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Isolation, structure elucidation, and total synthesis of bioactive natural products Forestieri, Roberto 2013

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ISOLATION, STRUCTURE ELUCIDATION, AND TOTAL SYNTHESIS OF BIOACTIVE NATURAL PRODUCTS  by Roberto Forestieri  B.Sc., The University of Naples Federico II, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   December 2013  ? Roberto Forestieri, 2013 ii  Abstract  Marine organisms are a potential source of lead compounds for drug discovery. Complex and unique chemical structures isolated from marine invertebrates and their associated  microorganisms can interact with specific cellular targets and selectively modulate biological pathways that play an important role in the pathogenesis of various diseases such as diabetes and tuberculosis (TB). The isolation, structure elucidation, and total synthesis of bioactive marine natural products are described herein.  Alotaketals A (2.1), B (2.3), D (2.4), and E (2.5) are a new class of sesterterpenoids isolated from the marine sponge Hamigera sp. that activate the cAMP cell signaling pathway. Here, the chemical structures of alotaketals and their unprecedented ?alotane? skeleton are described. The ability of alotaketal A (2.1) to activate cAMP signaling (EC50 = 18 nM) can be attributed to direct activation of adenylyl cylcase. Alotaketal A (2.1) is a chemical tool that can be useful for the study of cAMP signaling function in diabetes and other diseases. Clionamines A (5.1), B (5.2), C (5.3), and D (5.4) are a new class of aminosteroids isolated from the marine sponge Cliona celata that strongly stimulate autophagy in MCF-7 human breast cancer cells. Clionamine A (5.1) was tested for its ability to clear Mycobacterium tuberculosis (Mtb) from infected THP-1 cells and it gave a clear dose response with complete clearance at 5 ?M and an IC50 of ? 3 ?M. The anti-TB activity of clionamines confirms the role of autophagy in the response of human macrophages against Mtb infection. Clionamine B (5.2) was synthesized starting from the steroidal sapogenin tigogenin (5.30) in 12 steps with ? 2% overall yield. Synthetic clionamine B (5.2) strongly stimulates autophagy at 30 ?g/mL and inhibits Mtb proliferation in humane macrophages via autophagy activation. The clionamine pharmacophore iii  was identified by structure-activity analysis of unnatural clionamines analogues that were synthesized starting from the steroidal sapogenin sarsasapogenin (5.5). Among these synthetic analogues, N-benzyl-3,5-epi-clionamine B (5.24) was found to be a potent inhibitor (MIC = 5 ?g/mL) of Mtb proliferation in THP-1 human monocytic cells, indentifying N-benzyl-aminosteroids as a new class of potent antimicrobial compounds that are able to kill Mtb.  iv  Preface         A version of Chapter 2 has been reported in Organic Letters published by The American Chemical Society as: Forestieri, R.; Merchant, C. E.; de Voogd, N. G.; Matainaho, T.; Kieffer,  T. J. and Andersen R. J. ?Alotaketals A and B, Sesterterpenoids from the Marine Sponge Hamigera sp. that Activate the cAMP Cell Signaling Pathway? Org. Lett., 2009, 11, 5166-5169. I was responsible for the isolation and structure elucidation of alotaketals A (2.1), B (2.3), D (2.4), and E (2.5). Prof. Raymond J. Andersen wrote most of the manuscript. Catherine Merchant conducted in the Kieffer lab the screening of the Andersen?s natural products library and described the biological activity of alotaketals A (2.1), B (2.3), D (2.4), and E (2.5) in the cAMP assay. All the biological data reported in Chapter 2 were provided by Catherine Merchant and Dr. Timothy J. Kieffer. A version of Chapter 3 has been reported in PLoS ONE and in Exp. Cell. Res. as: 1) Khong, A.; Forestieri R.; Williams D. E.; Patrick B. O.; Olmstead A.; Svinti, V.; Schaeffer, E.; Jean, F.; Roberge, M.; Andersen, R. J.; Jan, E. ?A Daphnane Diterpenoid Isolated from Wikstroemia polyantha Induces an Inflammatory Response and Modulates miRNA Activity? PLoS ONE, 2012, 7 (6): e39621. 2) Zimmerman, C.; Austin P.; Khong A.; McLeod S.; Bean B.; Forestieri R.; Andersen R. J.; Jan E.; Roberge M.; Roskelley C. D. ?The Small Molecule Genkwanine M Induces Single Mode, Mesenchymal Tumor Cell Motility? Exp Cell Res, 2013, 319, 908-917. I was responsible for the isolation and structure elucidation of genkwanines M (3.1) and P (3.2). Dr. David E. Williams primarily collected the NMR spectroscopic data and identified the chemical structure of genkwanines M (3.1). I carried out the acetylation of genkwanines M (3.1) and the crystallization of diacetyl genkwanine M (3.3). Dr. Brian O. v  Patrick conducted the x-ray diffraction on the recrystallized sample of diacetyl genkwanine M (3.3) and interpreted the x-ray data to generate the ORTEP diagram of diacetyl genkwanine M (3.3). Anthony Khong conducted in the Jan lab the screening of Andersen?s natural products library and described the biological activity of genkwanines M (3.1) and P (3.2) in the miRNA assay. All the biological data reported in Chapter 3 were provided by Dr. Eric Jan and Anthony Khong. Carla Zimmerman identified in the Roberge lab genkwanines M (3.1) as an inducer of single mode, mesenchymal tumor cell motility (this part is not reported in this thesis). Regarding the work described in Chapter 4, I was responsible for the isolation and structure elucidation of virantmycin (4.1) and homoaerothionin (4.2). Elizabeth Donohue and Hilary J. Anderson conducted in the Roberge lab the screening of Andersen?s natural products library and described the biological activity of virantmycin (4.1) in the autophagy inhibition assay. Aruna Balgi conducted the screening of Andersen?s natural products library and described the biological activity of homoaerothionin (4.2) in the autophagy inhibition assay. All the biological data reported in Chapter 4 were provided by Elizabeth Donohue, Hilary J. Anderson, Aruna Balgi, and Prof. Michel Roberge. A version of Chapter 5 has been reported in Organic Letters published by The American Chemical Society as: Forestieri, R.; Donohue, E.; Balgi, A.; Roberge, M.; Andersen, R. J. ?Synthesis of Clionamine B, an Autophagy Stimulating Aminosteroid Isolated from the Sponge Cliona celata? Org. Lett., 2013, 15, 3918-3921. Robert A. Keyzers and Julie Daoust conducted the isolation and structure elucidation of natural clionamines A (5.1), B (5.2), C (5.3), and D (5.4) (this part is not reported in this thesis). Prof. Raymond J. Andersen wrote most of the manuscript. I was responsible for the total synthesis of clionamine B (5.2), 3,5-epi-clionamine B (5.25), N-benzyl-3,5-epi-clionamine B (5.24), compounds 5.12, 5.18, 5.23, 5.29, compounds 5.6-vi  5.11, 5.13-5.17, 5.19-5.22, 5.26-5.28, 5.30-5.42, and all the other compounds and reaction intermediates. The discovery, design, and optimization of all the chemical reactions are solely mine. Elizabeth Donohue, in cooperation with Aruna Balgi for synthetic clionamine B (5.2), described in the Roberge lab the biological activity of synthetic clionamine B (5.2), 3,5-epi-clionamine B (5.25), N-benzyl-3,5-epi-clionamine B (5.24), and compounds 5.12, 5.18, 5.23, 5.29 in the autophagy inhibition assay. Dennis Wong described in the Av-Gay lab the biological activity of synthetic clionamine B (5.2), 3,5-epi-clionamine B (5.25), N-benzyl-3,5-epi-clionamine B (5.24), and compounds 5.12, 5.18, 5.23, 5.29 in the Mtb proliferation and THP-1 viability assays. Figure 5.3, Figure 5.5, and Figure 5.6 and the data relative to the biological activity of natural clionamine A (5.1) in the Mtb proliferation and THP-1 viability assays were provided by Prof. Yossef Av-Gay. Figure 5.1 and Figure 5.8 and all the biological data reported in Chapter 5 and relative to the autophagy inhibition assay were provided by Elizabeth Donohue, Aruna Balgi, and Prof. Michel Roberge. All the biological data reported in Chapter 5 and relative to the Mtb proliferation and THP-1 viability assays were provided by Dennis Wong and Prof. Yossef Av-Gay. A version of Chapter 6 has been reported in PLoS Pathogens and in Journal of Biological Chemistry as: 1) Lam, K. K. Y.; Zheng, X.; Forestieri, R.; Balgi, A. D.; Nodwell, M.; Vollett, S.; Anderson, H. J.; Andersen, R. J.; Av-Gay, Y.; Roberge, M. ?Nitazoxanide Stimulates Autophagy and Inhibits mTORC1 Signaling and Intracellular Proliferation of Mycobacterium tuberculosis? PLoS Pathogens, 2012, 8 (5): e1002691. 2) Fonseca, B. D.; Diering, G. H.; Bidinosti, M. A.; Dalal, K.; Alain, T.; Balgi, A. D.; Forestieri, R.; Nodwell, M.; Rajadurai, C. V.; Gunaratnam, C.; Tee, A. R.; Duong, F.; Andersen, R. J.; Orlowski, J.; Numata, M.; Sonenberg, N.; Roberge, M. ?Structure-Activity Analysis of Niclosamide Reveals Potential Role for Cytoplasmic pH in vii  Control of Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling? Journal of Biological Chemistry, 2012, 287(21), 17530-17545. I was responsible for the synthesis of the niclosamide analogues 6.2-6.4 and nitazoxanide analogues 6.9-6.24 except niclosamide analogues 6.5 and 6.6 (synthesized by Matt Nodwell). Karen K. Y. Lam described in the Roberge lab the biological activity of nitazoxanide (6.7), tizoxanide (6.8), and analogues 6.9-6.24 in the autophagy activation and mTORC1 inhibition assays. Xingji Zheng described in the Av-Gay lab the biological activity of nitazoxanide (6.7), tizoxanide (6.8), and analogues 6.9-6.24 in the Mtb proliferation and THP-1 viability assays. All the biological data reported in Chapter 6 and relative to the autophagy activation and mTORC1 inhibition assays were provided by Karen K. Y. Lam, Aruna Balgi, Hilary J. Anderson, and Prof. Michel Roberge. All the biological data reported in Chapter 6 and relative to the Mtb proliferation and THP-1 viability assays were provided by Xingji Zheng and Prof. Yossef Av-Gay. All the biological data reported in Chapter 6 and relative to the mTORC1 signaling inhibition and protonophoric activities of niclosamide (6.1) and niclosamide analogues 6.2-6.6 were provided by Bruno D. Fonseca and Prof. Michel Roberge.    viii  Table of Contents  Abstract .................................................................................................................................... ii Preface ...................................................................................................................................... iv Table of Contents .................................................................................................................. viii List of Tables ...........................................................................................................................xii List of Figures........................................................................................................................ xiii List of Schemes ..................................................................................................................... xxiv List of Abbreviations ............................................................................................................. xxv Acknowledgements ................................................................................................................ xxx Dedication ............................................................................................................................. xxxi Chapter 1: Isolation, Structure Elucidation, and Total Synthesis of Bioactive Natural Products .....................................................................................................................................1 1.1 Bioactive Natural Products .......................................................................................1 1.2 Isolation and Structure Elucidation of Bioactive Natural Products ............................3 1.3 Total Synthesis of Bioactive Natural Products ..........................................................5 1.4 Structure Elucidation of Natural Products via Analysis of 1D and 2D NMR Spectroscopic Data ..............................................................................................................8 1.5 Research Summary ..................................................................................................9 Chapter 2: Alotaketals A, B, D, E, New Sesterterpenoids from the Marine Sponge Hamigera Sp. that activate the cAMP Cell Signaling Pathway ............................................. 11 2.1 Introduction ........................................................................................................... 11 2.2 Results and Discussion........................................................................................... 13 ix  2.2.1 Isolation and Structure Elucidation of Alotaketals A, B, D, E ............................. 15 2.2.2 Absolute Configuaration of the Alotaketals: Analysis of CD Spectra versus Mosher Analysis ............................................................................................................ 29 2.2.3 Biological Activity of the Alotaketals: Activation of the cAMP Signaling Pathway. ........................................................................................................................ 36 2.3 Conclusions ........................................................................................................... 37 2.4 Experimental Section ............................................................................................. 39 2.4.1 General Experimental Procedures ...................................................................... 39 2.4.2 Extraction of the Sponge .................................................................................... 40 2.4.3 Materials and Methods for cAMP Signaling Assay ............................................ 40 2.4.4 1H NMR, 13C NMR, HRESIMS and Optical Rotation Values for Alotaketal A (2.1), B (2.3), D (2.4), and E (2.5) .................................................................................. 41 Chapter 3: Genkwanines M and P, Daphnane Terpenoids from Wikstroemia polyantha that inhibit miRNA Activity ........................................................................................................... 60 3.1 Introduction ........................................................................................................... 60 3.2 Results and Discussion........................................................................................... 62 3.3 Conclusions ........................................................................................................... 69 3.4 Experimental Section ............................................................................................. 69 3.4.1 General Experimental Procedures ...................................................................... 69 3.4.2 Isolation Procedure ............................................................................................ 71 3.4.3 Derivatization of Genkwanine M (3.1) to Diacetyl Genkwanine M (3.3) ............ 71 3.4.4 1H NMR, 13C NMR, HRESIMS and Optical Rotation Values for Genkwanine M (3.1), Genkwanine P (3.2), and Diacetyl Genkwanine M (3.3) ....................................... 72 x  Chapter 4: Virantmycin and Homoaerothionin are Two New Autophagy Inhibitors ......... 83 4.1 Introduction ........................................................................................................... 83 4.2 Results and Discussion........................................................................................... 84 4.3 Conclusions ........................................................................................................... 87 4.4 Experimental Section ............................................................................................. 87 4.4.1 General Experimental Procedures ...................................................................... 87 4.4.2 Isolation Procedure ............................................................................................ 88 4.4.3 1H NMR, 13C NMR, HRESIMS and Optical Rotation Values for Virantmycin (4.1) and Homoaerothionin (4.2) .................................................................................... 89 Chapter 5: Total Synthesis of Clionamine B and Unnatural Analogues, Autophagy-Stimulating Aminosteroids that clear Mycobacterium tuberculosis from Human Macrophages ........................................................................................................................... 93 5.1 Introduction ........................................................................................................... 93 5.2 Total Synthesis of Clionamine B (5.2) and Unnatural Analogues ........................... 97 5.2.1 Synthesis of 3,5-epi-Clionamine B (5.25) and Analogues ................................... 97 5.2.2 Total Synthesis of Clionamine B (5.2) .............................................................. 106 5.3 Discussion ........................................................................................................... 110 5.4 Conclusions ......................................................................................................... 113 5.5 Experimental Section ........................................................................................... 115 5.5.1 General Experimental Procedures .................................................................... 115 5.5.2 1H NMR, 13C NMR, HRESIMS Values and Synthetic Methods for Clionamine B (5.2), 3,5-epi-Clionamine B (5.25), and Compounds 5.5-5.42 ...................................... 116 5.5.3 Inhibition Mtb Proliferation in THP-1 Macrophage: Bioassay Procedure ......... 181 xi  Chapter 6: Structural Requirements of Niclosamide and Nitazoxanide for the Stimulation of Autophagy Via Inhibition of mTORC1 ............................................................................ 183 6.1 Introduction ......................................................................................................... 183 6.2 Synthesis of Niclosamide and Nitazoxanide Analogues........................................ 185 6.2.1 Synthesis of Niclosamide Analogues: Inhibition of mTORC1 Signaling by Protonophoric Activity of Niclosamide (6.1) ............................................................... 185 6.2.2 Synthesis of Nitazoxanide Analogues: Stimulation of Autophagy, Inhibition of mTORC1 Signaling and Intracellular Proliferation of Mycobacterium tuberculosis ..... 187 6.3 Discussion and Conclusions ................................................................................. 189 6.4 Experimental Section ........................................................................................... 190 6.4.1 General Experimental Procedures .................................................................... 190 Chapter 7: Summary and Future Work .............................................................................. 222 7.1 Summary ............................................................................................................. 222 7.2 Future Work: Synthesis of New Analogues and Chemical Probes of Natural Clionamines .................................................................................................................... 225 7.3 Conclusions ......................................................................................................... 226 Bibliography .......................................................................................................................... 227 xii  List of Tables  Table 2.1 NMR spectroscopic data (600 MHz, benzene-d6) for alotaketal A (2.1). .................... 45 Table 2.2 NMR spectroscopic data (600 MHz, benzene-d6) for alotaketal B (2.3). .................... 46 Table 2.3 NMR spectroscopic data (600 MHz, benzene-d6) for alotaketal D (2.4). .................... 47 Table 2.4 NMR spectroscopic data (600 MHz, benzene-d6) for alotaketal E (2.5). .................... 48 Table 3.1 NMR spectroscopic data (600 MHz, DMSO-d6) for genkwanine M (3.1). ................. 75 Table 3.2 NMR spectroscopic data (600 MHz, DMSO-d6) for genkwanine P (3.2). .................. 76 Table 3.3 NMR spectroscopic data (600 MHz, DMSO-d6) for diacetyl genkwanine M (3.3). .... 77  xiii  List of Figures  Figure 1.1 Total synthesis of ecteinascidin (ET)-743 (1.2) from cyanosafracin B (1.1).1a-d ..........1 Figure 1.2 Chemical structures of E7389 (1.3)1e,f and nahuoic acid (1.4).8a .................................3 Figure 1.3 Chemical structures of triterpene monoglycoside (1.5)8b and anthracimycin (1.6).8c ...4 Figure 1.4 Total synthesis of alotaketal A (2.1).9a,b .....................................................................6 Figure 1.5 Viridicatumtoxin B (1.7) (revised structure).10a,b ........................................................7 Figure 2.1 Bioassay signaling pathway [adapted from Kieffer, T. J. and Habener, J. F. Endocrine Reviews, 1999, 20 (6), 876-913].14 ............................................................................................ 11 Figure 2.2 Chemical structures of alotaketal A (2.1)15 and forskolin (2.2).24 ............................. 12 Figure 2.3 1H NMR spectrum of alotaketal A (2.1) recorded in benzene-d6 at 600 MHz. .......... 13 Figure 2.4 13C NMR spectrum of alotaketal A (2.1) recorded in benzene-d6 at 150 MHz. ......... 14 Figure 2.5 Chemical structures of alotaketals B (2.3), D (2.4), E (2.5), and alotane skeleton 2.6. ................................................................................................................................................. 15 Figure 2.6 Key HSQC correlations of fragment A. ................................................................... 16 Figure 2.7 Key COSY correlations of fragment A. ................................................................... 17 Figure 2.8 Fragment A elucidated from COSY and HMBC data. .............................................. 18 Figure 2.9 Key HSQC and COSY correlations of fragment B. .................................................. 19 Figure 2.10 Fragment B elucidated from COSY and HMBC data. ............................................ 20 Figure 2.11 Key COSY correlations of fragment C. .................................................................. 21 Figure 2.12 Fragment C elucidated from COSY and HMBC data. ............................................ 22 Figure 2.13 Key HMBC correlations between fragments A, B, and C of alotaketal A (2.1). ...... 23 Figure 2.14 Key ROESY correlations of alotaketal (2.1). ......................................................... 24 xiv  Figure 2.15 Chemical structure of alotaketal B (2.3). COSY and HMBC correlations of the isovalerate fragment of alotaketal B (2.3) chemical structure. .................................................... 25 Figure 2.16 1H NMR spectrum of alotaketal B (2.3) recorded in benzene-d6 at 600 MHz. ........ 26 Figure 2.17 Proposed half-chair conformation for the 2-methyl-4-oxo-cyclohexenone ring of alotaketal A (2.1). ..................................................................................................................... 28 Figure 2.18 Chemical structures of phorbaketal A (2.7), phorbaketal B (2.8),20a,b ansellone A (2.9), and desacetylansellone A (2.10).21a,b ................................................................................ 30 Figure 2.19 CD spectrum of alotaketal A (2.1) recorded in methanol, and Snatzke?s sector rule for the the n??* transition in a cyclohexenone ring.18a-d, 19 ........................................................ 31 Figure 2.20 1H NMR chemical shift differences (??S-R) in ppm for S-/R-MTPA esters of phorbaketal B (2.8)  in CDCl3 (adapted from Rho, J. R.; Hwang, B. S.; Sim, C. J.; Joung, S.; Lee, H. Y. and Kim, H. J. Org. Lett., 2009, 11, 5590-5593).20a,b ........................................................ 32 Figure 2.21 CD spectrum of ansellone A (2.9) recorded in MeOH and ORTEP diagram for desacetylansellone A (2.10). The refined Flack parameter is -0.01(16)21c (adapted from Daoust, J.; Fontana, A.; Merchant, C. E.; de Voogd, N. J.; Kieffer, T. J.; Andersen, R. J. Org. Lett., 2010, 12, 3208-3211).21a,b ................................................................................................................... 33 Figure 2.22 Chemical structures of phorbasin C (2.11),20c (S)-carvone (2.12), and phorbasin C (2.13) after structural revision.20d............................................................................................... 35 Figure 2.23 Dose-response curve for cAMP activation by alotaketal A (2.1), forskolin (2.2),24,25 alotaketal B (2.3), alotaketal D (2.4), alotaketal E (2.5), and the crude extract of Hamigera. ..... 38 Figure 2.24 Numbered chemical structures of alotaketal A (2.1), B (2.3), D (2.4), and E (2.5). . 44 Figure 2.25 COSY spectrum of alotaketal A (2.1) recorded in benzene-d6 at 600 MHz. ............ 49 Figure 2.26 HSQC spectrum of alotaketal A (2.1) recorded in benzene-d6 at 600 MHz. ............ 49 xv  Figure 2.27 HMBC spectrum of alotaketal A (2.1) recorded in benzene-d6 at 600 MHz. ........... 50 Figure 2.28 ROESY spectrum of alotaketal A (2.1) recorded in benzene-d6 at 600 MHz. ......... 50 Figure 2.29 DEPT 135? of alotaketal A (2.1) recorded in benzene-d6 at 150 MHz. ................... 51 Figure 2.30 13C NMR spectrum of alotaketal B (2.3) recorded in benzene-d6 at 150 MHz. ........ 51 Figure 2.31 COSY spectrum of alotaketal B (2.3) recorded in benzene-d6 at 600 MHz. ............ 52 Figure 2.32 HSQC spectrum of alotaketal B (2.3) recorded in benzene-d6 at 600 MHz. ............ 52 Figure 2.33 HMBC spectrum of alotaketal B (2.3) recorded in benzene-d6 at 600 MHz. ........... 53 Figure 2.34 ROESY spectrum of alotaketal B (2.3) recorded in benzene-d6 at 600 MHz. .......... 53 Figure 2.35 DEPT 135? of alotaketal B (2.3) recorded in benzene-d6 at 150 MHz. ................... 54 Figure 2.36 CD spectrum of alotaketal B (2.3) recorded in methanol. ....................................... 54 Figure 2.37 1H NMR spectrum of alotaketal D (2.4) recorded in benzene-d6 at 600 MHz. ........ 55 Figure 2.38 13C NMR spectrum of alotaketal D (2.4) recorded in benzene-d6 at 150 MHz. ....... 55 Figure 2.39 COSY spectrum of alotaketal D (2.4) recorded in benzene-d6 at 600 MHz. ............ 56 Figure 2.40 HSQC spectrum of alotaketal D (2.4) recorded in benzene-d6 at 600 MHz. ............ 56 Figure 2.41 HMBC spectrum of alotaketal D (2.4) recorded in benzene-d6 at 600 MHz. ........... 57 Figure 2.42 1H NMR spectrum of alotaketal E (2.5) recorded in benzene-d6 at 600 MHz. ......... 57 Figure 2.43 13C NMR spectrum of alotaketal E (2.5) recorded in benzene-d6 at 150 MHz. ........ 58 Figure 2.44 COSY spectrum of alotaketal E (2.5) recorded in benzene-d6 at 600 MHz. ............ 58 Figure 2.45 HSQC spectrum of alotaketal E (2.5) recorded in benzene-d6 at 600 MHz. ............ 59 Figure 2.46 HMBC spectrum of alotaketal E (2.5) recorded in benzene-d6 at 600 MHz. ........... 59 Figure 3.1 Regulation of protein expression by miRNA (adapted from Mack, G. S. Nature Biotechnology, 2007, 25, 631-638).26 ........................................................................................ 60 xvi  Figure 3.2 Numbered chemical structures of genkwanine M (3.1), genkwanine P (3.2), diacetyl genkwanine M (3.3), and genkwanine A (3.4).28a,b..................................................................... 61 Figure 3.3 1H NMR spectrum of genkwanine M (3.1) recorded in DMSO-d6 at 600 MHz. ....... 62 Figure 3.4 ROESY correlations of genkwanine M (3.1). ........................................................... 63 Figure 3.5 1H NMR spectrum of genkwanine P (3.2) recorded in DMSO-d6 at 600 MHz. ......... 64 Figure 3.6 Key COSY and HSQC correlations of the cinnamic ester of genkwanine P (3.2). .... 65 Figure 3.7 Key HMBC correlations of the cinnamic ester of genkwanine P (3.2). ..................... 67 Figure 3.8 ORTEP diagram of diacetyl genkwanine M (3.3).29 ................................................. 68 Figure 3.9 13C NMR spectrum of genkwanine M (3.1) recorded in DMSO-d6 at 150 MHz. ....... 78 Figure 3.10 ROESY spectrum of genkwanine M (3.1) recorded in DMSO-d6 at 600 MHz. ....... 78 Figure 3.11 13C NMR spectrum of genkwanine P (3.2) recorded in DMSO-d6 at 150 MHz. ...... 79 Figure 3.12 COSY spectrum of genkwanine P (3.2) recorded in DMSO-d6 at 600 MHz. .......... 79 Figure 3.13 HSQC spectrum of genkwanine P (3.2) recorded in DMSO-d6 at 600 MHz. .......... 80 Figure 3.14 HMBC spectrum of genkwanine P (3.2) recorded in DMSO-d6 at 600 MHz. ......... 80 Figure 3.15 ROESY spectrum of genkwanine P (3.2) recorded in DMSO-d6 at 600 MHz. ........ 81 Figure 3.16 1H NMR spectrum of diacetyl genkwanine M (3.3) recorded in DMSO-d6 at 600 MHz. ......................................................................................................................................... 81 Figure 3.17 13C NMR spectrum of diacetyl genkwanine M (3.3) recorded in DMSO-d6 at 150 MHz. ......................................................................................................................................... 82 Figure 4.1 Chemical structures of virantmycin (4.1)33 and homoaerothionin (4.2).34a,b .............. 83 Figure 4.2 1H NMR spectrum of virantmycin (4.1) recorded in CDCl3 at 600 MHz. ................. 85 Figure 4.3 1H NMR spectrum of homoaerothionin (4.2) recorded in acetone-d6 at 600 MHz. ... 86 Figure 4.4 13C NMR spectrum of virantmycin (4.1) recorded in CDCl3 at 150 MHz. ................ 90 xvii  Figure 4.5 COSY spectrum of virantmycin (4.1) recorded in CDCl3 at 600 MHz. ..................... 90 Figure 4.6 HSQC spectrum of virantmycin (4.1) recorded in CDCl3 at 600 MHz. ..................... 91 Figure 4.7 HMBC spectrum of virantmycin (4.1) recorded in CDCl3 at 600 MHz..................... 91 Figure 4.8 13C NMR spectrum of homoaerothionin (4.2) recorded in acetone-d6 at 150 MHz. ... 92 Figure 5.1 Mechanism of autophagolysosome formation. ......................................................... 93 Figure 5.2 Chemical structures of clionamines A (5.1), B (5.2), C (5.3), and D (5.4).40 ............. 94 Figure 5.3 Natural clionamine A (5.1) clears Mtb from infected THP-1 at 5 ?M (IC50 ? 3 ?M). ................................................................................................................................................. 96 Figure 5.4 Autophagy stimulation by natural clionamine A (5.1) and synthetic clionamine B (5.2). MCF-7 cells expressing the autophagy marker EGFP-LC3 were incubated for 4 h with 5.1 or 5.2, and autophagosomes (green puncta) were measured. Cell nuclei are in blue (adapted from: Forestieri, R.; Donohue, E.; Balgi, A.; Roberge, M.; Andersen, R. J. Org. Lett., 2013, 15, 3918-3921).............................................................................................................................. 109 Figure 5.5 Effect of 3,5-epi-clionamine B (5.25) and compounds 5.12, 5.18, 5.23, 5.24, and 5.29, on Mtb proliferation and THP-1 viability. ....................................................................... 111 Figure 5.6 Minimum inhibitory concentrations (MICs) of 3,5-epi-clionamine B (5.25) and compounds 5.12, 5.18, 5.23, 5.24, and 5.29. ............................................................................ 112 Figure 5.7 SAR summary of clionamine analogues for autophagy activation. ......................... 113 Figure 5.8 Autophagy stimulation by natural clionamine A (5.1), 3,5-epi-clionamine B (5.25), and compounds 5.12, 5.18, 5.23, 5.24, and 5.29. ..................................................................... 114 Figure 5.9 1H NMR spectrum of compound 5.7 recorded in DMSO-d6 at 600 MHz. ............... 142 Figure 5.10 13C NMR spectrum of compound 5.7 recorded in DMSO-d6 at 150 MHz. ............ 142 Figure 5.11 1H NMR spectrum of compound 5.8 recorded in DMSO-d6 at 600 MHz. ............. 143 xviii  Figure 5.12 13C NMR spectrum of compound 5.8 recorded in DMSO-d6 at 150 MHz. ............ 143 Figure 5.13 1H NMR spectrum of compound 5.10 recorded in DMSO-d6 at 600 MHz. ........... 144 Figure 5.14 13C NMR spectrum of compound 5.10 recorded in DMSO-d6 at 150 MHz. .......... 144 Figure 5.15 1H NMR spectrum of compound 5.11 recorded in DMSO-d6 at 600 MHz. ........... 145 Figure 5.16 13C NMR spectrum of compound 5.11 recorded in DMSO-d6 at 150 MHz. .......... 145 Figure 5.17 1H NMR spectrum of compound 5.12 recorded in DMSO-d6 at 600 MHz. ........... 146 Figure 5.18 13C NMR spectrum of compound 5.12 recorded in DMSO-d6 at 150 MHz. .......... 146 Figure 5.19 1H NMR spectrum of compound 5.13 recorded in DMSO-d6 at 600 MHz. ........... 147 Figure 5.20 13C NMR spectrum of compound 5.13 recorded in DMSO-d6 at 150 MHz. .......... 147 Figure 5.21 1H NMR spectrum of compound 5.16 recorded in DMSO-d6 at 600 MHz. ........... 148 Figure 5.22 13C NMR spectrum of compound 5.16 recorded in DMSO-d6 at 150 MHz. .......... 148 Figure 5.23 ROESY spectrum of compound 5.16 recorded in DMSO-d6 at 600 MHz. ............ 149 Figure 5.24 1H NMR spectrum of compound 5.17 recorded in DMSO-d6 at 600 MHz. ........... 150 Figure 5.25 13C NMR spectrum of compound 5.17 recorded in DMSO-d6 at 150 MHz. .......... 150 Figure 5.26 ROESY spectrum of compound 5.17 recorded in DMSO-d6 at 600 MHz. ............ 151 Figure 5.27 1H NMR spectrum of compound 5.18 recorded in DMSO-d6 at 600 MHz. ........... 152 Figure 5.28 13C NMR spectrum of compound 5.18 recorded in DMSO-d6 at 150 MHz. .......... 152 Figure 5.29 1H NMR spectrum of compound 5.14 recorded in DMSO-d6 at 600 MHz. ........... 153 Figure 5.30 13C NMR spectrum of compound 5.14 recorded in DMSO-d6 at 150 MHz. .......... 153 Figure 5.31 1H NMR spectrum of compound 5.19 recorded in DMSO-d6 at 600 MHz. ........... 154 Figure 5.32 13C NMR spectrum of compound 5.19 recorded in DMSO-d6 at 150 MHz. .......... 154 Figure 5.33 1H NMR spectrum of compound 5.21 recorded in DMSO-d6 at 600 MHz. ........... 155 Figure 5.34 13C NMR spectrum of compound 5.21 recorded in DMSO-d6 at 150 MHz. .......... 155 xix  Figure 5.35 1H NMR spectrum of compound 5.22 recorded in benzene-d6 at 600 MHz. ......... 156 Figure 5.36 13C NMR spectrum of compound 5.22 recorded in benzene-d6 at 150 MHz. ......... 156 Figure 5.37 1H NMR spectrum of compound 5.23 recorded in DMSO-d6 at 600 MHz. ........... 157 Figure 5.38 13C NMR spectrum of compound 5.23 recorded in DMSO-d6 at 150 MHz. .......... 157 Figure 5.39 1H NMR spectrum of compound 5.24 recorded in DMSO-d6 at 600 MHz. ........... 158 Figure 5.40 13C NMR spectrum of compound 5.24 recorded in DMSO-d6 at 150 MHz. .......... 158 Figure 5.41 ROESY spectrum of compound 5.24 recorded in DMSO-d6 at 600 MHz. ............ 159 Figure 5.42 1H NMR spectrum of compound 5.25 recorded in DMSO-d6 at 600 MHz. ........... 160 Figure 5.43 13C NMR spectrum of compound 5.25 recorded in DMSO-d6 at 150 MHz. .......... 160 Figure 5.44 1H NMR spectrum of compound 5.25 recorded in MeOD at 600 MHz. ................ 161 Figure 5.45 13C NMR spectrum of compound 5.25 recorded in MeOD at 150 MHz. ............... 161 Figure 5.46 1H NMR spectrum of compound 5.27 recorded in DMSO-d6 at 600 MHz. ........... 162 Figure 5.47 13C NMR spectrum of compound 5.27 recorded in DMSO-d6 at 150 MHz. .......... 162 Figure 5.48 1H NMR spectrum of compound 5.28 recorded in DMSO-d6 at 600 MHz. ........... 163 Figure 5.49 13C NMR spectrum of compound 5.28 recorded in DMSO-d6 at 150 MHz. .......... 163 Figure 5.50 ROESY spectrum of compound 5.28 recorded in DMSO-d6 at 600 MHz. ............ 164 Figure 5.51 1H NMR spectrum of compound 5.29 recorded in DMSO-d6 at 600 MHz. ........... 165 Figure 5.52 13C NMR spectrum of compound 5.29 recorded in DMSO-d6 at 150 MHz. .......... 165 Figure 5.53 1H NMR spectrum of compound 5.31 recorded in DMSO-d6 at 600 MHz. ........... 166 Figure 5.54 13C NMR spectrum of compound 5.31 recorded in DMSO-d6 at 150 MHz. .......... 166 Figure 5.55 1H NMR spectrum of compound 5.33 recorded in DMSO-d6 at 600 MHz. ........... 167 Figure 5.56 13C NMR spectrum of compound 5.33 recorded in DMSO-d6 at 150 MHz. .......... 167 Figure 5.57 1H NMR spectrum of compound 5.34 recorded in DMSO-d6 at 600 MHz. ........... 168 xx  Figure 5.58 13C NMR spectrum of compound 5.34 recorded in DMSO-d6 at 150 MHz. .......... 168 Figure 5.59 1H NMR spectrum of compound 5.35 recorded in CD2Cl2 at 600 MHz. ............... 169 Figure 5.60 13C NMR spectrum of compound 5.35 recorded in CD2Cl2 at 150 MHz. .............. 169 Figure 5.61 1H NMR spectrum of compound 5.36 recorded in benzene-d6 at 600 MHz. ......... 170 Figure 5.62 13C NMR spectrum of compound 5.36 recorded in benzene-d6 at 150 MHz. ......... 170 Figure 5.63 1H NMR spectrum of compound 5.37 recorded in DMSO-d6 at 150 MHz. ........... 171 Figure 5.64 13C NMR spectrum of compound 5.37 recorded in DMSO-d6 at 150 MHz. .......... 171 Figure 5.65 ROESY spectrum of compound 5.37 recorded in DMSO-d6 at 600 MHz. ............ 172 Figure 5.66 1H NMR spectrum of compound 5.38 recorded in CD2Cl2 at 600 MHz. ............... 173 Figure 5.67 13C NMR spectrum of compound 5.38 recorded in CD2Cl2 at 150 MHz. .............. 173 Figure 5.68 1H NMR spectrum of compound 5.40 recorded in benzene-d6 at 600 MHz. ......... 174 Figure 5.69 13C NMR spectrum of compound 5.40 recorded in benzene-d6 at 150 MHz. ......... 174 Figure 5.70 1H NMR spectrum of compound 5.41 recorded in benzene-d6 at 600 MHz. ......... 175 Figure 5.71 13C NMR spectrum of compound 5.41 recorded in benzene-d6 at 150 MHz. ......... 175 Figure 5.72 1H NMR spectrum of compound 5.42 recorded in DMSO-d6 at 600 MHz. ........... 176 Figure 5.73 13C NMR spectrum of compound 5.42 recorded in DMSO-d6 at 150 MHz. .......... 176 Figure 5.74 ROESY spectrum of compound 5.42 recorded in DMSO-d6 at 600 MHz. ............ 177 Figure 5.75 1H NMR spectrum of synthetic clionamine B (5.2) recorded in MeOD at 600 MHz. ............................................................................................................................................... 178 Figure 5.76 13C NMR spectrum of synthetic clionamine B (5.2) recorded in MeOD at 150 MHz. ............................................................................................................................................... 178 Figure 5.77 1H NMR spectrum (enlarged images) of synthetic clionamine B (5.2) recorded in MeOD at 600 MHz. ................................................................................................................ 179 xxi  Figure 5.78 1H NMR spectrum of natural clionamine B (5.2) (bottom spectrum) recorded in MeOD at 600 MHz. (adapted from Keyzers, R. A.; Daoust, J.; Davies-Coleman, M. T.; Van Soest, R.; Balgi, A.; Donohue, E.; Roberge, M.; Andersen, R. J. Org. Lett. 2008, 10, 2959-2962).40 1H NMR spectrum of synthetic clionamine B (5.2) (top spectrum) recorded in MeOD at 600 MHz.49 ............................................................................................................................. 180 Figure 5.79 Minimum energy conformation of lactone 5.10 (ChemBio3D?). .......................... 181 Figure 6.1 mTORC1 and mTORC2 signaling pathways [adapted from Foster, K. G.; Fingar, D. C. Journal of Biological Chemistry, 2010, 285 (19), 14071-14077].66 ..................................... 183 Figure 6.2 Chemical structures of niclosamide (6.1), and niclosamide analogues 6.2-6.6. ....... 186 Figure 6.3 Chemical structures of nitazoxanide (6.7), tizoxanide (6.8), and nitazoxanide analogues 6.9-6.24. ................................................................................................................. 188 Figure 6.4 1H NMR spectrum of compound 6.2 recorded in DMSO-d6 at 600 MHz. ............... 203 Figure 6.5 13C NMR spectrum of compound 6.2 recorded in DMSO-d6 at 150 MHz. .............. 203 Figure 6.6 1H NMR spectrum of  compound 6.3 recorded in DMSO-d6 at 600 MHz. .............. 204 Figure 6.7 13C NMR spectrum of compound 6.3 recorded in DMSO-d6 at 150 MHz. .............. 204 Figure 6.8 1H NMR spectrum of  compound 6.4 recorded in DMSO-d6 at 600 MHz. .............. 205 Figure 6.9 13C NMR spectrum of compound 6.4 recorded in DMSO-d6 at 150 MHz. .............. 205 Figure 6.10 1H NMR spectrum of  compound 6.9 recorded in DMSO-d6 at 600 MHz. ............ 206 Figure 6.11 13C NMR spectrum of compound 6.9 recorded in DMSO-d6 at 150 MHz. ............ 206 Figure 6.12 1H NMR spectrum of  compound 6.10 recorded in DMSO-d6 at 600 MHz. .......... 207 Figure 6.13 13C NMR spectrum of compound 6.10 recorded in DMSO-d6 at 150 MHz. .......... 207 Figure 6.14 1H NMR spectrum of  compound 6.11 recorded in DMSO-d6 at 600 MHz. .......... 208 Figure 6.15 13C NMR spectrum of compound 6.11 recorded in DMSO-d6 at 150 MHz. .......... 208 xxii  Figure 6.16 1H NMR spectrum of  compound 6.12 recorded in DMSO-d6 at 600 MHz. .......... 209 Figure 6.17 13C NMR spectrum of  compound 6.12 recorded in DMSO-d6 at 150 MHz. ......... 209 Figure 6.18 1H NMR spectrum of  compound 6.13 recorded in DMSO-d6 at 600 MHz. .......... 210 Figure 6.19 13C NMR spectrum of  compound 6.13 recorded in DMSO-d6 at 150 MHz. ......... 210 Figure 6.20 1H NMR spectrum of  compound 6.14 recorded in DMSO-d6 at 600 MHz. .......... 211 Figure 6.21 13C NMR spectrum of  compound 6.14 recorded in DMSO-d6 at 150 MHz. ......... 211 Figure 6.22 1H NMR spectrum of  compound 6.15 recorded in DMSO-d6 at 600 MHz. .......... 212 Figure 6.23 13C NMR spectrum of  compound 6.15 recorded in DMSO-d6 at 150 MHz. ......... 212 Figure 6.24 1H NMR spectrum of  compound 6.16 recorded in DMSO-d6 at 600 MHz. .......... 213 Figure 6.25 13C NMR spectrum of  compound 6.16 recorded in DMSO-d6 at 150 MHz. ......... 213 Figure 6.26 1H NMR spectrum of  compound 6.17 recorded in DMSO-d6 at 600 MHz. .......... 214 Figure 6.27 13C NMR spectrum of  compound 6.17 recorded in DMSO-d6 at 150 MHz. ......... 214 Figure 6.28 1H NMR spectrum of  compound 6.18 recorded in DMSO-d6 at 600 MHz. .......... 215 Figure 6.29 13C NMR spectrum of  compound 6.18 recorded in DMSO-d6 at 150 MHz. ......... 215 Figure 6.30 1H NMR spectrum of  compound 6.19 recorded in DMSO-d6 at 600 MHz. .......... 216 Figure 6.31 13C NMR spectrum of  compound 6.19 recorded in DMSO-d6 at 150 MHz. ......... 216 Figure 6.32 1H NMR spectrum of  compound 6.20 recorded in DMSO-d6 at 600 MHz. .......... 217 Figure 6.33 13C NMR spectrum of  compound 6.20 recorded in DMSO-d6 at 150 MHz. ......... 217 Figure 6.34 1H NMR spectrum of  compound 6.21 recorded in DMSO-d6 at 600 MHz. .......... 218 Figure 6.35 13C NMR spectrum of  compound 6.21 recorded in DMSO-d6 at 150 MHz. ......... 218 Figure 6.36 1H NMR spectrum of  compound 6.22 recorded in DMSO-d6 at 600 MHz. .......... 219 Figure 6.37 13C NMR spectrum of  compound 6.22 recorded in DMSO-d6 at 150 MHz. ......... 219 Figure 6.38 1H NMR spectrum of compound 6.23 recorded in DMSO-d6 at 600 MHz. ........... 220 xxiii  Figure 6.39 13C NMR spectrum of  compound 6.23 recorded in DMSO-d6 at 150 MHz. ......... 220 Figure 6.40 1H NMR spectrum of  compound 6.24 recorded in DMSO-d6 at 600 MHz. .......... 221 Figure 6.41 13C NMR spectrum of  compound 6.24 recorded in DMSO-d6 at 150 MHz. ......... 221 Figure 7.1 Chemical structures of alotaketal A (2.1), genkwanine M (3.1), virantmycin (4.1), and homoaerothionin (4.2). ............................................................................................................ 223 Figure 7.2 Total synthesis of clionamine B (5.2) from the steroidal sapogenin tigogenin (5.30). ............................................................................................................................................... 224 Figure 7.3 Synthesis of new analogues and chemical probes of natural clionamines. .............. 226 xxiv  List of Schemes  Scheme 5.1 Retrosynthetic analysis for the total synthesis of clionamine B (5.2) and 3,5-epi-clionamine B (5.25). .................................................................................................................. 98 Scheme 5.2 Degradation of sarsasapogenin (5.5) side chain and synthesis of compound 5.12. 100 Scheme 5.3 Synthesis of compound 5.18 and ?-hydroxylation of lactone 5.13. ....................... 102 Scheme 5.4 Final steps in the synthesis of 3,5-epi-clionamine B (5.25). .................................. 104 Scheme 5.5 Synthesis of compound 5.29. ............................................................................... 105 Scheme 5.6 Degradation of tigogenin (5.30) side chain and ?-hydroxylation of 5.36.49 ........... 107 Scheme 5.7 Final steps in the synthesis of clionamine B (5.2). ................................................ 108   xxv  List of Abbreviations  ?C    degrees Celsius 13C NMR spectrum [1H]-decoupled 13C NMR spectrum 1D NMR one-dimensional nuclear magnetic resonance 2D NMR two-dimensional nuclear magnetic resonance ? angstrom Ac acetyl group acetone-d6 deuterated acetone AcOH acetic acid Atg autophagy-related  benzene-d6 or C6D6 deuterated benzene n-BuOH or n-butanol 1-butanol  calcd calculated CD circular dichroism  chloroform-d1 or CDCl3 deuterated chloroform  COSY  homonuclear correlated spectroscopy mCPBA meta-chloroperoxybenzoic acid CRE tyrosine recombinase enzyme ? chemical shift in parts per million DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DCE 1,2-dichloroethane xxvi  DCM or CH2Cl2  dichloromethane DEPT distortionless enhancement by polarization transfer DIPEA N,N-Diisopropylethylamine DMAP 4-dimethylaminopyridine DME 1,2-dimethoxyethane DMF  N,N-dimethylformamide DMP Dess-Martin periodinane DMSO  dimethyl sulfoxide DMSO-d6  deuterated dimethyl sulfoxide DNA deoxyribonucleic acid EC50  half maximal effective concentration equiv.  equivalent(s) ESIMS  electrospray ionization mass spectrometry Et  ethyl Et2O  diethyl ether EtOAc  ethyl acetate g  gram(s) GFP green fluorescent protein GFP-LC3  autophagy fusion gene in expression plasmid GIP glucose-dependent insulinotropic polypeptide  GLP-1 glucagon-like pepetide-1  h  hour(s) xxvii  HCV infection hepatitis C infection HEK cells human embryonic kidney 293 cells Hex hexane HMBC  heteronuclear multiple bond coherence HPLC  high-performance liquid chromatography HRESIMS  high resolution electrospray mass spectrometry HSQC  heteronuclear single quantum coherence HTS high-throughput screening Hz  hertz IC50  half maximal inhibitory concentration J  coupling constant in hertz K  degrees Kelvin lab laboratory LC-MS liquid chromatography?mass spectrometry LDA lithium diisopropylamide LRESIMS low resolution electrospray mass spectrometry M  molar concentration MCF-7 cells human breast adenocarcinoma cancer cells (Michigan Cancer Foundation) MDR-TB multidrug resistant TB Me  methyl MeCN or CH3CN or Acn acetonitrile xxviii  MeOH  methanol methanol-d4 or MeOD deuterated methanol MesCl mesyl chloride  methylene chloride-d2 or CD2Cl2 deuterated methylene chloride mRNA messenger RNA  miRNAs microRNAs  mg  milligram(s) MHz  megahertz MICs minimum inhibitory concentrations min  minute mL  millilitre(s) mm  millimetre(s) mmol  millimol(s) Mtb Mycobacterium tuberculosis  mTORC1 mammalian target of rapamycin complex 1  ?m  micromolar nM  nanomolar NMR  nuclear magnetic resonance NOE  nuclear Overhauser effect NOESY nuclear Overhauser effect spectroscopy ORTEP  Oak Ridge thermal ellipsoid plot PAC pancreatic adenocarcinoma  xxix  PCC pyridinium chlorochromate  pH  -log [H+] Ph  phenyl ROESY rotating frame nuclear Overhauser effect spectroscopy rt  room temperature RNA ribonucleic acid RNAi RNA interference  SAR  structure-activity relationship SCUBA self contained underwater breathing apparatus sp.  species TB tuberculosis  TDDFT time-dependent density functional theory  TEA triethylamine  TEP or P(OEt)3 triethyl phosphite TFA  trifluoroacetic acid THF  tetrahydrofuran THP-1 cells human acute monocytic leukemia cells p-TsOH p-toluenesulfonic acid  TLC  thin-layer chromatography TMSCl trimethylsilyl chloride TMSOTf trimethylsilyl trifluoromethanesulfonate UV ultraviolet  xxx  Acknowledgements  I would like to thank everybody that I have met in the Chemistry Department and in the Andersen?s group during my time in the PhD program at UBC. All my research and TA colleagues and all the chemistry professors for teaching the following courses: CHEM 568A, CHEM 563, CHEM 573, CHEM 566, CHEM 561, CHEM 540B, and EOSC 595A. All my research collaborators for being always available to use their expertise to test my samples in the best possible way. Finally, and most importantly, I need to thank my research supervisor Prof. Raymond J. Andersen for teaching me the isolation and structure elucidation of natural products, and Prof. Michel Roberge for being interested in the biological activity of my molecules. It has been a privilege for me to match my chemistry work with your well established research collaboration.    xxxi  Dedication  Dedicated to my parents, Anna and Ciro, for their long-term support from a long-distance.  1  Chapter 1: Isolation, Structure Elucidation, and Total Synthesis of Bioactive Natural Products  1.1 Bioactive Natural Products Marine invertebrates, such as sponges and tunicates, and their associated microorganisms are a source of new bioactive natural products with potential pharmacological applications. Ecteinascidin (ET)-743 (1.2),1a-d isolated from the Caribbean tunicate Ecteinascidia turbinate, is one of the most famous examples of a marine natural product inspired anticancer drug (Figure 1.1). Eribulin mesylate (1.3) (E7389), a synthetic macrocyclic analogue of the marine natural product halichondrin B, is a microtubule dynamics inhibitor with potent anticancer effects and it was recently approved for the treatment of metastatic breast cancer (Figure 1.2).1e,f Ziconotide, derived from the venom of Conus magus (a cone shell), is potent non-opioid analgesic agent used to treat severe and chronic pain.1g,h The isolation and structure elucidation of new bioactive compounds from natural sources is a pivotal step in drug discovery.     Figure 1.1 Total synthesis of ecteinascidin (ET)-743 (1.2) from cyanosafracin B (1.1).1a-d 2  Due to ongoing advances in the fields of Nuclear Magnetic Resonance (NMR) and x-ray crystallography,2a-c and to new improvements in chromatographic techniques, today we are able to isolate pure molecules on a microgram scale and elucidate their chemical structures with a high degree of certainty. Scientific SCUBA diving for the collection of marine invertebrate  samples and growth in culture of their associated microorganisms are the two preliminary steps for the preparation of a natural product extract library. The field of biology is directing its effort to the design of new and more selective bioassays in order to identify new natural products that can interact with specific cellular targets and selectively modulate biological pathways.3a,b High-throughput screening (HTS) allows rapid evaluation of a large number of chemical entities in a variety of cell-based and pure molecular target biological assays. The identification of new bioactive molecules from complex natural product mixtures is accomplished with a bioassay-guided fractionation approach.4 The total synthesis of bioactive natural products is another pivotal step in drug development.5a,b Total synthesis of natural products allows the production of a sufficient amount of material for further biological evaluation in vitro and in vivo, providing information about the structure-activity relationship and the pharmacophore. The synthesis of chemical probes for the identification of the molecular target is the ultimate and most challenging goal of chemical biology research. Click chemistry6a,b is a highly efficient and widely used chemical reaction for the identification of small-molecule protein targets.7a-e Cell biology signaling pathways are currently the object of multidisciplinary studies that involve collaborations between scientists of different research areas, including organic chemistry. The biological functions of small molecules are relevant to the pathogenesis of diseases. Chemical biology and drug discovery researchers are directing their attention to the 3  understanding of the molecular basis of these biological mechanisms and their potential applications for the cure of diseases. In this thesis we report chemical biology projects that involve the isolation, structure elucidation, and total synthesis of bioactive natural products.   1.2 Isolation and Structure Elucidation of Bioactive Natural Products  Due to the high biodiversity of life in the sea, marine organisms represent a potential source of new complex natural products with interesting bioactivities. Improvements in exploration technologies, such as scientific SCUBA diving and sediment collection, have allowed natural product chemists to explore the marine environment, leading to the identification of biologically active and chemically interesting marine natural products.8a-c In particular, the objects of natural product chemistry research are marine invertebrates, such as sponges and tunicates, and their associated microorganisms. The interactions between marine invertebrates and microorganisms, such as bacteria and fungi, facilitate the production of unique chemical structures that can be useful as tools for chemical biology studies.    Figure 1.2 Chemical structures of E7389 (1.3)1e,f and nahuoic acid (1.4).8a  4  Nahuoic acid A (1.4) (Figure 1.2), triterpene monoglycoside (1.5), and anthracimycin (1.6) (Figure 1.3), are three examples of recently isolated natural products with potential pharmacological applications.8a-c Nahuoic acid A (1.4), isolated by Andersen and coworkers in 2013,8a is a highly hydroxylated polyketide that is produced in culture by a Streptomyces sp. obtained from a marine sediment collected near the passage Padana Nahua in Papua New Guinea. Nahuoic acid A (1.4) is a selective SAM-competitive SETD8 inhibitor in vitro. Nahuoic acid A (1.4) is the first natural product known to inhibit SETD8 and the first selective SETD8 inhibitor known to be a competitive inhibitor of SAM-binding. Nahuoic acid A (1.4) has the potential to be a useful tool to act as a probe for defining a selective SAM-competitive binding site on SETD8 that can guide the design of more potent synthetic inhibitors for therapeutic or cell biology research applications.    Figure 1.3 Chemical structures of triterpene monoglycoside (1.5)8b and anthracimycin (1.6).8c  Triterpene monoglycoside (1.5),8b isolated from the black cohosh plant (Actaea racemosa) in 2012, was found to have an IC50 of 100 nM for selectively reducing the production of amyloidogenic A?42 while having a much smaller effect on the production of A?40 (IC50 = 6.3 5  ?M) in cultured cells overexpressing APP. Since the modulation of amyloid precursor protein (APP) and the consequent production of a lowered ratio of amyloid-beta peptide (A?42) relative to A?40 are potentially useful for the treatment for Alzheimer?s disease, triterpene monoglycoside (1.5) represents a new lead for the development of potential treatments for Alzheimer?s disease via modulation of gamma-secretase. Anthracimycin (1.6)8c is a potent antibiotic against Bacillus anthracis, showing a minimum inhibitory concentration (MIC) of 0.031 ?g mL-1. It was isolated by Fenical and coworkers in 2013 from cultures of a Streptomyces species collected from near-shore marine sediments found near Santa Barbara, CA. Anthracimycin (1.6) is a structurally unique compound and a potent antibacterial metabolite with potential for the treatment of Gram-positive pathogens such as Bacillus anthracis and methicillin-resistant Staphylococcus aureus (MRSA). All of the above examples of bioactive natural products with potential pharmacological applications support the hypothesis that the isolation and structure elucidation of new bioactive molecules from natural sources is a pivotal step in drug discovery.   1.3 Total Synthesis of Bioactive Natural Products Synthetic chemists are directing their research efforts to the total synthesis of natural products with useful biological properties aiming for practical syntheses that are able to produce sufficient amounts of material for further biological evaluation in vitro and in vivo. Structure-activity relationship (SAR) studies via biological evaluation of new synthetic analogues and molecular target identification via Click chemistry6a,b are two important goals of chemical biology research. The ?Click reaction? is a copper(I)-catalyzed azide-alkyne cycloaddition that selectively gives 1,2,3-triazoles.6b This reaction is a more efficient version of the thermal 1,3-6  dipolar cycloaddition which was first developed by Huisgen.6a In 2001, Sharpless fully described the copper(I)-catalyzed azide-alkyne cycloaddition and he named it as ?Click reaction? for its high efficiency and wide scope. The use of Click chemistry allows the identification of small-molecules protein targets7a-e without the need for chromatography or recrystallization.  The total synthesis of natural products is also inspired by the complexity of the chemical structures requiring the design of elegant and efficient synthetic routes and the discovery of new chemical reactions. The total synthesis of alotaketal A (2.1) (Chapter 2) and viridicatumtoxin B (1.7) are two recent examples of total synthesis of complex natural products (Figure 1.4 and Figure 1.5, respectively).    Figure 1.4 Total synthesis of alotaketal A (2.1).9a,b  In 2012, Yang9a,b and coworkers developed a convergent synthetic route to the potent cAMP signaling agonist alotaketal A (2.1). Alotaketal A (2.1), isolated by Andersen and coworkers and subsequently by Rho and coworkers in 2009, is a complex natural product with a new sesterterpene skeleton. The simultaneous substitution of the spiroketal center by both allyl and vinyl groups is unprecedented in a natural spiroketal. Yang?s total synthesis of alotaketal A (2.1) involves two SmI2-mediated reductive allylation reactions for assembling the polycycle and fragment coupling. Furthermore, two key steps are a Hg(OAc)2-mediated selective alkene 7  oxidation and the subtlety of the spiroketalization/isomerization of the unprecedented spiroketal ring system. The total synthesis of alotaketal A (2.1) and unnatural analogues provided extensive information about the structure-activity relationship for its cAMP signaling agonist activity.    Figure 1.5 Viridicatumtoxin B (1.7) (revised structure).10a,b  In 2013, Nicolaou  and coworkers synthesized the potent antibiotic viridicatumtoxin B (1.7)10a,b aiming to fully elucidate its chemical structure and establish the foundation for the synthesis and biological evaluation of designed analogues within this family of antibiotics. Using a highly convergent strategy, they obtained racemic viridicatumtoxin B (1.7) from four easily accessible building blocks. Based on this synthetic work, the previously proposed chemical structure of viridicatumtoxin B (1.7) turned out to be wrong, and the originally assigned epoxy hemiacetal structure of viridicatumtoxin B (1.7) was revised to its hydroxy ketone form. Their current research efforts aim to achieve an enantioselective total synthesis of viridicatumtoxin B (1.7) and confirm its absolute configuration. The chemistry developed in this total synthetic work allows the synthesis and the biological evaluation of new analogues of the viridicatumtoxins that can be potential leads for drug discovery to combat bacterial infections. The total synthesis of alotaketal A (2.1) and viridicatumtoxin B (1.7) are two examples of 8  total syntheses that facilitate the pharmacological evaluation of natural products, supporting the hypothesis that the total synthesis of new bioactive natural products is a pivotal step in drug discovery.  1.4 Structure Elucidation of Natural Products via Analysis of 1D and 2D NMR Spectroscopic Data 1D NMR experiments (1H and 13C) and 2D NMR experiments (COSY, HSQC, HMBC, NOESY, ROESY) are widely used in organic chemistry.2c The analysis of NMR spectroscopic data allows synthetic and natural product chemists to elucidate the chemical structure of complex organic molecules. Advances in high-resolution NMR technologies, such as multidimensional pulse methods and sensitivity improvements, have enhanced the use of NMR experiments in the areas of organic synthesis and natural products isolation. Furthermore, stronger magnetic fields provided by cryogenic probe electronics have dramatically lowered the amount of material needed for organic structural determinations, allowing the execution of synthetic routes on small scale and the identification of minor components from complex natural products mixtures.  Homonuclear correlated spectroscopy (COSY) is a 2D NMR experiment that provides correlations (cross peaks) between proton resonances that are coupled to each other throughout two or three covalent bonds, establishing the connectivity among different groups in the molecule. Heteronuclear single quantum coherence (HSQC) is another widely used 2D NMR experiment that correlates each proton resonance to its attached heteroatom resonance, providing cross peaks between protons and heteroatoms (13C, 15N) that are directly bound via a covalent bond. Heteronuclear multiple bond coherence (HMBC) is a particularly useful 2D NMR experiment that detects long range correlations (throughout two or three covalent bonds, 9  typically) between proton resonances and heteroatom resonances (13C, 15N), identifying the carbon-carbon bonds in the molecule. Nuclear Overhauser effect spectroscopy (NOESY) and rotating frame nuclear Overhauser effect spectroscopy (ROESY) are 2D NMR experiments that correlate resonances relative to protons that are close in space (dipolar interaction), indicating the relative configuration of molecules.  The work reported in this thesis is largely based on the analysis of 1D and 2D NMR spectroscopic data.   1.5 Research Summary The research projects reported herein have focused on the isolation, structure elucidation, and total synthesis of bioactive natural products. In our efforts to isolate new bioactive natural products, we have identified alotaketal A (2.1) and analogues, a new class of potent cAMP signaling agonists isolated from the marine sponge Hamigera sp. To the best of our knowledge, the new alotane sesterterpene alotaketal A (2.1) and the labdane diterpene forskolin (2.2) are the only naturally-occurring small molecules that can strongly and selectively activate cAMP signaling pathway. In our interdisciplinary research project involving the study of miRNA functions, we have isolated genkwanines M (3.1) and P (3.2) from the Indonesian plant Wikstroemia polyantha. Genkwanines M (3.1) and P (3.2) are two known daphnane diterpenoids that modulate miRNA activity. Genkwanines M (3.1) induces an early inflammatory response and can moderately inhibit miR-122 activity in the Huh-7 liver cell line. Genkwanines are potential chemical tools for the study of miRNA regulation and signaling in cellular diseases. We have completed the first total synthesis of the new aminosteroid clionamine B (5.2), 10  previously isolated from the marine sponge Cliona celata. Synthetic clionamine B (5.2) strongly stimulates autophagy at 30 ?g/mL in human breast cancer MCF-7 cells and inhibits Mycobacterium tuberculosis (Mtb) proliferation in human macrophages via autophagy activation. Clionamine B (5.2) and unnatural analogues are potent anti-Mtb compounds and have potential application for the cure of multidrug resistant-tuberculosis (MDR-TB). We have synthesized five niclosamide analogues 6.2-6.6 and sixteen nitazoxanide analogues 6.9-6.24 for structure-activity studies. Niclosamide (6.1) and nitazoxanide (6.7) are two new autophagy stimulators/mTORC1 signaling inhibitors with potential applications against tuberculosis infection. The goals of the chemical biology studies described in this thesis were the isolation and structure elucidation of bioactive molecules from natural sources via a bioassay-guided fractionation approach. Total synthesis of natural products provided more material for further biological investigation and for structure-activity analysis. We have used an interdisciplinary stepwise process that can connect the research activities conducted in the fields of organic chemistry and molecular biology. Such a scientific process can potentially lead to the identification of new drug candidates for the cure of diseases.  11  Chapter 2: Alotaketals A, B, D, E, New Sesterterpenoids from the Marine Sponge Hamigera Sp. that activate the cAMP Cell Signaling Pathway  2.1 Introduction Diabetes is a debilitating disorder that affects more than 200 million people worldwide.11 Because of the growing incidence and the costs associated with diabetes, new therapeutic approaches are strongly required to fight the disease.     Figure 2.1 Bioassay signaling pathway [adapted from Kieffer, T. J. and Habener, J. F. Endocrine Reviews, 1999, 20 (6), 876-913].14  Type 2 diabetes is characterized by insufficient stimulation of insulin secretion by incretin hormones. At present, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like pepetide-1 (GLP-1) are the two incretin hormones that have been characterized. Intravenous 12  administration of GLP-1 seems to have beneficial effects in individuals who are affected with type 2 diabetes and obesity.12a-d However, anti-diabetic therapy with GLP-1 would require daily intravenous administrations of the incretin hormone (GLP-1 can only be administrated directly into the bloodstream), negatively affecting the patients quality of life. Therefore, the identification of new small molecules that can stimulate the GLP-1 receptor is a central interest in anti-diabetic drug discovery.   Figure 2.2 Chemical structures of alotaketal A (2.1)15 and forskolin (2.2).24  In order to discover new small molecules that can stimulate the GLP-1 receptor and increase blood insulin level,13 a large number of marine sponge extracts have been screened in a cell-based luciferase reporter assay for the activation of the GLP-1 receptor (bioassay mechanism is shown in Figure 2.1).14 Agonists binding to the GLP-1 receptor turn on the cAMP pathway by stimulating adenylyl cyclase activity. Activation of cAMP signaling leads to cytoplasmic responses by protein kinases, resulting in CRE-dependant gene transcription of the luciferase reporter. Bioluminescence reaction of luciferin (previously injected in the cells) and molecular oxygen is then catalyzed by the luciferase generating light emission. In this bioassay, designed to 13  monitor the activity of cAMP signaling pathway, the relative light output is proportional to the stimulation of the cAMP pathway and to the biological activity of the GLP-1 agonist.  2.2 Results and Discussion       Catherine Merchant in the Kieffer lab screened our library of marine sponge extracts using both HEK-GLP-1 cells (GLP-1 Receptor-expressing Human Embryonic Kidney 293 cells) and HEK-GLP-1-knockout cells (GLP-1 Receptor-knockout HEK 293 cells) to identify new selective GLP-1 agonists with potential applications for the study of cAMP signaling and the treatment of diabetes. The use of two different HEK cell lines, one with GLP-1 receptors, another without GLP-1 receptors, was intended to distinguish between selective stimulation of GLP-1 receptors and activation of cAMP signaling by the marine sponge extracts.    Figure 2.3 1H NMR spectrum of alotaketal A (2.1) recorded in benzene-d6 at 600 MHz.  14  The crude extract RJA 03-398 (from Raymond J. Andersen?s library of marine sponge extracts) from the marine sponge Hamigera sp. collected in Papua New Guinea showed promising activity in the screening assay and it was therefore subjected to assay-guided fractionation leading to the isolation of four sesterterpenoids, alotaketals A (2.1), B (2.3), D (2.4), and E (2.5) (Figure 2.2 and Figure 2.5). Positive modulation of the cAMP signaling pathway by the alotaketals was confirmed by testing the pure compounds, with alotaketal A (2.1) showing the strongest activity in the assay.15 However, pure alotaketal A (2.1) (likewise the crude extract RJA 03-398 after it was retested) activated the cAMP signaling pathway equally in HEK-GLP-1 and HEK-GLP-1-knockout cells, indicating that the alotaketals are cAMP signaling pathway stimulators with no activity on the GLP-1 receptors. Therefore, the alotaketals aren?t GLP-1 agonists but strongly stimulate cAMP signaling via interaction with an unidentified molecular target(s).      Figure 2.4 13C NMR spectrum of alotaketal A (2.1) recorded in benzene-d6 at 150 MHz. 15  2.2.1 Isolation and Structure Elucidation of Alotaketals A, B, D, E The crude extract RJA 03-398 was first fractionated on a Sephadex? LH-20 column with 100% methanol as the eluent to afford three major fractions. Following a bioassay-guided fractionation approach, these three fractions were tested by Merchant in the Kieffer lab at UBC. The bioactive metabolites were present in the middle fraction. Reversed-phase HPLC was used as the next step for the isolation of pure compounds. Using a mixture of acetonitrile/water (8/2) as an isocratic eluent, alotaketal A (2.1) (5.3 mg), alotaketal B (2.3) (2.1 mg), alotaketal D (2.4) (1.1 mg), and alotaketal E (2.5) (1.0 mg), were isolated as pure white solids.    Figure 2.5 Chemical structures of alotaketals B (2.3), D (2.4), E (2.5), and alotane skeleton 2.6. 16  The first analysis of the proton spectrum of the major compound alotaketal A (2.1) recorded in methanol-d4 showed the presence of aliphatic methyls which are highly diagnostic for terpenoids. In order to investigate the presence of exchangeable protons and to achieve better resolution in the aliphatic zone, 1D and 2D NMR experiments were carried out on the major compound using benzene-d6 as a solvent [1H NMR and 13C NMR spectra of alotaketal A (2.1) are shown in Figure 2.3 and Figure 2.4, respectively]. The HRESIMS spectrum gave a [M + Na]+ ion at m/z 421.2346 (calcd for C25H34O4Na, 421.2355), which was appropriate for a molecular formula of C25H34O4, with nine sites of unsaturation. The characteristic NMR resonance at ? 0.53, along with the ?one mass unit shift? observed in the deuterium exchange LRESIMS (m/z at 422), indicated the presence of an alcohol functionality. Extensive analysis of 1D and 2D NMR spectroscopic data allowed the elucidation of the alotaketal A structure (2.1). Fragments A, B, and C of alotaketal A (2.1), were elucidated from COSY and HMBC data.15     Figure 2.6 Key HSQC correlations of fragment A.  17  A 1H NMR resonance at ? 6.32 (H-2) was correlated in the HSQC spectrum (Figure 2.6 and Figure 2.26) to a carbon resonance at ? 139.6 (C-2) and showed HMBC correlations (Figure 2.8) to carbon resonances at ? 139.0 (C-3) and ? 197.7 (C-4). A methyl resonance at ? 1.71 (C-21) showed HMBC correlations to the resonances at ? 139.6 (C-2), ? 197.7 (C-4), and ? 139.0 (C-3), as well as COSY correlations (Figure 2.7 and Figure 2.8) to the resonance at ? 6.32 (H-2). Based on this preliminary NMR analysis, an ?,?-unsaturated carbonyl with a methyl substituent (methyl C-21 attached to C-3) in the alpha position was identified as a part of fragment A (Figure 2.8).     Figure 2.7 Key COSY correlations of fragment A.  In the HMBC spectrum (Figure 2.13 and Figure 2.27) proton resonances assigned to H-5a (? 2.47) and H-5b (? 2.39) had strong correlations to the carbon resonance at ? 197.7 assigned to the carbonyl (C-4), and they were correlated to the same resonance at ? 38.6 (C-5) in the HSQC 18  spectrum (Figure 2.6 and Figure 2.26), indicating that C-5 was a methylene attached to the carbonyl C-4. Furthermore, the proton resonance at ? 2.06 (H-6) showed COSY correlations (Figure 2.7) to resonances at ? 4.36 (H-1), ? 2.47 and ? 2.39 (H-5a and H-5b), and HMBC correlations to carbon resonances at ? 63.6 (C-1) and ? 38.6 (C-5).     Figure 2.8 Fragment A elucidated from COSY and HMBC data.  Since the resonance at ? 4.36 assigned to H-1, which is attached to an sp3 carbon bearing an oxygen atom as indicated by its deshielded chemical shift, showed correlations to the resonance at ? 6.32 (assigned to H-2) in the COSY spectrum (Figure 2.7 and Figure 2.25) and correlations to the resonance at ? 139.6 (C-2) in the HMBC spectrum, it was possible to identify a 2-methyl-4-oxo-cyclohexenone ring in alotaketal A (2.1). Moreover, the proton resonance at ? 3.53 (H2-22) showed HMBC correlations to the carbon resonance at ? 125.1 (C-8), which was correlated to the olefinic proton resonance at ? 5.56 (H-8) in the HSQC spectrum, and the alcoholic proton resonance (? 0.53) showed HMBC correlations to the carbon resonance at ? 63.8 (C-22), indicating the presence of an allylic alcohol. Additionally, HMBC correlations from 19  the proton resonance at ? 3.53 (H2-22) to the carbon resonance at ? 34.0 (C-6), and from proton resonance at ? 2.06 (H-6) to the carbon resonance at ? 142.5 (C-7), indicated that the allylic alcohol was linked to the 2-methyl-4-oxo-cyclohexenone ring via a covalent bond between the quaternary carbon C-7 (? 142.5) and C-6 (? 34.0). Additional weak COSY correlations between both of the proton resonances at ? 3.53 (H2-22) and ? 5.56 (H-8) and the resonance at ? 2.06 (H-6) were consistent with this assignment.     Figure 2.9 Key HSQC and COSY correlations of fragment B.  The proton resonances at ? 5.56 (H-8) and ? 4.36 (H-1) showed strong HMBC correlations to the carbon resonance at ? 97.2 (C-9), indicating that an olefinic methine (C-8, ? 125.1) was directly attached to the quaternary carbon (C-9, ? 97.2) which was linked through an ether bond to C-1 (? 63.6) based on 2D NMR correlations (HMBC correlations are shown in Figure 2.13). Considering its chemical shift value at ? 97.2, C-9 was classified as a ketal carbon. Fragment A 20  (Figure 2.8) was therefore identified as the oxo-decalin ring (with a cis ring junction as indicated by ROESY correlations between H-1 and H-6 proton resonances, see Figure 2.14 later in this Chapter). The structure elucidation analysis continued with the identification of the two remaining fragments (B and C) of the alotaketal A (2.1) chemical structure. The two proton resonances at ? 4.87 and ? 4.89 (H2-23) were correlated to the same carbon resonance at ? 111.4 (C-23) in the HSQC spectrum, and showed COSY correlations to resonances of the two diastereotopic protons of both methylenes H2-10 (? 2.29/ ? 2.34) and H2-12 (? 2.20/ ? 2.31), identifying the presence of an exocyclic double bond (CH2-23) (Figure 2.9).   Figure 2.10 Fragment B elucidated from COSY and HMBC data.  Moreover, the H2-10 resonances (? 2.29/ ? 2.34) showed HMBC correlations to the resonance at ? 97.2 of the ketal carbon C-9. Additionally, COSY correlations of the methylene H2-12 resonances (? 2.20/ ? 2.31) to the resonance of H-13 (? 4.85), which is clearly attached to a carbon bearing an oxygen atom, and HMBC correlations of the H-13 resonance (? 4.85) to the 21  resonance of the ketal carbon C-9 (? 97.2), led the elucidation of fragment B (Figure 2.10) which is linked to fragment A via a covalent bond between C-10 and C-9, identifying the presence of a spiroketal in the chemical structure of alotaketal A (2.1) (Figure 2.13).  The structure elucidation continued with the identification of the third and last fragment C (Figure 2.12) of the alotaketal A (2.1) chemical structure. The proton resonance at ? 1.62 assigned to H3-25 is very characteristic for an olefinic methyl and it showed HMBC correlations to the carbon resonances of the olefinic methylene C-20 at ? 110.9 (H2-20 at ? 4.80/ ? 4.81) and the aliphatic methylene C-18 at ? 38.0 (H2-18 at ? 1.94).    Figure 2.11 Key COSY correlations of fragment C.  Furthermore, a proton resonance at ? 1.68, assigned to the olefinic methyl H3-24, showed HMBC correlations to the resonance of C-14 (? 126.8), directly attached to the olefinic proton H-14 (? 5.48), and to the resonance of the aliphatic methylene C-16 at ? 39.7 (H-16 at ? 1.98). 22  Additional COSY correlations (Figure 2.11) between the aliphatic methylene resonance H2-17 (? 1.55) and the two aliphatic methylenes resonances H2-16 (? 1.98) and H2-18 (? 1.94), as well as the quintet multiplet assigned to H2-17, identified the isoprenoid side chain as fragment C of alotaketal A (2.1) (Figure 2.12).  Fragment C is linked to fragment B via a covalent bond between carbon C-13 and C-14, as indicated by very diagnostic COSY and HMBC correlations. In particular, in the HMBC spectrum (Figure 2.13 and Figure 2.27) the H-12 resonance (? 2.20/ ? 2.31) correlates to the C-13 (? 68.5) and C-14 (? 126.8) resonances, the H-13 (? 4.85) resonance to the C-14 (? 126.8) and C-15 (? 139.2) resonances, and H-14 (? 5.48) resonance to C-13 (? 68.5) resonance.    Figure 2.12 Fragment C elucidated from COSY and HMBC data.  The observation of key HMBC correlations between the atoms of these three fragments (A, B, C) allowed the complete structure elucidation of the alotaketal A (2.1) carbon skeleton. Particularly important were the HMBC correlations (Figure 2.13) between the H-13 (? 4.85) and H-1 (? 4.36) resonances to the C-9 resonance (the ketal carbon at ? 97.2), and the H-14 resonance (? 5.48) to the C-12 resonance (? 40.7). 23  The structure elucidation continued with the assignment of the configuration of the ?14, 15 double bond, and the relative configuration of the oxo-decalin ring, spiroketal, and substituents of alotaketal A (2.1). 1D NOESY and 2D ROESY correlations were interpreted in order to describe the relative configuration of alotaketal A (2.1) (Figure 2.14 and Figure 2.28). 1D NOESY and 2D ROESY correlations between the olefinic proton resonance at ? 5.48 (H-14) and the aliphatic methylene resonance at ? 1.98 (H2-16) indicated that the ?14,15 double bond has an E configuration.    Figure 2.13 Key HMBC correlations between fragments A, B, and C of alotaketal A (2.1). 24  Additionally, the resonance assigned to H-1 (? 4.36) showed strong correlations to the H-6 resonance (? 2.06) in the ROESY spectrum, which was confirmed by the irradiation of both H-1 and H-6 in 1D NOESY experiments. Therefore the oxo-decalin fragment has a cis ring junction. 1D NOESY and ROESY correlations between the proton resonances assigned to H-1 (? 4.36) and H-13 (? 4.85) identified the relative configuration of the spiroketal carbon C-9 and C-13 (1S*/6R*/9S*/13S*), as shown in Figure 2.14.   Figure 2.14 Key ROESY correlations of alotaketal (2.1). 25  Fractionation of the crude extract of the sponge Hamigera sp. also led to the isolation of alotaketal B (2.3) (Figure 2.15), an analogue of the major compound alotaketal A (2.1). The HRESIMS spectrum of alotaketal B gave a [M + Na]+ ion at m/z 523.3024 (calcd for C30H44O6Na, 523.3036), which was appropriate for a molecular formula of C30H44O6, indicating a C5H10O2 unit of difference with alotaketal A (2.1). Analysis of 1D and 2D NMR spectroscopic data obtained for this new analogue, and comparison with the data obtained for alotaketal A (2.1), allowed the elucidation of the alotaketal B structure (2.3) (Figure 2.15).    Figure 2.15 Chemical structure of alotaketal B (2.3). COSY and HMBC correlations of the isovalerate fragment of alotaketal B (2.3) chemical structure.  In particular, the carbon spectrum indicated the presence of a resonance at ? 172.4 (C-26), a typical value for an ester carbonyl, and three other signals at ? 45.0 (C-27), ? 25.6 (C-28), and ? 23.1 (C-29/C-30), typical values for aliphatic carbons [13C NMR spectrum of alotaketal B (2.3) is shown in Figure 2.30]. Additionally, the proton spectrum of alotaketal B (2.3) (Figure 2.16) showed a resonance at ? 0.89, which was assigned to the two aliphatic methyls H3-29 and H3-30, 26  and another resonance at ? 2.12, assigned to the methine H-28, whose resonance was overlapped with the resonance assigned to the methylene H2-27. Since the methylene H2-27 resonance at ? 2.12 had a strong HMBC correlation with the carbonyl resonance at ? 172.4 (C-26), it was possible to elucidate the isovalerate unit as a fragment of alotaketal B (2.3) (Figure 2.15). In order to determine the linkage of the isovalerate in the alotaketal B (2.3) structure, the NMR spectroscopic data of alotaketal A (2.1) and B (2.3) were compared. The resonances at ? 4.87/ ? 4.89 assigned to the exocyclic olefinic methylene (H2-23) in alotaketal A (2.1) were not present in the alotaketal B (2.3) proton spectrum (Figure 2.16), which showed a new resonance at ? 1.48 (H3-23), a typical value for a deshielded aliphatic methyl. In the HMBC spectrum (Figure 2.33), the resonance of the methyl H3-23 was correlated with the resonance of the quaternary carbon C-11 (? 77.4), which is clearly attached to an oxygen atom. Therefore, the oxygen of the isovalerate unit is linked to carbon 11 in alotaketal B (2.3) (Figure 2.15).     Figure 2.16 1H NMR spectrum of alotaketal B (2.3) recorded in benzene-d6 at 600 MHz. 27  Alotaketal B (2.3) was used as a model compound to further investigate the relative configuration of  C-13. In order to confirm the relative configuration of C-13, [1H-1H] decoupling experiments were carried out on alotaketal B (2.3). A coupling constant of 10.1 Hz observed between H-13 (? 5.15) and H-12axial (? 1.95) indicates that they are antiperiplanar. Proton H-14 (? 5.45) was then selectively irradiated while the proton spectrum was acquired. While H-14 was irradiated, the multiplicity of H-13 collapsed from a triplet to a doublet, with a coupling constant value of 10.8 Hz, due to the coupling with the antiperiplanar H-12axial. Therefore, the isoprenoid side chain, attached to C-13, is in an equatorial orientation. Further conformational studies on alotaketal A (2.1) were carried out on the cyclohexenone ring using NMR coupling constants. The diasereotopic H-5a and H-5b resonances were well enough resolved in benzene-d6 to allow the measurement of their coupling constants. Analysis of the 1H NMR spectrum showed that H-5b is a double-doublet with coupling constants of 13.2 and 15.6 Hz, and H-5a is a double-doublet with coupling constants of 4.8 and 15.6 Hz. H-6 is a double-triplet with coupling constant values of 4.2 and 13.2 Hz. The data indicated that H-5b is antiperiplanar to H-6, with a coupling constant of 13.2 Hz, and geminal with H-5a with a coupling constant of 15.6 Hz. H-5a is geminal with H-5b with a coupling constant of 15.6 Hz, and is gauche to H-6 with a coupling constant of 4.8 Hz. H-6 is coupled to both H-1 and H-5a with a coupling constant of 4.8 Hz, and antiperiplanar with H-5b with a coupling constant of 13.2 Hz. Therefore its multiplicity is a double-triplet. ROESY and 1D NOESY correlations between H-5a and H-6, as well as W-coupling observed between H-5a and H-1 in the COSY spectrum, indicated the half-chair type conformation to be a reasonable proposal for the 2-methyl-4-oxo-cyclohexenone ring of alotaketal A (2.1) (Figure 2.17). Alotaketal D (2.4) and E (2.5) (Figure 2.5) were isolated from the crude extract of the 28  sponge Hamigera sp., using the same isolation procedure described above. The structures of alotaketals D (2.4) and E (2.5) were elucidated by comparing their MS and NMR spectroscopic data with the data for alotaketal A (2.1). The HRESIMS spectrum of alotaketal D (2.4) gave a [M + Na]+ ion at m/z 419.2204 (calcd for C25H32O4Na, 419.2198), which was appropriate for a molecular formula of C25H32O4Na, indicating two mass units less compared with alotaketal A (2.1). The chemical structure of alotaketal D (2.4) seemed to be very similar to the alotaketal A (2.1) structure, just possessing one more site of unsaturation. Analysis of the NMR spectroscopic data revealed that the structure of alotaketal D (2.4) contains an allyl aldehyde in place of the allylic alcohol of alotaketal A (2.1). Indeed, the carbon spectrum (Figure 2.38) indicated the presence of a resonance at ? 191.8 (C-22), a typical value for a conjugated aldehyde carbonyl, and the proton spectrum (Figure 2.37) showed a new resonance at ? 9.01, which is appropriate for an aldehyde proton, and no allylic alcohol resonance was present.    Figure 2.17 Proposed half-chair conformation for the 2-methyl-4-oxo-cyclohexenone ring of alotaketal A (2.1).  The HRESIMS spectrum of alotaketal E (2.5) gave a [M + Na]+ ion at m/z 453.2624 (calcd for C26H38O5Na, 453.2617), which was appropriate for a molecular formula of C26H38O5Na, 29  indicating an additional CH4O unit compared with alotaketal A (2.1). Analysis of the NMR spectroscopic data revealed that the structure of alotaketal E (2.5) lacks the enone system, which has been replaced with a ketone having a methyl ether in the beta position. The carbon spectrum (Figure 2.43) contained a resonance at ? 207.2 (C-4), a typical value for a saturated ketone carbonyl, and second carbon resonance at ? 58.7 (C-26), which is appropriate for a methyl ether. Furthermore, the proton spectrum (Figure 2.42) showed two new resonances at ? 3.59 (H1-2) and 2.98 (H3-26), typical values for the methine and methyl groups of a secondary methyl ether. The relative configuration of this new 2-methyl-3-methoxy-4-oxo-cyclohexanone A ring was studied via NOESY and ROESY correlations. However, the small amount of sample did not allow a complete elucidation of the relative configuration (not enough sample for 1D NOESY experiments), which was therefore based on the hypothesis that alotaketal E (2.5) can be considered as an artifact of alotaketal A (2.1). Thus, it was assumed that upon extracting the frozen sponge with methanol, alotaketal A (2.1) underwent Michael addition to the beta carbon of the enone by nucleophilic attack of methanol (pH values of the methanolic solution of the sponge extract can be different from neutral pH leading to the base/acid-catalyzed reactions) from the bottom face, which is the less hindered face in this case, followed by the protonation of the enolate from the top face. In this hypothesis the relative configuration of alotaketal E (2.5) is the one shown in Figure 2.5.  2.2.2 Absolute Configuaration of the Alotaketals: Analysis of CD Spectra versus Mosher Analysis   Alotaketal A (2.1) is an optically active compound with four stereocenters ([?]25D  = -38.9, c = 0.01, MeOH). In order to elucidate the absolute configuration of alotaketal A (2.1), 30  crystallization of the natural product was attempted, but without success. The absolute configuration was therefore proposed based on the interpretation of circular dichroism spectra (CD spectra), by analyzing the sign of the Cotton effect16a-d assigned to the enone functionality of the alotaketals. The two components of the circularly polarized light are absorbed unequally in an optically active medium which exhibits circular dichroism. A CD curve (an ellipse) is obtained by measuring the differential dichroic absorption ?? (?? = ?L ? ?R, ?L and ?R are the molecular extinction coefficients for the right- and left-handed circularly polarized light) or the molar elipticity [?] as a function of the wavelength. The magnitude of circular dichroism becomes relevant in the immediate vicinity of the UV (ultraviolet) absorption band.    Figure 2.18 Chemical structures of phorbaketal A (2.7), phorbaketal B (2.8),20a,b ansellone A (2.9), and desacetylansellone A (2.10).21a,b 31  The enone functionality possesses characteristic UV transitions,16e and in the presence of vicinal stereocenters, shows diagnostic bands17 characterized by Cotton effects18a-d (positive or negative). The Cotton effects in the CD spectrum of alotaketals A (2.1) and B (2.3) (Figure 2.19 and Figure 2.36) recorded in methanol were compared to the literature, using cyclohexenone as a model compound. Applying Snatze?s rule18a-d, the absolute configuration of alotaketal A (2.1) was proposed to be 1R,6S,9R,13R,14E. The n??* (~330 nm, Figure 2.19) transition showed a positive Cotton effect in the CD spectra of both alotaketals A (2.1) and B (2.3). The contribution to the sign of the Cotton effect is due to the intrinsic helicity of the chromophore and/or to the vicinal asymmetric centers (extrachromophoric perturbations).17-19 The half-chair type conformation proposed for the cyclohexenone ring requires the enone chromophore to be planar (Figure 2.17). Assuming that the torsion angle ? of the enone approaches to 0? or 180?, the contribution of its helicity to the Cotton effect drops to zero. Therefore the contribution of the vicinal asymmetric centers to the Cotton effect should dominate versus the intrinsic helicity.        Figure 2.19 CD spectrum of alotaketal A (2.1) recorded in methanol, and Snatzke?s sector rule for the the n??* transition in a cyclohexenone ring.18a-d, 19 32  Subsequently, a series of closely related sesterterpenoids called phorbaketals (alotaketals and phobaketals are closely related natural products and have the same sesterterpenoid skeleton) were isolated from a Korean marine sponge by Rho and coworkers20a,b The absolute configuration of the phorbaketals was elucidated via the modified Mosher?s method (Figure 2.20). The phorbaketals were isolated in gram quantities, allowing semisynthetic modification of the enone to the allylic alcohol. In particular, phorbaketal A (2.7) was converted to phorbaketal B (2.8) which was then converted to the Mosher ester. The Mosher ester analysis of phorbaketal B (2.8) revealed the opposite absolute configuration of the phorbaketals compared with that assigned to the alotaketals via CD analysis, although the signs of the optical rotations of alotaketal A (2.1) and phorbaketal A (2.7) are the same. The Mosher analysis conducted on phorbaketal B (2.8) by Rho and coworkers appeared to be correct and more certain than our absolute configuration analysis based on CD spectra.   Figure 2.20 1H NMR chemical shift differences (??S-R) in ppm for S-/R-MTPA esters of phorbaketal B (2.8)  in CDCl3 (adapted from Rho, J. R.; Hwang, B. S.; Sim, C. J.; Joung, S.; Lee, H. Y. and Kim, H. J. Org. Lett., 2009, 11, 5590-5593).20a,b  Subsequently, ansellone A (2.9)21a,b was isolated by Andersen and coworkers from a 33  marine sponge collected in British Columbia. Ansellone A (2.9) has a new ?ansellane? sesterterpenoid carbon skeleton containing the same oxo-decalin ring (with the same cis ring junction) as the alotaketals and phorbaketals. The absolute configurations of alotaketal A (2.1), phorbaketal A (2.7), and ansellone A (2.9) must be the same, considering that natural products are biosynthesized in a chiral environment (enzymes) and must have the same absolute configuration when they belong to the same category.    Figure 2.21 CD spectrum of ansellone A (2.9) recorded in MeOH and ORTEP diagram for desacetylansellone A (2.10). The refined Flack parameter is -0.01(16)21c (adapted from Daoust, J.; Fontana, A.; Merchant, C. E.; de Voogd, N. J.; Kieffer, T. J.; Andersen, R. J. Org. Lett., 2010, 12, 3208-3211).21a,b  A single crystal x-ray diffraction analysis of desacetylansellone A (2.10),21a,b a semisynthetic derivative of ansellone A (2.9), established the absolute configuration of ansellone 34  A (2.9), as shown in the ORTEP diagram in Figure 2.21. Considering that the CD spectra of alotaketal A (2.1) and ansellone A (2.9) (Figure 2.19 and Figure 2.21) are identical, the absolute configuration of alotaketal A (2.1) was revised to its enantiomeric structure (wrong: 1R,6S,9R,13R,14E; correct: 1S,6R,9S,13S,14E).21a,b The absolute configuration proposal based on the interpretation of CD spectra of alotaketals turned out to be wrong and the proposal of Rho and coworkers20a,b for the absolute configuration of phorbaketals based on the Mosher?s analysis was correct. The isolation of small amounts of compound is a major issue in the natural product research, limiting the possibility of biological testing as well as the success of the structure elucidation process. Recrystallization and chemical derivatizations are often required for the determination of the absolute configuration of natural products. However, they are not always possible when molecules are isolated in small amounts, as was the case for the alotaketals. We weren?t able to crystallize alotaketal A (2.1). At the same time we didn?t have enough material for derivatization and, therefore, we predicted the absolute configuration of the alotaketals via CD analysis using empirical rules. It turns out that the application of the Snatzke?s rules to the determination of the absolute configuration of alotaketal A (2.1) failed and we had to correct our absolute configuration proposal. We propose two hypotheses to explain the failure: either the conformation of the enone chromophore is not completely planar and/or the oxygen attached to C-1 via an ether bond could interact with the enone chromophore, changing the sign of the Cotton effect.17-19 Computational studies are required in order to establish the conformation of the enone and its relationship with the sign of the Cotton effect in the CD specrum of alotaketal A (2.1).   A similar case of an anomalous sign of the Cotton effect relative to the predicted sign 35  based on empirical rules for the n??* transition in cyclohexenone rings has been reported. In 2008, Capon and coworkers20c isolated phorbasin C (2.11) and analogues (Figure 2.22) from the southern Australian marine sponge Phorbas sp. A cyclohexenone ring along with an isoprenoid chain in position 6, an alcohol is position 5, and an oxygen atom in position 1 of the ring are present in the chemical structures of phorbasins. The absolute configuration of phorbasin C (2.11) was assigned based on analysis of CD spectra. The sign of the Cotton effect for the n??* transition of the enone present in phorbasin C (2.11) was compared to the model compound (S)-carvone (2.12) (Figure 2.22). Since phorbasin C (2.11) and (S)-carvone (2.12) had the same sign of the Cotton effect, it was proposed that they had the same absolute configuration. In 2009, Micalizio and coworkers synthesized phorbasin C (2.13), showing that the absolute configuration is opposite to the one proposed by Capon and coworkers based on CD analysis.20d   Figure 2.22 Chemical structures of phorbasin C (2.11),20c (S)-carvone (2.12), and phorbasin C (2.13) after structural revision.20d  Aloaketal A (2.1) and phorbasin C (2.11) have an anomalous sign of the Cotton effect for the n??* transition of the enone. The sign is opposite to the one expected from the comparisons 36  with simple cyclohexenone rings, such as (S)-carvone/(R)-carvone. Both cyclohexenone rings of alotaketal A (2.1) and phorbasin C (2.11) have an oxygen atom in the ? position of the cyclohexenone ring. It is possible that this oxygen atom interacts with the enone chromophore causing a change in the sign of the Cotton effect.   2.2.3 Biological Activity of the Alotaketals: Activation of the cAMP Signaling Pathway   Glucagon-like peptide-1 (GLP-1) analogues and dipeptidyl peptidase-IV (DPP-IV) inhibitors have been recently developed as new antidiabetic drugs.22 Their mechanism of action involves the activation of the cAMP signaling pathway. cAMP signaling stimulation in pancreatic ?-cells leads to the increase of insulin secretion, which can be useful to combat diabetes. The identification of new cAMP signaling activators is central in antidiabetic drug discovery. After stimulation, adenylyl cylcase produces cAMP, thus activating cAMP signaling. Forskolin24,25 is a known natural product that activates adenylyl cyclase. In our research effort to isolate new small molecules that can stimulate cAMP signaling pathway, we have identified from the marine sponge Hamigera sp. four sesterterpenoids, alotaketals A (2.1), B (2.3), D (2.4), and E (2.5) (Figure 2.2 and Figure 2.5). Alotaketal A (2.1), the most potent among them (EC50 = 18 nM), activates cAMP signaling with an unknown mechanism. Alotaketals represent a new class of sesterterpenoids that are able to activate cAMP signaling (Figure 2.23). These new molecules are potential chemical tools that can be useful for the study of cAMP signaling functions in diabetes and in other diseases. The identification of the molecular target of the alotaketals is the future goal of this project. The isolation of alotaketals is an example of success in natural product research. Natural products represent a valid source for the discovery of new compounds with interesting 37  bioactivity. The activation of cAMP signaling by alotaketals is even more relevant, considering that there are only a few molecules that can stimulates this cellular pathway. The selective biological activity is consistent with the chemical novelty of alotaketals since they are molecules that belong to one of the rarest class of terpenes (sesterterpenes). Two total synthesis of alotaketal A (2.1) have been recently published.23 In the first total synthesis, Yang et al. have confirmed the activation of cAMP signaling pathway by alotaketal A (2.1) and also reported structure-relationship data for the pharmacophore.9a,b   Alotaketals D (2.4), E (2.5) are new alotaketal analogues that have been recently isolated in the Andersen lab. They represent a new group of cAMP activators that are currently being tested in the cAMP assay providing further information on the structural requirements of alotaketals for the activation of cAMP signaling pathway. Modification of the allylic alcohol to the allyl aldehyde in alotaketal D (2.4) and of the enone to the ?-methoxy ketone in alotaketal E (2.5) resulted into partial loss of activity indicating that both the enone and the allylic alcohol are not essential for the cAMP activity of alotaketals. However, the decreased biological activity of alotaketal D (2.4) and alotaketal E (2.5) indicated that both the allylic alcohol and enone are required for full potency in the cAMP agonist activity and they may be part of the alotaketal pharmacophore.    2.3 Conclusions  The alotaketals are new sesterterpenoids containing a spiroketal substructure. They have a regular monocyclic sesterterpenoid carbon skeleton that has not been previously encountered in a natural product (Figure 2.5).15 The alotaketals show an anomalous sign of the cotton effect in the CD spectra. They are an 38  exception to Snatzke?s rules for enones and therefore they represent an interesting example for the study of circular dichroism for the determination of the absolute configuration in organic molecules. The absolute configuration of alotaketal (2.1), previously proposed based on CD analysis, was revised based on a single crystal x-ray diffraction analysis of desacetylansellone A (2.10) (Figure 2.21). Time-dependent density functional theory (TDDFT) calculations for further studies of the relationship between the absolute configuration and the CD spectra of the alotaketals are also part of our ongoing and future research.    Figure 2.23 Dose-response curve for cAMP activation by alotaketal A (2.1), forskolin (2.2),24,25 alotaketal B (2.3), alotaketal D (2.4), alotaketal E (2.5), and the crude extract of Hamigera. 39  Alotaketals activated the cAMP signaling pathway equally in HEK-GLP-1 and HEK-GLP-1-knockout cells. Therefore the alotaketals aren?t GLP-1 agonists but strongly stimulate cAMP signaling via interaction with an unidentified molecular target(s). Alotaketal A (2.1) is the most potent and activates the cAMP signaling pathway with an EC50 of 18 nM. Alotaketal A (2.1) is 170 times more potent than forskolin (2.2),24 an activator of the adenylyl cylcase,25 which has an EC50 of 3 ?M in the same assay. However, forskolin (2.2) elicits a much stronger response than alotaketal A (2.1) (Figure 2.23). The identification of the molecular target of alotaketal A (2.1) is still ongoing. There is preliminary evidence, from both enzymatic and cellular based assays, that alotaketal A (2.1) activates adenylyl cyclase. In that case, alotaketal A (2.1) and forskolin (2.2) would be the only two known natural products that are able to activate this enzyme directly.   2.4 Experimental Section 2.4.1 General Experimental Procedures Optical rotations were measured using a Jasco P-1010 spectrophotometer. The 1H and 13C NMR spectra were recorded on a Bruker AV-600 spectrometer with a 5 mm CPTCI cryoprobe. 1H chemical shifts are referenced to the residual benzene-d6 signal (? 7.16 ppm) and 13C chemical shifts are referenced to the benzene-d6 solvent peak (? 128.39 ppm). Low resolution ESI +/- were recorded on Bruker Esquire LC ion trap mass spectrometer equipped with an electrospray ion source. The solvent for ESI-MS experiments was methanol. The sample solution concentration was 10 ?M. It was infused into the ion source by a syringe pump at flow rate of 10 ?L/min. High resolution ESI+ were recorded on a Micromass LCT time-of-flight (TOF) mass spectrometer equipped with an electrospray ion source. The samples were dissolved in MeOH. The working solutions were 20 ?M. Flow rate: 20 ?L min-1; sample cone: 40  90 V; source temperature: 120 ?C; desolvation temperature: 120 ?C. For accurate mass measurement, arg-ser-arg was used as reference compound. The mass of arg-ser-arg was used as a lock mass. Merck Type 5554 silica gel plates and Whatman MKC18F plates were used for analytical thin layer chromatography. Sephadex? LH-20 column packed and elueted with 100% MeOH was used for size separation chromatography. Reversed-phase HPLC purifications were performed on a Waters 600E System Controller liquid chromatography attached to a Waters 996 photodiode array detector. All solvents used for HPLC were Fisher HPLC grade.  2.4.2 Extraction of the Sponge The frozen sponge (24 g) was extracted repeatedly with MeOH (3 x 150 mL) at room temperature. The combined methanolic extracts were concentrated in vacuo to afford 285 mg of brown solid. This extract (38.2 mg of it) were chromatographed on a Sephadex LH-20 column in 100% MeOH as eluent to give 3 fractions A-C (A: 15.0 mg; B: 16.0 mg; C: 7.2 mg). Pure samples of alotaketal A (2.1) (5.3 mg), alotaketal B (2.3) (2.1 mg), alotaketal D (2.4) (1.1 mg), and alotaketal E (2.5) (1.0 mg), were obtained from fraction B (16.0 mg) via C18 reversed phase HPLC using a CSC-Inertsil 150A/ODS2, 5 ?m 25 x 0.94 cm column, with 8:2 acetonitrile/H2O as eluent over 50 min (flow rate 2 mL/min).  2.4.3 Materials and Methods for cAMP Signaling Assay HEK-pHTS-CRE Cell Line Derivation HEK293 cells were transfected with the pHTS-CRE plasmid (Biomyx, San Diego, CA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer?s instructions. Stable clones were generated by growing the cells in HG-DMEM 41  (10% FBS, 1 x pen-strep) with 200 ?g/ml hygromycin (Invitrogen) as a selection agent. Clones were then assessed for responsiveness to forskolin induction in a luciferase assay. One clone was selected based on its high sensitivity to forskolin and favorable growth characteristics (HEK-pHTSCRE). Luciferase Assay HEK-pHTS-CRE cells were plated in 96 well flat bottom white polystyrene plates (BD Falcon, Mississauga, ON) at a density of 5 x 104 cells/well and incubated overnight at 37 ?C in a 5% CO2 atmosphere. The next day, the medium was aspirated, cells were washed with PBS (Invitrogen), and 100 ?l of sample (extract in media) was added. Following a 5-h incubation at 37 ?C in a 5% CO2 atmosphere, the samples were removed and wells washed with PBS (Invitrogen). A luciferase assay was then performed using the Bright-Glo luciferase assay kit (Promega, Madison, MI) according to the manufacturer?s instructions. Luminescence was measured using a Tecan Infinite M1000 luminometer (Tecan, M?nnedorf, Switzerland).   2.4.4 1H NMR, 13C NMR, HRESIMS and Optical Rotation Values for Alotaketal A (2.1), B (2.3), D (2.4), and E (2.5) Alotaketal A (2.1). [?]25D -38.9? (c = 0.01, MeOH). 1H NMR (benzene-d6, 600 MHz) ? 6.32 (1H, dq, J = 6.0, 1.2 Hz, H-2), 5.56 (1H, s, H-8), 5.48 (1H, d, J = 8.4 Hz, H-14), 4.89 (1H, bs, H-23a), 4.87 (1H, bs, H-23b), 4.85 (1H, ddd, J =  12.6, 8.4, 3.0 Hz, H-13), 4.81 (1H, bs, H-20a), 4.80 (1H, bs, H-20b), 4.36 (1H, dd, J = 6.0, 4.9 Hz, H-1), 3.53 (2H, m, H-22), 2.47 (1H, dd, J = 15.6, 4.9 Hz, H-5a), 2.39 (1H, dd, J = 15.6, 13.2 Hz, H-5b), 2.34 (1H, d, J = 13.2 Hz, H-10a), 2.31 (1H, m, H-12a), 2.29 (1H, d, J = 13.2 Hz, H-10b), 2.20 (1H, t, J = 12.6 Hz, H-12b), 2.06 (1H, dt, J = 13.2, 4.9 Hz, H-6), 1.98 (2H, t, J = 7.2 Hz, H-16), 1.94 (2H, t, J = 7.2 Hz, H-18), 1.71 (3H, s, H-21), 1.68 (3H, s, H-24), 1.62 (3H, s, H-25), 1.55 (2H, quin, J = 7.2 Hz, H-17), 0.53 (1H, t, J = 5.4 Hz, 22-OH). 13C NMR (benzene-d6, 150 MHz) ? 197.7 (C, C-4), 145.9 (C, C-42  19), 142.5 (C, C-7), 141.5 (C, C-11), 139.6 (CH, C-2), 139.2b (C, C-15), 139.0b (C, C-3), 126.8 (CH, C-14), 125.1 (CH, C-8), 111.4 (CH2, C-23), 110.9 (CH2, C-20), 97.2 (C, C-9), 68.5 (CH, C-13), 63.8 (CH2, C-22), 63.6 (CH, C-1), 44.1 (CH2, C-10), 40.7 (CH2, C-12), 39.7 (CH2, C-16), 38.6 (CH2, C-5), 38.0 (CH2, C-18), 34.0 (CH, C-6), 26.3 (CH2, C-17), 22.8 (CH3, C-25), 17.1 (CH3, C-24), 16.4 (CH3, C-21). HRESIMS [M + Na]+ m/z 421.2346 (calcd for C25H34O4Na, 421.2355). b) may be interchanged Alotaketal B (2.3). [?]25D -10.0? (c = 0.01, MeOH). 1H NMR (benzene-d6, 600 MHz) ? 6.32 (1H, d, J = 6.0 Hz, H-2), 5.45 (1H, d, J = 8.4 Hz, H-14), 5.43 (1H, s, H-8), 5.15 (1H, t, J = 10.8 Hz, H-13), 4.82 (1H, bs, H-20a), 4.81 (1H, bs, H-20b), 4.33 (1H, bs, H-1), 3.51 (2H, m, H-22), 3.07 (1H, d, J = 15.0 Hz, H-10a), 2.37 (2H, bd, J = 7.8 Hz, H-5), 2.12 (1H, m, H-28), 2.12 (2H, m, H-27), 2.01 (2H, t, J = 7.2 Hz, H-16), 2.00 (1H, m, H-6), 1.96 (2H, t, J = 7.2 Hz, H-18), 1.95 (1H, m, H-12a), 1.83 (3H, s, H-21), 1.77 (3H, s, H-24), 1.63 (3H, s, H-25), 1.58 (2H, quin, J = 7.2 Hz, H-17), 1.48 (3H, s, H-23), 1.24 (1H, m, H-12b), 1.23 (1H, d, J = 15.0 Hz, H-10b), 0.89 (3H, d, J = 5.4 Hz, H-29), 0.89 (3H, d, J = 5.4 Hz, H-30), 0.51 (1H, t, J = 5.4 Hz, 22-OH). 13C NMR (benzene-d6, 150 MHz) ? 197.7 (C, C-4), 172.4 (C, C-26), 145.8 (C, C-19), 142.5 (C, C-7), 139.6 (CH, C-2), 139.1b (C, C-15), 138.9b (C, C-3), 126.5 (CH, C-14), 125.3 (CH, C-8), 110.9 (CH2, C-20), 96.9 (C, C-9), 77.4 (C, C-11), 63.9 (CH, C-13), 63.7 (CH2, C-22), 63.4 (CH, C-1), 45.0 (CH2, C-27), 43.9 (CH2, C-12), 40.5 (CH2, C-10), 39.7 (CH2, C-16), 38.4 (CH2, C-5), 38.0 (CH2, C-18), 34.0 (CH, C-6), 27.6 (CH3, C-23), 26.4 (CH2, C-17), 25.6 (CH, C-28), 23.1 (CH3, C-29), 23.1 (CH3, C-30), 22.8 (CH3, C-25), 17.2 (CH3, C-24), 16.6 (CH3, C-21). HRESIMS [M + Na]+ m/z 523.3024 (calcd for C30H44O6Na, 523.3036). b) may be interchanged Alotaketal D (2.4). 1H NMR (benzene-d6, 600 MHz) ? 9.01 (1H, s, H-22), 6.18 (1H, d, J = 43  4.2 Hz, H-2), 5.85 (1H, s, H-8), 5.42 (1H, d, J = 9.6 Hz, H-14), 4.87 (1H, bs, H-23a), 4.83 (1H, bs, H-23b), 4.82 (1H, bs, H-20a), 4.81 (1H, bs, H-20b), 4.76 (1H, dt, J = 9.6, 2.4 Hz, H-13), 4.11 (1H, bs, H-1), 2.83 (1H, dd, J = 16.2, 4.2 Hz, H-5a), 2.66 (1H, dt, J = 12.6, 4.2 Hz, H-6), 2.27 (1H, d, J = 12.0 Hz, H-12a), 2.21 (1H, dd, J = 16.2, 12.6 Hz, H-5b), 2.17 (1H, d, J = 15.0 Hz, H-10a), 2.17c (1H, m, H-12b), 2.13 (1H, d, J = 15.0 Hz, H-10b), 1.97 (2H, t, J = 7.2 Hz, H-16), 1.95 (2H, t, J = 7.2 Hz, H-18), 1.66 (3H, s, H-21), 1.63 (3H, s, H-25), 1.61 (3H, s, H-24), 1.56 (2H, quin, J = 7.2 Hz, H-17). 13C NMR (benzene-d6, 150 MHz) ? 196.6 (C, C-4), 191.8 (CH, C-22), 146.6 (CH, C-8), 145.8 (C, C-19), 143.1(C, C-7), 140.2b (C, C-11), 140.1b (C, C-15), 139.5 (C, C-3), 138.3 (CH, C-2), 126.1 (CH, C-14), 112.1 (CH2, C-23), 111.0 (CH2, C-20), 96.8 (C, C-9), 68.7 (CH, C-13), 63.3 (CH, C-1), 42.9 (CH2, C-10), 40.5 (CH2, C-12), 39.6 (CH2, C-16), 38.2 (CH2, C-5), 37.9 (CH2, C-18), 31.2 (CH, C-6), 26.3 (CH2, C-17), 22.8 (CH3, C-25), 17.0 (CH3, C-24), 16.4 (CH3, C-21). HRESIMS [M + Na]+ m/z 419.2204 (calcd for C25H32O4Na, 419.2198). b) may be interchanged; c) partially obscured Alotaketal E (2.5). 1H NMR (benzene-d6, 600 MHz) 5.55 (1H, s, H-8), 5.46 (1H, d, J = 8.4 Hz, H-14), 4.89 (2H, bs, H-23), 4.84c (1H, m, H-13), 4.81 (1H, bs, H-20a), 4.80 (1H, bs, H-20b), 4.34 (1H, bs, H-1), 3.59 (1H, bs, H-2), 3.51 (1H, d, J = 13.8 Hz, H-22a), 3.46 (1H, d, J = 13.8 Hz, H-22b), 2.98 (3H, s, H-26), 2.85 (1H, m, H-3), 2.48 (1H, dd, J = 13.2, 4.8 Hz, H-5a), 2.41 (1H, m, H-6), 2.33c (2H, m, H-10), 2.30c (2H, m, H-12a), 2.22 (1H, t, J = 12.0 Hz, H-12b), 2.08 (1H, t, J = 13.2 Hz, H-5b), 1.98 (2H, t, J = 7.2 Hz, H-16), 1.94 (2H, t, J = 7.2 Hz, H-18), 1.65 (3H, s, H-24), 1.62 (3H, s, H-25), 1.55 (2H, quin, J = 7.2 Hz, H-17), 1.18 (3H, d, J = 6.6 Hz, H-21). 13C NMR (benzene-d6, 150 MHz) ? 207.2 (C, C-4), 145.9 (C, C-19), 143.6 (C, C-7), 141.9 (C, C-11), 139.8 (C, C-15), 126.7 (CH, C-14), 124.0 (CH, C-8), 111.1 (CH2, C-23), 110.9 (CH2, C-20), 97.4 (C, C-9), 86.6 (CH, C-2), 68.2 (CH, C-13), 66.3 (CH, C-1), 63.7 (CH2, C-22), 44  58.7 (CH3, C-26), 44.9 (CH, C-3), 44.4 (CH2, C-10), 42.8 (CH2, C-5), 40.8 (CH2, C-12), 39.6 (CH2, C-16), 38.0 (CH2, C-18), 34.2 (CH, C-6), 26.3 (CH2, C-17), 22.8 (CH3, C-25), 17.0 (CH3, C-24), 10.8 (CH3, C-21). HRESIMS [M + Na]+ m/z 453.2624 (calcd for C26H38O5Na, 453.2617). c) partially obscured   Figure 2.24 Numbered chemical structures of alotaketal A (2.1), B (2.3), D (2.4), and E (2.5).    45  alotaketal A (2.1) Position ?C, multi. ?H, (J in Hz) HMBCa  1 63.6, CH 4.36, dd (6.0, 4.9) 3, 5, 9 2 139.6, CH 6.32, dq (6.0, 1.2) 1, 3, 4, 6 3 139.0b, C   4 197.7, C   5 38.6, CH2 2.47, dd (15.6, 4.9) 1, 4, 6, 7   2.39, dd (15.6, 13.2)  6 34.0, CH 2.06, dt (13.2, 4.9) 5, 7 7 142.5, C   8 125.1, CH 5.56, s 6, 7, 9, 10, 22 9 97.2, C   10 44.1, CH2 2.34, d (13.2) 8, 9, 11, 12, 23   2.29, d (13.2)  11 141.5, C   12 40.7, CH2 2.31, m 10, 11, 13, 14, 23   2.20, t (12.6)  13 68.5, CH 4.85, ddd (12.6, 8.4, 3.0)  9, 12, 14, 15 14 126.8, CH 5.48, d (8.4) 13, 16, 24 15 139.2b, C   16 39.7, CH2 1.98, t (7.2) 14, 15, 17, 18, 24 17 26.3, CH2 1.55, quin (7.2) 15, 16, 18, 19 18 38.0, CH2 1.94, t (7.2) 16, 17, 19, 20, 25 19 145.9, C   20 110.9, CH2 4.81, bs 18, 19, 25   4.80, bs  21 16.4, CH3 1.71, s 2, 3, 4 22 63.8, CH2 3.53, m 6, 7, 8 23 111.4, CH2 4.89, bs 10, 11, 12   4.87, bs  24 17.1, CH3 1.68, s 14, 15, 16 25 22.8, CH3 1.62, s 18, 19, 20 22-OH  0.53, t (5.4) 7, 22 aHMBC correlations, optimized for 10 Hz, are from proton(s) stated to the indicated carbon. bMay be interchanged.   Table 2.1 NMR spectroscopic data (600 MHz, benzene-d6) for alotaketal A (2.1).       46  alotaketal B (2.3) position ?C, multi. ?H, (J in Hz) HMBCa  1 63.4, CH 4.33, bs 2 2 139.6, CH 6.32, d (6.0) 4, 6, 21 3 138.9b, C   4 197.7, C   5 38.4, CH2 2.37, bd (7.8) 1, 4, 6, 7 6 34.0, CH 2.00, m 2, 5, 7 7 142.5, C   8 125.3, CH 5.43, s 6, 9, 22 9 96.9, C   10 40.5, CH2 3.07, d (15.0) 8, 9, 11, 12, 23   1.23, d (15.0)  11 77.4, C   12 43.9, CH2 1.95, m 11, 13, 23   1.24, m  13 63.9, CH 5.15, t (10.8)  15 14 126.5, CH 5.45, d (8.4) 12, 16, 24 15 139.1b, C   16 39.7, CH2 2.01, t (7.2) 14, 15, 17, 18 17 26.4, CH2 1.58, quin (7.2) 15, 16, 18, 19 18 38.0, CH2 1.96, t (7.2) 16, 17, 19, 20, 25 19 145.8, C   20 110.9, CH2 4.82, bs 18, 25   4.81, bs  21 16.6, CH3 1.83, s 2, 4 22 63.7, CH2 3.51, m 7, 8 23 27.6, CH3 1.48, s 10, 11, 12 24 17.2, CH3 1.77, s 14, 15, 16 25 22.8, CH3 1.63, s 18, 19, 20 26 172.4, C   27 45.0, CH2 2.12, m 26, 29, 30 28 25.6, CH 2.12, m 26, 27, 29, 30 29 23.1, CH3 0.89, d (5.4) 27, 28, 30 30 23.1, CH3 0.89, d (5.4) 27, 28, 29 22-OH  0.51, t (5.4)  aHMBC correlations, optimized for 10 Hz, are from proton(s) stated to the indicated carbon. bMay be interchanged.   Table 2.2 NMR spectroscopic data (600 MHz, benzene-d6) for alotaketal B (2.3).    47  alotaketal D (2.4) position ?C, multi. ?H, (J in Hz) HMBCa  1 63.3, CH 4.11, bs 3, 6 2 138.3, CH 6.18, d (5.4) 1, 4, 6, 21 3 139.5, C   4 196.6, C   5 38.2, CH2 2.83, dd (16.2, 4.2) 1, 4, 6    2.21, dd (16.2, 12.6)  6 31.2, CH 2.66, dt (12.6, 4.2) 1, 5, 7 7 143.1, C   8 146.6, CH 5.85, s 6, 9, 10, 22 9 96.8, C   10 42.9, CH2 2.17, d (15.0) 9, 11, 23   2.13, d (15.0)  11 140.2b, C   12 40.5, CH2 2.27, d (12.0) 11, 13, 23   2.17c, m  13 68.7, CH 4.76, dt (9.6, 2.4)  12, 15 14 126.1, CH 5.42, d (9.6) 13, 16, 24 15 140.1b, C    16 39.6, CH2 1.97, t (7.2) 14, 15, 17, 18, 24  17 26.3, CH2 1.56, quin (7.2) 16, 18 18 37.9, CH2 1.95, t (7.2) 16, 17, 19, 20, 25  19 145.8, C   20 110.0, CH2 4.82, bs 18, 25   4.81, bs  21 16.4, CH3 1.66, s 2, 3, 4 22 191.8, CH 9.01, s 6, 7, 8 23 112.1, CH2 4.87, bs 10, 12   4.83, bs  24 17.0, CH3 1.61, s 14, 15, 16 25 22.8, CH3 1.63, s 18, 19, 20 aHMBC correlations, optimized for 10 Hz, are from proton(s) stated to the indicated carbon. bMay be interchanged.  cPartially obscured.  Table 2.3 NMR spectroscopic data (600 MHz, benzene-d6) for alotaketal D (2.4).    48  alotaketal E (2.5) position ?C, multi. ?H, (J in Hz) HMBCa  1 66.3, CH 4.34, bs 2, 6 2 86.6, CH 3.59, bs 3, 6 3 44.9, CH 2.85, m 4  4 207.2, C   5 42.8, CH2 2.48, dd (13.2, 4.8) 4, 6   2.08, t (13.2)  6 34.2, CH 2.41, m 1, 5 7 143.6, C   8 124.0, CH 5.55, s 6, 9, 22 9 97.4, C   10 44.4, CH2 2.33c, m 9, 11, 12, 23 11 141.9, C   12 40.8, CH2 2.30c, m 11, 13, 10, 23   2.22, t (12.0)  13 68.2, CH 4.84c, m  12, 14 14 126.7, CH 5.46, d (8.4) 16, 24 15 139.8, C   16 39.6, CH2 1.98, t (7.2) 14, 15, 17, 18, 24  17 26.3, CH2 1.55, quin (7.2) 16, 18 18 38.0, CH2 1.94, t (7.2) 16, 17, 19, 20, 25 19 145.9, C   20 110.9, CH2 4.82, bs 18, 25   4.81, bs  21 10.8, CH3 1.18, d (6.6) 2, 3, 4 22 63.7, CH2 3.51, d (13.8) 7, 8   3.46, d (13.8)  23 111.1, CH2 4.89, bs 10, 12 24 17.0, CH3 1.65, s 14, 15, 16 25 22.8, CH3 1.62, s 18, 19, 20 26 58.7, CH3 2.98, s 2 aHMBC correlations, optimized for 10 Hz, are from proton(s) stated to the indicated carbon. cPartially obscured.  Table 2.4 NMR spectroscopic data (600 MHz, benzene-d6) for alotaketal E (2.5).    49   Figure 2.25 COSY spectrum of alotaketal A (2.1) recorded in benzene-d6 at 600 MHz.  Figure 2.26 HSQC spectrum of alotaketal A (2.1) recorded in benzene-d6 at 600 MHz. 50   Figure 2.27 HMBC spectrum of alotaketal A (2.1) recorded in benzene-d6 at 600 MHz.  Figure 2.28 ROESY spectrum of alotaketal A (2.1) recorded in benzene-d6 at 600 MHz. 51    Figure 2.29 DEPT 135? of alotaketal A (2.1) recorded in benzene-d6 at 150 MHz.      Figure 2.30 13C NMR spectrum of alotaketal B (2.3) recorded in benzene-d6 at 150 MHz. 52   Figure 2.31 COSY spectrum of alotaketal B (2.3) recorded in benzene-d6 at 600 MHz.   Figure 2.32 HSQC spectrum of alotaketal B (2.3) recorded in benzene-d6 at 600 MHz. 53   Figure 2.33 HMBC spectrum of alotaketal B (2.3) recorded in benzene-d6 at 600 MHz.  Figure 2.34 ROESY spectrum of alotaketal B (2.3) recorded in benzene-d6 at 600 MHz. 54    Figure 2.35 DEPT 135? of alotaketal B (2.3) recorded in benzene-d6 at 150 MHz.   Figure 2.36 CD spectrum of alotaketal B (2.3) recorded in methanol. 55    Figure 2.37 1H NMR spectrum of alotaketal D (2.4) recorded in benzene-d6 at 600 MHz.        Figure 2.38 13C NMR spectrum of alotaketal D (2.4) recorded in benzene-d6 at 150 MHz. 56   Figure 2.39 COSY spectrum of alotaketal D (2.4) recorded in benzene-d6 at 600 MHz.  Figure 2.40 HSQC spectrum of alotaketal D (2.4) recorded in benzene-d6 at 600 MHz. 57   Figure 2.41 HMBC spectrum of alotaketal D (2.4) recorded in benzene-d6 at 600 MHz.   Figure 2.42 1H NMR spectrum of alotaketal E (2.5) recorded in benzene-d6 at 600 MHz. 58   Figure 2.43 13C NMR spectrum of alotaketal E (2.5) recorded in benzene-d6 at 150 MHz.   Figure 2.44 COSY spectrum of alotaketal E (2.5) recorded in benzene-d6 at 600 MHz. 59   Figure 2.45 HSQC spectrum of alotaketal E (2.5) recorded in benzene-d6 at 600 MHz.  Figure 2.46 HMBC spectrum of alotaketal E (2.5) recorded in benzene-d6 at 600 MHz. 60  Chapter 3: Genkwanines M and P, Daphnane Terpenoids from Wikstroemia polyantha that inhibit miRNA Activity  3.1 Introduction MicroRNAs (miRNAs) are small endogenous ribonucleic acids (RNAs), typically 21-23 nucleotides long. miRNAs are post-transcriptional non-coding regulators that bind imperfectly the 3? untranslated region (3? UTR) of mRNAs. The miRNAs binding activity at the mRNAs level results in translational repression and/or mRNA decay induction. Therefore, miRNAs play a central role in the regulation of protein expression (Figure 3.1) and their malfunction has been associated with many diseases, such as cancer, viral infections, and diabetes.26    Figure 3.1 Regulation of protein expression by miRNA (adapted from Mack, G. S. Nature Biotechnology, 2007, 25, 631-638).26   The goal of this project was the isolation of small molecules that can inhibit miRNA activity. In order to identify compounds that interfere with miRNA regulation and/or processing, 61  Dr. Eric Jan and Anthony Khong have used a cell-based reporter system that monitors miRNA activity. A library of crude marine and plant extracts (~12,000) was screened using a cell-based high throughput approach, leading to several hits. The crude extract of the Indonesian plant Wikstroemia polyantha was active in the miRNA inhibition assay. Bioassay-guided fractionation of this extract led to the identification of the daphnane diterpenoid genkwanine M (3.1) (GENK). GENK (3.1) (Figure 3.2) can moderately inhibit miR-122 activity in the liver Huh-7 cell line. The diterpenoid (3.1) does not alter miR-122 levels nor does it directly inhibit siRNA activity in an in vitro cleavage assay. Finally, GENK (3.1) can inhibit HCV infection in Huh-7 cells.27   Figure 3.2 Numbered chemical structures of genkwanine M (3.1), genkwanine P (3.2), diacetyl genkwanine M (3.3), and genkwanine A (3.4).28a,b 62  3.2 Results and Discussion  Wikstroemia polyantha (family Thymelaeaceae) was collected on the centerline of Peninsula Malaysia approximately 50 miles south of the Thai border by E. Soepadmo and M Suhaimi under contract with the University of Chicago at Illinois. The plant material was transferred to the NCI Open Repository where it was dried and then extracted with dichloromethane and methanol. Three grams of the crude methanolic extract were supplied to UBC by D. Newman of the NCI Open Repository.     Figure 3.3 1H NMR spectrum of genkwanine M (3.1) recorded in DMSO-d6 at 600 MHz.  Three grams of the crude methanolic extract were separated between water and ethyl acetate. The combined ethyl acetate extract was active in the miRNA inhibition assay. This extract was fractionated on a Sephadex? LH-20 column, using methanol/dichloromethane (1/1) 63  as an eluent, to afford several fractions. Only one fraction was active in the assay and it was chromatographed on silica gel using a hexane/ethyl acetate step gradient to furnish several fractions. One of the fractions showed miRNA inhibition activity and interesting NMR signals. The fraction was further purified on reversed phase-HPLC, with acetonitrile/water (8/2) as an isocratic eluent, to afford genkwanines M (3.1), and P (3.2) (Figure 3.2). 27, 28a,b    Figure 3.4 ROESY correlations of genkwanine M (3.1).  The structure elucidation of the genkwanines was based on the analysis of HRESIMS and NMR spectroscopic data. The major compound genkwanine M (3.1) [1H NMR spectrum of genkwanine M (3.1) is shown in Figure 3.3] was isolated as a white amorphous solid. GENK 64  (3.1) gave a [M + Na]+ ion at m/z 613.2421 (calcd for C34H38O9Na, 613.2414) in the HRESIMS consistent with a molecular formula of C34H38O9, requiring 16 sites of unsaturation. The deterpenoid skeleton was easily identified by 1D and 2D NMR analysis. However, some of the relative configurations in the region C-1 to C-7 could not be assigned from spectroscopic data [ROESY correlations and ROESY spectrum of genkwanine M (3.1) are shown in Figure 3.4 and Figure 3.10, respectively].     Figure 3.5 1H NMR spectrum of genkwanine P (3.2) recorded in DMSO-d6 at 600 MHz.  Thus, the crystallization of GENK (3.1) seemed to be necessary and it was attempted using several different solvents without success. GENK (3.1) was then converted to its diacetyl derivative (3.3), which gave crystals from a carbon tetrachloride/hexane (9/1) solution that were 65  suitable for single crystal x-ray diffraction analysis.29 An ORTEP diagram (Figure 3.8) confirms the structure and allowed a statistical prediction of the absolute configuration. The structure of genkwanine M (3.1)28a-b has been previously reported, and the assignment of proton (Figure 3.3) and carbon (Figure 3.9) resonances was based on comparisons with literature NMR spectroscopic data (see experimental section for NMR spectroscopic data in DMSO-d6).     Figure 3.6 Key COSY and HSQC correlations of the cinnamic ester of genkwanine P (3.2).  Unlike genkwanine M (3.1), genkwanine P (3.2) is a new compound with a cinnamate ester moiety. It gave a [M + Na]+ ion at m/z 639.2554 (calcd for C36H40O9Na, 639.2570) in the HRESIMS consistent with a molecular formula corresponding to C36H40O9. The chemical structure of genkwanine P (3.2) was elucidated via analysis of 1D and 2D NMR spectroscopic data as described below. 1H NMR spectrum of genkwanine P (3.2) recorded in DMSO-d6 (Figure 3.5) was 66  compared with the 1H spectrum of genkwanine M (3.1) recorded in the same solvent (Figure 3.3), revealing almost identical 1H NMR resonances. However, genkwanine P (3.2) has an additional C2H2 fragment as indicated from analysis of HRESIMS data, in comparison with genkwanine M (3.1). The proton spectrum of genkwanine P (3.2) showed the presence of two resonances at ? 7.67 (H-3?) and ? 6.67 (H-2?), which appeared to be diagnostic for olefinic protons. Indeed, ? 7.67 (H-3?) and ? 6.67 (H-2?) showed correlations to resonances at ? 144.6 (C-3?) and ? 118.1 (C-2?), respectively, in the HSQC spectrum (Figure 3.6 and Figure 3.13), typical 13C chemical shifts of olefinic carbons.  Additionally, the resonances at ? 7.67 (H-3?) and ? 6.67 (H-2?) showed strong correlations between themselves in the COSY spectrum (Figure 3.6 and Figure 3.12) indicating that C-3? (? 144.6) and C-2? (? 118.1) are two vicinal olefinic methines. These two methines appeared to be the additional fragment (C2H2) of the genkwanine P (3.2) chemical structure in comparison with the genkwanine M (3.1) structure. In the HMBC spectrum (Figure 3.14) the proton resonance at ? 7.67 (H-3?), already assigned to the methine C-3? (? 144.6), showed strong correlations to the carbon resonances at ? 166.1 (C-1?) and ? 128.4, which integrates for two aromatic resonances C-5? and C-9? of the previously elucidated benzene ring (Figure 3.7). Furthermore, the proton resonance at ? 6.67 (H-2?), assigned to the methine C-2? (? 118.1), showed strong HMBC correlations to a resonance at ? 134.0, which integrates for the quaternary aromatic carbon C-4? directly attached to carbon C-3? via a covalent bond (Figure 3.7). Our structure elucidation analysis led to the identification of the cinnamate ester moiety of genkwanine P (3.2). At this point of our structure elucidation analysis, it clearly appeared that the cinnamic ester fragment of genkwanine P (3.2) was replacing the benzoate ester of genkwanine M (3.1). 67  The cinnamate ester fragment was directly attached to C-20 (? 67.1), via a covalent bond between the oxygen atom of the ester and C-20.    Figure 3.7 Key HMBC correlations of the cinnamic ester of genkwanine P (3.2).  The structure elucidation process was then completed with the assignment of the configuration of the ?2?,3? double bond, based on ROESY correlations and coupling constant calculations. The H-3? (? 7.67) and H-2? (? 6.67) resonances didn?t show any ROESY correlations between them, indicating that the conjugated double bond has an E configuration (trans cinnamic ester). This interpretation was also supported by the value of the scalar coupling constant (J = 16.2 Hz), between the H-3? and H-2? resonances in the proton spectrum of genkwanine P (3.2) (Figure 3.5). This value of 16.2 Hz is typical of trans olefinic protons, whereas cis olefinic protons have a smaller coupling constants (typically, ~10 Hz). Moreover, trans cinnamate esters are very common moieties in naturally occurring compounds. Thus, the 68  chemical structure of genkwanine P (3.2) is fully elucidated. miRNAs are evolutionary conserved regulators of gene expression.30 They play a central role in cellular homeostasis, growth and diseases. Regulation and signaling pathways that influence miRNA function are poorly understood and characterized.  In their effort to elucidate miRNA functions, Dr. Eric Jan and Anthony Khong have developed a cell-based miRNA sensor system for the identification of genkwanines from the extract. Such a screening system was created using genkwanines as chemical tools to monitor miRNA activity, and it might be useful in the future for the identification of new molecules that affect miRNA/siRNA activity.   Figure 3.8 ORTEP diagram of diacetyl genkwanine M (3.3).29 69  3.3 Conclusions Genkwanines M (3.1) and P (3.2) are daphnane diterpenoids isolated from the Indonesian plant Wikstroemia polyantha. Genkwanine M (3.1)28a,b was previously isolated from a Chinese plant, whereas genkwanine P (3.2) is a new compound with a cinnamate ester moiety. The ortho-ester of benzoic acid is a rare functional group that is present in the chemical structure of genkwanines. Structure elucidation of the genkwanines was based on the interpretation of 1D and 2D NMR spectroscopic data as well as on x-ray diffraction analysis of the diacetyl derivative of genkwanine M (3.3). We have confirmed the abosolute configuration of this category of daphnane diterpenoids using x-ray analysis. The ORTEP diagram of diacetyl genkwanine M (3.3) (Figure 3.8) is consistent with the previously reported absolute configuration of closely related terpenoids.28c Genkwanines M (3.1) and P (3.2) are weak inhibitors of miR-122 activity in the liver Huh-7 cell line. Furthermore, genkwanine M (3.1) can inhibit HCV infection in Huh-7 cells. The study of miRNA signaling in relation with cellular diseases using genkwanines as chemical tools and the isolation of more potent miRNA inhibitors are the future goals of this project.   3.4 Experimental Section 3.4.1 General Experimental Procedures Optical rotations were measured using a Jasco P-1010 spectrophotometer. The 1H and 13C NMR spectra were recorded on a Bruker AV-600 spectrometer with a 5 mm CPTCI cryoprobe. 1H chemical shifts are referenced to the residual DMSO-d6 signal (? 2.50 ppm) and 13C chemical shifts are referenced to the DMSO-d6 solvent peak (? 39.51 ppm). The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = 70  multiplet, quin = quintuplet, sext = sextet, sep = septet, b = broad. Low resolution ESI +/- were recorded on Bruker Esquire LC ion trap mass spectrometer equipped with an electrospray ion source. The solvent for ESI-MS experiments was methanol. The sample solution concentration was 10 ?M. It was infused into the ion source by a syringe pump at flow rate of 10 ?L/min. High resolution ESI+ were recorded on a Micromass LCT time-of-flight (TOF) mass spectrometer equipped with an electrospray ion source. The samples were dissolved in MeOH. The working solutions were 20 ?M. Flow rate: 20 ?L min-1; sample cone: 90 V; source temperature: 120 ?C; desolvation temperature: 120 ?C. The mass of arg-lys-asp-val-tyr was used as lock mass for genkwanines M (3.1), and the mass of relative and erythromycin as lock mass for diacetyl genkwanines M (3.3). Sephadex? LH-20 column packed and elueted with a mixture of 1:1 MeOH/CH2Cl2 was used for size separation chromatography. Reversed-phase HPLC purifications were performed on a Waters 600E System Controller liquid chromatography attached to a Waters 996 photodiode array detector using a C18 reversed-phase column (CSC-Inertsil 150A/ODS2, 5 ?m 25 x 0.94 cm). All solvents used for HPLC were Fisher HPLC grade. The acetylation reaction of the polyanthoxide A was carried out under a nitrogen atmosphere with dry solvents under anhydrous conditions. Commercially available anhydrous tetrahydrofuran (THF) was used to perform the reaction. Yield refer to chromatographically and spectroscopically (1H NMR) homogeneous materials. Reagents were purchased at the highest commercial quality and used without further purification. The reaction was monitored by thin layer chromatography (TLC) carried out on Merck Type 5554 silica gel plates using UV light as visualizing agent and either an ethanolic solution of cerium sulfate or vanillin in ethanol/aqueous H2SO4, and heat as developing agents. 71  3.4.2 Isolation Procedure Leaves and twigs from a 0.5 m tall shrub of Wikstroemia polyantha (family Thymelaeaceae) were collected on the centerline of Peninsula Malaysia approximately 50 miles south of the Thai border by E. Soepadmo and M. Suhaimi under contract with the University of Chicago at Illinois. A voucher numbered Q66O4184 was deposit in the Field Museum in Chicago and at the National Herbarium at the Smithsonian. The plant material was transferred to the NCI Open Repository where it was dried and then extracted with CH2Cl2 and MeOH. The crude extract was entered into the Open Repository screening plates as sample number NO44759. Three grams of the crude extract were supplied to UBC by D. Newman of the NCI Open Repository. Three grams of the crude extract were then partitioned between EtOAc (3 ? 50mL) and H2O (100 mL). The combined EtOAc extract was evaporated to dryness to give 2.100 g of dark green oil; 1.000 g of this was chromatographed on a Waters 10g Sep-Pak?s for direct-phase flash chromatography, employing a step gradient from 95:5 hexane/EtOAc to EtOAc, and from 90:10 EtOAc/MeOH to MeOH, to give fractions A-E. Fraction C (38.0 mg), eluting with 60:40 hexane/EtOAc, was chromatographed on a Sephadex? LH-20 column in 1:1 MeOH/CH2Cl2 as an eluent to give fractions C-(A-G). Pure genkwanines M (3.1) (4.9 mg) and P (3.2) (2.1 mg) were obtained as a white amorphous solid from fraction C-E (15 mg) via C18 reversed-phase HPLC using 8:2 MeOH/H2O as an eluent over 70 min. (flow rate 2 mL/min).  3.4.3 Derivatization of Genkwanine M (3.1) to Diacetyl Genkwanine M (3.3) To a stirred solution of acetic anhydride (4?L, 4.24 10-5 mol) and DMAP (5.18 10-5 g, 4.24 10-7 mol) in THF (0.5 mL) was added genkwanine M (3.1) (2.5 10-3g, 4.24 10-6 mol), previously dissolved in THF (0.5 mL), and the mixture was stirred for 24 h at rt. Then the mixture of 72  acetylated products is concentrated to dryness, and purified by C18 reversed-phase HPLC using a CSC-Inertsil 150A/ODS2, 5 ?m 25 x 0.94 cm column and 80:20 MeOH/H2O as an eluent to yield pure diacetyl as major product (1.1 mg) of the reaction. NMR spectroscopic data obtained for the diacetyl product was consistent with acetylation at C-3 (3-OH; ? 5.56), C-5 (5-OH; ? 4.94), of the proposed diacetyl genkwanine M structure (3.3). Diacetyl genkwanine M (3.3) gave needle-shaped crystals from a mixture of 9:1 carbon tetrachloride/hexane.  3.4.4 1H NMR, 13C NMR, HRESIMS and Optical Rotation Values for Genkwanine M (3.1), Genkwanine P (3.2), and Diacetyl Genkwanine M (3.3) Genkwanine M (3.1). [?]25D -6.0? (c = 0.02, MeOH). 1H NMR (DMSO-d6, 600 MHz) ? 8.01 (2H, d, J = 7.8 Hz, H-3?, H-7?), 7.66 (1H, t, J = 7.8 Hz, H-5?), 7.59-7.57 (2H, m, H-3?, H-7?), 7.54 (2H, t, J = 7.8 Hz, H-4?, H-6?), 7.39-7.37 (3H, m, H-4?, H-5?, H-6?), 5.56 (1H, d, J = 5.4 Hz, 3-OH), 5.03 (1H, bs, H-16a), 4.95 (1H, d, J = 12.0 Hz, H-20a), 4.94 (1H, d, J = 9.6 Hz, 5-OH), 4.87 (1H, bs, H-16b), 4.70 (1H, d, J = 3.0 Hz, H-14), 4.25 (1H, s, 4-OH), 3.92 (1H, d, J = 12.0 Hz, H-20b), 3.73 (1H, d, J = 9.6 Hz, H-5), 3.68 (1H, t, J = 5.4 Hz, H-3), 3.49 (1H, s, H-7), 3.04 (1H, d, J = 3.0 Hz, H-8), 2.52b (1H, m, H-10), 2.41 (1H, quin, J = 7.2, H-11), 2.18 (1H, dd, J = 14.4, 7.2 Hz, H-12a), 1.79 (3H, s, H-17), 1.58 (1H, d, J = 14.4, H-12b), 1.52 (1H, m, H-2), 1.49 (2H, m, H-1), 1.17 (3H, d, J = 7.2 Hz, H-18), 0.94 (3H, d, J = 6.6 Hz, H-19). 13C NMR (DMSO-d6, 150 MHz) ? 165.5 (C, C-1?), 146.4 (C, C-15), 136.6 (C, C-2?), 133.3 (CH, C-5?), 129.8 (C, C-2?), 129.3 (2CH, C-3?, C-7?), 129.1 (CH, C-5?), 128.7 (2CH, C-4?, C-6?), 127.8 (2CH, C-4?, C-6?), 125.8 (2CH, C-3?, C-7?), 116.3 (C, C-1?), 110.5 (CH2, C-16), 84.0 (C, C-13), 81.2 (CH, C-14), 80.1 (C, C-9), 79.5 (C, C-4), 76.4 (CH, C-3), 71.2 (CH, C-5), 67.8 (CH2, C-20), 63.4 (CH, C-7), 60.0 (C, C-6), 48.1 (CH, C-10), 36.1 (CH, C-2), 35.9 (CH, C-8), 35.6 (CH2, C-73  12), 34.5 (CH2, C-1), 34.4 (CH, C-11), 20.8 (CH3, C-18), 19.0 (CH3, C- 17), 13.5 (CH3, C-19). HRESIMS [M + Na]+ m/z 613.2421 (calcd for C34H38O9Na, 613.2414). b) partially obscured  Genkwanine P (3.2). [?]25D +4.7? (c = 0.02, MeOH). 1H NMR (DMSO-d6, 600 MHz) ? 7.74-7.73 (2H, m, H-5?, H-9?), 7.67 (1H, d, J = 16.2 Hz, H-3?), 7.59-7.57 (2H, m, H-3?, H-7?), 7.43-7.42 (3H, m, H-6?, H-7?, H-8?), 7.40-7.39 (3H, m, H-4?, H-5?, H-6?), 6.67 (1H, d, J = 16.2 Hz, H-2?), 5.58 (1H, d, J = 5.4 Hz, 3-OH), 5.03 (1H, bs, H-16a), 4.87 (1H, d, J = 9.6 Hz, 5-OH), 4.87 (1H, bs, H-16b), 4.79 (1H, d, J = 11.4 Hz, H-20a), 4.69 (1H, d, J = 2.4 Hz, H-14), 4.24 (1H, s, 4-OH), 3.85 (1H, d, J = 11.4 Hz, H-20b), 3.67 (1H, m, H-3), 3.66 (1H, m, H-5), 3.45 (1H, s, H-7), 3.02 (1H, d, J = 2.4 Hz, H-8), 2.53b (1H, m, H-10), 2.41 (1H, quin, J = 7.8, H-11), 2.18 (1H, dd, J = 13.8, 7.8 Hz, H-12a), 1.78 (3H, s, H-17), 1.58 (1H, d, J = 13.8, H-12b), 1.52 (1H, m, H-2), 1.49 (2H, m, H-1), 1.17 (3H, d, J = 7.2 Hz, H-18), 0.94 (3H, d, J = 6.6 Hz, H-19). 13C NMR (DMSO-d6, 150 MHz) ? 166.1 (C, C-1?), 146.4 (C, C-15), 144.6 (CH, C-3?), 136.6 (C, C-2?), 134.0 (C, C-4?), 130.5 (CH, C-7?), 129.1 (CH, C-5?), 128.9 (2CH, C-6?, C-8?), 128.4 (2CH, C-5?, C-9?), 127.8 (2CH, C-4?, C-6?), 125.8 (2CH, C-3?, C-7?), 118.1 (CH, C-2?), 116.3 (C, C-1?), 110.5 (CH2, C-16), 84.0 (C, C-13), 81.2 (CH, C-14), 80.1 (C, C-9), 79.4 (C, C-4), 76.4 (CH, C-3), 71.2 (CH, C-5), 67.1 (CH2, C-20), 63.3 (CH, C-7), 59.9 (C, C-6), 48.1 (CH, C-10), 36.2 (CH, C-2), 35.9 (CH, C-8), 35.6 (CH2, C-12), 34.5 (CH2, C-1), 34.4 (CH, C-11), 20.7 (CH3, C-18), 19.0 (CH3, C-17), 13.5 (CH3, C-19). HRESIMS [M + Na]+ m/z 639.2554 (calcd for C36H40O9Na, 639.2570). b) partially obscured Diacetyl Genkwanine M (3.3). 1H NMR (DMSO-d6, 600 MHz) ? 7.98 (2H, d, J = 7.2 Hz, H-3?, H-7?), 7.67 (1H, t, J = 7.2 Hz, H-5?), 7.60-7.58 (2H, m, H-3?, H-7?), 7.54 (2H, t, J = 7.2 Hz, H-4?, H-6?), 7.40-7.39 (3H, m, H-4?, H-5?, H-6?), 5.33 (1H, s, H-5), 5.04 (1H, bs, H-16a), 74  4.90 (1H, bs, H-16b), 4.77 (1H, d, J = 4.8 Hz, H-3), 4.75 (1H, d, J = 2.4 Hz, H-14), 4.68 (1H, d, J = 12.0 Hz, H-20a), 4.68 (1H, s, 4-OH), 3.93 (1H, d, J = 12.0 Hz, H-20b), 3.61 (1H, s, H-7), 3.16 (1H, m, H-8), 2.62 (1H, m, H-10), 2.48b (1H, m, H-11), 2.19 (1H, dd, J = 14.1, 8.4 Hz, H-12a), 2.05 (3H, s, H-24), 2.00 (3H, s, H-22), 1.80 (3H, s, H-17), 1.79b (1H, m, H-1a), 1.69 (1H, q, J = 4.8 Hz, H-2), 1.64 (1H, d, J = 14.1, H-12b), 1.56 (1H, dd, J = 12.6, 12.0 Hz, H-1b), 1.21 (3H, d, J = 6.6 Hz, H-18), 0.83 (3H, d, J = 4.8 Hz, H-19). 13C NMR (DMSO-d6, 150 MHz) ? 170.1 (C, C-23), ? 170.0 (C, C-21), 165.4 (C, C-1?), 146.2 (C, C-15), 136.3 (C, C-2?), 133.5 (CH, C-5?), 129.4 (2CH, C-3?, C-7?), 129.2 (C, C-2?), 129.2 (CH, C-5?), 128.7 (2CH, C-4?, C-6?), 127.9 (2CH, C-4?, C-6?), 125.7 (2CH, C-3?, C-7?), 116.4 (C, C-1?), 110.7 (CH2, C-16), 84.1 (C, C-13), 81.0 (CH, C-14), 80.9 (C, C-4), 79.6 (C, C-9), 77.7 (CH, C-3), 71.4 (CH, C-5), 68.1 (CH2, C-20), 64.0 (CH, C-7), 58.7 (C, C-6), 48.4 (CH, C-10), 36.0 (CH, C-8), 35.6 (CH2, C-12), 35.3 (CH2, C-1), 35.0 (CH, C-2), 34.4 (CH, C-11), 20.9c (CH3, C-24), 20.8c (CH3, C-22); 20.7 (CH3, C-18), 18.9 (CH3, C-17), 12.9 (CH3, C-19). HRESIMS [M + Na]+ m/z 697.2620 (calcd for C38H42O11Na, 697.2625. b) partially obscured; c) maybe interchanged          75  genkwanine M (3.1) position ?C, multi. ?H, (J in Hz) HMBCa  1 34.5, CH2 1.49, m 2, 3, 9, 10 2 36.1, CH 1.52, m 1, 4, 10, 19 3 76.4, CH 3.68, t (5.4) 1, 5, 10 4 79.5, C   5 71.2, CH 3.73, d (9.6) 4, 6, 7 6 60.0, C   7 63.4, CH 3.49, s 6, 8, 9, 14, 20 8 35.9, CH 3.04, d (3.0) 6, 7, 11 9 80.1, C   10 48.1, CH 2.52, mb 1, 4, 5, 11 11 34.4, CH 2.41, quin (7.2) 9, 10, 12, 13, 18 12 35.6, CH2 2.18, dd (14.4, 7.2) 9, 11, 13, 18   1.58, d (14.4)  13 84.0, C   14 81.2, CH 4.70, d (3.0) 1?, 7, 9, 15 15 146.4, C   16 110.5, CH2 5.03, bs 13, 15, 17   4.87, bs  17 19.0, CH3 1.79, s 13, 15, 16 18 20.8, CH3 1.17, d (7.2) 9, 11, 12 19 13.5, CH3 0.94, d (6.6) 1, 2, 3 20 67.8, CH2 4.95, d (12.0) 1?, 5, 6, 7   3.92, d (12.0)  1? 116.3, C   2? 136.6, C   3? 7? 125.8, CH 7.58, m 1?, 5? 4? 6? 127.8, CH 7.38, m 2?, 3?, 7? 5? 129.1, CH 7.38, m 2?, 3?, 7? 1? 165.5, C   2? 129.8, C   3? 7? 129.3, CH 8.01, d (7.8) 1?, 2?, 5? 4? 6? 128.7, CH 7.54, t (7.8) 2?, 3?, 7? 5? 133.3, CH 7.66, t (7.8) 3?, 7? 3-OH  5.56, (5.4) 2, 3, 4 4-OH  4.25, s 3, 4, 5, 10 5-OH  4.94, d (9.6) 4, 5 ,6 aHMBC correlations, optimized for 10 Hz, are from proton(s) stated to the indicated carbon. bPartially obscured.   Table 3.1 NMR spectroscopic data (600 MHz, DMSO-d6) for genkwanine M (3.1).  76  genkwanine P (3.2) position ?C, multi. ?H, (J in Hz) HMBCa  1 34.5, CH2 1.49, m 2, 3, 9, 10 2 36.2, CH 1.52, m 1, 4, 19 3 76.4, CH 3.67, m 1, 5, 10 4 79.4, C   5 71.2, CH 3.66, m 4, 6, 7 6 59.9, C   7 63.3, CH 3.45, s 6, 8, 9, 20 8 35.9, CH 3.02, d (2.4) 7, 11 9 80.1, C   10 48.1, CH 2.53, mb 1, 4, 5, 11 11 34.4, CH 2.41, quin (7.8) 9, 12, 13, 18 12 35.6, CH2 2.18, dd (13.8, 7.8) 9, 11, 13, 18   1.58, d (13.8)  13 84.0, C   14 81.2, CH 4.69, d (2.4) 1?, 7, 9, 15 15 146.4, C   16 110.5, CH2 5.03, bs 13, 15, 17   4.87, bs  17 19.0, CH3 1.78, s 13, 15, 16 18 20.7, CH3 1.17, d (7.2) 9, 11, 12 19 13.5, CH3 0.94, d (6.6) 1, 2, 3 20 67.1, CH2 4.79, d (11.4) 1?, 5, 6, 7   3.85, d (11.4)  1? 116.3, C   2? 136.6, C   3? 7? 125.8, CH 7.58, m 1?, 5? 4? 6? 127.8, CH 7.40, m 2?, 3?, 7? 5? 129.1, CH 7.40, m 2?, 3?, 7? 1? 166.1, C   2? 118.1, CH 6.67, d (16.2) 1?, 3?, 4? 3?  144.6, CH 7.67, d (16.2) 1?, 4? 4?  134.0, C   5? 9? 128.4, CH 7.74, m 3?, 7? 6? 8? 128.9, CH 7.43, m 4?, 5?, 9? 7? 130.5, CH 7.43, m 4?, 5?, 9? 3-OH  5.58, (5.4) 2, 3, 4 4-OH  4.24, s 3, 4, 5, 10 5-OH  4.87, d (9.6) 4, 5 ,6 aHMBC correlations, optimized for 10 Hz, are from proton(s) stated to the indicated carbon. bPartially obscured.   Table 3.2 NMR spectroscopic data (600 MHz, DMSO-d6) for genkwanine P (3.2). 77  diacetyl genkwanine M (3.3) position ?C, multi. ?H, (J in Hz) HMBCa     1 35.3, CH2 1.79, mb 2, 10   1.56, dd (12.6, 12.0)  2 35.0, CH 1.69, q (4.8) 1, 19 3 77.7, CH 4.77, d (4.8) 2, 4, 5, 10, 23 4 80.9, C   5 71.4, CH 5.33, s 3, 6, 7, 21 6 58.7, C   7 64.0, CH 3.61, s 6, 8, 9, 14, 20 8 36.0, CH 3.16, m 6, 7, 9, 11, 14 9 79.6, C   10 48.4, CH 2.62, m 1, 2, 4, 5, 9, 11 11 34.4, CH 2.48, mb 9, 12, 13, 18 12 35.6, CH2 2.19, dd (14.1, 8.4) 9, 11, 13, 14, 15   1.64, d (14.1)  13 84.1, C   14 81.0, CH 4.75, d (2.4) 7, 9, 15 15 146.2, C   16 110.7, CH2 5.04, bs, 4.90, bs 13, 15, 17 17 18.9, CH3 1.80, s 13, 15, 16 18 20.7, CH3 1.21, d (6.6) 11, 12 19 12.9, CH3 0.83, d (4.8) 1, 2, 3 20 68.1, CH2 4.68, d (12.0) 1?, 5, 6   3.93, d (12.0)  21 170.0, C   22 20.8, CH3c 2.00, s 21 23 170.1, C   24 20.9, CH3c 2.05, s 23 1? 116.4, C   2? 136.3, C   3? 7? 125.7, CH 7.59, m 1?, 5? 4? 6? 127.9, CH 7.39, m 2?, 3?, 7? 5? 129.2, CH 7.39, m 2?, 3?, 7? 1? 165.4, C   2? 129.2, C   3? 7? 129.4, CH 7.98, d (7.2) 1?, 2?, 5? 4? 6? 128.7, CH 7.54, t (7.2) 2?, 3?, 7? 5? 133.5, CH 7.67, t (7.2) 3?, 7? 4-OH  4.68, s 3, 4, 5  aHMBC correlations, optimized for 10 Hz, are from proton(s) stated to the indicated carbon. bPartially obscured. cMay be interchanged.   Table 3.3 NMR spectroscopic data (600 MHz, DMSO-d6) for diacetyl genkwanine M (3.3). 78   Figure 3.9 13C NMR spectrum of genkwanine M (3.1) recorded in DMSO-d6 at 150 MHz.  Figure 3.10 ROESY spectrum of genkwanine M (3.1) recorded in DMSO-d6 at 600 MHz. 79   Figure 3.11 13C NMR spectrum of genkwanine P (3.2) recorded in DMSO-d6 at 150 MHz.  Figure 3.12 COSY spectrum of genkwanine P (3.2) recorded in DMSO-d6 at 600 MHz. 80   Figure 3.13 HSQC spectrum of genkwanine P (3.2) recorded in DMSO-d6 at 600 MHz.  Figure 3.14 HMBC spectrum of genkwanine P (3.2) recorded in DMSO-d6 at 600 MHz. 81   Figure 3.15 ROESY spectrum of genkwanine P (3.2) recorded in DMSO-d6 at 600 MHz.   Figure 3.16 1H NMR spectrum of diacetyl genkwanine M (3.3) recorded in DMSO-d6 at 600 MHz. 82   Figure 3.17 13C NMR spectrum of diacetyl genkwanine M (3.3) recorded in DMSO-d6 at 150 MHz.           83  Chapter 4: Virantmycin and Homoaerothionin are Two New Autophagy Inhibitors   4.1 Introduction Autophagy is a complex cellular process that involves the degradation and recycling of cytoplasmic components in autolysosomes which are formed with the fusion of autophagosomes and lysosomes. Human cells use basal autophagy to maintain normal cellular homeostasis and energy balance.31 Numerous literature reports showed that cancer cells can use autophagy as a cell survival mechanism to combat stresses associated with hypoxia and nutrient deprivation resulting from poor tumour vascularization. Furthermore, treatments with chemotherapeutic drugs and ionizing radiation lead to induction of autophagy in human cancer cell lines. Autophagy activation and the consequent production of autolysosomes enhance the clearance of damaged organelles and proteins increasing the resistance of tumor cells against chemotherapies.32a-c    Figure 4.1 Chemical structures of virantmycin (4.1)33 and homoaerothionin (4.2).34a,b  Preclinical studies have demonstrated that inhibition of autophagy by RNAi-mediated 84  knockdown of autophagy (Atg) genes or with small molecules can sensitize the response to anticancer treatments, both in cell culture and in vivo. Pancreatic adenocarcinoma (PAC) is unresponsive to current chemotharapies with a 5-years survival rate of ? 6% . New anticancer drugs that can overcome current treatment failures are needed in order to fight PAC. The identification of autophagy inhibitors can potentially lead to the discovery of new chemotherapeutics that may be more effective against PAC, thus developing new strategies to fight the disease. The antiviral agent virantmycin33 (4.1) and the bromotyrosine-derived alkaloid homoaerothionin34a,b (4.2) (Figure 4.1) were isolated from cultures of a red marine-derived actinomycete and a marine sponge extract, respectively. These two extracts were active in an automated cell-based assay for the inhibition of autophagy. The two pure compounds were identified as new autophagy inhibitors using a bioassay-guided fractionation approach.  4.2 Results and Discussion The crude extract of cultures of a red marine-derived actinomycete (RJA2505) was active in an automated cell-based assay for the inhibition of autophagy. In this assay, cells were treated with chloroquine, which causes the accumulation of autophagosomes by inhibiting their fusion with lysosomes. In the presence of an inhibitor of autophagy, the accumulation of autophagosomes is not observed. The active extract (RJA2505) was fractionated in order to isolate the pure active compound(s). The ethyl acetate extract of the actinomycete (RJA2505) culture was fractionated on a Sephadex? LH-20 column using 100% methanol as an eluent. A major red fraction was further chromatographed on a reversed phase-Sep Pack column using methanol/water step gradient to 85  afford three fractions. Only one fraction was active in the autophagy assay. Reversed phase-HPLC of this fraction, using acetonitrile/water (8/2) as an isocratic eluent, led to the isolation of a white amorphous solid (3.0 mg).    Figure 4.2 1H NMR spectrum of virantmycin (4.1) recorded in CDCl3 at 600 MHz.  The analysis of the LRESIMS provided clear evidence for the presence of a chlorine atom, because of the [M + 2] ion relative to the molecular ion at m/z 374. HRESIMS [M ? H]- was consistent with the molecular formula C19H25NO3Cl (m/z 350.1523). A search on Scifinder? identified the virantmycin chemical structure (4.1). The NMR spectroscopic data (1H NMR spectrum is shown in Figure 4.2) in CDCl3 matched the literature values reported for the potent antiviral compound.33 Virantmycin (4.1) (Figure 4.1) is a known antibiotic with antiviral activity.35 However, there is no information in the literature about its mechanism of action. Inhibition of autophagy by virantmycin (4.1) could be related to its antiviral activity.  86    Figure 4.3 1H NMR spectrum of homoaerothionin (4.2) recorded in acetone-d6 at 600 MHz.  Pure homoaerothionin (4.2) (4.0 mg) was isolated as a white solid from a marine sponge extract which was active in an automated cell-based assay for the inhibition of autophagy. Homoaerothionin (4.2) (Figure 4.1) is a known bromotyrosine-derived alkaloid previously isolated from the sponge V. aerophoba. It is reported to have antiproliferative activities against the human breast adenocarcinoma cancer cell line MCF-7.34a,b We have identified homoaerothionin (4.2) (1H NMR spectrum is shown in Figure 4.3) as a new autophagy inhibitor. As for virantmycin (4.1), the autophagy inhibitory activity of homoaerothionin (4.2) was not previously reported. Virantmycin (4.1) and homoaerothionin (4.2) represent two major findings considering the limited number of autophagy inhibitors available.  87  4.3 Conclusions Tumor cells use induction of autophagy36a,b as a defensive mechanism for their survival in extreme conditions.37a-d It has been reported that cancer cells can become addicted to autophagy in response to the metabolic stress caused by antitumor chemotherapies.38a,b The identification of the autophagy inhibitors virantmycin (4.1) and homoaerothionin (4.2) is relevant to the field of drug discovery. Virantmycin (4.1) and homoaerothionin (4.2) represent chemical tools for the study of autophagy functions in cancer cells and can be considered as new potential drug leads for the cure of pancreatic adenocarcinoma (PAC).  4.4 Experimental Section 4.4.1 General Experimental Procedures 1H and 13C NMR spectra were recorded on a Bruker AV-600 spectrometer with a 5 mm CPTCI cryoprobe. 1H chemical shifts are referenced to the residual chloroform-d1 (? 7.24 ppm) and acetone-d6 (? 2.05 ppm) signals. 13C chemical shifts are referenced to the chloroform-d1 peak (? 77.23 ppm) and acetone-d6 (? 29.92 ppm) solvent peaks. The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, quin = quintuplet, sext = sextet, sep = septet, b = broad. Low resolution ESI +/- were recorded on Bruker Esquire LC ion trap mass spectrometer equipped with an electrospray ion source. The solvent for ESI-MS experiments was methanol. The sample solution concentration was 10 ?M. It was infused into the ion source by a syringe pump at flow rate of 10 ?L/min. High resolution ESI+ were recorded on a Micromass LCT time-of-flight (TOF) mass spectrometer equipped with an electrospray ion source. The samples were dissolved in MeOH. The working solutions were 20 ?M. Flow rate: 20 ?L min-1; sample cone: 88  90 V; source temperature: 120 ?C; desolvation temperature: 120 ?C. Sephadex? LH-20 column packed and elueted with a mixture of 100 % MeOH was used for size separation chromatography. Reversed-phase HPLC purifications were performed on a Waters 600E System Controller liquid chromatography attached to a Waters 996 photodiode array detector using a C18 reversed-phase column (CSC-Inertsil 150A/ODS2, 5 ?m 25 x 0.94 cm). All solvents used for HPLC were Fisher HPLC grade. The isolation procedure was monitored by thin layer chromatography (TLC) carried out on Merck Type 5554 silica gel plates using UV light as visualizing agent and either an ethanolic solution of cerium sulfate or vanillin in ethanol/aqueous H2SO4, and heat as developing agents.  4.4.2 Isolation Procedure  Five grams of the crude extract of the red actinomycete #RJA2505 were then partitioned between EtOAc (3 ? 50mL) and H2O (100 mL). The combined EtOAc extract was evaporated to dryness to give 2.100 g of dark red solid; 2.100 g of this was chromatographed on a Sephadex? LH-20 column in 100% MeOH as an eluent to give fractions A-D. Fraction C (510.0 mg) was chromatographed on a reversed phase-Sep Pack column, employing a step gradient from 80:20 H2O/MeOH to MeOH, to afford fractions A-E. Pure virantmycin (4.1) (3.0 mg) was obtained as a white amorphous solid from the combined fraction B-C (30.0 mg), via C18 reversed- phase HPLC using 6:4 acetonitrile/H2O as an eluent over 40 min. (flow rate 2 mL/min). Four grams of the crude extract of a marine sponge were then partitioned between EtOAc (3 ? 50mL) and H2O (100 mL). The combined EtOAc extract was evaporated to dryness to give 1.100 g of dark brown oil; 1.100 g of this was chromatographed on a Sephadex? LH-20 column in 100% MeOH as an eluent to give fractions A-E. Fraction B (210.0 mg) was chromatographed 89  on a Waters 10g Sep-Pak?s for direct-phase flash chromatography, employing a step gradient from 95:5 hexane/EtOAc to EtOAc, and from 90:10 EtOAc/MeOH to MeOH, to give fractions A-E. Pure homoaerothionin (4.2) (4.0 mg) was obtained as a white amorphous solid from the combined fraction B-C (20.0 mg) via C18 reversed-phase HPLC using 7:2 acetonitrile/H2O as an eluent over 50 min. (flow rate 2 mL/min).  4.4.3 1H NMR, 13C NMR, HRESIMS and Optical Rotation Values for Virantmycin (4.1) and Homoaerothionin (4.2) Virantmycin (4.1). [?]25D -11.0 ? (c 0.01, CDCl3). 1H NMR (CDCl3, 600 MHz) ? 7.77 (1H, dd, J = 8.3, 2.0 Hz), 7.76 (1H, d, J = 2.0 Hz), 6.53 (1H, d, J = 8.3 Hz), 4.63 (1H, bs), 4.36 (1H, dd, J = 5.9, 4.9 Hz), 3.58 (1H, d, J = 9.2 Hz), 3.55 (1H, d, J = 9.2 Hz), 3.39 (3H, s), 3.37 (1H, dd, J = 17.1, 4.9 Hz), 3.11 (1H, dd, J = 17.1, 5.9 Hz), 2.09 (1H, dt, J = 12.2, 4.9 Hz), 2.01 (1H, dt, 12.2, 4.9 Hz), 1.81 (1H, ddd, J = 13.7, 12.2, 4.9 Hz), 1.63 (1H, ddd, J = 13.7, 12.2, 4.9 Hz), 1.63 (3H, s), 1.61 (6H, s). 13C NMR (CDCl3, 150 MHz) ? 171.9, 147.2, 132.4, 130.4, 126.5, 124.8, 117.7, 116.0, 113.5, 74.1, 59.4, 58.0, 56.2, 33.6, 33.5, 27.8, 20.6, 19.9, 18.4. HRESIMS [M    H]    m/z 350.1606 (calcd for C19H25NO3Cl, 350.1523). Homoaerothionin (4.2). 1H NMR (acetone-d6, 600 MHz) and 13C NMR (acetone-d6, 150 MHz) were consistent with the literature data.34a,b HRESIMS [M + H]+ m/z 850.8539 (calcd for C25H28N4O8Br4, 850.8538). 90   Figure 4.4 13C NMR spectrum of virantmycin (4.1) recorded in CDCl3 at 150 MHz.     Figure 4.5 COSY spectrum of virantmycin (4.1) recorded in CDCl3 at 600 MHz. 91   Figure 4.6 HSQC spectrum of virantmycin (4.1) recorded in CDCl3 at 600 MHz.    Figure 4.7 HMBC spectrum of virantmycin (4.1) recorded in CDCl3 at 600 MHz. 92   Figure 4.8 13C NMR spectrum of homoaerothionin (4.2) recorded in acetone-d6 at 150 MHz. 93  Chapter 5: Total Synthesis of Clionamine B and Unnatural Analogues, Autophagy-Stimulating Aminosteroids that clear Mycobacterium tuberculosis from Human Macrophages   5.1 Introduction Autophagy (Greek for ?self-eating?) is complex multistep process that is under the control of more than 35 autophagy-related (Atg) proteins.39a-c Although basic knowledge about the role of autophagy in cell biology and disease progression is increasing, the roles of individual Atg proteins remain unknown. The identification of new small molecules that can selectively modulate Atg protein function should be extremely useful for chemical genetics studies of autophagy.    Figure 5.1 Mechanism of autophagolysosome formation.  In order to discover new small molecule stimulators of autophagy, a library of marine 94  organism crude extracts was screened in a cell-based assay for the activation of autophagy. Clionamines A (5.1), B (5.2), C (5.3), and D (5.4) were isolated from the methanol extract of the sponge Cliona celata collected on the Wild Coast of South Africa (Figure 5.2).40 Clionamine A (5.1), the major component in the extract, strongly stimulated autophagy in human breast cancer MCF-7 cells in the screening assay. The combination of structural features present in the clionamines was not previously encountered in naturally occurring steroids. In particular, the 3?-amine group and the E ring ?-lactone with the C-20 ?-hydroxyl are present in clionamines A (5.1), B (5.2), and C (5.3), whereas the spirobislactone side chain was found only in clionamine D (5.4).     Figure 5.2 Chemical structures of clionamines A (5.1), B (5.2), C (5.3), and D (5.4).40 95  Autophagy (Figure 5.1) is an important defensive mechanism used by cells to clear pathogenic viruses41 and bacteria.42a-c It is well documented that human immune cells, such as macrophages, activate autophagy as a response to Mycobacterium tuberculosis (Mtb).43 Roberge and coworkers have recently shown that the antiprotozoal drug nitazoxanide (6.7) strongly stimulates autophagy and inhibits Mtb growth in infected THP-1 cells.44 Similarly, Schreiber and coworkers have discovered a group of small molecule activators of autophagy that enhance killing of intracellular mycobacteria by human macrophages.45 Tuberculosis (TB) is caused by infection with the microbial pathogen Mycobacterium tuberculosis. The World Health Organization estimated that in 2011 there were 1.4 million deaths and 8.7 million new cases of TB worldwide. Reported cases of multidrug resistant TB (MDR-TB) are on the increase, numbering almost 60,000 worldwide in 2011. There is an urgent need to find new approaches to treating MDR-TB and latent TB infections that are not responsive to current antimycobacterial chemotherapies. The primary site of Mtb infections are the human alveolar macrophage cells. This route of infection exposes Mtb to the innate immune system defenses that human cells deploy to destroy invading pathogens. To counter this threat, Mtb has developed sophisticated mechanisms that enable it to thwart the macrophage innate immune defenses and replicate.46a-d One of the most important components of the macrophage innate immune system is autophagy.47a-c Autophagy functions as an effective defense mechanism adopted by macrophages against microbial pathogens. Mtb avoids clearance via autophagy by interfering with membrane trafficking, which prevents phagosome maturation and ultimately phagosome-lysosome fusion. It is generally accepted that Mtb can reside and multiply in macrophages due to its ability to inhibit autophagy and recent evidence suggests that Mtb?s ability to escape autophagy-mediated death 96  determines its pathogenicity.48a,b Based on this knowledge, it has been proposed that stimulation of autophagy by small molecule drugs might be a promising approach to the clearance of Mtb from colonized macrophages. Natural clionamine A (5.1) was tested for its ability to clear Mtb from infected THP-1 cells (human monocytic cells) and it gave a clear dose response with complete clearance at 5 ?M and an IC50 of ? 3 ?M with no observed cytotoxicity to the host THP-1 cells (Figure 5.3). These biological results confirmed the potential of autophagy-stimulating compounds to promote clearance of Mtb from macrophages and prompted us to further investigate the ability of the clionamines to clear Mtb from macrophages.    Figure 5.3 Natural clionamine A (5.1) clears Mtb from infected THP-1 at 5 ?M (IC50 ? 3 ?M).  The total synthesis of clionamine B (5.2) and unnatural analogues was undertaken in order to provide sufficient material for further biological evaluation in vitro and in vivo.49 The synthesis also provided a number of unnatural clionamine analogues that revealed SAR for this 97  new autophagy-activating pharmacophore. Since the ultimate goal was to be able to generate sufficient quantities of a natural clionamines for in vivo studies in animal models, the synthesis was designed to use cheap starting materials and reagents. The steroidal sapogenins tigogenin (5.30) and sarsasapogenin (5.5) were used as the starting materials for the total synthesis of clionamine B (5.2) and unnatural analogues, respectively.    5.2 Total Synthesis of Clionamine B (5.2) and Unnatural Analogues 5.2.1 Synthesis of 3,5-epi-Clionamine B (5.25) and Analogues Aglycones of steroidal saponins (also called sapogenins) are common plant metabolites and have been extensively used as starting materials for the total synthesis of steroidal hormone drugs.50a-e Tigogenin (5.30) and sarsasapogenin (5.5) are two of the most common sapogenins which are readily available in kg quantities at low costs. Furthermore, degradation reactions of sapogenin side chains to give the E ring ?-lactone found in the clionamines are well described in the literature.51 For these reasons, tigogenin (5.30) and sarsasapogenin (5.5) were selected as the starting materials for the total synthesis of clionamine B (5.2) and 3,5-epi-clionamine B (5.25), respectively (Scheme 5.1). It has been reported that the bromination of sarsasapogenin (5.5), which has the opposite configurations at both C-5 and C-25 compared with tigogenin (5.30), only gives the desired equatorial bromide 5.7, due to steric interactions of bromine with C-25 axial methyl during the bromination step (Scheme 5.2). The axial C-23 bromide 5.32, which is obtained in a mixture with the equatorial bromide after bromination of tigogenin (5.30), cannot be converted to bistetrahydrofuran intermediates, so its formation drastically reduces the overall yield of the side chain degradation. Therefore, considering its higher efficiency, we have used sarsapogenin (5.5) 98  as the starting material to explore reaction conditions and synthesize 3,5-epi-clionamine B (5.25) and unnatural analogues. Subsequently, having optimized reaction conditions, we planned to use tigogenin (5.30) as the starting material for the total synthesis of clionamine B (5.2), since the C-5 configuration in tigogenin (5.30) structure is the same of natural clionamine B (5.2). Contrarily, in sarsapogenin (5.5) the C-5 configuration is opposite compared to natural clionamines, therefore by using sarsasapogenin (5.5) as the starting material it is possible to generate only 5-epi-clionamines (Scheme 5.1).    Scheme 5.1 Retrosynthetic analysis for the total synthesis of clionamine B (5.2) and 3,5-epi-clionamine B (5.25).  Acetylation of sarsasapogenin (5.5) with acetic anhydride and a catalytic amount of DMAP followed by bromination with Br2 in glacial acetic acid52 proceeded in good yield (85% over two steps) to give the single stereoisomer 5.7 with the bromine atom in the equatorial orientation on the tetrahydropyran ring (Scheme 5.2). Treatment of equatorial bromide 5.7 with aqueous ammonia in 1-butanol at reflux for 7 days led to the formation of hemiketal 5.8. The published 99  conditions51 for this reaction (aqueous ammonia in 1-butanol at reflux for 7 days, followed by evaporation under vacuum of the reaction mixture without any aqueous workup) led to significant epimerization at the tetrahydrofuran methine center C-23 in the hemiketal product 5.8. Hemiketal 5.8 was found to readily epimerize at basic pH, but this can be completely suppressed by using a buffer solution (pH = 7.0) to neutralize the reaction during workup and obtain the desired product 5.8 (70% yield) and unreacted starting material 5.7 (see experimental section). The acetate ester in 5.8 was cleaved with K2CO3 in MeOH prior to simultaneous oxidation of the secondary alcohol to the ketone and oxidative degradation of the hemiketal with pyridinium chlorochromate (PCC) in CH2Cl2 to give the ?-lactone 5.10. The use of crushed 4 ? molecular sieves improved the yield and facilitated the isolation of product 5.10.53 Attempts to transform the C-23 epimer of 5.8 using the same hydrolysis and PCC oxidation conditions gave none of the desired ?-lactone product 5.10. Thus, the relative configuration of C-23 in 5.9 is critical for the efficiency of the oxidative degradation step, which highlights the importance of the suppressing the epimerization of 5.8 during its production from 5.7. The optimized yield for the 5 step conversion of 5.5 to 5.10 was 43% (Scheme 5.2). Reductive amination of the C-3 ketone in lactone 5.10 with benzylamine and sodium triacetoxyborohydride [NaBH(OAc)3] gave only the benzylamine 5.11 with the ? configuration at C-3 in 72% yield (Scheme 5.2).54a,b Sodium triacetoxyborohydride is a bulky hydride donor that can preferentially approach from the less hindered face (steric approach control). The top face of ring A in lactone 5.10 is less hindered compared with the bottom face which is blocked by the presence of ring B (see Figure 5.79 in the experimental section for minimum energy conformation of lactone 5.10, page 180). The alternative diastereomeric benzylamine with the ? 100  configuration at C-3 was not observed. Furthermore, there was no evidence for the benzylamine reacting with the ?-lactone. N-debenzylation of 5.11 using ammonium formate (NH4HCO2) and palladium on charcoal (Pd/C) catalysis removed the benzyl group to give the primary amine 5.12 in 90% yield.55 Compound 5.12 has the 3-amino and E ring ?-lactone moieties found in the clionamines, but differs from the natural products by having a truncated alkyl side chain, the absence of a C-20 hydroxyl, the 3-amino group with the ? configuration at C-3, and C-5 with the opposite configuration.    Scheme 5.2 Degradation of sarsasapogenin (5.5) side chain and synthesis of compound 5.12.  The further elaboration of lactone 5.10 towards the synthesis of 3,5-epi-clionamine B (5.25) and analogues started with transformation of the C-3 ketone to a cyclic ketal by reaction 101  with 1,3-propandediol and p-toluenesulfonic acid (TsOH) to give the key intermediate 5.13 in 90% yield (Scheme 5.3). A pivotal step in the synthesis of a clionamine involved introducing an ?-hydroxyl group at C-20 in 5.13. The initial synthetic plan envisioned using a Rubottom oxidation56a-c to carry out this hydroxylation. Numerous attempts were made to convert the lactone carbonyl in 5.13 into the corresponding silyl enol ether intermediate required for the subsequent mCPBA oxidation in the Rubottom sequence. However, standard conditions using LDA and TMSCl in THF from -78 ?C to room temperature returned only starting lactone 5.13 and TMSOTf and DIPEA in DCM at 0 ?C only removed the C-3 ketal to give lactone 5.10.57a,b The observed difficulty in forming the desired silyl enol from 5.13 was attributed to two factors. First, the lactone ?-hydrogen in 5.13 is close in space to the axial C-18 methyl in the energy-minimized conformation of 5.13 (2.707 ? is the calculated distance between the ?-hydrogen and C-18 using ChemBio3D?), making deprotonation with a sterically hindered base (either LDA or DIPEA) potentially difficult. Second, after deprotonation the resultant lactone enolate anion might be relatively unstable due to ring strain. Faced with these challenges, we decided to explore alternate approaches. Alkyl enol ethers are relatively easy to synthesize and methyl enol ethers can undergo ?-hydroxylation upon exposure to a variety of oxidation methods.58a,b Lactone 5.13 was treated with a large excess of t-BuOK and (CH3)2SO4 in dimethoxyethane (DME) at 0 ?C in an attempt to convert it to the corresponding methyl enol ether 5.15. However, the reaction only produced a low yield of the ?-methoxy lactone 5.14 (Scheme 5.3). It has been reported that t-BuOK and DME at low temperatures is a very efficient base-solvent combination to generate lactone enolates that can subsequently react with molecular oxygen.59a-c Therefore, it appeared that the 102  lactone enolate of 5.13 had reacted with oxygen to give alpha hydroxylation, which was followed by methylation with (CH3)2SO4 to generate the ?-methoxy lactone 5.14. Capitalizing on this serendipitous observation, conditions for just the ?-hydroxylation of 5.13 were developed and optimized taking advantage of literature precedents for related transformations.     Scheme 5.3 Synthesis of compound 5.18 and ?-hydroxylation of lactone 5.13.  The best conditions for the transformation of 5.13 to 5.16 involved bubbling oxygen gas (O2) into a cold solution of 5.13 in DME containing excess triethyl phosphite [P(OEt)3] and t-BuOK in DME. The O2 was bubbled through the solution for 4 hours starting at -41 ?C and warming to 0 ?C, followed by an acidic quench with 10% HCl to simultaneously neutralize the excess base and hydrolyze the C-3 ketal to the ketone. Using these optimized conditions, the 103  transformation of 5.13 to 5.16, which gave only the clionamine ?-methyl configuration at C-20, proceeded in 70% yield. Reductive amination with benzyl amine followed by N-debenzylation converted ketone 5.16 to the clionamine analogue 5.18 (Scheme 5.3). The remaining requirement to complete the synthesis of a natural clionamine was to develop an approach to elaborating the C-20 alkyl substituent into one of the naturally occurring side chains. Conjugate addition of an alkyl anion to an E ring ?-methylene lactone offered the flexibility needed to add the different side chains required to synthesize 3,5-epi-clionamine B (5.25) and analogues for SAR studies. Towards this end, ?-hydroxylactone 5.16 was first reacted with mesyl chloride (MesCl) and triethylamine (TEA) in CH2Cl2 at 0 ?C to give the mesylate 5.19 in 85% yield (Scheme 5.4).60 The plan was to form the exocyclic alkene via anti elimination of the mesylate by using a sterically hindered base such as DBU or t-BuOK,61a-c which requires an anti-periplanar orientation of the ? bonding orbital of the C-H bond that is being deprotonated with the ?* orbital of the C-O mesyl bond that is breaking. Since the mesylate group in 5.19 is syn to the lactone ?-methine, the required anti-periplanar orientation is only possible between the mesylate and the C-H bonds of the adjacent methyl, which predicted that only the exocyclic alkene would be formed. In order to prevent side reactions of the C-3 ketone with the base that was to be used to catalyze the elimination reaction, 5.19 was reacted with 1,3-propanediol and TsOH in refluxing toluene containing molecular sieves in order to protect it as the cyclic ketal.  Under these conditions, not only did the C-3 ketal form, but the mesylate also cleanly eliminated to give the desired ?-methylene lactone 5.21 in one step in 68% yield. No endocyclic double bond formation was observed (Scheme 5.4). Isopentylmagnesium bromide 5.20 was prepared using standard conditions and then added 104  to a THF solution of 5.21 in the presence of copper(I) iodide at 0 ?C. The conjugate addition62a-d worked efficiently, but gave a mixture of the C-20 ? and ? alkyl products 5.22 in a ratio of 4:1, indicating that protonation of the enolate anion intermediate preferentially occurred from the less sterically hindered ? face (the bottom face). Attempts to scale up the reaction to tens of milligram scales using the original conditions led to a significant decrease in the yield of 5.22. Slowly adding a large excess of Grignard reagent 5.20 to lactone 5.21 in the presence of a catalytic amount of copper(I) iodide at 0 ?C gave lactone-ring opening as a side reaction (isopentylmagnesium bromide 5.20 attacks the carbonyl of the lactone). Lowering the reaction temperature to ? 78 ?C and using an excess of copper(I) iodide rather than a catalytic amount suppressed the lactone-opening side reaction and gave good yields on larger scales.     Scheme 5.4 Final steps in the synthesis of 3,5-epi-clionamine B (5.25).  105  The mixture of C-20 epimers 5.22 was ?-hydroxylated using the same conditions developed for the hydroxylation of 5.13 [bubbling O2 into a cold solution of 5.22 in DME containing excess P(OEt)3 and t-BuOK] to give the single C-20 ?-hydroxy product 5.23 in 60% yield. Ketone 5.23 was then converted into 3,5-epi-clionamine B (5.25) (63% over two steps) via the reductive amination followed by N-debenzylation (Scheme 5.4). Furthermore, ketal 5.22 was converted to the clionamine analogue 5.29 over 3 steps in order to probe the role of the C-20 alcohol in the autophagy activating properties of the clionamine pharmacophore (Scheme 5.5).    Scheme 5.5 Synthesis of compound 5.29.  3,5-epi-Clionamine B (5.25) and compounds 5.12, 5.18, 5.23, 5.24, 5.29, were tested for both activation of autophagy and inhibition of Mtb proliferation in MCF-7 cells and THP-1 cells, respectively. The analysis of their biological activity describes the clionamine pharmacophore and defines the role of autophagy in the response of human macrophages against Mtb infection 106  (Figure 5.5, Figure 5.6, Figure 5.7, and Figure 5.8).          5.2.2 Total Synthesis of Clionamine B (5.2) The total synthesis of clionamine B (5.2) (Scheme 5.1) starting from the plant sapogenin tigogenin (5.30) involved the same chemical reactions and synthetic methodologies used for the synthesis of 3,5-epi-clionamine B (5.25) from sarsasapogenin (5.5). However, there are some remarkable differences between the two synthetic pathways. In the clionamine B (5.2) synthetic pathway the bromination step of 5.31 gave a mixture of bromides 5.32, drastically decreasing the yield of the hemiketal formation step to 26%. Furthermore, the solubility of the tigogenin intermediates, especially compounds 5.32-5.37, turned out to be extremely low at cold temperatures making the execution of every single step much more difficult. We were able to overcome this issue by making appropriate adjustments to the reaction conditions previously established in the sarsasapogenin synthetic series.49 The synthesis of clionamine B (5.2) started with acetylation of tigogenin (5.30) to give the acetate 5.31 in nearly quantitative yield.51 Treatment of 5.31 with Br2 in glacial acetic acid led to bromination at C-23 on the dihydropyran ring adjacent to the spiro ketal (Scheme 5.6).52 A mixture of the axial and equatorial brominated products 5.32 was obtained in 80% combined yield. We were not able to calculate the diastereomeric ratio of the mixture since it was impossible to separate the two diastereoisomers. Treatment of the epimeric mixture of bromides 5.32 with aqueous ammonia in 1-butanol at reflux for 7 days led to the formation of the hemiketal product 5.33 in a modest 26% yield.51 The low yield was anticipated because it was known that the C-23 axial bromides do not effectively rearrange to give C-22 hemiketals 5.33 under these conditions. Hydrolysis of the acetate 5.33 with K2CO3 in MeOH gave the alcohol 107  5.34 in 91% yield. Oxidative degradation of the hemiacetal 5.34 was carried out by treatment with PCC in DCM at room temperature in the presence of 4 ? molecular sieves to give the ketone 5.35, containing the E ring ?-lactone found in the clionamines, in 90% yield.51       Scheme 5.6 Degradation of tigogenin (5.30) side chain and ?-hydroxylation of 5.36.49  Installation of the C-20 hydroxyl group was expected to be a key transformation in the synthesis of clionamine B (5.2). To set the stage for this transformation, the C-3 ketone in 5.35 was protected as the cyclic ketal 5.36 in 92% yield, to prevent unwanted base catalyzed condensation and ketone ?-oxidation side reactions (Scheme 5.6). The reaction conditions used for the ?-hydroxylation of E ring ?-lactone in 5.36 were slightly different compared with those used in the 3,5-epi-clionamine B (5.25) synthetic route. The temperature of -41 ?C 108  (acetonitrile/dry ice) made the solubility of 5.36 in 1,2-dimethoxyethane extremely low, and the first attempt to oxidize 5.36 to 5.37 led to the recovery of starting material 5.36 with no product formation. By optimizing reaction conditions, formation of the ?-hydroxy-lactone 5.37 was achieved by using t-BuOK in DME at -10 ?C (brine/ice) in the presence of a stream of oxygen gas and triethyl phosphite followed by a 10% HCl workup with 51% yield. The hydroxyl group at C-20 has the same configuration (?-orientation) as the natural clionamines.    Scheme 5.7 Final steps in the synthesis of clionamine B (5.2).  With the C-20 hydroxyl group in hand, we turned our attention to dehydration as a route to an E ring ?-methylene lactone intermediate. Mesylation60 of 5.37 gave the sulfonate ester 5.38 in 96% yield. During the ketalization reaction on 5.38, the mesylate also eliminated as happened in the sarsasapogenin synthetic series to give the desired E ring ?-methylene lactone 5.40 in 92% yield. Copper-mediated addition62a-d of the isopentyl Grignard reagent 5.39 to the ?-methylene lactone 5.40 gave compound 5.41 as a 4:1 mixture of ? and ? epimers in 50% overall yield. 109  Hydroxylation of the mixture of C-20 epimers 5.41 using the same enolate oxidation conditions developed for lactone 5.36 gave the C-20 hydroxy derivative 5.42 as a single epimer having the clionamine configuration in 65% yield. Approach of molecular oxygen to the ? face of C-20 is completely blocked by steric interaction with methyl-18. Reductive amination of 5.42 using ammonium acetate (CH3CO2NH4) and sodium cyanoborohydride63 [NaBH3(CN)] gave clionamine B (5.2) in 90% yield. The 1H NMR spectrum recorded for synthetic clionamine B (5.2) at 600 MHz in MeOD was identical to the 1H spectrum of natural clionamine B (5.2) recorded in the same solvent (Figure 5.78).49 The total synthesis of clionamine B (5.2) confirms the proposed structure of the natural product. Furthermore, we were able to show for the first time that clionamine B (5.2) strongly stimulates autophagy, with similar potency compared with clionamine A (5.1) (Figure 5.4).40, 49     Figure 5.4 Autophagy stimulation by natural clionamine A (5.1) and synthetic clionamine B (5.2). MCF-7 cells expressing the autophagy marker EGFP-LC3 were incubated for 4 h with 5.1 or 5.2, and autophagosomes (green puncta) were measured. Cell nuclei are in blue (adapted from: Forestieri, R.; Donohue, E.; Balgi, A.; Roberge, M.; Andersen, R. J. Org. Lett., 2013, 15, 3918-3921). 110  5.3 Discussion Autophagy activation is a promising strategy to fight tuberculosis infection. Human macrophages, after being infected by Mtb, activate autophagy as a clearance mechanism to eliminate the pathogenic bacteria. However, Mtb responds by generating biological responses that thwart autophagosome functions, resulting into inhibition of autophagy in macrophages. Small molecules that can activate autophagy in human macrophages infected by Mtb are potential anti-TB drug candidates. Clionamine B (5.2) and unnatural analogues are new autophagy-stimulating aminosteroids that clear Mycobacterium tuberculosis from human macrophages at low concentrations showing no cytotoxicity to the macrophages. Clionamines represent a new category of naturally-occurring molecules with promising anti-TB properties. The total synthesis of clionamine B (5.2) and unnatural analogues from steroidal sapogenins and the consequent production of a sufficient amount of compounds for biological testing allow further studies on the role of autophagy in Mtb-infected human macrophages. The use of tigogenin (5.30) and sarsasapogenin (5.5) as the starting materials for the total synthesis of natural and unnatural clionamines turned out to be a very efficient synthetic strategy. Furthermore, the stereoselective ?-hydroxylation of the E ring ?-lactone followed by mesylation of the obtained tertiary alcohol and thermal elimination of the mesylate group represent high yielding synthetic methodologies for both the oxidation of sterically hindered and strained lactones and the synthesis of E ring ?-methylene lactones. The copper-mediated conjugate addition to the E ring ?-methylene lactone using Grignard reagents and CuI that generate in situ organo-copper intermediates is also an efficient method for the formation of carbon-carbon bonds between aliphatic chains and enones with low electrophilic character. All these synthetic steps may find new applications in the total synthesis of other natural products. 111   Figure 5.5 Effect of 3,5-epi-clionamine B (5.25) and compounds 5.12, 5.18, 5.23, 5.24, and 5.29, on Mtb proliferation and THP-1 viability.  The identification of new small molecules that selectively activate autophagy in Mtb-infected human macrophages is central in the discovery of anti-TB drug. Clionamines are new 112  naturally-occurring molecules that strongly activate autophagy leading to Mtb-clearance from human macrophages. We have described an efficient synthetic route for the production of these autophagy activators, solving the supply issue and allowing further biological evaluation.  In our effort of discovering potential anti-TB drugs with selectivity and low cytotoxicity, we have indentified the N-benzyl-aminosteroid (5.24) as a new potent antimicrobial compound (Figure 5.5 and Figure 5.6). Compounds 5.24 and other new N-benzyl-aminosteroids, that we are currently synthesizing in our lab, are showing a strong ability of killing TB in human macrophages. The role of autophagy activation in this potent anti-TB activity is under investigation.  Compound MICs (?g / mL) 3,5-epi-Clionamine B (5.25) 60 Compound 5.12 > 60 Compound 5.18 > 60 Compound 5.23 > 60 Compound 5.24 5 Compound 5.29 30  Figure 5.6 Minimum inhibitory concentrations (MICs) of 3,5-epi-clionamine B (5.25) and compounds 5.12, 5.18, 5.23, 5.24, and 5.29.  The ability of activating autophagy in Mtb-infected human macrophages is clearly related to the TB-clearance activity by the clionamines. The high efficiency of the clionamine synthetic route allows the generation of photoaffinity probes for both the clionamines that are able to kill TB via autophagy activation and the N-benzyl-aminosteroids that are antimicrobial compounds 113  with anti-TB properties. The evaluation of the biological activity of these chemical probes in both the autophagy activation and anti-TB assays is preliminary to the identification of the molecular protein target(s) of the clionamines via Click chemistry.6a,b        5.4 Conclusions The naturally occurring aminosteroid clionamine B (5.2) was synthesized starting from the steroidal sapogenin tigogenin (5.30) in 12 steps with ? 2 % overall yield. Synthetic clionamine B (5.2) strongly stimulates autophagy at 30 ?g/mL and inhibits Mtb proliferation in human macrophages via autophagy activation.49 The clionamine pharmacophore (SAR summary of clionamine analogues for autophagy activation is shown in Figure 5.7) was identified by structure-activity analysis of unnatural clionamine analogues that were synthesized from the steroidal sapogenin sarsasapogenin (5.5).    Figure 5.7 SAR summary of clionamine analogues for autophagy activation.  114  Among these synthetic analogues, N-benzyl-3,5-epi-clionamine B (5.24) was found to be a potent inhibitor (MIC = 5 ?g/mL) of Mtb proliferation in THP-1 human acute monocytic leukemia cells, indentifying N-benzyl-aminosteroids as a new category of potent antimicrobial compounds that are able to kill TB. The synthesis of more potent clionamine analogues and photoaffinity probes for the identification of the molecular targets involved in the activation of autophagy in Mtb-infected human macrophages is part of our current and future research.    Figure 5.8 Autophagy stimulation by natural clionamine A (5.1), 3,5-epi-clionamine B (5.25), and compounds 5.12, 5.18, 5.23, 5.24, and 5.29.  115  5.5 Experimental Section 5.5.1 General Experimental Procedures  All reactions were carried out with dry solvents under argon atmosphere in anhydrous conditions, unless otherwise noted. Commercially available anhydrous 1-butanol (n-BuOH), dichloromethane (DCM), diethyl ether, 1,2-dimethoxyethane (DME), methanol (MeOH), tetrahydrofuran (THF) and toluene were used to the perform the reactions, unless otherwise stated. Yields refer to chromatographically and spectroscopically (1H NMR, 13C NMR) homogeneous materials, unless otherwise stated. Reagents were purchased at the highest commercial quality and used without further purification, unless otherwise stated. Reactions were monitored by thin layer chromatography (TLC) carried out on Merck Type 5554 silica gel plates using UV light as visualizing agent and a solution of p-anisaldehyde in ethanol/aqueous H2SO4, and heat as developing agents. Flash chromatography was performed using Silicycle Ultra Pure silica gel (230-400 mesh). Reversed-phase HPLC purifications were performed on a Waters 600E System Controller liquid chromatography attached to a Waters 996 photodiode array detector. All solvents used for HPLC were Sigma Aldrich HPLC grade. The 1H and 13C NMR spectra were recorded on a Bruker AV-600 spectrometer with a 5 mm TCI or QNP cryoprobe . 1H and 13C chemical shifts are referenced to the residual solvent signals (benzene-d6: ?H 7.16, ?C 128.39; methylene chloride-d2: ?H 5.32, ?C 54.00; DMSO-d6: ?H 2.50, ?C 39.51; methanol-d4: ?H 3.31, ?C 49.15). The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, quin = quintuplet, sext = sextet, sep = septet, b = broad. Low resolution ESI +/- were recorded on Bruker Esquire LC ion trap mass spectrometer equipped with an electrospray ion source. The solvent for ESI-MS experiments was methanol. 116  The sample solution concentration was 10 ?M. It was infused into the ion source by a syringe pump at flow rate of 10 ?L/min. High resolution ESI+ were recorded on a Micromass LCT time-of-flight (TOF) mass spectrometer equipped with an electrospray ion source. The samples were dissolved in methanol. The working solutions were 20 ?M. Flow rate: 20 ?L min-1; sample cone: 90 V; source temperature: 120 ?C; desolvation temperature: 120 ?C. The purity of compounds was evaluated via LC-MS: 400 ?L/min; dissolved in 0.1 mL of MeOH; dil 2x; XDB C18 column; 15%-100% acetonitrile 0.1% FA gradient for 20 minutes; ESI+. Samples of all compounds tested for autophagy activation in the cell-based assay were >95% pure.  5.5.2 1H NMR, 13C NMR, HRESIMS Values and Synthetic Methods for Clionamine B (5.2), 3,5-epi-Clionamine B (5.25), and Compounds 5.5-5.42  Compound 5.6. To a solution of sarsasapogenin (5.5) (100 mg, 2.4 ? 10-4 mol) in THF (10 mL) were added acetic anhydride (110 ?L, 1.2 ? 10-3 mol) and 4-(dimethylamino)pyridine (2.9 mg, 2.4 ? 10-5 mol). The solution was stirred for 24 hours. TLC analysis of the reaction shows a complete disappearance of the starting material. The reaction mixture was quenched with water (50 mL) and then extracted with dichloromethane (3 ? 50 mL). The combined organic phase was washed with water (2 ? 100 mL), dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.6 (105 mg, 95%) as a white solid. HRESIMS [M + H]+ m/z 459.3484 (calcd for C29H47O4, 459.3474). Compound 5.7. To a solution of compound 5.6 (100 mg, 2.2 ? 10-4 mol) in glacial acetic acid (4.0 mL) was added dropwise a solution of bromine (13 ?L, 2.6 ? 10-4 mol) in glacial acetic acid (1.0 ml). The solution was heated to 35 ?C and stirred for 1 hour. TLC analysis of the 117  reaction shows a complete disappearance of the starting material. The reaction mixture was quenched with water (50 mL) and then extracted with dichloromethane (3 ? 50 mL). The combined organic phase was washed with water (2 ? 100 mL), dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.7 (104 mg, 89%) as a yellow gum. 1H NMR (600 MHz, DMSO-d6) ? 4.94 (bs, 1H), 4.50 (dd, J = 13.2, 4.8 Hz, 1H), 4.31 (m, 1H), 3.82 (dd, J = 10.8, 2.4 Hz, 1H), 3.24 (d, J = 11.4 Hz, 1H), 2.44 (t, J = 6.6 Hz, 1H), 2.36 (m, 1H), 1.99 (s, 3H), 1.95 (m, 1H), 1.93 (m, 2H), 1.86 (m, 1H), 1.84 (m, 1H), 1.71 (m, 1H), 1.69 (m, 1H), 1.59 (m, 1H), 1.57 (m, 1H), 1.49 (m, 2H), 1.48 (m, 1H), 1.38 (m, 4H), 1.22 (m, 5H), 1.14 (m, 1H), 1.06 (d, J = 7.2 Hz, 3H), 1.05 (m, 1H), 0.94 (s, 3H), 0.93 (d, J = 9.0 Hz, 3H), 0.81 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 169.9, 108.9, 80.9, 69.9, 63.6, 60.8, 55.3, 48.5, 40.9, 39.6a, 39.5a, 39.4a, 38.0, 37.9, 36.9, 34.6, 31.2, 31.1, 30.5, 30.0, 26.0, 25.9, 24.4, 23.7, 21.2, 20.5, 16.5, 16.4, 13.9. HRESIMS [M + H]+ m/z 537.2576 (calcd for C29H46O4Br, 537.2579). a: partially obscured                                                                                                                                                                            Compound 5.8. To a solution of compound 5.7 (100 mg, 1.9 ? 10-4 mol) in 1-butanol (50 mL) was added ammonium hydroxide solution (7.0 mL, 28% NH3 in H2O). The solution was stirred and refluxed for 7 days. Additional aqueous ammonium hydroxide solution (3 ? 5.0 mL) was added during 7 days period. TLC analysis of the reaction shows the presence of unreacted starting material along with a new product. The reaction was cooled to room temperature and the top layer and the bottom layer were separated. The top layer was quenched with a buffer solution (50 mL, pH = 7.0) and the resultant solution was stirred for 20 minutes. The mixture was then extracted with ethyl acetate (100 mL) and water (50 mL). The ethyl acetate layer was washed with water (2 ? 50 mL), dried over MgSO4 and evaporated under vacuum. The product was 118  purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.8 (62 mg, 70%) as a yellow gum. 1H NMR (600 MHz, DMSO-d6) ? 4.99 (s, OH), 4.94 (bs, 1H), 4.45 (q, J = 7.8 Hz, 1H), 3.86 (m, 1H), 3.83 (m, 1H), 3.17 (t, J = 7.8 Hz, 1H), 2.25 (sext, J = 7.2 Hz, 1H), 2.07 (m, 1H), 2.03 (m, 1H), 1.98 (s, 3H), 1.94 (d, J = 12.0 Hz, 1H), 1.86 (m, 1H), 1.83 (m, 1H), 1.67 (m,1H), 1.63 (m, 1H), 1.57 (m, 1H), 1.50 (m, 4H), 1.34 (m, 5H), 1.21 (m, 2H), 1.15 (m, 5H), 0.95 (d, J = 7.2 Hz, 3H), 0.94 (d, J = 6.6 Hz, 3H), 0.93 (s, 3H), 0.73 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 169.9, 109.1, 81.7, 80.3, 74.5, 69.9, 63.1, 55.6, 40.6, 39.7a, 39.5a, 38.3, 36.9, 34.8, 34.6, 34.3, 32.9, 31.6, 30.5, 30.0, 26.0, 25.9, 24.4, 23.7, 21.2, 20.4, 17.9, 16.4, 16.1. HRESIMS [M + Na]+ m/z 497.3250 (calcd for C29H46O5Na, 497.3243). a: partially obscured                                                                                                                                                                                Compound 5.9. To a solution of compound 5.8 (100 mg, 2.1 ? 10-4 mol) in methanol (20 mL) was added anhydrous K2CO3 (152 mg, 1.1 ? 10-3 mol). The resultant solution was stirred for 48 hours. TLC analysis of the reaction shows a complete disappearance of the starting material. The reaction mixture was quenched with a saturated solution of ammonium chloride (100 mL) and extracted with ethyl acetate (2 ? 100 mL). The combined organic phase was washed with water (2 ? 100 mL), dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.9 (73 mg, 80%) as a yellow gum. HRESIMS [M + Na]+ m/z 455.3129 (calcd for C27H44O4Na, 455.3137). Compound 5.10. To a solution of compound 5.9 (100 mg, 2.3 ? 10-4 mol) in dichloromethane (50 mL) were added pyridinium chlorochromate (496 mg, 2.3 ? 10-3 mol) and 4 ? molecular sieves (250 mg, powder). The solution was vigorously stirred for 3 days. TLC analysis of the reaction shows a complete disappearance of the starting material. The reaction 119  mixture was diluted with diethyl ether (3 ? 50 mL). The resultant solution was then filtered through a column (silica gel, 100% diethyl ether), dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.10 (72 mg, 90%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 4.99 (m, 1H), 2.74 (t, J = 14.4 Hz, 1H), 2.59 (q, J = 7.8 Hz, 1H), 2.35 (dt, J = 14.4, 6.0 Hz, 1H), 2.22 (quin, J = 6.0 Hz, 1H), 1.95 (m, 2H), 1.88 (d, J = 7.2 Hz, 1H), 1.84 (m, 2H), 1.75 (m, 2H), 1.61 (m, 1H), 1.54 (m, 1H), 1.44 (m, 2H), 1.35 (m, 1H), 1.30 (m, 2H), 1.20 (d, J = 7.8 Hz, 3H), 1.18 (m, 4H), 0.97 (s, 3H), 0.67 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 211.7, 180.7, 82.2, 58.1, 53.4, 43.5, 41.8, 41.4, 39.6a, 37.4, 36.7, 36.4, 35.3, 34.6, 34.5, 32.5, 26.1, 25.4, 22.2, 20.1, 17.5, 13.3. HRESIMS [M + Na]+ m/z 367.2245 (calcd for C22H32O3Na, 367.2249). a: partially obscured                                                                                                                                                                             Compound 5.11. To a solution of compound 5.10 (100 mg, 2.9 ? 10-4 mol) in 1,2-dichloroethane (10 mL) was added benzylamine (38 ?L, 3.5 ? 10-4 mol), the resultant solution was stirred for 1 hour. Glacial acetic acid (17 ?L, 2.9 ? 10-4 mol) was then added to the solution which was stirred for 1 hour. Sodium triacetoxyborohydride (61 mg, 2.9 ? 10-4 mol) was subsequently added to the solution in two portions (50% of the amount after the first 2 hours of the reaction and 50% after 5 additional hours). The solution was stirred for 24 hours. TLC analysis of the reaction shows a complete disappearance of the starting material. The reaction mixture was quenched with a solution of sodium bicarbonate (24 mg, 2.9 ? 10-4, in 30 mL of water) and then extracted with ethyl acetate (2 ? 60 mL). The combined organic phase was dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ ethyl acetate to 100% ethyl acetate, and from 9:1 ethyl acetate/MeOH to 100% MeOH) to furnish compound 5.11 (91 mg, 72%) as a 120  white solid. 1H NMR (600 MHz, DMSO-d6) ? 7.32 (d, J = 7.2 Hz, 2H), 7.28 (t, J = 7.8 Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 4.96 (m, 1H), 3.64 (m, 2H), 2.80 (bs, 1H), 2.57, (q, J = 7.2 Hz, 1H), 2.18 (quin, J = 6.6 Hz, 1H), 1.87 (d, J = 7.2 Hz, 1H), 1.82 (m, 2H), 1.72 (m, 2H), 1.48 (m, 1H), 1.41 (m, 8H), 1.31 (m, 1H), 1.23 (d, J = 14.4 Hz, 1H), 1.19 (d, J = 7.2 Hz, 3H), 1.15 (m, 4H), 0.91 (s, 3H), 0.63 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 180.7, 141.6, 128.0, 127.9, 126.3, 82.2, 58.0, 53.8, 51.2, 50.5, 41.3, 39.5a, 37.6, 36.1, 35.3, 35.0, 34.7, 32.6, 30.6, 30.0, 26.5, 26.1, 24.4, 23.8, 19.9, 17.5, 13.3. HRESIMS [M + H]+ m/z 436.3216 (calcd for C29H42NO2, 436.3216). a: partially obscured                                                                                                                                                                               Compound 5.12. To a solution of compound 5.11 (100 mg, 2.3 ? 10-4 mol) in methanol (10 mL) were added 10 wt. % Pd-C (100 mg) and anhydrous ammonium formate (76 mg, 1.2 ? 10-3 mol). The resulting reaction mixture was stirred at reflux temperature for 10 minutes. TLC analysis of the reaction shows a complete disappearance of the starting material. The solution was filtered through a celite pad which was then washed with chloroform (10 mL). The resultant solution was dried over MgSO4 and evaporated under vacuum to furnish compound 5.12 (71 mg, 90%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 4.97 (m, 1H), 3.41a (bs, 1H), 2.58 (q, J = 7.8 Hz, 1H), 2.19 (quin, J = 6.6 Hz, 1H), 2.02 (dt, J = 14.1, 3.6 Hz, 1H), 1.87 (d, J = 7.2 Hz, 1H), 1.84 (m, 1H), 1.73 (d, J = 12.0 Hz, 1H), 1.67 (d, J = 13.8 Hz, 1H), 1.59 (m, 1H), 1.48 (m, 2H), 1.46 (m, 1H), 1.32 (m, 6H), 1.19 (d, J = 7.2 Hz, 3H), 1.18 (m, 1H), 1.12 (m, 4H), 0.94 (s, 3H), 0.63 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 180.7, 82.2, 58.0, 53.6, 46.6, 41.3, 39.5a, 37.5, 35.3, 35.0, 34.7, 34.5, 32.5, 28.8, 28.7, 25.8, 25.7, 23.1, 23.0, 19.8, 17.5, 13.3. HRESIMS [M + H]+ m/z 346.2752 (calcd for C22H36NO2, 346.2746). a: partially obscured                                                                                                                                                                               Compound 5.13. To a solution of compound 5.10 (100 mg, 2.9 ? 10-4 mol) in toluene (10 mL) were added 1,3-propanediol (210 ?L, 2.9 ? 10-3 mol), p-toluenesulfonic acid monohydrate 121  (5.5 mg, 2.9 ? 10-5 mol) and 3 ? molecular sieves (15-20 beads). The resultant solution was stirred and refluxed for 6 hours. TLC analysis of the reaction shows a complete disappearance of the starting material. The solution was cooled to room temperature and then filtered through a column (silica gel, 100% toluene), dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.13 (105 mg, 90%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 4.97 (m, 1H), 3.81 (m, 2H), 3.76 (t, J = 5.4 Hz, 2H), 2.57 (q, J = 7.2 Hz, 1H), 2.19 (quin, J = 6.6 Hz, 1H), 1.98 (d, J = 13.8 Hz, 1H), 1.88 (d, J = 7.8 Hz, 1H), 1.80 (m, 1H), 1.74 (m, 2H), 1.64 (t, J = 13.8 Hz, 1H), 1.56 (m, 2H), 1.54 (m, 1H), 1.46 (m, 1H), 1.37 (m, 5H), 1.19 (d, J = 7.8 Hz, 3H), 1.18 (m, 1H), 1.16 (m, 2H), 1.10 (m, 4H), 0.89 (s, 3H), 0.63 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 180.7, 98.0, 82.2, 58.4, 58.2, 58.0, 53.6, 41.3, 39.4a, 38.7, 37.5, 35.3, 34.6, 34.5, 33.5, 32.5, 32.3, 27.2, 26.1, 26.0, 25.3, 23.1, 19.9, 17.5, 13.3. HRESIMS [M + H]+ m/z 403.2852 (calcd for C25H39O4, 403.2848). a: partially obscured                                                                                                                                                                              Compound 5.16. To a solution of compound 5.13 (100 mg, 2.5 ? 10-4 mol) in 1,2-dimethoxyethane (12 mL) was added triethyl phosphite (860 ?L, 5.0 ? 10-3 mol), the resultant solution was stirred for 10 minutes at room temperature. The reaction mixture was cooled to -41 ?C (acetonitrile/dry ice) and oxygen gas was bubbled through the solution for 10 minutes. Potassium tert-butoxide (281 mg, 2.5 ? 10-3 mol) was then added to the mixture and oxygen was bubbled through the solution for 3 hours at -41 ?C and for 1 hour at 0 ?C. The reaction mixture was slowly warmed to room temperature and water (100 ?L) was added to the solution which was stirred for 1 additional hour while oxygen was constantly bubbled through the solution. TLC analysis of the reaction shows the presence of unreacted starting material along with a new product. The reaction mixture was quenched with a 10% HCl solution (200 mL, water solution) 122  and extracted with ethyl acetate (200 + 100 mL). The combined organic phase was dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.16 (63 mg, 70%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 5.90 (s, OH), 4.99 (m, 1H), 2.73 (t, J = 14.4 Hz, 1H), 2.36 (m, 1H), 2.18 (quin, J = 6.6 Hz, 1H), 2.00 (d, J = 6.6 Hz, 1H), 1.94 (m, 2H), 1.84 (m, 2H), 1.81 (m, 1H), 1.73 (m, 1H), 1.60 (m, 1H), 1.55 (m, 1H), 1.41 (m, 2H), 1.38 (s, 3H), 1.35 (m, 1H), 1.27 (m, 4H), 1.18 (m, 1H), 1.13 (m, 1H), 0.96 (s, 3H), 0.70 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 211.7, 177.5, 81.7, 73.2, 63.2, 54.6, 43.4, 41.8, 40.0a, 39.5a, 38.0, 36.7, 36.4, 34.5, 34.1, 31.3, 26.0, 25.2, 22.2, 19.8, 19.0, 13.3. HRESIMS [M + Na]+ m/z 383.2212 (calcd for C22H32O4Na, 383.2198). a: partially obscured                                                                                                                                                                               Compound 5.14. To a solution of compound 5.13 (100 mg, 2.5 ? 10-4 mol) in 1,2-dimethoxyethane (12 mL), previously cooled to 0 ?C, was added potassium tert-butoxide (281 mg, 2.5 ? 10-3 mol). The resultant solution was stirred for 10 minutes at 0 ?C. Dimethyl sulfate (235 ?L, 2.5 ? 10-3 mol) was then added to the solution which was stirred for 1 hour at 0 ?C and 3 hours at room temperature. TLC analysis of the reaction shows the presence of unreacted starting material along with a new product. The reaction mixture was quenched with water (50 mL) and extracted with dichloromethane (2 ? 50 mL). The combined organic phase was dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.14 (11 mg, 10%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 4.94 (m, 1H), 3.80 (m, 2H), 3.75 (t, J = 5.4 Hz, 2H), 3.14 (s, 3H), 2.15 (m, 1H), 2.10 (d, J = 6.6 Hz, 1H), 1.98 (m, 1H), 1.82 (m, 1H), 1.77 (m, 2H), 1.63 (t, J = 13.8 Hz, 1H), 1.55 (m, 2H), 1.47 (m, 2H), 1.44 (m, 1H), 1.36 (s, 3H), 1.34 (m, 5H), 1.20 (m, 4H), 1.09 (m, 1H), 1.04 (m, 1H), 0.88 (s, 123  3H), 0.70 (m, 3H). 13C NMR (150 MHz, DMSO-d6) ? 174.6, 97.9, 82.1, 78.7, 61.7, 58.4, 58.2, 54.6, 50.4, 39.9a, 39.5a, 38.7, 37.8, 34.5, 34.1, 33.5, 32.3, 31.2, 27.1, 26.1, 25.8, 25.3, 23.1, 19.7, 13.5, 13.3. HRESIMS [M + Na]+ m/z 455.2778 (calcd for C26H40O5Na, 455.2773). a: partially obscured                                                                                                                                                                            Compound 5.17. To a solution of compound 5.16 (100 mg, 2.8 ? 10-4 mol) in 1,2-dichloroethane (10 mL) was added benzylamine (37 ?L, 3.4 ? 10-4 mol), the resultant solution was stirred for 1 hour. Glacial acetic acid (16 ?L, 2.8 ? 10-4 mol) was then added to the solution which was stirred for 1 hour. Sodium triacetoxyborohydride (59 mg, 2.8 ? 10-4 mol) was subsequently added to the solution in two portions (50% of the amount after the first 2 hours of the reaction and 50% after 5 additional hours). The solution was stirred for 24 hours. TLC analysis of the reaction shows a complete disappearance of the starting material. The reaction mixture was quenched with a solution of sodium bicarbonate (24 mg, 2.8 ? 10-4, in 30 mL of water) and then extracted with ethyl acetate (2 ? 60 mL). The combined organic phase was dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ ethyl acetate to 100% ethyl acetate, and from 9:1 ethyl acetate/MeOH to 100% MeOH) to furnish compound 5.17 (89 mg, 71%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 7.32 (d, J = 7.2 Hz, 2H), 7.28 (t, J = 7.8 Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 5.88 (s, OH), 4.96 (m, 1H), 3.64 (m, 2H), 2.80 (bs, 1H), 2.14 (quin, J = 6.6 Hz, 1H), 1.99 (d, J = 6.0 Hz, 1H), 1.82 (m, 2H), 1.81 (m, 1H), 1.70 (m, 1H), 1.48 (m, 1H), 1.40 (m, 4H), 1.37 (s, 3H), 1.34 (m, 4H), 1.23 (m, 2H), 1.17 (m, 2H), 1.07 (m, 2H), 0.90 (s, 3H), 0.67 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 177.5, 141.6, 128.0, 127.9, 126.3, 81.7, 73.2, 63.1, 54.9, 51.2, 50.5, 39.9a, 39.1a, 38.1, 36.1, 34.9, 34.3, 31.4, 30.5, 30.0, 26.5, 25.9, 24.4, 23.8, 19.6, 19.0, 13.3. HRESIMS [M + H]+ m/z 452.3177 (calcd for C29H42NO3, 452.3165). a: partially 124  obscured                                                                                                                                                                              Compound 5.18. To a solution of compound 5.17 (100 mg, 2.2 ? 10-4 mol) in methanol (10 mL) were added 10 wt. % Pd-C (100 mg) and anhydrous ammonium formate (69 mg, 1.1 ? 10-3 mol). The resulting reaction mixture was stirred at reflux temperature for 10 minutes. TLC analysis of the reaction shows a complete disappearance of the starting material. The solution was filtered through a celite pad which was then washed with chloroform (10 mL). The resultant solution was dried over MgSO4 and evaporated under vacuum to furnish compound 5.18 (72 mg, 90%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 5.92 (bs, OH), 4.97 (m, 1H), 3.35a (bs, 1H), 2.15 (quin, J = 6.6 Hz, 1H), 2.00 (m, 1H), 1.99 (d, J = 6.6 Hz, 1H), 1.83 (m, 2H), 1.66 (m, 1H), 1.58, (m, 1H), 1.48 (m, 1H), 1.43 (m, 2H), 1.37 (s, 3H), 1.34 (m, 5H), 1.23 (m, 2H), 1.18 (m, 2H), 1.08 (m, 2H), 0.92 (s, 3H), 0.67 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 177.5, 81.7, 73.2, 63.1, 54.8, 46.3, 39.9a, 38.8, 38.1, 35.1, 34.7, 34.1, 31.3, 29.4, 28.9, 25.9, 25.5, 23.9, 23.1, 19.5, 19.0, 13.3. HRESIMS [M + H]+ m/z 362.2700 (calcd for C22H36NO3, 362.2695). a: partially obscured                                                                                                                                                           Compound 5.19. To a solution of compound 5.16 (100 mg, 2.8 ? 10-4 mol) in dichloromethane (8.0 mL) was added triethylamine (60 ?L, 4.2 ? 10-4 mol), the resultant solution was stirred for 5 minutes at room temperature. The reaction mixture was then cooled to 0 ?C and methanesulfonyl chloride (26 ?L, 3.4 ? 10-4 mol) was added to the solution over a period of 10 minutes. The resultant solution was slowly warmed to room temperature and stirred for 1 hour. TLC analysis of the reaction shows a complete disappearance of the starting material. The reaction mixture was quenched with cold water (100 mL) and extracted with dichloromethane (2 ? 100 mL). The combined organic phase was then washed a 10% HCl solution (100 mL, water solution), water (2 ? 100 mL), dried over MgSO4 and evaporated under vacuum. The product 125  was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.19 (103 mg, 85%) as a yellow gum. 1H NMR (600 MHz, DMSO-d6) ? 5.14 (m, 1H), 3.29 (s, 3H), 2.80 (d, J = 6.0 Hz, 1H), 2.72 (t, J = 14.4 Hz, 1H), 2.38 (dt, J = 14.4, 5.4 Hz, 1H), 2.26 (quin, J = 6.6 Hz, 1H), 1.95 (m, 2H), 1.90 (m, 1H), 1.83 (s, 3H), 1.81 (m, 2H), 1.74 (m, 1H), 1.63 (m, 1H), 1.58 (m, 1H), 1.46 (m, 4H), 1.40 (m, 1H), 1.32 (m, 2H), 1.16 (m, 2H), 0.96 (s, 3H), 0.72 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 211.7, 171.6, 89.5, 82.6, 60.2, 54.4, 43.4, 41.8, 40.9, 40.8, 39.1a, 37.5, 36.7, 36.4, 34.4, 33.9, 31.0, 25.9, 25.1, 22.1, 19.7, 17.2, 13.1. HRESIMS [M + Na]+ m/z 461.1961 (calcd for C23H34O6SNa, 461.1974). a: partially obscured                                                                                                                                                                    Compound 5.21. To a solution of compound 5.19 (100 mg, 2.3 ? 10-4 mol) in toluene (10 mL) were added 1,3-propanediol (165 ?L, 2.3 ? 10-3 mol), p-toluenesulfonic acid monohydrate (4.4 mg, 2.3 ? 10-5 mol) and 3 ? molecular sieves (15-20 beads). The resultant solution was stirred and refluxed for 9 hours. TLC analysis of the reaction shows a complete disappearance of the starting material. The reaction mixture was cooled to room temperature and then filtered through a column (silica gel, 100% toluene), dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.21 (62 mg, 68%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 6.11 (s, 1H), 5.67 (s, 1H), 4.89 (m, 1H), 3.81 (m, 2H), 3.76 (t, J = 5.4 Hz, 2H), 2.88 (m, 1H), 2.23 (quin, J = 6.0 Hz, 1H), 1.99 (m, 1H), 1.80 (m, 1H), 1.78 (m, 2H), 1.65 (t, J = 13.8 Hz, 1H), 1.56 (m, 2H), 1.53 (m, 1H), 1.44 (m, 6H), 1.30 (m, 2H), 1.22 (m, 1H), 1.20 (m, 2H), 1.10 (m, 2H), 0.89 (s, 3H), 0.54 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 170.6, 136.8, 122.1, 98.0, 81.3, 58.4, 58.2, 54.2, 53.7, 43.6, 39.5a, 38.7, 37.5, 34.8, 34.6, 33.6, 32.5, 32.3, 27.1, 26.1, 26.0, 25.3, 23.1, 20.0, 14.2. HRESIMS [M + Na]+ m/z 126  423.2519 (calcd for C25H36O4Na, 423.2511). a: partially obscured                                                                                                                                                                          Compound 5.22. To a flame dried flask containing magnesium turnings (180 mg, 7.4 ? 10-3 mol), previously scraped with sand paper, and iodine (2 small crystals) was added a THF solution (4.0 mL) of 1-bromo-3-methylbutane (600 ?L, 5.0 ? 10-3 mol) over a period of 10 minutes. The dark violet solution was stirred for 3 minutes until it became colorless. Spontaneous bubbling was observed and the solution became dark grey within 5 minutes. The resultant solution was stirred and refluxed for 1 hour and then cooled to room temperature before it was ready to be used. To a solution of compound 5.21 (100 mg, 2.5 ? 10-4 mol) in THF (8.0 mL) was added copper(I) iodide (800 mg, 4.2 ? 10-3 mol), the resultant solution was stirred for 10 minutes at room temperature and then cooled to -78 ?C. The isopentylmagnesium bromide solution was then added to the reaction mixture in portions of 100 ?L at -78 ?C. The reaction was constantly monitored with TLC analysis while adding an excess of isopentylmagnesium bromide solution. TLC analysis of the reaction showed a complete disappearance of the starting material. The reaction mixture was then filtered through a column (silica gel, 100% ethyl acetate), dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.22 (85 mg, 72%) as a yellow gum. 1H NMR (600 MHz, benzene-d6) ? 4.26 (m, 1H), 3.72 (t, J = 5.4 Hz, 2H), 3.59 (t, J = 5.4 Hz, 2H), 2.21 (m, 1H), 2.13 (m, 1H), 2.10 (m, 1H), 1.98 (m, 1H), 1.87 (t, J = 13.8 Hz, 1H), 1.79 (m, 2H), 1.73 (m, 2H), 1.67 (m, 1H), 1.60 (m, 4H), 1.55 (m, 2H), 1.35 (m, 6H), 1.15 (m, 7H), 0.95 (d, J = 1.8 Hz, 3H), 0.94 (d, J = 1.8 Hz, 3H), 0.84 (s, 3H), 0.82 (m, 2H), 0.73 (s, 3H). 13C NMR (150 MHz, benzene-d6) ? 178.1, 99.1, 82.3, 59.6, 59.3, 55.6, 55.0, 44.4, 42.6, 40.4, 39.8, 39.7, 39.6, 35.3, 35.1, 34.8, 33.6, 33.3, 33.0, 28.7, 28.5, 27.7, 27.3, 26.7, 26.5, 23.8, 23.2, 23.1, 20.9, 14.3. HRESIMS [M + Na]+ m/z 495.3442 127  (calcd for C30H48O4Na, 495.3450). Compound 5.23. To a solution of compound 5.22 (100 mg, 2.1 ? 10-4 mol) in 1,2-dimethoxyethane (12 mL) was added triethyl phosphite (720 ?L, 4.2 ? 10-3 mol), the resultant solution was stirred for 10 minutes at room temperature. The reaction mixture was cooled to -41 ?C (acetonitrile/dry ice) and oxygen gas was bubbled through the solution for 10 minutes. Potassium tert-butoxide (236 mg, 2.1 ? 10-3 mol) was then added to the solution and oxygen was bubbled through the solution for 3 hours at -41 ?C and for 1 hour at 0 ?C. The reaction mixture was slowly warmed to room temperature, water (100 ?L) was added to the solution which was stirred for 1 additional hour while oxygen was constantly bubbled through the solution. TLC analysis of the reaction shows the presence of unreacted starting material along with a new product. The reaction mixture was quenched with a 10% HCl solution (200 mL, water solution) and extracted with ethyl acetate (200 + 100 mL). The combined organic phase was dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.23 (55 mg, 60%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 5.72 (s, OH), 4.99 (m, 1H), 2.73 (t, J = 14.4 Hz, 1H), 2.37 (m, 1H), 2.17 (quin, J = 6.6 Hz, 1H), 2.03 (d, J = 6.0 Hz, 1H), 1.95 (m, 2H), 1.80 (m, 5H), 1.63 (m, 2H), 1.54 (m, 2H), 1.52 (m, 1H), 1.35 (m, 4H), 1.33 (m, 2H), 1.27 (m, 2H), 1.18 (m, 4H), 0.95 (s, 3H), 0.88 (d, J = 3.6 Hz, 3H), 0.86 (d, J = 3.0 Hz, 3H), 0.71 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 211.8, 177.4, 82.0, 75.7, 61.4, 54.7, 43.5, 41.8, 39.5a, 39.0a, 38.9, 38.1, 36.7, 36.4, 34.5, 34.0, 31.4, 31.2, 27.5, 26.0, 25.1, 22.8, 22.4, 22.2, 21.0, 20.0, 13.3. HRESIMS [M + Na]+ m/z 453.2992 calcd for C27H42O4Na, 453.2981). Compound 5.24. To a solution of compound 5.23 (100 mg, 2.3 ? 10-4 mol) in 1,2-dichloroethane (10 mL) was added benzylamine (31 ?L, 2.8 ? 10-4 mol), the resultant solution 128  was stirred for 1 hour. Glacial acetic acid (13 ?L, 2.3 ? 10-4 mol) was then added to the solution which was stirred for 1 hour. Sodium triacetoxyborohydride (49 mg, 2.3 ? 10-4 mol) was subsequently added to the solution in two portions (50% of the amount after the first 2 hours of the reaction and 50% after 5 additional hours). The solution was stirred for 24 hours. TLC analysis of the reaction shows a complete disappearance of the starting material. The reaction mixture was quenched with a solution of sodium bicarbonate (19 mg, 2.3 ? 10-4, in 30 mL of water) and then extracted with ethyl acetate (2 ? 60 mL). The combined organic phase was dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ ethyl acetate to 100% ethyl acetate, and from 9:1 ethyl acetate/MeOH to 100% MeOH) to furnish compound 5.24 (85 mg, 70%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 7.33 (d, J = 7.2 Hz, 2H), 7.30 (t, J = 7.2 Hz, 2H), 7.20 (t, J = 7.2 Hz, 1H), 5.69 (s, OH), 4.96 (m, 1H), 3.65 (m, 2H), 2.81 (bs, 1H), 2.13 (quin, J = 6.6 Hz, 1H), 2.03 (d, J = 6.0 Hz, 1H), 1.81 (m, 1H), 1.79 (m, 1H), 1.72 (m, 2H), 1.63 (m, 1H), 1.53 (quin, J = 6.6 Hz, 2H), 1.47 (m, 2H), 1.40 (m, 4H), 1.39 (m, 2H), 1.35 (m, 2H), 1.21 (m, 7H), 1.06 (m, 2H), 0.89 (s, 3H), 0.87 (d, J = 3.0 Hz, 3H), 0.86 (d, J = 3.0 Hz, 3H), 0.67 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 177.4, 141.4, 128.0, 127.9, 126.4, 82.1, 75.7, 61.3, 55.1, 51.2, 50.5, 39.9a, 39.0, 38.9, 38.2, 36.0, 34.9, 34.3, 31.4, 31.2, 30.4, 29.9, 27.5, 26.5, 25.8, 24.3, 23.8, 22.7, 22.4, 20.9, 19.8, 13.3. HRESIMS [M + H]+ (calcd for C34H52O3Na, 522.3947). a: partially obscured 3,5-epi-clionamine B (5.25). To a solution of compound 5.24 (100 mg, 1.9 ? 10-4 mol) in methanol (10 mL) were added 10 wt. % Pd-C (100 mg) and anhydrous ammonium formate (60 mg, 9.5 ? 10-4 mol). The resulting reaction mixture was stirred at reflux temperature for 10 minutes. TLC analysis of the reaction shows a complete disappearance of the starting material. 129  The solution was filtered through a celite pad which was then washed with chloroform (10 mL). The resultant solution was dried over MgSO4 and evaporated under vacuum to furnish 3,5-epi-clionamine B (5.25) (74 mg, 90%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 5.72 (bs, OH), 4.97 (m, 1H), 3.39a (bs, 1H), 2.14 (quin, J = 6.6 Hz, 1H), 2.02 (d, J = 6.0 Hz, 1H), 2.00 (m, 1H), 1.85 (m, 1H), 1.79 (m, 2H), 1.73 (d, J = 12.0 Hz, 1H), 1.65 (m, 1H), 1.63 (m, 2H), 1.53 (quin, J = 6.6 Hz, 1H), 1.45 (m, 4H), 1.35 (m, 4H), 1.23 (m, 4H), 1.18 (m, 2H), 1.07 (m, 2H), 0.92 (s, 3H), 0.87 (d, J = 3.0 Hz, 3H), 0.86 (d, J = 3.0 Hz, 3H), 0.67 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 177.4, 82.0, 75.7, 61.3, 54.9, 46.4, 39.5a, 38.9, 38.7, 38.1, 35.0, 34.6, 34.1, 31.4, 31.1, 29.0, 28.8, 27.5, 25.8, 25.4, 23.3, 23.0, 22.7, 22.4, 21.0, 19.7, 13.3. 1H NMR (600 MHz, methanol-d4) ? 5.07 (m, 1H), 3.57 (bs, 1H), 2.28 (m, 1H), 2.25 (m, 1H), 2.12 (d, J = 6.0 Hz, 1H), 2.02 (m, 1H), 1.93 (m, 1H), 1.85 (m, 2H), 1.73, (m, 2H), 1.60 (m, 5H), 1.47 (m, 4H), 1.38 (m, 4H), 1.30 (m, 2H), 1.28 (m, 1H), 1.22 (m, 2H), 1.18 (m, 1H), 1.05 (s, 3H), 0.93 (d, J = 4.2 Hz, 3H), 0.92 (d, J = 3.6 Hz, 3H), 0.81 (s, 3H). 13C NMR (150 MHz, methanol-d4) ? 180.1, 84.6, 78.1, 63.4, 57.0, 49.2a, 41.8, 40.8, 40.7, 40.3, 37.6, 36.3, 36.1, 33.1, 32.6, 30.6, 30.1, 29.4, 27.3, 27.0, 24.5, 24.0, 23.4, 23.0, 22.7, 21.4, 14.3. HRESIMS [M + H]+ m/z 432.3477 (calcd for C27H46NO3, 432.3478). a: partially obscured Compound 5.26. To a solution of compound 5.23 (100 mg, 2.3 ? 10-4 mol) in absolute methanol (10 mL) were added anhydrous ammonium acetate (177 mg, 2.3 ? 10-3 mol) and sodium cyanoborohydride (10 mg, 1.6 ? 10-4 mol). The solution was heated to 35 ?C and stirred for 12 hours. TLC analysis of the reaction shows a complete disappearance of the starting material. Concentrated HCl was added until pH ? 2 and the resultant solution was evaporated under vacuum. A 1/1 mixture of 3-alpha/beta amines 5.26 (90 mg, 90%, white solid) was obtained from the reaction mixture via C18 reversed phase HPLC using a CSC-Inertsil 130  150A/ODS2, 5 ?m 25 x 0.94 cm column, with 3:7 acetonitrile/H2O as eluent over 20 min (flow rate 2 mL/min). HRESIMS [M + H]+ m/z 432.3477 (calcd for C27H46NO3, 432.3478). Compound 5.27. To a solution of compound 5.22 (100 mg, 2.0 ? 10-4 mol) in dichloromethane (8.0 mL), previously cooled to 0 ?C, was added dropwise a 1.0 M solution of boron tribromide in dichloromethane (1.0 mL, 1.0 ? 10-3 mol). The resultant solution was stirred for 30 minutes at 0 ?C and for 2 hours at room temperature. TLC analysis of the reaction shows a complete disappearance of the starting material. The reaction mixture was quenched with methanol (10 mL) and the resultant solution was evaporated under vacuum. The residual gum was then dissolved in ethyl acetate (50 mL). The organic phase was washed with water (2 ? 50 mL), dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ ethyl acetate to 100% ethyl acetate) to furnish compound 5.27 (61 mg, 70%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 4.78 (m, 1H), 2.82 (m, 1H), 2.73 (t, J = 14.4 Hz, 1H), 2.36 (m, 1H), 2.28 (t, J = 7.2 Hz, 1H), 2.17 (quin, J = 6.6 Hz, 1H), 1.95 (m, 2H), 1.84 (m, 2H), 1.78 (m, 1H), 1.72 (m, 2H), 1.59 (m, 1H), 1.52 (m, 4H), 1.42 (m, 4H), 1.32 (m, 2H), 1.23 (m, 2H), 1.16 (m, 4H), 0.96 (s, 3H), 0.87 (s, 3H), 0.86 (s, 3H), 0.73 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 211.8, 178.5, 82.0, 54.3, 53.9, 43.5, 42.9, 41.8, 41.7, 39.5a, 38.4, 38.3, 36.7, 36.4, 34.5, 34.2, 32.0, 27.3, 26.1, 26.0, 25.4, 25.2, 22.6, 22.5, 22.2, 20.0, 13.5. HRESIMS [M + Na]+ m/z 437.3043 (calcd for C27H42O3Na, 437.3032). a: partially obscured Compound 5.28. To a solution of compound 5.27 (100 mg, 2.4 ? 10-4 mol) in 1,2-dichloroethane (10 mL) was added benzylamine (32 ?L, 2.9 ? 10-4 mol), the resultant solution was stirred for 1 hour. Glacial acetic acid (14 ?L, 2.4 ? 10-4 mol) was then added to the solution which was stirred for 1 hour. Sodium triacetoxyborohydride (51 mg, 2.4 ? 10-4 mol) was 131  subsequently added to the solution in two portions (50% of the amount after the first 2 hours of the reaction and 50% after 5 additional hours). The solution was stirred for 24 hours. TLC analysis of the reaction shows a complete disappearance of the starting material. The reaction mixture was quenched with a solution of sodium bicarbonate (20 mg, 2.4 ? 10-4, in 30 mL of water) and then extracted with ethyl acetate (2 ? 60 mL). The combined organic phase was dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate, and from 9:1 ethyl acetate/MeOH to 100% MeOH) to furnish compound 5.28 (88 mg, 72%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 7.32 (d, J = 7.2 Hz, 2H), 7.28 (t, J = 7.8 Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 4.74 (m, 1H), 3.64 (m, 2H), 2.79 (m, 1H), 2.78 (m, 1H), 2.27 (t, J = 7.2 Hz, 1H), 2.13 (quin, J = 6.6 Hz, 1H), 1.81 (m, 4H), 1.72 (m, 2H), 1.51 (m, 2H), 1.48 (m, 2H), 1.46 (m, 1H), 1.39 (m, 8H), 1.24 (m, 2H), 1.22 (m, 2H), 1.09 (m, 1H), 1.06 (m, 2H), 0.90 (s, 3H), 0.86 (s, 3H), 0.85 (s, 3H), 0.69 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 178.4, 141.5, 128.0, 127.9, 126.3, 82.0, 54.7, 53.8, 51.2, 50.5, 42.9, 41.7, 39.2a, 38.5, 38.4, 36.0, 34.9, 34.4, 32.0, 30.5, 30.0, 27.3, 26.5, 26.1, 26.0, 25.4, 24.4, 23.8, 22.5, 22.4, 19.9, 13.5. HRESIMS [M + H]+ m/z 506.3995 (calcd for C34H52NO2, 506.3998). Compound 5.29. To a solution of compound 5.28 (100 mg, 2.0 ? 10-4 mol) in methanol (10 mL) were added 10 wt. % Pd-C (100 mg) and anhydrous ammonium formate (63 mg, 1.0 ? 10-3 mol). The resulting reaction mixture was stirred at reflux temperature for 10 minutes. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The solution was filtered through a celite pad which was then washed with chloroform (10 mL). The resultant solution was dried over MgSO4 and evaporated under vacuum to furnish compound 5.29 (78 mg, 95%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 4.75 (m, 1H), 3.37 (bs, 132  1H), 2.80 (m, 1H), 2.27 (t, J = 7.2 Hz, 1H), 2.14 (quin, J = 6.6 Hz, 1H), 1.99 (m, 1H), 1.84 (m, 1H), 1.74, (m, 1H), 1.69 (m, 2H), 1.58 (m, 1H), 1.52 (m, 2H), 1.47 (m, 4H), 1.40 (m, 2H), 1.35 (m, 5H), 1.23 (m, 2H), 1.19 (m, 2H), 1.15 (m, 1H), 1.08 (m, 2H), 0.92 (s, 3H), 0.86 (s, 3H), 0.85 (s, 3H), 0.70 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 178.5, 82.0, 54.5, 53.8, 46.3, 42.9, 41.7, 38.9, 38.5, 38.4, 35.0, 34.6, 34.3, 32.0, 29.0, 28.8, 27.3, 26.1, 25.8, 25.6, 25.4, 23.5, 23.0, 22.5, 22.4, 19.7, 13.5. HRESIMS [M + H]+ m/z 416.3521 (calcd for C27H46NO2, 416.3529).  Compound 5.31. To a solution of tigogenin 5.30 (100 mg, 2.4 ? 10-4 mol) in THF (10 mL) were added acetic anhydride (110 ?L, 1.2 ? 10-3 mol) and 4-(dimethylamino)pyridine (2.9 mg, 2.4 ? 10-5 mol). The solution was stirred for 24 hours. TLC analysis of the reaction shows a complete disappearance of the starting material. The reaction mixture was quenched with water (50 mL) and then extracted with dichloromethane (3 ? 50 mL). The combined organic phase was washed with water (2 ? 100 mL), dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.31 (106 mg, 96%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 4.56 (m, 1H), 4.26 (q, J = 7.2 Hz, 1H), 3.33a (m, 1H), 3.20 (t, J = 10.8 Hz, 1H), 1.96 (s, 3H), 1.89 (m, 1H), 1.80 (quin, J = 6.6 Hz, 1H), 1.72 (m, 1H), 1.66 (m, 2H), 1.65 (m, 1H), 1.61 (m, 1H), 1.50 (m, 8H), 1.41 (m, 2H), 1.27 (m, 2H), 1.23 (m, 2H), 1.21 (m, 1H), 1.12 (m, 1H), 1.10 (m, 1H), 0.98 (m, 1H), 0.89 (d, J = 7.2 Hz, 3H), 0.87 (m, 1H), 0.79 (s, 3H), 0.73 (d, J = 6.0 Hz, 3H), 0.71 (s, 3H), 0.66 (m, 1H). 13C NMR (150 MHz, DMSO-d6) ? 169.8, 108.4, 80.2, 72.9, 65.9, 61.9, 55.6, 53.5, 44.0, 41.1, 39.5a, 39.4a, 36.1, 35.1, 34.6, 33.7, 31.7, 31.4, 30.9, 29.8, 28.5, 28.0, 27.1, 21.1, 20.5, 17.1, 16.2, 14.7, 12.0. HRESIMS [M + H]+ m/z 459.3468 (calcd for C29H47O4, 459.3474). a: partially obscured Compound 5.32. To a solution of compound 5.31 (100 mg, 2.2 ? 10-4 mol) in glacial 133  acetic acid (5.0 mL) was added dropwise a solution of bromine (11 ?L, 2.1 ? 10-4 mol) in glacial acetic acid (1.25 ml). The solution was stirred for 1 hour at room temperature. TLC analysis of the reaction shows a complete disappearance of the starting material. The reaction mixture was quenched with water (50 mL) and then extracted with dichloromethane (3 ? 50 mL). The combined organic phase was washed with water (2 ? 100 mL), dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.32 (mixture of bromides, 94 mg, 80%) as a white solid. HRESIMS [M + H]+ m/z 537.2571 (calcd for C29H46O4Br, 537.2579). Compound 5.33. To a solution of compound 5.32 (mixture of bromides, 100 mg, 1.9 ? 10-4 mol) in 1-butanol (50 mL) was added ammonium hydroxide solution (7.0 mL, 28% NH3 in H2O). The solution was stirred and refluxed for 7 days. Additional aqueous ammonium hydroxide solution (3 ? 5.0 mL) was added during 7 days period. TLC analysis of the reaction shows the presence of unreacted starting material along with a new product. The reaction was cooled to room temperature and the top layer and the bottom layer were separated. The top layer was quenched with a buffer solution (50 mL, pH = 7.0) and the resultant solution was stirred for 20 minutes. The mixture was then extracted with ethyl acetate (100 mL) and water (50 mL). The ethyl acetate layer was washed with water (2 ? 50 mL), dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound (5.33) (23 mg, 26%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 5.01 (bs, OH), 4.56 (m, 1H), 4.45 (m, 1H), 3.81 (dd, J = 6.6, 6.3 Hz, 1H), 3.76 (t, J = 7.8 Hz, 1H), 3.18 (t, J = 8.4 Hz, 1H), 2.17 (m, 1H), 2.03 (m, 1H), 1.96 (s, 3H), 1.94 (m, 1H), 1.86 (m, 1H), 1.73 (m, 1H), 1.63 (m, 4H), 1.46 (m, 5H), 1.22 (m, 134  4H), 1.07 (m, 4H), 0.96 (d, J = 6.6 Hz, 6H), 0.95 (m, 1H), 0.87 (m, 1H),0.78 (s, 3H), 0.73 (s, 3H), 0.66 (m, 1H). 13C NMR (150 MHz, DMSO-d6) ? 169.8, 109.1, 82.8, 80.3, 74.2, 72.8, 63.0, 55.6, 53.5, 44.0, 40.5, 39.5a, 39.4a, 38.4, 36.1, 35.1, 34.6, 33.8, 33.7, 31.7, 31.6, 28.1, 27.1, 21.1, 20.5, 16.5, 16.5, 16.1, 12.0. HRESIMS [M + Na]+ m/z 497.3239 (calcd for C29H46O5Na, 497.3243). a: partially obscured                                                                                                                                                                            Compound 5.34. To a solution of compound 5.33 (100 mg, 2.1 ? 10-4 mol) in methanol (20 mL) was added anhydrous K2CO3 (152 mg, 1.1 ? 10-3 mol). The resultant solution was stirred for 48 hours. TLC analysis of the reaction shows a complete disappearance of the starting material. The reaction mixture was quenched with a saturated solution of ammonium chloride (100 mL) and extracted with ethyl acetate (2 ? 100 mL). The combined organic phase was washed with water (2 ? 100 mL), dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.34 (83 mg, 91%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 5.01 (bs, OH), 4.45 (m, 1H), ? 4.42 (d, J = 4.8 Hz, OH), 3.81 (t, J = 7.5 Hz, 1H), 3.76 (t, J = 7.8 Hz, 1H), 3.33a (m, 1H), 3.18 (t, J = 8.4 Hz, 1H), 2.17 (m, 1H), 2.02 (m, 1H), 1.94 (m, 1H), 1.85 (m, 1H), 1.62 (m, 5H), 1.43 (m, 4H), 1.20 (m, 4H), 1.12 (m, 1H), 1.10 (m, 4H), 0.96 (d, J = 6.6 Hz, 6H), 0.85 (m, 2H), 0.75 (s, 3H), 0.72 (s, 3H), 0.61 (m, 1H). 13C NMR (150 MHz, DMSO-d6) ? 109.1, 82.8, 80.3, 74.2, 69.3, 63.0, 55.7, 53.8, 44.3, 40.5, 39.5a, 38.4, 38.2, 36.6, 35.2, 34.7, 34.6, 33.8, 31.9, 31.6, 31.4, 28.3, 20.6, 16.5, 16.5, 16.1, 12.2. HRESIMS [M + Na]+ m/z 455.3138 (calcd for C27H44O4Na, 455. 3137). a: partially obscured                                                                                                                                                                          Compound 5.35. To a solution of compound 5.34 (100 mg, 2.3 ? 10-4 mol) in dichloromethane (50 mL) were added pyridinium chlorochromate (496 mg, 2.3 ? 10-3 mol) and 4 ? molecular sieves (250 mg, powder). The solution was vigorously stirred for 3 days. TLC 135  analysis of the reaction shows a complete disappearance of the starting material. The reaction mixture was diluted with diethyl ether (3 ? 50 mL). The resultant solution was then filtered through a column (silica gel, 100% diethyl ether), dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.35 (72 mg, 90%) as a white solid. 1H NMR (600 MHz, methylene chloride-d2) ? 4.92 (m, 1H), 2.54 (q, J = 7.8 Hz, 1H), 2.37 (dt, J = 14.4, 6.6 Hz, 1H), 2.28 (m, 1H), 2.26 (m, 2H), 2.01 (m, 2H), 1.87 (d, J = 7.8 Hz, 1H), 1.77 (dt, J = 12.6, 3.6 Hz, 1H), 1.72 (m, 1H), 1.57 (m, 2H), 1.52 (m, 1H), 1.44 (m, 5H), 1.28 (d, J = 7.8 Hz, 3H), 1.10 (m, 2H), 1.02 (s, 3H), 0.95 (m, 1H), 0.78 (m, 1H), 0.75 (s, 3H). 13C NMR (150 MHz, methylene chloride-d2) ? 211.6, 181.6, 83.2, 59.5, 54.9, 54.4a, 47.1, 45.1, 42.2, 39.0, 38.7, 38.6, 36.6, 36.3, 35.3, 33.5, 32.4, 29.2, 21.3, 18.3, 14.2, 11.7. HRESIMS [M + H]+ m/z 345.2430 (calcd for C22H33O3, 345.2430). a: partially obscured                                                                                                                                                                           Compound 5.36. To a solution of compound 5.35 (100 mg, 2.9 ? 10-4 mol) in toluene (10 mL) were added 1,3-propanediol (210 ?L, 2.9 ? 10-3 mol), p-toluenesulfonic acid monohydrate (5.5 mg, 2.9 ? 10-5 mol) and 3 ? molecular sieves (15-20 beads). The resultant solution was stirred and refluxed for 8 hours. TLC analysis of the reaction shows a complete disappearance of the starting material. The solution was cooled to room temperature and then filtered through a column (silica gel, 100% toluene, then washed with 7:3 dichloromethane/ethyl acetate), dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 95:5 dichloromethane/ethyl acetate to 7:3 dichloromethane/ethyl acetate) to furnish compound 5.36 (108 mg, 92%) as a white solid. 1H NMR (600 MHz, benzene-d6) ? 4.34 (m, 1H), 3.69 (t, J = 5.4 Hz, 2H), 3.60 (m, 2H), 2.33 (q, J = 7.8 Hz, 1H), 2.25 (m, 1H), 2.04 (dt, J = 13.2, 3.0 Hz, 1H), 1.82 (quin, J = 6.0 Hz, 1H), 1.53 (m, 136  2H), 1.42 (m, 4H), 1.36 (m, 1H), 1.33 (m, 2H), 1.24 (m, 2H), 1.21 (m, 1H), 1.16 (m, 1H), 1.10 (m, 2H), 1.05 (m, 1H), 1.01 (d, J = 7.8 Hz, 3H), 0.71 (m, 2H), 0.65 (s, 3H), 0.54 (s, 3H), 0.53 (m, 2H). 13C NMR (150 MHz, benzene-d6) ? 180.3, 98.5, 82.2, 59.5, 59.4, 59.3, 54.9, 54.8, 42.5, 42.0, 38.7, 36.7, 36.6, 36.4, 35.4, 35.2, 33.5, 32.8, 29.7, 29.0, 26.5, 21.1, 18.3, 14.2, 12.1. HRESIMS [M + H]+ m/z 403.2846 (calcd for C25H39O4, 403.2848). Compound 5.37. To a solution of compound 5.36 (100 mg, 2.5 ? 10-4 mol) in 1,2-dimethoxyethane (12 mL) was added triethyl phosphite (860 ?L, 5.0 ? 10-3 mol), the resultant solution was stirred for 10 minutes at room temperature. The reaction mixture was cooled to -10 ?C (brine/ice) and oxygen gas was bubbled through the solution for 10 minutes. Potassium tert-butoxide (280 mg, 2.5 ? 10-3 mol) was then added to the mixture and oxygen was bubbled through the solution for 3 hours at -10 ?C and for 1 hour at 0 ?C. The reaction mixture was slowly warmed to room temperature and water (100 ?L) was added to the solution which was stirred for 1 additional hour while oxygen was constantly bubbled through the solution. TLC analysis of the reaction shows the presence of unreacted starting material along with a new product. The reaction mixture was quenched with a 10% HCl solution (200 mL, water solution) and extracted with ethyl acetate (200 + 100 mL). The combined organic phase was dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.37 (46 mg, 51%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 5.89 (s, OH), 4.97 (m, 1H), 2.41 (dt, J = 15.0, 6.6 Hz, 1H), 2.30 (t, J = 14.4 Hz, 1H), 2.16 (quin, J = 6.6 Hz, 1H), 2.08 (m, 1H), 2.00 (d, J = 6.6 Hz, 1H), 1.90 (m, 2H), 1.83 (m, 1H), 1.63 (m, 1H), 1.51 (m, 2H), 1.43 (m, 1H), 1.38 (s, 3H), 1.33 (m, 2H), 1.24 (m, 4H), 1.10 (m, 1H), 0.97 (s, 3H), 0.90 (m, 1H), 0.74 (m, 1H), 0.70 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 210.4, 177.5, 81.6, 73.2, 137  63.0, 54.7, 52.7, 45.9, 44.1, 39.9a, 37.9, 37.8, 37.6, 35.3, 33.8, 31.4, 31.2, 28.2, 20.0, 19.0, 13.3, 11.1. HRESIMS [M + Na]+ m/z 383.2195 (calcd for C22H32O4Na, 383.2198). a: partially obscured                                          Compound 5.38. To a solution of compound 5.37 (100 mg, 2.8 ? 10-4 mol) in dichloromethane (8.0 mL) was added triethylamine (60 ?L, 4.2 ? 10-4 mol), the resultant solution was stirred for 5 minutes at room temperature. The reaction mixture was then cooled to 0 ?C and methanesulfonyl chloride (26 ?L, 3.4 ? 10-4 mol) was added to the solution over a period of 10 minutes. The resultant solution was slowly warmed to room temperature and stirred for 1 hour. TLC analysis of the reaction shows a complete disappearance of the starting material. The reaction mixture was quenched with cold water (100 mL) and extracted with dichloromethane (2 ? 100 mL). The combined organic phase was then washed a 10% HCl solution (100 mL, water solution), water (2 ? 100 mL), dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.38 (117 mg, 96%) as a white solid. 1H NMR (600 MHz, methylene chloride-d2) ? 5.10 (m, 1H), 3.06 (s, 3H), 2.76 (d, J = 6.0 Hz, 1H), 2.37 (dt, J = 14.4, 6.6 Hz, 1H), 2.28 (m, 1H), 2.24 (m, 2H), 2.15 (m, 1H), 1.96 (m, 2H), 1.91 (s, 3H), 1.71 (m, 1H), 1.57 (m, 4H), 1.35 (m, 5H), 1.21 (m, 1H), 1.01 (s, 3H), 0.96 (m, 1H), 0.81 (s, 3H), 0.77 (m, 1H). 13C NMR (150 MHz, methylene chloride-d2) ? 211.4, 172.4, 90.8, 83.3, 62.0, 56.0, 53.8a, 47.0, 45.0, 41.9, 41.4, 38.9, 38.8, 38.5, 36.2, 34.9, 32.0, 32.0, 29.1, 20.9, 17.8, 14.1, 11.7. HRESIMS [M + Na]+ m/z 461.1976 (calcd for C23H34O6SNa, 461.1974). a: partially obscured                                             Compound 5.40. To a solution of compound 5.38 (100 mg, 2.3 ? 10-4 mol) in toluene (10 mL) were added 1,3-propanediol (165 ?L, 2.3 ? 10-3 mol), p-toluenesulfonic acid monohydrate (4.4 mg, 2.3 ? 10-5 mol) and 3 ? molecular sieves (15-20 beads). The resultant solution was 138  stirred and refluxed for 9 hours. TLC analysis of the reaction shows a complete disappearance of the starting material. The reaction mixture was cooled to room temperature and then filtered through a column (silica gel, 100% toluene), dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.40 (84 mg, 92%) as a white solid. 1H NMR (600 MHz, benzene-d6) ? 6.25 (s, 1H), 5.06 (s, 1H), 4.24 (m, 1H), 3.69 (t, J = 5.4 Hz, 2H), 3.61 (m, 2H), 2.25 (m, 1H), 2.14 (m, 1H), 2.03 (dt, J = 12.6, 3.0 Hz, 1H), 1.80 (quin, J = 6.0 Hz, 1H), 1.53 (m, 1H), 1.43 (m, 4H), 1.33 (m, 4H), 1.25 (m, 2H), 1.20 (m, 1H), 1.15 (m, 1H), 1.05 (m, 2H), 0.79 (dt, J = 12.9, 3.6 Hz, 1H), 0.69 (dt, J = 12.3, 4.8 Hz, 1H), 0.62 (s, 3H), 0.58 (m, 1H), 0.51 (m, 1H), 0.49 (s, 3H). 13C NMR (150 MHz, benzene-d6) ? 170.8, 138.1, 121.4, 98.5, 81.3, 59.5, 59.3, 55.6, 54.9, 54.5, 44.4, 42.4, 38.7, 36.7, 36.6, 35.4, 35.4, 33.4, 32.7, 29.7, 28.9, 26.5, 21.2, 14.8, 12.0. HRESIMS [M + H]+ m/z 401.2698 (calcd for C25H37O4, 401.2692). Compound 5.41. To a flame dried flask containing magnesium turnings (180 mg, 7.4 ? 10-3 mol), previously scraped with sand paper, and iodine (2 small crystals) was added a THF solution (4.0 mL) of 1-bromo-3-methylbutane (600 ?L, 5.0 ? 10-3 mol) over a period of 10 minutes. The dark violet solution was stirred for 3 minutes until it became colorless. Spontaneous bubbling was observed and the solution became dark grey within 5 minutes. The resultant solution was stirred and refluxed for 1 hour and then cooled to room temperature before it was ready to be used. To a solution of compound 5.40 (100 mg, 2.5 ? 10-4 mol) in THF (8.0 mL) was added copper(I) iodide (800 mg, 4.2 ? 10-3 mol), the resultant solution was stirred for 10 minutes at room temperature and then cooled to -78 ?C. The isopentylmagnesium bromide solution was then added to the reaction mixture in portions of 500 ?L at -78 ?C. The reaction was 139  constantly monitored with TLC analysis while adding an excess of isopentylmagnesium bromide solution. TLC analysis of the reaction showed a complete disappearance of the starting material. The reaction mixture was then filtered through a column (silica gel, 100% ethyl acetate), dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.41 (59 mg, 50%) as a yellow oil. 1H NMR (600 MHz, benzene-d6) ? 4.21 (m, 1H), 3.69 (t, J = 6.0 Hz, 2H), 3.60 (m, 2H), 2.25 (m, 1H), 2.16 (m, 1H), 2.07 (m, 2H), 1.81 (quin, J = 6.6 Hz, 1H), 1.63 (m, 1H), 1.58 (m, 1H), 1.55 (m, 5H), 1.44 (m, 4H), 1.33 (m, 4H), 1.21 (m, 2H), 1.10 (m, 5H), 0.93 (d, J = 1.8 Hz, 3H), 0.92 (d, J = 1.8 Hz, 3H), 0.87 (m, 1H), 0.75 (s, 3H), 0.73 (m, 1H), 0.65 (m, 3H), 0.60 (m, 1H), 0.51 (m, 1H). 13C NMR (150 MHz, benzene-d6) ? 178.1, 98.5, 82.2, 59.5, 59.3, 55.9, 55.0, 54.6, 44.4, 42.6, 42.5, 39.7, 39.6, 36.7, 36.5, 35.4, 35.0, 33.0, 32.6, 29.8, 28.9, 28.6, 27.7, 26.7, 26.5, 23.2, 23.1, 21.0, 14.3, 12.1. HRESIMS [M + Na]+ m/z 495.3454 (calcd for C30H48O4Na, 495.3450). Compound 5.42. To a solution of compound 5.41 (100 mg, 2.1 ? 10-4 mol) in 1,2-dimethoxyethane (12 mL) was added triethyl phosphite (720 ?L, 4.2 ? 10-3 mol), the resultant solution was stirred for 10 minutes at room temperature. The reaction mixture was cooled to -10 ?C (brine/ice) and oxygen gas was bubbled through the solution for 10 minutes. Potassium tert-butoxide (235 mg, 2.1 ? 10-3 mol) was then added to the solution and oxygen was bubbled through the solution for 3 hours at -10 ?C and for 1 hour at 0 ?C. The reaction mixture was slowly warmed to room temperature, water (100 ?L) was added to the solution which was stirred for 1 additional hour while oxygen was constantly bubbled through the solution. TLC analysis of the reaction shows the presence of unreacted starting material along with a new product. The reaction mixture was quenched with a 10% HCl solution (200 mL, water solution) and extracted 140  with ethyl acetate (200 + 100 mL). The combined organic phase was dried over MgSO4 and evaporated under vacuum. The product was purified by column chromatography (silica gel, step gradient from 9:1 hexane/ethyl acetate to 100% ethyl acetate) to furnish compound 5.42 (59 mg, 65%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 5.69 (s, OH), 4.97 (m, 1H), 2.41 (dt, J = 15.0, 6.6 Hz, 1H), 2.29 (t, J = 14.4 Hz, 1H), 2.15 (quin, J = 6.6 Hz, 1H), 2.07 (m, 1H), 2.04 (d, J = 6.0 Hz, 1H), 1.90 (m, 2H), 1.80 (m, 1H), 1.74 (m, 1H), 1.63 (m, 2H), 1.51 (m, 7H), 1.35 (m, 2H), 1.23 (m, 5H), 1.12 (m, 1H), 0.96 (s, 3H), 0.89 (m, 1H), 0.87 (d, J = 4.2 Hz, 3H), 0.86 (d, J = 4.2 Hz, 3H), 0.72 (m, 1H), 0.71 (s, 3H). 13C NMR (150 MHz, DMSO-d6) ? 210.4, 177.4, 82.0, 75.7, 61.2, 54.9, 52.6, 45.9, 44.1, 39.5a, 38.9, 38.0, 37.8, 37.7, 35.3, 33.8, 31.4, 31.2, 31.1, 28.2, 27.5, 22.8, 22.3, 20.9, 20.2, 13.4, 11.1. HRESIMS [M + Na]+ m/z 453.2985 (calcd for C27H42O4Na,453.2981). a:partially obscured                                                                                                                                                                        Clionamine B (5.2). To a solution of compound 5.42 (100 mg, 2.3 ? 10-4 mol) in absolute methanol (10 mL) were added anhydrous ammonium acetate (177 mg, 2.3 ? 10-3 mol) and sodium cyanoborohydride (10 mg, 1.6 ? 10-4 mol). The solution was heated to 30 ?C and stirred for 6 hours. TLC analysis of the reaction shows a complete disappearance of the starting material. Concentrated HCl was added until pH?2 and the resultant solution was evaporated under vacuum. Pure sample of clionamine B (5.2) (90 mg, 90%, white solid) was obtained from the reaction mixture via C18 reversed phase HPLC using a CSC-Inertsil 150A/ODS2, 5 ?m 25 x 0.94 cm column, with 1:9 acetonitrile/H2O as eluent over 15 min (flow rate 2 mL/min). 1H NMR (600 MHz, methanol-d4) ? 5.06 (m, 1H), 3.09 (m, 1H), 2.23 (quin, J = 7.2 Hz, 1H), 2.10 (d, J = 6.0 Hz, 1H), 1.92 (m, 2H), 1.85 (m, 2H), 1.73 (m, 2H), 1.60 (m, 7H), 1.48 (m, 1H), 1.41 (m 1H), 1.37 (m, 2H), 1.34 (m, 2H), 1.30 (m, 1H), 1.25 (m, 2H), 1.23 (m, 1H), 1.10 (m, 1H), 0.94 (m, 1H), 0.92 (d, J = 3.6 Hz, 3H), 0.91 (d, J = 3.6 Hz, 3H), 0.87 (s, 3H), 0.80 (s, 3H), 0.77 (m, 1H). 141  13C NMR (150 MHz, methanol-d4) ? 180.1, 84.5, 78.1, 63.4, 57.1, 55.1, 51.9, 46.1, 41.7, 40.8, 40.2, 37.8, 36.6, 35.9, 34.1, 33.1, 33.0, 32.6, 29.5, 29.4, 27.7, 23.3, 23.0, 22.6, 21.5, 14.4, 12.6. HRESIMS [M + H]+ m/z 432.3476 (calcd for C27H46NO3, 432.3478).             142    Figure 5.9 1H NMR spectrum of compound 5.7 recorded in DMSO-d6 at 600 MHz.         Figure 5.10 13C NMR spectrum of compound 5.7 recorded in DMSO-d6 at 150 MHz. 143    Figure 5.11 1H NMR spectrum of compound 5.8 recorded in DMSO-d6 at 600 MHz.       Figure 5.12 13C NMR spectrum of compound 5.8 recorded in DMSO-d6 at 150 MHz. 144    Figure 5.13 1H NMR spectrum of compound 5.10 recorded in DMSO-d6 at 600 MHz.          Figure 5.14 13C NMR spectrum of compound 5.10 recorded in DMSO-d6 at 150 MHz. 145    Figure 5.15 1H NMR spectrum of compound 5.11 recorded in DMSO-d6 at 600 MHz.      Figure 5.16 13C NMR spectrum of compound 5.11 recorded in DMSO-d6 at 150 MHz. 146    Figure 5.17 1H NMR spectrum of compound 5.12 recorded in DMSO-d6 at 600 MHz.      Figure 5.18 13C NMR spectrum of compound 5.12 recorded in DMSO-d6 at 150 MHz. 147    Figure 5.19 1H NMR spectrum of compound 5.13 recorded in DMSO-d6 at 600 MHz.      Figure 5.20 13C NMR spectrum of compound 5.13 recorded in DMSO-d6 at 150 MHz. 148    Figure 5.21 1H NMR spectrum of compound 5.16 recorded in DMSO-d6 at 600 MHz.      Figure 5.22 13C NMR spectrum of compound 5.16 recorded in DMSO-d6 at 150 MHz. 149        Figure 5.23 ROESY spectrum of compound 5.16 recorded in DMSO-d6 at 600 MHz.  150    Figure 5.24 1H NMR spectrum of compound 5.17 recorded in DMSO-d6 at 600 MHz.      Figure 5.25 13C NMR spectrum of compound 5.17 recorded in DMSO-d6 at 150 MHz. 151        Figure 5.26 ROESY spectrum of compound 5.17 recorded in DMSO-d6 at 600 MHz.   152    Figure 5.27 1H NMR spectrum of compound 5.18 recorded in DMSO-d6 at 600 MHz.      Figure 5.28 13C NMR spectrum of compound 5.18 recorded in DMSO-d6 at 150 MHz. 153    Figure 5.29 1H NMR spectrum of compound 5.14 recorded in DMSO-d6 at 600 MHz.      Figure 5.30 13C NMR spectrum of compound 5.14 recorded in DMSO-d6 at 150 MHz. 154    Figure 5.31 1H NMR spectrum of compound 5.19 recorded in DMSO-d6 at 600 MHz.      Figure 5.32 13C NMR spectrum of compound 5.19 recorded in DMSO-d6 at 150 MHz. 155    Figure 5.33 1H NMR spectrum of compound 5.21 recorded in DMSO-d6 at 600 MHz.      Figure 5.34 13C NMR spectrum of compound 5.21 recorded in DMSO-d6 at 150 MHz. 156    Figure 5.35 1H NMR spectrum of compound 5.22 recorded in benzene-d6 at 600 MHz.      Figure 5.36 13C NMR spectrum of compound 5.22 recorded in benzene-d6 at 150 MHz. 157    Figure 5.37 1H NMR spectrum of compound 5.23 recorded in DMSO-d6 at 600 MHz.      Figure 5.38 13C NMR spectrum of compound 5.23 recorded in DMSO-d6 at 150 MHz. 158    Figure 5.39 1H NMR spectrum of compound 5.24 recorded in DMSO-d6 at 600 MHz.      Figure 5.40 13C NMR spectrum of compound 5.24 recorded in DMSO-d6 at 150 MHz. 159         Figure 5.41 ROESY spectrum of compound 5.24 recorded in DMSO-d6 at 600 MHz.  160    Figure 5.42 1H NMR spectrum of compound 5.25 recorded in DMSO-d6 at 600 MHz.      Figure 5.43 13C NMR spectrum of compound 5.25 recorded in DMSO-d6 at 150 MHz. 161    Figure 5.44 1H NMR spectrum of compound 5.25 recorded in MeOD at 600 MHz.      Figure 5.45 13C NMR spectrum of compound 5.25 recorded in MeOD at 150 MHz. 162    Figure 5.46 1H NMR spectrum of compound 5.27 recorded in DMSO-d6 at 600 MHz.      Figure 5.47 13C NMR spectrum of compound 5.27 recorded in DMSO-d6 at 150 MHz. 163    Figure 5.48 1H NMR spectrum of compound 5.28 recorded in DMSO-d6 at 600 MHz.      Figure 5.49 13C NMR spectrum of compound 5.28 recorded in DMSO-d6 at 150 MHz. 164         Figure 5.50 ROESY spectrum of compound 5.28 recorded in DMSO-d6 at 600 MHz.  165    Figure 5.51 1H NMR spectrum of compound 5.29 recorded in DMSO-d6 at 600 MHz.      Figure 5.52 13C NMR spectrum of compound 5.29 recorded in DMSO-d6 at 150 MHz. 166    Figure 5.53 1H NMR spectrum of compound 5.31 recorded in DMSO-d6 at 600 MHz.      Figure 5.54 13C NMR spectrum of compound 5.31 recorded in DMSO-d6 at 150 MHz. 167    Figure 5.55 1H NMR spectrum of compound 5.33 recorded in DMSO-d6 at 600 MHz.      Figure 5.56 13C NMR spectrum of compound 5.33 recorded in DMSO-d6 at 150 MHz. 168    Figure 5.57 1H NMR spectrum of compound 5.34 recorded in DMSO-d6 at 600 MHz.      Figure 5.58 13C NMR spectrum of compound 5.34 recorded in DMSO-d6 at 150 MHz. 169    Figure 5.59 1H NMR spectrum of compound 5.35 recorded in CD2Cl2 at 600 MHz.      Figure 5.60 13C NMR spectrum of compound 5.35 recorded in CD2Cl2 at 150 MHz. 170    Figure 5.61 1H NMR spectrum of compound 5.36 recorded in benzene-d6 at 600 MHz.      Figure 5.62 13C NMR spectrum of compound 5.36 recorded in benzene-d6 at 150 MHz. 171    Figure 5.63 1H NMR spectrum of compound 5.37 recorded in DMSO-d6 at 150 MHz.      Figure 5.64 13C NMR spectrum of compound 5.37 recorded in DMSO-d6 at 150 MHz. 172        Figure 5.65 ROESY spectrum of compound 5.37 recorded in DMSO-d6 at 600 MHz.   173    Figure 5.66 1H NMR spectrum of compound 5.38 recorded in CD2Cl2 at 600 MHz.      Figure 5.67 13C NMR spectrum of compound 5.38 recorded in CD2Cl2 at 150 MHz. 174    Figure 5.68 1H NMR spectrum of compound 5.40 recorded in benzene-d6 at 600 MHz.      Figure 5.69 13C NMR spectrum of compound 5.40 recorded in benzene-d6 at 150 MHz. 175    Figure 5.70 1H NMR spectrum of compound 5.41 recorded in benzene-d6 at 600 MHz.      Figure 5.71 13C NMR spectrum of compound 5.41 recorded in benzene-d6 at 150 MHz. 176    Figure 5.72 1H NMR spectrum of compound 5.42 recorded in DMSO-d6 at 600 MHz.      Figure 5.73 13C NMR spectrum of compound 5.42 recorded in DMSO-d6 at 150 MHz. 177         Figure 5.74 ROESY spectrum of compound 5.42 recorded in DMSO-d6 at 600 MHz. 178    Figure 5.75 1H NMR spectrum of synthetic clionamine B (5.2) recorded in MeOD at 600 MHz.     Figure 5.76 13C NMR spectrum of synthetic clionamine B (5.2) recorded in MeOD at 150 MHz. 179   Figure 5.77 1H NMR spectrum (enlarged images) of synthetic clionamine B (5.2) recorded in MeOD at 600 MHz.    180    Figure 5.78 1H NMR spectrum of natural clionamine B (5.2) (bottom spectrum) recorded in MeOD at 600 MHz. (adapted from Keyzers, R. A.; Daoust, J.; Davies-Coleman, M. T.; Van Soest, R.; Balgi, A.; Donohue, E.; Roberge, M.; Andersen, R. J. Org. Lett. 2008, 10, 2959-2962).40 1H NMR spectrum of synthetic clionamine B (5.2) (top spectrum) recorded in MeOD at 600 MHz.49 181   Figure 5.79 Minimum energy conformation of lactone 5.10 (ChemBio3D?).  5.5.3 Inhibition Mtb Proliferation in THP-1 Macrophage: Bioassay Procedure Effect of 3,5-epi-clionamine B (5.25) and compounds 5.12, 5.18, 5.23, 5.24, and 5.29 on Mtb proliferation and THP-1 viability. Differentiated THP-1 cells infected with Mtb H37Rv harboring a luciferase-expressing plasmid were treated with various concentrations of 3,5-epi-clionamine B (5.25) or its analogs at the indicated times. DMSO served as negative control. Intracellular Mtb proliferation was measured as luciferase activity at 24 h (A), 48 h (C) and 72 h (E). (B, D, F) Viability of THP-1 cells treated with drugs was measured with the MTT assay at 24 h (B), 48 h (D) and 72 h (F). To assess the effect of 3,5-epi-clionamine B (5.25) and compounds 5.12, 5.18, 5.23, 5.24, 182  and 5.29 on the intracellular survival of Mtb during macrophage infection, we infected differentiated THP-1 human acute monocytic leukemia cells with an Mtb strain harboring a plasmid that expresses luciferase constitutively under the control of the hsp60 promoter. After removal of non-internalized bacteria, 3,5-epi-clionamine B (5.25) or its analogs was added, and Mtb proliferation was assessed at 24, 48 and 72 hours by measuring luminescence signal. 3,5-epi-clionamine B (5.25) (at 60 ?g/mL), and compound 5.29 (at 60 ?g/mL) showed greater than 99% inhibition of Mtb proliferation at 24, 48 and 72 hours post-infection. However, the reduction in luminescence is likely due to the cytotoxicity of the compounds instead of macrophage elimination of Mtb as less than 10% THP-1 cells were viable when incubated with the compounds at the aforementioned concentrations. Compound 5.12 (at 60, 30, 10 and 5 ?g/mL) showed ~50% inhibition of Mtb proliferation at 24 hours post-infection. However, by 72 hours post-infection, the same efficacy was not observed as there was no significant reduction in luminescence signal when compared to the DMSO control. Furthermore, at 60 ?g/mL, compound 5.12 also showed cytotoxicity (~40% viable THP-1) by 72 hours. The same trend was found for compound 5.18 where reduction of luminescence was observed only at toxic concentrations of the compounds. Compound 5.23 showed no significant effect on Mtb proliferation as compared to the DMSO control.  Interestingly, compound 5.24 (at 5 ?g/mL) showed greater than 98% inhibition of Mtb proliferation by 72 hours post-infection with minimal cytotoxicity (greater than 85% THP-1 viable). In this regard, Compound 5.24 is the most effective and promising compound in reducing the intracellular growth of Mtb during macrophage infection.    183  Chapter 6: Structural Requirements of Niclosamide and Nitazoxanide for the Stimulation of Autophagy Via Inhibition of mTORC1  6.1 Introduction Autophagy is a cellular process that involves the degradation of cytoplasmic components in double membrane vesicles, called autophagosomes, upon fusion with lysosomal compartments.64 The process is activated in response to cellular stresses, such as lack of nutrients and bacteria infection.65a,b     Figure 6.1 mTORC1 and mTORC2 signaling pathways [adapted from Foster, K. G.; Fingar, D. C. Journal of Biological Chemistry, 2010, 285 (19), 14071-14077].66  184  Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb).67 TB is a major cause of mortality in the world today, with 1.4 millions of deaths in 2011. The ability of Mtb to develop resistance against antibiotics is dramatically increasing, therefore there is a major need for new therapeutic agents to curb the disease.68a,b The activation of autophagy in macrophages plays a key role in stopping Mtb proliferation. The fusion of autophagosomes and lysosomes generates phagolysosomes that can subsequently kill Mtb in macrophages. Mtb is able to respond to this defense mechanism by manipulating intracellular membrane trafficking events in macrophages. Precisely, Mtb stops the formation of phagolysosomes by secreting a protein phosphatase (pTpA) that blocks the acquisition of the vacuolar-type H+-ATPase required for acidification of the lumen, limiting the acquisition of lysosomal hydrolases and depleting the phagosome of phosphatidylinositol 3-phosphate. There are published reports showing that induction of autophagy by small molecules reduces the survival of intracellular Mtb in macrophages.69a-e Autophagy is regulated by the mammalian target of rapamycin complex 1 (mTORC1), a major regulator of cell growth and metabolism (Figure 6.1). Stimulation of mTORC1 by growth factors and nutrients leads to the activation of anabolic processes such as protein synthesis. Conversely, inhibition of mTORC1 by lack of nutrients and cellular stresses, such as bacteria infections, leads to the suppression of anabolic processes and induces autophagy as a protective response.70a-d The activation of autophagy in macrophages is a valid strategy to combat Mtb infection. Induction of autophagy can be achieved by selective inhibition of mTORC1, which becomes an attractive target of new treatments for tubercolosis. Prof. Michel Roberge and coworkers have recently identified two new autophagy stimulators/mTORC1 signaling inhibitors: niclosamide 185  (6.1) and nitazoxanide (6.7) (Figure 6.2 and Figure 6.3). A library of new analogues (Figure 6.2 and Figure 6.3) was synthesized to describe the structural requirements of these two synthetic compounds for activity in mTORC1 inhibition and autophagosome induction. Niclosamide (6.1) and nitazoxanide (6.7) are two potential drug candidates for the treatment of Mtb.71a,b  6.2 Synthesis of Niclosamide and Nitazoxanide Analogues 6.2.1 Synthesis of Niclosamide Analogues: Inhibition of mTORC1 Signaling by Protonophoric Activity of Niclosamide (6.1) Niclosamide (6.1) is a salicylanilide derivative used in the treatment of parasitic infections. In this work, we found that niclosamide (6.1) inhibits mTORC1 signaling.71a,b We also studied its unknown mechanism of action, identifying a linkage between the ability of this drug to transport protons across biological membranes, defined as protonophoric activity, and its biological activity. We were able to demonstrate that niclosamide (6.1) is an effective protonophore, by showing that niclosamide (6.1) has protonophoric activity in a cell-free system. Niclosamide (6.1) is already well characterized as a protonophore in the literature. Based on a previously reported model,72a-e the weakly acidic OH, the electron-withdrawing NO2 and the NH groups are the structural groups that confer protonophoric activity to niclosamide (6.1). In particular, the OH group has a pKa in the physiological pH range; the electron-withdrawing NO2 group helps delocalize the negative charge of the anionic form of the molecule to maintain its hydrophobicity and membrane association; the NH group participates in an intramolecular hydrogen bond. Five niclosamide analogues 6.2-6.6 were synthesized to describe structural requirements of niclosamide (6.1) for its biological activity associated with protonophoric activity (Figure 6.2). Compound 6.6 without the OH group did not show protonophoric activity. 186  Compound 6.5 with the OH group at the meta position to prevent hydrogen bonding with the NH group also showed no protonophoric activity. Furthermore, compound 6.4 lacking the electron-withdrawing NO2 group that makes the dissociable proton more acidic was also completely inactive as well as compound 6.2. Finally, compound 6.3 with an N-methyl substituent incapable of forming the intramolecular hydrogen bond did not show protonophoric activity in vitro. Our description of structural requirements for protonophoric activity of niclosamide (6.1) completely matches the previously reported model by Terada and coworkers.72b    Figure 6.2 Chemical structures of niclosamide (6.1), and niclosamide analogues 6.2-6.6.   Each analogue 6.2-6.6 was then tested in a mTORC1 signaling inhibition assay, showing no activity. It turns out that the structural requirements for protonophoric activity of niclosamide (6.1) are the same as those for mTORC1 inhibition. Niclosamide analogues 6.2-6.6 were then tested for their ability to modulate autophagy and inhibit cell proliferation, showing again no activity. Thus, the structural requirements of niclosamide (6.1) for inhibition of mTORC1 signaling are also required for autophagy activation and inhibition of proliferation, confirming 187  the function of mTORC1 in autophagy and cell proliferation.  6.2.2 Synthesis of Nitazoxanide Analogues: Stimulation of Autophagy, Inhibition of mTORC1 Signaling and Intracellular Proliferation of Mycobacterium tuberculosis The antiprotozoal drug nitazoxanide (6.7) and its active metabolite tizoxanide (6.8) (Figure 6.3) strongly stimulate autophagy and inhibit mTORC1, a major negative regulator of autophagy.71a,b Nitazoxanide (6.7) inhibits Mtb proliferation in infected human THP-1 cells (human monocytic cells) by autophagosome induction, EGFP-LC3 (autophagy fusion gene in expression plasmid) processing and mTORC1 inhibition. The human quinone oxidoreductase NQO1 was identified as a possible nitazoxanide (6.7) target. NQO1 inhibition might be partly responsible for mTORC1 inhibition and for autophagy activation. Sixteen nitazoxanide analogues 6.9-6.24 (Figure 6.3) were synthesized to describe nitazoxanide (6.7) structural requirements for both anti-TB activity and autophagy induction in macrophages as a response to tuberculosis infection.71a The sixteen nitazoxanide analogues 6.9-6.24 were tested for their effects on autophagosome accumulation, EGFP-LC3 processing and inhibition of mTORC1 activity. Compounds 6.13-6.15 without the OH group were completely inactive in all three assays, implying that the free OH group, generated after hydrolysis of nitazoxanide (6.7) by esterases, is essential for activity. Additionally, compounds 6.9-6.11 with a non-cleavable ether bond were also inactive in all three assays. Furthermore, compounds 6.17-6.19, bearing more bulky propionate, isobutyrate and pivalate esters that are cleaved more slowly by esterases, were also active in all three assays, confirming that nitazoxanide (6.7) is the active prodrug of tizoxanide (6.8) and that the OH group is critical for activity.  188    Figure 6.3 Chemical structures of nitazoxanide (6.7), tizoxanide (6.8), and nitazoxanide analogues 6.9-6.24.  Methylation of the NH group led to the loss of activity of compound 6.12, suggesting that 189  intramolecular hydrogen bonding of nitazoxanide (6.7) are required for activity. Nitazoxanide (6.7) also possesses a nitro group attached to the thiazole ring. Compound 6.22 lacking the nitro group was completely inactive in all three assays, showing this group is also required for activity. Furthermore, compounds 6.23 and 6.24 with one or two chlorine substituents on the 6-membered ring of nitazoxanide (6.7) were inactive as well as compounds 6.20 and 6.21. This structure-activity study on nitazoxanide (6.7) shows that the OH, NH and NO2 groups are all essential for induction of autophagosome accumulation, EGFP-LC3 processing and mTORC1 inhibition. Considering that the activity of nitazoxanide (6.7) and its 16 analogues is the same in all these three assays, we hypothesize that these biological responses are linked and probably result from inhibition of a single target rather than multiple independent targets.  6.3 Discussion and Conclusions Tuberculosis (TB) is a contagious disease caused by a bacterium called Mycobacterium tuberculosis. TB usually infects the lungs and can be easily transmitted among people through the air. In the last decade, TB cases have increased by about 20%. It has been estimated by Canadian Lung Association that there will be a total of 36 million deaths from TB by 2020. Multidrug resistant tuberculosis (MDR-TB) is becoming a common and extremely dangerous problem that is threatening public health, therefore getting the attention of governments in many countries. Medical sciences field is directing its attention and effort to the discovery of new anti TB-drugs. Niclosamide (6.1) and nitazoxanide (6.7) are two new autophagy stimulators/mTORC1 signaling inhibitors. Therefore niclosamide (6.1) and nitazoxanide (6.7) are potential drug candidates for the treatment of Mtb. Five niclosamide analogues 6.2-6.6 and sixteen nitazoxanide analogues 6.9-6.24 (Figure 190  6.2 and Figure 6.3) were synthesized for structure-activity studies. Identifying the structural requirements for these two potential anti TB-drugs, we were able to advance our knowledge of  their mechanisms of action. It turns out that EGFP-LC3 processing, mTORC1 inhibition and activation of autophagy are strictly related biological processes, considering that the activity of niclosamide and nitazoxanide analogues is consistent in these three biological assays.  Niclosamide (6.1) and nitazoxanide (6.7) are two known drugs that activates autophagy via inhibition of mTORC1. The biological activity of niclosamide (6.1) and nitazoxanide (6.7) synthetic analogues (Figure 6.2 and Figure 6.3) reveals the structural requirements of these two drugs for autophagy activation and Mtb clearance in human macrophages.   6.4 Experimental Section 6.4.1 General Experimental Procedures  All reactions were carried out with dry solvents under a nitrogen atmosphere in anhydrous conditions, unless otherwise noted. Commercially available anhydrous tetrahydrofuran (THF), dimethylformamide (DMF), and dichloromethane (CH2Cl2) were used to the perform the reactions, unless otherwise stated. Yields refer to chromatographically and spectroscopically (1H NMR, 13C NMR) homogeneous materials, unless otherwise stated. Reagents were purchased at the highest commercial quality and used without further purification, unless otherwise stated. Reactions were monitored by thin layer chromatography (TLC) carried out on Merck Type 5554 silica gel plates using UV light as visualizing agent and a solution of p-anisaldehyde in ethanol/aqueous H2SO4, and heat as developing agents. Flash chromatography was performed using Silicycle Ultra Pure silica gel (230-400 mesh). The 1H and 13C NMR spectra were recorded on a Bruker AV-600 spectrometer with a 5 191  mm CPTCI cryoprobe. 1H chemical shifts are referenced to the residual DMSO- d6 signal (? 2.50 ppm) and 13C chemical shifts are referenced to the DMSO- d6 solvent peak (? 39.51 ppm). The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, quin = quintuplet, sext = sextet, sep = septet, b = broad. Low resolution ESI +/- were recorded on Bruker Esquire LC ion trap mass spectrometer equipped with an electrospray ion source.  The solvent for ESI-MS experiments was methanol. The sample solution concentration was 10 ?M. It was infused into the ion source by a syringe pump at flow rate of 10 ?L/min. High resolution ESI+ were recorded on a Micromass LCT time-of-flight (TOF) mass spectrometer equipped with an electrospray ion source. The samples were dissolved in methanol. The working solutions were 20 ?M. Flow rate: 20 ?L min-1; sample cone: 90 V; source temperature: 120 ?C; desolvation temperature: 120 ?C.  Compounds 6.9, 6.10, 6.11, 6.13, 6.14, 6.15, 6.21, 6.22: Method A (aromatic acyl chloride formation and coupling with heteroaromatic primary amine). To a solution of aromatic carboxylic acid (0.001 mol) in dichloromethane (10 mL) was added thionyl chloride (144 ?L, 0.002 mol) and a catalytic amount of DMF (50 ?L). The solution was stirred for 1 hour. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The solvent was removed under nitrogen flow to give the aromatic acid chloride as a yellow oil. The aromatic acyl chloride was dissolved in THF (10 mL) and the heteroaromatic primary amine (0.001 mol) was added to the resultant solution. The solution was stirred for 10 minutes then triethylamine (279 ?L, 0.002 mol) was added slowly to the mixture. The reaction was stirred for 1 hour. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The reaction was then quenched with 40 mL of 10% hydrochloric acid solution. The 192  solution was extracted with dichloromethane (2 x 30 mL). The combined organic phase was washed with a saturated sodium bicarbonate solution (2 x 20 mL), dried and evaporated under vacuum. The resulting residue was purified by flash column chromatography  (silica gel, step gradient from 9:1 dichloromethane/MeOH to MeOH) to obtain analytically pure product. Compounds 6.16, 6.17, 6.18, 6.19, 6.23, 6.24: Method B (synthesis of salicylic acid esters, acyl chloride formation and coupling with heteroaromatic primary amine). To a solution of salicylic acid (0.001 mol) in THF (10 mL) was added the aliphatic anhydride (0.005 mol) and a catalytic amount of 4-dimethylaminopyridine (12 mg, 0.0001 mol). The solution was stirred for 1 hour. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The reaction was then quenched with 40mL of water. The solution was extracted with dichloromethane (2 x 30 mL). The combined organic phase was washed with water (2 x 20 mL), dried  and evaporated under vacuum to give salicylic acid ester as a white solid. The salicylic acid ester was dissolved in dichloromethane (10 mL). Then thionyl chloride (144 ?L, 0.002 mol) and a catalytic amount of DMF (50 ?L) were added to the solution. The reaction was stirred for 1 hour. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The solvent was removed under nitrogen flow to give the salicylic acyl chloride as a yellow oil. The salicylic acid chloride was dissolved in THF (10 mL) and the heteroaromatic primary amine (0.001 mol) was added to the resultant solution. The solution was stirred for 10 minutes then triethylamine (279 ?L, 0.002 mol) was added slowly to the mixture. The reaction was stirred for 1 hour. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The reaction was then quenched with 40mL of 10% hydrochloric acid solution. The solution was extracted with dichloromethane (2 x 30 mL). The combined organic phase was washed with a saturated sodium bicarbonate solution (2 x 20 mL), dried  and 193  evaporated under vacuum. The resulting residue was purified by flash column chromatography (silica gel, step gradient from 9:1 dichloromethane/MeOH to MeOH) to obtain analytically pure product. 5-chloro-N-(2-chloro-4-nitrophenyl)-2-methoxybenzamide (6.2). Niclosamide (6.1) (327 mg, 0.001 mol) was added to a stirred suspension of K2CO3 (138 mg, 0.001 mol) in DMF (10 mL). The methylating agent dimethyl sulfate (95 ?L, 0.001 mol) was then added slowly to the reaction mixture. The solution was stirred for 24 hours. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The reaction was then quenched with 40mL of water. The solution was extracted with dichloromethane (2 x 30 mL). The combined organic phase was washed with water (2 x 20 mL), dried and evaporated under vacuum. The resulting residue was purified by column chromatography (silica gel, step gradient from 9:1 dichloromethane/MeOH to 100% MeOH) to afford the pure product 6.2 (85 mg, 25 %) as a yellow solid. 1H NMR (600 MHz, DMSO-d6) ? 4.12 (s, 3H), 7.40 (d, J = 9.0 Hz, 1H,), 7.73 (dd, J = 9.0, 3.0 Hz, 1H), 8.02 (d, J = 3.0 Hz, 1H), 8.32 (dd, J = 9.0, 2.4 Hz, 1H), 8.48 (d, J = 3.0 Hz, 1H), 8.77 (d, J = 9.0 Hz, 1H), 10.93 (s, 1H). 13C NMR (150 MHz, DMSO-d6) ? 164.7a, 156.2, 142.8, 140.0a, 134.1, 130.6, 125.3, 124.9, 123.9, 122.8, 121.8, 121.0, 115.2, 57.4  HRESIMS [M    H]    m/z 338.9934 (calcd for C14H9Cl2N2O4, 338.9939). a: partially obscured 5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxy-N-methylbenzamide (6.3). To a solution of niclosamide (6.1) (327 mg, 0.001 mol) in THF (10 mL) was added acetic anhydride (472 ?L, 0.005 mol) and a catalytic amount of DMAP (12 mg, 0.0001 mol). The solution was stirred for 1 hour. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The reaction was then quenched with 40mL of water. The solution was extracted with dichloromethane (2 x 30 mL). The combined organic phase was washed with 194  water (2 x 20 mL), dried and evaporated under vacuum to give O-acetyl niclosamide as a yellow solid. O-acetyl niclosamide was added to a stirred suspension of K2CO3 (690 mg, 0.005 mol) in DMF (10 mL). The methylating agent dimethyl sulfate (95 ?L, 0.001 mol) was then added slowly to the reaction mixture. The solution was stirred for 24 hours. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The reaction was then quenched with 40mL of 10% hydrochloric acid solution. The solution was extracted with dichloromethane (2 x 30 mL). The combined organic phase was washed with water (2 x 20 mL), dried and evaporated under vacuum. The resulting residue was purified by column chromatography (silica gel, step gradient from 9:1 dichloromethane/MeOH to 100% MeOH) to afford the O-acetyl analogue of 6.3, which was then added to a stirred solution of K2CO3 (138 mg, 0.001 mol) in MeOH (10 mL). The reaction was stirred for 12 hours. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The reaction was then quenched with 40mL of 10% hydrochloric acid solution. The solution was extracted with dichloromethane (2 x 30 mL). The combined organic phase was washed with water (2 x 20 mL), dried and evaporated under vacuum. The resulting residue was purified by flash column chromatography (silica gel, step gradient from 9:1 dichloromethane/MeOH to MeOH) to obtain analytically the pure product 6.3 (204 mg, 60%) as a yellow solid. 1H NMR (600 MHz, DMSO-d6) ? 3.54 (s, 3H), 7.60 (dd, J = 8.1, 1.2 Hz, 1H,), 7.73 (m, 4H), 8.18 (dd, J = 9.0, 2.4 Hz, 1H), 8.35 (d, J = 2.4 Hz, 1H). 13C NMR (150 MHz, DMSO-d6) ? 36.6, 117.1, 121.8, 122.5, 124.1, 126.9, 130.1, 131.2, 134.3, 141.4, 144.5, 151.8, 157.7, 163.6. HRESIMS [M  H]    m/z 338.9930 (calcd for C14H9Cl2N2O4, 338.9939). 5-chloro-N-(2-chlorophenyl)-2-hydroxybenzamide (6.4). To a solution of 5-chloro-2-hydroxybenzoic acid (172 mg, 0.001 mol) in THF (10 mL) was added acetic anhydride (472 ?L, 195  0.005 mol) and a catalytic amount of DMAP (12 mg, 0.0001 mol). The solution was stirred for 1 hour. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The reaction was then quenched with 40mL of water. The solution was extracted with dichloromethane (2 x 30 mL). The combined organic phase was washed with water (2 x 20 mL), dried  and evaporated under vacuum to give 2-acetoxy-5-chlorobenzoic acid as a white solid. 2-acetoxy-5-chlorobenzoic acid was dissolved in dichloromethane (10 mL). Then thionyl chloride (144 ?L, 0.002 mol) and a catalytic amount of DMF (50 ?L) were added to the solution. The reaction was stirred for 1 hour. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The solvent was removed under nitrogen flow to give 2-acetoxy-5-chlorobenzoic acyl chloride as a yellow oil. 2-acetoxy-5-chlorobenzoic acyl chloride was dissolved in THF (10 mL) and 2-chloroaniline (105 ?L, 0.001 mol) was added to the resultant solution. The solution was stirred for 10 minutes then triethylamine (279 ?L, 0.002 mol) was added slowly to the mixture. The reaction was stirred for 1 hour. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The reaction was then quenched with 40mL of 10% hydrochloric acid solution. The solution was extracted with dichloromethane (2 x 30 mL). The combined organic phase was washed with a saturated sodium bicarbonate solution (2 x 20 mL), dried and evaporated under vacuum. The resulting residue was purified by flash column chromatography (silica gel, step gradient from 9:1 dichloromethane/MeOH to MeOH) to obtain the O-acetyl analogue of 6.4, which was then added to a stirred solution of K2CO3 (138 mg, 0.001 mol) in MeOH (10 mL). The reaction was stirred for 12 hours. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The reaction was then quenched with 40mL of 10% hydrochloric acid solution. The solution was extracted with dichloromethane (2 x 30 mL). The combined organic phase was 196  washed with water (2 x 20 mL), dried and evaporated under vacuum. The resulting residue was purified by flash column chromatography (silica gel, step gradient from 9:1 dichloromethane/MeOH to MeOH) to obtain analytically pure product 6.4 (56 mg, 20%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 7.08 (d, J = 8.4 Hz, 1H), 7.20 (t, J = 7.8 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.51 (dd, J = 8.7, 2.4 Hz, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.99 (d, J = 2.4 Hz, 1H), 8.39 (d, J = 8.4 Hz, 1H), 10.89 (s, 1H), 12.27 (s, 1H). 13C NMR (150 MHz, DMSO-d6) ? 119.1, 119.6, 122.9, 123.5, 123.6, 125.5, 127.9, 129.4, 129.7, 133.5, 135.0, 155.5, 162.7. HRESIMS [M + H]+ m/z 282.0084 (calcd for C13H10Cl2NO2, 282.0089). 2-Ethoxy-N-(5-nitrothiazol-2-yl)benzamide (6.9). Method A yielded compound 6.9 (129 mg, 41%) as a yellow solid. 1H NMR (600 MHz, DMSO-d6) ? 1.39 (t, J = 7.2 Hz, 3H), 4.20 (q, J = 7.2 Hz, 2H), 7.11 (t, J = 7.2 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.60 (dt, J = 7.5, 1.8 Hz, 1H), 7.74 (dd, J = 7.5, 1.8 Hz, 1H), 8.69 (s, 1H), 12.73 (bs, 1H). 13C NMR (150 MHz, DMSO-d6) ? 14.4, 64.5, 113.2, 120.6, 120.7, 130.5, 134.3, 142.0, 142.8, 156.8, 161.4, 165.6. HRESIMS [M + Na]+ m/z 316.0363 (calcd for C12H11N3O4SNa, 316.0368). 2-Ethoxy-N-(5-formylthiazol-2-yl)benzamide (6.10). Method A yielded compound 6.10 (160 mg, 58%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 1.41 (t, J = 7.2 Hz, 3H), 4.22 (q, J = 7.2 Hz, 2H), 7.11 (t, J = 7.8 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.59 (dt, J = 7.8, 1.8 Hz, 1H), 7.77 (dd, J = 7.5, 1.8 Hz, 1H), 8.48 (s, 1H), 10.00 (s, 1H), 12.37 (bs, 1H). 13C NMR (150 MHz, DMSO-d6) ? 14.5, 64.6, 113.3, 120.8, 120.9, 130.5, 132.4, 134.1, 150.8, 156.8, 163.5, 164.8, 184.3. HRESIMS [M + Na]+ m/z 299.0473 (calcd for C13H12N2O3SNa, 299.0466). 2-Ethoxy-N-(thiazol-2-yl)benzamide (6.11). Method A yielded compound 6.11 (149 mg, 60%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 1.43 (t, J = 6.6 Hz, 3H), 4.24 (q, J = 6.6 Hz, 2H), 7.11 (t, J = 7.5 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 3.6 Hz, 1H), 7.53 (d, J = 197  3.6 Hz, 1H), 7.57 (dt, J = 7.5, 1.8 Hz, 1H), 7.82 (dd, J = 7.8, 1.8 Hz, 1H), 11.78 (s, 1H). 13C NMR (150 MHz, DMSO-d6) ? 14.5, 64.7, 113.3, 114.0, 120.8, 120.9, 130.7, 133.7, 138.0, 156.6, 157.5, 163.3. HRESIMS [M + Na]+ m/z 271.0515 (calcd for C12H12N2O2SNa, 271.0517). 2-(Methyl(5-nitrothiazol-2-yl)carbamoyl)phenyl acetate (6.12). To a solution of acetylsalicylic acid (180 mg, 0.001 mol) in dichloromethane (10 mL) was added thionyl chloride (144 ?L, 0.002 mol) and a catalytic amount of DMF (50 ?L). The solution was stirred for 1 hour. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The solvent was removed under nitrogen flow to give acetylsalicylic acyl chloride as a yellow oil. The acetylsalicylic acyl chloride was dissolved in THF (10 mL) and 2-amino-5-nitrothiazole (145 mg, 0.001 mol)  was added to the resultant solution. The solution was stirred for 10 minutes then triethylamine (279 ?L, 0.002 mol) was added to the mixture. The reaction was stirred for 1 hour. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The reaction was then quenched with 40 mL of 10% hydrochloric acid solution. The solution was extracted with dichloromethane (2 x 30 mL). The combined organic phase was washed with a saturated sodium bicarbonate solution (2 x 20 mL), dried and evaporated under vacuum. The resulting residue was purified by flash column chromatography (silica gel, step gradient from 9:1 dichloromethane/MeOH to MeOH) to obtain pure nitazoxanide (6.7). Nitazoxanide (6.7) was added to a stirred suspension of K2CO3 (690 mg, 0.005 mol) in DMF (10 mL). The methylating agent iodomethane (62 ?L, 0.001 mol) was then added slowly to the reaction mixture. The solution was stirred for 24 hours. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The reaction was then quenched with 40mL of 10% hydrochloric acid solution. The solution was extracted with dichloromethane (2 x 30 mL). The combined organic phase was washed with water (2 x 20 mL), dried and evaporated 198  under vacuum.  The resulting residue was purified by column chromatography (silica gel, step gradient from 9:1 dichloromethane/MeOH to 100% MeOH) to afford the the pure product 6.12 (177 mg, 55%) as a yellow solid. 1H NMR (600 MHz, DMSO-d6) ? 2.32 (s, 3H), 3.83 (s, 3H), 7.22 (d, J = 8.4 Hz, 1H), 7.43 (t, J = 7.2 Hz, 1H), 7.65 (dt, J = 7.8, 1.2 Hz, 1H), 8.30 (dd, J = 7.8, 1.2 Hz, 1H), 9.19 (s, 1H). 13C NMR (150 MHz, DMSO-d6) ? 21.1, 36.7, 123.9, 126.0, 128.0, 131.6, 133.5, 133.6, 136.1, 150.3, 165.1, 169.2, 172.8. HRESIMS [M + Na]+ m/z 344.0312 (calcd for C13H11N3O5SNa, 344.0317). N-(5-Nitrothiazol-2-yl)benzamide (6.13). Method A yielded compound 6.13 (142 mg, 57%) as a yellow solid. 1H NMR (600 MHz, DMSO-d6) ? 7.59 (t, J = 7.8 Hz, 2H), 7.70 (t, J = 7.8 Hz, 1H), 8.13 (d, J = 7.8 Hz, 2H), 8.73 (s, 1H), 13.61 (bs, 1H). 13C NMR (150 MHz, DMSO-d6) ? 128.6, 128.8, 130.9, 133.5, 142.0, 142.7, 162.8, 166.6. HRESIMS [M  +  H] + m/z 248.0124 (calcd for C10H6N3O3S, 248.0130). N-(5-Formylthiazol-2-yl)benzamide (6.14). Method A yielded compound 6.14 (142 mg, 61%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 7.58 (t, J = 7.8 Hz, 2H), 7.68 (t, J = 7.8 Hz, 1H), 8.12 (d, J = 7.2 Hz, 2H), 8.51 (s, 1H), 10.00 (s, 1H), 13.26 (bs, 1H). 13C NMR (150 MHz, DMSO-d6) ? 128.5, 128.7, 131.4, 132.3, 133.2, 150.6, 164.8, 166.0, 184.3. HRESIMS [M + H]+ m/z 233.0387 (calcd for C11H9N2O2S, 233.0385). N-(Thiazol-2-yl)benzamide (6.15). Method A yielded compound 6.15 (139 mg, 68%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 7.29 (d, J = 3.6 Hz, 1H), 7.55 (t, J = 7.8 Hz, 2H), 7.57 (d, J = 3.6 Hz, 1H), 7.63 (t, J = 7.2 Hz, 1H), 8.09 (d, J = 7.2 Hz, 2H), 12.64 (bs, 1H). 13C NMR (150 MHz, DMSO-d6) ? 113.9, 128.1, 128.6, 132.2, 132.6, 137.8, 158.7, 165.1. HRESIMS [M + Na]+ m/z 227.0259 (calcd for C10H8N2OSNa, 227.0255). 4-(5-nitrothiazol-2-ylcarbamoyl)phenyl acetate (6.16). Method B yielded compound 199  6.16 (100 mg, 33%) as a yellow solid. 1H NMR (600 MHz, DMSO-d6) ? 2.31 (s, 3H), 7.35 (d, J = 8.4 Hz, 2H), 8.18 (d, J = 8.4 Hz, 2H), 8.72 (s, 1H), 13.62 (bs, 1H). 13C NMR (150 MHz, DMSO-d6) ? 20.9, 122.3, 128.8, 130.3, 141.7, 142.9, 154.2, 163.3, 166.1, 168.9. HRESIMS [M + Na]+ m/z 330.0167 (calcd for C12H9N3O5SNa 330.0161). 2-(5-Nitrothiazol-2-ylcarbamoyl)phenyl propionate (6.17). Method B yielded compound 6.17 (132 mg, 41%) as a yellow solid. 1H NMR (600 MHz, DMSO-d6) ? 1.03 (t, J = 7.2 Hz, 3H), 2.57 (q, J = 7.2 Hz, 2H), 7.32 (d, J = 7.8 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.69 (dt, J = 7.8, 1.2 Hz, 1H), 7.84 (dd, J = 7.8, 1.2 Hz, 1H), 8.70 (s, 1H), 13.62 (bs, 1H). 13C NMR (150 MHz, DMSO-d6) ? 8.8, 27.0, 123.4, 125.6, 125.9, 129.8, 133.5, 142.1, 142.6, 148.7, 161.9, 165.4, 172.2. HRESIMS [M + H]+ m/z 322.0493 (calcd for C13H12N3O5S 322.0498). 2-(5-Nitrothiazol-2-ylcarbamoyl)phenyl isobutyrate (6.18). Method B yielded compound 6.18 (124 mg, 37%) as a yellow solid. 1H NMR (600 MHz, DMSO-d6) ? 1.18 (d, J = 6.6 Hz, 6H), 2.79 (sep, J = 7.2 Hz, 1H), 7.32 (d, J = 7.8 Hz, 1H), 7.45 (t, J = 7.8 Hz, 1H), 7.68 (dt, J = 7.8, 1.2 Hz, 1H), 7.82 (dd, J = 7.8, 1.2 Hz, 1H), 8.70 (s, 1H), 13.62 (bs, 1H). 13C NMR (150 MHz, DMSO-d6) ? 18.5, 33.4, 123.3, 125.9, 126.0, 129.7, 133.4, 142.1, 142.6, 148.5, 161.8, 165.4, 174.5. HRESIMS [M + Na]+ m/z 358.0467 (calcd for C14H13N3O5SNa 358.0474). 2-(5-Nitrothiazol-2-ylcarbamoyl)phenyl pivalate (6.19). Method B yielded compound 6.19 (119 mg, 34%) as a yellow solid. 1H NMR (600 MHz, DMSO-d6) ? 1.25 (s, 9H), 7.29 (d, J = 7.8 Hz, 1H), 7.44 (t, J = 7.2 Hz, 1H), 7.67 (dt, J = 7.2, 1.2 Hz, 1H), 7.81 (dd, J = 7.8, 1.2 Hz, 1H), 8.69 (s, 1H), 13.60 (bs, 1H). 13C NMR (150 MHz, DMSO-d6) ? 26.7, 38.5, 123.2, 126.0, 126.2, 129.6, 133.2, 142.0, 142.7, 148.6, 162.0, 165.5, 175.9. HRESIMS [M + Na]+ m/z 372.0623 (calcd for C15H15N3O5SNa 372.0630). Methyl 2-(5-nitrothiazol-2-ylcarbamoyl)benzoate (6.20). To a solution of phthalic 200  anhydride (148 mg, 0.001 mol) in THF (10 mL) was added a catalytic amount of 4-dimethylaminopyridine (12 mg, 0.0001 mol). The solution was stirred for 30 minutes. Then methanol (5 mL) was added to the reaction mixture which was stirred for 1 hour. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The reaction was then quenched with 40mL of water. The solution was extracted with chloroform (2 x 30 mL). The combined organic phase was washed with water (2 x 20 mL), dried and evaporated under vacuum to give 2-(methoxycarbonyl)benzoic acid as a white solid. 2-(methoxycarbonyl)benzoic acid was dissolved in dichloromethane (10 mL). Then thionyl chloride (144 ?L, 0.002 mol) and a catalytic amount of DMF (50 ?L) were added to the solution. The reaction was stirred for 1 hour. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The solvent was removed under nitrogen flow to give 2-(methoxycarbonyl)benzoic acyl chloride as a yellow oil. The 2-(methoxycarbonyl)benzoic acyl chloride was dissolved in THF (10 mL) and 2-amino-5-nitrothiazole (145 mg, 0.001 mol) was added to the resultant solution. The solution was stirred for 10 minutes then triethylamine (279 ?L, 0.002 mol) was added to the mixture. The reaction was stirred for 1 hour. TLC analysis of the reaction mixture shows a complete disappearance of the starting material. The reaction was then quenched with 40mL of 10% hydrochloric acid solution. The solution was extracted with dichloromethane (2 x 30 mL). The combined organic phase was washed with a saturated sodium bicarbonate solution (2 x 20 mL), dried  and evaporated under vacuum. The resulting residue was purified by column chromatography (silica gel, step gradient from 9:1 dichloromethane/MeOH to MeOH) to afford the pure product 6.20 (92 mg, 30%) as a yellow solid. 1H NMR (600 MHz, DMSO-d6) ? 3.78 (s, 3H), 7.73 (m, 3H), 7.94 (d, J = 7.8 Hz, 1H), 8.67 (s, 1H), 13.59 (bs, 1H). 13C NMR (150 MHz, DMSO-d6) ? 52.6, 124.0, 128.4, 129.0, 129.5, 201  131.0, 132.4, 135.2, 135.3, 142.9, 166.1, 168.8. HRESIMS [M + Na]+ m/z 330.0164 (calcd for C12H9N3O5SNa, 330.0161). 2-(5-Formylthiazol-2-ylcarbamoyl)phenyl acetate (6.21). Method A yielded the title compound 6.21 (157 mg, 54%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 2.23 (s, 3H), 7.30 (d, J = 7.8 Hz, 1H), 7.43 (t, J = 7.8 Hz, 1H), 7.66 (dt, J = 7.2, 1.2 Hz, 1H), 7.82 (d, J = 7.2 Hz, 1H), 8.49 (s, 1H), 10.00 (s, 1H), 13.25 (bs, 1H). 13C NMR (150 MHz, DMSO-d6) ? 20.7, 117.2, 123.4, 125.9, 126.1, 129.7, 132.4, 133.2, 148.6, 150.5, 164.0, 168.9, 184.3. HRESIMS [M + Na]+ m/z 313.0261 (calcd for C13H10N2O4SNa 313.0259). 2-(Thiazol-2-ylcarbamoyl)phenyl acetate (6.22). Method A yielded compound 6.22 (131 mg, 50%) as a white solid. 1H NMR (600 MHz, DMSO-d6) ? 2.22 (s, 3H), 7.27 (d, J = 8.4 Hz, 1H), 7.28 (d, J = 3.6 Hz, 1H), 7.40 (t, J = 7.8 Hz, 1H), 7.54 (d, J = 3.6 Hz, 1H), 7.62 (dt, J = 7.8, 1.2 Hz, 1H), 7.77 (d, J = 7.8 Hz, 1H), 12.58 (bs, 1H). 13C NMR (150 MHz, DMSO-d6) ? 20.7, 113.9, 123.3, 125.9, 126.9, 129.6, 132.6, 137.8, 148.5, 158.0, 163.9, 168.9. HRESIMS [M + Na]+ m/z 285.0316 (calcd for C12H10N2O3SNa, 285.0310). 2-Chloro-6-(5-nitrothiazol-2-ylcarbamoyl)phenyl acetate (6.23). Method B yielded compound 6.23 (109 mg, 32%) as a yellow solid. 1H NMR (600 MHz, DMSO-d6) ? 2.32 (s, 3H), 7.50 (t, J = 7.8 Hz, 1H), 7.84 (dd, J = 7.8, 1.2 Hz, 1H), 7.87 (dd, J = 8.4, 1.2 Hz, 1H), 8.72 (s, 1H), 13.76 (bs, 1H).13C NMR (150 MHz, DMSO-d6) ? 20.2, 127.3, 127.5, 127.8, 128.8, 133.7, 142.2, 142.5, 144.9, 161.9, 164.6, 167.9. HRESIMS [M + Na]+ m/z 363.9767 (calcd for C12H8ClN3O5SNa, 363.9771). 2,4-Dichloro-6-(5-nitrothiazol-2-ylcarbamoyl)phenyl acetate (6.24). Method B yielded compound 6.24 (112 mg, 30%) as a yellow solid. 1H NMR (600 MHz, DMSO-d6) ? 2.33 (s, 3H), 8.00 (d, J = 2.4 Hz, 1H), 8.10 (d, J = 2.4 Hz, 1H), 8.72 (s, 1H), 13.80 (bs, 1H). 13C NMR (150 202  MHz, DMSO-d6) ? 20.2, 128.7, 128.9, 128.9, 130.7, 132.9, 142.2, 142.5, 144.0, 162.0, 163.6, 167.8. HRESIMS [M + Na]+ m/z 397.9389 (calcd for C12H7Cl2N3O5SNa, 397.9381).               203    Figure 6.4 1H NMR spectrum of compound 6.2 recorded in DMSO-d6 at 600 MHz.         Figure 6.5 13C NMR spectrum of compound 6.2 recorded in DMSO-d6 at 150 MHz. 204    Figure 6.6 1H NMR spectrum of  compound 6.3 recorded in DMSO-d6 at 600 MHz.       Figure 6.7 13C NMR spectrum of compound 6.3 recorded in DMSO-d6 at 150 MHz. 205    Figure 6.8 1H NMR spectrum of  compound 6.4 recorded in DMSO-d6 at 600 MHz.      Figure 6.9 13C NMR spectrum of compound 6.4 recorded in DMSO-d6 at 150 MHz. 206    Figure 6.10 1H NMR spectrum of  compound 6.9 recorded in DMSO-d6 at 600 MHz.         Figure 6.11 13C NMR spectrum of compound 6.9 recorded in DMSO-d6 at 150 MHz. 207    Figure 6.12 1H NMR spectrum of  compound 6.10 recorded in DMSO-d6 at 600 MHz.       Figure 6.13 13C NMR spectrum of compound 6.10 recorded in DMSO-d6 at 150 MHz. 208    Figure 6.14 1H NMR spectrum of  compound 6.11 recorded in DMSO-d6 at 600 MHz.       Figure 6.15 13C NMR spectrum of compound 6.11 recorded in DMSO-d6 at 150 MHz. 209    Figure 6.16 1H NMR spectrum of  compound 6.12 recorded in DMSO-d6 at 600 MHz.       Figure 6.17 13C NMR spectrum of  compound 6.12 recorded in DMSO-d6 at 150 MHz. 210    Figure 6.18 1H NMR spectrum of  compound 6.13 recorded in DMSO-d6 at 600 MHz.        Figure 6.19 13C NMR spectrum of  compound 6.13 recorded in DMSO-d6 at 150 MHz. 211    Figure 6.20 1H NMR spectrum of  compound 6.14 recorded in DMSO-d6 at 600 MHz.          Figure 6.21 13C NMR spectrum of  compound 6.14 recorded in DMSO-d6 at 150 MHz. 212    Figure 6.22 1H NMR spectrum of  compound 6.15 recorded in DMSO-d6 at 600 MHz.         Figure 6.23 13C NMR spectrum of  compound 6.15 recorded in DMSO-d6 at 150 MHz. 213    Figure 6.24 1H NMR spectrum of  compound 6.16 recorded in DMSO-d6 at 600 MHz.       Figure 6.25 13C NMR spectrum of  compound 6.16 recorded in DMSO-d6 at 150 MHz. 214    Figure 6.26 1H NMR spectrum of  compound 6.17 recorded in DMSO-d6 at 600 MHz.       Figure 6.27 13C NMR spectrum of  compound 6.17 recorded in DMSO-d6 at 150 MHz. 215    Figure 6.28 1H NMR spectrum of  compound 6.18 recorded in DMSO-d6 at 600 MHz.       Figure 6.29 13C NMR spectrum of  compound 6.18 recorded in DMSO-d6 at 150 MHz. 216    Figure 6.30 1H NMR spectrum of  compound 6.19 recorded in DMSO-d6 at 600 MHz.        Figure 6.31 13C NMR spectrum of  compound 6.19 recorded in DMSO-d6 at 150 MHz. 217    Figure 6.32 1H NMR spectrum of  compound 6.20 recorded in DMSO-d6 at 600 MHz.       Figure 6.33 13C NMR spectrum of  compound 6.20 recorded in DMSO-d6 at 150 MHz. 218    Figure 6.34 1H NMR spectrum of  compound 6.21 recorded in DMSO-d6 at 600 MHz.       Figure 6.35 13C NMR spectrum of  compound 6.21 recorded in DMSO-d6 at 150 MHz. 219    Figure 6.36 1H NMR spectrum of  compound 6.22 recorded in DMSO-d6 at 600 MHz.       Figure 6.37 13C NMR spectrum of  compound 6.22 recorded in DMSO-d6 at 150 MHz. 220    Figure 6.38 1H NMR spectrum of compound 6.23 recorded in DMSO-d6 at 600 MHz.       Figure 6.39 13C NMR spectrum of  compound 6.23 recorded in DMSO-d6 at 150 MHz. 221    Figure 6.40 1H NMR spectrum of  compound 6.24 recorded in DMSO-d6 at 600 MHz.       Figure 6.41 13C NMR spectrum of  compound 6.24 recorded in DMSO-d6 at 150 MHz. 222  Chapter 7: Summary and Future Work  7.1 Summary The work reported in this thesis is largely focused on the chemical biology studies of bioactive molecules that have potential therapeutic applications in the cure of certain diseases. The isolation of new compounds from natural sources, both marine and terrestrial organisms, and the understanding of their biological function in cellular pathways were the two main goals of our interdisciplinary research projects. The use of reported chemical reactions and the discovery of their new applications in the total synthesis of natural products allowed the production of a sufficient amount of the identified bioactive compounds as well as the synthesis of more potent analogues for biological activity evaluation both in vitro and in vivo. In Chapter 2, alotaketal A (2.1) and analogues (Figure 7.1), a new class of potent cAMP signaling agonists, were identified from the marine sponge Hamigera sp. collected in Papua New Guinea. The alotaketals are new sesterterpenoids containing a spiroketal substructure. They have a regular monocyclic sesterterpenoid carbon skeleton that has not been previously encountered in a natural product. Alotaketal A (2.1) is the most potent and activates the cAMP signaling pathway with an EC50 of 18 nM. To the best of our knowledge, the new alotane sesterterpene alotaketal A (2.1) and the labdane diterpene forskolin (2.2) are the only naturally-occurring small molecules that can strongly and selectively activate cAMP signaling pathway. Chapter 3 involves the study of miRNA pathway and its regulation by genkwanines M (3.1) and P (3.2), two daphnane diterpenoids isolated from the Indonesian plant Wikstroemia polyantha (Figure 7.1). Genkwanines M (3.1) induces an early inflammatory response and can moderately inhibit miR-122 activity in the Huh-7 liver cell line. Genkwanines are potential 223  chemical tools for the study of miRNA regulation and signaling in cellular diseases. Chapter 4 describes the isolation of the antiviral agent virantmycin33 (4.1) and the bromotyrosine-derived alkaloid homoaerothionin (4.2) from cultures of a red marine-derived actinomycete and a marine sponge extract, respectively (Figure 7.1). The two pure compounds were identified as new autophagy inhibitors using a bioassay-guided fractionation approach. Virantmycin (4.1) and homoaerothionin (4.2) represent chemical tools for the study of autophagy functions in cancer cells and can be considered as new potential drug leads for the cure of pancreatic adenocarcinoma (PAC).    Figure 7.1 Chemical structures of alotaketal A (2.1), genkwanine M (3.1), virantmycin (4.1), and homoaerothionin (4.2).  224  In Chapter 5, the naturally occurring aminosteroid clionamine B (5.2) was synthesized starting from the steroidal sapogenin tigogenin (5.30) in 12 steps with ? 2 % overall yield (Figure 7.2). Synthetic clionamine B (5.2) strongly stimulates autophagy at 30 ?g/mL in human breast cancer MCF-7 cells and inhibits Mycobacterium tuberculosis (Mtb) proliferation in human macrophages via autophagy activation. Clionamine B (5.2) and unnatural analogues are potent anti-Mtb compounds and have potential application for the cure of multidrug resistant-tuberculosis (MDR-TB). The clionamine pharmacophore was identified by structure-activity analysis of unnatural clionamine analogues that were synthesized from the steroidal sapogenin sarsasapogenin (5.5). Among these analogues, N-benzyl-3,5-epi-clionamine B (5.24) was found to be a potent inhibitor (MIC = 5 ?g/mL) of Mtb proliferation in THP-1 human acute monocytic leukemia cells. Thus N-benzyl-aminosteroids is a new category of potent antimicrobial compounds that are able to kill TB.     Figure 7.2 Total synthesis of clionamine B (5.2) from the steroidal sapogenin tigogenin (5.30).  Chapter 6 describes the synthesis of five niclosamide analogues 6.2-6.6 and sixteen nitazoxanide analogues 6.9-6.24 for structure-activity studies. Niclosamide (6.1) and nitazoxanide (6.7) are two new autophagy stimulators/mTORC1 signaling inhibitors with 225  potential applications against tuberculosis infection. The biological activity of niclosamide (6.1) and nitazoxanide (6.7) synthetic analogues reveals the structural requirements of these two drugs for autophagy activation and Mtb clearance in human macrophages.   7.2 Future Work: Synthesis of New Analogues and Chemical Probes of Natural Clionamines Future work should involve the synthesis of clionamine chemical probes for molecular protein target(s) identification. We have reported very efficient synthetic methodologies which were designed to use cheap reagents in order to easily generate natural and unnatural clionamines starting from either tigogenin (5.30) or sarsasapogenin (5.5) (Figure 7.3). In particular the ?-methylene lactones 5.21 and 5.40 are key intermediates for the synthesis of chemical probes with photophore and alkyne/azide Click chemistry components. Copper(I)-mediated conjugate addition to the ?-methylene lactone can facilitate the incorporation of different types of side chains into the clionamine structure. SAR studies reported in Chapter 5 reveal that the tertiary alcohol attached to C-20 in the natural clionamines is not required for activity, making the synthesis of chemical probes that would retain biological activity more achievable. After conjugate addition, the C-3 ketone can be easily generated by ketal cleavage using standard conditions. Furthermore, stereoselective reductive amination conducted on the C-3 ketone gives the possibility of incorporating different types of amines into the clionamine structure.  Finally, the optimization of key steps in the total synthesis of clionamines is also desirable.  The PCC oxidation step should be avoided due to the high toxicity of chromium(VI). The use of Dess-Martin periodinane (DMP) could be a valid alternative to this step. Moreover, the use of a 226  Lewis acid in the conjugate addition step could be useful to overcome the low reactivity of ?-methylene lactone and improve the yield. The choice of the Lewis acid should be made taking into consideration the sensitivity of the ketal towards certain types of Lewis acids.       Figure 7.3 Synthesis of new analogues and chemical probes of natural clionamines.  7.3 Conclusions In conclusion, the reaserch presented in this thesis will aid in the identification of new therapeutic strategies to combat diseases such as multidrug resistant-tuberculosis (MDR-TB), type 2 diabetes, and pancreatic adenocarcinoma (PAC). The synthetic methodologies reported herein will facilitate the synthesis of chemical probes for the molecular target identification via Click chemistry. 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