux 2 hrSNOMe hydrazine (6.0 eq.)EtOH, reflux 30 minSNNHNH21 41 125  Figure 64.  Literature procedure for the preparation of 3-hydrazino-1,2-benzisothiazole 125.98      3.3.1.1 Observed Formation of the Rearrangement Products 125 and 82 In our hands, the reaction of 3-methoxy-1,2-benizothiazole 41 with excess hydrazine hydrate did not give the reported 3-hydrazino substituted product 125.  Indeed, the only non-polar compound detected by TLC, and isolated in 35-47% yield by crystallization from MeOH was the ring-opened disulfide 9 (Figure 65).   67  SNOMehydrazine (6.0 eq.)MeOH, reflux 30 minSNNHNH241 125CNSSNC9EXPECTED product(NOT formed)Isolated product (35 - 47% yield)  Figure 65. Reaction of the non-NO2-substituted 3-methoxy-1,2-benzisothiazole 41 with excess hydrazine hydrate according to known literature procedure: formation of disulfide 9.  Further, when these conditions were then applied to the 5-nitro substituted 3-methoxy-1,2-benzisothiazole 119, a non-polar component was similarly detected, and isolated as an intense yellow coloured solid by crystallization from MeOH.  From the 1H/13C NMR spectrum for this compound it was clear that it did not correspond to either the related 5-nitro substituted ring opened disulfide, or, to the expected 3-hydrazino substituted product 45. Indeed, a peak was still present in the 1H NMR spectrum (DMSO-d6) for the -OMe group (δ 3.71 ppm; shifted upfield by 0.7 ppm compared to 119).  Ultimately, a single crystal X-ray diffraction study showed that it corresponded to the ring opened compound (Z)-methyl 2-amino-5-nitrobenzohydrazonate 126.  On repeating the experiment at room temperature, it was observed that the reaction flask became very warm at around the 15-20 minute mark, and that the starting material (119) was consumed within 30 minutes, being converted to the rearranged product 126 in high (77%) yield (crystallization in methanol).  In the structure of 126 two features, in particular, stood out: i) the benzisothiazole sulfur atom has been extruded, and replaced by NH2 at C-8,  and  ii) the -OMe group in the starting material (the expected leaving group) had seemingly been retained at C-3. This latter point was confirmed by the absence of -OMe  -OEt exchange when the reaction was run in EtOH, and the absence of OCH3  OCD3 exchange when the experiment was carried out using deuterated methanol as the solvent. The implications these observations have on the mechanism of formation of compound 126 are discussed in the following section (3.3.1.2). 68   Figure 66. Formation of the rearrangement product 126 from the reaction of 3-methoxy-5-nitro-1,2-benzisothiazole 119 with excess hydrazine hydrate.  In an effort to favour formation of the desired SNAr substitution product 45 by accentuating the leaving group character of the C-3 substituent, the corresponding reaction of hydrazine with 3-chloro-5-nitrobenzisothiazole 42 was studied. When compound 42 was reacted with 6.0 eq. of hydrazine in MeOH at 60 °C for 30 minutes, an orange coloured multicomponent mixture was produced, which decomposed into unidentifiable components upon attempted separation by column chromatography. However, by gradually lowering the amount of hydrazine used to 0.5 eq., a new and structurally distinct rearrangement product 82 was detected (Figure 67). Note that in this case, 1.0 eq. of DIEA was added at the start of the reaction to neutralize the HCl produced. Note, further, that it was expected that this hindered tertiary amine would not compete with hydrazine for reaction with benzisothiazole 42.  This compound was the only isolatable product in this reaction mixture. It is worth noting that, through comparison of NMR spectra, it was deduced that it corresponded to the same rearrangement product formed in the reaction between 3-chloro-5-nitrobenzisothiazole 42 and the 4-pyridinone amine 49 (see Chapter 2, page 46).  In this other experiment, the structure of the reaction product was determined by single crystal X-ray diffraction to correspond to 82, a “dimer” in which two 2,1-benzisothiazole subunits are joined by a sulfur atom bridge. In light of the structure of compound 82, it was determined that it was formed in up to 33% yield.  69  Compared to the starting 1,2-benzisothiazole 42, it was noted that the 3-chloro group was absent in product 82. Further, the S and N positions were swapped in position, such that the -NO2 is now para to the N atom at C-8, i.e. the same position as the amine nitrogen atom in the rearrangement product 126.  This observation suggested that repositioning of the nitrogen atom could be a common mechanistic element in the formation of both rearrangement products 126 and 82.   Figure 67.  Summary of the rearrangement products formed in the reactions of 3-substituted 5-nitro-1,2-benzisothiazoles 42 and 119 with hydrazine hydrate.  3.3.1.2 Proposed Mechanism for the Formation of the Rearrangement Products 126 and 82  Why these deep-seated transformations occurred, rather than the expected C-3 substitution (SNAr) process, stems from the inherent dual reactivity of 1,2-benzisothiazoles (Figure 68).  As 70  alluded to in Chapter 1 (Section 1.10.2), a nucleophile (hydrazine in our case) can react at the C-3 position of 1,2-benzisothiazole to give the 3-substituted benzisothiazole a (pathway 1).  Alternatively, it can attack the sulfur atom, breaking the weak N-S bond, resulting in the formation of compound c and ultimately disulphide 9, which has been considered in many literature articles to be a “dead-end” product (pathway 2).   SNXSNXNuSNNuSNXNu NXSNuCNSNuCNSSCNPathway 1 Pathway 21,2-benzisothiazole9cab Figure 68.  Reactivity of benzisothiazole with a nucleophile.  X = Cl, -OCH3.   On the basis of the information available, a mechanism is proposed for the conversion of 119 to the ring opened hydrazonate product 126, which in the first step involves hydrazine induced ring opening of the isothiazole ring through attack at sulfur to give the ring opened imidate anion 127 (Figure 69).  One might expect that this intermediate would rapidly eliminate OMe- anion to produce a neutral nitrile containing product (b  c in Figure 68). However, it is known that methoxy imidates, derived from the reaction of methanol with nitriles in either acidic or basic conditions can be relatively stable.100-101  Taking this factor into account, the opportunity would be provided for the NO2 group on the phenyl ring of 127 to enable an alternate process resulting in the  formation of the nitro group stabilized Meisenheimer complex 128. In essence, this transformation corresponds to the first step in an intramolecular SNAr reaction. Subsequent loss of NH2NHS- from this Meisenheimer species would then produce the neutral aza-bicyclo [6,4] intermediate 129. Formation of 129, would account for the migration of the nitrogen atom to the carbon centre on the phenyl ring para to NO2, and for the absence of the sulfur atom in the product ultimately formed. Supporting the idea that 129 may be an actual intermediate in the formation of 126 - the compound missing the -OMe and -NO2 substituents (7-azabicyclo[4.2.0]octa-1(6),2,4,7-71  tetraene), can, in fact, be prepared by different routes and  isolated.102  Subsequent 1,2-addition of hydrazine to the “imine” carbon in 129 to generate 130 would set the stage for participation of the lone pair of electrons on the hydrazine nitrogen in opening of the strained four-membered (1,2-dihydroazete) ring and formation of the observed product 126.    Figure 69.  Proposed mechanism for the formation of rearrangement product 126.102  Concerning the formation of product 82, its structure strongly hinted that the reaction of 3-chloro-5-nitrobenzisothiazole 42 with hydrazine generated multiple reaction pathways, one of which provided the third “bridging” sulfur atom.  Taking as a premise that integrating this sulfur atom would result from the reaction of two C-3 activated 2,1-benzisothiazole components with elemental sulfur, or some equivalent species, two mechanisms were proposed (Pathways a and b; Figure 70) for the formation of 82, both of which converge on the formation of a Meisenheimer complex as the pivotal intermediate that enables the observed migration of the benzisothiazole ring nitrogen atom to the aromatic C-8 centre.    SNH3CONO2119NH2NH2H3CON SHNNH2NO2NH3CONO2SHNNH2Meisenheimer complex(SHNHNH2)NNO2MeOH2NNH2HNNO2HNH2NMeONH2NO2NMeOH2N127 12812913012672  In Pathway a, it was expected that SN2 reaction of hydrazine at the sulfur centre in 3-chlorobenzisothiazole 42 would lead to formation of the nitrile compound 132 via a two-step process where the chloroimidate 131 is initially formed as a reactive/transient intermediate.  Although the cyano group generated on ring-opened benzisothiazoles generally does not engage in further reaction, to access the rearrangement product 82 compound 132 would have to react with hydrazine to give the amidrazone (“hydrazino imidate”) 133. This intermediate, being more stable than chloroimidate 131, could undergo reaction with a second molecule of hydrazine at sulfur, effecting ring opening and concomitant ring closure to the Meisenheimer complex 135. Under the reaction conditions where hydrazine was present as the limiting reagent (0.5 equiv.), it was envisaged that 135 would undergo a 1,3-sulfur migration of the pendant -SNHNH2 side chain to give the new/alternate, and probably less strained, Meisenheimer complex 135 (represented along with its “rearomatized” resonance form 135’).103  Displacement of hydrazine (in its protonated form) from 135 would result in formation of the three-membered thiaziridine ring in the [3:4:6] tricyclic species 136.  Spontaneous electron reorganization in this [3:4:6] tricyclic species would reveal the more stable isothiazole motif in the 2,1-benziosthiazole product 137. As mentioned above, further conversion of this intermediate to the final product 82 is envisaged to involve its reaction with a sulfur-based nucleophile (presumably elemental sulfur, released through some other reaction pathway by which 3-chloro-5-nitro benzisothiazole 42 decomposes).    73   Figure 70.  Proposed mechanism for the formation of rearrangement product 82. 74   In the other proposed mechanism (pathway b), involving the reaction of hydrazine at the sulfur atom of 42 in the first step, it was envisaged that a simultaneous/concerted migration of benzisothiazole nitrogen to the C-8 centre on the aromatic ring would occur, producing the Meisenheimer intermediate 139 In this way the C-3 chloro substituent in 42 is conserved. At this point, a 1,3-sulfur migration of the -SNHNH2 motif would produce the Meisenheimer complex 140, which would evolve to the [3:4:6] tricyclic species 141. Like its counterpart 136, this transient intermediate can undergo an energetically favourable ring expansion, producing, in this case, the isomeric 3-chloro substituted 2,1-benzisothiazole 142. Although there is literature precedent, suggesting that several of the intermediates leading to the rearrangement product 125 could be stable entities, there are several steps in the proposed mechanism leading to this product, and compound 126, that could easily be considered tenuous. For this reason, we sought to gain more insight into the energetics of the individual steps in this proposed mechanism, and the mechanism leading to compounds 125/82 using computational methods (Density Function Theory).    3.3.1.3 Predicted Mechanism for the Formation of the Rearrangement Products 125 and 82 In collaboration with Pierre Kennepohl and Xing Tong (Department of Chemistry, UBC), Density Functional Theory (DFT) calculations (Gaussian09 software) were employed to predict the relative energies of potential species evoked in the proposed mechanisms summarized in Figure 69 and Figure 70.  DFT is derived from the N-particle Schrodinger equation and is entirely expressed in terms of the density distribution of the ground state ρGS(r) and the single particle wave function Ψ. DFT reduces the calculations of the ground state properties of systems of interacting particles exactly to the solution of single-particle Hartree-type equations, which is computationally more scalable than other methods for comparable accuracy. This is why it has been the most widely used computational method for large chemical systems. The use of DFT methods, however, still requires the use of significant approximations. Most notably, solvation effects – of critical importance in condensed phase processes - are generally approximated using dielectric corrections. The inclusion of explicit solvation (i.e. the inclusion of solvent molecules in the quantum mechanical model) is still a major challenge. 75  To begin with, DFT-derived electrostatic potential (ESP) maps for 3-methoxy-5-nitrobenzisothiazole 119 and 3-chloro-5-nitro-1,2-benzisothiazole 42 were determined (Figure 71). For both compounds, the ESP map indicated that the C-3 and the S atoms are electrophilic and thus prone to nucleophilic attack by hydrazine. The nature of the substituent, however, is seen to affect the relative electrophilicity of the C-3 and S positions. Note that the Cl substituent leads to a more positive electrostatic potential than the electron-rich OMe. As the two centers are susceptible to nucleophilic attack by hydrazine, nucleophilic attack at either site was considered in the calculations.  Figure 71.  Electrostatic potential map of 119 and 42 showing relative potential on the two different reactants. Electrostatic potentials were generated with Gaussian09 using the M062X theory and Def2TZVP basis set and then visualised with WebMO. While bright blue and bright red represent -0.15V and 0.15V, respectively, the change in electrostatic potential depicted by change in hue as depicted by the scale is approximate for all electrostatic potential maps visualised with WebMO.  For benzisothiazole 119, nucleophilic attack of a hydrazine at the sulfur atom was anticipated to proceed via three distinct reaction pathways (Figure 72): the first potential pathway (P1) involves an initial SN2 reaction at the sulfur leading to ring opening (via S-N bond cleavage) to give the methoxy imidate intermediate 127, followed by its conversion to the Meisenheimer complex 128; the second pathway (P2) is a concerted variant of P1 wherein S-N bond cleavage leads to a shift of the nitrogen atom to give the Meisenheimer complex 128 directly; and the third pathway (P3) would result from S-C bond cleavage with concerted formation of the alternate Meisenheimer complex 143. From the calculated Gibbs free energies (∆G0), the formation of 143 76  is highly endergonic (∆G0 = + 39.5 kcal/mol) and hence highly unfavourable. However, the formation of 128 (via P1 or P2) is significantly more favourable (∆G0 = +13.4 kcal/mol) albeit still endergonic. Note, the imidate intermediate formed from the stepwise reaction (P1) is essentially isoenergetic with 119 (∆G0 = -1.7 kcal/mol).  Transition states connecting each of the potential intermediates in pathways P1 and P2 were investigated to confirm that such processes were feasible. Appropriate transition states for the stepwise process P1 were optimized and were shown to connect 119𝑇𝑇𝑆𝑆1��127 and 127𝑇𝑇𝑆𝑆2��128 (see Figure 73). Importantly, the energies of both TS1 (25.8 kcal/mol) and TS2 (30.2 kcal/mol) are significantly lower than the predicted energy of 143, which, again, suggests that P3 is unfeasible. Unfortunately, we have been unable to find an appropriate transition state for P2 and thus cannot conclusively compare the stepwise and concerted processes leading to 128. In any case, the available information shows that the formation of 128 is both kinetically and thermodynamically feasible.   Figure 72.  Three proposed pathways where hydrazine attacks the S atom of 119. The colored arrows represent thermodynamic change in the first step for each pathway. Red represents an endergonic process while green signifies an exergonic step. The Gibbs free energy change is indicated between brackets (unit: kcal/mol).   77   Figure 73.  Reaction coordinate diagram of the first step in P1and P2 in 119 system. The pink are represent an approximation of where the transition state in the P2 pathway lies in.  The total Gibbs free energy of all reactants was set as baseline (0 kcal/mol). Each plateau represents the total Gibbs free energy of all existing compounds (i.e. reactants, transition state TS, and products) at each stage.  To complete the analysis of the first steps in the formation of hydrazonate 126, a further study was carried out to determine whether the SNAr-type addition of hydrazine at C-3 of 3-methoxy-5-nitrobenzisothiazole 119 (PC pathway) was energetically favourable/competitive with the stepwise P1/concerted P2 pathways. Taking into consideration that the OMe group in 119 is retained in the rearranged product 126, the addition of hydrazine to the “imine” double bond in 119 was stopped at the stage where the initial adduct 144 was formed (Figure 74). Note, that the next step in the PC pathway involved reaction of 144 with a second molecule of hydrazine at the sulfur centre, i.e. a return to the P1/P2 reaction manifold.   Figure 74.  Proposed pathway PC. The colored arrows represent thermodynamic change in each pathway. Red represents an endergonic process while green means an exergonic step. Every point (pt) of the linewidth stands for 5 kcal/mol. 78   Comparison of the PC versus P1/P2 pathways is illustrated in the reaction coordinate diagram (Figure 75). By setting the reactants’ total energy as reference, the Gibbs free energy change in each pathway was plotted. In general, PC is less thermodynamically favorable when compared to P1 and P2, especially in the step of 144 to 145 versus 128 to 129.  Figure 75.  Reaction coordinate diagram of P1, P2 and PC in the 119 reaction system. The total Gibbs free energy of all reactants was set as baseline (0 kcal/mol). Each plateau represents the total Gibbs free energy of all existing compounds at each stage.  On the basis of these results it is concluded that the P1/P2 reaction pathways predominate in the reaction of 119 with hydrazine. Also included in this predicted reaction coordinate diagram are the energies for the steps leading from the Meisenheimer complex 128 to intermediates 129 and 130, and from there to the observed product 126. These steps are significantly exothermic, supporting strongly our initial proposed mechanism. This mechanism in its revised form is presented in Figure 76.   79   Figure 76.  Revised pathway for the conversion of 3-methoxy-5-nitrobenzisothiazole 119 to the observed hydrazonate product 126. The coloured arrows represent thermodynamic change in the first step for each pathway. Green represents an exergonic step. Every point (pt) of the linewidth stands for 5 kcal/mol.   In the study of the reaction between hydrazine and 3-chloro-5-nitrobenzisothiazole 42, we again looked at the three different ways in which hydrazine can react at the S atom (P1, P2, and P3 pathways) and compared the findings with that found for the SNAr reaction of hydrazine at C-3 (PC pathway; (Figure 77).  Immediately clear was the finding that the concerted pathways P2 (∆G = +13.96 kcal/mol) and P3 (∆G = +24.28 kcal/mol), leading to direct formation of the Meisenheimer complexes 139 and 146, were endergonic, and consequently far less favourable relative to the highly exergonic pathway P1 (∆G = -61.38 kcal/mol), which leads to formation of stable nitrile product 132. This suggested, that, as proposed (Figure 70; pathway a) formation of nitrile 132 may be a key step in the process giving the observed rearrangement product 82.   80   Figure 77.  Four proposed pathways showing reaction between hydrazine and 42.  Pathways P1, P2, and P3 show proposed pathway when hydrazine attacks S atom.  PC shows the proposed pathway when hydrazine attacks at C-3 giving 45.  The colored arrows represent thermodynamic change in the first step for each pathway. Red represents an endergonic process while green means an exergonic step. Every point (pt) of the linewidth stands for 5 kcal/mol. The Gibbs free energy change is labeled in the bracket (unit: kcal/mol).  However, the seminal observation in the computational study was the finding that the hydrazine would react with 42 via the PC pathway is an even more highly exergonic reaction to give the C-3 hydrazino substituted product 45 (∆G = -79.9 kcal/mol).  The further study to identify the transition states in the P1 and PC pathways (Figure 78), “confirmed” that thermodynamically and kinetically, benzisothiazole 42 would react with hydrazine preferentially via the SNAr manifold. Looking further down the reaction coordinate, it is noteworthy that if both P1 and PC contributed to formation of 82, through the reaction of a second molecule of hydrazine with the cyano group in 132 or the sulfur atom in 45 the same  amidrazone intermediate 133 would be generated (Figure 79); a common intermediate joining the two proposed reaction mechanisms  (see Figure 70.  In this context, it’s important to note that neither nitrile 132, or the disulfide resulting 81  from its dimerization, were detected in the reaction mixture giving 126, suggesting that nitrile 132 was not present in the reaction medium.  Figure 78.  Reaction coordinate diagram of the first step in P1 and PC in 42 system. The total Gibbs free energy of all reactants was set as baseline (0 kcal/mol). Each plateau represents the total Gibbs free energy of all existing compounds (i.e. reactants, transition state TS) at each stage.  82   Figure 79.  Reaction of intermediates 132 and 45 with hydrazine. Formation of amidrazone 133/Meisenheimer complex 134 as the common products issuing from both reactions. Note the calculated energy for the conversion of 45 to 134 (+10.68 kcal/mol).  ` Having determined that the PC pathway would predominate over P1 in the reaction of benzisothiazole 42 with hydrazine, it remained to compare the concerted P2 pathway to PC for the steps leading from the Meisenheimer intermediates 139 and 134 to the 2,1-benziosothiazole components 142 and 137, which are proposed as the building blocks to the sulfur bridged dimer 82 (Figure 80). According to the proposed mechanism (Figure 70), a 1,3-sulfur migration of the SNHNH2 side chain in 134 and 139 can occur, leading to the formation of the alternate Meisenheimer complexes 135 and 140.103 This transformation was found to be energetically favourable in both cases, as were the subsequent steps wherein the tricyclic intermediates 136 and 141 are generated and undergo spontaneous ring expansion to the target 2,1-benzisothiazole products. Note that in all steps, starting with the initial conversion of 42 to the 3-hydrazino-benzisothiazole 45 and the Meisenheimer complex 139, the PC pathway via 45 is significantly more energetically favourable.  83   Figure 80.  Reaction coordinate diagram of P2 and PC in 42 system. The total Gibbs free energy of all reactants was set as baseline (0 kcal/mol). Each plateau represents the total Gibbs free energy of all existing compounds at each stage.  Both pathways start from 42, and structures involved in each step of transformations are shown.  On the basis of this computational study, the predicted mechanism for the formation of the rearranged product 82 is presented in (Figure 81). This mechanism incorporates certain elements in the proposed pathways found in Figure 70, and importantly it shows that the 1,3-sulfur migration, anticipated under the reaction conditions where hydrazine is used as the limiting reagent, is energetically feasible.103 Further, it is noteworthy that the molecule of hydrazine consumed during the ring opening process 45134, is liberated during formation of the tricyclic intermediate 136, and that the hydrazine molecule initially introduced at C-3 in 45 is recovered during the reaction of the 2,1-benzisothiazole subunits 137 with the proposed sulfur species that completes formation of 82.  84   Figure 81. Predicted pathway PC to compound 82. The colored arrows represent thermodynamic change in the first step for each pathway. Red represents an endergonic process while green means an exergonic step. Every point (pt) of the linewidth stands for 5 kcal/mol. The reagents above the arrow are reactants in the step, while the reagents below represent products formed in the step. To better compare the energy of each compound, two states of hydrazine were used (NHNH2- and NH2NH2).  3.3.2 Conclusions The predicted pathways, issuing from the computational studies, suggest that the formation of a Meisenheimer complex is an energetically accessible process, key to the migration of the benzisothiazole nitrogen to C-8. Subsequent loss of (SNHNH2)- anion and formation of the observed ring opened hydrazonate product 126, of alternatively the 1.3 sulfur migration of -SNHNH2, and the steps leading to formation of the isomeric 2,1-benzisothiazole system in 82 are also energetically accessible. Most interesting, was the indication that the reaction of 3-chloro-5-nitro-1,2-benzisothiazole 42 with hydrazine occurred preferentially through the SNAr mechanism involving displacement of the 3-chloro group and formation of the 3-hydrazino-5-nitrobenzisothiazole intermediate 45. This suggests that if, the subsequent reaction of 45 with 85  hydrazine at the sulfur centre in this compound could be suppressed, then compound 45 could be isolated in yields permitting us to continue with the investigation of approach 2a to the construction of the N-benzisothiazole substituted pyridopyrazolone analog of 1C8 44. Further, it suggested that the development of the alternate (Approach 2b) to the synthesis of our target 1C8 analog may be feasible.  3.4 Approach 2b(1):  The projected synthesis of key intermediate 117.  Literature/Model studies involving the reaction 3-substituted-5-nitrobenzisothiazoles 42 and 119 with phenylhydrazine  Figure 82.  (i) Approach 2b for the preparation of the key intermediate 117 from 3-substituted-5-nitrobenzisothiazoles 42 and 119.  (ii) Illustrated also is a literature precedent for this transformation (147  148), and (iii) our successful conversion of benzisothiazole 41 to 3-phenylhydrazinobenzisothiazole 149.  The alternate route envisaged (Approach 2b) to synthesize the key intermediate 117, needed for preparation of the constrained amide 44, was to react the 3-subsituted-5-nitro-1,2-benzisothiazoles 42/119 with 4-hydrazino-nicotinic acid methyl ester 118 (Figure 82a).  In support 86  of this approach, it has been reported that the reaction between the 5-acetyl-3-methoxy-1,2-benzisothiazole 147 and excess phenylhydrazine gives 3-phenylhydrazino-benzisothiazole 148 in 67% yield (Figure 82b).104  In keeping with this observation, we  similarly found that 3-methoxybenzisothiazole 41 reacted with phenylhydrazine to give the 3-hydrazino substitution product 149 in good (57%) yield after 1.5 day of reflux in MeOH (0.5 mmol scale). However, when these conditions were applied to 3-methoxy-5-nitro-1,2-benzisothiazole 119 we did not obtain the expected SNAr product. 150 (Figure 83).  Instead, we obtained the rearranged ring-opened, sulfur-extruded product 151.  Note that, compared to the reaction between 119 and hydrazine described in the previous section, the reaction between 119 and phenylhydrazine took a much longer time at room temperature.  Even after 24 hours, the starting material 119 was recovered in 80%.    Figure 83.  Rearranged product 151 from the reaction between phenylhydrazine and 5-nitro-3-methoxy-1,2-benzisothiazole 119.  In light of this result, we looked next at the reaction of 3-chloro-5-nitro-1,2-benzisothiazole 42 with phenylhydrazine (Figure 84).  The reaction was carried out in DMF to avoid the possibility of the formation of products containing a methoxy group at C-3. When an excess of phenylhydrazine was used (5.0 eq.), the reaction mixture showed up as a series of orange bands on TLC, none of which could be isolate/identified.  In contrast, when compound 42 was reacted with 1.5 eq. of phenylhydrazine in the presence of 1.2 eq. of DBU in DMF at room temperature for one hour, an orange coloured product could be isolated in 11% yield (based on the presumed formation of compound 150) by column chromatography (hexane 100% to hexane 65% / ethyl acetate 35%).  NO2SNH3CO O2N NOCH3NHPhNH2PhNHNH2 (6.0 eq.)EtOH, r.t. 24 hr6.2%119 151NSHN NHO2N15087   Figure 84.  The reaction between 3-chloro-5-nitrobenzisothiazole 42 with 1.5 eq. phenylhydrazine in the presence of DBU in DMF: Possible product structures 150 and 152a-b.  The different conditions subsequently studied to improve the yield for this reaction are outlined in Table 6.  Note that when DBU was added in a catalytic amount, a complex product mixture (black tar) was produced.  Note also, when phenylhydrazine was not added to the reaction, there was also formation of a complex mixture (all starting material was consumed).   Table 6.  Conditions tried for the reaction between 3-chloro-5-nitro-1,2-benzisothiazole and phenylhydrazine.    88  The 1H NMR spectrum (Figure 85a) for this orange coloured product showed signals corresponding to the phenyl ring of phenylhydrazine and the three aromatic protons with chemical shifts typical for the phenyl ring of a 1,2-benzisothiazole component. Further, in the 13C NMR (Figure 85b), the C-8 and C-9 carbon peaks occur at 151.0 and 116.2 ppm, respectively.  Note that the C-8 13C NMR signal is much more upfield shifted compared to that of the 2,1-benzisothiazole dimer 82 we obtained before (Figure 85d), indicating that the new compound does not correspond to a 2,1-benzisothiazole derivative.  Indeed, at first site it appeared that compound 150 was the product formed (as predicted from the computational studies described in the previous section).  However, the signal at 7.82 ppm indicated the presence of a proton at C-3, which would not have been observed in the spectrum of compound 150.  Further, in the 13C NMR (Figure 85b), C-3 of the new product is at 139.6 ppm.  Note also that the HSQC spectrum (Figure 85c) shows a correlation between the 1H signal at 7.82 ppm and the 13C NMR signal at 139.6 ppm.  Based on the NMR evidence we have proposed two alternative structures (152a and 152b, Figure 84), both of which are benzopyrazoles, as possible products from this reaction (chemical shifts for the quaternary carbon atoms of 1- and 2-methylbenzopyrazoles reported in literature are shown in Figure 85d).  However, although the NMR data is consistent with the proposed structures, it wasn’t obvious how or why one or the other of these two benzopyrazoles were formed.  For that reason, efforts were taken to obtain suitable crystals of this product for structure confirmation by X-ray diffraction.     89   90   Figure 85. (a) 1H NMR and (b) 13C NMR of proposed structure 152.  (c) HSQC of compound obtained from the reaction between 42 and 1.5 eq. phenylhydrazine in the presence of DBU in DMF.  (d) quaternary 13C chemical shifts of 149, 82, 1- and 2-methylbenzopyrazole.  NMR solvent is CDCl3.33   From the X-ray crystal diffraction data (obtained by Dr. Brian Patrick, UBC Chemistry department), the structure of the mystery compound formed in the reaction of 42 with 1.5 eq. of phenylhydrazine (in the presence of DBU in DMF at RT) was determined to correspond to the ring opened hydrazine compound 152c (Figure 86), which bears an amino group rather than a sulfur 91  atom at C-8 (benzisothiazole numbering).  Importantly, upon repeating the experiment on a larger scale (1.4 mmol of 42), we were able to isolate a second product.  By comparing its 13C NMR with values reported by Still and coworkers105 , the identity of this second product turned out to be 5-nitro-2,1-benzisothiazole (152e) (Figure 86). Initially, we were perplexed by this result, as C-3 of 152e no longer bears a substituent, i.e. it has a lower (aldehyde-type) oxidation state compared to the “carboxylic acid-type” oxidation state for C-3 in the starting 1,2-benzisothiazole compound 42.  However, with our knowledge of the reaction between benzisothiazole 42 and hydrazine leading to the sulfur bridged 2,1-BIT dimer 82 described earlier (Figure 81), we were able to deduce a possible pathway for the formation of this compound and 152c (Figure 86).  In the first step of the reaction, the Cl atom at C-3 of 42 was displaced by phenylhydrazine to form the desired SNAr substitution product 150. Subsequently compound 150 underwent the rearrangement process, leading to 3-phenylhydrazino-5-nitro-2,1-benzisothiazole 152d.  In the presence of DBU in the reaction medium 152d, it is proposed that the elimination reaction leading to formation of phenyldiimide and vinyl anion intermediate a occurred. Protonation of anion a resulted in the formation of 2,1-benzisothiazole 152e, lacking the C-3 substituent.  Consistent with the known reaction of 2,1-benzisothiazole with amine nucleophiles, phenylhydrazine could then react with 152e in a 1,4-addition manner to give b.  Elimination of sulfur (likely in the form of hydrogen sulfide) from the thioaminal in the five-membered ring in c lead to ring-opening and generated the observed hydrazone product 152c.106-108   92    Figure 86.  Proposed mechanism for the formation of 152c and 152e in the reaction between 42 and 1.5 eq. of phenylhydrazine in DMF under the presence of DBU at room temperature.   3.5 Approach 2b(2) The projected synthesis of key intermediate 117.  Model studies involving the reaction between non-nitro substituted 3-methoxy/triflate-1,2-benzisothiazoles and phenylhydrazine In view of these results, our subsequent efforts to construct the pivotal intermediate 117, reverting back to the study of the reaction of the non-nitro substituted 3-OMe substituted benzisothiazole 41, and its 3-OTf congener 2, with phenylhydrazine.  In this way the unwanted contribution of the nitro group to the reactivity of the benzisothiazole system could be avoided, and the needed nitro group could be introduced in the last steps of the synthesis. To this end, we decided to make the key intermediate 44 by reacting 41 with 118 to form 153, followed by (1) cyclization and methylation, (2) and nitration (Figure 87).     NO2SNCl42DBU (1.2 eq.)PhNHNH2 (1.5 eq.)DMF, room T, 1hrNSHN NHO2N150SNHN NO2N152dSNO2N152e (7%)HDBUPhN NHH DBUSNO2NHNH2NHPhaSNO2NH NH2NHPhSNHO2NH N O2NNH2HNNHPhNHPhH-"S"b c 152c (13%)1,4-addition93   Figure 87.  Reaction between phenylhydrazine and 3-methoxy-1,2-benzisothiazole 41.  Observation 1:  Formation of 3-phenylhydrazino-1,2-BIT from the “dead-end” 2,2’-dithiobis-benzonitrile 9 As pointed out in the previous section, the reaction between 41 and 5.0 eq. of phenylhydrazine (on a 3.0 mmol scale of 41) in refluxing MeOH for 5 days gave the desired product 149 in 57% yield. The crude product was concentrated, and the pure final compound (in the form of orange solid) was obtained by crystallization from methanol/hexane.  This opened up the possibility to react 3-methoxybenzisothiazole with the 4-hydrazino nicotinic acid methyl ester. Following the successful preparation of 149, the decision was taken to replace the 3-OMe function in the starting benzisothiazole by a more reactive leaving group in order to improve the reaction yield and reduce the reaction time.  It was hoped that by having a better leaving group at C-3, the substitution reaction would be favoured over the opening of the benzisothiazole ring.  In this optic, the 3-OTf (triflate) substituted benzisothiazole 2 was prepared and its reaction with phenylhydrazine was studied. Indeed, triflate is >10,000 times better as a leaving group compared to Cl/OMe in substitution reactions. This is a consequence of the presence of the three highly electronegative fluorine atoms in its structure.109 Further, the 3-triflate derivative of benzisothiazole has been used with success during the synthesis of lurasidone (see Figure 9). 94  Triflate 2, prepared by reaction of benzisothiazalone 37 with triflic anhydride in CDM, was obtained in 53% yield as a stable white solid.  Following the progress of the reaction of triflate 2 with excess phenylhydrazine in refluxing MeOH by TLC (9 hexane: 1 EtOAc), it was observed that the starting material 2 disappeared completely after about 3 hours, and a new and much more polar spot appeared. This new compound was isolated and found to correspond to the dead-end disulfide compound 9 (Figure 88). However, in experiments where the heating was continued for a longer reaction time (> 3 hr), additional spots appeared on TLC, one of which corresponded to the target compound 149.  More precisely, when the reaction was stopped after 8 hr, and the residue obtained after work-up was column chromatographed using hexane/EtOAc mixtures (100% hexane to 85 hexane: 15 EtOAc), compound   149 was isolated as a yellow solid in 19% yield.  Note that when the reaction period was extended beyond 9 hours the yield of 149 decreased significantly.    Figure 88.  Synthesis of 149 using 3-OTf-benzisothiazole 2 as the starting material.  To find out the differences between the reactions of phenylhydrazine and 3-methoxy-1,2-benzisothiazole 41 vs. 3-OTf-1,2-benzisothiazole 2, we then used 1H NMR and 19F NMR to monitor these experiments.  Based on 1H NMR, we found that the two reactions occurred through two different manners.  Figure 89a illustrates the reaction between 41 and phenylhydrazine at different time points over the course of 5 days.  The starting 3-methoxy-1,2-benzisothiazole 41 gave the target compound 149 directly, and the formation of disulfide 9 was not observed during 95  the course of the reaction.   Figure 89b, the expanded aromatic region, shows that the formation of 149 (indicated by the rise of the signal at 9.39 ppm) occurred at around 3 days.  The signals corresponding to the formation of 149 are marked by blue *.  Note that from the 1H NMR, another species that was formed during the reaction was aniline (triplet at 6.48 ppm corresponding to aniline is marked by an orange arrow).  This will be discussed in Observation 2 of this experiment.  Figure 89.  (a)1H NMR (DMSO-d6) of samples taken at different time points during the reaction between 41 and 5.0 eq. phenylhydrazine in MeOH (reflux).  Spectra of 149, aniline, and phenylhydrazine are shown on the top for purpose of comparison.  (b) The expanded aromatic region on 1H NMR of the 73 hr sample and the target compound 149.  Peaks for product 149 are marked by *, and the signal for aniline is marked indicated by an orange arrow.  96  For the reaction of 2 and phenylhydrazine the 1H NMR spectra for samples taken at different time intervals are shown in Figure 90.  Shown at the bottom is the 1H NMR of the starting material 2.  Note that compound 2 was totally consumed by 2hr 50 min.  This is supported by the loss of the signal at -72.34 ppm in the 19F spectra for the 3-OTf group in 2 and the appearance of the signal at -77.76 ppm for the triflate anion present in the solution (Figure 91).  Further, as indicated in the boxed area in the 1H NMR by the 2 hr 50 min mark peaks belonging to the disulfide 9 were present, and near maximum intensity.  As the reaction proceeded, more spots appeared on TLC and the aromatic region in the 1H NMR shows a range of broad peaks (see spectrum at 6.5 hr).   Although it was difficult to tell with certainty whether product 149 had been formed on the basis from theses 1H NMR peaks, the spot corresponding to product 149 was seen on TLC of a sample taken from the reaction medium.  Note, that at the 6.5 h mark the reaction medium had turned deep purple and contained gluey substances that did not dissolve in any organic solvents.  At 8 hours, the reaction was finished/stopped and worked-up. Two products were isolated, product 149 (19%) and disulfide 9 (up to 13% yield).  Note that aniline formation was also observed in this reaction (starting at 2hr 50 min) and continues throughout the course of the reaction (Figure 90).   Figure 90.  1H NMR (DMSO-d6) of samples taken at different time points during the reaction between 3-OTf-1,2-benzisothiazole 2 and 5.0 eq. phenylhydrazine in MeOH (reflux).  The 97  formation of the disulfide species 9 at 2 hr 50 min is indicated by the black box.  1H NMR of 9, 149, phenylhydrazine, and aniline are shown at the top for comparison. Note the formation of aniline started at 2 hr 50 min.   Figure 91.  19F NMR (DMSO-d6) of samples taken at different time points during the reaction between 3-OTf-1,2-benzisothiazole 2 and 5.0 eq. phenylhydrazine in MeOH (reflux).  Note that the signal at -72.34 ppm from the starting triflate 2 disappeared completely at 2 hr 50 min of the reaction, indicating all the starting material has been consumed.  The signal at -77.76 ppm corresponds to the triflate anion.  These observations indicate that formation of compound 149 occurred subsequently to the conversion of 2 to disulfide 9.  In other terms, as illustrated in Figure 88, rather than being a “dead-end product”, disulfide 9 serves as a precursor to 3-phenyhydrazino substituted benzisothiazole 149. This conclusion was surprising, in view of the fact that our intention in reacting 3-OTf benzisothiazole 2 with phenyl hydrazine was to favour the direct formation of 149.  At this time, our current understanding is that the greater electron attracting effect of the triflate group in 2 versus the OMe group in 41, influences/enhances the electropositive nature of the benzisothiazole sulfur atom, favouring the SN2 pathway to 9 over the direct 3-substitution (SNAr) pathway to 149. The build-up of disulfide 9 in the reaction medium via the SN2 pathway (Figure 92), is envisaged to involve initial formation of the nitrile intermediate 155, followed by its conversion to thiol/sulfide 7 and dimerization by air oxidation and/or through its reaction with a molecule of 2.  As indicated in the Figure, conversion of 155 to thiol/sulfide 7 could occur by an elimination process generating a molecule of phenyldiimide. The subsequent formation of 3-hydrazino 98  benzisothiazole 149 from disulfide 9 is believed to occur by addition of phenylhydrazine to the nitrile function in 9, followed by ring closure of the derived amidrazone intermediate 156. In this cyclization step a molecule of 7 in the disulfide motif acts as a leaving group.  Figure 92.  Proposed reaction pathway for the conversion of 3-OTf benzisothiazole 2 to compound 149, via the intermediacy of disulfide 9.  To test whether disulfide 9 could act as a precursor to compound 149 it was reacted with 5.0 equivalents of phenylhydrazine in refluxing MeOH for 8 hours. Interestingly, formation of compound 149 was not detected by TLC. Instead, we did isolate a new product along with residual disulfide 9 and determined that it corresponded to 3-(2-cyanophenylthio)-1,2-benzisothiazole 157. As illustrated in Figure 93, formation of this side product may result from the unwanted reaction of sulfide 9 with compound 149, as it is liberated during cyclization of intermediate 156 (Figure 92). This would result (despite the presence of phenylhydrazine in excess) in reformation of disulfide 9, which in turn reacts with sulfide anion 7. Note that in this experiment, no aniline production was observed. SSCNNSNHHNNSSCNHN NNH9156149SNOTfPhNHNH22NOTfSNHNHPhCNSHN-OTf-S-N bond cleavageCNS154 155 7SNTfOPhNHNH2NPhHHN NPhH2NNHPhreductionCNS799  SNNHHNNCSCSSCNNNCSNSSNCSSNSCNCN149 91581577 7  Figure 93.  Proposed pathway for the formation of 3-(2-cyanophenylthio)-1,2-benzisothiazole 157 from the reaction between disulfide 9 and excess phenylhydrazine.  These observations bear several features in common with earlier work by Walinsky and coworkers, who showed that 3-chlorobenzisothiazole 1 reacts with excess piperazine to give 3-piperazinylbenzisothiazole 3 via a complex and temperature dependent pathway involving the ring-opened intermediates 13 and 14 (Figure 15 and Figure 94).45  Importantly, when their reaction was carried out at 60-65 °C in THF for 17 hours the ring-opened sulfenamide species 13 in 85% yield.  However, continued reaction of 13 with piperazine for 12 hours at 112 °C gave the target compound 3 in 36% yield (along with small amounts of disulfide 9).  Presumably, the disulfide was formed by conversion of intermediate 13 to sulfide 7. In separate experiments, they further showed that on heating a highly concentrated solution of disulfide 9 at higher temperature (120-130 °C) for 24 h in isopropanol, the target compound 3 was formed in 28% yield. The side product, 3-(2-cyanophenylthio)-1,2-benzisothiazole 157 was also formed during the first two hours of the reaction conducted at 120-130 °C but was then consumed (longer reaction time) as product 3 started to form.   Interestingly, the yield of 3 in this reaction can be increased to 78% by adding 2 equivalents of DMSO (oxidant), which was used to oxidize the thiolate species present in the reaction mixture back to the disulfide form.  Walinsky et al. further noted that excess of piperazine was required in order to trap the sulfenamide species 13 and to accelerate the reaction from 13 to 3.  It has also been indicated that the desired 3-piperizino-1,2-benzisothiazole can be formed at yields comparable to the transformation from 8 to 3 by treating 3-(2-cyanophenylthio)-1,2-100  benzisothiazole 157 under the same conditions.  Details of this experiment has not been provided by Walinsky.  NCSCNSSCNNCSNNHN NSNNHHNSNNHNslowHN NHSNSCN+NHHNNHHNno experimental data for the reverse process(4.4 eq.)DMSO (2.0 eq)>100 °C1379157143140-145°C, 11 hr, 38%SNCl117 hr at 60-65°C Figure 94.  Pathway for the formation of 3-piperizinyl-1,2-benzisothiazole from disulfide 9 proposed by Walinsky.45  101  Unfortunately, the importance of Walinsky’s work to our experiments with disulfide 9, and in particular the ability of DMSO to remove the unwanted sulfide 7 from the reaction medium, was not sufficiently well appreciated while our study was in progress. However, we did explore an alternate means using the thiosulfonate 161 to both enhance the reactivity of disulfide 9 during the cyclization step to the target molecule 149, and to eliminate the parasite reaction leading to destruction of 149 (Figure 95).  Indeed, the cyclization of intermediate 162 would liberate a phenylsulfone anion, which would be expected to be less reactive/nucleophilic than sulfide 7.110 NSNHHNPhNHNSSO ONHRSSO ORPhNHNH2CNSHSOClON159161162149 Figure 95.  Proposed preparation of 3-phenylhydrazino-1,2-benzisothiazole 149 through the thiosulfonate 161 pathway.  Several different strategies were explored to prepare thiosulfonate 161.  Initially, effort was directed toward the mono-oxidation of the starting disulfide 9 using mCPBA (Figure 96a).  However, the reaction did not proceed at room temperature (no reaction), and at higher temperature   multiple components (>10) were visible on TLC.  Next, we tried to synthesize the thiosulfonate intermediate by reacting phenylsulfonyl chloride with 2-mercaptobenzonitrile 159 (Figure 96b).  However, dimerization of the starting thiol to form the disulfide 9 was preferred over the formation of the desired product 161.  Based on precedent in our lab concerning the reaction of acid fluorides and acid chlorides with amines, we decided to react the silylated thiol 164 with sulfonyl fluoride in acetonitrile using 0.2 eq. of TBAF (Figure 96c).77  The reaction didn’t work as we expected even upon reflux.  As we looked into literatures later on, the homolytic bond dissociation energy for S-F in sulfonyl fluoride is quite high (~81 kcal/mol) compared to S-Cl bond in sulfonyl chlorides (~46 kcal/mol).111  In fact, sulfonyl fluorides have very high thermostability (no reactivity in refluxing aniline at 130°C.111 In light of the negative results obtained during efforts to prepare compound 161, this aspect of my research was abandoned.  102   Figure 96.  (a) Mono-oxidation of disulfide 9. (b) Attempt to generate thiosulfonate from benzenesulfonyl chloride and 2-mercaptobenzonitrile. (c) Attempt to generate thiosulfonate 161 from benzenesulfonyl fluoride and silylated 2-mercaptobenzonitrile 164. The dimerization to form 9 was preferred, and no thiosulfonate formation was observed even upon heating the reaction at reflux.  Observation 2:  Aniline production in the formation of 3-phenylhydrazino-1,2-benzisothiazole (formal cleavage of N-N bond)   As already mentioned, by following the reactions of 3-methoxy and 3-OTf -benzisothiazole with phenylhydrazine by 1H NMR (Figure 89 and Figure 90) it was noticed that,  in addition to formation of the desired product 149, in both cases aniline was being produced. This was surprizing, as it indicated that at some point in these reactions an intermediate was being generated that promoted/allowed the N-N bond in phenylhydrazine to be cleaved. Further, from the 1H NMR 103  spectra for the two reactions it appeared that the quantity of aniline being produced was significant. A further detail was the observation that during the progress of the reactions, a gas was being evolved (indicated by bubbling in the bubbler connected to the reaction vessel).  Also, in the experiment in which we failed to produce 149 by treating disulfide 9 with phenylhydrazine, no gas formation nor aniline was observed. Taking into consideration that phenylhydrazine was used in excess in these reactions, it was pertinent to quantify the amount of aniline produced, relative to the limiting reagents in the reactions, the benzisothiazole starting materials. To quantify the amount of aniline formed in each of the two cases, we used quantitative NMR with ethylene carbonate (EC) as the internal standard.  To avoid the loss of solvent due to gas formation, these reactions were performed on 0.5 mmol (of starting 1,2-benzisothiazole) in 1.6 mL of MeOH in microwave vials (using the microwave as a heating source at 120 °C).  The 3-OMe-benzisothiazole reaction was heated for 7 hours, while the 3-OTf-benzisothiazole reaction was heated for 2 hours.  For each reaction, 0.04mL of the reaction mixture was taken with a syringe at the start and at the end of the reaction.  The solvent was removed by rotary evaporation (25 torr for 3-4 minutes), and the dried mixture was weighed.  A weighed amount of ethylene carbonate was added to this portion of the crude, and the mixture was dissolved in DMSO-d6.  From the 1H NMR spectra (Figure 97), the molar ratio of the two species is calculated from the ratio of the integrals corrected for the number (N) of nuclei represented by the peaks chosen: r (aniline/EC) = (integralaniline/Naniline) / (integralEC/NEC) The triplet (C-H at 6.48 ppm) from aniline C-4 proton indicated by the arrow in both (a) and (b) of Figure 97 was used for this calculation (Naniline = 1).  The signal for ethylene carbonate at 4.5 ppm corresponds to 4 C-H hydrogen atoms (NEC) = 4.  The amount of EC in mole (NEC) present in the mixture (in the NMR tube) can be calculated since we know the amount of EC we put in.  Using these two values, we can then calculate the amount of aniline present in the NMR tube:  = (nEC)( r (aniline/EC)) This then allow us to calculate the amount of aniline present in the mixture in the NMR tube: Mass of aniline in NMR tube = (naniline)(MWaniline) We can calculate the fraction of the NMR mixture to reaction (rxn) mixture by Weight fraction (NMR) = (weight of mixtureNMR) / (weight of mixturerxn) The amount of aniline present in the reaction mixture would be: Mass of aniline (rxn) = (mass of anilineNMR tube) / (weight fraction of NMR) 104  Using this method, the yield of aniline was determined to 131% and 203% when 3-OMe and 3-OTf benzisothiazole, respectively, was the limiting reagents.  Noteworthy was the fact that the amount of aniline produced was greater than the amount of starting benzisothiazole used.  Also, more aniline was generated when 3-OTf-benzisothiazole was used as the starting material.  Also, as mentioned in the previous section, when 3-OTf-1,2-benzisothiazole was the starting material, prolonged reaction time (over 2 hours in the microwave at 120 °C) resulted in loss of the product (149) yield.  At this point, no more aniline was produced in the reaction vessel.  The observation suggested that somewhere in the pathway to 149, an intermediate is produced that generates aniline, and in this process is reverted back to something that could react further with phenylhydrazine to produce more aniline. In fact, as the quantity of 149 produced is low when 3-OTf-benzisothiazole was used as the starting material, more aniline was generated, suggesting that the aniline pathway may be independent to the formation of 149.  At this point, we are unsure of the mechanism of the formation of aniline. 105   Figure 97.  1H NMR (DMSO-d6) for q-NMR of aniline (using ethylene carbonate as the internal standard) in reactions between phenylhydrazine and (a) 41; (b) 2.  Spectra of aniline and phenylhydrazine are shown for the purpose of comparison.  106  Approach 2b conclusions: In our studies, regarding 3-methoxy-1,2-benzisothiazole, we were able to generate the desired 3-phenylhydrazino-1,2-benzisothiazole 149 using the literature procedures available.104  In the attempt to optimize the yield of the reaction by replacing the 3-methoxy group on 1,2-benzisothiazole with 3-OTf, we found that the reaction undertook a different pathway by first forming the ring-opened disulfide 9 before the formation of the desired product 149.  The formation of 9 to 149 has been described in Walinsky’s study on the generation of 3 from 9.45  Further investigations into these reactions revealed the formation of aniline from phenylhydrazine at a yield of over 100%.  More experiments are required for further investigation into the aniline formation.107  Chapter 4: Conclusions In the absence of any structural-mechanistic data concerning the putative protein target of 1C8, and the precise mechanism whereby it blocks HIV replications, classical Structure-Activity Relationship (SAR) studies have been carried out in an effort to: i) optimize the biological activity of 1C8, and explore 1C8- protein target interactions. The initial SAR studies on 1C8 focused on different structural modifications. These included reduction of the potentially bioactivatable NO2 group to the corresponding amine, oxidation of the benzisothiazole sulfur atom, preparation of the inversed amides 46a-c and replacement of the central amide function by an amino-ether motif. All new 1C8 analogs were all inactive when tested in the anti-HIV assay. From these results, it was of particular note that the orientation of the amide bond plays an important role in the binding interaction between 1C8 and its putative target.  Molecular modeling using Chemdraw 3D revealed that in the lowest energy conformations, the C=O of the inverse amide 46b points in a different direction to that in 1C8.  Furthermore, 1C8 is held together in a defined conformation by a six-membered ring hydrogen bonding interaction, while 46b possesses a weaker five-membered ring hydrogen bonding interaction.   In our attempt to prepare the aminoether analog of 1C8 (excision of C=O in the amide bond), it was observed that the benzisothiazole motif underwent a deep seated rearrangement giving the sulfur bridged 2,1-benzisotiazole dimer 82  (identified by single crystal X-ray diffraction). The SAR study was also directed to the preparation and evaluation of a series of 4,6-hihetero aryl pyrimidine-based analogs of 1C8 (compounds 92a-f), obtained using a 3-component one-pot strategy.  In this series of molecules, the isothiazole ring in benzisothiazole was opened, and the S atom was extruded.  Further, the C=O in the amide bond and the C=N from the isothiazole ring was incorporated into the pyrimidine ring.  Unfortunately, although this strategy would have readily allowed variation in the nature of the aryl substituents attached to the pyrimidine ring, compounds 92a-f tested negative in anti-HIV assay. This result also suggested that the presence of the amide function and the correct orientation of the amide C=O in 1C8 is crucial for target binding/ anti-HIV activity.   Given these results, we next directed attention to the preparation of the pyridopyrazolone-benzisothiazole-based 1C8 analog 44. This analog was designed to mimic the hydrogen bonding structure in the rotational conformer of 1C8 in which the amide and 4-pyridinone carbonyls are pointing away from each other. A key building block for the proposed route to construct compound 108  44 was the 3-hydrazino-5-nitrobenzisothiazole 45.  However, efforts to prepare this intermediate through reaction of 3-methoxy-5-nitrobenzisothiazole 119 with 6.0 equivalents of hydrazine led to formation of the unexpected rearranged product 126.  More surprising, the corresponding reaction of 3-chloro-5-nitrobenzisothiazole 42 with a sub-stoichiometric quantity of hydrazine led to formation of the sulfur bridged dimeric compound 82, previously observed. To obtain support for the proposed mechanisms for the formation of these rearrangement products, a collaboration was established with Pr. Pierre Kennepohl and Ms. Xing Tong (UBC Chemistry department) to study/calculate the energetics for the different mechanistic steps using computational methods (Density Function Theory). It was predicted that the formation of the pivotal Meisenheimer complexes 128 and 134, stabilized by the presence of the nitro group, was an energetically favourable transformation toward these two rearranged compounds. Further, the calculations suggested that, whereas the formation of compound 126 involved an initial nucleophilic attack on the benzisothiazole sulfur atom, the initial step in the reaction of 3-chloro-5-nitrobenzisothiazole 42 with hydrazine involved formation of the targeted 3-hydrazino-5-nitrobenzisothiazole 45 through displacement of the 3-chloro group (SNAr reaction pathway). Interestingly, the prediction that the conversion of 42 to dimer 82 on reaction with hydrazine actually starts with the formation of the compound 45 provides a strong motivation to further study this reaction under conditions (lower reaction temperature, flow techniques, etc) where this valuable intermediate is “captured” before it is destroyed. This would open the possibility to continue the planned synthesis of the pyridopyrazolone-benzisothiazole analog 44 of 1C8.   In the final part of my thesis research, the reaction of the non-nitro substituted benzisothiazole   41 with phenylhydrazine was briefly explored.  As hoped, we managed to reproduce the literature procedure and obtain 3-phenylhydrazino-1,2-benzisothiazole 149 in 57% yield. Interestingly, in subsequent attempts to improve the yield for this reaction using the “more reactive 3-OTf substituted benzisothiazole 2 it was observed that the reaction proceeded via the intermediary of the disulfide compound 9 (overall yields 10-19%). This observation was novel, in that it is generally considered that conversion of benzisothiazole to disulfide 9 upon reaction with nucleophiles is a “dead-end” pathway. A second, and entirely unexpected observation in the reactions of benzisothiazole 41 and 2 with phenylhydrazine was the formation of aniline as a by-product in >100% yield relative to benzisothiazoles 41 / 2.  At this moment it is not understood how this occurs, but it seems possible that an intermediate that can be regenerated in the process 109  leading from 41 / 2 to 149 may promote/allow this N-N bond cleaving process to occur. We have proposed a pathway for the possible route of aniline formation, but more experimentation will needed to determine what is actually happening in this transformation. In conclusion, our work on the SAR of 1C8 has revealed the complexity of the chemistry of the benzisothiazole ring system.      110  Experiments  All reactions were performed under nitrogen atmosphere unless otherwise indicated.  The glasswares have been flame- or oven-dried glassware. All chemicals were purchased from commercial sources (Aldrich, Alfa Aesar, Oakwood, TCI America) unless otherwise described. THF was pre-dried by refluxing over sodium in benzophenone under nitrogen atmosphere and distilling prior to use.  Microwave experiments were carried in a Biotage Initiator Robot Eight System. Thin layer chromatography (TLC) used for monitoring experiments was performed using EMD Silica gel 60 F254 plates.  TLC plates were visualized by exposing them to UV light at a wavelength of 254 nm.  NMR experiments were performed on a Bruker Ascend-400 spectrometer (400 MHz for 1H, 100 MHz for 13C, and 376.5 MHz for 19F).  All spectra were measured at room temperature.  For NMR spectra, chemical shifts are measured in ppm (δ scale).  The coupling constant are reported in Hertz (Hz).  Multiplicities are reported as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublet), dt (doublet of triplet), p (pentet), m (multiplet), and br (broad singlet).  High resolution mass spectrometry was performed on Thermo Scientific Q Exactive™ Orbitrap High Resolution Mass Spectrometer.  Low resolution mass spectra were collected from AB Sciex QTRAP 5500-Agilent 1290.  Single X-ray diffraction experiments were carried out by Dr. Brian Patrick from UBC Chemistry using a Bruker APEX II area detector diffractometer.  Preliminary Library Screen for Anti-HIV-1 Activity4 The anti-HIV-1 activity of the library molecules was measured using a human T-cell reporter assay based on CEM-GXR cells.29 These cells naturally express the HIV-1 receptor CD4 and coreceptor CXCR4 and have been engineered to expresses the other HIV-1 coreceptor, CCR5.  Upon HIV-1 infection, CEM-GXR cells express GFP due to the activation of an exogenous Tat-driven LTR-GFP expression cassette, and the level of infection was monitored using flow cytometric analysis (guavaSoft 2.2 software, Guava HT8, Millipore). Cells were grown in RPMI-1640 media (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) and 100 U per ml penicillin-streptomycin. During propagation, CEM-GXR cells were selected with 0.2 mg/mL G418 (Invitrogen) and 0.1 mg/mL hygromycin B (Calbiochem). Antiviral activity was evaluated in the assay that measures inhibition of HIV-1 spread in a coculture of 111  CEM-GXR cells containing 1% of HIV-1NL4−3 infected (GFP positive) cells. Infection was performed in T25 flasks or in 96-well plates containing 1 × 106 or 8 × 104 CEM-GXR cells, respectively. The library molecules at 2 μM concentration and commercially available anti-HIV-1 reagents zidovudine 2 μM and nelfinavir 100 nM were added to the culture immediately after the inoculation by infected cells. All compounds were prepared in DMSO at the final concentration in culture that were <0.1% DMSO. The percent of infected cells in the culture was determined by flow cytometry on day 3.  Chapter 2 experiments: Preparation of 1-methyl-3-nitro-4(1H)-pyridinone (48)70: NONO2 4-hydroxy-3-nitro-pyridine (576 mg, 4.11 mmol) was dissolved in 25 mL dichloromethane.  2.75 mL of diisopropylethylamine and 0.63 mL of DMF was added to the reaction followed by addition of 0.7 mL of methyl iodide.  The reaction was stirred at room temperature for 18 hours.  Precipitate from the reaction was filtered and recrystallized from methanol to give yellow needle-like crystals (50%). 1H-NMR (400 MHz, methanol-d4) δ 8.92 (d, J = 2.0 Hz, 1H), 7.78 (dd, J = 7.6, 2.1 Hz, 1H), 6.66 (d, J = 7.6Hz, 1H), 3.86 (s, 3H).  Preparation of 3-amino-1-methyl-4(1H)-pyridinone (49)70: NONH2 To a flask containing 60.1 mg of 10% Pd/C, 1-methyl-3-nitro-4(1H)-Pyridinone (400.5 mg, 2.60 mmol) partially dissolved in 20 mL of methanol was added.  The reaction proceeded at 1 atm of H2 and was stopped when no starting material remained (by TLC).  Approximately 219 mL of H2 was used.  The reaction mixture was filtered through Celite, and the filtrate was concentrated in vacuo to afford a dark brown wax. The dark brown wax was stirred in 8 ether: 2 112  MeOH for 45 minutes.  A light grey solid precipitated out and was collected by suction filtration.  This light grey solid was then redisolved in MeOH, and 0.1 mmol of edetate disodium (EDTA) was added to the solution.  The mixture was stirred for 30 minutes at room temperature and filtered over Celite.  The filtrate was concentrated to give 49 as an orange wax (123 mg, 38% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.15 (dd, J = 6.8, 2.0 Hz, 1H), 6.94 (d, J = 2.1Hz, 1H), 6.00 (d, J = 6.8 Hz, 1H), 3.34 (s, 3H), 2.91 (br, 2H). MS ESI (m/z): 124.9 [M+H]+  Preparation of benzothiophene-2,3-dione (53)72: SOO Oxalyl chloride (1.88 mL, 21.4 mmol) was added in portions to a solution of thiophenol (2.0 mL, 19.4 mmol) in diethyl ether (60 mL) at 0°C.  The mixture was heated to reflux for 2 hours, and the solvent was evaporated under reduced pressure.  The crude was dissolved in dichloromethane (100 mL), and AlCl3 (9.5 g, 70 mmol) was added portion-wise to the stirring reaction mixture at 0°C.  The reaction was refluxed for 1 hour, poured onto ice, and stirred until clear separations were obtained.  The aqueous layer was extracted 3 times with dichloromethane.  The organic phases were washed with brine, dried over sodium sulfate, and filtered.  The solvent was removed by rotary evaporation, and the crude was recrystallized from ethyl acetate.  The orange crystals from the recrystallization was collected and identified to be the target compound (1.58 g, 45%).  1H NMR (400 MHz, Chloroform-d) δ 7.83 (dd, J = 7.6, 1.5 Hz, 1H ), 7.67 (dt, J = 7.7, 1.5 Hz, 1H ), 7.4 (d, J = 7.8 Hz, 1H ), 7.36 (t, J = 7.5 Hz, 1H ).  Preparation of 1,2-benzisothiazole-3-carboxamide (54)72: SNONH2 113  600 mg of benzothiophene-2,3-dione (3.65 mmol) was dissolved in 34 mL methanol.  20.1 mL of NH4OH (1.7 mmol) and 1.67 mL of 35% H2O2 (1.7 mmol) were added to the stirring reaction mixture.  After 16 hours, the white precipitate formed during the reaction was filtered and washed with cold water.  Yield = 46%.   1H NMR (400 MHz, Chloroform-d) δ 8.95 (d, J = 7.7 Hz, 1H), 7.94 (d, J = 7.6 Hz, 1H), 7.58 (t, J = 7.7 Hz, 1H), 7.54 (t, J = 7.7 Hz, 1H), 7.26 (br, 2H). Preparation of 1,2-benzisothiazole-3-carboxylic acid (55)72: SNOOH 1,2-Benzisothiazole-3-carboxamide (500 mg, 2.8 mmol) was dissolved in 55 mL of methanol.  5.5 mL of 4M NaOH was added dropwise to the stirring mixture.  The reaction was refluxed for 18 hours.   The reaction was stopped and cooled down to room temperature.  Methanol was removed by rotary evaporation.  50 mL of H2O was added to the crude.  The mixture was brought to 0°C and acidified to pH 2-3 using concentrated HCl.  The flask containing the crude was left at 0°C for 2 hours.  The precipitate was filtered and stirred in dichloromethane for 30 min.  The solid was filtered off, and the filtrate was concentrated to give a white solid, which is the target compound.  Yield = 85%.   1H NMR (400 MHz, Chloroform-d) δ 8.84 (d, J = 7.9 Hz, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.62 (t, J = 7.7 Hz, 1H), 7.59 (d, J = 7.7 Hz, 1H).   Preparation of nitro-1,2-benzisothiazole-3-carboxylic acid compounds (56a-c)73: SNOOHNO256a             SNOOHO2N56b                         SNOOHNO256c                           1,2-benzisothiazole-3-carboxylic acid (2.24 g, 12.5 mmol) was dissolved in 13.4 mL of concentrated sulfuric acid at 0°C under nitrogen.  0.8 mL of 68% nitric acid was added dropwise to the reaction mixture.  The reaction was kept below 5°C and stirred for three hours, time after which the reaction was poured over ice and stirred for 30 minutes.  The precipitate was filtered 114  and dried to afford a mixture of 4-, 5-, and 7-nitro-1,2-benzisothiazole-3-carboxylic acid.  The crude was then left in ethyl acetate in the fridge for three days.  The precipitate was filtered.  The filtrate was concentrated and found to be a mixture containing 1.0 5-nitro: 1.3 7-nitro: 2.5 4-nitro isomers based on their 1H NMR spectra.  The precipitate collected is a mixture containing 1.0 5-nitro: 1.2 7-nitro compounds.  To isolate the 5-nitro and 7-nitro compounds from this mixture separately, the precipitate was recrystallized from ethyl acetate.  Both the filtrate and crystals collected from each recrystallization, and their 1H NMR spectra were collected for the determination of relative ratios of 5- vs. 7-nitro.  7-nitro-1,2-benzisothiazole-3-carboxylic acid can be obtained in its pure form through repeat recrystallization using ethyl acetate.  For the remaining fractions, ones with a 5-nitro: 7-nitro ratio higher than 1.5 are recrystallized repeatedly from acetonitrile until pure 5-nitrobenzisothiazole-3-carboxylic acid can be obtained.  5-nitro-1,2-benzisothiazole-3-carboxylic acid (56b): Isolated yield of pure 56b:  2.90% 1H NMR (400 MHz, acetone-d6) δ 9.61 (d, J = 2.3 Hz, 1H), 8.58 (d, J = 9.0 Hz, 1H), 8.50    (dd, J = 9.0 Hz, 2.4, 1H).  7-nitro-1,2-benzisothiazole-3-carboxylic acid (56c): Isolated yield of pure 56c:  7.37% 1H NMR (400 MHz, acetone-d6) δ 9.20 (d, J = 7.7 Hz, 1H), 8.71 (d, J = 9.1 Hz, 1H),          7.99 (t, J = 8.0 Hz, 1H).  4-nitro-1,2-benzisothiazole-3-carboxylic acid (from mixture) (56a) (in mixture with 56 b     and c): The reported peaks are for 56a only: 1H NMR (400 MHz, acetone-d6) δ 8.70 (d, J = 7.0 Hz, 1H), 8.21 (d, J = 7.57 Hz, 1H),         7.95 (5, J = 7.9 Hz, 1H).     115   Preparation of N-(1-methyl-4-oxo-1,4-dihydropyridin-3-yl)-4-nitrobenzo[d]isothiazole-3-carboxamide (46a): NONHOSNO2N 65.7 mg of a mixture containing 4-, 5-, and 7-nitro-1,2-benzisothiazole-3-carboxylic acid (0.29 mmol) was dissolved in 1 mL of DMF in a dry round bottome flask under nitrogen.  0.06 mL DeoxoFluor (0.32 mmol) was added to the reaction mixture.  The peak on 19F NMR corresponding to the formation of the acyl fluoride of the 4-nitro compound was 37.0 ppm.  After 18 hours, 42.4 mg of 3-amino-1-methyl-4(1H)-pyridinone (0.34 mmol) was added to the reaction.  The reaction was stopped after 1 day and diluted with 10 mL of dichloromethane.  The yellow precipitate filtered was washed 3 more times with acetone to afford the target compound.  The product is a mixture of the 3 different isomers.  The signals for 46a on the 1H NMR spectrum were deduced by superimposing the spectrum with 46b and 46c. 1H NMR (400 MHz, methanol-d4) δ 8.98 (d, J = 2.2Hz, 1H), 8.51 (d, J = 9.1 Hz, 1H), 8.09 (d, J = 7.4 Hz, 1H), 7.84 (m, 1H), 7.77 (d, J = 7.3 Hz, 1H), 6.55 (d, J =7.3 Hz, 1H,), 3.89 (s, 3H).  Preparation of N-(1-methyl-4-oxo-1,4-dihydropyridin-3-yl)-5-nitrobenzo[d]isothiazole-3-carboxamide (46b): NONHOSNNO2 65.7 mg of 5-nitro-1,2-benzisothiazole-3-carboxylic acid (0.293 mmol) was dissolved in 1 mL of N,N-dimethylformamide in a dry round bottom flask under nitrogen.  0.06 mL DeoxoFluor (0.322 mmol) was added to the reaction mixture.  A peak on 19F NMR spectrum indicated formation of the acyl fluoride species (δ = 26.2 ppm).  After 18 hours, 42.4 mg of 3-116  amino-1-methyl-4(1H)-pyridinone (0.34 mmol) was added to the reaction.  The reaction was stopped after 1 day and diluted with 10 mL of dichloromethane.  The orange waxy precipitate filtered was washed 3 more times with dichloromethane and recrystallized from methanol to the orange target compound.  Yield = 4.1% 1H-NMR (400 MHz, methanol-d4) δ 9.83 (d, J = 1.9 Hz, 1H), 9.15 (d, J = 2.2 Hz, 1H), 8.49 (dd,  J = 9.1, 2.1 Hz, 1H), 8.41 (d, J = 8.8 Hz, 1H), 7.78 (dd, J= 7.2, 2.1 Hz, 1H), 6.55 (d, J=7.2Hz, 1H), 3.92 (s, 3H).  HRMS (HESI) m/z:  [M+H]+ Calcd for C14H10N4O4S 331.04955; found 331.04953.  Preparation of N-(1-methyl-4-oxo-1,4-dihydropyridin-3-yl)-7-nitrobenzo[d]isothiazole-3-carboxamide (46c): NONHOSNNO2  65.7 mg of 7-nitro-1,2-benzisothiazole-3-carboxylic acid (0.293 mmol) was dissolved in 1 mL of DMF in a dry round bottome flask under nitrogen.  0.06 mL DeoxoFluor (0.322 mmol) was added to the reaction mixture.  A peak on 19F NMR indicated formation of the acyl fluoride species (δ = 26.9 ppm).  After 18 hours, 42.4 mg of 3-amino-1-methyl-4(1H)-pyridinone (0.34 mmol) was added to the reaction.  The reaction was stopped after 1 day and diluted with 10 mL of dichloromethane.  The yellow precipitate filtered was washed 3 more times with acetone to afford the target compound. Yield = 3% 1H NMR (400 MHz, methanol-d4) δ 9.42 (d, J = 8.1 Hz, 1H,), 9.13 (d, J = 2.2 Hz, 1H) , 8.69 (d, J = 7.8 Hz, 1H), 7.92 (t, J = 8.0 Hz, 1H), 7.77 (dd, J = 7.2, 21 Hz, 1H), 6.55 (d, J = 7.2Hz, 1H), 3.91 (s, 3H). HRMS (HESI) m/z:  [M+H]+ Calcd for C14H10N4O4S 331.04955; found 331.04935.     117   Synthesis of reduced 1C8 hydrochloride salt (58): NOOHN NSH2NHCl 50mg (0.15 mmol) of 1C8 (previously prepared by other members from the Grierson Lab) was dissolved in 0.2mL of concentrated HCl in a 5mL round bottom flask.  165.0 mg (0.87 mmol) of SnCl2 dissolved in 0.2 mL of concentrated HCl was added dropwise to the round bottom flask containing 1C8.  The reaction was stirred at room temperature for 7 days, and the white precipitate was filtered and dried under high vacuum (35.0 mg, 69% yield).   1H NMR (400 MHz, DMSO-d6) δ 15.20 (broad s, 1H), 8.83 (d, J = 2.40 Hz, 1H), 8.02 (dd, J = 7.5, 2.3 Hz, 1H), 7.8 (d, J = 2.1 Hz, 1H), 7.75 (d, J = 9.2 Hz, 1H), 7.43 (dd, J = 9.6, 2.1 Hz, 1H), 6.72 (d, 1H, J = 7.5 Hz ), 3.01(s, 3H). HRMS (HESI) m/z:  [M+H]+ Calcd for C14H12N4O2S 301.075373; found 301.07565.  Preparation of ethyl-2-nitrophenylacetate (63)83: NO2OEtO 2-nitrophenylacetic acid (724.6 mg, 4 mmol) was dissolved in 8.9 mL of ethanol.  0.15 mL of concentrated sulfuric acid was added to the reaction.  The mixture was refluxed overnight, concentrated, and diluted with ethyl acetate.  The organic layer was washed with 10% K2CO3 until the wash became basic, dried over Na2SO4, and concentrated in vacuo.  The target compound was a light yellow solid.  Yield = 85%. 1H NMR (400 MHz, methanol-d4) δ 8.11 (dd, J= 8.2, 1.0 Hz, 1H), 7.67 (dt, J = 7.5, 1.2 Hz, 1H), 7.54 (t, J = 7.8Hz, 1H), 7.48 (d, J = 7.6Hz, 1H), 4.14 (q, J = 7.1Hz, 2H), 1.23 (t, J = 7.1 Hz, 3H).  118   Preparation of ethyl 2-(hydroxyimino)-2-(2-nitrophenyl)acetate (64)83: NO2OEtONOH Ethyl-2-nitrophenylacetate (6.833 g, 32.66 mmol) and isoamyl nitrite (5.0 mL, 37.2 mmol) were dissolved in 68 mL of ethanol and warmed to 60°C.  12.2 mL of 21% sodium ethoxide solution in ethanol (32.66 mmol) was added dropwise to the stirring reaction mixture.  The reaction was allowed to proceed for 2.5 hours.  It was stopped, cooled to room temperature, and acidified to pH 7.0 using 2N HCl.  The mixture was extracted with ethyl acetate, water, and brine.  The crude was recrystallized from dichloromethane to give pale yellow crystals (32%).   1H NMR (400 MHz, acetone-d6) δ 11.66 (s, 1H), 8.24 (d, J = 8.1 Hz, 1H), 7.87 (t, J = 7.5 Hz, 1H), 7.75 (t, J = 7.9Hz, 1H), 4.26 (q, J = 7.1 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H).  Preparation of ethyl-1,2-benzisoxazole-3-carboxylate (65)83: ONOOEt 260.28 mg of NaH (60% in oil) was added to a dry round bottom flask and washed 6 times with hexane.  13 mL of N,N-dimethylformamide was added to this flask followed by the addition of ethyl 2-(hydroxyimino)-2-(2-nitrophenyl)acetate (1.55 g, 6.51 mmol) dissolved in 9.3 mL of N,N-dimethylformamide.  The light of the fumehood was turned off, and the reaction was brought to reflux gradually.  The reaction was stopped after 6 hours, cooled down to room temperature, and diluted with 60 mL of water.  The mixture was extracted with ethyl acetate (2 × 60 mL).  The organic layer was washed with water (2 × 60 mL), brine, and dried over sodium sulfate.  The solvent was removed in vacuo.  The crude was chromatographed using hexane: ethyl acetate (100: 0 to 92: 8).  The target compound is a white solid.  Yield = 49%. 1H NMR (400 MHz, acetone-d6) δ 8.15 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.76 (t, J = 7.7 Hz, 1H), 7.55 (t, J = 7.5Hz, 1H), 4.54 (q, J = 7.1 Hz, 2H), 1.46 (t, J = 7.1 Hz, 3H).  119   Preparation of ethyl-5-nitro-1,2-benzisoxazole-3-carboxylate (66)83: ONOOEtO2N Ethyl-1,2-benzisoxazole-3-carboxylate (191.2 mg, 1.0 mmol) was dissolved in 1.4 mL of concentrated sulfuric acid at 0°C under nitrogen.  0.33 mL of 68% nitric acid was added dropwise to the reaction, which was kept under 5°C.  The reaction was stopped after 2.5 hours and poured over ice.  The yellow precipitate was collected and dried under high vacuum overnight.  Yield = 77.2%. 1H NMR (400 MHz, acetone-d6) δ 8.97 (d, J =2.2 Hz, 1H), 8.64 (dd, J = 9.2, 2.3 Hz, 1H), 8.10 (d, J = 9.2Hz, 1H), 4.60 (q, J = 7.1Hz, 2H), 1.49 (t, J = 7.1Hz, 3H).  Preparation of 5-nitro-1,2-benzisoxazole-3-carboxylic acid (67)83: ONOOHO2N Ethyl-5-nitro-1,2-benzisoxazole-3-carboxylate (118.1 mg, 0.5 mmol) was heated to 80°C in 1.9 mL of 70% H2SO4 for 4 hours.  The reaction was cooled to room temperature and poured onto ice.  The resulting mixture was stirred for 40 minutes and filtered.  The white precipitate collected was dried under high vacuum.  Yield = 85%. 1H NMR (400 MHz, acetone-d6) δ 8.99 (d, J = 2.2Hz, 1H), 8.64 (dd, J = 9.2, 2.3 Hz, 1H), 8.10 (d, J = 9.2 Hz, 1H).        120  Preparation of N-(1-methyl-4-oxo-1,4-dihydropyridin-3-yl)-5-nitrobenzo[d]isoxazole-3-carboxamide (69):   ONO2NONHNO 87.9 mg of 5-nitro-1,2-benzisoxazole-3-carboxylic acid (0.422 mmol) was dissolved in 1 mL of N,N-dimethylformamide.  0.08 mL DeoxoFluor (0.464 mmol) was added to the reaction mixture.  A peak on 19F NMR indicated formation of the acyl fluoride species (δ = 32.0 ppm).  After 18 hours, 62.9 mg of 3-amino-1-methyl-4(1H)-pyridinone (0.506 mmol) was added to the reaction.  The reaction was stopped after 1 day and filtered.  The precipitate was washed 3 times with dichloromethane and chromatographed using dichloromethane: methanol (100: 0 to 95: 5).  Fractions containing the target compound were combined, and the solvent was removed in vacuo to give a yellow solid.  Yield = 8%. 1H NMR (400 MHz, methanol-d4) δ 9.15 (d, 1H, J=2.1Hz), 9.08 (d, J = 2.1Hz, 1H,), 8.62 (dd, J = 9.2, 2.2 Hz, 1H), 8.00 (d, J = 9.0 Hz, 1H), 7.79 (dd, J = 7.2, 2.3 Hz, 1H,), 6.55 (d, J = 7.3Hz, 1H,), 3.91 (s, 3H).  Preparation of 4-chloronicotinic acid (72)4: NClCOOH 2.33 g of 4-chloropyridine hydrochloride (15.5 mmol) was put in a round bottom flask containing 5 mL of water at 0˚C under nitrogen.  823 mg of Na2CO3 dissolved in 5 mL of water was added dropwise to the stirring mixture.  The solution was stirred at 0C for 30 minutes, and the aqueous layer was extracted 3 times with pentane.  The organic layer was dried over Na2SO4 in the fridge for 45 minutes.  Na2SO4 was removed by filtration, and the organic layer was concentrated in vacuo.  The colourless oil (4-chloropyridine) was then dissolved in 12.7 mL.  Lithium diisopropylamine (LDA) was prepared in situ using 3.3 mL of diisopropylamine and 14.4 mL of n-butyllithium (1M in hexane) in tetrahydrofuran (THF) at -78˚C.  The 4-121  chlorpyridine solution in THF was added dropwise to the LDA solution at -78˚C and stirred for 50 minutes.  CO2 was then bubbled into the reaction flask for 45 minutes.  The reaction was brought up to room temperature gradually and stirred overnight.  The reaction was stopped, concentrated and partially dissolved in 10 mL of water.  The white suspension was filtered through Celite and glass wool packed in a syringe.  The filtrate was then acidified to pH 3-4 using concentrated HCl.  The white crystals were collected by suction filtration and dried under high vacuum for 72 hours.  Yield = 33%. 1H NMR (400 MHz, DMSO-d6) δ 13.83 (br, 1H), 8.94 (s, 1H), 8.65 (d, J = 5.4 Hz, 1H), 7.66 (d, J = 5.4 Hz, 1H).  Preparation of methyl-4-methoxynicotinate (73)4: NOCH3CO2CH3 4-Chloronicotinic acid (50 mg, 0.318 mmol) in 1.25 M methanolic HCl (1.2 mL) was stirred under reflux for 5 hours.  The reaction mixture was concentrated and dissolved in minimal amount of water.  Na2CO3 was added to the solution until pH was 10.  The solution was extracted 5 times with dichloromethane, dried over Na2SO4, and concentrated to afford a white solid.  Yield = 71%. 1H NMR (400 MHz, DMSO-d6) δ 8.71 (s, 1H), 8.59 (d, J = 5.9 Hz, 1H), 7.22 (d, J = 5.9 Hz, 1H), 3.91 (s, 3H), 3.81 (s, 3H).  Preparation of 1,4-dihydro-1-methyl-4-oxo-3-pyridinecarboxylic acid methyl ester (74)4: NOOCH3O Methyl-4-methoxynicotinate (127.2 mg, 0.76 mmol) was dissolved in 2 mL of anhydrous acetonitrile in a microwave reaction vial under nitrogen.  Methyl iodide (11.8 μL, 0.19 mmol) was added to the stirring solution dropwise.  The vial was heated at 120°C for 1 hour 15 minutes 122  in the microwave machine.  The reaction mixture was concentrated to give the target compound as a white solid (quantitative yield). 1H NMR (400 MHz, methanol-d4) δ 8.45 (s, 1H), 7.71 (d, J = 7.5 Hz, 1H), 6.50 (d, J = 7.6 Hz, 1H), 3.83 (s, 1H), 3.80 (s, 1H).  Preparation of 2-(1-methyl-4-oxo-1,4-dihydropyridin-3-yl)-2-oxoacetic acid (75)4: NOCOOH Compound 74 (1.00g, 5.98mmol) was dissolved in THF:MeOH:H2O (2:2:1, 120ml). Lithium hydroxide (0.340g, 14.20mmol) was then added. The transparent, yellow solution was stirred overnight at room temperature. The reaction mixture was concentrated, acidified with concentrated HCl, and the flask was left in an ice/water bath.  A white precipitate formed, and the mixture was suction filtered.  This white solid was identified as the desired product (782mg, 72% yield) 1H NMR (400 MHz, DMSO-d6) δ 8.70 (d, J = 2.3Hz, 1H), 8.07 (dd, J = 7.4, 2.2 Hz, 1H ), 6.75 (d, J =  7.4 Hz, 1H ), 3,88 (s, 3H )  Synthesis of 3-chloro-5-nitro-1,2-benzisothiazole (42)88: SNO2NCl To a warm solution (65 °C) of copper (II) chloride (anhydrous, 1.65 g, 12.3 mmol), isoamyl nitrite (2.1 mL, 15.6 mmol) in anhydrous acetonitrile (80 mL), a solution of 3-amino-5-nitrobenzo[d]isothiazole (2.0 g, 10.2 mmol) in acetonitrile (20 mL) was added dropwise. The resulting brown reaction mixture was allowed to stir at 65 °C for 1h after which time it was poured into 20% HCl aqueous solution, neutralized to pH 8 with solid sodium bicarbonate and extracted with methylene chloride. The combined organic layers were then dried over anhydrous sodium sulfate, filtered and concentrated to give 2.8 g of a brown solid. Purification by column chromatography, on silica gel, eluting with 9:1 hexane/ethyl acetate provided 3-chloro-5-nitro-1,2-benzisothiazole (0.7 g, 31.9%) as a light-yellow solid.  123  1H NMR (400 MHz, Chloroform-d): δ 8.69 (s, 1H), 8.22 (d, J = 9.8 Hz, 1H), 7.84 (d, J = 9.7 Hz, 1H)  Synthesis of bis(5-nitrobenzo[c]isothiazol-3-yl)sulfane (82): NSO2NSS NNO2  A mixture of 51.7 mg (0.42 mmol) of 49 and 74.5mg (0.35 mmol) of 3-chloro-5-nitro-benzisothiazole in 1.5mL of acetonitrile is heated at 125 °C for 2.5 hour.  The resulting dark purple mixture was concentrated and chromatographed over hexane/ethyl acetate (100:0 to 85:15).  Compound 82 is obtained as a yellow solid (16.0mg, 35% yield).  The structure of 82 has been confirmed with X-ray diffraction. 1H NMR (400 MHz, Chloroform-d) δ 8.86 (s, 2H), 8.30 (d, J = 9.7 Hz, 2H), 7.95 (d, J = 9.6 Hz, 2H) 13C NMR (101 MHz, Chloroform-d) δ 162.97, 157.40, 146.16, 133.64, 124.34, 123.31, 117.82  Synthesis of diheteroaryl aminoether (83) and isolation of C-H activated product (84):  NONHSN83                      NONH2SN84   135.7 (0.8 mmol) of 3-chloro-1,2-benzisothiazole and 198.6mg (1.6 mmol) of 49 were dissolved in 1.5 mL of anhydrous DMF under nitrogen in a 5mL microwave reaction vessel.  The reaction was heated at 125 °C for 2 hours and 30 minutes.  The reaction was concentrated, and the crude was purified by column chromatography (95 DCM/ 5 MeOH).  After purification, the target diheteroaryl aminoether (83) was isolated as a white solid (26.5 mg, 13% yield).   124   1H NMR (400 MHz, Chloroform-d) δ 9.10 (s, 1H), 8.72 (br, 1H), 8.01 (dd, J = 8.1, 2.4 Hz, 1H), 7.82 (dd, J = 8.2, 2.4 Hz, 1H), 7.53 (td, J = 7.6, 2.4 Hz, 1H), 7.44 (td, J = 7.6, 2.4 Hz, 1H), 7.25 (d, J = 7.1 Hz, 1H), 6.46 (dt, J = 7.2, 1.7 Hz, 1H), 3.78 (s, 3H).  MS ESI (m/z): 257.9 [M+H]+, 279.8 [M + Na]+  The more polar, C-H activated product (84) was isolated as a white solid (75.0 mg, 36.5%) by gradually increasing the polarity of the column to 100% MeOH.  1H NMR (400 MHz, Chloroform-d) δ 7.68 (d, J = 8.2 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.30 (d, J = 7.8 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 6.33 (d, J = 6.8 Hz, 1H), 4.97 (br, 2H), 3.72 (s, 3H).  13C NMR (101 MHz, Chloroform-d) δ 169.83, 146.41, 138.86, 138.38, 134.32, 133.94, 127.05, 126.19, 116.33, 113.34, 111.03, 110.36, 43.38.  MS ESI (m/z): 258.2 [M+H]+  Synthesis of nitrated diheteroaryl aminoether (85): SNHN NONO2  23.5mg (0.09 mmol) of 83 was dissolved in 0.4mL of concentrated sulfuric acid at 0 °C.  0.01 mL of 70% nitric acid was added to the stirring reaction.  The reaction was stirred at this temperature for two hours and then poured onto ice.  No precipitate was formed.  The reaction mixture was neutralized by adding sodium carbonate.  The crude was concentrated and treated with MeOH.  The precipitate was filtered, and the filtrate was concentrated to afford 6.0mg of orange solid (yield = 22%).  1H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H), 7.88 (d, J = 7.2 Hz, 1H), 7.68 (d, J = 7.2 Hz, 1H), 7.45 (m, 3H), 6.06 (d, J = 7.4 Hz, 2H), 3.66 (s, 3H).   MS ESI (m/z):  301.0 (M-H)-    125  Preparation of 4-methoxy-3-pyridinemethanol (99)92: NOCH3OH Methyl-4-methoxynicotinate (100 mg, 0.598 mmol) was dissolved in 3 mL of dry dichloromethane under nitrogen.  1.78 mL of 1.0 M di-isobutalaluminum hydride (DIBAL-H) in hexane (2.98 mmol) was added dropwise to the stirring solution at -78°C.  The reaction was gradually warmed up to room temperature through the course of 3.5 hours.  It was stirred at room temperature for another 1 hour, and cooled down to -78 °C again, during which 5 mL of methanol was added.  The reaction was brought back to room temperature and stirred for an additional 90 minutes.  It was suction filtered through silica, which was washed several times with methanol.  The filtrate was concentrated and dried under high vacuum overnight to afford a light yellow solid.  This solid was stirred in dichloromethane for 30 minutes and filtered through Celite.  The filtrate was concentrated to give the target compound.  Yield = 70%. 1H NMR (400 MHz, Chloroform-d) δ 8.43 (d, J = 5.6 Hz, 1H), 8.39 (s, 1H), 6.80 (d, J = 5.6 Hz, 1H), 4.69 (s, 2H), 3.90 (s, 3H), 2.76 (broad s, 1H).  Preparation of 4-methoxy-3-pyridinecarboxaldehyde (100): NOCH3O 4-methoxy-3-pyridinemethanol (2.16 g, 15.5 mmol) was dissolved in 100 mL of dichloromethane.  Manganese dioxide (13.50 g, 155.2 mmol) was added to the solution.  The reaction as stirred overnight at room temperature.  The reaction mixture was filtered through Celite, which was washed with dichloromethane three times.  The filtrate was concentrated to afford the title compound (light-yellow solid).  Yield = 86%. 1H NMR (400 MHz, Chloroform-d) δ 10.36 (s, 1H), 8.78 (s, 1H), 8.55 (d, J = 5.9 Hz, 1H), 6.87 (d, J = 5.9 Hz, 1H), 3.93 (s, 3H).  Preparation of 1,4-dihydro-1-methyl-4-oxo-3-pyridinecarboxaldehyde (97): 126  NOHO 4-Methoxy-3-pyridinecarboxaldehyde (60 mg, 0.438 mmol) was dissolved in 0.5 mL of anhydrous acetonitrile in a microwave reaction vial under nitrogen.  Methyl iodide (10.9 μL, 0.175 mmol) was added to the stirring solution dropwise.  The vial was heated at 120°C for 2 hours in the microwave machine.  The reaction mixture was concentrated, redissolved in dichloromethane, and filtered through silica.  The silica was washed with dichloromethane/methanol (98:2) mixture.  The filtrate was concentrated to give the target compound.  Yield= 60%.   1H-NMR (400 MHz, Chloroform-d) δ 10.32 (s, 1H), 7.98 (s, 1H), 7.27 (d, J = 9.7 Hz, 1H), 6.60 (d, J = 7.6 Hz, 1H), 3.74 (s, 1H).  Synthesis of 3-(4-methoxypyridin-2-yl)-1,5-diphenylpentane-1,5-dione (102): OONOMe 4-Methoxy-3-pyridinecarboxaldehyde (35.0 mg, 0.255 mmol) and acetophenone (30 μL, 0.255 mmol) was dissolved in 1.28 mL of ethanol.  0.34 mL of 1M NaOH was added dropwise to the reaction mixture at 0°C.  The reaction was then warmed up to room temperature and stirred overnight.  The reaction was stopped and concentrated.  The 1,5-diketone compound was obtained by using column chromatography in dichloromethane/methanol (100:0 to 97:3).  Corresponding fractions were combined and concentrated to give a yellow oil.  Yield = 50%. 1H NMR (400 MHz, Chloroform-d) δ 8.34 (s, 2H), 7.95 (d, J = 7.8 Hz, 4H), 7.54 (t, J = 7.3 Hz, 2H), 7.44 (t, J = 7.4 Hz, 4H), 6.74 (d, J = 5.6 Hz, 1H), 4.27 ( p, J = 7.0 Hz, 1H), 3.84 (s, 3H), 3.55 (dd, J = 16.7, 6.9 Hz, 2H), 3.42 (dd, J = 16.7, 7.0 Hz, 2H).    127    Synthesis of 3-(1-methyl-4-oxo-1,4-dihydropyridin-2-yl)-1,5-diphenylpentane-1,5-dione (103): OONO 1,4-dihydro-1-methyl-4-oxo- 3-pyridinecarboxaldehyde (20.0 mg, 0.146 mmol) and acetophenone (17 μL, 0.146 mmol) was dissolved in 0.73 mL of ethanol.  0.19 mL of 1M NaOH was added dropwise to the reaction mixture at 0°C.  The reaction was then warmed up to room temperature and stirred overnight.  The reaction was stopped and concentrated.  The 1,5-diketone compound was obtained by using column chromatography in dichloromethane/methanol (100:0 to 95:5).  Corresponding fractions were combined and concentrated to give a yellow oil.  Yield = 51%. 1H NMR (400 MHz, Chloroform-d) δ 7.95 (d, J = 7.7 Hz, 4H), 7.54 (d, J = 8.2 Hz, 1H), 7.50 (d, J = 7.2 Hz, 2H), 7.40 (t, J = 7.5 Hz, 4H), 7.13 (d, J = 7.2 Hz, 1H), 6.31 (d, J = 7.3 Hz, 1H), 3.95 (dd, J = 17.4, 7.9 Hz, 2H), 3.87 (m, 1H), 3.62 (s, 3H), 3.44 (dd, J = 17.3, 4.6 Hz, 2H).  Synthesis of azachalcone (104): NOCH3 ONO2  100 (54.9 mg, 0.4 mmol) and 3-nitroacetophenone (33.0 mg, 0.2 mmol) was dissolved in 1 mL of anhydrous acetonitrile.  DBU (29.9 µL, 0.2 mmol) was added to this mixture, and the reaction was stirred for 1.5 hours. The crude was concentrated and column chromatographed using dichloromethane/methanol (100:0 to 95:5).  The desired product was eluded out as a mixture with the starting aldehyde 100.  The following reported 1H NMR signals are for 104 only. 128   1H NMR (400 MHz, Chloroform-d) δ 8.82 (s, 1H), 8.70 (s, 1H), 8.51 (d, J = 5.3 Hz, 1H), 8.46 – 8.39 (m, 2H), 8.33 (d, J = 7.7 Hz, 1H), 7.98 (d, J = 15.8 Hz, 1H), 7.76 (d, J = 15.8 Hz, 1H), 7.71 (t, J = 8.0 Hz, 1H), 6.90 (d, J = 6.0 Hz, 1H), 4.01 (s, 3H).  Synthesis of (E)-1-methyl-3-(3-oxo-3-phenylprop-1-en-1-yl)pyridin-4(1H)-one (105): NO O 1,4-dihydro-1-methyl-4-oxo-3-pyridinecarboxaldehyde (54.9 mg, 0.4 mmol) and acetophenone (23.3 µL, 0.2 mmol) was dissolved in 1 mL of anhydrous acetonitrile.  1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) (29.9 µL, 0.2 mmol) was added to this mixture, and the reaction was stirred overnight. The crude was concentrated and column chromatographed using dichloromethane/methanol (100:0 to 93:7).   Yield = 32% 1H NMR (400 MHz, Chloroform-d) δ 8.75 (d, J = 15.2 Hz, 1H), 8.08 (d, J = 7.7 Hz, 2H), 7.55 (m, 2H), 7.47 (d, J = 14.8 Hz, 2H), 7.45 (d, J = 15.0 Hz, 1H), 7.21 (d, J = 7.5 Hz, 1H), 6.48 (d, J = 7.5 Hz, 1H), 3.72 (s, 3H).  3-(1-hydroxy-3-oxo-3-phenylpropyl)-1-methylpyridin-4(1H)-one (106): NO OOH This compound is another product obtained during the synthesis of 105. It is eluded from the column along with unreacted 1,4-dihydro-1-methyl-4-oxo-3-pyridinecarboxaldehyde as a mixture.  The reported 1H NMR spectrum does not include signals from the starting aldehyde.  1H-NMR (400 MHz, Chloroform-d) δ 7.96 (d, J = 7.8 Hz, 2H), 7.55 (m, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.27 (d, J = 7.9 Hz, 1H), 6.39 (d, J = 7.4 Hz, 1H), 5.21 (t, J = 6.0 Hz, 1H), 3.94 (dd, J = 17.4, 5.1 Hz, 1H), 3.69 (s, 3H), 3.47 (s, 1H), 3.22 (dd, J = 17.4, 7.0 Hz, 1H).  129    Synthesis of (E)-1-methyl-3-(3-(3-nitrophenyl)-3-oxoprop-1-en-1-yl)pyridin-4(1H)-one (98): NO ONO2  1,4-dihydro-1-methyl-4-oxo-3-pyridinecarboxaldehyde (54.9 mg, 0.4 mmol) and 3-nitroacetophenone (33.0 mg, 0.2 mmol) was dissolved in 1 mL of anhydrous acetonitrile.  DBU (29.9 µL, 0.2 mmol) was added to this mixture, and the reaction was stirred for 2 hours. The crude was concentrated and column chromatographed using dichloromethane/methanol (100:0 to 92:8).  Yield = 10%. 1H NMR (400 MHz, CD3CN) δ 8.73 (s, 1H), 8.59 (d, J = 15.3 Hz, 1H), 8.42 (d, J = 7.4 Hz, 1H), 8.37 (d, J = 7.7 Hz, 1H), 7.93 (s, 1H), 7.78 (t, J = 7.9 Hz, 1H), 7.60 (d, J = 15.2 Hz, 1H), 7.41 (d, J = 7.5 Hz, 1H), 6.32 (d, J = 7.5 Hz, 1H), 3.68 (s, 3H).  3-(1-hydroxy-3-(3-nitrophenyl)-3-oxopropyl)-1-methylpyridin-4(1H)-one (107): NO ONO2OH This compound is another product obtained during the synthesis of 98. It is eluded from the column along with unreacted 1,4-dihydro-1-methyl-4-oxo-3-pyridinecarboxaldehyde as a mixture.  The reported 1H NMR peaks do not include signals from the starting aldehyde. 1H NMR (400 MHz, Chloroform-d) δ 8.73 (s, 1H), 8.34 (t, J = 8.1 Hz, 1H), 7.64 (t, J = 8.0 Hz, 1H), 7.59 (s, 1H), 7.30 (d, J = 8.9 Hz, 1H), 6.35 (d, J = 7.4 Hz, 1H), 5.20 (dd, J = 7.1, 4.7 Hz, 1H), 3.85 (dd, J = 17.0, 4.3 Hz, 1H), 3.70 (s, 3H), 3.42 (s, 1H), 3.23 (dd, J = 17.0, 7.6 Hz, 1H)   130   Synthesis of 3-(2-amino-6-(3-nitrophenyl)pyrimidin-4-yl)-1-methylpyridin-4(1H)-one (92a): NO NNNH2NO2  1,4-Dihydro-1-methyl-4-oxo-3-pyridinecarboxaldehyde (80 mg, 0.583 mmol), guanidine hydrochloride (66.9 mg, 0.700 mmol), 3-nitroacetophenone (96.3 mg, 0.583 mmol), and sodium carbonate (185.5 mg, 1.750 mmol) were put in a 0.5-2.0 mL size microwave reaction vessel.  1.5 mL of N,N-dimethylformamide was added to this mixture.  The reaction proceeded for 2 hours at 120°C in the microwave.  After the reaction was finished, the mixture was filtered, concentrated, and put in a small amount of water.  It was acidified to pH~3, concentrated, and loaded onto silica gel.  Column chromatography of 9 dichloromethane: 1 methanol gives the target compound.  Yield = 6% 1H NMR (400 MHz, Chloroform-d) δ 9.02 (s, 1H), 8.81 (s, 1H), 8.62 (s, 1H), 8.42 (d, J = 7.6 Hz, 1H), 8.30 (d, J = 8.0 Hz, 1H), 7.64 (t, J = 7.9 Hz, 1H), 7.28 (d, 1H), 6.57 (d, J = 7.6 Hz, 1H), 5.05 (br, 2H), 3.80 (s, 3H).  Synthesis of 3-(2-amino-6-(3-(trifluoromethyl)phenyl)pyrimidin-4-yl)-1-methylpyridin-4(1H)-one (92b): NO NNNH2CF3  1,4-Dihydro-1-methyl-4-oxo-3-pyridinecarboxaldehyde (67.2 mg, 0.49 mmol), guanidine hydrochloride (56.2 mg, 0.59 mmol), 1-(3-(trifluoromethyl)phenyl)ethanone (76 µL, 0.49 mmol), and sodium carbonate (155.8 mg, 1.46 mmol) were put in a 0.5-2.0 mL size microwave reaction vessel.  1.5 mL of DMF was added to this mixture.  The reaction proceeded for 3.5 hours at 120°C 131  in the microwave.  After the reaction was finished, the mixture was filtered, concentrated, and put in a small amount of water.  It was acidified to pH~3, concentrated, and loaded onto silica gel.  Column chromatography of 93 dichloromethane: 7 methanol gives the target compound.  Yield = 7%. HRMS (HESI) m/z:  [M+H]+ Calcd for C17H13F3N4O 347.11142; found 347.11105. 1H NMR (400 MHz, DMSO-d6) δ 9.47 (s, 1H), 8.56 (d, J = 12.1 Hz, 2H), 8.32 (br, 2H), 8.23 (m, 2H), 8.03 (d, J = 7.5 Hz, 1H), 7.88 (t, J = 7.8 Hz, 1H), 6.98 (d, J = 6.7 Hz, 1H), 4.03 (s, 3H). 19F NMR (376 MHz, DMSO-d6) δ 61.09 (s, 3F).  Synthesis of ethyl 3-(2-amino-6-(1-methyl-4-oxo-1,4-dihydropyridin-3-yl)pyrimidin-4-yl)benzoate (92c): NOCOOEtNNNH2 1,4-Dihydro-1-methyl-4-oxo-3-pyridinecarboxaldehyde (67.2 mg, 0.49 mmol), ethyl 3-acetylbenzoate (94.6 mg, 0.49 mmol), guanidine hydrochloride (56.2 mg, 0.59 mmol), sodium carbonate (155.8 mg, 1.47 mmol) were put in a 0.5-2.0 mL size microwave reaction vessel.  1.5 mL of N,N-dimethylformamide was added to this mixture.  The reaction proceeded for 8 hours at 120°C in the microwave.  After the reaction was finished, the mixture was filtered, concentrated, and loaded onto silica gel.  Column chromatography of 92 dichloromethane: 8 methanol gives the target compound.  Yield = 14%. HRMS (HESI) m/z:  [M+H]+ Calcd for C11H12N2O3 351.14517; found 351.14523. 1H NMR (400 MHz, Chloroform-d) δ 8.73 (s, 1H), 8.62 (s, 1H), 8.54 (s, 1H),8.25 (d, J = 7.7 Hz, 1H), 8.12 (d, J = 7.4 Hz, 1H), 7.53 (t, J = 7.7 Hz, 1H), 7.27 (d, J = 6.8 Hz, 1H), 6.55 (d, J = 7.4 Hz, 1H), 5.09 (broad s, 2H), 4.40 (q, J = 7.1 Hz, 2H), 3.77 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H).   132  Synthesis of 2-amino-4-(1-methyl-4-oxo-1,4-dihydropyridin-3-yl)-6-phenylpyrimidine-5-carbonitrile (92d): NO NNNH2CN 1,4-Dihydro-1-methyl-4-oxo-3-pyridinecarboxaldehyde (67.2 mg, 0.49 mmol), guanidine hydrochloride (56.2 mg, 0.59 mmol), 3-oxo-3-phenylpropanenitrile (71.1 mg, 0.49 mmol), and sodium carbonate (155.8 mg, 1.46 mmol) were put in a 0.5-2.0 mL size microwave reaction vessel.  1.5 mL of N,N-dimethylformamide was added to this mixture.  The reaction proceeded for 2 hours at 120°C in the microwave.  After the reaction was finished, the mixture was filtered, concentrated, and put in a small amount of water.  It was acidified to pH~3, concentrated, and loaded onto silica gel.  Column chromatography of 9 dichloromethane: 1 methanol gives the target compound.  Yield = 15%. HRMS (HESI) m/z:  [M+H]+ Calcd for C19H15N5O 304.11929; found 304.11954. 1H NMR (400 MHz, Chloroform-d) δ 7.93 (d, J = 7.4 Hz, 2H), 7.67 (s, 1H), 7.49 (m, 3H), 7.29 (d, J = 7.5 Hz, 1H), 6.56 (d, J = 7.5 Hz, 1H), 5.97 (broad s, 1H), 3.69 (s, 3H).  Synthesis of 3-(2-amino-6-(thiophen-2-yl)pyrimidin-4-yl)-1-methylpyridin-4(1H)-one (92e): NO NNNH2S 1,4-Dihydro-1-methyl-4-oxo-3-pyridinecarboxaldehyde (67.2 mg, 0.49 mmol), guanidine hydrochloride (56.2 mg, 0.59 mmol), 1-(thiophen-2-yl)ethanone (52.8 µL, 0.49 mmol), and sodium carbonate (155.8 mg, 1.46 mmol) were put in a 0.5-2.0 mL size microwave reaction vessel.  1.5 mL of DMF was added to this mixture.  The reaction proceeded for 3.5 hours at 120°C in the microwave.  After the reaction was finished, the mixture was filtered, concentrated, and put in a small amount of water.  It was acidified to pH~6, and the precipitate was collected.  Yield = 42%. 133   HRMS (HESI) m/z:  [M+H]+ Calcd for C14H12N4OS 285.08046; found 285.08014. 1H NMR (400 MHz, DMSO-d6) δ 9.46 (s, 1H), 8.39 (br 2H), 8.30 (s, 1H), 8.14 (d, J = 6.3 Hz, 2H), 8.00 (d, J = 4.8 Hz, 1H), 7.36 (m, 1H), 6.83 (d, J = 7.2 Hz, 1H), 3.99 (s, 3H).  Synthesis of 3-(2-amino-6-(4-nitrothiophen-2-yl)pyrimidin-4-yl)-1-methylpyridin-4(1H)-one (92f): NO NNSNO2NH2 Preparation of 1-(4-nitrothiophen-2-yl)ethanone: OSNO2  At 0˚C under nitrogen atmosphere, 0.602 mL of 68% nitric acid was added dropwise to a stirring solution of 1.26 g (9.99 mmol) of 1-(thiophen-2-yl)ethanone.  After 3 hours, the reaction was poured over ice/water mixture and stirred for one hour.  The light brown precipitate was collected by suction filtration and dried under high vacuum overnight.  The crude was then stirred in ether for 1 hour.  The precipitate was collected.  Yield = 29%. 1H-NMR (400 MHz, Chloroform-d) δ 8.54 (s, 1H), 8.15 (s, 1H), 2.62 (s, 3H).  1,4-Dihydro-1-methyl-4-oxo-3-pyridinecarboxaldehyde (67.2 mg, 0.49 mmol), guanidine hydrochloride (56.2 mg, 0.59 mmol), 1-(4-nitrothiophen-2-yl)ethanone (83.9 mg, 0.49 mmol), and sodium carbonate (155.8 mg, 1.46 mmol) were put in a 0.5-2.0 mL size microwave reaction vessel.  1.5 mL of DMF was added to this mixture.  The reaction proceeded for 3.5 hours at 120°C in the microwave.  After the reaction was finished, the mixture was filtered, concentrated, and put in a small amount of water.  It was acidified to pH~3, concentrated, and loaded onto silica gel.  Column chromatography of 92 dichloromethane: 8 methanol gives the target compound.  Yield = 12%. 134  1H NMR (400 MHz, DMSO-d6) δ 8.90 (d, J = 1.4 Hz, 1H), 8.56 (d, J = 2.3 Hz, 1H), 8.45 (s, 1H), 8.17 (d, J = 1.4 Hz, 1H), 7.70 (dd, J = 7.5, 2.3 Hz, 1H), 6.75 (br, 2H), 6.33 (d, J = 7.5 Hz, 1H), 3.76 (s, 3H).  Chapter 3 experiments: Synthesis of 1H-pyrazolo[4,3-c]pyridin-3(2H)-one (122)74:  NNHHNO  0.2mL of DMF was added to 4-chloronicotinic acid (2.0g, 12.7 mmol) in 50mL of dichloromethane. At 0°C, 1.6mL (19.0 mmol) of oxalyl chloride was added to the reaction mixture. The resulting mixture was stirred at 0°C for 2 hours and then concentrated in vacuo. The solid was then dissolved in 50.8 mL of 1.0M hydrazine in tetrahydrofuran (THF) and refluxed for four hours. The reaction was cooled down to room temperature and concentrated in vacuo. The crude product was chromatographed with ethyl acetate/methanol to afford the desired product as a light-yellow solid in 55% yield.   1H NMR (400 MHz, methanol-d4): δ 8.60 (s, 1H), 7.59 (d, J = 7.26 Hz, 1H), 7.11 (d, J = 7.29 Hz, 1H) Synthesis of 5-methyl-2H-pyrazolo[4,3-c]pyridin-3(5H)-one (115)74:  NNHNO  530 mg (3.92mmol) of 122 was added to NaH (195mg, 4.86mmol, 60% in oil) in 25 mL of dry THF. The reaction was refluxed for two hours and the cooled to room temperature. A second batch of NaH (149mg, 3.72 mmol, 60% in oil) was added to the reaction mixture, which was then refluxed for an additional two hours. The reaction was cooled to room temperature, and 0.34mL of methyl iodide was added to the reaction. The resulting mixture was stirred at room temperature for 24 hours, concentrated in vacuo, and chromatographed with ethyl acetate/methanol to afford the desired product in 47.2% yield.  135   1H NMR (400 MHz, methanol-d4): δ 8.50 (s, 1H), 7.47 (d, J = 7.5 Hz, 1H), 7.03 (d, J = 7.5 Hz, 1H), 3.89 (s, 3H).  Synthesis of 3-methoxy-1,2-benzisothiazole (41)68: SNOMe  0.25 mL of 25 wt. % sodium methoxide in methanol was added to 3-chloro-1,2-benzisothiazole (170.0mg, 1.0 mmol) in 3 mL anhydrous methanol. The reaction was refluxed for two hours, cooled down to room temperature, and concentrated in vacuo. The crude was chromatographed over hexane/ethyl acetate (100% hexane to 88% hexane: 12% ethyl acetate) to give the title product as pale-yellow liquid (104.3 mg, 63.2% yield).   1H NMR (400 MHz, Chloroform-d) δ 7.92 (d, J = 8.0 Hz, 1H), 7.77 (d, J = 8.2 Hz, 1H), 7.52 (t, J = 7.7 Hz, 1H), 7.39 (dd, J = 45.1, 7.6 Hz, 1H), 4.20 (s, 3H).  Synthesis of 3-methoxy-5-nitro-1,2-benzisothiazole (119)68:  NO2SNH3CO 0.5 mL of 25 wt. % sodium methoxide in methanol was added to 3-chloro-5-nitro-benzisothiazole (429mg, 2.0 mmol) in 4mL anhydrous methanol. The reaction was refluxed for one hour, cooled down to room temperature, and concentrated in vacuo. The crude was chromatographed over hexane/ethyl acetate (100% hexane to 85% hexane: 15% ethyl acetate) to give the title product as yellow needles (280.6 mg, 67% yield).  1H NMR (400 MHz, DMSO-d6) δ 8.61 (d, J = 2.31 Hz, 1H), 8.12 (dd, J = 9.7 Hz, 2.4 Hz, 1H), 7.68 (dd, J = 9.7 Hz, 1H), 4.39 (s, 3H)     136  Synthesis of (Z)-2-amino-5-nitrobenzohydrazonamide (126):  O2N NOCH3NH2NH2   0.1 mL of hydrazine hydrate (50-60%) was added to 52.5mg (0.25mmol) of 3-methoxy-5-nitro-benzisothiazole in 1mL methanol. The mixture was refluxed for 30 minutes and then cooled to room temperature. The yellow precipitate formed was filtered to give product 126 as yellow needles (40.3 mg, 77% yield).  1H NMR (400 MHz, DMSO-d6) δ 8.15 (d, J = 2.7 Hz, 1H), 7.92 (dd, J = 9.1 Hz, 2.8 Hz, 1H), 7.89 (br, 2H), 6.79 (d, J = 9.1 Hz, 1H), 6.39 (br, 1H), 3.71 (s, 3H) 13C NMR (400 MHz, DMSO-d6) δ 152.74, 146.42, 135.38, 124.92, 123.16, 114.59, 111.75, 56.48.  HRMS (HESI) m/z: calcd for C8H10N4O3 [M + H]+ 211.08257, found 211.08101.  Synthesis of benzo[d]isothiazol-3-yl trifluoromethanesulfonate (2)37:  NSOTf 0.17 mL of diisopropyl ethylamine was added to a stirring mixture of 1,2-Benzisothiazol-3(2H)-one (151.0mg, 1.0 mmol) in 4mL of dichloromethane at 0°c. 1.0mL of 1.0M triflic anhydride in DCM was added dropwise to the stirring mixture. The reaction was stirred in ice bath for two hours after the addition of triflic anhydride, poured into an ice water bath, and extracted with DCM. The organic layer was concentrated and passed over a layer of silica (95% hexane: 5% ethyl acetate) under suction filtration. The filtrate was collected and concentrated to give the title compound as an off white solid (151.2mg, 53% yield).  1H NMR (400 MHz, Chloroform-d) δ 7.96 (dt, J = 8.2 Hz, 0.9 Hz, 1H ), 7.91 (d, J = 8.2 Hz, 0.8 Hz, 1H ), 7.65 (td, J = 7.7 Hz, 1.0 Hz, 1H ), 7.55 (td, J = 7.6 Hz, 0.8 Hz, 1H )   19F NMR (400 MHz, Chloroform-d) δ -72.35     137  Synthesis of 2,2'-disulfanediyldibenzonitrile (9):  CNSSNC   From the reaction between 41 and hydrazine: 0.24 mL of hydrazine monohydrate was added to a stirring solution of 100 mg (0.6 mmol) of 3-methoxybenzisothiazole in methanol.  The resulting mixture was refluxed for 30 minutes in methanol and allowed to cool to room temperature.  The solvent was removed by rotary evaporation, and the light yellow crude solid was extracted with DCM/water.  The organic layer was concentrated and afforded 9 as a white solid in 36% yield. The experiment conducted in refluxing EtOH also resulted in the formation of 9 in similar yields. 1H NMR (400 MHz, DMSO-d6) δ 7.95 (dd, J = 7.65, 1.47 Hz, 2H), 7.81 (dd, J = 8.0, 0.9 Hz, 2H), 7.74 (td, J = 7.7, 1.3 Hz, 2H), 7.58 (td, J = 7.7, 1.2 Hz, 2H).   MS ESI (m/z):  269.1 [M + H]+, 291.0 [M+Na]+   From the reaction between 2 and phenylhydrazine: 0.25 mL (2.5 mmol) of phenylhydrazine was added to 142 mg (0.5mmol) of 2 in 1.5mL of methanol at room temperature. The reaction was monitored by TLC. After the reaction was refluxed for 1 hour, a new spot besides compound 2 and phenylhydraine appeared on TLC. The reaction was stopped, cooled to room temperature, and concentrated. The crude was extracted with dichloromethane/water, and the organic layer was dried over anhydrous sodium sulfate. The organic layer was then concentrated and chromatographed with hexane/ethyl acetate. A white solid was isolated and identified to be 2,2'-disulfanediyldibenzonitrile (37.5mg, 28% yield).         138  Synthesis of 3-(2-phenylhydrazinyl)benzo[d]isothiazole (149):  NSNHHN  From reaction between 1 and phenylhydrazine: 0.25 mL (2.5 mmol) of phenylhydrazine was added to 82.6 mg (0.5 mmol) of 41, which was stirred in 1.6 mL of MeOH.  The reaction was refluxed for 1.5 days, cooled to room temperature, and concentrated.  The crude was extracted with DCM/water, and the organic layer was concentrated.  149 was obtained from recrystallization in MeOH as yellow needles (68.9 mg, 57% yield). 1H NMR (400 MHz, Chloroform-d) δ 7.89 (d, J = 8.1 Hz, 1H), 7.76 (d, J = 8.2 Hz, 1H), 7.49 (td, J = 7.6 Hz, 1.1 Hz, 1H), 7.34 (td, J = 7.6 Hz, 1.1 Hz, 1H), 7.24 (s, 1H), 7.2 (t, J = 6.7 Hz, 2H), 7.05 (br, 1H), 6.97 (d, J = 8.4 Hz, 2H), 6.86 (t, J = 7.3 Hz, 1H)  13C NMR (400 MHz, Chloroform-d) δ 159.58, 152.64, 148.88, 129.44, 128.59, 125.63, 124.44, 122.40, 120.97, 120.52, 113.54, 77.55, 77.24, 76.91   MS ESI (m/z):  242.1 [M + H]+.  From reaction between 2 and phenylhydrazine: 0.84 mL (8.5 mmol) of phenylhydrazine was added to 505 mg (1.78mmol) of 2 in 5.28mL of methanol at room temperature. The reaction was refluxed overnight, cooled to room temperature, and concentrated. The crude was extracted with dichloromethane/water, and the organic layer was dried over anhydrous sodium sulfate. The concentrated organic layer was chromatographed with hexane/ethyl acetate. The desire product was obtained as a yellow solid (127.8 mg, 19% yield).      139  Synthesis of bis(5-nitrobenzo[c]isothiazol-3-yl)sulfane (82):  NSO2NSS NNO2 From the reaction between hydrazine and 42: 10µL of 98% hydrazine hydrate (0.5 eq.) was added to a stirring solution containing 0.09 mL of DIEA (1.0 eq.) and 107.3 mg (0.5 mmol) of 3-chloro-5-nitro-benzisothiazole 42 (in 1.6mL of anhydrous EtOH).  The reaction mixture was stirred at room temperature for 30 minutes.  The resulting dark purple mixture was concentrated and chromatographed over hexane/ethyl acetate. Compound 82 is obtained as a yellow solid (20.5 mg, 31.5% yield).   Synthesis of methyl (Z)-2-amino-5-nitro-N-phenylbenzohydrazonate (151): O2N NOCH3NHPhNH2  In a 5mL round bottom flask, 0.3mL of phenylhydrazine (3.0 mmol) was added to 105.0mg (0.5 mmol) of 3-methoxy-5-nitro-1,2-benzisothiazole in 0.6 mL ethanol.  The mixture was stirred at room temperature for 24 hours.  The brown mixture was concentrated and chromatographed using hexane/ethyl acetate (100: 0 to 70: 30).  The fractions containing the product were collected and concentrated in vacuo to give yellow powder (8.9 mg, 6.2% yield). 80% of the starting 3-methoxy-5-nitro-benzisothiazole was recovered.  1H NMR (400 MHz, Chloroform-d) δ 8.36 (d, J = 2.6 Hz, 1H), 8.02 (dd, J = 9.0, 2.6 Hz, 1H), 7.33 – 7.25 (m, 2H), 7.02 – 6.96 (m, 2H), 6.93 – 6.85 (m, 1H), 6.70 (d, J = 9.0 Hz, 1H), 3.87 (s, 3H).  MS ESI (m/z): 285.1 [M - H]-.  Synthesis of compound 152c: O2NNH2NHNH 140  In a 5mL round bottom flask, 50 µL of DBU (0.3 mmol, 1.2 eq.) was added to a stirring solution of 53.7 mg (0.25 mmol, 1.0 eq.) of 42 in 1mL of dry DMF at room temperature.  40µL of phenylhydrazine (0.38 mmol, 1.5 eq.) was then added to this reaction mixture.  The brown mixture was stirred at room temperature for 1 hour and poured into water.  It was extracted three times with DCM, and the organic layers were combined and concentrated to afford a dark solid.  The dark solid was chromatographed using hexane/ethyl acetate (100: 0 to 70: 30).  The fractions containing 152c were collected and concentrated in vacuo to give yellow powder (8.1 mg, 12.7% yield).   1H NMR (400 MHz, Chloroform-d) δ 8.01 (d, J = 2.6 Hz, 1H), 7.93 (dd, J = 9.1, 2.6 Hz, 1H), 7.8 (s, 1H), 7.24 (t, J = 7.9 Hz, 2H), 6.92 (d, J = 7.99 Hz, 2H), 6.86 (t, J = 7.39 Hz, 1H), 6.62 (d, J = 9.04 Hz, 1H).  13C NMR (400 MHz, Chloroform-d) δ 151.04, 143.89, 139.55, 138.10, 129.73, 127.47, 125.24, 121.03, 116.22, 114.93, 112.83.  Synthesis of compound 152e: SNO2NH  Upon repeating the procedure for synthesis of 152c on a larger scale (starting with 1.4 mmol of 42), 152e (orange solid, 12 mg, 7% yield) was isolated at hexane/ethyl acetate (75: 25) along with 152c(isolated at 70 hexane: 30 ethyl acetate).   1H NMR (400 MHz, Chloroform-d) δ 9.61 (s, 1H), 8.82 (s, 1H), 8.23 (dd, J = 9.6, 2.3 Hz, 1H), 7.94 (d, J = 9.7 Hz, 1H),   13C NMR (400 MHz, Chloroform-d) δ 162.11, 151.08, 145.32, 133.15, 122.96, 122.42, 120.22.      141  Bbliography  1.  Boone, L. R.; Koszalka, G. W., Antiretroviral drug development for HIV: challenges for the future. Curr. Opin. Investig. Drugs. 2010, 11 (8), 863-67. 2.  Hughes, J. P.; Rees, S.; Kalindjian, S. B.; Philpott, K. L., Principles of early drug discovery. Br. J. Pharmacol. 2011, 162 (6), 1239-1249. 3.  Bakkour, N.; Lin, Y.-L.; Maire, S.; Ayadi, L.; Mahuteau-Betzer, F.; Nguyen, C. 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Int. Ed. 2014, 53 (36), 9430-9448.  150  Appendices  Sing crystal X-ray diffraction data for compounds 126 and 82  X-ray crystal data for compound 126 O2N NOCH3NH2NH2                                Empirical Formula C8H10N4O3 Formula Weight 210.20 Crystal Colour, Habit orange, prism Crystal Dimensions 0.07 x 0.09 x 0.15 mm  Crystal System monoclinic Lattice Type Primitive Lattice Parameters a = 3.8206(06) Å  b = 11.726(2) Å  c = 10.475(2) Å  α = 90o  β = 100.091(3)o  γ = 90o  V = 462.03(13) Å3 Space Group P 21 (#4) Z value 2 Dcalc 1.511 g/cm3 F000 220.00 µ(Mo-Kα) 1.19 cm-1 Diffractometer Bruker APEX DUO  Radiation Mo-Kα (λ = 0.71073 Å)  Data Images 1065 exposures @ 15.0 seconds Detector Position 38.22 mm 2θmax 60.0o  No. of Reflections Measured Total: 10778   Unique: 2700 (Rint = 0.038) Corrections Absorption (Tmin = 0.917, Tmax= 0.992) 151   Lorentz-polarization Structure Solution Direct Methods (XT) Refinement Full-matrix least-squares on F2 Function Minimized Σ w (Fo2 - Fc2)2  Least Squares Weights w=1/(σ2(Fo2)+(0.0465P)2 + 0.1048P) Anomalous Dispersion All non-hydrogen atoms No. Observations (I>0.00σ(I)) 2700 No. Variables 153 Reflection/Parameter Ratio 17.65 Residuals (refined on F2, all data): R1; wR2 0.045; 0.095 Goodness of Fit Indicator 1.07 No. Observations (I>2.00σ(I)) 2804  Residuals (calculated on F2): R1; wR2 0.038; 0.091 Max Shift/Error in Final Cycle 0.00 Maximum peak in Final Diff. Map 0.29 e-/Å3 Minimum peak in Final Diff. Map -0.20 e-/Å3    152  X-ray crystal data for compound 82: NSO2NSS NNO2                                                  Empirical Formula C14H6N4O4S3 Formula Weight 390.41 Crystal Colour, Habit orange, needle Crystal Dimensions 0.02 x 0.04 x 0.20 mm Crystal System monoclinic Lattice Type C-centred Lattice Parameters a = 35.719(4) Å  b = 4.8236(4) Å  c = 26.797(2) Å  α = 90o  β = 104.704(4)o  γ = 90o  V = 4465.7(7) Å3 Space Group C 2/c  (#15) Z value 12 Dcalc 1.742 g/cm3 F000 2376.00 µ(MoKα) 5.29 cm-1 Diffractometer Bruker X8 APEX II Radiation MoKα (λ = 0.71073 Å)  graphite monochromated Data Images 847 exposures @ 30.0 seconds Detector Position 37.90 mm 2θmax 50.8o  No. of Reflections Measured Total: 17601   Unique: 4076 (Rint = 0.051) Corrections Absorption (Tmin = 0.911, Tmax= 0.989)  Lorentz-polarization Structure Solution Direct Methods (SIR97) Refinement Full-matrix least-squares on F2 Function Minimized Σ w (Fo2 - Fc2)2  Least Squares Weights w=1/(σ2(Fo2)+(0.0572P)2 + 0.2730P) 153  Anomalous Dispersion All non-hydrogen atoms No. Observations (I>0.00σ(I)) 4076 No. Variables 339 Reflection/Parameter Ratio 12.02 Residuals (refined on F2, all data): R1; wR2 0.058; 0.105 Goodness of Fit Indicator 1.04 No. Observations (I>2.00σ(I)) 3047  Residuals (refined on F2): R1; wR2 0.037; 0.089 Max Shift/Error in Final Cycle 0.00 Maximum peak in Final Diff. Map 0.34 e-/Å3 Minimum peak in Final Diff. Map -0.27 e-/Å3    154  X-ray crystal data for compound 152c:                                           Formula  C13H12N4O2  Dcalc./ g cm-3  1.443  µ/mm-1 0.102  Formula Weight  256.27  Colour  orange  Shape  blade  Size/mm3  0.22×0.04×0.02  T/K  100(2)  Crystal System  orthorhombic  Flack Parameter  -0.2(5)  Hooft Parameter  -0.1(6)  Space Group  Pca21  a/Å  28.0998(16)  b/Å  5.8800(3)  c/Å  7.1391(4)  α/° 90  β/° 90  γ/°   90  V/Å3  1179.57(11)  Z  4  Z'  1  Wavelength/Å  0.71073  Radiation type  MoK  Θmin/° 2.900  Θmax/° 26.502  Measured Refl's.  8843  Indep't Refl's  2404  Refl's I≥2 σ(I) 2214  Rint  0.0298  Parameters  180  Restraints  1   O2NNH2NHNH155  Largest Peak  0.180  Deepest Hole  -0.183  GooF  1.050  wR2 (all data)  0.0820  wR2  0.0801  R1 (all data)  0.0379  R1  0.0336   

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