Multiplicative Expansion of the Pool of Fully Synthetic Tetracycline Antibiotics. A dissertation presented by Peter Maughan Wright to The Department of Chemistry and Chemical Biology in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Chemistry Harvard University Cambridge, Massachusetts May, 2012 © 2012 – Peter Maughan Wright All rights reserved. Dissertation Adviser: Professor Andrew G. Myers Peter Maughan Wright Multiplicative Expansion of the Pool of Fully Synthetic Tetracycline Antibiotics Abstract This thesis describes the development of chemical pathways for the preparation of more than 80 novel fully synthetic tetracyclines with structural variability at positions C5 and C5a. Progress toward the synthesis of 5-hetero-tetracyclines, another new class of tetracycline antibiotics, is also described. The results detailed herein – including successful C-ring-forming Michael–Claisen cyclizations of numerous modified AB precursors with just a few of the extraordinarily diverse D-ring precursors known to be effective nucleophiles in this key coupling reaction – represent the first steps toward a multiplicative expansion of the pool of fully synthetic tetracyclines. Novel and versatile -functionalization reaction sequences employing tris(methylthio)methyllithium and 2-lithio-1,3-dithiane have been developed to transform the AB enone 10 (the key precursor to fully synthetic tetracyclines) into a diverse range of -substituted AB enone products, including a highly efficient, single-operation method for the synthesis of a methyl ester-substituted AB enone (20). It is demonstrated that the six-membered C ring of tetracyclines comprising a C5a quaternary carbon center (e.g. 29) can be assembled by stereocontrolled coupling reactions of -substituted AB enones and o-toluate ester anion D-ring precursors. A C5a–C11a-bridged cyclopropane tetracycline precursor (37) was found to undergo efficient and regioselective ring-opening reactions with a range of nucleophiles in the presence iii of magnesium bromide, thus providing another avenue for the preparation of fully synthetic tetracyclines containing an all-carbon quaternary center at position C5a. The AB enone 10 has also been transformed into structurally diverse -substituted AB precursors, which in turn have been converted into fully synthetic tetracyclines with unprecedented modifications at position C5, including 5-fluorotetracyclines such as 94. Numerous fully synthetic tetracyclines and tetracycline precursors have been shown to serve as diversifiable branch-points, allowing maximally expedient synthesis of C5- and C5a-substituted tetracyclines by late-stage diversification. The substrate scope of the Michael–Claisen cyclization reaction has been expanded to include new heterocyclic enone electrophiles such as dihydro-4-pyridones, affording cycloadducts such as 142. In this way, the viability of an iterative Michael–Claisen strategy for constructing 5-hetero-tetracyclines has been established. Numerous examples in this thesis serve to further demonstrate the broad applicability of the Michael–Claisen cyclization reaction as a powerful method for the assembly of stereochemically complex six-membered rings. N(CH3)2 O A O OTBS 10 N OBn CH3O O B O N(CH3)2 O A O OTBS 20 F H N O N H F 5 H B O H N(CH3)2 D BocO C CH3 H B N(CH3)2 O A N OBn N OBn 5a O HO 29 O OTBS N(CH3)2 5a 11a H N(CH3)2 O N t-Bu H H N(CH3)2 OH NH2 F O N OBn BnO O O 37 O OTBS OBn OH O HO O O H 94 O BocO O HO 142 iv Table of Contents Abstract Table of Contents Acknowledgements List of Abbreviations Chapter 1 Introduction Introduction Michael–Claisen Reaction Sequences Antibacterial Action of Tetracyclines Tetracycline Resistance Chapter 2 Synthesis of C5a-Substituted Tetracyclines Introduction Results Antibacterial Activities Conclusion Experimental Section iii v vii ix 1 2 10 14 24 31 32 36 52 63 64 v Catalog of Spectra Chapter 3 Synthesis of C5-Substituted Tetracyclines Introduction Results Antibacterial Activities Conclusion Experimental Section Catalog of Spectra Chapter 4 Progress Toward the Synthesis of 5-Hetero-Tetracyclines Introduction Retrosynthetic Strategy and Background Results Conclusion Experimental Section Catalog of Spectra 128 153 154 158 171 185 186 254 276 277 282 286 295 296 333 vi Acknowledgements First, I would like to thank my thesis advisor, Professor Andrew Myers. I am extremely grateful for Andy’s mentorship, his incredible commitment to the tetracycline project, his ability to pay such close attention to the smallest details (of experiments and written communication) while never losing sight of the big picture, and his singular talent for inspiring metaphors. I would also like to thank Professors Eric Jacobsen and Tobias Ritter for serving on my Graduate Advising Committee. I have always enjoyed our interactions and it has been a privilege to carry out chemistry research in the same department as academics of their caliber. Throughout my tenure in the Myers group I have been fortunate to be surrounded by brilliant chemists and great people. I would particularly like to thank Jon Mortison and Evan Hecker for being extremely helpful and entertaining baymates. I also thank the other recent members of “Team Tetracycline” – Fan Liu, Robin Sussman, David Kummer, and Amelie Dion – for being fantastic co-workers. I especially acknowledge Simon Lewis for his incredible patience while teaching me basic experimental techniques during my first stint in the Myers group as an undergraduate summer student. I would like to express heartfelt thanks to Diana Eck and Dorothy Austin, the Masters of Lowell House, for giving me the opportunity to live and work in their wonderful community as a resident tutor. Lowell House breathed new life into my graduate school experience and has provided me with so many friends and memories I will cherish for a long time to come. I thank my mother, Angela, for giving me a fantastic start in life and for her constant love and support, even from such a long distance. Finally, I would like to thank Professor George vii Fleet, my organic chemistry tutor at St. John’s College, Oxford, a wonderful teacher and mentor who opened my eyes to where chemistry could take me. viii List of Abbreviations Å AB BC Bn Boc DBU DCE DEAD DMAP DMF DMSO EC EF equiv ESI angstrom Acinetobacter baumannii Burkholderia cenocepacia benzyl tert-butyl carbonate 1,8-diazabicyclo[5.4.0]undec-7-ene 1,2-dichloroethane diethyl azodicarboxylate 4-dimethylaminopyridine dimethylformamide dimethyl sulfoxide Escherichia coli Enterococcus faecalis equivalent electrospray ionization ix Et FDA FTIR g HMPA HRMS Hz IBX J KHMDS KP LDA LiHMDS M mg MHz ethyl Food and Drug Administration Fourier transform infrared gram hexamethylphosphoramide high-resolution mass spectrometry hertz 2-iodoxybenzoic acid coupling constant (in Hz) potassium hexamethyldisilazide Klebsiella pneumoniae lithium diisopropylamide lithium hexamethyldisilazide molar (moles/liter) milligram megahertz x MIC mL mmol NaHMDS NBS NCS NIS NMR nOe PA Pd Ph PM ppm Pr R minimum inhibitory concentration milliliter millimole sodium hexamethyldisilazide N-bromosuccinimide N-chlorosuccinimide N-iodosuccinimide nuclear magnetic resonance nuclear Overhauser effect Pseudomonas aeruginosa palladium phenyl Proteus mirabilis parts per million propyl rectus (Cahn-Ingold-Prelog system) xi Rf S SA SM SP TBS TBSOTf Tf2O TFA THF TLC TMEDA TMSCl UV retention factor sinister (Cahn-Ingold-Prelog system) Staphlococcus aureus Stenotrophomonas maltophilia Streptococcus pneumoniae tert-butyldimethylsilyl tert-butyldimethylsilyl trifluoromethanesulfonate trifluoromethanesulfonic anhydride trifluoroacetic acid tetrahydrofuran thin-layer chromatography N,N,N’,N’-tetramethylethylenediamine chlorotrimethylsilane ultraviolet xii Chapter 1 Introduction 1 Introduction The tetracyclines are a class of broad-spectrum antibiotics that have been widely used in human and veterinary medicine for more than 50 years.1 The first tetracycline antibiotic was discovered in 1948 when Benjamin Duggar of Lederle Laboratories isolated the natural product chlorotetracycline (Aureomycin®, 1, Figure 1.1) from the culture broth of a novel species of Streptomyces.2 Within two years a research team from Chas. Pfizer and Co. had isolated a second natural tetracycline, oxytetracycline (Terramycin®, 2),3 and in 1953 tetracycline itself (3) was prepared from chlorotetracycline by catalytic hydrogenolysis of the carbon-chlorine bond, a transformation discovered by Lloyd Conover of Pfizer.4 Subsequently, tetracycline was found to be a natural product and,5 later still, Lederle researchers isolated 6demethyltetracyclines (see structures 4 and 5) from culture broths of a mutant strain of Streptomyces.6 Tetracyclines in Biology, Chemistry and Medicine; Nelson, M.; Hillen, W.; Greenwald, R. A., Eds.; Birkhauser: Boston, 2001. (a) Duggar, B. M. Ann. N. Y. Acad. Sci. 1948, 51, 177–181. (b) Duggar, B. M. Aureomycin and Preparation of Same. U.S. Patent 2,482,055, Sept 13, 1949. 3 2 1 (a) Finlay, A. C.; Hobby, G. L.; P’an, S. Y.; Regna, P. P.; Routien, J. B.; Seeley, D. B.; Shull, G. M.; Sobin, B. A.; Solomons, I. A.; Vinson, J. W.; Kane, J. H. Science 1950, 111, 85. (b) Sobin, B. A.; Finlay, A. C. Terramycin and its Production. U.S. Patent 2,516,080, July 18, 1950. (a) Booth, J. H.; Morton, J.; Petisi, J. P.; Wilkinson, R. G.; Williams, J. H. J. Am. Chem. Soc. 1953, 75, 4621. (b) Conover, L. H.; Moreland, W. T.; English, A. R.; Stephens, C. R.; Pilgrim, F. J. J. Am. Chem. Soc. 1953, 75, 4622–4623. 5 4 Minieri, P. P.; Sokol, H.; Firman, M. C. Process for the Preparation of Tetracycline and Chlorotetracycline. U. S. Patent 2,734,018, Feb 7, 1956. McCormick, J. R. D.; Sjolander, N. O.; Hirsch, U.; Jensen, E. R.; Doerschuk, A. P. J. Am. Chem. Soc. 1957, 79, 4561-4563. 6 2 Figure 1.1 Natural tetracycline antibiotics. All tetracyclines approved for human or veterinary use are fermentation products or are derived from fermentation products by semisynthesis. This is also true of most beta-lactam and all macrolide antibiotics. Tracing the paths of human efforts to produce new antibiotics from natural products not accessible by synthesis reveals an evolutionary process marked by specific, impactful discoveries. In the case of the tetracyclines, Pfizer scientists achieved a major enabling advance approximately 10 years after the class had been identified when they demonstrated that the C6-hydroxyl group of the natural products oxytetracycline (2), tetracycline (3) and 6-demethyltetracycline (4) could be removed reductively.7 The 6-deoxytetracyclines produced, including 6-deoxy-6- (a) Stephens, C. R.; Murai, K.; Rennhard, H. H.; Conover, L. H.; Brunings, K. J. J. Am. Chem. Soc. 1958, 80, 5324–5325. (b) McCormick, J. R. D.; Jensen, E. R.; Miller, P. A.; Doerschuk, A. P. J. Am. Chem. Soc. 1960, 82, 3381–3386. (c) Stephens, C. R.; Beereboom, J. J.; Rennhard, H. H.; Gordon, P. N.; Murai, K.; 7 3 demethyltetracycline (sancycline, 6, Figure 1.2), were found to be more stable than the parent compounds, yet retained broad-spectrum antibacterial activity. The important and now generic antibiotics doxycycline (Pfizer, 1967, 7) and minocycline (Lederle, 1972, 8) followed as a consequence, the latter arising from the additional discovery that electrophilic aromatic substitution at C7 becomes possible when the more stable 6deoxytetracyclines are used as substrates.8 N(CH3)2 OH NH2 OH O HO O O H 1958 ( Discovery ) (–)-Sancycline (6) O OH O HO O O H O H CH3 OH N(CH3)2 H H OH 6 5 H 6 H NH2 1958 ( Discovery ) 1967 ( FDA approval ) (–)-Doxycycline (7) N(CH3)2 7 H H N(CH3)2 OH NH2 t–Bu H N 7 N(CH3)2 O N H 9 H H N(CH3)2 OH NH2 OH O HO O O H O OH O HO O O H O 1967 ( Discovery ) 1972 ( FDA approval ) (–)-Minocycline (8) 1993 ( Discovery ) 2005 ( FDA approval ) (–)-Tigecycline (9) Figure 1.2 Semisynthetic tetracycline antibiotics. Decades later, a team of Wyeth scientists led by Frank Tally synthesized 7,9disubstituted tetracycline derivatives, leading to the discovery of the antibiotic tigecycline Blackwood, R. K.; Schach von Wittenau, M. J. Am. Chem. Soc. 1963, 85, 2643–2652. (d) Blackwood, R. K.; Stephens, C. R. J. Am. Chem. Soc. 1962, 84, 4157–4159. (a) Martell, M. J.; Boothe, J. H. J. Med. Chem. 1967, 10, 44–46. (b) Church, R. F. R.; Schaue, R. E.; Weiss, M. J. J. Org. Chem. 1971, 36, 723–725. (c) Spencer, J. L.; Hlavka, J. J.; Petisi, J.; Krazinski, H. M.; Boothe, J. H. J. Med. Chem. 1963, 6, 405–407. (d) Zambrano, R. T. U.S. Patent 3,483,251, Dec 9, 1969. 8 4 (Tigacyl®, US approval 2005, 9).9 Tigecycline is the most important member of a class of tetracyclines known as glycylcyclines which retain activity against many tetracyclineresistant bacteria (vide infra). As shown in Scheme 1.1 below, tigecycline (9) is synthesized from minocycline (7) by an efficient three-step sequence comprising: (1) nitration at the C9 position of minocycline upon addition of potassium nitrate to a solution of minocycline in concentrated sulfuric acid, (2) palladium-catalyzed reduction of the nitro group, and (3) acylation of the resulting aniline with 2-(tertbutylamino)acetyl chloride hydrochloride. Scheme 1.1. Synthesis of tigecycline (9) from minocycline (8). (a) Sum, P.-E.; Lee, V. J.; Testa, R. T.; Hlavka, J. J.; Ellestad, G. A.; Bloom, J. D.; Gluzman, Y.; Tally, F. P. J. Med. Chem. 1994, 37, 184–188. (b) Sum, P.-E.; Petersen, P. Bioorg. Med. Chem. Lett. 1999, 9, 1459– 1462. 9 5 A generalized structure-activity relationship profile of tetracyclines was formulated following extensive semisynthetic investigations (Figure 1.3). Structural modification of the D ring and upper periphery of tetracyclines provided compounds with enhanced, equal or reduced antibacterial activity; this region is therefore considered “variable”. Antibacterial activity was diminished or abolished by modification of the lower periphery and A ring, indicating that the functional groups at these positions are essential for the antibiotic action of tetracyclines. These observations were rationalized by X-ray crystal structures of tetracycline bound to its target, the bacterial ribosome (vide infra).10 The limitations of semisynthesis have meant that relatively few structural variations have been explored along the upper periphery of tetracyclines (C4, C4a, C5, C5a and C6). This region is considered variable largely on the basis that natural tetracyclines have different substitution patterns at C5 and C6. Like many “old” classes of antibiotics, tetracyclines have never been systematically modified by de novo synthesis and knowledge of structure-activity relationships is limited or non-existent at many positions on the scaffold.11 10 (a) Brodersen, D. E.; Clemons, W. M., Jr.; Carter, A. P.; Morgan-Warren, R. J.; Wimberly, B. T.; Ramakrishnan, V. Cell 2000, 103, 1143–1154. (b) Pioletti, M.; Schlünzen, F.; Harms, J.; Zarivach, R.; Glühmann, M.; Avila, H.; Bashan, A.; Bartels, H.; Auerbach, T.; Jacobi, C.; Hartsch, T.; Yonath, A.; Franceschi, F. EMBO J. 2001, 20, 1829–1839. Von Nussbaum, F.; Brands, M.; Hinzen, B.; Weigand, S.; Häbich, D. Angew. Chem. Int. Ed. 2006, 45, 5072-5129. 11 6 Figure 1.3 Summary of 50 years of structure-activity relationships data. From the time that the structures of the tetracycline antibiotics were first revealed by Woodward and collaborators,12 many laboratories have sought to develop a practical route for their synthesis. Strategically, the original route developed by Woodward and collaborators for the synthesis of sancycline employed a "left-to-right" or D A mode of construction. The Shemyakin and Muxfeldt research groups adopted a similar directionality in their remarkable syntheses of tetracycline (3, 1967) and oxytetracycline (2, 1968), respectively, using a bicyclic CD-ring precursor as starting material.13,14 (a) Hochstein, F.A.; Stephens, C. R.; Conover, L. H.; Regna, P. P.; Pasternack, R.; Gordon, P. N.; Pilgrim, F. J.; Brunings, K. J.; Woodward, R. B. J. Am. Chem. Soc. 1953, 75, 5455-5475. (b) Stephens, C. R.; Conover, L. H.; Hochstein, F. A.; Regna, P. P.; Pilgrim, F. J.; Brunings, K. J. J. Am. Chem. Soc. 1952, 74, 4976-4977. (a) Gurevich, A. I.; Karapetyan, M. G.; Kolosov, M. N.; Korobko, V. G.; Onoprienko, V. V.; Popravko, S. A.; Shemyakin, M. M. Tetrahedron Lett. 1967, 8, 131-134. (b) Kolosov, M. N.; Popravko, S. A.; Shemyakin, M. M. Lieb. Ann. 1963, 668, 86-91. 14 13 12 (a) Muxfeldt, H.; Hardtmann, G.; Kathawala, F.; Vedejs, E.; Mooberry, J. B. J. Am. Chem. Soc. 1968, 90, 6534–6536. (b) Muxfeldt, H.; Haas, G.; Hardtmann, G.; Kathawala, F.; Mooberry, J. B.; Vedejs, E. J. Am. Chem. Soc. 1979, 101, 689–701. 7 The Myers laboratory adopted a completely different synthetic approach to the tetracyclines, aiming to form the C ring by convergent coupling of D- and AB-ring precursors.15,16 This strategy was devised following consideration of structure-activity relationship data and X-ray crystal structures of tetracycline bound to the bacterial ribosome (its putative target).10 Much of the functionality known to be required for binding to the ribosome was contained (in protected form) in the AB precursor 10 (Scheme 1.2 below). It was intended that structural variation would be permitted in the D-ring portion of tetracyclines, which had emerged as the most promising site for the attachment of new substituents. To date more than 3,000 fully synthetic molecules of the tetracycline class, broadly defined, have been prepared by a general and convergent process that involves a Michael–Claisen coupling of the AB enone 10 with structurally diverse D-ring precursors followed by deprotection, a route of typically 3-4 steps (Scheme 1.2).17,18 Most of the candidate antibiotics prepared in this way would have been difficult if not impossible to obtain by semisynthesis. Thus, the development of a practical, convergent synthesis of tetracyclines has dramatically expanded the pool of accessible compounds, allowing unprecedented modifications at positions C6, C7, C8, C9 and C10. 15 16 Charest, M. G.; Lerner, C. D.; Brubaker, J. D.; Siegel, D. R.; Myers, A. G. Science 2005, 308, 395–398. Sun, C.; Wang, Q.; Brubaker, J. D.; Wright, P. M.; Lerner, C. D.; Noson, K.; Charest, M. G.; Siegel, D. R.; Wang, Y.-M.; Myers, A. G. J. Am. Chem. Soc. 2008, 130, 17913–17927. Clark, R. B.; He, M.; Fyfe, C.; Lofland, D.; O’Brien, W. J.; Plamondon, L.; Sutcliffe, J. A.; Xiao, X.-Y. J. Med. Chem. 2011, 54, 1511–1528. Sun, C.; Hunt, D. K.; Clark, R. B.; Lofland, D.; O’Brien, W. J.; Plamondon, L.; Xiao, X.-Y. J. Med. Chem. 2011, 54, 3704–3731. 17 18 8 Scheme 1.2. The Myers synthetic approach to tetracyclines. Three distinct synthetic routes to the AB enone 10 have been reported.15,19,20 The convergency and scalability of the second- and third-generation routes have enabled preparation of large quantities of this key intermediate. The third-generation route (see Chapter 4 Introduction for complete discussion) is characterized by a highly diastereoselective A-ring-forming Michael–Claisen coupling reaction, meaning that all four carbon-carbon bonds needed to assemble tetracyclines from three readily available components – a D-ring precursor, B-ring precursor 11 and isoxazole precursor 12 (Figure 1.4) – are formed using this powerful method for bond-pair construction (see Scheme 1.4 for details of these cyclization reactions). 19 20 Brubaker, J. D.; Myers, A. G. Org. Lett. 2007, 9, 3523–3526. Kummer, D. A.; Li, D.; Dion, A.; Myers, A. G. Chem Sci. 2011, 2, 1710–1718. 9 Figure 1.4. Construction of tetracyclines by iterative Michael–Claisen cyclizations; bonds and rings formed by Michael–Claisen reactions are highlighted in orange. Michael–Claisen Reaction Sequences Michael–Claisen and Michael–Dieckmann reaction sequences have been widely employed in organic synthesis to construct naphthalene derivatives and non-aromatic sixmembered rings.21 The origins of this method for bond-pair construction can be traced to 1978, when three different cyclization protocols were introduced by independent research groups. Hauser and Rhee used a sulfoxide-stabilized o-toluate ester anion as the nucleophilic component in a Michael–Dieckmann cyclization reaction with methyl crotonate (Scheme 1.3, eq 1). In this case, aromatization occurred upon thermal elimination of phenylsulfenic acid.22 The use of phthalide and cyanophthalide anions as (a) Mal, D.; Pahari, P. Chem. Rev. 2007, 107, 1892–1918. (b) Mitchell, A. S.; Russell, R. A. Tetrahedron 1995, 51, 5207–5236. (c) Rathwell, K.; Brimble, M. A. Synthesis 2007, 5, 643–662. 22 21 Hauser, F. M.; Rhee, R. P. J. Org. Chem. 1978, 43, 178–180. 10 nucleophilic components was described by Broom and Sammes (eq 2),23 and Kraus and Sugimoto (eq 3),24 respectively. Formal loss of water and hydrogen cyanide, respectively, led to naphthoate ester products in these procedures. 1. LDA, THF, –78 °C; CH3 CO2CH3 CH3 (1) CO2Et 2. toluene, 115 °C OH 70% 1. LDA, THF, –78 °C; CH3 O O 2. CF3CO2H CO2CH3 OH 32% CN LDA, THF, –78 °C; O O CO2Et , –78 0 °C OH CO2Et (3) OH CO2CH3 CH3 (2) CO2CH3 SOAr CH3O CH3 CO2CH3 LDA, THF, –78 °C; O CH3O CH3 CH3 (4) CH3O CH3 –78 O CH3 CH3O OH O 79% O 23 °C Scheme 1.3. Early examples of Michael–Claisen and Michael–Dieckmann cyclizations. In 1979, the Weinreb and Staunton research groups first reported that simple otoluate ester anions (unsubstituted at the benzylic position) undergo Michael–Claisen 23 24 Broom, N. J. P.; Sammes, P. G. J. Chem. Soc., Chem. Comm. 1978, 162–164. Kraus, G. A.; Sugimoto, H. Tetrahedron Lett. 1978, 26, 2263–2266. 11 cyclization reactions with -methoxycyclohexenones and -pyrones to form naphthyl ketones (eq 4, Scheme 1.3), a sequence sometimes referred to as Staunton–Weinreb annulation.25 Experimental evidence indicates that the mechanism of this reaction sequence involves sequential conjugate addition, -elimination of alkoxide, re-lithiation at the benzylic position (in the presence of excess base), followed by Claisen cyclization. There are numerous examples of the formation of non-aromatic 6-membered rings by Michael–Claisen and Michael–Dieckmann reaction sequences.26 However, the stereochemical features of these cyclization reactions have only rarely been discussed,27 frequently because they were of little consequence (aromatization followed cyclization). The Michael–Claisen cyclizations developed as part of Myers’ synthesis of tetracyclines are unusual in their stereochemical complexity, stereocontrol and efficiency. The C-ringforming cyclizations of D-ring precursors with AB enone 10 appear to proceed with complete stereocontrol at C5a (attack upon a single diastereoface of the enone; see Scheme 1.4 for an example).16 This selectivity may arise as a consequence of stereoelectronic factors (pseudoaxial addition to the enone) and/or steric effects (addition 25 (a) Dodd, J. H.; Weinreb, S. M. Tetrahedron Lett. 1979, 38, 3593–3596. (b) Dodd, J. H.; Starrett, J. E.; Weinreb, S. M. J. Am. Chem. Soc. 1984, 106, 1811–1812. (c) Leeper, F. J.; Staunton, J. J. Chem. Soc., Chem. Comm. 1979, 5, 206-207. (d) Leeper, F. J.; Staunton, J. J. Chem. Soc. Perkin Trans. 1 1984, 1053– 1059. 26 (a) Tarnchompoo, B.; Thebtaranonth, C.; Thebtaranonth, Y. Synthesis 1986, 9, 785-786. (b) Boger, D. L.; Zhang, M. J. Org. Chem. 1992, 57, 3974-3977. (c) Nishizuka, T.; Hirosawa, S.; Kondo, S.; Ikeda, D.; Takeuchi, T. J. Antibiotics 1997, 50, 755-764. (d) Hill, B.; Rodrigo, R. Org. Lett. 2005, 7, 5223-5225. (e) Tatsuta, K.; Yoshimoto, T.; Gunji, H.; Okado, Y.; Takahashi, M. Chem. Lett. 2000, 646–647. 27 For examples of stereocontrol in Michael–Claisen and Michael–Dieckmann cyclization reactions, see: (a) Franck, R. W.; Bhat, V.; Subramaniam, C. S. J. Am. Chem. Soc. 1986, 108, 2455-2457. (b) Tatsuta, K.; Yamazaki, T.; Mase, T.; Yoshimoto, T. Tetrahedron Lett. 1998, 39, 1771-1772. (c) White, J. D.; Demnitz, F. W. J.; Qing, X.; Martin, W. H. C. Org. Lett. 2008, 10, 2833-2836. 12 of the nucleophile to the -face opposite the tert-butyldimethylsilyloxy substituent, which is also axial; see Figure 1.5 below). The C-ring-forming cyclization depicted in Scheme 1.4 occurs with >20:1 stereoselectivity at C6. Scheme 1.4. Diastereoselective Michael–Claisen cyclizations to form the C ring and the A ring of tetracyclines. As noted above, the key step of the third-generation synthesis of the AB enone is another highly diastereoselective Michael–Claisen coupling, in this case forming the A ring of tetracyclines (Scheme 1.4). Conjugate addition of the sodium enolate of isoxazole precursor 12 to the B-ring precursor 11 occurs with complete control of relative stereochemistry at C4 and C4a, affording cycloadduct 13 in 80% yield as a single 13 diastereomer (following Claisen cyclization). The stereochemistry at C4a is believed to result from approach of the enolate from the less sterically hindered diastereoface of cyclohexenone 11 (opposite the cyclopentene cage). Figure 1.5. X-ray crystal structure of AB enone 10. Antibacterial Action of Tetracyclines The 70S bacterial ribosome is made up of two subunits – the small subunit, 30S, through which mRNA tunnels and which contains on its surface the decoding site where the mRNA sequence is read in blocks of three nucleotides; and the large subunit, 50S, which possesses catalytic ribozyme peptidyltransferase activity and is the site of peptide chain elongation.28,29 As mRNA tunnels through the 30S subunit, a short stretch of the 28 Antibiotics – Actions, Origins, Resistance; Walsh, C.; ASM Press – Washington, D.C., 2003. 14 molecule protrudes through to the interface with the 50S subunit. This stretch consists of six nucleotides which occupy two codon sites: the aminoacyl (A) site, and the peptidyl (P) site. Upstream of the peptidyl site is the exit codon (E) site, where deacylated tRNA moves from the P site following peptidyl transfer. Aminoacyl-tRNAs arrive at the unoccupied A site in complex with EF-Tu (elongation factor thermo unstable). The existence of Watson-Crick base pairs between the A site mRNA codon and the anti-codon loop of the aminoacyl-tRNA leads to appropriate orientation and complex formation. The P site is occupied by a tRNA molecule carrying the nascent peptide chain. The aminoacylated ends of the tRNA molecules occupying the A and P sites are approximately 75 Å away from the tRNA binding sites, in the catalytic center of the 50S subunit. In each chain elongation step, the aminoacyl moiety of the A site tRNA attacks the adjacent peptide chain of the peptidyltRNA. The peptidyl chain (lengthened by one amino acid) is now tethered to the tRNA docked in the A site, and the P site is occupied by a deacylated tRNA molecule. Before the next chain elongation step occurs, the deacylated tRNA moves from the P site to the E site and the peptidyl-tRNA relocates to the P site, opening up the A site for docking of an aminoacyl-tRNA possessing an anticodon loop complementary to the three mRNA nucleotides now being presented in the A site. The translocation process is catalyzed by EF-G (elongation factor G). 29 Wilson, D. N. Crit. Rev. Biochem. Mol. Biol. 2009, 44, 393-433. 15 Tetracyclines inhibit bacterial protein synthesis by binding to the 30S subunit near the A site, thereby blocking accommodation of aminoacyl-tRNAs into the A site. Following an initial decoding event involving interaction of the anticodon loop of aminoacyl-tRNA (complexed at this stage with EF-Tu and GTP) with the mRNA codon, the release of aminoacyl-tRNA from EF-Tu is inhibited as the anticodon loop clashes with bound tetracycline during rotation into the A site.29,10a This explanation fits with the observation that tetracycline does not affect the level of ribosomal binding of the ternary complex of aminoacyl-tRNA with EF-Tu and GTP (although binding occurs more slowly). Selective inhibition of bacterial protein synthesis is possible because of structural differences between the ribosomal RNA of bacterial and eukaryotic ribosomes, as well as selective concentration in susceptible bacterial cells.30 Tetracycline binds to the ribosome in complex with Mg2+, as depicted in Figure 1.6 below.10a The C11-C12 keto-enol portion of tetracycline binds to Mg2+, which is then also coordinated by the phosphate oxygens of RNA nucleotides G1197, G1198 and C1054 in helix 34 of the 30S ribosomal subunit. In addition, the phenolic hydroxyl group at C10 and tertiary carbinol at C12a appear to engage in hydrogen bonding interactions with the ribose portion of C1054 and a phosphate oxygen of G1198, respectively. Furthermore, there is an apparent interaction between the A ring of tetracycline and a phosphate oxygen of residue G966 in helix 31, as well as a hydrophobic interaction between the aromatic D ring of tetracycline and the base of C1054 in helix 31. 30 Chopra, I.; Roberts, M. Microbiol. Mol. Biol. Rev. 2001, 65, 232-260. 16 G1053 G1198 G966 C1054 C1195 U1196 Figure 1.6. Tetracycline bound to its primary binding site in the bacterial ribosome.10a Tetracycline and glycylcyclines (such as tigecycline, 9) share a common ribosomal binding site, to which selected glycylcyclines appear to bind five times more efficiently.31 It has been proposed that the bulky C9 side-chain of tigecycline either causes the antibiotic to bind to the ribosome in a different orientation to tetracycline, or the ribosomal RNA conformation is altered to accommodate the bulkier drug molecule.32 One (or both) of these effects probably leads to the higher affinity binding of tigecycline 31 Bergeron, J; Ammirati, M.; Danley, D.; James, L.; Norcia, M.; Retsema, J.; Strick, C. A.; Su, W. G.; Sutcliffe, J.; Wondrack, L. Antimicrob. Agents Chemother. 1996, 40, 2226-2228. Bauer, G.; Berens, C.; Projan, S. J.; Hillen, W. J. Antimicrob. Chemother. 2004, 53, 592-599. 32 17 compared to tetracycline and also helps explain the fact that tigecycline retains activity against many tetracycline-resistant bacteria (vide infra). All bacterial cells are bounded by a cytoplasmic membrane, a symmetric lipid bilayer which is permeable to uncharged, lipophilic molecules. In addition, Gramnegative bacterial cells are also bounded by a second barrier – the outer membrane – which is significantly less permeable to lipophilic molecules than the cytoplasmic membrane. Unlike the cytoplasmic membrane, the bilayer of the outer membrane is asymmetric.33 The outer leaflet (of the outer membrane) consists of lipopolysaccharides, while the inner leaflet is made up of phospholipids. The “gel-like” nature of the lipopolysaccharide leaflet makes the outer membrane a more effective permeability barrier than the inner membrane. Donnan potential is the electric potential arising between two solutions of unequal ionic solute concentration separated by a partially permeable membrane. In all mediums containing tetracycline, an equilibrium exists between a net uncharged, metal-free form of tetracycline (denoted “tc” in Figure 1.7 below) and a complex in which deprotonated tetracycline coordinates a magnesium dication, [M-tc]+.34 The position of the equilibrium in any medium depends on both pH and magnesium ion concentration. The higher the pH and the higher the magnesium concentration, the higher will be the concentration of the 33 34 Nikaido, H. Microbio. Mol. Biol. Rev. 2003, 67, 593-656. Key references for bacterial cell penetration of tetracyclines: (a) Yamaguchi, A.; Ohmori, H.; KanekoOhdera, M.; Nomura, T.; Sawai, T. Antimicrob. Agents Chemother. 1991, 35, 53-56. (b) Nikaido, H.; Thanassi, D. G. Antimicrob. Agents Chemother. 1993, 37, 1393-1399. (c) Schnappinger, D.; Hillen, W. Arch. Microbiol. 1996, 165, 359-369. 18 hydrophilic chelate complex, to which the cytoplasmic membrane is impermeable. In E. coli, the cytoplasmic pH is higher than the external pH by about 1.7 pH units, and the cytoplasmic magnesium ion concentration is also higher. Therefore, the proportion of tetracycline in its uncharged (permeable) form is significantly higher in the periplasm than in the cytoplasm. The pH difference is dependent on the proton motive force and explains why tetracycline accumulation is energy dependent. Figure 1.7. Diagrammatic representation of Gram-negative cell penetration by tetracycline (tc). A high concentration of acidic groups at the surface of the outer membrane means that Mg2+ ions are abundant at this location. Tetracyclines, like most antibacterials, are 19 thought to penetrate the outer membrane of Gram-negative cells predominantly by passing through aqueous channels provided by porin proteins imbedded in the outer membrane. Porin proteins favor passage of charged, hydrophilic solutes, and tetracyclines pass through these channels in complex with Mg2+ (denoted [M-tc]+ in the Figure 1.7).34,35 The Donnan potential across the outer membrane leads to accumulation of [Mtc]+ in the periplasm (relative to the exterior) and, following equilibration, passage of neutral and metal-free tetracycline across the cytoplasmic membrane, which is permeable to uncharged, lipophilic small molecules. Tetracycline has pKa values of 3.3, 7.7, and 9.7, and at pH 7.4, 7.1% of tetracycline exists in uncharged form. The uncharged form of tetracycline is weakly lipophilic. Although diffusion across the outer membrane is relatively slow, it may still occur at a significant rate with more lipophilic tetracyclines such as minocycline.34b O’Shea and Moser found that antibacterial agents with activity against Gramnegative bacteria (which, with few exceptions, are a subset of compounds with Grampositive activity) are significantly more polar (less lipophilic) than “Gram-positive only” agents.36 Antibacterials with Gram-negative activity have a mean relative polar surface area 12% higher than compounds which are only active against Gram-positive bacteria, as well as significantly lower values for clogD7.4 (the calculated log of the octanol-water partition coefficient based on the distribution of charged and uncharged forms of the 35 36 Koebnik, R.; Locher, K. P.; Van Gelder, P. Mol. Microbiol. 2000, 37, 239-253. O’Shea, R.; Moser, H. E. J. Med. Chem. 2008, 51, 2871-2878. 20 compound at pH 7.4). Furthermore, Gram-negative agents have a smaller mean molecular mass, with a seemingly strict upper limit of 600 Da. This analysis of physicochemical properties was modified by Silver to differentiate between antibacterials whose targets are located in the cytoplasm (and which reach their targets by diffusion) and those which need not penetrate the inner membrane to exert their antibacterial effect.37 Specifically, antibacterials were divided into two groups: (i) drugs with cytoplasmic targets which enter the cytoplasm by diffusion (including tetracyclines); (ii) drugs with no requirement for diffusion across the lipid bilayer of the cytoplasmic membrane, i.e. antibacterials with extracytoplasmic targets plus those with cytoplasmic targets which are known to move across the cytoplasmic membrane by means other than diffusion (such as the aminoglycosides, whose transport is energy dependent). The difference between these two groups is even more significant than for Gram-positive-only agents versus drugs with Gram-negative activity. “Extracytoplasmic and transported” antibacterials are even more polar (on average) than Gram-negative agents as a whole. 37 Silver, L. L. Clinical Microbio. Rev. 2011, 24, 71–109. 21 Figure 1.8. Analyses of physicochemical properties of antibacterials (divided into two groups) and drugs in other therapeutic areas; A: antibacterials divided into Gramnegative agents and Gram-positive-only agents; B: antibacterials divided into those with cytoplasmic targets and those which act extracytoplasmically or are actively transported across the cytoplasmic membrane. The requirements that Gram-negative antibacterials have higher polarity and lower molecular weight are believed to be driven by the properties of porin proteins. All known porins have a characteristic -barrel structure and significantly conserved transmembrane -strands (according to analysis of amino acid sequence and a limited number of crystal structures).38 The presence of charged amino acid side chains on opposite sides of the porin channels leads to highly directional orienting of water 38 (a) Cowan, S. W.; Schirmer, T.; Rummel, G.; Steiert, M.; Ghosh, R.; Pauptit, R. A.; Jansonius, J. N.; Rosenbusch, J. P. Nature 1992, 358, 727–733. (b) Dutzler, R.; Rummel, G.; Alberti, S.; Hernández-Allés, S.; Phale, P. S.; Rosenbusch, J. P.; Benedi, V. J.; Schirmer, T. Structure 1999, 7, 425–434. 22 molecules, and disruption of this ordered structure is thermodynamically disfavored. Passage of lipophilic molecules is most strongly disfavored because the energy cost for removal of the hydration sphere of channel-lining amino acids and temporary replacement with the small molecule is prohibitively high.39 When hydrophilic solutes come into the channel, the broken hydrogen bonds between water molecules and channellining amino acids are temporarily replaced by hydrogen bonds between the hydrophilic solute and polar amino acid side chains. Passage of an antibacterial through these openings requires that the activation energy for removal of the hydration sphere of channel-lining amino acids and temporary replacement with the drug molecule is not prohibitively high. Charged, hydrophilic molecules are therefore more likely to penetrate the outer membrane of Gram-negative cells. Cell penetration is clearly a crucial factor in determining antibacterial activity of any tetracycline, and the conflict between the broad requirements for passage through the two membranes of Gram-negative bacteria presents a very significant challenge in tetracycline drug discovery. Modified tetracyclines with higher influx rates could hold significant advantages over existing tetracyclines, but as the conditions that mediate compound uptake through the bacterial cell envelope are still incompletely understood, rational design with respect to this feature is extremely difficult.40 39 40 Nikaido, H.; Rosenberg, E. Y.; Foulds, J. J. Bacteriology 1983, 153, 232–240. Brotz-Oesterhelt, H.; Sass, P. Future Microbiology 2010, 5, 1553–1579. 23 Tetracycline Resistance Decades of clinical and agricultural use have led to widespread bacterial resistance to tetracyclines. The tetracycline “resistome” may be the largest assortment of resistance genes acting against an individual class of antibiotics. A 2010 review stated that there were over 1,189 reported tetracycline genes that had been classified into 41 resistance determinant classes (of which 26 are efflux pumps and 11 are ribosomal protection proteins).41 The majority of tetracycline efflux proteins are members of the major facilitator superfamily of integral membrane transporters.42 Although no crystal structure is currently available, tetracycline efflux proteins are predicted to be water-filled channels surrounded by six transmembrane helices. The flow of protons down a pH gradient provides the energy required to pump tetracycline out of the cytoplasm.43 Export of tetracycline protects ribosomes within the cell by reducing cytoplasmic drug concentration. Each tetracycline efflux protein has an associated tetracycline repressor protein responsible for regulating expression of the efflux pump. Expression of TetA (a widely distributed and clinically important efflux pump found in Gram-negative bacteria) is regulated by TetR, a protein which forms a dimer and binds to DNA operator sequences 41 42 43 Thaker, M.; Spanogiannopoulos; Wright, G. D. Cell. Mol. Life Sci. 2010, 67, 419–431. Paulsen, I. T.; Brown, M. H.; Skurray, R. A. Microbiol. Rev. 1996, 60, 575–608. Yamaguchi, A.; Ono, N.; Akasaka, T.; Noumi, T.; Sawai, T. J. Biol. Chem. 1990, 265, 15525–15530. 24 tetO1 and tetO2. Upon entering the cytoplasm, tetracycline forms a complex with Mg2+ and the complex in turn binds to the TetR–DNA complex, leading to a conformational change within the TetR dimer which dramatically reduces its affinity for the DNA operator sequences, leading to dissociation of the protein–DNA complex and transcription of both TetA and TetR. A crystal structure of the complex formed between TetR and tetracycline–Mg2+ provided a detailed view of the binding interactions between the antibiotic and the repressor protein (Figure 1.9A below).44 Although there is significant sequence variation amongst the repressor proteins responsible for regulating the expression of different efflux proteins, the amino acid residues involved in hydrogen bonding to tetracycline and Mg2+ coordination are largely conserved. The crystal structure of the complex formed between tetracycline and TetR revealed that the repressor protein completely surrounds the antibiotic, forming an extensive network of contacts around the tetracycline scaffold (tetracycline has a Kd of ~ 1 nM for Tet(R), compared with ~1 µM for the bacterial ribosome). In contrast, the interactions between tetracycline and its primary binding site in the 30S ribosomal subunit are largely confined to the highly oxygenated “lower” periphery of the molecule, while the upper periphery of the antibiotic projects away from the binding site into free aqueous solution (see Figure 1.9B). As a result of this difference, it is conceivable that the attachment of new substituents in this region (C4a, C5, C5a and C6) could disrupt the interaction with TetR (and potentially combat 44 (a) Hinrichs, W.; Kisker, C.; Duvel, M.; Saenger, W. Science 1994, 264, 418–420. (b) Kisker, C.; Hinrichs, W.; Tovar, K.; Hillen, W.; Saenger, W. J. Mol. Biol. 1995, 247, 260–280. (c) Orth, P.; Schnappinger, D.; Hillen, W.; Saenger, W.; Hinrichs, W. Nature Struct. Biol. 2000, 7, 215–219. 25 resistance due to tetracycline efflux) without compromising the ribosomal binding required for antibacterial activity.41 It has been demonstrated that natural and non-natural tetracyclines with diverse substitution patterns along the upper periphery have different binding affinities for TetR.45 A. B. Figure 1.9. A. Tetracycline bound to the tet repressor (TetR). B. Tetracycline bound to the 30S subunit of the bacterial ribosome. 45 Degenkolb, J.; Takahashi, M.; Ellestad, G. A.; Hillen, W. Antimicrob. Agents Chemother. 1991, 35, 1591-1595. 26 Multi-drug efflux pumps expel a range of structurally dissimilar compounds from bacterial cells, including antibiotics of many different classes.46 The non-selective export of multiple antibacterial agents means that these pumps are often associated with multidrug resistance. Any given bacterial species may express a number of different multidrug efflux pumps; some are constitutively expressed (conferring intrinsic multi-drug resistance) while the expression of others may be induced by the presence of certain substrates. Across different species of bacteria there is significant variation in the structure and substrate scope of efflux pumps, so the impact of multi-drug efflux systems on antibacterial susceptibility is highly species-dependent. Multi-drug efflux pumps can be either single component or multi-component systems. Those expressed in some Gramnegative bacteria are tripartite systems, with a constituent inner membrane transporter protein, periplasmic accessory protein (or membrane-fusion protein) and outer membrane protein channel.46 Tetracyclines are substrates for some multi-drug efflux pumps, including those expressed in Pseudomonas aeruginosa, a particularly problematic pathogen. Resistance of Pseudomonas strains to multiple classes of otherwise effective antibiotics is a result of both slow permeation (poor influx) and highly efficient efflux of drug molecules.47 Tigecycline is exported from Pseudomonas by multi-drug pumps less efficiently than (a) Piddock, L. J. V. Clin. Microbiol. Rev. 2006, 19, 382-402. (b) Piddock, L. J. V. Nature Rev. Microbiol. 2006, 4, 629-636. 47 46 Li, X. Z.; Livermore, D. M.; Nikaido, H. Antimicrob. Agents Chemother. 1994, 38, 1732-1741. 27 older tetracyclines, but efflux is still sufficient to confer resistance.48 Given that glycylcyclines are exported less efficiently and that the relative contributions of different multi-drug pumps to net efflux varies significantly between different tetracyclines, it is possible that efflux pump-mediated drug resistance could be overcome through chemical modification of existing, otherwise effective drugs. Ribosomal protection proteins (RPPs) are thought to give rise to tetracycline resistance by binding to tetracycline–ribosome complex at a distinct site (i.e. away from the primary tetracycline binding site), causing a ribosomal conformation change which weakens the interaction between tetracycline and the ribosome.49 Tetracycline is thus “dislodged” from its primary binding site (Figure 1.10 below). Upon tetracycline release, GTP is hydrolyzed and the RPP dissociates from the ribosome.50 RPPs have significant sequence homology with elongation factors EF-Tu and EF-G, both of which are also GTPases.51 The mechanism by which RPPs “recognize” ribosomes bound by tetracycline has not been conclusively established. It is possible that tetracycline binding induces a conformational change which promotes RPP binding. Alternatively, since Tet(O) (a prevalent and well-studied RPP) cannot bind to a ribosome with an occupied A site, the ability of tetracycline to block the ribosome in a state with an open A site may explain the 48 Dean, C. R.; Visalli, M. A.; Projan, S. J.; Sum, P.-E.; Bradford, P. A. Antimicrob. Agents Chemother. 2003, 47, 972-978. Connell, S. R.; Tracz, D. M.; Nierhaus, K. H.; Taylor, D. E. Antimicrob. Agents Chemother. 2003, 47, 3675-3681. 50 51 49 Burdett, V. J. Bacteriol. 1996, 178, 3246-3251. (a) Taylor, D. E.; Chau, A. Antimicrob. Agents Chemother. 1996, 40, 1-5. (b) Kobayashi, T.; Nonaka, L.; Maruyama, F.; Suzuki, S. J. Mol. Evol. 2007, 65, 228-235. 28 binding preference of RPPs.52 It is not known whether RPPs prevent rebinding of tetracycline after causing tetracycline release. Figure 1.10. Ribosomal protection resistance mechanism.41 Glycylcycline antibiotics such as tigecycline retain activity against bacteria expressing ribosomal protection proteins. Tigecycline and older tetracyclines all bind to the same primary binding site in the ribosome, but tigecycline binds more strongly and in a slightly different orientation. It is possible that RPPs bind less strongly to the tigecycline–ribosome complex than to the tetracycline–ribosome complex, or that RPP Connell, S. R.; Trieber, C. A.; Einfeldt, E.; Dinos, G. P.; Taylor, D. E.; Nierhaus, K. EMBO J. 2003, 22, 945-953. 52 29 binding to the tigecycline–ribosome complex causes little or no conformational change (i.e. tigecycline is simply harder to dislodge). Glycylcyclines also show a high level of activity against organisms carrying tetracycline efflux resistance determinants. Someya and co-workers studied the glycylcycline antibiotic 9-(N,N-dimethylaminoglycylamido)-6-demethyl-6-deoxy- tetracycline (DMG-DMDOT) and found that it does induce expression of the TetA efflux protein, indicating that this glycylcycline is a substrate for TetR.53 There are therefore two possible explanations for the potent activity of this glycylcycline in the presence of tetracycline efflux determinants: (1) the efflux protein is not able to recognize the glycylcycline, or (2) the efflux protein–glycylcycline complex cannot be translocated across the bacterial membrane. Indirect results suggest that the glycylcycline does not bind to the efflux protein TetA (at least not to the tetracycline binding site), thus explaining the ability of this antibiotic to overcome resistance caused by tetracyclinespecific efflux. 53 Someya, Y.; Yamaguchi, A.; Sawai, T. Antimicrobial Agents and Chemotherapy 1995, 39, 247-249. 30 Chapter 2 Synthesis of C5a-Substituted Tetracyclines 31 Introduction Previous work has demonstrated that our fully synthetic approach to tetracyclines allows modifications at positions C6, C7, C8, C9 and C10 that are not feasible by semisynthesis (Scheme 2.1).54,55 Given that the AB plus D strategy for tetracycline synthesis had proven to be robust across a range of different carbocyclic and heterocyclic D-ring precursors, we questioned whether the key C-ring-forming coupling reaction would still be effective with new AB precursors of wide structural variability, potentially enabling modifications at positions on the A and B rings of tetracycline that have never previously been modified. The development of chemical pathways that expand our synthetic platform to allow efficient preparation of tetracyclines of variable composition at positions C5 and C5a is the subject of this thesis. 7 8 6 7 6 H D B CO2Ph PO O N(CH3)2 O A N OBn 2. Deprotection 9 1. C–Ring Construction 8 H D C 5a 5 H N(CH3)2 OH A O NH2 B 9 O OTBS OH O HO O O H Hundreds of individual D-ring precursors AB enone (10) Fully synthetic tetracyclines with variable D-ring portions Scheme 2.1. Synthesis of tetracyclines by coupling of AB enone 10 with D-ring precursors of wide structural variability followed by deprotection. 54 (a) Charest, M. G.; Lerner, C. D.; Brubaker, J. D.; Siegel, D. R.; Myers, A. G. Science 2005, 308, 395– 398. (b) Sun, C.; Wang, Q.; Brubaker, J. D.; Wright, P. M.; Lerner, C. D.; Noson, K.; Charest, M. G.; Siegel, D. R.; Wang, Y.-M.; Myers, A. G. J. Am. Chem. Soc. 2008, 130, 17913–17927. (a) Clark, R. B.; He, M.; Fyfe, C.; Lofland, D.; O’Brien, W. J.; Plamondon, L.; Sutcliffe, J. A.; Xiao, X.Y. J. Med. Chem. 2011, 54, 1511–1528. (b) Sun, C.; Hunt, D. K.; Clark, R. B.; Lofland, D.; O’Brien, W. J.; Plamondon, L.; Xiao, X.-Y. J. Med. Chem. 2011, 54, 3704–3731. 55 32 Position C5a is on of two po ne oints of fusi of the B and C rings of tetracyc ion clines an as such substitutio of this p nd, h, on position giv rise to a quaternary carbon ce ves y enter. In nspection of X-ray cryst f tallographic data of tetra acycline bou to the 30 subunit o the und 0S of ri ibosome of Thermus The T ermophilus s suggests that substitution of position C5a (situat in t n n ted th “variable or hydro he e” ophobic reg gion of the tetracycline skeleton, away from the e m fu unctionality required for binding to the bacterial ribosome) would not o r l obviously im mpede ri ibosome bin nding and th could pr hus resent an int teresting and unexplored avenue fo the d or discovery of potential ne antibiotics to addres problems such as ba ew ss acterial resistance 5 (F Figure 2.1).56 Figure 2.1. Tetracycline bound to the 30S subuni of the bact F T e it terial ribosom 56a me. 56 (a) Brodersen D. E.; Clem n, mons, W. M., Jr.; Carter, A P.; Morgan-Warren, R. J Wimberly, B. T.; A. J.; Ramakrishnan, V. Cell 2000, 103, 1143–1154. (b) Piolet M.; Schlün R , tti, nzen, F.; Harm J.; Zarivac R.; ms, ch, Glühmann, M.; Avila, H.; Ba G ashan, A.; Ba artels, H.; Aue erbach, T.; Jac cobi, C.; Harts sch, T.; Yonat A.; th, Fr ranceschi, F. EMBO J. 2001, 20, 1829–1839. E 33 The limitations of semisynthesis have meant that modification of C5a has not previously been viable. Unpublished research by W. Rogalski and R. Kirchlechner led to the synthesis of analogs such as racemic 6-demethyl-6-deoxy-7-chloro-5a- methyltetracyclines, with both the natural and unnatural stereochemistry at C5a (compounds 14 and 15, Figure 2.2 below).57 The racemic stereoisomer with natural C5a stereochemistry (14) showed good activity against Gram-positive bacteria (including some tetracycline-resistant strains of S. aureus, in which it was more active than minocycline) but was completely inactive against Gram-negative organisms tested. The corresponding C5a-epimer 15 was less active across all strains tested but did retain poor activity against tetracycline-resistant Gram-positive organisms. 6-Demethyl-6-deoxy-7methoxy-epi-5a-methyltetracycline (compound 16, unnatural stereochemistry at C5a) was found to be completely inactive. Figure 2.2. Racemic, fully synthetic tetracyclines possessing methyl substituents at C5a, prepared by W. Rogalski and R. Kirchlechner.57 To apply our general route for tetracycline synthesis to C5a-substituted analogs it was first necessary to develop methodology to prepare AB enones containing different substituents, and then to determine if these modified AB enones would successfully 57 Cunha, B. A. Clinical Uses of Tetracyclines. In Handbook of Experimental Pharmacology; Hlavka, J. J. and Boothe, J. H., Eds.; Springer-Verlag: New York, 1985; pp 393–403. 34 undergo Michael–Claisen cyclization reactions with D-ring precursors, transformations that would give rise to a quaternary, stereogenic center at position C5a (Scheme 2.2). Scheme 2.2. Synthesis of fully synthetic tetracyclines with an all-carbon quaternary center at C5a from -substituted AB enones. The most rapid and straightforward approach to the synthesis of -substituted AB enones appeared to be the direct functionalization of the AB enone 10. While introduction of simple alkyl groups such as -methyl proved to be relatively - straightforward (though low-yielding), introduction of more highly oxidized substituents was less so, and provided us with the opportunity to pursue a number of chemical innovations. The subsequent stereocontrolled construction of the C ring, comprising an all-carbon quaternary center at position C5a, was viewed to be a challenging transformation, and its successful implementation we view to be a significant 35 advance. During the course of our investigations we also serendipitously discovered a C5a–C11a-bridged cyclopropane-containing tetracycline intermediate that has proven to be an extraordinarily versatile precursor to a diverse array of C5a-substituted tetracyclines, providing another avenue for the synthesis of this novel class of substituted tetracycline antibiotics. Results As a first step toward the stereocontrolled construction of all-carbon quaternary, C5a-substituted tetracyclines we first sought to develop methods to transform the AB enone 10 into -substituted AB enones as novel cyclization substrates. A conventional reaction sequence served for the synthesis of the simple -methyl-substituted AB enone 18 (Scheme 2.3). Specifically, conjugate addition of lithium dimethylcuprate to the AB enone 10 in the presence of trimethylsilylchloride58 afforded the corresponding -methylsubstituted trimethylsilyl enol ether 17 in 85% yield (1D-NOESY experiments support the 5a-S-stereochemistry depicted, in accord with all precedent in this system);54b oxidation of this intermediate with palladium acetate in dimethyl sulfoxide (DMSO) at 80 °C then afforded the -methyl-substituted AB enone 18 in modest yield (44% yield on 160-mg scale, and 34% yield on 2.8-g scale).59,60 Attempted oxidation of the intermediate 58 59 60 Corey, E. J.; Boaz, N. W. Tetrahedron Lett. 1985, 26, 6019–6022. Ito, Y.; Hirao, T.; Saegusa, T. J. Org. Chem. 1978, 43, 1011–1013. The highest yield of the -methyl-substituted enone 18 was obtained when the oxidation was performed on a small scale (160 mg, 1.15 equiv Pd(OAc)2, DMSO, 80 °C, 16 h) and the work-up was carried out before the starting material had been completely consumed. Mass recovery from this reaction was consistently poor. 36 trimethylsilyl enol ether 17 with the alternative oxidant o-iodoxybenzoic acid (IBX) was not successful.61 Scheme 2.3. Synthesis of -methyl-substituted AB enone 18 from the -unsubstituted AB enone 10. To transform the AB enone 10 into -substituted AB enones with more highly oxidized -substituents we were led to explore novel chemistry, as precedented methods were deemed to be too indirect or were impracticable in the present application.62,63,64 First, we developed a versatile sequence for -functionalization of the AB enone 10 61 Nicolaou, K. C.; Gray, D. L. F.; Montagnon, T.; Harrison, S. T. Angew. Chem. Int. Ed. 2002, 41, 996– 1000. 62 Enones with ester, aldehyde and alkoxymethyl substituents at the -position can be prepared indirectly from unsubstituted enone starting materials via -cyano-substituted enones: (a) Fukuta, Y.; Mita, T.; Fukuda, N.; Kanai, M.; Shibasaki, M. J. Am. Chem. Soc. 2006, 128, 6312–6313. (b) Nicolaou, K. C.; Li, A. Angew. Chem. Int. Ed. 2008, 47, 6579–6582. (c) Isobe, M.; Nishikawa, T.; Pikul, S.; Goto, T. Tetrahedron Lett. 1987, 28, 6485–6488. Though not successful in the current application (using the AB enone 10 as substrate), phosphoniosilylation methodology has been used to introduce -alkoxycarbonyl substituents in enone substrates: (a) Kim, S.; Lee, P. H.; Kim, S. S. Bull. Korean Chem Soc. 1989, 10, 218–219. (b) Kim, J. H.; Jung, H. K. Bull. Korean Chem Soc. 2004, 25, 1729–1732. 63 Alternative synthetic sequences for the preparation of enones with -alkoxymethyl and -aldehyde substituents from unsubstituted enone starting materials: (a) Kienzle, F.; Minder, R. E. Helv. Chim. Acta 1976, 59, 439–452. (b) Heguaburu, V.; Schapiro, V.; Pandolfi, E. Tetrahedron Lett. 2010, 51, 6921–6923. 64 37 initiated by conjugate addition of the Seebach reagent tris(methylthio)methyllithium65 (THF, –78 –45 °C) followed by trapping of the resulting enolate at –45 °C with (Scheme 2.4 below).66,67 The corresponding - chlorotrimethylsilane tris(methylthio)methyl trimethylsilyl enol ether (19) was isolated as a single stereoisomer (stereochemistry not determined) in 89% yield after purification by flash-column chromatography. We found that oxidation of the trimethylsilyl enol ether function and transformation of the tris(methylthio)methyl group occurred simultaneously upon treatment of a solution of 19 in the solvent mixture 500:1 methanol–water with an excess of N-bromosuccinimide (NBS, 5 equiv) at 23 °C, affording the AB enone containing a methyl ester substituent (20) in 85% yield. The detailed mechanism of the transformation of the intermediate 19 into the -substituted AB enone 20 is not known, but presumably involves some variation of a sequence involving -bromination of the trimethylsilyl enol ether, bromonium-induced conversion of the tris(methylthio)methyl substituent into the corresponding methyl ester (with incorporation of one molar equivalent each of methanol and water), and elimination of hydrogen bromide. 65 (a) Seebach, D. Angew. Chem. Int. Ed. 1967, 6, 442–443. (b) Bürstinghaus, R.; Seebach, D. Chem. Ber. 1977, 110, 841–851. (c) Gröbel, B.-T.; Seebach, D. Synthesis 1977, 357–402. (d) “Tris(methylthio)methane” Electronic Encyclopedia of Reagents for Organic Synthesis 2007. Other examples of conjugate addition–enolate trapping reactions of tris(methylthio)methyllithium with , -unsaturated carbonyl compounds: (a) Damon, R. E.; Schlessinger, R. H. Tetrahedron Lett. 1976, 19, 1561–1564. (b) Mikolajczyk, M.; Kielbasi ski, P.; Wieczorek, M. W.; Blaszczyk, J.; Kolbe, A. J. Org. Chem. 1990, 55, 1198–1203. 66 67 The regioselectivity observed upon reaction of tris(methylthio)methyllithium with , -unsaturated carbonyl compounds is substrate-dependent. For an example of a 1,2-addition reaction, see: Dailey, O. D., Jr.; Fuchs, P. L. J. Org. Chem. 1980, 45, 216-236. 38 Scheme 2.4. Two-step syntheses of modified AB enones with methyl ester, thioester and trifluoroethyl ester groups at the -position, starting from the -unsubstituted AB enone 10. By modification of the reaction solvent we found that different esters could be synthesized, including active esters. For example, treatment of a solution of the trimethylsilyl enol ether 19 in tert-butanol with NBS (4 equiv) at 23 °C afforded the AB enone 21 bearing an S-methyl thioester at the -position in 82% yield (Scheme 2.4 above).68 Alternatively, addition of NBS (5 equiv) to a solution of substrate 19 in 2,2,2trifluoroethanol and water (500:1 mixture) at 23 °C afforded the corresponding - trifluoroethyl ester-substituted AB enone 22 in 87% yield. Interestingly, treatment of a solution of 19 in 2,2,2-trifluoroethanol (no added water) with NBS (5 equiv) afforded - 68 Degani, I.; Dughera, S.; Fochi, R.; Gatti, A. Synthesis 1996, 4, 467–469. 39 trifluoroethyl ortho ester-substituted enone 23 as a major by-product (37% yield, Scheme 2.5). N(CH3)2 O N TMSO O OTBS 19 OBn CF3CH2OH O O OTBS 22, 48% NBS ( 5 equiv ) CF3CH2O O N(CH3)2 O N OBn CF3CH2O OCH2CF3 N(CH3)2 H O CF3CH2O N O O OTBS 23, 37% OBn (CH3S)3C H H H Scheme 2.5. Formation of a -trifluoroethyl ortho ester-substituted AB enone (23). In a further optimization, we found that transformation of the -unsubstituted AB enone 10 to the -methyl ester-substituted AB enone 20 could be achieved directly, and most efficiently (90% yield), in a single operation (Scheme 2.6 below). Scheme 2.6. Synthesis of the -methyl ester-substituted AB enone 20 from the - unsubstituted AB enone 10 in a single operation. The transformations of Schemes 2.4–2.6 represent novel, direct and highly efficient -functionalization reactions of an enone substrate. Important foundational precedents include the conversion of a -cyano-substituted trimethylsilyl enol ether to the corresponding -cyano enone upon sequential treatment with NBS and triethylamine62a as 40 well as the original discovery that dithianes undergo oxidative hydrolysis in the presence of N-halosuccinimides.69 A related strategy was effective for the synthesis of the AB enone 25 bearing a carbaldehyde substituent, which in turn provided an expedient route to AB enones with alkoxymethyl substituents (Scheme 2.7). Thus, conjugate addition of the Corey–Seebach reagent 2-lithio-1,3-dithiane70 to the AB enone 10 in the presence of hexamethylphosphoramide (HMPA)71 at –78 °C followed by quenching of the resulting enolate intermediate with chlorotrimethylsilane provided the -(1,3-dithian-2-yl) trimethylsilyl enol ether 24 as a single stereoisomer (90% yield; stereochemistry not determined). Treatment of the latter product with NBS (6 equiv) in the solvent mixture 100:1 tert-butanol–water at 23 °C afforded the -carbaldehyde AB enone 25 in 90% yield. Selective reduction of the aldehyde group occurred upon warming a solution of 25 and sodium triacetoxyborohydride in benzene at 40 °C. The resulting primary alcohol was then protected as a methoxymethyl ether in the presence of chloromethyl methyl ether and N,N-diisopropylethylamine in benzene at 50 °C to afford the methoxymethoxymethyl AB enone 26 (85% yield over two steps). - 69 70 Corey, E. J.; Erickson, B. W. J. Org. Chem. 1971, 36, 3553–3560. (a) Corey, E. J.; Seebach, D. Angew. Chem. Int. Ed. 1965, 4, 1075–1077. (b) Seebach, D.; Corey, E. J. J. Org. Chem. 1975, 40, 231–237. (c) Page, P. C. B.; van Niel, M. B.; Prodger, J. C. Tetrahedron 1989, 45, 7643–7677. (a) Brown, C. A.; Yamaichi, A. J. Chem. Soc., Chem Commun. 1979, 100–101. (b) Lucchetti, J.; Dumont, W.; Krief, A. Tetrahedron Lett. 1979, 20, 2695–2696. (c) Mukhopadhyay, T.; Seebach, D. Helv. Chim. Acta 1982, 65, 385–391. 71 41 Scheme 2.7. Synthesis of the -methoxymethoxymethyl AB enone 26 from the AB enone 10 via the -carbaldehyde AB enone 25. -Methoxymethoxymethyl AB enone 26 could also be prepared in fewer steps but much lower (and highly variable) yield by reaction of the AB enone 10 with (methoxymethoxy)methyllithium (prepared in situ from tri-n- butyl[(methoxymethoxy)methyl]stannane72 and n-butyllithium) in the presence of HMPA at –78 °C, trapping of the resulting enolate with chlorotrimethylsilane (affording the methoxymethoxymethyl trimethylsilyl enol ether 27 in 40–55% yield), and then oxidation of the intermediate 27 with palladium acetate in DMSO at 50 °C (providing AB enone 26 in 25–52% yield; Scheme 2.8). For preparation of tri-n-butyl[(methoxymethoxy)methyl]stannane, see: Danheiser, R. L.; Romines, K. R.; Koyama, H.; Gee, S. K.; Johnson, C. R.; Medich, J. R. Org. Synth. 1993, 71, 133; Org. Synth. 1998, Coll. Vol. 9, 704. 72 42 Scheme 2.8. Alternative synthesis of the -methoxymethoxymethyl AB enone 26 from the AB enone 10. Having established versatile methodology for the synthesis of -substituted AB enone substrates, we next investigated the feasibility of constructing the C ring of tetracyclines with an all-carbon quaternary C5a stereocenter by a Michael–Claisen cyclization reaction (Schemes 2.9 and 2.10). The D-ring precursor 28 was chosen for initial cyclization experiments. This precursor comprises the D-ring functionality of minocycline (8) and was known from prior research to be an effective substrate in Michael–Claisen cyclization reactions.54b Addition of the -methyl-substituted AB enone 18 (1 equiv) to a bright red solution of the o-toluate ester anion formed by deprotonation of the minocycline D-ring precursor 28 (3 equiv) with lithium diisopropylamide (LDA, 3 equiv) in the presence of N,N,N’,N’-tetramethylethylenediamine (TMEDA, 6 equiv) at – 78 °C, followed by warming to –10 °C, provided the Michael–Claisen cyclization product 29 in 80% yield as a single stereoisomer after purification by flash-column chromatography. A minor by-product (compound 30, 4%), thought to be the product of 1,2-addition-cyclization (by lactonization), was isolated separately.73 73 Michael–Claisen cyclization product 29 was obtained in 60% yield and 1,2-addition-cyclization product 30 was not observed when the cyclization reaction was performed without TMEDA as an additive. 43 Scheme 2.9. Stereocontrolled formation of an all-carbon quaternary center by Michael– Claisen cyclization of an o-toluate ester anion with -methyl-substituted AB enone 18. The stereochemical assignment of the Michael–Claisen cyclization product (29), with C5a-R configuration, is supported by nOe studies; this stereochemistry is homologous with that of Michael–Claisen cyclization products derived from the nonsubstituted AB enone 10.54b In both cases, addition appears to occur from a single diastereoface of the enone, that opposite the C12a tert-butyldimethylsilyloxy substituent. There are two examples in the literature of Michael–Claisen cyclization reactions of achiral -methyl cyclohexenones with o-toluate ester anions,74 but we are unaware of any examples beyond those described in this thesis of the stereocontrolled construction of a six-membered ring containing a quaternary center by a Michael–Claisen cyclization reaction. 74 Hill, B.; Rodrigo, R. Org. Lett. 2005, 7, 5223–5225. 44 Michael–Claisen reaction of the -methoxymethoxymethyl AB enone 26 and the minocycline D-ring precursor 31 (with OBn protection at C10, chosen so as to allow later deprotection of the methoxymethyl ether at C5a without concomitant cleavage of the C10 phenoxy protective group) was also efficient, affording the cyclization product 32, with a protected C5a-hydroxymethyl-substituted quaternary center, in 72% yield (Scheme 2.10). Reaction of the -methyl ester AB enone 20 with the anion formed from the minocycline D-ring precursor 28 afforded a complex mixture of products containing the desired Michael–Claisen cyclization product 33 as one component. Cycloadduct 33 was isolated in 23% yield after purification by sequential flash-column chromatography and reversephase high-performance liquid chromatography (rp-HPLC). N(CH3)2 CH3 MOMO N(CH3)2 O N O O OTBS 26 OBn LDA, TMEDA, THF –78 –10 °C 72% N(CH3)2 CH3 CH3 O CH3O O O OTBS 20 H N(CH3)2 O N OBn LDA, TMEDA, THF –78 –10 °C 23% BocO O HO 33 O OTBS CO2Ph BocO 28 (CH3)2N O O H N(CH3)2 O N OBn BnO CO2Ph BnO 31 N(CH3)2 OMOM N(CH3)2 H O N O OTBS OBn H O HO 32 Scheme 2.10. Stereocontrolled formation of an all-carbon quaternary center by Michael– Claisen cyclization reactions of o-toluate ester anions with -substituted AB enones. 45 Two-step deprotection of cyclization products 29 and 33 under typical conditions54b,75 provided C5a-methylminocycline (34, 100% yield, Scheme 2.11) and C5a-carbomethoxyminocycline (35, 89% yield), respectively, after purification by rpHPLC. Scheme 2.11. Synthesis of C5a-methylminocycline (34) and C5a-carbomethoxyminocycline (35) by two-step deprotection of cyclization products 29 and 33, respectively. In a search for a versatile branch point for the synthesis of various C5a-substituted tetracyclines we were led to a serendipitous but highly effective solution (Scheme 2.12). Removal of the methoxymethyl ether protective group within the Michael–Claisen cyclization product 32 was achieved by treatment with perchloric acid76 providing the substituted neopentyl alcohol 36 (73% yield). Addition of phosgene to a solution of 36 in 75 76 Stork, G.; Hagedorn, A. A. III. J. Am. Chem. Soc. 1978, 100, 3609–3611. Perchloric acid is a strong oxidizing agent and can form explosive mixtures. For safety information, see: (a) Wolsey, W. C. J. Chem. Educ. 1973, 50, A335–A337. (b) Muse, L. A. J. Chem. Educ. 1972, 49, A463– A466. 46 dichloromethane–pyridine (10:1) at 0 °C unexpectedly afforded the C5a–C11a-bridged cyclopropane tetracycline precursor 37 in 81% yield. Scheme 2.12. Discovery of a C5a–C11a-bridged cyclopropane tetracycline precursor (37). Scheme 2.13. Bridged tetracycline product formed by an internal nucleophilic addition involving the (C11–C12) 1,3-diketone of tetracycline.77 After the fact, the formation of cyclopropane 37 is easily rationalized. In this context it is interesting to note that Barton and co-workers had previously reported that the (C11–C12) 1,3-diketone of tetracycline can participate in an internal nucleophilic 47 addition, albeit in that case with addition to an electrophilic carbonyl group that had been introduced at position C4 (Scheme 2.13 above).77 Cyclopropanes with geminal electron-withdrawing substituents are known to undergo nucleophilic ring-opening,78 a transformation often enhanced in the presence of Lewis acids.79 We were led to explore the use of magnesium salts as Lewis acid activators in this system in view of the well documented affinity of Mg2+ for binding to the (C11– C12) 1,3-diketone function of tetracyclines, complexation which is critical for inhibition of the ribosome.56 We observed that the bridged cyclopropane intermediate 37 underwent regioselective ring-opening at the bridging carbon atom in the presence of various nucleophiles and magnesium bromide as Lewis acid (Chart 2.1). The ring-opened products were readily deprotected in the typical two-step sequence to furnish the corresponding C5a-substituted tetracyclines. For example, reaction of the C5a–C11abridged cyclopropane tetracycline precursor 37 with pyrrolidine (10 equiv) in the presence of a stoichiometric amount of anhydrous magnesium bromide in THF at 23 °C, followed by direct deprotection of the crude ring-opened product (38), provided C5apyrrolidinomethylminocycline (39) in 74% yield over the three steps after purification by rp-HPLC. 77 Barton, D. H. R.; Ley, S. V.; Meguro, K.; Williams, D. J. J. Chem. Soc., Chem. Commun. 1977, 790– 791. For an overview, see: “Electrophilic Cyclopropanes in Organic Synthesis” Danishefsky, S. Acc. Chem. Res. 1979, 12, 66–72. Examples of Lewis acid-promoted ring-opening of activated cyclopropanes: (a) Swain, N. A.; Brown, R. C. D.; Bruton, G. J. Org. Chem. 2004, 69, 122–129. (b) Tanimori, S.; He, M.; Nakayama, M. Synth. Commun. 1993, 23, 2861–2868. (c) Lifchits, O.; Charette, A. B. Org. Lett. 2008, 10, 2809–2812. 78 79 48 Chart 2.1. Fully synthetic tetracyclines prepared from the C5a–C11a-bridged cyclopropane intermediate 37 by magnesium bromide-promoted ring-opening followed by two-step deprotection. A number of different amines and alcohols were found to function effectively as nucleophiles in the magnesium bromide-promoted cyclopropane ring-opening reaction (Chart 2.1). Ring-opening reactions were typically performed with a large excess of nucleophile ( 7 equiv) and stoichiometric or superstoichiometric quantities of anhydrous magnesium bromide in THF (in the cases of low molecular weight alcohols as nucleophiles, the alcohol was used as solvent) at a range of temperatures (23–75 °C, see experimental section for details). Partial (and inconsequential) loss of the benzyl ether 49 phenolic protective group was observed to occur in the cyclopropane ring-opening reaction in some instances. The C5a–C11a-bridged cyclopropane intermediate 37 also served as a precursor to tetracyclines with aminomethyl (49) and piperazinylmethyl (51) substituents at position C5a (Scheme 2.14), highly versatile compounds which functioned as further branch-points for the synthesis of C5a-substituted tetracyclines (Figures 2.3 and 2.4). Thus, treatment of cyclopropane 37 with sodium azide in dimethylformamide at 23 °C afforded the azido-substituted ring-opened product 48 in 78% yield after purification by flash-column chromatography. Addition of trimethylphosphine (2 equiv) to a solution of the azide 48 and 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (Boc-ON, 2 equiv) in THF at –10 °C followed by warming to 23 °C afforded the corresponding tertbutyl carbamate (51% yield).80 Two-step deprotection of the tert-butyl carbamate intermediate was best achieved by an inverted deprotection sequence (hydrogenolysis followed by treatment with hydrofluoric acid),81 providing C5a-aminomethylminocycline 49 after purification by rp-HPLC (69% yield over two steps). In addition, magnesium bromide-promoted ring-opening of cyclopropane 37 with N-tert-butoxycarbamylpiperazine followed by deprotection of the ring-opened product 50 provided C5apiperazinylmethylminocycline (51, 58% yield over three steps). 80 81 Ariza, X.; Urpí, F.; Viladomat, C.; Vilarrasa, J. Tetrahedron Lett. 1998, 39, 9101–9102. The hydrogenolysis deprotection step (typically the final step) was slow and low-yielding in the presence of primary and secondary amines. To synthesize the amines 49 and 51 most efficiently the usual order of deprotection steps was reversed and the hydrogenolysis reaction was performed on substrates in which primary or secondary amines were protected as tert-butyl carbamates. For discussion of the inhibitory effects of amines on Pd-catalyzed hydrogenolysis, see: Sajiki, H.; Hirota, K. Tetrahedron 1998, 54, 13981– 13996. 50 Scheme 2.14. Synthesis of substrates for final-step diversification from cyclopropane 37. Figure 2.3. Selected fully synthetic tetracyclines prepared by final-step diversification of C5a-aminomethylminocycline (49). Final-step diversification of 49 and 51 was readily achieved, affording a range of novel tetracyclines with C5a substituents incorporating amides, sulfonamides and amines 51 (Figures 2.3 and 2.4). In this manner the C5a–C11a-bridged cyclopropane 37 served as a common precursor for the synthesis of more than 25 structural variants of minocycline with a diverse range of substituents at C5a. Figure 2.4. Fully synthetic tetracyclines prepared by final-step diversification of C5apiperazinylmethylminocycline (51). Antibacterial Activities Minimum inhibitory concentrations (MICs) were determined in whole-cell antibacterial assays using a panel of tetracycline-sensitive and tetracycline-resistant Gram-positive and Gram-negative bacteria. Initial experiments were performed with assistance from members of the Kahne research group, and more thorough analyses of antibacterial activity were conducted at Tetraphase Pharmaceuticals. In summary, analogs of minocycline possessing angular substituents at C5a were found to be significantly less active than the parent compound against both Gram-positive and Gramnegative bacteria. 52 MIC assays for C5a-methylminocycline (34), minocycline (8) and tetracycline (3) against tetracycline-susceptible E. coli and leaky E. coli strains are depicted in Figure 2.5 below. Leaky E. coli has a less asymmetric and more permeable outer membrane than the corresponding E. coli strain due to knockout of a lipopolysaccharide gene. As discussed in Chapter 1, the outer membrane of Gram-negative bacteria is asymmetric and consists of both lipopolysaccharides and phospholipids.82 The “gel-like” nature of the lipopolysaccharide component makes the outer membrane a more effective permeability barrier than the inner membrane, and most antibacterials (including tetracyclines) penetrate the outer membrane predominantly by passing through aqueous channels provided by porin proteins. To determine MIC values, 96-well plates containing the bacterial strain and an appropriate medium were treated with different concentrations of drug molecules. Each pair of rows corresponds to a different small molecule and the drug concentration decreases uniformly across each plate (from left to right, as depicted). Following incubation, the wells were treated with a stain which (in the examples shown in Figure 2.5 below) turned black in the presence of bacteria, indicating that the drug concentration was insufficient to prevent bacterial growth. 82 Nikaido, H. Microbio. Mol. Biol. Rev. 2003, 67, 593-656. 53 E. E coli C5a-Methy ylminocycline (Rows 1 & 2 e 2) Mino ocycline (Row 3 & 4) ws Tetra acycline (Row 5 & 6) ws 16 2 2 Leak E. coli ky 1 0.5 0.5 Figure 2.5. Minimum in F M nhibitory con ncentration ( (MIC) assay and value in µg / mL for ys es C5a-methylm C minocycline (34), minoc cycline (8) a tetracyc and cline (3) aga ainst E. coli and le eaky E. coli strains. The re esults of the MIC ass ese says revealed that C5a-m d methylminoc cycline poss sesses an ntibacterial activity but is 8-fold les active tha minocycl ss an line against the E. coli s strain an 2-fold less active in leaky E. co Since the is a sma nd oli. ere aller differen in poten in nce ncy le eaky E. coli where the dr molecule are better able to pene rug es r etrate the ou membran by uter ne diffusion (as well as by passage thr rough porin channels), t reduced activity of C5athe methylminocy m ycline relativ to minocy ve ycline in E. coli could b partly due to reduced outer be e membrane pe m ermeability. 54 Complete antibacterial activity data for C5a-substituted analogs is presented in the tables at the end of this chapter (pages 57–62). The C5a-modified compounds synthesized in this study were found to be significantly less active than minocycline (the parent compound) against both Gram-positive and Gram-negative bacteria. Analogs with smaller substituents (e.g. methyl) at C5a exhibited modest activity against some bacterial strains, while most of the compounds with larger substituents appended at C5a were almost completely inactive as antibiotics. The detrimental effect of C5a-substitution on activity did not seem to be dependent on the tetracycline resistance determinants possessed by a given bacterial strain. -Methyl-substituted AB enone 18 was also transformed into C5a- methyltigecycline (54) via the four-step synthetic sequence presented in Scheme 2.15 below. 5a-Methyltigecycline was found to have greatly reduced potency relative to tigecycline. Indeed, the detrimental effect on activity (C5a-methyl vs. C5a-unsubstituted) was even more pronounced for tigecycline than for minocycline, indicating that the introduction of substituents at this position diminishes (or eliminates) antibiotic activity regardless of the D-ring substitution pattern. 55 Scheme 2.15. Synthesis of C5a-methyltigecycline (54). 56 57 58 59 60 61 62 Conclusion Synthetic methodological advances have permitted efficient and stereocontrolled construction of fully synthetic tetracyclines containing an all-carbon quaternary, stereogenic center at position C5a, a structurally novel class of compounds in this important family of therapeutic agents. We anticipate that the new strategies presented herein for the introduction of ester, thioester and aldehyde substituents at the -position of cyclohexenones will be of value in many contexts. The discovery of a highly diversifiable bridged cyclopropane-containing tetracycline intermediate enabled efficient synthesis of numerous C5a-substituted tetracyclines. Although it is conceivable that many of these structures could also have been accessed by Michael–Claisen cyclization reactions of individually prepared -substituted AB enones, the discovery of a diversifiable late-stage intermediate greatly expedited the process of synthesis, in that a single Michael–Claisen cycloadduct served as a precursor to large numbers of C5asubstituted tetracyclines. The C5a-modified analogs synthesized in this study were found to be significantly less active than the corresponding C5a-unsubstituted tetracyclines against both Gram-positive and Gram-negative bacteria. 63 Experimental Section General experimental procedures: All reactions were performed in round-bottom flasks fitted with rubber septa under a positive pressure of argon, unless otherwise noted. Air- and moisture-sensitive liquids were transferred via syringe or stainless steel cannula. Organic solutions were concentrated by rotary evaporation (house vacuum, ca. 25–40 Torr) at ambient temperature, unless otherwise noted. Analytical thin-layer chromatography (TLC) was performed using glass plates pre-coated with silica gel (0.25 mm, 60 Å pore-size, 230–400 mesh, Merck KGA) impregnated with a fluorescent indicator (254 nm). TLC plates were visualized by exposure to ultraviolet light, then were stained with either an aqueous sulfuric acid solution of ceric ammonium molybdate (CAM) or an aqueous sodium carbonate solution of potassium permanganate (KMnO4) followed by brief heating on a hot plate. Flash-column chromatography was performed as described by Still et al.,83 employing silica gel (60 Å, 32–63 Dynamic Adsorbents, Inc.). Materials: Commercial solvents and reagents were used as received with the following exceptions. Diisopropylamine, trimethylsilyl chloride, and hexamethylphosphoramide were distilled from calcium hydride under an atmosphere of argon or dinitrogen. Tetrahydrofuran was purified by the method of Pangborn et al.84 The molarity of nM, standard grade, 83 W. C. Still, M. Khan, A. Mitra, J. Org. Chem. 1978, 43, 2923–2925. A. B. Pangborn, M. A. Giardello, R. H. Grubbs, R. K. Rosen, F. J. Timmers, Organometallics 1996, 15, 1518–1520. 84 64 butyllithium solutions was determined by titration against a standard solution of diphenylacetic acid in tetrahydrofuran (average of three determinations).85 Instrumentation: Proton magnetic resonance (1H NMR) spectra were recorded on Varian INOVA 500 (500 MHz) or 600 (600 MHz) NMR spectrometers at 23 °C. Proton chemical shifts are expressed in parts per million (ppm, residual protium in the NMR solvent (CHCl3, scale) and are referenced to 7.26). Data are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet and/or multiple resonances, br = broad, app = apparent), integration, and coupling constant (J) in Hertz. Carbon nuclear magnetic resonance spectra (13C NMR) were recorded on Varian INOVA 500 (125 MHz) NMR spectrometers at 23 °C. Carbon chemical shifts are expressed in parts per million (ppm, carbon resonances of the NMR solvent (CDCl3, scale) and are referenced to the 77.0). Fluorine nuclear magnetic resonance spectra (19F NMR) were recorded on a Varian INOVA 400 NMR spectrometer at 23 °C. Infrared (IR) spectra were obtained using a Shimadzu 8400S FT-IR spectrometer and were referenced to a polystyrene standard. Data are represented as follows: frequency of absorption (cm–1), intensity of absorption (s = strong, m = medium, w = weak, br = broad). High-resolution mass spectra were obtained at the Harvard University Mass Spectrometry Facility. X-ray crystallographic analyses were performed at the Harvard University X-ray Crystallographic Laboratory by Dr. Shao-Liang Zheng. 85 W. G. Kofron, L. M. Baclawski, J. Org. Chem. 1976, 41, 1879 1880. 65 Minimum Inhibitory Concentration (MIC) Values. MIC values were used to determine the efficacy of a particular antibiotic by measuring its ability to inhibit growth at a range of different concentrations. A 96-well plate (Corning Costar 3596) was prepared by adding 100 µL of media to all wells and an additional 87.2 µL of media to the first column of wells. 12.8 µL of drug solution in dimethyl sulfoxide (DMSO) was then added to the first well of each row (2.0 mg/mL drug solution provides final concentration of 64 µg/mL in the first column of wells). A serial dilution was performed across the plate, discarding the final 100 µL of media. Cells were prepared by reinoculating an overnight culture into fresh lysogeny broth (LB) until they reached an OD600 = ~ 0.6, then by diluting the cells 100-fold in fresh LB in a media reservoir. 100 µL of cells from the media reservoir were then added to each row of the plate. Cells were incubated for 24 h at 37 °C. A 1 mg/mL aqueous solution of thiazolyl blue tetrazolium bromide (MTT) was prepared and 50 µL of this solution was added to each well. Following incubation for a further 1 h, MICs were determined by measuring the first well that stained successfully (indicating respiration by the organism). The drug concentration present in the last well in which the stain did not appear provided the MIC value for a particular drug molecule in this bacterial strain. The MIC value for each drug was verified by a duplicate (two rows for each small molecule) and the growth of bacteria in the absence of small molecule was confirmed using a DMSO control. 66 -Methyl-substituted trimethylsilyl enol ether 17. A round-bottomed flask charged with copper (I) iodide (1.89 g, 9.95 mmol, 1.6 equiv) was flame-dried under high vacuum. After cooling to room temperature, the flask was blanketed with dry argon. Tetrahydrofuran (50 mL) was added and the resulting suspension was cooled to 0 °C. A solution of methyllithium in ethyl ether (1.6 M, 12.2 mL, 19.6 mmol, 3.15 equiv) was added dropwise via syringe over 5 min. The resulting solution was stirred at 0 °C for 20 min, then was cooled to –78 °C. A solution of the AB enone 10 (3.0 g, 6.22 mmol, 1 equiv) and chlorotrimethylsilane (1.25 mL, 9.95 mmol, 1.6 equiv) in tetrahydrofuran (10 mL) was added to the cuprate solution dropwise via syringe at –78 °C. After stirring at – 78 °C for 90 min, the cooling bath was removed and the product solution was diluted with ethyl acetate (100 mL) and hexanes (100 mL). A mixture of saturated aqueous ammonium chloride solution and saturated aqueous ammonium hydroxide solution (19:1, 100 mL) was then added carefully. The phases were separated and the organic phase was washed sequentially with saturated aqueous ammonium chloride solution (100 mL) and saturated aqueous sodium chloride solution (2 x 100 mL). The organic phase was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The product was purified by flash-column chromatography (7% ethyl acetate-hexanes), providing -methyl-substituted trimethylsilyl enol ether 17 as a white solid (3.01 g, 85%). 67 Rf = 0.57 (15% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 7.48 (dd, 2H, J = 8.0, 1.5 Hz), 7.36-7.31 (m, 3H), 5.36 (AB quartet, 2H), 4.69 (d, 1H, J = 3.0 Hz), 3.76 (d, 1H, J = 10.0 Hz), 2.55-2.52 (m, 1H), 2.44 (s, 6H), 2.32-2.25 (m, 2H), 1.84 (d, 1H, J = 13.5 Hz), 1.18 (d, 3H, J = 7.5 Hz), 0.87 (s, 9H), 0.22 (s, 3H), 0.11 (s, 3H), –0.04 (s, 9H); 13 C NMR (125 MHz, CDCl3) 189.7, 181.5, 167.4, 147.2, 135.2, 128.6, 128.5, 128.4, 110.7, 108.3, 80.7, 72.2, 61.1, 46.3, 41.9, 26.2, 26.0, 25.1, 24.0, 18.8, –0.5, –2.8, –3.6; FTIR (neat film), 2955 (w), 1721 (m), 1653 (w), 1614 (w), 1510 (m), 1471 (w), 1250 (m), 1198 (m), 1148 (m), 936 (m), 833 (s) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C30H47N2O5Si2, 571.3018; found, 571.3041. 68 -Methyl-substituted AB enone 18.60 Palladium (II) acetate (75 mg, 0.329 mmol, 1.15 equiv) was added to a solution of -methyl-substituted trimethylsilyl enol ether 17 (163 mg, 0.286 mmol, 1 equiv) in anhydrous dimethyl sulfoxide (3.0 mL) at 23 °C. The resulting mixture was heated to 80 °C. After stirring at this temperature for 16 h, the reaction mixture was allowed to cool to 23 °C. The cooled suspension was diluted with ethyl acetate (20 mL) and the whole was filtered through a pad of Celite. Hexanes (20 mL) were added to the filtrate and the resulting solution was washed sequentially with saturated aqueous sodium bicarbonate solution (20 mL) and saturated aqueous sodium chloride solution (2 x 20 ml). The organic phase was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The product was purified by flash-column chromatography (10% ethyl acetate-hexanes), affording the methyl-substituted AB enone 18 as a pale yellow solid (63 mg, 44%). Rf = 0.26 (15% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 7.50 (d, 2H, J = 7.0 Hz), 7.40-7.32 (m, 3H), 5.93 (s, 1H), 5.35 (AB quartet, 2H), 3.74 (d, 1H, J = 11.0 Hz), 2.81-2.70 (m, 3H), 2.46 (s, 6H), 2.01 (s, 3H), 0.82 (s, 9H), 0.26 (s, 3H), 0.05 (s, 3H); 13 C NMR (125 MHz, CDCl3) 193.1, 188.1, 181.2, 167.5, 161.2, 135.0, 128.5, 128.5, 124.7, 108.4, 82.5, 72.5, 59.8, 47.5, 42.0, 30.4, 25.9, 24.4, 19.0, –2.5, –4.2; FTIR (neat film), 2930 (w), 1719 (s), 1672 (m), 1510 (m), 1472 (w), 1175 (m), 1045 (m), 936 (s), 829 (m) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C27H37N2O5Si, 497.2466; found, 497.2494. 69 -Tris(methylthio)methyl trimethylsilyl enol ether 19. A solution of n-butyllithium in hexanes (2.5 M, 518 µL, 1.30 mmol, 1.25 equiv) was added dropwise via syringe to a solution of tris(methylthio)methane (176 µL, 1.30 mmol, 1.25 equiv) in tetrahydrofuran (11 mL) at –78 °C. The resulting colorless solution was stirred at this temperature for 20 min, whereupon a solution of the AB enone 10 (500 mg, 1.04 equiv, 1 equiv) in tetrahydrofuran (3.0 mL) was added dropwise via syringe, forming a bright orangeyellow solution. The reaction solution was allowed to warm slowly to –45 °C over 60 min, then chlorotrimethylsilane (199 µL, 1.55 mmol, 1.5 equiv) was added. The (yellow) reaction mixture was stirred at –45 °C for 30 min, then was partitioned between aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 20 mL) and dichloromethane (30 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (20 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording an orange-yellow oil. The crude product was purified by flash-column chromatography (6% ethyl acetate–hexanes), providing - tris(methylthio)methyl trimethylsilyl enol ether 19 as a pale yellow foam (654 mg, 89%). Rf = 0.69 (20% ethyl acetate–hexanes); 1H NMR (500 MHz, CDCl3) 7.49-7.47 (m, 2H), 7.37-7.32 (m, 3H), 5.37 (AB quartet, 2H), 5.36 (d, 1H, J = 2.0 Hz), 4.00 (d, 1H, J = 9.3 Hz), 3.15-3.12 (m, 1H), 2.52 (dd, 1H, J = 14.4, 4.2 Hz), 2.45 (s, 6H), 2.42-2.39 (m, 1H), 2.18 (s, 9H), 2.14-2.06 (m, 1H), 0.86 (s, 9H), 0.22 (s, 3H), 0.12 (s, 3H), –0.01 (s, 9H); 13C 70 NMR (125 MHz, CDCl3) 189.6, 181.6, 167.2, 150.5, 135.2, 128.6, 128.5, 128.4, 108.7, 103.8, 81.6, 75.1, 72.2, 61.7, 46.6, 42.1, 41.7, 26.1, 21.2, 19.0, 14.1, –0.3, –2.6, –3.3; FTIR (neat film), 1722 (m), 1651 (w), 1614 (w), 1510 (m), 1472 (w), 1254 (m), 1206 (w), 903 (m), 839 (s) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C33H53N2O5S3Si2, 709.2650; found, 709.2617. 71 -Methyl ester-substituted AB enone 20. N-Bromosuccinimide (151 mg, 0.846 mmol, 5.0 equiv) was added in one portion to a stirring solution of -tris(methylthio)methyl trimethylsilyl enol ether 19 (120 mg, 0.169 mmol, 1 equiv) in methanol (4.5 mL) and water (9.0 µL, 3.0 equiv, 500:1 mixture of methanol and water) at 23 °C. The pale yellow reaction solution was allowed to stir at 23 °C for 30 min, then was concentrated. The resulting yellow oil was dissolved in dichloromethane (20 mL) and the resulting solution was washed with saturated aqueous sodium bicarbonate solution (20 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (20 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude product was purified by flash-column chromatography (11% ethyl acetate-hexanes, grading to 15%), providing the -methyl ester-substituted AB enone 20 as a yellow solid (78 mg, 85%). Rf = 0.26 (15% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 7.50 (d, 2H, J = 6.9 Hz), 7.40-7.33 (m, 3H), 6.82 (d, 1H, J = 1.8 Hz), 5.36 (AB quartet, 2H), 3.87 (s, 3H), 3.65 (d, 1H, J = 10.5 Hz), 3.21 (d, 1H, J = 18.8 Hz), 2.91-2.84 (m, 2H), 2.46 (s, 6H), 0.80 (s, 9H), 0.26 (s, 3H), 0.03 (s, 3H); 13 C NMR (125 MHz, CDCl3) 194.2, 187.0, 181.1, 167.4, 166.1, 147.1, 134.9, 131.4, 128.6, 128.5, 128.5, 108.3, 82.5, 72.6, 59.3, 52.9, 47.2, 41.9, 25.9, 24.6, 19.0, –2.5, –4.1; FTIR (neat film), 1721 (s), 1684 (m), 1609 (w), 1510 72 (m), 1252 (m), 1173 (m), 1030 (m), 831 (m), 737 (m) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C28H37N2O7Si, 541.2365; found, 541.2368. 73 Single-operation synthesis of -methyl ester-substituted AB enone 20. A solution of n-butyllithium in hexanes (2.5 M, 104 µL, 0.259 mmol, 1.25 equiv) was added dropwise via syringe to a solution of tris(methylthio)methane (35.2 µL, 0.259 mmol, 1.25 equiv) in tetrahydrofuran (2.0 mL) at –78 °C. The resulting colorless solution was stirred at this temperature for 20 min, whereupon a solution of the AB enone 10 (100 mg, 0.207 mmol, 1 equiv) in tetrahydrofuran (0.4 mL) was added dropwise via syringe, forming a bright orange-yellow solution. The reaction solution was allowed to warm slowly to –45 °C over 30 min, then chlorotrimethylsilane (39.7 µL, 0.311 mmol, 1.5 equiv) was added. The resulting (yellow) mixture was allowed to warm to 23 °C over 30 min, whereupon methanol (4.0 mL), water (8.0 µL, 2.1 equiv, 500:1 mixture of methanol and water) and N-bromosuccinimide (221 mg, 1.24 mmol, 6.0 equiv) were added in sequence. The reaction mixture was allowed to stir at 23 °C for 30 min, then was concentrated. The resulting oily (yellow) suspension was dissolved in dichloromethane (15 mL) and the resulting solution was washed with saturated aqueous sodium bicarbonate solution (15 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (15 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude product was purified by flash-column chromatography (7% acetone-hexanes), providing the enone 20 as a yellow solid (101 mg, 90%). 74 -methyl ester-substituted AB -S-Methyl thioester-substituted AB enone 21. N-Bromosuccinimide (48 mg, 0.271 mmol, 4.0 equiv) was added in one portion to a stirring solution of - tris(methylthio)methyl trimethylsilyl enol ether 19 (48 mg, 0.068 mmol, 1 equiv) in tertbutanol (3.0 mL) at 23 °C. The resulting bright yellow suspension was allowed to stir at 23 °C for 45 min (slowly clearing to give a yellow solution). The reaction mixture was diluted with dichloromethane (20 mL) and the resulting solution was washed with saturated aqueous sodium bicarbonate solution (20 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (20 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude product was purified by flash-column chromatography (8% ethyl acetate-hexanes), providing the -Smethyl thioester-substituted AB enone 19 as a pale yellow solid (31 mg, 82%). Rf = 0.51 (20% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 7.51-7.50 (m, 2H), 7.41-7.35 (m, 3H), 6.72 (d, 1H, J = 2.4 Hz), 5.36 (AB quartet, 2H), 3.70 (d, 1H, J = 10.7 Hz), 3.30 (d, 1H, J = 19.5 Hz), 2.98-2.93 (m, 1H), 2.89 (dd, 1H, J = 10.5, 3.7 Hz), 2.49 (s, 6H), 2.45 (s, 3H), 0.82 (s, 9H), 0.26 (s, 3H), 0.05 (s, 3H); CDCl3) 13 C NMR (125 MHz, 194.2, 192.9, 186.8, 181.0, 167.4, 153.2, 134.9, 128.6, 128.5, 128.5, 127.8, 108.3, 82.6, 72.6, 59.3, 47.3, 41.9, 25.9, 25.9, 24.4, 19.0, 11.9, –2.5, –4.0; FTIR (neat 75 film), 1721 (m), 1684 (w), 1661 (w), 1510 (m), 1136 (w), 1040 (m), 937 (s), 735 (s) cm– 1 ; HRMS–ESI (m/z): [M+H]+ calcd for C28H37N2O6SSi, 557.2136; found, 557.2109. 76 -Trifluoroethyl ester-substituted AB enone 22. N-Bromosuccinimide (100 mg, 0.564 mmol, 5.0 equiv) was added in one portion to a stirring solution of - tris(methylthio)methyl trimethylsilyl enol ether 19 (80 mg, 0.113 mmol, 1 equiv) in 2,2,2-trifluoroethanol (3.0 mL) and water (6.0 µL, 500:1 mixture of 2,2,2-trifluoroethanol and water) at 23 °C. The bright orange reaction solution was allowed to stir at 23 °C for 45 min, then was concentrated. The resulting yellow oil was dissolved in dichloromethane (20 mL) and the resulting solution was washed with saturated aqueous sodium bicarbonate solution (20 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (20 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude product was purified by flash-column chromatography (10% ethyl acetate-hexanes), providing the substituted AB enone 22 as a yellow solid (60 mg, 87%). Rf = 0.33 (15% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 7.50 (d, 2H, J = -trifluoroethyl ester- 6.9 Hz), 7.41-7.35 (m, 3H), 6.90 (d, 1H, J = 2.3 Hz), 5.36 (AB quartet, 2H), 4.68-4.62 (m, 2H), 3.65 (d, 1H, J = 11.0 Hz), 3.22 (d, 1H, J = 18.8 Hz), 2.95-2.87 (m, 2H), 2.47 (s, 6H), 0.80 (s, 9H), 0.27 (s, 3H), 0.04 (s, 3H); 13C NMR (125 MHz, CDCl3) 194.0, 186.7, 181.0, 167.4, 164.1, 145.2, 134.9, 132.7, 128.6, 128.5, 128.5, 122.5 (q, J = 276.5 Hz), 108.3, 82.4, 72.7, 61.3 (q, J = 37.5 Hz), 59.3, 47.0, 41.9, 25.9, 24.5, 18.9, –2.5, –4.1; 77 FTIR (neat film), 1742 (m), 1721 (s), 1688 (m), 1607 (w), 1510 (m), 1167 (s), 936 (s) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C29H36F3N2O7Si, 609.2238; found, 609.2299. 78 -Trifluoroethyl ortho ester-substituted enone 23. N-Bromosuccinimide (100 mg, 0.564 mmol, 5.0 equiv) was added in one portion to a stirring solution of - tris(methylthio)methyl trimethylsilyl enol ether 19 (80 mg, 0.113 mmol, 1 equiv) in 2,2,2-trifluoroethanol (3.0 mL) at 23 °C. The bright orange reaction solution was allowed to stir at 23 °C for 45 min, then was concentrated. The resulting yellow oil was dissolved in dichloromethane (20 mL) and the resulting solution was washed with saturated aqueous sodium bicarbonate solution (20 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (20 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude product was purified by flash-column chromatography (4% acetone-hexanes, grading to 6%), affording the trifluoroethyl ester-substituted AB enone 22 as a yellow solid (33 mg, 48%). Further purification by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 250 nm, Solvent A: water, Solvent B: methanol, injection volume: 10.0 mL (8.5 mL water, 1.5 mL methanol), gradient elution with 85 100% B over 40 min, flow rate: 15.0 mL/min, fractions eluting at 27-29 min collected and concentrated] provided the -trifluoroethyl ortho ester-substituted enone 23 as a white solid (33 mg, 37%). 79 Rf = 0.48 (15% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 7.50 (d, 2H, J = 7.3 Hz), 7.41-7.35 (m, 3H), 6.31 (d, 1H, J = 2.3 Hz), 5.37 (s, 2H), 4.04-3.97 (m, 6H), 3.61 (d, 1H, J = 10.5 Hz), 2.93 (d, 1H, J = 17.4 Hz), 2.87-2.82 (m, 2H), 2.46 (s, 6H), 0.84 (s, 9H), 0.25 (s, 3H), 0.11 (s, 3H); 13 C NMR (125 MHz, CDCl3) 192.5, 186.4, 180.9, 167.4, 151.9, 134.9, 128.6, 128.6, 128.5, 126.8, 122.9 (q, J = 277.4 Hz), 112.0, 108.3, 82.3, 72.7, 61.3 (q, J = 36.6 Hz), 59.4, 47.4, 41.7, 25.9, 23.8, 18.9, -2.4, -3.7; FTIR (neat film), 1724 (m), 1688 (w), 1611 (w), 1514 (m), 1288 (s), 1175 (s) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C33H40F9N2O8Si, 791.2405; found, 791.2459. 80 -(1,3-Dithian-2-yl) trimethylsilyl enol ether 24. A solution of n-butyllithium in hexanes (2.5 M, 2.91 mL, 7.27 mmol, 1.15 equiv) was added to a solution of 1,3-dithiane (862 mg, 6.95 mmol, 1.1 equiv) in tetrahydrofuran (60 mL) at –78 °C. The resulting solution was stirred at this temperature for 30 min, at which point hexamethylphosphoramide (2.44 mL, 13.9 mmol, 2.2 equiv) was added dropwise. After stirring at –78 °C for a further 2 min, a solution of the AB enone 10 (3.05 g, 6.32 mmol, 1 equiv) in tetrahydrofuran (15 mL) was added to the reaction solution dropwise via syringe. The brownish-yellow reaction mixture was stirred at –78 °C for 40 min whereupon chlorotrimethylsilane (1.20 mL, 9.48 mmol, 1.5 equiv) was added. After stirring at –78 °C for 40 min, aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 100 mL) was added to the reaction solution. The resulting mixture was allowed to warm to 23 °C, then was extracted with dichloromethane (3 x 100 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The product was purified by flash-column chromatography (8% ethyl acetate-hexanes), affording -(1,3dithian-2-yl) trimethylsilyl enol ether 24 as a white foam (3.85 g, 90%). Rf = 0.53 (30% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 7.48 (d, 2H, J = 7.0 Hz), 7.38-7.31 (m, 3H), 5.36 (AB quartet, 2H), 4.98 (d, 1H, J = 3.0 Hz), 4.12 (d, 1H, J = 5.0 Hz), 3.89 (d, 1H, J = 9.5 Hz), 2.96-2.82 (m, 5H), 2.46 (s, 6H), 2.34-2.29 (m, 1H), 81 2.28-2.23 (m, 2H), 2.15-2.09 (m, 1H), 1.90-1.80 (m, 1H), 0.86 (s, 9H), 0.21 (s, 3H), 0.10 (s, 3H), –0.01 (s, 9H); 13C NMR (125 MHz, CDCl3) 189.4, 181.5, 167.3, 149.5, 135.1, 128.7, 128.5, 128.4, 108.5, 104.8, 81.0, 72.3, 61.3, 54.9, 46.1, 41.9, 37.1, 31.1, 30.8, 26.1, 25.7, 21.8, 18.9, –0.4, –2.7, –3.6; FTIR (neat film), cm–1 2953 (w), 1721 (s), 1653 (w), 1614 (w), 1510 (s), 1472 (w), 1454 (w), 1254 (s), 1204 (w), 1150 (w), 1024 (w), 934 (s), 901 (s), 835 (s); HRMS–ESI (m/z): [M+H]+ calcd for C33H51N2O5S2Si2, 675.2772; found, 675.2783. 82 -Carbaldehyde AB enone 25. N-Bromosuccinimide (158 mg, 0.889 mmol, 6.0 equiv) was added in one portion to a stirring solution of -(1,3-dithian-2-yl) trimethylsilyl enol ether 24 (100 mg, 0.148 mmol, 1 equiv) in tert-butanol (4.0 mL) and water (40 µL) at 23 °C. The reaction mixture was stirred at this temperature for 1 h, then was partitioned between dichloromethane (20 mL) and saturated aqueous sodium bicarbonate solution (20 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (20 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude product was purified by flash-column chromatography (12% ethyl acetate-hexanes), affording the -carbaldehyde AB enone 25 as a yellow solid (68 mg, 90%). Rf = 0.18 (15% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 9.82, (s, 1H), 7.50 (d, 2H, J = 6.9 Hz), 7.41-7.35 (m, 3H), 6.66 (d, 1H, J = 2.7 Hz), 5.36 (AB quartet, 2H), 3.58 (d, 1H, J = 11.0 Hz), 3.17 (d, 1H, J = 19.7 Hz), 2.90 (dd, 1H, J = 10.8, 4.8 Hz), 2.77-2.71 (m, 1H), 2.44 (s, 6H), 0.78 (s, 9H), 0.27 (s, 3H), 0.05 (s, 3H); 13C NMR (125 MHz, CDCl3) 194.6, 193.4, 186.5, 181.2, 167.4, 152.5, 137.2, 134.9, 128.6, 128.5, 128.5, 108.3, 83.2, 72.7, 59.4, 47.0, 41.8, 25.9, 21.5, 19.0, –2.5, –4.0; FTIR (neat film), 1721 (m), 1694 (m), 1607 (w), 1510 (m), 1173 (w), 1036 (m), 937 (s), 737 (s) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C27H35N2O6Si, 511.2259; found, 511.2286. 83 -Methoxymethoxymethyl AB enone 26. Sodium triacetoxyborohydride (205 mg, 0.918 mmol, 3.5 equiv) was added in one portion to a solution of the -carbaldehyde AB enone 25 (134 mg, 0.262 mmol, 1 equiv) in benzene (2.0 mL) at 23 °C. The resulting solution was heated to 40 °C. After stirring at 40 °C for 5 ½ h, the reaction mixture was allowed to cool to 23 °C. The cooled solution was diluted with dichloromethane (30 mL), and the resulting solution was added slowly and carefully to saturated aqueous sodium bicarbonate solution (30 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (30 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. N,N-Diisopropylethylamine (229 µL, 1.31 mmol, 5.0 equiv) and chloromethyl methyl ether (59.8 µL, 0.787 mmol, 3.0 equiv) were added sequentially to a solution of the crude reduction product in benzene (1.5 mL) at 23 °C. The reaction flask was sealed and the solution was heated to 50 °C. After stirring at 50 °C for 24 h, the reaction mixture was allowed to cool to 23 °C. The cooled solution was partitioned between dichloromethane (30 mL) and saturated aqueous sodium bicarbonate solution (30 mL). The layers were separated and the aqueous phase was extracted with dichloromethane (30 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording an orange oil. The product was purified by flash-column chromatography (15% ethyl acetate-hexanes, grading to 17% 84 ethyl acetate-hexanes), providing the -methoxymethoxymethyl AB enone 26 as a pale yellow solid (124 mg, 85% yield, two steps). Rf = 0.40 (30% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 7.50 (d, 2H, J = 7.0 Hz), 7.40-7.32 (m, 3H), 6.21 (s, 1H), 5.35 (AB quartet, 2H), 4.67 (AB quartet, 2H), 4.16 (m, 2H), 3.74 (d, 1H, J = 10.0 Hz), 3.38 (s, 3H), 2.80-2.74 (m, 3H), 2.45 (s, 6H), 0.82 (s, 9H), 0.26 (s, 3H), 0.05 (s, 3H); 13 C NMR (125 MHz, CDCl3) 193.1, 187.7, 181.1, 167.5, 159.7, 135.0, 128.5, 128.5, 128.5, 122.4, 108.4, 96.0, 83.0, 72.6, 68.4, 59.6, 55.5, 47.5, 41.9, 25.9, 25.8, 19.0, –2.5, –4.1; FTIR (neat film), cm–1 2951 (w), 2930 (w), 1719 (s), 1674 (m), 1510 (s), 1175 (m), 1152 (m), 1038 (s), 934 (s), 829 (s), 735 (s); HRMS–ESI (m/z): [M+H]+ calcd for C29H41N2O7Si, 557.2678; found, 557.2690. 85 Michael–Claisen cyclization product 29. A freshly prepared solution of lithium diisopropylamide in tetrahydrofuran (1.0 M, 1.21 mL, 1.21 mmol, 3.0 equiv) was added dropwise via syringe to a solution of phenyl ester 28 (449 mg, 1.21 mmol, 3.0 equiv) and TMEDA (365 µL, 2.42 mmol, 6.0 equiv) in tetrahydrofuran (15 mL) at –78 °C, forming a bright red solution. After stirring at –78 °C for 40 min, a solution of the -methylsubstituted AB enone 18 (200 mg, 0.403 mmol, 1 equiv) in tetrahydrofuran (3.0 ml) was added to the reaction solution dropwise via syringe. The resulting mixture was allowed to warm slowly to –10 °C over 80 min, then was partitioned between aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 60 mL) and dichloromethane (60 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (2 × 40 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording an orange-yellow oil. The product was purified by flash-column chromatography (15% ethyl acetate-hexanes, grading to 20%), providing the Michael– Claisen cyclization product 29 as a yellow solid (249 mg, 80%). Rf = 0.28 (20% ethyl acetate-hexanes); 1H NMR (600 MHz, CDCl3) 15.96 (s, 1H), 7.49 (d, 2H, J = 7.8 Hz), 7.39-7.33 (m, 3H), 7.26-7.24 (m, 1H), 7.04 (d, 1H, J = 8.5 Hz), 5.36 (s, 2H), 4.16 (d, 1H, J = 10.0 Hz), 3.20 (d, 1H, J = 16.1 Hz), 2.75 (d, 1H, J = 16.1 Hz), 2.66 (s, 6H), 2.54-2.50 (m, 7H), 2.37 (d, 1H, J = 14.4 Hz), 2.16 (dd, 1H, J = 14.8, 4.5 86 Hz), 1.56 (s, 9H), 1.12 (s, 3H), 0.90 (s, 9H), 0.25 (s, 3H), 0.21 (s, 3H); MHz, CDCl3) 13 C NMR (125 186.8, 185.6, 181.7, 178.3, 167.6, 152.3, 150.4, 145.4, 136.4, 135.1, 128.5, 128.4, 128.3, 124.2, 123.9, 122.3, 112.0, 108.1, 83.8, 81.7, 72.4, 60.7, 47.0, 44.2, 41.9, 40.6, 32.4, 32.1, 29.8, 27.7, 26.4, 19.2, –1.9, –2.3; FTIR (neat film), 1759 (w), 1721 (m), 1613 (w), 1510 (m), 1456 (w), 1265 (m), 1152 (m), 737 (s) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C42H55N3O9Si, 774.3780; found, 774.3796. 87 C5a-Methylminocycline (34). Concentrated aqueous hydrofluoric acid solution (48 wt%, 2.0 mL) was added to a solution of the Michael–Claisen cyclization product 14 (249 mg, 0.322 mmol, 1 equiv) in acetonitrile (3.0 mL) in a polypropylene reaction vessel at 23 °C. The reaction solution was stirred vigorously at 23 °C for 17 h, then was poured into water (100 mL) containing dipotassium hydrogenphosphate trihydrate (20.0 g). The resulting mixture was extracted with ethyl acetate (100 mL, then 2 x 50 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording an orange-brown solid. Methanol (3.0 mL) and dioxane (3.0 mL) were added to the crude product, forming an orange-brown solution. Palladium black (13.7 mg, 0.129 mmol, 0.4 equiv) was added in one portion at 23 °C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The reaction mixture was stirred at 23 °C for 1 h, then was filtered through a plug of Celite. The filtrate was concentrated, affording a brownish yellow solid. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, 2 batches, injection volume: 5.0 mL (4.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 24-31 min were collected and concentrated, affording C5amethylminocycline trifluoroacetate 17 as a yellow solid (188 mg, 100%, two steps). 88 1 H NMR (600 MHz, CD3OD, trifluoroacetate) 7.90 (d, 1H, J = 9.4 Hz), 7.07 (d, 1H, J = 9.4 Hz), 4.13 (d, 1H, J = 1.0 Hz), 3.25 (s, 6H), 3.17 (d, 1H, J = 15.8 Hz), 3.07-3.02 (m, 1H), 3.04 (s, 6H), 2.80 (d, 1H, J = 15.7 Hz), 2.05 (dd, 1H, J = 13.8, 2.9 Hz), 1.93 (dd, 1H, J = 14.1, 13.9 Hz), 1.26 (s, 3H); HRMS–ESI (m/z): [M+H]+ calcd for C24H29N3O7, 472.2078; found, 472.2087. 89 Michael–Claisen cyclization product 33. A freshly prepared solution of lithium diisopropylamide in tetrahydrofuran (1.0 M, 0.416 mL, 0.416 mmol, 3.0 equiv) was added dropwise via syringe to a solution of phenyl ester 28 (155 mg, 0.416 mmol, 3.0 equiv) and TMEDA (126 µL, 0.832 mmol, 6.0 equiv) in tetrahydrofuran (6 mL) at –78 °C, forming a bright red solution. After stirring at –78 °C for 40 min, a solution of the methyl ester-substituted AB enone 20 (75 mg, 0.139 mmol, 1 equiv) in tetrahydrofuran (1.5 ml) was added to the reaction solution dropwise via syringe. The resulting mixture was allowed to warm slowly to –10 °C over 75 min, then was partitioned between aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 25 mL) and dichloromethane (25 mL). The phases were separated and the aqueous phase was further extracted with dichloromethane (2 × 20 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording a yellow solid. The crude product was purified first by flash-column chromatography (15% acetone-hexanes), then by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: water, Solvent B: methanol, injection volume: 6.0 mL (5.0 mL methanol, 1.0 mL water), gradient elution with 85 100% B over 40 min, flow rate: 15 mL/min]. Fractions eluting at 25-28 min were collected and concentrated, providing the Michael–Claisen cyclization product 33 as a yellow solid (26 mg, 23%). 90 Rf = 0.34 (25% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 15.97 (s, 1H), 7.49 (d, 2H, J = 7.3 Hz), 7.39-7.33 (m, 3H), 7.24 (d, 1H, J = 8.3 Hz), 7.03 (d, 1H, J = 8.3 Hz), 5.37 (s, 2H), 4.11 (d, 1H, J = 9.3 Hz), 3.84 (d, 1H, J = 16.1 Hz), 3.36 (s, 3H), 2.80 (d, 1H, J = 16.1 Hz), 2.67-2.64 (m, 1H), 2.64 (s, 6H), 2.57-2.53 (m, 2H), 2.51 (s, 6H), 1.57 (s, 9H), 0.82 (s, 9H), 0.23 (s, 3H), 0.17 (s, 3H); 13C NMR (125 MHz, CDCl3) 187.6, 186.2, 181.5, 176.3, 175.2, 167.6, 152.4, 150.0, 145.4, 135.0, 134.2, 128.5, 128.5, 128.3, 124.6, 124.4, 123.1, 108.0, 106.8, 83.8, 80.9, 72.5, 60.6, 52.4, 46.6, 44.2, 44.1, 41.9, 38.2, 27.7, 26.2, 19.1, –2.1, –2.9; FTIR (neat film), 1761 (w), 1722 (m), 1512 (m), 1234 (s), 1150 (s), 833 (m), 733 (s) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C43H56N3O11Si, 818.3679; found, 818.3760. 91 C5a-Carbonyloxymethylminocycline (35). Concentrated aqueous hydrofluoric acid solution (48 wt%, 0.8 mL) was added to a solution of the HPLC-purified product 33 from the cyclization step above (25.0 mg, 0.031 mmol, 1 equiv) in acetonitrile (1.2 mL) in a polypropylene reaction vessel at 23 °C. The reaction solution was stirred vigorously at 23 °C for 17 h, then was poured into water (30 mL) containing dipotassium hydrogenphosphate trihydrate (10.0 g). The resulting mixture was extracted with ethyl acetate (30 mL, then 2 x 20 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording an orange solid. Palladium black (4.9 mg, 0.046 mmol, 1.5 equiv) was added in one portion to a solution of the crude product in methanol (1.5 mL) and dioxane (1.5 mL) at 23 °C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The reaction mixture was stirred at 23 °C for 30 min, then was filtered through a plug of Celite. The filtrate was concentrated, affording a yellow solid. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, injection volume: 5.0 mL (4.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 27-32 min were collected and concentrated, affording C5a-carbomethoxyminocycline trifluoroacetate 35 as a yellow solid (17.1 mg, 89%). 92 1 H NMR (600 MHz, CD3OD) 7.81 (d, 1H, J = 9.2 Hz), 7.03 (d, 1H, J = 9.2 Hz), 4.14 (s, 1H), 3.66 (d, 1H, J = 15.7 Hz), 3.61 (s, 3H), 3.14 (s, 6H), 2.97 (s, 6H), 2.87-2.83 (m, 2H), 2.53 (dd, 1H, J = 14.3, 2.6 Hz), 2.04 (dd, 1H, J = 14.2, 14.1 Hz); HRMS–ESI (m/z): [M+H]+ calcd for C26H30N3O9, 516.1977; found, 516.2011. 93 Michael–Claisen cyclization product 32. A freshly prepared solution of lithium diisopropylamide (1.0 M, 7.86 mL, 7.86 mmol, 3.6 equiv) was added dropwise via syringe to a solution of phenyl ester 31 (2.84 g, 7.86 mmol, 3.6 equiv) and TMEDA (2.27 mL, 15.1 mmol, 7.0 equiv) in tetrahydrofuran (60 mL) at –78 °C, forming a bright red solution. After stirring at –78 °C for 40 min, a solution of the -methoxymethoxymethyl AB enone 26 (1.20 g, 2.16 mmol, 1 equiv) in tetrahydrofuran (15 mL) was added to the reaction solution dropwise via syringe. The resulting mixture was allowed to warm slowly to –10 °C over 80 min, then was partitioned between aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 100 mL) and dichloromethane (100 mL). The phases were separated and the aqueous phase was further extracted with dichloromethane (2 x 75 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording an orange-yellow oil. The product was purified by flash-column chromatography (3.5% ethyl acetate-dichloromethane), providing the Michael–Claisen cyclization product 32 as a yellow solid (1.29 g, 72%). Rf = 0.31 (30% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 16.77 (s, 1H), 7.51 (d, 4H, J = 8.0 Hz), 7.41-7.28 (m, 6H), 7.21 (d, 1H, J = 9.0 Hz), 6.90 (d, 1H, J = 9.0 Hz), 5.38 (s, 2H), 5.17 (AB quartet, 2H), 4.47 (d, 1H, J = 6.5 Hz), 4.34 (d, 1H, J = 6.5 Hz), 4.15 (d, 1H, J = 9.5 Hz), 3.78 (d, 1H, J = 16.5 Hz), 3.38 (d, 1H, J = 9.0 Hz), 3.27 (d, 1H, 94 J = 9.5 Hz), 3.12 (s, 3H), 2.63 (s, 6H), 2.65-2.58 (m, 1H), 2.51 (s, 6H), 2.51-2.41 (m, 2H), 2.32 (dd, 1H, J = 14.5, 2.0 Hz), 0.93 (s, 9H), 0.29 (s, 3H), 0.20 (s, 3H); 13 C NMR (125 MHz, CDCl3) 186.9, 184.6, 183.0, 181.6, 167.7, 154.7, 145.7, 136.8, 136.1, 135.1, 128.5, 128.4, 128.4, 128.3, 127.7, 127.0, 125.0, 120.7, 113.7, 108.1, 107.2, 96.1, 82.3, 72.9, 72.4, 71.4, 61.1, 54.6, 46.4, 44.4, 41.9, 35.8, 34.7, 28.4, 26.5, 19.3, –2.0, –2.1; FTIR (neat film), 2932 (w), 1721 (s), 1611 (w), 1510 (m), 1472 (m), 1452 (m), 1269 (w), 1148 (w), 1107 (w), 1040 (s), 1020 (s), 922 (w), 831 (s), 733 (s) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C46H58N3O9Si, 824.3937; found, 824.3885. 95 Substituted neopentyl alcohol 36. Perchloric acid76 (13.0 mL, 70% solution) was added dropwise over 5 min to a solution of the Michael–Claisen cyclization product 32 (1.04 g, 1.26 mmol, 1 equiv) in tetrahydrofuran (130 mL) at 23 °C. After stirring at this temperature for 10 min, the reaction solution was slowly and carefully poured into icecold saturated aqueous sodium bicarbonate solution (300 mL). The resulting mixture was extracted with dichloromethane (2 x 250 mL, then 50 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing an orange-yellow oil. The product was purified by flash-column chromatography (55% ethyl acetate-hexanes, grading to 75% ethyl acetate-hexanes), affording the substituted neopentyl alcohol 36 as a yellow solid (720 mg, 73%). Rf = 0.26 (65% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 16.76 (s, 1H), 7.53-7.49 (m, 4H), 7.41-7.28 (m, 6H), 7.22 (d, 1H, J = 9.0 Hz), 6.90 (d, 1H, J = 9.0 Hz), 5.38 (s, 2H), 5.17 (AB quartet, 2H), 4.11 (d, 1H, J = 9.5 Hz), 3.66 (d, 1H, J = 16.0 Hz), 3.48 (d, 1H, J = 11.0 Hz), 3.32 (d, 1H, J = 11.0 Hz), 2.64 (s, 6H), 2.68-2.59 (m, 1H), 2.56-2.48 (m, 1H), 2.51 (s, 6H), 2.38 (dd, 1H, J = 14.5, 4.5 Hz), 2.23 (brd, 1H, J = 14.0 Hz), 0.92 (s, 9H), 0.25 (s, 3H), 0.18 (s, 3H); 13C NMR (125 MHz, CDCl3) 186.7, 184.7, 182.7, 181.4, 167.7, 154.9, 145.7, 136.8, 135.9, 135.1, 128.5, 128.5, 128.5, 128.3, 127.8, 126.9, 125.3, 120.7, 113.7, 108.2, 107.3, 82.3, 72.4, 71.4, 68.2, 61.3, 46.2, 44.7, 42.0, 36.8, 34.5, 28.2, 26.5, 19.3, –1.8, –2.0; FTIR (neat film), 2938 (w), 1719 (m), 1609 (w), 96 1510 (s), 1452 (s), 1265 (m), 1020 (m), 829 (s), 733 (s) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C44H54N3O8Si, 780.3675; found,780.3654. 97 C5a–C11a-Bridged cyclopropane tetracycline precursor 37. 4Å molecular sieves (2.4 g, small chunks) were added to a solution of the substituted neopentyl alcohol 36 (720 mg, 0.923 mmol, 1 equiv) in dichloromethane (72 mL) and pyridine (7.2 mL) at 23 °C. The resulting mixture was stirred at 23 °C for 1 h, then was cooled to 0 °C. A solution of phosgene in toluene (20 wt%, 537 µL, 1.02 mmol, 1.1 equiv) was added dropwise to the cooled mixture. The resulting solution was stirred at 0 °C for 1 h, whereupon aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 20 mL) was added. The resulting mixture was allowed to warm to 23 °C, then was filtered to remove the molecular sieves. Dichloromethane (60 mL) and aqueous potassium phosphate buffer solution (pH 7.0, 0.2M, 60 mL) were added and the phases were separated. The aqueous phase was further extracted with dichloromethane (2 x 60 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, providing an orange-yellow oil. The product was purified by flash-column chromatography (20% ethyl acetate-hexanes, grading to 30% ethyl acetate-hexanes), affording the C5a–C11a-bridged cyclopropane tetracycline precursor 37 as a white solid (572 mg, 81%). Rf = 0.25 (30% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 7.52 (d, 2H, J = 7.3 Hz), 7.44-7.24 (m, 8H), 7.13 (d, 1H, J = 9.0 Hz), 6.86 (d, 1H, J = 9.0 Hz), 5.35 (s, 2H), 5.05 (AB quartet, 2H), 4.01 (d, 1H, J = 10.5 Hz), 3.85 (d, 1H, J = 17.4 Hz), 2.77 (d, 1H, J = 17.4 Hz), 2.68-2.57 (m, 3H), 2.62 (s, 6H), 2.49 (s, 6H), 2.25 (d, 1H, J = 5.0 Hz), 98 1.71 (d, 1H, J = 5.0 Hz), 0.89 (s. 9H), 0.28, (s, 3H), 0.12 (s, 3H); 13C NMR (125 MHz, CDCl3) 194.4, 191.8, 185.3, 180.9, 167.6, 152.6, 144.8, 136.7, 135.0, 132.2, 128.6, 128.5, 128.5, 128.4, 127.6, 127.1, 123.3, 123.2, 113.5, 107.9, 84.0, 72.6, 71.2, 58.8, 49.0, 44.8, 43.1, 41.8, 32.1, 31.1, 30.9, 26.6, 26.3, 19.5, –2.0, –2.6; FTIR (neat film), 2938 (w), 1728 (s), 1711 (m), 1670 (w), 1510 (m), 1474 (m), 1452 (m), 1362 (w), 1258 (m), 916 (m), 827 (s), 733 (s) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C44H52N3O7Si, 762.3569; found,762.3569. 99 C5a-Pyrrolidinomethylminocycline (39). Anhydrous magnesium bromide (8.2 mg, 0.045 mmol, 2.0 equiv) was added to a solution of the C5a-C11a-bridged cyclopropane 37 (17.0 mg, 0.022 mmol, 1 equiv) and pyrrolidine (18 µL, 0.223 mmol, 10 equiv) in tetrahydrofuran (0.5 mL) at 23 °C. The reaction mixture was stirred at 23 °C for 16 h, then was partitioned between dichloromethane and saturated aqueous sodium bicarbonate solution (10 mL each). The phases were separated and the aqueous phase was extracted with dichloromethane (10 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude ring-opened product (38) was dissolved in acetonitrile (1.2 mL). The resulting solution was transferred to a polypropylene reaction vessel and concentrated aqueous hydrofluoric acid solution (48 wt%, 0.8 mL) was added. The reaction mixture was stirred vigorously at 23 °C for 20 h, then was poured into water (30 mL) containing dipotassium hydrogenphosphate (8.0 g). The resulting mixture was extracted with ethyl acetate (3 x 40 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. Palladium black (5.0 mg, 0.047 mmol, 2.8 equiv) was added in one portion to a solution of the crude product in methanol (1.0 mL) and dioxane (1.0 mL) at 23 °C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The reaction mixture was stirred at 23 °C for 1 ¼ h, then was filtered through a plug of Celite. The filtrate was 100 concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, injection volume: 5.0 mL (4.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 35% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 39-43 min were collected and concentrated, affording C5a-pyrrolidinomethylminocycline bistrifluoroacetate 39 as a yellow solid (12.5 mg, 74%, three steps). 1 H NMR (600 MHz, CD3OD, bistrifluoroacetate) 7.55 (d, 1H, J = 9.0 Hz), 6.95 (d, 1H, J = 9.0 Hz), 4.05 (s, 1H), 3.84 (brs, 1H), 3.75-3.71 (brs, 1H), 3.73 (d, 1H, J = 14.4 Hz), 3.62 (d, 1H, J = 16.8 Hz), 3.20 (d, 1H, J = 13.2 Hz), 3.14 (d, 1H, J = 15.0 Hz), 3.13-3.03 (brs, 1H), 3.10 (s, 6H), 2.70 (s, 6H), 2.67 (d, 1H, J = 16.8 Hz), 2.59 (dd, 1H, J = 15.0, 3.0 Hz), 2.50 (brs, 1H), 2.02-1.90 (m, 5H); HRMS–ESI (m/z): [M+H]+ calcd for C28H37N4O7, 541.2657; found, 541.2684. 101 O N(CH3)2 N(CH3)2 O N BnO O O 37 O OTBS OBn 2. HF (aq), CH3CN 3. H2, Pd black CH3OH–dioxane O OH O HO H O 41 O 1. Morpholine, MgBr2 THF, 55 °C N(CH3)2 N H N(CH3)2 OH NH2 H C5a-Morpholinomethylminocycline (41). Anhydrous magnesium bromide (6.3 mg, 0.034 mmol, 2.0 equiv) was added to a solution of the C5a-C11a-bridged cyclopropane 37 (13.0 mg, 0.017 mmol, 1 equiv) and morpholine (15 µL, 0.17 mmol, 10 equiv) in tetrahydrofuran (0.5 mL) at 23 °C. The reaction flask was sealed and the reaction mixture was heated to 55 °C. After stirring at 55 °C for 14 h, the reaction mixture was allowed to cool to 23 °C. The cooled mixture was partitioned between dichloromethane and saturated aqueous sodium bicarbonate solution (10 mL each). The phases were separated and the aqueous phase was further extracted with dichloromethane (10 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude ringopened product was dissolved in acetonitrile (1.2 mL). The resulting solution was transferred to a polypropylene reaction vessel and concentrated aqueous hydrofluoric acid solution (48 wt%, 0.8 mL) was added. The reaction mixture was stirred vigorously at 23 °C for 16 ½ h, then was poured into water (30 mL) containing dipotassium hydrogenphosphate (8.0 g). The resulting mixture was extracted with ethyl acetate (3 x 40 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. Palladium black (5.0 mg, 0.047 mmol, 2.8 equiv) was added in one portion to a solution of the crude product in methanol (1.0 mL) and dioxane (1.0 mL) at 23 °C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing 102 with pure hydrogen (1 atm). The reaction mixture was stirred at 23 °C for 1 ¾ h, then was filtered through a plug of Celite. The filtrate was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, injection volume: 5.0 mL (4.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 25% B over 50 min, then 25 100% B over 20 min, flow rate: 7.5 mL/min]. Fractions eluting at 49-52 min were collected and concentrated, affording C5a-morpholinomethylminocycline bistrifluoroacetate 41 as an orange-yellow solid (7.5 mg, 56%, three steps). 1 H NMR (600 MHz, CD3OD, bistrifluoroacetate) 7.56 (d, 1H, J = 9.0 Hz), 6.95 (d, 1H, J = 9.0 Hz), 4.04 (s, 1H), 3.77-3.69 (m, 4H), 3.53 (d, 1H, J = 16.8 Hz), 3.24 (d, 1H, J = 13.2 Hz), 3.18-3.14 (m, 1H), 3.08 (s, 6H), 2.99-2.94 (m, 2H), 2.88 (d, 1H, J = 15.0 Hz), 2.81-2.72 (m, 2H), 2.74 (s, 6H), 2.62 (d, 1H, J = 16.8 Hz), 2.53 (brd, 1H, J = 14.4 Hz), 1.92 (dd, 1H, J = 14.2, 14.0 Hz); HRMS–ESI (m/z): [M+H]+ calcd for C28H37N4O8, 557.2606; found, 557.2611. 103 C5a-Piperidinylmethylminocycline (40). Anhydrous magnesium bromide (8.2 mg, 0.045 mmol, 2.0 equiv) was added to a solution of the C5a–C11a-bridged cyclopropane 37 (17.0 mg, 0.022 mmol, 1 equiv) and piperidine (22 µL, 0.223 mmol, 10 equiv) in tetrahydrofuran (0.5 mL) at 23 °C. The reaction mixture was stirred at 23 °C for 21 h, then was heated to 45 °C. After stirring at this temperature for 14 h, the reaction mixture was allowed to cool to 23 °C. The cooled mixture was partitioned between dichloromethane (15 mL) and saturated aqueous sodium bicarbonate solution (10 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (10 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude ring-opened product was dissolved in acetonitrile (1.2 mL). The resulting solution was transferred to a polypropylene reaction vessel and concentrated aqueous hydrofluoric acid solution (48 wt%, 0.8 mL) was added. The reaction mixture was stirred vigorously at 23 °C for 13 h, then was poured into water (30 mL) containing dipotassium hydrogenphosphate (8.0 g). The resulting mixture was extracted with ethyl acetate (3 x 40 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. Palladium black (5.0 mg, 0.047 mmol, 2.8 equiv) was added in one portion to a solution of the crude product in methanol (1.0 mL) and dioxane (1.0 mL) at 23 °C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing 104 with pure hydrogen (1 atm). The reaction mixture was stirred at 23 °C for 1 ½ h, then was filtered through a plug of Celite. The filtrate was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, injection volume: 5.0 mL (4.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 35% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 40-44 min were collected and concentrated, affording C5apiperidinylmethylminocycline bistrifluoroacetate 40 as a yellow solid (12.0 mg, 70%, three steps). 1 H NMR (600 MHz, CD3OD, bistrifluoroacetate) 7.56 (d, 1H, J = 9.0 Hz), 6.96 (d, 1H, J = 9.0 Hz), 4.10 (d, 1H, J = 2.4 Hz), 3.66 (d, 1H, J = 16.8 Hz), 3.46 (d, 1H, J = 14.4 Hz), 3.40-3.35 (brm, 1H), 3.27-3.23 (m, 1H), 3.23-3.18 (m, 1H), 3.10 (s, 6H), 3.10-3.00 (m, 2H), 2.71 (s, 6H), 2.71-2.62 (m, 2H), 2.59 (dd, 1H, J = 15.0, 3.0 Hz), 2.02 (dd, 1H, J = 15.1, 12.7 Hz), 1.95-1.88 (brm, 1H), 1.85-1.78 (brm, 2H), 1.72-1.61 (brm, 2H), 1.43-1.36 (brm, 1H); HRMS–ESI (m/z): [M+H]+ calcd for C29H39N4O7, 555.2813; found, 555.2788. 105 C5a-Diethylaminomethylminocycline (42). Anhydrous magnesium bromide (7.2 mg, 0.039 mmol, 2.0 equiv) was added to a solution of the C5a-C11a-bridged cyclopropane 37 (15.0 mg, 0.020 mmol, 1 equiv) and diethylamine (102 µL, 0.987 mmol, 50 equiv) in tetrahydrofuran (0.5 mL) at 23 °C. The reaction flask was sealed and the reaction mixture was heated to 45 °C. After stirring at this temperature for 20 h, the reaction mixture was allowed to cool to room temperature. The reaction flask was opened briefly and a second portion of diethylamine (204 µL, 1.97 mmol, 100 equiv) was added. The flask was resealed and the reaction mixture was heated to 45 °C. After stirring at this temperature for a further 55 h, the reaction mixture was allowed to cool to 23 °C. The cooled mixture was partitioned between dichloromethane and saturated aqueous sodium bicarbonate solution (10 mL each). The phases were separated and the aqueous phase was extracted with dichloromethane (10 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude ring-opened product was dissolved in acetonitrile (1.2 mL). The resulting solution was transferred to a polypropylene reaction vessel and concentrated aqueous hydrofluoric acid solution (48 wt%, 0.8 mL) was added. The reaction mixture was stirred vigorously at 23 °C for 10 ½ h, then was poured into water (30 mL) containing dipotassium hydrogenphosphate (8.0 g). The resulting mixture was extracted with ethyl acetate (3 x 40 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was 106 filtered and the filtrate was concentrated. Palladium black (5.0 mg, 0.047 mmol, 2.8 equiv) was added in one portion to a solution of the crude product in methanol (1.0 mL) and dioxane (1.0 mL) at 23 °C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The reaction mixture was stirred at 23 °C for 1 ¾ h, then was filtered through a plug of Celite. The filtrate was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, injection volume: 5.0 mL (4.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 36-39 min were collected and concentrated, affording C5a-diethylaminomethylminocycline bistrifluoroacetate 42 as a yellow solid (12.0 mg, 79%, three steps). 1 H NMR (600 MHz, CD3OD, bistrifluoroacetate) 7.54 (d, 1H, J = 9.0 Hz), 6.95 (d, 1H, J = 9.0 Hz), 4.10 (d, 1H, J = 3.0 Hz), 3.61 (d, 1H, J = 16.8 Hz), 3.42 (d, 1H, J = 15.0 Hz), 3.25-3.20 (m, 2H), 3.16-3.02 (brm, 3H), 3.10 (s, 6H), 2.93 (brs, 1H), 2.73-2.69 (m, 1H), 2.70 (s, 6H), 2.52 (dd, 1H, J = 15.0, 3.0 Hz), 2.07 (dd, 1H, J = 14.1, 13.3 Hz), 1.28 (brs, 3H), 1.04 (brs, 3H); HRMS–ESI (m/z): [M+H]+ calcd for C28H39N4O7, 543.2813; found, 543.2821. 107 C5a-N-Imidazolylmethylminocycline (43). Anhydrous magnesium bromide (7.2 mg, 0.039 mmol, 3.0 equiv) was added to a solution of the C5a-C11a-bridged cyclopropane 37 (10.0 mg, 0.013 mmol, 1 equiv) and imidazole (6.2 mg, 0.091 mmol, 7.0 equiv) in tetrahydrofuran (0.5 mL) at 23 °C. The reaction flask was sealed and the reaction mixture was heated to 60 °C. After stirring at this temperature for 60 h, the reaction mixture was allowed to cool to 23 °C. The cooled mixture was partitioned between dichloromethane and saturated aqueous sodium bicarbonate solution (15 mL each). The phases were separated and the aqueous phase was extracted with dichloromethane (15 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude ring-opened product was dissolved in acetonitrile (1.2 mL). The resulting solution was transferred to a polypropylene reaction vessel and concentrated aqueous hydrofluoric acid solution (48 wt%, 0.8 mL) was added. The reaction mixture was stirred vigorously at 23 °C for 18 h, then was poured into water (30 mL) containing dipotassium hydrogenphosphate (8.0 g). The resulting mixture was extracted with ethyl acetate (3 x 40 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. Palladium black (5.0 mg, 0.047 mmol, 3.6 equiv) was added in one portion to a solution of the crude product in methanol (1.0 mL) and dioxane (1.0 mL) at 23 °C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing 108 with pure hydrogen (1 atm). The reaction mixture was stirred at 23 °C for 1 ¼ h, then was filtered through a plug of Celite. The filtrate was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, injection volume: 5.0 mL (4.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 25-27 min were collected and concentrated, affording C5aimidazolylmethylminocycline bistrifluoroacetate 43 as a yellow solid (7.3 mg, 73%, three steps). 1 H NMR (600 MHz, CD3OD, bistrifluoroacetate) 8.42 (t, 1H, J = 1.3 Hz), 7.51 (d, 1H, J = 9.0 Hz), 7.40 (dd, 1H, J = 1.8, 1.5 Hz), 7.23 (dd, 1H, J = 1.8, 1.6 Hz), 6.86 (d, 1H, J = 9.0 Hz), 4.48 (AB quartet, 2H), 4.13 (s, 1H), 3.42 (d, 1H, J = 16.8 Hz), 3.25 (dd, 1H, J = 13.8, 1.2 Hz), 3.11 (s, 6H), 2.73 (s, 6H), 2.69 (d, 1H, J = 16.2 Hz), 2.14 (dd, 1H, J = 15.0, 3.0 Hz), 1.98 (dd, 1H, J = 14.5, 14.2 Hz); HRMS–ESI (m/z): [M+H]+ calcd for C27H32N5O7, 538.2296; found, 538.2285. 109 C5a-Cyclopropylaminomethylminocycline (44). Anhydrous magnesium bromide (8.2 mg, 0.045 mmol, 2.0 equiv) was added to a solution of the C5a-C11a-bridged cyclopropane 37 (17.0 mg, 0.022 mmol, 1 equiv) and cyclopropylamine (15 µL, 0.223 mmol, 10 equiv) in tetrahydrofuran (0.5 mL) at 23 °C. The reaction mixture was stirred at 23 °C for 16 h, then was heated to 40 °C. After stirring at this temperature for 22 h, the reaction mixture was allowed to cool to room temperature. The reaction flask was opened briefly and a second portion of cyclopropylamine (15 µL, 0.223 mmol, 10 equiv) was added. The flask was sealed and the reaction mixture was heated to 40 °C. After stirring at this temperature for a further 13 h, the reaction mixture was allowed to cool to 23 °C. The cooled mixture was partitioned between dichloromethane (15 mL) and saturated aqueous sodium bicarbonate solution (10 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (10 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude ring-opened product was dissolved in acetonitrile (1.2 mL). The resulting solution was transferred to a polypropylene reaction vessel and concentrated aqueous hydrofluoric acid solution (48 wt%, 0.8 mL) was added. The reaction mixture was stirred vigorously at 23 °C for 12 h, then was poured into water (30 mL) containing dipotassium hydrogenphosphate (8.0 g). The resulting mixture was extracted with ethyl acetate (3 x 40 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The 110 dried solution was filtered and the filtrate was concentrated. Palladium black (5.0 mg, 0.047 mmol, 2.8 equiv) was added in one portion to a solution of the crude product in methanol (1.0 mL) and dioxane (1.0 mL) at 23 °C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The reaction mixture was stirred at 23 °C for 3 ½ h, then was filtered through a plug of Celite. The filtrate was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, injection volume: 5.0 mL (4.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 35% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 35-36 min were collected and concentrated, affording C5a-cyclopropylaminomethylminocycline bistrifluoroacetate 44 as a yellow solid (3.0 mg, 18%, three steps). 1 H NMR (600 MHz, CD3OD, bistrifluoroacetate) 7.58 (d, 1H, J = 9.0 Hz), 6.96 (d, 1H, J = 9.0 Hz), 4.00 (d, 1H, J = 2.4 Hz), 3.60 (d, 1H, J = 14.4 Hz), 3.55 (d, 1H, J = 16.8 Hz), 3.19-3.10 (m, 1H), 3.13 (s, 6H), 3.03 (d, 1H, J = 14.4 Hz), 2.76 (s, 6H), 2.76-2.70 (m, 1H), 2.62-2.58 (m, 1H), 2.25 (dd, 1H, J = 14.8, 2.8 Hz), 1.98 (dd, 1H, J = 14.2, 13.8 Hz), 0.89-0.82 (m, 1H), 0.77-0.69 (m, 3H); HRMS–ESI (m/z): [M+H]+ calcd for C27H35N4O7, 527.2500; found, 527.2502. 111 C5a-Methoxymethylminocycline (45). Anhydrous magnesium bromide (9.1 mg, 0.049 mmol, 2.5 equiv) was added to a solution of the C5a-C11a-bridged cyclopropane 37 (15.0 mg, 0.020 mmol, 1 equiv) in methanol (1.5 mL) at 23 °C. The reaction flask was sealed and the reaction mixture was heated to 65 °C. After stirring at 65 °C for 24 h, the reaction mixture was allowed to cool to 23 °C. The cooled mixture was partitioned between aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 10 mL) and dichloromethane (10 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (10 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude ring-opened product was dissolved in acetonitrile (1.2 mL). The resulting solution was transferred to a polypropylene reaction vessel and concentrated aqueous hydrofluoric acid solution (48 wt%, 0.8 mL) was added. The reaction mixture was stirred vigorously at 23 °C for 15 h, then was poured into water (30 mL) containing dipotassium hydrogenphosphate trihydrate (10.0 g). The resulting mixture was extracted with ethyl acetate (3 x 30 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. Palladium black (6.0 mg, 0.057 mmol, 2.8 equiv) was added in one portion to a solution of the crude product in methanol (1.0 mL) and dioxane (1.0 mL) at 23 °C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The reaction 112 mixture was stirred at 23 °C for 2 ½ h, then was filtered through a plug of Celite. The filtrate was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, injection volume: 7.0 mL (6.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 29-33 min were collected and concentrated, affording C5a-methoxymethylminocycline trifluoroacetate 45 as a yellow solid (7.1 mg, 58%, three steps). 1 H NMR (600 MHz, CD3OD, hydrochloride) 7.82 (d, 1H, J = 9.4 Hz), 7.05 (d, 1H, J = 9.2 Hz), 4.15 (s, 1H), 3.49 (d, 1H, J = 10.3 Hz), 3.36 (d, 1H, J = 15.6 Hz), 3.25 (s, 3H), 3.14 (s, 6H), 3.12 (d, 1H, J = 10.3 Hz), 3.08-3.04 (m, 1H), 2.99 (s, 6H), 2.56 (d, 1H, J = 15.6 Hz), 2.41 (dd, 1H, J = 14.1, 2.9 Hz), 1.69 (dd, 1H, J = 14.1, 13.9 Hz); HRMS–ESI (m/z): [M+H]+ calcd for C25H32N3O8, 502.2184; found, 502.2186. 113 C5a-n-Butoxymethylminocycline (46). Anhydrous magnesium bromide (7.2 mg, 0.039 mmol, 2.0 equiv) was added to a solution of the C5a-C11a-bridged cyclopropane 37 (15.0 mg, 0.020 mmol, 1 equiv) in n-butanol (1.0 mL) at 23 °C. The resulting mixture was heated to 75 °C. After stirring at 75 °C for 14 h, the reaction mixture was allowed to cool to 23 °C. The cooled mixture was partitioned between aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 10 mL) and dichloromethane (10 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (10 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude ring-opened product was dissolved in acetonitrile (1.2 mL). The resulting solution was transferred to a polypropylene reaction vessel and concentrated aqueous hydrofluoric acid solution (48 wt%, 0.8 mL) was added. The reaction mixture was stirred vigorously at 23 °C for 13 ½ h, then was poured into water (30 mL) containing dipotassium hydrogenphosphate (8.0 g). The resulting mixture was extracted with ethyl acetate (3 x 40 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. Palladium black (5.0 mg, 0.047 mmol, 2.4 equiv) was added in one portion to a solution of the crude product in methanol (1.0 mL) and dioxane (1.0 mL) at 23 °C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The reaction mixture was stirred at 23 °C for 3 ¾ h, then was 114 filtered through a plug of Celite. The filtrate was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, injection volume: 5.0 mL (4.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 41-44 min were collected and concentrated, affording C5a-nbutoxymethylminocycline trifluoroacetate 46 as a yellow solid (7.3 mg, 56%, three steps). 1 H NMR (600 MHz, CD3OD, trifluoroacetate) 7.81 (d, 1H, J = 9.0 Hz), 7.04 (d, 1H, J = 9.6 Hz), 4.16 (s, 1H), 3.50 (d, 1H, J = 10.2 Hz), 3.38-3.30 (m, 3H), 3.21 (d, 1H, J = 9.6 Hz), 3.13 (s, 6H), 3.11-3.06 (m, 1H), 2.98 (s, 6H), 2.58 (d, 1H, J = 15.6 Hz), 2.42 (dd, 1H, J = 13.8, 3.0 Hz), 1.71 (dd, 1H, J = 13.9, 13.8 Hz), 1.50-1.39 (m, 2H), 1.34-1.24 (m, 2H), 0.87 (t, 3H, J = 7.2 Hz); HRMS–ESI (m/z): [M+H]+ calcd for C28H38N3O8, 544.2653; found, 544.2655. 115 C5a-Methoxyethoxymethylminocycline (47). Anhydrous magnesium bromide (6.2 mg, 0.034 mmol, 2.0 equiv) was added to a solution of the C5a-C11a-bridged cyclopropane 37 (13.0 mg, 0.017 mmol, 1 equiv) in 2-methoxyethanol (0.5 mL) at 23 °C. The resulting mixture was heated to 60 °C. After stirring at 60 °C for 26 h, the reaction mixture was allowed to cool to 23 °C. The cooled mixture was partitioned between aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 10 mL) and dichloromethane (10 mL). The phases were separated and the aqueous phase was further extracted with dichloromethane (10 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude ring-opened product was dissolved in acetonitrile (1.2 mL). The resulting solution was transferred to a polypropylene reaction vessel and concentrated aqueous hydrofluoric acid solution (48 wt%, 0.8 mL) was added. The reaction mixture was stirred vigorously at 23 °C for 10 ½ h, then was poured into water (30 mL) containing dipotassium hydrogenphosphate (8.0 g). The resulting mixture was extracted with ethyl acetate (3 x 40 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. Palladium black (5.0 mg, 0.047 mmol, 2.8 equiv) was added in one portion to a solution of the crude product in methanol (1.0 mL) and dioxane (1.0 mL) at 23 °C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The reaction mixture was stirred at 23 °C for 1 ¾ h, 116 then was filtered through a plug of Celite. The filtrate was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, injection volume: 5.0 mL (4.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 32-34 min were collected and concentrated, affording C5amethoxyethoxymethylminocycline trifluoroacetate 47 as a yellow solid (5.8 mg, 52%, three steps). 1 H NMR (600 MHz, CD3OD, trifluoroacetate) 7.83 (d, 1h, J = 9.0 Hz), 7.05 (d, 1H, J = 9.0 Hz), 4.13 (s, 1H), 3.54-3.45 (m, 4H), 3.44-3.40 (m, 1H), 3.34 (d, 1H, J = 15.6 Hz), 3.27-3.25 (m, 1H), 3.26 (s, 3H), 3.18 (s, 6H), 3.11 (brd, 1H, J = 14.4. Hz), 2.99 (s, 6H), 2.61 (d, 1H, J = 16.2 Hz), 2.45 (dd, 1H, J = 14.4, 3.0 Hz), 1.71 (t, 1H, J = 14.4 Hz); HRMS–ESI (m/z): [M+H]+ calcd for C27H36N3O9, 546.2446; found, 546.2459. 117 Azido ring-opened product 48. Sodium azide (50.0 mg, 0.763 mmol, 3.0 equiv) was added to a solution of the C5a-C11a-bridged cyclopropane 37 (194 mg, 0.255 mmol, 1 equiv) in dimethylformamide (7.5 mL) at 23 °C. The resulting solution was stirred at this temperature for 14 h, then was partitioned between saturated aqueous sodium chloride solution and diethyl ether (60 mL each). The phases were separated and the aqueous phase was further extracted with diethyl ether (60 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The product was purified by flashcolumn chromatography (12% ethyl acetate-hexanes), providing the azido-substituted ring-opened product 48 as a yellow solid (160 mg, 78%). = 0.41 (30% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 16.72 (s, 1H), 7.51 (brd, 4H, J = 8.0 Hz), 7.41-7.26 (m, 7H), 6.95 (d, 1H, 8.5 Hz), 5.38 (s, 2H), 5.18 (AB quartet, 2H), 4.11 (d, 1H, J = 10.5 Hz), 3.72 (d, 1H, J = 16.5 Hz), 3.30 (d, 1H, J = 11.5 Hz), 3.13 (d, 1H, J = 11.5 Hz), 2.65 (s, 6H), 2.65-2.45 (m, 2H), 2.51 (s, 6H), 2.35-2.25 (m, 2H), 0.92 (s, 9H), 0.27 (s, 3H), 0.19 (s, 3H); 13C NMR (125 MHz, CDCl3) 186.5, 184.0, 183.6, 181.5, 167.7, 154.9, 145.9, 136.7, 135.3, 135.0, 128.5, 128.5, 128.5, 128.4, 127.9, 127.0, 125.8, 120.2, 114.0, 108.3, 107.3, 82.3, 72.5, 71.5, 61.1, 59.1, 46.6, 44.6, 41.9, 36.4, 35.2, 28.3, 26.5, 19.3, -1.9, -2.1; FTIR (neat film), 2099 (s), 1721 (s), 1609 (m), 1510 (s), 1258 (m), 829 (s) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C44H53N6O7Si, 805.3740; found, 805.3722. 118 C5a-Aminomethylminocycline (49).80 A solution of trimethylphospine in tetrahydrofuran (1.0 M, 398 µL, 0.398 mmol, 2.0 equiv) was added dropwise via syringe to a solution of the azido-substituted ring-opened product 48 (160 mg, 0.199 mmol, 1 equiv) and 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (98.0 mg, 0.398 mmol, 2.0 equiv) in tetrahydrofuran at –10 °C. The reaction mixture was allowed to warm to 23 °C over 15 min. After stirring at this temperature for 15 h, the product solution was partitioned between dichloromethane and water (60 mL each). The phases were separated and the organic phase was washed sequentially with water (60 mL) and saturated aqueous sodium chloride solution (2 x 60 mL). The organic solution was then dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The product was purified by flash-column chromatography (25% ethyl acetate-hexanes), providing the desired tert-butyl carbamate as a yellow solid (90 mg, 51%). Methanol (2.5 mL) and dioxane (2.5 mL) were added to this product, forming a yellow solution. Palladium black (25 mg, 0.235 mmol, 2.3 equiv) was added in one portion at 23 °C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The reaction mixture was stirred at 23 °C for 1 h, whereupon more palladium black (25 mg) was added. The resulting mixture was stirred at 23 °C for a further 2 h, then was filtered through a plug of Celite. The filtrate was concentrated, providing an orange solid. Concentrated aqueous hydrofluoric acid (48 119 wt%, 1.4 mL) was added to a solution of the crude product in acetonitrile (2.0 mL) in a polypropylene reaction vessel at 23 °C. The reaction mixture was stirred vigorously at 23 °C for 15 h. Excess hydrofluoric acid was quenched by the careful addition of methoxytrimethylsilane (9.0 mL). The resulting mixture was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, injection volume: 5.0 mL (4.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 22-27 min were collected and concentrated, affording C5aaminomethylminocycline bistrifluoroacetate 49 as a yellow solid (50 mg, 69%, two steps). 1 H NMR (500 MHz, CD3OD, bistrifluoroacetate) 7.88 (d, 1H, J = 9.0 Hz), 7.10 (d, 1H, J = 9.0 Hz), 4.09 (d, 1H, J = 2.5 Hz), 3.60 (d, 1H, J = 17.0 Hz), 3.34 (d, 1H, J = 14.5 Hz), 3.18 (s, 6H), 3.16 (s, 6H), 3.20-3.14 (m, 1H), 3.02 (d, 1H, J = 14.5 Hz), 2.95 (d, 1H, J = 17.0 Hz), 2.33 (dd, 1H, J = 15.0, 3.0 Hz), 1.96 (dd, 1H, J = 14.6, 13.7 Hz); HRMS–ESI (m/z): [M+H]+ calcd for C24H31N4O7, 487.2187; found 487.2181. 120 C5a-Piperazinylmethylminocycline (51). Anhydrous magnesium bromide (51.0 mg, 0.276 mmol, 2.0 equiv) was added to a solution of the C5a-C11a-bridged cyclopropane 37 (105 mg, 0.138 mmol, 1 equiv) and tert-butyl 1-piperazine carboxylate (186 mg, 1.00 mmol, 7.2 equiv) in tetrahydrofuran (2.0 mL) at 23 °C. The reaction flask was sealed and the reaction mixture was heated to 45°C. After stirring at 45 °C for 36 h, the reaction mixture was allowed to cool to 23 °C. The cooled mixture was partitioned between dichloromethane and saturated aqueous sodium bicarbonate solution (25 mL each). The phases were separated and the aqueous phase was extracted with dichloromethane (25 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The product mixture was filtered through a short pad of silica gel (eluting with 40% ethyl acetate-hexanes) and the filtrate was concentrated, affording an orangeyellow oil. Methanol (2.5 mL) and dioxane (2.5 mL) were added to the crude ring-opened product (50), forming an orange-yellow solution. Palladium black (25 mg, 0.235 mmol, 1.7 equiv) was added in one portion at 23 °C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The reaction mixture was stirred at 23 °C for 2 h, then was filtered through a plug of Celite. The filtrate was concentrated, providing an orange solid. Concentrated aqueous hydrofluoric acid (48 wt%, 1.5 mL) was added to a solution of the crude product in acetonitrile (2.0 mL) in a polypropylene reaction vessel at 23 °C. The reaction mixture was stirred 121 vigorously at 23 °C for 14 h. Excess hydrofluoric acid was quenched by the careful addition of methoxytrimethylsilane (10.0 mL). The resulting mixture was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, 2 batches, injection volume (for each batch): 5.0 mL (4.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 35% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 22-29 min were collected and concentrated, affording C5a-piperazinylmethylminocycline bistrifluoroacetate 51 as a yellow solid (63 mg, 58%, three steps). 1 H NMR (600 MHz, CD3OD, bistrifluoroacetate) 7.58 (d, 1H, J = 9.6 Hz), 6.93 (d, 1H, J = 9.0 Hz), 4.06 (s, 1H), 3.44 (d, 1H, J = 16.2 Hz), 3.11 (brd, 1H, J = 12.6 Hz), 3.083.02 (m, 2H), 3.05 (s, 6H), 3.01-2.95 (m, 2H), 2.82 (s, 6H), 2.61-2.56 (m, 3H), 2.50 (d, 1H, J = 16.2 Hz), 2.48-2.42 (m, 3H), 2.20 (dd, 1H, J = 13.8, 3.0 Hz), 1.74 (dd, 1H, J = 14.1, 13.9 Hz); HRMS–ESI (m/z): [M+H]+ calcd for C28H38N5O7, 556.2766; found, 556.2771. 122 C5a-N-Acetylaminomethylminocycline (52). Acetyl chloride (0.9 µL, 0.013 mmol, 2.3 equiv) was added to a solution of C5a-aminomethylminocycline bistrifluoroacetate (49, 4.0 mg, 0.0056 mmol, 1 equiv) and N,N-diisopropylethylamine (4.6 µL, 0.027 mmol, 4.8 equiv) in methanol (200 µL) at 0 °C. The resulting solution was allowed to warm to 23 °C over 5 min. The reaction mixture was stirred at this temperature for 1 h, then was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, injection volume: 5.0 mL (4.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 25-28 min were collected and concentrated, affording C5a-N-acetylaminomethylminocycline trifluoroacetate 52 as a yellow solid (3.2 mg, 89%). 1 H NMR (600 MHz, CD3OD, trifluoroacetate) 7.80 (d, 1H, J = 9.0 Hz), 7.03 (d, 1H, J = 9.0 Hz), 3.89 (s, 1H), 3.46 (d, 1H, J = 14.4 Hz), 3.27 (d, 1H, J = 13.8 Hz), 3.23 (d, 1H, J = 16.8 Hz), 3.15-3.08 (m, 13H), 2.63 (d, 1H, J = 16.2 Hz), 2.11 (dd, 1H, J = 14.4, 2.4 Hz), 1.91 (s, 3H), 1.69 (t, 1H, J = 14.4 Hz); HRMS–ESI (m/z): [M+H]+ calcd for C26H33N4O8, 529.2293; found 529.2299. 123 C5a-N-Methanesulfonylaminomethylminocycline (55). Methanesulfonic anhydride (2.3 mg, 0.013 mmol, 2.3 equiv) was added to a solution of C5a- aminomethylminocycline bistrifluoroacetate (49, 4.0 mg, 0.0056 mmol, 1 equiv) and N,N-diisopropylethylamine (4.6 µL, 0.027 mmol, 4.8 equiv) in methanol (200 µL) at 23 °C. The reaction mixture was stirred at this temperature for 2 h, then was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, injection volume: 5.0 mL (4.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 28-30 min were collected and concentrated, affording C5aN-methanesulfonylaminomethyl-minocycline trifluoroacetate 55 as a yellow solid (1.5 mg, 39%). 1 H NMR (600 MHz, CD3OD, trifluoroacetate) 7.77 (d, 1H, J = 9.0 Hz), 7.01 (d, 1H, J = 9.0 Hz), 3.91 (s, 1H), 3.34 (d, 1H, J = 16.2 Hz), 3.26-3.21 (m, 2H), 3.08 (s, 6H), 3.03 (s, 6H), 2.99 (d, 1H, J = 13.8 Hz), 2.86 (s, 3H), 2.60 (d, 1H, J = 16.2 Hz), 2.35 (dd, 1H, J = 14.4, 3.0 Hz), 1.70 (t, 1H, J = 14.4 Hz); HRMS–ESI (m/z): [M+H]+ calcd for C25H32N4O9S, 565.1963; found 565.1973. 124 C5a-N-Methoxyacetylaminomethylminocycline (56). Methoxyacetyl chloride (1.2 µL, 0.013 mmol, 2.3 equiv) was added to a solution of C5a-aminomethylminocycline bistrifluoroacetate (49, 4.0 mg, 0.0056 mmol, 1 equiv) and N,N-diisopropylethylamine (4.6 µL, 0.027 mmol, 4.8 equiv) in methanol (200 µL) at 23 °C. The reaction mixture was stirred at this temperature for 2 h, then was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, injection volume: 5.0 mL (4.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 28-30 min were collected and concentrated, affording C5a-N-methoxyacetylaminomethylminocycline trifluoroacetate 56 as a yellow solid (2.0 mg, 53%). 1 H NMR (600 MHz, CD3OD, trifluoroacetate) 7.75 (d, 1H, J = 9.0 Hz), 7.00 (d, 1H, J = = 10.2 Hz), 3.59 (d, 1H, J = 9.0 Hz), 3.92 (s, 1H), 3.84 (AB quartet, 2H, J = 15.0 Hz, 14.4 Hz), 3.40 (s, 3H), 3.32-3.28 (m, 1H), 3.27 (d, 1H, J = 15.6 Hz), 3.10 (s, 6H), 3.103.07 (m, 1H), 3.03 (s, 6H), 2.58 (d, 1H, J = 16.2 Hz), 2.09 (dd, 1H, J = 14.4, 3.0 Hz), 1.69 (t, 1H, J = 13.8 Hz); HRMS–ESI (m/z): [M+H]+ calcd for C27H34N4O9, 559.2399; found 559.2435. 125 O N(CH3)2 NH2 N(CH ) 3 2 H OH NH2 O OH O HO H O 49 O t-Bu Cl O N(CH3)2 t-Bu NH N(CH3)2 H OH NH2 i-Pr2NEt, CH3OH 23 °C O OH O HO H O 57 O C5a-N-Trimethylacetylaminomethylminocycline (57). Trimethylacetyl chloride (1.6 µL, 0.013 mmol, 2.3 equiv) was added to a solution of C5a-aminomethylminocycline bistrifluoroacetate (49, 4.0 mg, 0.0056 mmol, 1 equiv) and N,N-diisopropylethylamine (4.6 µL, 0.027 mmol, 4.8 equiv) in methanol (200 µL) at 23 °C. The reaction mixture was stirred at this temperature for 1 ½ h, then was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, injection volume: 5.0 mL (4.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 38-40 min were collected and concentrated, affording C5a-N- trimethylacetylaminomethylminocycline trifluoroacetate 57 as a yellow solid (3.0 mg, 78%). 1 H NMR (600 MHz, CD3OD, trifluoroacetate) 7.73 (d, 1H, J = 9.0 Hz), 6.99 (d, 1H, J = 9.6 Hz), 3.92 (s, 1H), 3.54 (d, 1H, J = 14.4 Hz), 3.30-3.25 (m, 2H), 3.11 (s, 6H), 3.063.00 (m, 1H), 3.03 (s, 6H), 2.59 (d, 1H, J = 16.2 Hz), 2.03 (dd, 1H, J = 14.4, 3.0 Hz), 1.71 (t, 1H, J = 14.4 Hz), 1.11 (s, 9H); HRMS–ESI (m/z): [M+H]+ calcd for C29H39N4O8, 571.2762; found 571.2771. 126 O N(CH3)2 NH2 N(CH ) 3 2 H OH NH2 O OH O HO H O 49 O Ph Cl O N(CH3)2 NH N(CH3)2 H OH NH2 O OH O HO H O 58 O i-Pr2NEt, CH3OH 23 °C C5a-N-Benzoylaminomethylminocycline (58). Benzoyl chloride (1.5 µL, 0.013 mmol, 2.3 equiv) was added to a solution of C5a-aminomethylminocycline bistrifluoroacetate (49, 4.0 mg, 0.0056 mmol, 1 equiv) and N,N-diisopropylethylamine (4.6 µL, 0.027 mmol, 4.8 equiv) in methanol (200 µL) at 23 °C. The reaction mixture was stirred at this temperature for 1 ½ h, then was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, injection volume: 5.0 mL (4.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 38-40 min were collected and concentrated, affording C5a-N-benzoylaminomethylminocycline trifluoroacetate 58 as a yellow solid (1.6 mg, 41%). 1 H NMR (600 MHz, CD3OD, trifluoroacetate) 7.75 (d, 2H, J = 7.2 Hz), 7.70 (d, 1H, J = 9.6 Hz), 7.54 (t, 1H, J = 7.8 Hz), 7.45 (t, 2H, J = 7.8 Hz), 6.95 (d, 1H, J = 9.0 Hz), 3.94 (s, 1H), 3.74 (d, 1H, J = 14.4 Hz), 3.37 (d, 1H, J = 15.6 Hz), 3.36-3.27 (m, 2H), 3.13 (s, 6H), 3.02 (s, 6H), 2.63 (d, 1H, J = 16.2 Hz), 2.21 (dd, 1H, J = 14.4, 2.4 Hz), 1.76 (t, 1H, J = 14.4 Hz); HRMS–ESI (m/z): [M+H]+ calcd for C31H35N4O8, 591.2449; found 591.2459. 127 Catalog of spectra CH3 H H N(CH3)2 O N TMSO O OTBS 17 OBn CH3 H H N(CH3)2 O N TMSO O OTBS 17 OBn 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 N(CH3)2 On-Bu N(CH3)2 H OH NH2 O OH O HO H O 46 O 147 N(CH3)2 NH2 N(CH3)2 H OH NH2 O OH O HO H O 49 O 148 149 150 151 O N(CH3)2 t-Bu NH N(CH3)2 H OH NH2 O OH O HO H O 57 O 152 Chapter 3 Synthesis of C5-Substituted Tetracyclines 153 Introduction Oxytetracycline (2, Scheme 3.1) is the only naturally-occurring tetracycline with substitution at C5.86 Methacycline (65) and doxycycline (7), semisynthetic tetracycline antibiotics which are derived from oxytetracycline, retain the C5 hydroxyl substituent found in the natural product. The synthesis of methacycline is presented in Scheme 3.1 below. Treatment of oxytetracycline with N-chlorosuccinimide (NCS) in 50% aqueous 1,2-dimethoxyethane in the presence of triethylamine affords chlorinated product 63.87 The 11a-chloro substituent prevents formation of anhydrotetracyclines (which contain an aromatic C ring) in subsequent steps. Exocyclic dehydration of 63 in the presence of anhydrous hydrogen fluoride provides exo-methylene 64, which is then converted into methacycline (65) upon reductive removal of the 11a-chloro substituent with sodium hydrosulfite. Doxycycline (7) was originally isolated from the mixture of products obtained by palladium-catalyzed hydrogenolysis of oxytetracycline. It is now prepared on an industrial scale by hydrogenation of methacycline (65) in the presence of a rhodium metal complex (Scheme 3.2 below).88 Following its approval in 1967, doxycycline became Pfizer’s first once-a-day, broad-spectrum antibiotic. The utility of doxycycline has declined due to increasingly widespread bacterial resistance, but it is still used frequently to treat acne, rosacea, chlamydia, syphilis and rickettsial infections. Tetracyclines in Biology, Chemistry and Medicine; Nelson, M., Hillen, W., Greenwald, R. A., Eds.; Birkhauser: Boston, 2001. 87 86 (a) Blackwood, R. K.; Beereboom, J. J.; Rennhard, H. H.; Schach Von Wittenau, M.; Stephen, C. R. J. Am. Chem. Soc. 1961, 83, 2773–2775. (b) Blackwood, R. K.; Beereboom, J. J.; Rennhard, H. H.; Schach Von Wittenau, M.; Stephen, C. R. J. Am. Chem. Soc. 1963, 85, 3943–3953. Villax, I.; Page, P. U.S. Patent 4,500,458, 1985. 88 154 CH3 OH OH N(CH3)2 H H OH 5 H3C NCS, Et3N H2O–DME (1:1) 50% O OH N(CH ) 3 2 H H OH 11a NH2 O OH O HO H O Oxytetracycline (2) O NH2 O Cl OH O HO O O H 63 liquid HF 47% H OH N(CH3)2 H OH 5 Na2S2O4 H2O–CH3OH (1:1) 62% H OH N(CH3)2 H OH NH2 NH2 O OH O HO H O Methacycline (65) O OH O Cl O O H O 64 O Scheme 3.1. Synthesis of methacycline (65) from oxytetracycline (2). Scheme 3.2. Synthesis of doxycycline (7) from methacycline (65). A limited range of tetracyclines with modified C5 substituents has been prepared by semisynthesis.89 The C5 hydroxyl group of doxycycline can be oxidized using dimethyl sulfoxide and acetic anhydride to provide the corresponding ketone (5-deoxy-5oxo-doxycycline), which was found to be inactive. In addition, selective esterification of 89 Cunha, B. A. Clinical Uses of Tetracyclines. In Handbook of Experimental Pharmacology; Hlavka, J. J. and Boothe, J. H., Eds.; Springer-Verlag: New York, 1985; pp 393–403. 155 the C5 secondary carbinol can be achieved by treatment of tetracyclines with carboxylic acids in the presence of strong acids such as HF and CH3SO3H (esterification of the C12a tertiary carbinol does not occur under these conditions). Tetracyclines with ester substituents at C5 were generally found to be less active than the corresponding C5hydroxy compounds, although certain C5-esters exhibited notable activity against tetracycline-resistant Gram-positive organisms. The 5-formyl derivatives of methacycline and doxycycline were reported to be more active than their parent compounds. The first-generation synthesis of the AB enone 10 also provided access to an AB precursor with a protected hydroxyl group at the -position (enone 66, Scheme 3.3).90 Dr. Qui Wang, a postdoctoral researcher in the Myers laboratory, converted oxygenated AB enone 66 to 5-hydroxyminocycline (67). This fully synthetic tetracycline was found to be 2- to 4-fold less active than minocycline against most bacterial strains tested (and significantly less active against others). Scheme 3.3. Synthesis of 5-hydroxyminocycline (67) from oxygenated AB enone 66. The lack of knowledge of structure-activity relationships at position C5 led us to consider expansion of our fully synthetic platform to enable a thorough analysis of this 90 Charest, M. G.; Lerner, C. D.; Brubaker, J. D.; Siegel, D. R.; Myers, A. G. Science 2005, 308, 395–398. 156 chemical space. The availability of large quantities of the AB enone 10 meant that this compound could be used as starting material for these investigations. We envisioned preparing fully synthetic tetracyclines with unprecedented modifications at C5 by Michael–Claisen cyclizations of diverse -substituted AB precursors with D-ring precursors, followed by deprotection (Scheme 3.4). In this chapter, the development of chemical pathways for transformation of the AB enone 10 into a range of -substituted AB enones and then C5-substituted tetracyclines will be described. Scheme 3.4. Synthesis of fully synthetic C5-substituted tetracyclines from -substituted AB enones. 157 Results The chemical reactivity that has formed the foundation of our efforts to prepare substituted AB precursors was discovered by Dr. Evan Hecker, a post-doctoral researcher in the Myers laboratory. The AB enone 10 was first converted to tert-butyldimethylsilyl dienol ether 68 in 78% yield by heating a solution of 10, tert-butyldimethylsilyl trifluoromethanesulfonate (1.5 equiv) and triethylamine (3 equiv) in 1,2-dichloroethane at reflux for 18 h (Scheme 3.5). Treatment of a solution of tert-butyldimethylsilyl dienol ether 68 in the solvent mixture 4:1 tetrahydrofuran–water with N-bromosuccinimide (NBS) at 23 °C then afforded -( )-bromo AB precursor 69 as a single diastereomer in 92% yield. This stereochemical assignment was supported by NOE studies and confirmed by X-ray crystallography (Figure 3.1). The regio- and stereo-selectivity of this bromination reaction are striking. The -selectivity of electrophilic addition may be governed by electronic effects,91 but could also be augmented by significant steric hindrance on both faces of the silyl dienol ether 68 at the -position (presumably 68 adopts a similar conformation to the AB enone 10). The ( )-stereochemistry of the bromo substituent results from selective electrophilic addition to the “lower” convex face of silyl dienol ether 68. 91 For a discussion of the factors governing the regioselectivity of vinylogous aldol reactions of silyl dienol ether nucleophiles, see: Denmark, S. E.; Heemstra Jr., J, R.; Beutner, G. L. Angew. Chem. Int. Ed. 2005, 44, 4682–4698. 158 Scheme 3.5. Two-step synthesis of -( )-bromo AB precursor 69 from the AB enone 10. Figure 3.1. X-ray crystal structure of -( )-bromo AB precursor 69. Building upon this result, I chose to pursue the synthesis of an intermediate which could be more easily diversified than -bromo enone 69. Addition of N-iodosuccinimide to a solution of silyl dienol ether 68 in acetonitrile and water (19:1 mixture) at –10 °C, followed by warming to 0 °C afforded -( )-iodo AB enone 70 (Scheme 3.6). Exclusion of light during reaction, work-up and purification enabled isolation of -iodo enone 70 in 159 93% yield (11-g batch). Crucially, treatment of a solution of -iodo enone 70 in dioxane and water (5:1 mixture) with silver (I) trifluoroacetate (1 equiv) and heating of this mixture at 45 °C provided -( )-hydroxy AB enone 71 in 51% yield. Scheme 3.6. Two-step synthesis of butyldimethylsilyl dienol ether 68. -( )-hydroxy AB enone 71 from tert- -( )-Hydroxy enone 71 was then transformed into a diverse range of substituted AB precursors (Scheme 3.7). Activation of the - -hydroxy group of 71 followed by nucleophilic displacement provided enone products with various substituents on the “lower” face at the -position. Treatment of a solution of 71 in dichloromethane with diethylamino sulfur trifluoride (DAST) at 0 °C following by warming to 23 °C afforded -fluoro AB enone 72 in 67% yield. Similarly, sequential addition of triphenylphosphine, diethyl azodicarboxylate (DEAD) and diphenyl phosphoryl azide (DPPA) to a solution of 71 in THF at 23 °C provided -azido AB enone 73 (72% yield). Furthermore, the -hydroxy substituent of enone 71 could be straightforwardly inverted by Mitsunobu reaction with formic acid followed by deformylation of the formate ester intermediate upon treatment with ammonium hydroxide in methanol at 0 °C, providing - 160 ( )-hydroxy AB enone 74. The hydroxyl group of 74 was then straightforwardly alkylated and acylated to provide new AB precursors 75 and 76 (Scheme 3.7). Scheme 3.7. Synthesis of diverse -substituted AB precursors from -( )-hydroxy enone 71. tert-Butyldimethylsilyl dienol ether 68 also served as a precursor to AB enones with one-carbon extensions at the -position (Scheme 3.8). Addition of N,N- dimethylmethyleneiminium chloride (Eschenmoser’s salt, 1.2 equiv) to a solution of silyl dienol ether 68 in 1,2-dichloroethane and heating of this mixture at reflux for 14 h afforded a mixture of -dimethylaminomethyl AB enone 77 (40%) and -exo-methylene enone 78 (48%). This useful reactivity was then harnessed to provide an enone with a protected aminomethyl substituent at the -position (80). In this case, aminoalkylation 161 was achieved using a diallylimmonium trifluoroacetate salt (79) developed by Knochel.92 Formation of the -exo-methylene enone by-product (78) in this reaction was minimized by: (1) adding electrophile 79 (4 equiv) in a portionwise manner, and (2) employing a lower reaction temperature (60–65 °C). These procedural modifications enabled isolation of -diallylaminomethyl AB precursor 80 in 70% yield. Scheme 3.8. Aminoalkylation reactions of tert-butyldimethylsilyl dienol ether 68. With a diverse collection of modified AB precursors in hand, the next task was to transform these substrates into the corresponding fully synthetic tetracyclines. The D-ring precursors corresponding to minocycline and 7-fluorotetracyclines (28 and 81, respectively) were chosen as suitable substrates for these investigations.93 AB enones 92 93 Millot, N.; Piazza, C.; Avolio, S.; Knochel, P. Synthesis 2000, 7, 941–948. 7-Fluorotetracyclines were discovered and developed by Tetraphase Pharmaceuticals using the fully synthetic platform developed in the Myers laboratory. The synthesis and utility of fluorinated D-ring 162 with “lower” face -substituents were found to undergo highly efficient Michael–Claisen cyclizations with benzylic anions formed by LDA deprotonation of 28 and 81 in the presence of TMEDA, affording tetracycline precursors with a range of substituents at C5. A selection of these cyclization reactions is presented in Scheme 3.9. N(CH3)2 CH3 CO2Ph BocO 28 O O OTBS 72 F N(CH3)2 O N OBn 66% BocO O HO 82 O OTBS LDA, TMEDA, THF –78 –10 °C N(CH3)2 F N(CH3)2 O N OBn H H H N(CH3)2 CH3 CO2Ph BocO 28 CH3O H N(CH3)2 O N LDA, TMEDA, THF –78 –10 °C 87% N(CH3)2 H O CH3 N(CH3)2 H O N O OTBS OBn O O OTBS 76 OBn BocO O HO 83 CH3O F CH3 CO2Ph BocO 81 O O OTBS 75 O O H N(CH3)2 O N OBn 77% BocO LDA, TMEDA, THF –10 °C –78 F CH3O O H O H N(CH3)2 O N O HO 84 O OTBS OBn Scheme 3.9. Michael–Claisen cyclizations of o-toluate ester anions with AB precursors possessing fluoro (72), methoxy (76) and methyl carbonate (75) substituents at the position. Two-step deprotection of the Michael–Claisen cyclization products shown in Scheme 3.9 under typical conditions provided fully synthetic tetracyclines with precursor 81 is described here: C7-Fluoro Substituted Tetracycline Compounds. PCT International Application Serial No. US2009/053142. 163 unprecedented modifications at C5.54b For example, treatment of 5-methoxy cycloadduct 83 with hydrofluoric acid in acetonitrile at 23 °C, followed by hydrogenolysis of the crude reaction product (85) in the presence of palladium black under an atmosphere of hydrogen in methanol–dioxane at 23 °C and subsequent purification by rp-HPLC afforded 5-methoxyminocycline (86, 100% over two steps, Scheme 3.10). CH3 N(CH3)2 H O N O OTBS OBn CH3 N(CH3)2 H O N OBn CH3 N(CH3)2 H OH NH2 O OH O HO H O O N(CH3)2 H O N(CH3)2 HF (aq) CH3CN H O N(CH3)2 H2, Pd black CH3OH–dioxane 100% over 2 steps 9 7 H O 5 BocO O HO 83 O OH O HO H O 85 5–Methoxyminocycline (86) KNO3, H2SO4 0 °C 63% N(CH3)2 t-Bu H N O HCl Cl H2N H O CH3 N(CH3)2 H OH NH2 N(CH3)2 H2, Pd black CH3OH O2N H O CH3 N(CH3)2 H OH NH2 DMF CH3CN ( 1 : 1 ) 49% over 2 steps OH O HO O O H 88 O OH O HO O O H 87 O N(CH3)2 H N O N H H O CH3 N(CH3)2 H OH NH2 t-Bu OH O HO O O H O 5–Methoxytigecycline (89) Scheme 3.10. Synthesis of 5-methoxyminocycline (86) by two-step deprotection of Michael–Claisen product 83 and transformation of 86 into 5-methoxytigecycline (89). 164 The goal of multiplicatively expanding the pool of fully synthetic tetracyclines was then efficiently advanced by employing “semisynthetic” strategies.94 Selective nitration at C9 of 5-methoxyminocycline (86) was achieved by addition of potassium nitrate (1.1 equiv) to an ice-cold solution of 86 in concentrated sulfuric acid, affording 9nitro-5-methoxyminocycline (87) in 63% yield after purification by rp-HPLC (Scheme 3.10 above). Reduction of the nitro group of 87 by palladium-catalyzed hydrogenation followed by treatment of a solution of the crude aniline product (88) in dimethylformamide and acetonitrile (1:1 mixture) with 2-(tert-butylamino)acetyl chloride hydrochloride afforded 5-methoxytigecycline (89, 49% yield over two steps). A number of glycylcyclines with different C9 side chains were straightforwardly prepared by reaction of 9-amino “branch points” such as 88 with various different electrophiles (Scheme 3.11 below). Furthermore, the three-step sequence described above for transformation of a C5-substituted minocycline analog into the corresponding tigecycline compound (nitration–nitro reduction–side chain attachment) was also effective for the synthesis of 9-glycylamido derivatives of 7-fluorotetracyclines from the corresponding C9-unsubstituted compounds.93 For example, numerous 5,7- difluorotetracyclines (compounds 93–96) with different substitution patterns at C9 were conveniently prepared from a single Michael–Claisen cycloadduct (92) by deprotection followed by C9 functionalization (Scheme 3.12). 94 (a) Sum, P.-E.; Lee, V. J.; Testa, R. T.; Hlavka, J. J.; Ellestad, G. A.; Bloom, J. D.; Gluzman, Y.; Tally, F. P. J. Med. Chem. 1994, 37, 184–188. (b) Sum, P.-E.; Petersen, P. Bioorg. Med. Chem. Lett. 1999, 9, 1459–1462. 165 Scheme 3.11. Synthesis of diverse glycylcyclines by final step diversification of 9amino-5-methoxyminocycline (88). Scheme 3.12. 5,7-Difluorotetracyclines (93–96) prepared from a single Michael–Claisen cyclization product (92) by deprotection followed by C9 functionalization (nitration–nitro reduction–side chain attachment). 166 Fully synthetic tetracyclines possessing amino and aminomethyl substituents at C5 were also targeted for synthesis. We anticipated that these compounds could serve as substrates for late-stage diversification, thus allowing maximally expedient exploration of chemical space at C5. 5-Aminominocycline (99) was prepared in five synthetic operations from -azido AB enone 73 (Scheme 3.13). C-ring-forming cyclization of 73 with D-ring precursor 31 afforded the desired Michael–Claisen product (97) in 50% yield. Staudinger reduction was achieved by treatment of a solution of azide 97 in THF with trimethylphosphine followed by hydrolysis of the iminophosphorane intermediate with 1M aqueous sodium hydroxide solution. The resulting primary amine was then protected by treatment with di-tert-butyl dicarbonate and triethylamine, providing tertbutyl carbamate 98 in 60% yield from azide 97.95 Two-step deprotection using the inverted sequence afforded 5-aminominocycline (99) after purification by rp-HPLC (100% yield over two steps). Fully synthetic 5-amido derivatives of 5-aminominocycline (99) are presented in Scheme 3.14 below. 95 Prior research had shown that the hydrogenolysis deprotection step is slow and low-yielding in the presence of free primary amines. For this reason, the hydrogenolysis reaction was performed on a substrate in which the primary amine was protected as a tert-butyl carbamate. 167 Scheme 3.13. Synthesis of 5-aminominocycline (99) from -azido AB enone 73. Scheme 3.14. Fully synthetic 5-amido derivatives of 5-aminominocycline (99). 168 5-Aminomethylminocycline (103) was prepared in five steps from - diallylaminomethyl AB enone 80 (Scheme 3.15). Michael–Claisen reaction of 80 and the minocycline D-ring precursor 28 provided the cyclization product 101 in 70% yield. Bisdeallylation of 101 was achieved by treatment with a catalytic amount of palladium tetrakis-(triphenylphosphine) and an excess of dimethylbarbituric acid (DCE, 35 °C). The resulting primary amine (102) was protected as a tert-butyl carbamate prior to two-step deprotection (hydrogenolysis followed by hydrofluoric acid treatment), affording 5aminomethylminocycline (103). With this fully synthetic tetracycline in hand, it was straightforward to rapidly synthesize a collection of related analogs possessing extended substituents at C5 (Scheme 3.16). N(CH3)2 CH3 N H N(CH3)2 O N O O OTBS 80 OBn LDA, TMEDA, THF –78 –10 °C 70% O CH3 N O NH2 N(CH3)2 H OH NH2 OH O HO O O H O N O CH3 BocO O HO 101 O OTBS BocO 28 CO2Ph N N(CH3)2 H H N(CH3)2 O N OBn Pd(PPh3)4, 1,2–DCE 35 °C 77% NH2 N(CH3)2 H O N O OTBS OBn N(CH3)2 H 5 1. Boc2O , Et3N , CH2Cl2 2. H2, Pd black CH3OH–dioxane 3. HF (aq), CH3CN N(CH3)2 H BocO O HO 102 5 Aminomethylminocycline (103) Scheme 3.15. Synthesis of 5-aminomethylminocycline (103) from -diallylaminomethyl AB enone 80. 169 Scheme 3.16. Fully synthetic 5-amido derivatives of 5-aminomethylminocycline (103). Finally, it is worth noting that the C-ring-forming Michael–Claisen cyclization of an AB precursor possessing an “upper” face (or -face) substituent at the -position can also be stereoselective, albeit to a lesser extent (Scheme 3.17). Michael–Claisen reaction of enone 104 (with a protected hydroxy substituent on the upper face at the -position) with minocycline D-ring precursor 31 afforded the desired cyclization product 105 in 40% yield. The stereochemistry of 105 was confirmed by X-ray crystallography (Figure 3.2 below). A minor diastereomer, believed to be epimeric at C5a, was isolated separately (15% yield). Two-step deprotection of Michael–Claisen product 105 provided 5-( )hydroxyminocycline (106, 58% over two steps). 170 Scheme 3.17. Three-step synthesis of 5-( )-hydroxyminocycline (106) from an AB enone with a protected hydroxy substituent on the upper face at the -position (104). Figure 3.2. X-ray crystal structure of Michael–Claisen cyclization product 105. Antibacterial Activities Minimum inhibitory concentrations (MIC) values were determined for all C5substituted tetracyclines against a broad panel of tetracycline-sensitive and tetracyclineresistant Gram-positive and Gram-negative bacteria. In summary, numerous fully 171 synthetic C5-modified tetracyclines exhibited good activity against Gram-positive bacteria (including tetracycline-resistant organisms) and weak activity against some Gram-negative organisms, however C5 substitution provided compounds which were less active (to greater and lesser extents) than the corresponding C5-unsubstituted compounds (e.g. minocycline, tigecycline). Specific examples are discussed here and complete antibacterial activity data for all C5-substituted analogs is presented in the tables below (pages 176–184). MIC assays for tigecycline (9), 5-fluoro-TP-434 (96)96 and 5-methoxytigecycline (89) against tetracycline-susceptible E. coli and leaky E. coli strains are depicted in Figure 3.3 below. As discussed in Chapter 2, leaky E. coli has a more permeable outer membrane than E. coli (due to knockout of a lipopolysaccharide gene) and so provides some insight as to the effect of outer membrane penetration on antibacterial activity. The results of these MIC assays revealed that both synthetic analogs possess antibacterial activity and that 5-fluoro-glycylcycline 96 has similar activity to tigecycline (9) in this E. coli strain and is 16-fold more active than tigecycline in leaky E. coli. Thus, fully synthetic C5-fluoro-substituted tetracycline 96 was found to exhibit significantly greater potency than tigecycline against a tetracycline-susceptible strain with a permeabilized outer membrane. Unfortunately this improvement in activity (vs. tigecycline) was not common to other bacteria. Determination of antibacterial activities against a broad panel of Gram-positive and Gram-negative bacteria (including tetracycline-resistant organisms) 96 TP-434 is a fully synthetic tetracycline antibiotic discovered and developed by Tetraphase Pharmaceuticals that is currently undergoing a Phase 2 clinical trial. 172 showed that 96 is significantly less active than tigecycline against most bacterial strains (see tables below for complete data). E. coli Tigecycline (9, rows 1 & 2) 5-Fluoro-TP-434 (96, rows 3 & 4) 5-Methoxytigecycline (89, rows 5 & 6) 1 1 4 Leaky E. coli 1 0.063 1 Figure 3.3. Minimum inhibitory concentration (MIC) assays and values in µg / mL for tigecycline (9), 5-fluoro-TP-434 (96) and 5-methoxytigecycline (89) against E. coli and leaky E. coli strains. 173 GP SA101 SA158 tetK EF404 tetM SP160 tetM EC107 tetM EC155 tetA GN EC878 tolC KP457 PA555 Minocycline N(CH3)2 H F H N(CH3)2 OH NH2 O OH O HO H O O 0.031 0.016 16 4 0.25 8 0.25 4 32 0.063 0.016 16 8 0.25 16 0.125 16 32 CH3 N(CH3)2 N(CH3)2 O H H OH NH2 O OH O HO H O N(CH3)2 H O 0.5 0.5 16 16 1 >32 0.25 16 32 NH2 N(CH3)2 H OH NH2 2 2 32 32 1 >32 1 8 16 O OH O HO H O N(CH3)2 H O NH2 N(CH3)2 H OH NH2 32 16 >32 >32 32 >32 16 >32 >32 O OH O HO H O O Chart 3.1. MIC values in µg/mL for minocycline and C5-substituted minocycline analogs. Abbreviations: GP, Gram-positive; GN, Gram-negative; SA, S. aureus; EF, E. faecalis; SP, S. pneumoniae; EC, E. coli; KP, K. pneumoniae; PA, P. aeruginosa; tetM, ribosomal protection proteins; tetA, tetK, tetracycline efflux proteins; tolC, multiple efflux pump knockout. Antibacterial activities for selected C5-substituted variants of minocycline are displayed in Chart 3.1 above. 5-Fluorominocycline and minocycline exhibited identical activity in many bacterial strains, while 5-fluorominocycline was found to be 2-fold less 174 active in others. 5-Methoxyminocycline and 5-aminominocycline were substantially less active against Gram-positive bacteria such as S. aureus and slightly less active than minocycline against Gram-negative organisms. 5-Aminomethylminocycline and its derivatives were found to be almost completely inactive as antibiotics, indicating that the introduction of extended substituents at C5 is not a promising strategy for the discovery of new tetracycline antibiotics. The discovery that 5-fluorominocycline has similar antibacterial activity to minocycline led to optimism regarding the activity of 5fluorotigecycline. Unfortunately this compound was found to be significantly less potent than tigecycline (the corresponding 5-unsubstituted compound, Chart 3.2 below). The finding that fluoro-substitution has different effects on the activity of minocycline and tigecycline is one of many “non-linear” effects we have observed. In the case of tigecycline, 5-methoxy and 5-fluoro substitution were found to be similarly detrimental to antibacterial activity. Chart 3.2. MIC values in µg/mL for tigecycline and selected C5-substituted tigecycline analogs (see Chart 3.1 for abbreviations). 175 176 177 178 179 180 181 182 183 184 Conclusion In conclusion, we have developed new chemical pathways for the synthesis of diverse -substituted AB enones from the -unsubstituted AB enone 10. These modified AB precursors were then transformed into more than 40 fully synthetic tetracyclines with unprecedented substitutions at C5 by coupling with various D-ring precursors followed by deprotection and late-stage “branch point” diversification. Many of the modified AB precursors served as precursors to small libraries of tetracyclines with a given substituent at C5 and different D-ring portions, thereby multiplicatively expanding the pool of fully synthetic tetracyclines. Many fully synthetic C5-substituted tetracyclines demonstrated good activity against Gram-positive bacteria (including tetracycline-resistant organisms), however most compounds were significantly less active than the corresponding C5unsubstituted tetracyclines against both Gram-positive and Gram-negative bacteria. 185 C5–Substituted Tetracyclines: Experimental Section tert–Butyldimethylsilyl dienol ether 68. Triethylamine (13.0 mL, 93.0 mmol, 3.0 equiv) and tert–butyldimethylsilyl trifluoromethanesulfonate (10.7 mL, 46.6 mmol, 1.5 equiv) were added sequentially to a stirring solution of AB enone 10 (15.0 g, 31.1 mmol, 1 equiv) in 1,2-dichloroethane (120 mL) at 23 °C. The reaction mixture was heated to reflux and stirred at this temperature for 18 h. The reaction solution was allowed to cool, then was poured into saturated aqueous sodium bicarbonate solution (350 mL). The resulting mixture was extracted with ethyl acetate (2 x 350 mL). The organic extracts were combined and the combined extracts were dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude product was purified by flash-column chromatography (4% ethyl acetate–hexanes, grading to 6%), affording tert-butyldimethylsilyl dienol ether 68 as an off-white solid (14.5 g, 78%). Rf = 0.68 (20% ethyl acetate–hexanes); 1H NMR (500 MHz, CDCl3) 7.48 (d, 2H, J = 7.8 Hz), 7.35 (m, 3H), 5.98 (dd, 1H, J = 9.3, 5.9 Hz), 5.87 (dd, 1H, J = 9.3, 5.9 Hz), 5.32 (m, 2H), 5.25 (d, 1H, J = 5.8 Hz), 3.78 (d, 1H, J = 10.3 Hz), 2.83 (dd, 1H, J = 9.8, 5.9 Hz), 2.48 (s, 6H), 0.82 (s, 9H), 0.77 (s, 9H), 0.15 (s, 6H), 0.13 (s, 3H), –0.04 (s, 3H); 13C NMR (500 MHz, CDCl3) 188.7, 181.8, 167.7, 151.0, 139.3, 135.1, 128.9, 128.5, 128.4, 123.0, 122.9, 107.9, 103.4, 94.7, 81.7, 72.3, 64.2, 48.1, 42.4, 25.8, 25.4, 18.9, 17.8, –2.8, 186 –3.4, –4.5, –5.3; FTIR (neat film) 2929, 1716, 1510, 1247 cm–1; HRMS–ESI (m/z) calcd for C32H49N2O5Si2, 597.3175; found, 596.3179. 187 -( )-Iodo AB enone 70. Light was excluded throughout the synthesis, work-up and purification of -( )-iodo AB enone 70. N-Iodosuccinimide (4.83 g, 20.4 mmol, 1.05 equiv) was added in one portion to a stirring solution of tert-butyldimethylsilyl dienol ether 68 (11.6 g, 19.4 mmol, 1 equiv) in acetonitrile (171 mL) and water (9.0 mL) at –10 °C. The resulting mixture was stirred at this temperature for 5 min, then was allowed to warm to 0 °C. After stirring at 0 °C for 4 h, the reaction solution was partitioned between saturated aqueous sodium thiosulfate solution and ethyl acetate (300 mL each). The phases were separated and the aqueous phase was extracted with ethyl acetate (300 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude product was purified by flash-column chromatography (12% ethyl acetate–hexanes, grading to 15%), providing the -( )-iodo AB enone 70 as a yellow solid (11.0 g, 93%). Rf = 0.59 (30% ethyl acetate–hexanes); 1H NMR (500 MHz, CDCl3) 7.49 (d, 2H, J = 7.3 Hz), 7.40–7.34 (m, 3H), 6.91 (ddd, 1H, J = 10.1, 4.6, 1.4 Hz), 6.03 (d, 1H, J = 10.1 Hz), 5.74 (d, 1H, J = 4.6 Hz), 5.36 (AB quartet, 2H), 3.52 (d, 1H, J = 11.0 Hz), 3.23 (d, 1H, J = 11.0 Hz), 2.50 (s, 6H), 0.99 (s, 9H), 0.20 (s, 3H), 0.11 (s, 3H); MHz, CDCl3) 13 C NMR (125 193.1, 186.4, 179.0, 167.4, 147.7, 134.9, 128.6, 128.5, 128.4, 126.1, 108.3, 83.1, 72.6, 60.8, 53.9, 41.9, 26.8, 19.5, 11.9, –2.2, –2.2; HRMS–ESI (m/z): [M+H]+ calcd for C26H34IN2O5Si, 609.1276; found, 609.1304. 188 -( )-Hydroxy AB enone 71. Light was excluded throughout the reaction and work–up in this procedure. Silver (I) trifluoroacetate (2.59 g, 11.5 mmol, 1 equiv) was added in one portion to a solution of the -( )-iodo AB enone 70 (7.0 g, 11.5 mmol, 1 equiv) in dioxane (85 mL) and water (17 mL) at 23 °C. The resulting mixture was heated to 45 °C and stirred at this temperature for 14 h. The reaction mixture was allowed to cool to 23 °C, then was partitioned between saturated aqueous sodium thiosulfate solution and ethyl acetate (250 mL each). The phases were separated and the organic phase was washed with saturated aqueous sodium bicarbonate solution (100 mL). The aqueous phases were combined and the combined aqueous mixture was extracted with ethyl acetate (250 mL, then 100 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude product was purified by flash-column chromatography (20% ethyl acetate–hexanes, grading to 25%), affording the -( )-hydroxy AB enone 71 as an off–white solid (2.90 g, 51%). Rf = 0.40 (30% ethyl acetate–hexanes); 1H NMR (500 MHz, CDCl3) 7.48 (d, 2H, J = 7.3 Hz), 7.39–7.34 (m, 3H), 7.07 (d, 1H, J = 10.5 Hz), 6.05 (dd, 1H, J = 10.5, 2.3 Hz), 5.35 (AB quartet, 2H), 5.20 (brs, 1H), 4.37 (d, 1H, J = 11.0 Hz), 3.26–3.24 (m, 1H), 2.53 (brs, 6H), 0.83 (s, 9H), 0.26 (s, 3H), 0.03 (s, 3H); 13C NMR (125 MHz, CDCl3) 191.9, 186.0, 178.7, 167.4, 154.2, 134.7, 128.6, 128.5, 128.4, 126.4, 108.6, 83.8, 72.7, 68.3, 189 59.0, 50.7, 25.9, 19.0, –2.5, –4.1; FTIR (neat film), 2930 (w), 2857 (w), 1722 (s), 1684 (m), 1613 (w), 1512 (m), 1022 (s), 837 (s) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C26H35N2O6Si, 499.2259; found, 499.2278. 190 -( )-Fluoro AB enone 72. Diethylaminosulfur trifluoride (74 µL, 0.562 mmol, 1.1 equiv) was added dropwise to a solution of the -( )-hydroxy enone 71 (255 mg, 0.511 mmol, 1 equiv) in dichloromethane (10 mL) at 0 °C. The resulting orange solution was allowed to warm to 23 °C. After stirring at this temperature for 10 min, the reaction mixture was diluted with dichloromethane (10 mL) and washed with saturated aqueous sodium bicarbonate solution (20 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (20 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and filtrate was concentrated. The product was purified by flash-column chromatography (15% ethyl acetate–hexanes), providing the -( )-fluoro AB enone 72 as a yellow solid (171 mg, 67%). Rf = 0.40 (25% ethyl acetate–hexanes); 1H NMR (500 MHz, CDCl3) 7.51 (d, 2H, J = 7.0 Hz), 7.41–7.35 (m, 3H), 6.95–6.91 (m, 1H), 6.18 (dd, 1H, J = 10.5, 2.0 Hz), 5.43 (dd, 1H, J = 43.5, 4.0 Hz), 5.37 (AB quartet, 2H), 3.47 (dd, 1H, J = 11.0, 1.5 Hz), 3.08–3.01 (m, 1H), 2.51 (s, 6H), 0.85 (s, 9H), 0.25 (s, 3H), 0.04 (s, 3H); CDCl3) 13 C NMR (125 MHz, 193.9 (d, J = 2.7 Hz), 186.6, 180.0 (d, J = 3.7 Hz), 167.4, 141.0 (d, J = 17.4 Hz), 134.8, 129.7 (d, J = 8.2 Hz), 128.6, 128.5, 128.5, 108.3, 83.8, 82.1 (d, J = 73.2 Hz), 72.7, 59.1 (d, J = 7.3 Hz), 50.4 (d, J = 17.4 Hz), 42.0, 25.9, 19.1, –2.4, –3.9; (300 MHz, CDCl3) 19 F NMR –170.1; FTIR (neat film), 2930 (w), 1721 (s), 1692 (m), 1512 (m), 191 1474 (m), 1188 (w), 1051 (m), 936 (s), 839 (s) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C26H34FN2O5Si, 501.2216; found, 501.2245. 192 -( )-Azido AB enone 73. Triphenylphosphine (631 mg, 2.41 mmol, 1.2 equiv), a solution of diethyl azodicarboxylate (40% in toluene, 1.10 mL, 2.41 mmol, 1.2 equiv) and diphenyl phosphoryl azide (535 µL, 2.41 mmol, 1.2 equiv) were added sequentially to a solution of -( )-hydroxy AB enone 71 (1.00 g, 2.01 mmol, 1 equiv) in dichloromethane (25 mL) at 23 °C. After stirring at this temperature for 1 h, the reaction mixture was concentrated. The crude product was purified by flash-column chromatography (7% acetone–hexanes), affording the -( )-azido AB enone 73 as an offwhite solid (802 mg, 76%). Rf = 0.08 (8% acetone–hexanes); 1H NMR (500 MHz, CDCl3) 7.50 (d, 2H, J = 7.8 Hz), 7.40–7.34 (m, 3H), 6.74 (dd, 1H, J = 9.8, 3.9 Hz), 6.14 (d, 1H, J = 9.8 Hz), 5.37 (AB quartet, 2H), 4.83 (d, 1H, J = 3.9 Hz), 3.54 (d, 1H, J = 11.7 Hz), 2.86 (d, 1H, J = 10.7 Hz), 2.50 (s, 6H), 0.89 (s, 9H), 0.27 (s, 3H), 0.03 (s, 3H); 13C NMR (125 MHz, CDCl3) 193.4, 186.5, 179.9, 167.3, 142.4, 134.8, 128.8, 128.6, 128.5, 128.4, 108.3, 81.9, 72.6, 59.7, 55.0, 49.4, 41.8, 25.8, 19.1, –2.5, –3.6; FTIR (neat film), 2099 (s), 1722 (s), 1688 (m), 1611 (w), 1506 (m), 1248 (m), 839 (s), 737 (s) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C26H34N5O5Si, 524.2324; found, 524.2324. 193 -( )-Hydroxy AB enone 74. A solution of diethyl azodicarboxylate (40% in toluene, 396 µL, 1.01 mmol, 1.4 equiv) was added dropwise via syringe to a stirred solution of the -( )-hydroxy AB enone 71 (360 mg, 0.722 mmol, 1 equiv), triphenylphosphine (265 mg, 1.01 mmol, 1.4 equiv) and formic acid (38.1 µL, 1.01 mmol, 1.4 equiv) in tetrahydrofuran (2.0 mL) at 23 °C. After stirring at this temperature for 5 h, the reaction mixture was diluted with dichloromethane (20 mL) and washed with saturated aqueous sodium bicarbonate solution (20 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (20 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and filtrate was concentrated, providing a green oil. The crude formate ester product was dissolved in methanol (15 mL) and the resulting solution was cooled to 0 °C. Aqueous ammonia (30% solution, 127 µL, 0.978 mmol, 1.35 equiv) was added to the cooled solution. The reaction was stirred at 0 °C for 25 min, then was diluted with dichloromethane (80 mL) and washed with saturated aqueous sodium bicarbonate solution (60 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (80 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and filtrate was concentrated, affording a brown-red oil. The product was purified by flashcolumn chromatography (17% ethyl acetate–hexanes), providing the -( )-hydroxy AB enone 74 as a white solid (302 mg, 84%, two steps). 194 Rf = 0.23 (25% ethyl acetate–hexanes); 1H NMR (500 MHz, CDCl3) 7.51–7.50 (m, 2H), 7.41–7.35 (m, 3H), 7.02–6.98 (m, 1H), 6.11 (d, 1H, J = 10.2 Hz), 5.37 (AB quartet, 2H), 4.71–4.67 (m, 1H), 3.63 (d, 1H, J = 11.2 Hz), 3.57 (d, 1H, J = 11.2 Hz), 2.93–2.91 (m, 1H), 2.49 (s, 6H), 0.88 (s, 9H), 0.32 (s, 3H), 0.08 (s, 3H); CDCl3) 13 C NMR (125 MHz, 193.2, 186.5, 180.7, 167.3, 146.9, 134.9, 128.6, 128.5, 128.5, 127.2, 108.2, 84.3, 72.7, 64.5, 59.6, 51.8, 41.9, 26.2, 19.1, –2.3, –3.6; HRMS–ESI (m/z): [M+H]+ calcd for C26H35N2O6Si, 499.2259; found, 499.2270. 195 -( )-Methoxy AB enone 76. Trimethylsilyldiazomethane (320 µL, 0.64 mmol, 2.5 equiv) was added dropwise in three portions (2 x 120 µL, then 80 µL) over 10 min to a solution of the -( )-hydroxy AB enone 74 (127 mg, 0.255 mmol, 1 equiv) and tetrafluoroboric acid (60 µL, 0.458 mmol, 1.8 mmol) in dichloromethane (3.0 mL) at 0 °C. The resulting mixture was allowed to warm to 23 °C. After stirring at this temperature for 3 h, more trimethylsilyldiazomethane (120 µL) was added to the reaction mixture. The resulting solution was stirred at 23 °C for a further 1 h, then was diluted with dichloromethane (20 mL) and poured into water (20 mL). Saturated aqueous sodium bicarbonate solution (10 mL) was added and the phases were separated. The aqueous phase was then extracted with dichloromethane (20 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude product was purified by flash-column chromatography (13% ethyl acetate–hexanes), affording the -( )-methoxy AB enone 76 as a white solid (43 mg, 33%). Rf = 0.32 (25% ethyl acetate–hexanes); 1H NMR (500 MHz, CDCl3) 7.50 (d, 2H, J = 7.3 Hz), 7.40–7.34 (m, 3H), 6.91 (dd, 1H, J = 10.1, 4.1 Hz), 6.08 (d, J = 10.1 Hz), 5.37 (AB quartet, 2H), 4.21 (s, 1H), 3.47 (d, 1H, J = 10.5 Hz), 3.41 (s, 3H), 2.88 (d, 1H, J = 9.6 Hz), 2.50 (s, 6H), 0.84 (s, 9H), 0.22 (s, 3H), 0.03 (s, 3H); CDCl3) 13 C NMR (125 MHz, 194.5, 187.5, 180.4, 167.4, 144.7, 135.0, 128.5, 128.5, 128.4, 127.8, 108.2, 196 82.3, 73.9, 72.6, 59.6, 57.0, 48.3, 42.2, 25.8, 19.1, –2.4, –3.7; HRMS–ESI (m/z): [M+H]+ calcd for C27H37N2O6Si, 513.2415; found, 513.2414. 197 -( )-Oxycarbonyloxymethyl AB Enone 75. Methyl chloroformate (233 µL, 3.00 mmol, 2.2 equiv) and 4-dimethylaminopyridine (367 mg, 3.00 mmol, 2.2 equiv) were added sequentially to a solution of the -( )-hydroxy AB enone 74 (680 mg, 1.36 mmol, 1 equiv) in dichloromethane (15 mL) at 23 °C. The reaction mixture was stirred at this temperature for 13 h, then was diluted with dichloromethane (50 mL) and poured into saturated aqueous sodium bicarbonate solution (50 mL). The phases were separated and the aqueous phase was extracted with dichloromethane (50 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude product was purified by flash-column chromatography, providing methyl carbonate 75 (517 mg, 68%). Rf = 0.18 (20% ethyl acetate–hexanes); 1H NMR (500 MHz, CDCl3) 7.51–7.50 (m, 2H), 7.41–7.35 (m, 3H), 6.93 (ddd, 1H, J = 10.3, 4.7, 1.8 Hz), 6.19 (dd, 1H, J = 10.3, 1.0 Hz), 5.71 (m, 1H), 5.37 (AB quartet, 2H), 3.82 (s, 3H), 3.57 (d, 1H, J = 11.3 Hz), 3.02– 2.99 (m, 1H), 2.51 (s, 6H), 0.86 (s, 9H), 0.26 (s, 3H), 0.02 (s, 3H); 13C NMR (125 MHz, CDCl3) 193.8, 186.6, 180.3, 167.4, 155.4, 141.4, 134.9, 129.7, 128.6, 128.5, 128.5, 108.3, 82.0, 72.7, 69.1, 59.7, 55.0, 49.9, 42.0, 25.9, 19.1, –2.4, –3.7. 198 -( )-N,N-Diallylaminomethyl-substituted AB enone 80. A freshly prepared solution of immonium trifluoroacetate salt 79 in anhydrous 1,2-dichloroethane (0.6 M, 4.3 mL, 2.58 mmol, 1.5 equiv)92 was added dropwise to a solution of tert-butyldimethylsilyl dienol ether 68 (1.01 g, 1.69 mmol, 1 equiv) in anhydrous 1,2-dichloroethane (10 mL) at 23 °C. The reaction mixture was heated to 60–65 °C. After stirring at this temperature for 1 h, a second portion of immonium trifluoroacetate solution (0.6 M, 4.3 mL, 2.58 mmol, 1.5 equiv) was added dropwise to the reaction solution. The resulting solution was stirred at 60–65 °C for 1 h, whereupon a final portion of immonium trifluoroacetate solution (0.6 M, 2.8 mL, 1.68 mmol, 1 equiv) was added dropwise. After stirring at 60–65 °C for a further 30 min, the reaction solution was allowed to cool to 23 °C. The cooled solution was poured into saturated aqueous sodium bicarbonate solution (60 mL). Dichloromethane (60 mL) was added and the phases were separated. The aqueous phase was extracted with dichloromethane (60 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording a brown oil. The crude product was purified by flash-column chromatography (dichloromethane flush, then 1% ether– dichloromethane, grading to 3% ether–dichloromethane), affording the -( )-N,N- diallylaminomethyl-substituted AB enone 80 as a pale yellow solid (699 mg, 70%). 199 1 H NMR (500 MHz, CDCl3) 7.50 (d, 2H, J = 6.8 Hz), 7.40–7.34 (m, 3H), 6.94 (ddd, 1H, J = 10.3, 4.9, 1.5 Hz), 6.06 (dd, 1H, J = 10.3, 2.0 Hz), 5.87–5.79 (m, 2H), 5.36 (2H, AB quartet), 5.18–5.13 (m, 4H), 3.57 (d, 1H, J = 11.2 Hz), 3.21–3.16 (m, 5H), 2.89 (dd, 1H, J = 12.7, 7.3 Hz), 2.83 (dd, 12.7, 7.3 Hz), 2.76 (d, 1H, J = 11.2 Hz), 2.48 (s, 9H), 0.86 (s, 9H), 0.24 (s, 3H), 0.01 (s, 3H); HRMS–ESI (m/z): [M+H]+ calcd for C33H46N3O5Si, 592.3201; found, 592.3212. 200 Michael–Claisen cyclization product 82. A freshly prepared solution of lithium diisopropylamide (1.0 M, 3.30 mL, 3.30 mmol, 3.0 equiv) was added dropwise via syringe to a solution of minocycline D-ring precursor 28 (1.22 g, 3.30 mmol, 3.0 equiv) and N,N,N’,N’-tetramethylethylenediamine (TMEDA, 995 µL, 6.59 mmol, 6.0 equiv) in tetrahydrofuran (40 mL) at –78 °C, forming a dark red solution. After stirring at –78 °C for 35 min, a solution of -( )-fluoro AB enone 72 (550 mg, 1.10 mmol, 1 equiv) in tetrahydrofuran (5 mL) was added dropwise via syringe to the reaction solution. The resulting mixture was allowed to warm slowly to –10 °C over 100 min, then was partitioned between aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 100 mL) and dichloromethane (100 mL). The phases were separated and the aqueous phase was further extracted with dichloromethane (2 x 50 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording a dark green oil. The product was purified by flash-column chromatography (13% ethyl acetate–hexanes), providing the Michael–Claisen cyclization product 82 as a yellow solid (560 mg, 66%). Rf = 0.43 (25% ethyl acetate–hexanes); 1H NMR (500 MHz, CDCl3) 15.70 (s, 1H), 7.51 (d, 2H, J = 6.8 Hz), 7.41–7.35 (m, 3H), 7.28 (d, 1H, J = 8.8 Hz), 7.05 (d, 1H, J = 8.8 Hz), 5.38 (AB quartet, 2H), 5.07 (dd, 1H, J = 47.9, 2.0 Hz), 3.83 (dd, 1H, J = 15.6, 5.9 Hz), 201 3.69 (d, 1H, J = 11.7 Hz), 3.37 (ddd, 1H, J = 30.3, 15.7, 4.9, 3.9 Hz), 2.94 (dd, 1H, J = 20.5, 10.8 Hz), 2.68 (s, 6H), 2.52 (s, 6H), 2.34 (dd, 1H, J = 15.7, 14.7 Hz), 1.54 (s, 9H), 0.83 (s, 9H), 0.24 (s, 3H), 0.09 (s, 3H); 13 C NMR (125 MHz, CDCl3) 186.4, 185.8, 180.8, 180.1 (d, J = 3.7 Hz), 167.5, 152.1, 149.7, 145.3, 135.8, 135.0, 128.6, 128.5, 128.5, 124.7, 123.1, 122.5, 108.4, 104.4 (d, J = 7.3 Hz), 92.4 (d, J = 179.4 Hz), 83.8, 81.8, 72.6, 59.5 (d, J = 10.1 Hz), 52.5 (d, J = 19.2 Hz), 44.3, 41.9, 37.5 (d, J = 28.4 Hz), 31.4 (d, J = 1.8 Hz), 27.7, 25.9, 19.0, –2.4, –3.4; FTIR (neat film), 1759 (m), 1722 (m), 1614 (w), 1512 (m), 1234 (s), 1148 (w), 735 (w) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C41H53FN3O9Si, 778.3530; found, 778.3533. 202 5-( )-Fluorominocycline (107). Concentrated aqueous hydrofluoric acid solution (48 wt%, 3.0 mL) was added to a solution of the Michael–Claisen cyclization product 82 (522 mg, 0.671 mmol, 1 equiv) in acetonitrile (4.5 mL) in a polypropylene reaction vessel at 23 °C. The reaction solution was stirred vigorously at 23 °C for 13 ½ h, then was poured into water (150 mL) containing dipotassium hydrogenphosphate trihydrate (30 g). The resulting mixture was extracted with ethyl acetate (150 mL, then 2 x 100 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording a brownish yellow solid. Methanol (4.0 mL) and dioxane (4.0 mL) were added to the crude product. Palladium black (28.6 mg, 0.268 mmol, 0.4 equiv) was added in one portion to the resulting solution at 23 °C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The reaction mixture was stirred at 23 °C for 75 min, then was filtered through a plug of Celite. The filtrate was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, 2 batches, injection volume: 8.0 mL (6.0 mL 0.1% trifluoroacetic acid in water, 2.0 mL acetonitrile), gradient elution with 20 40% B over 40 min, flow rate: 15 mL/min]. Fractions eluting at 6–13 min were collected and concentrated, then re–purified by preparative HPLC [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: 203 acetonitrile, 2 batches, injection volume: 8.0 mL (7.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting 23–30 min were collected and concentrated, affording 5-( )fluorominocycline trifluoroacetate (107) as a yellow solid (321 mg, 81%, two steps). 1 H NMR (500 MHz, CD3OD, trifluoroacetate) 7.83 (d, 1H, J = 9.3 Hz), 7.03 (d, 1H, J = 9.3 Hz), 4.59 (ddd, 1H, J = 48.8, 11.7, 9.3 Hz), 4.57 (d, 1H, J = 1.0 Hz), 3.66 (dd, 1H, J = 15.6, 4.4 Hz), 3.36–3.26 (m, 2H), 3.13 (s, 6H), 2.94 (s, 6H), 2.65 (dd, 1H, J = 14.6, 14.2 Hz); HRMS–ESI (m/z): [M+H]+ calcd for C23H27FN3O7, 476.1828; found, 476.1843. 204 Michael–Claisen cyclization product 83. A freshly prepared solution of lithium diisopropylamide (1.0 M, 2.06 mL, 2.06 mmol, 3.0 equiv) was added dropwise via syringe to a solution of the minocycline D-ring precursor 28 (765 mg, 2.06 mmol, 3.0 equiv) and TMEDA (622 µL, 4.12 mmol, 6.0 equiv) in tetrahydrofuran (17 mL) at –78 °C, forming a dark red solution. After stirring at –78 °C for 45 min, a solution of -( )methoxy AB enone 76 (352 mg, 0.687 mmol, 1 equiv) in tetrahydrofuran (2.5 mL) was added dropwise via syringe to the reaction solution. The resulting mixture was allowed to warm slowly to –10 °C over 100 min, then was partitioned between aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 70 mL) and dichloromethane (70 mL). The phases were separated and the aqueous phase was further extracted with dichloromethane (70 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The product was purified by flash-column chromatography (15% ethyl acetate–hexanes), providing the Michael–Claisen cyclization product 83 as a yellow solid (473 mg, 87%). Rf = 0.24 (20% ethyl acetate–hexanes); 1H NMR (500 MHz, CDCl3) 15.70 (s, 1H), 7.52–7.50 (m, 2H), 7.41–7.34 (m, 3H), 7.26 (d, 1H, J = 8.8 Hz), 7.02 (d, 1H, J = 8.3 Hz), 5.37 (AB quartet, 2H), 3.80 (d, 1H, J = 3.9 Hz), 3.72 (dd, 1H, J = 15.6, 5.4 Hz), 3.64 (d, 205 1H, J = 10.7 Hz), 3.38 (s, 3H), 3.17 (ddd, 1H, J = 14.6, 5.4, 4.4 Hz), 2.79 (d, 1H, J = 10.3 Hz), 2.67 (s, 6H), 2.51 (s, 6H), 2.34 (dd, 1H, J = 15.1, 14.6 Hz), 1.53 (s, 9H), 0.83 (s, 9H), 0.23 (s, 3H), 0.05 (s, 3H); 13 C NMR (125 MHz, CDCl3) 187.3, 186.3, 180.6, 180.5, 167.4, 152.1, 149.6, 145.2, 136.4, 135.0, 128.5, 128.5, 128.4, 124.4, 123.2, 122.2, 108.5, 106.0, 83.6, 82.5, 81.7, 72.5, 60.4, 56.2, 50.1, 44.4, 41.9, 37.5, 32.4, 27.7, 25.9, 19.0, –2.4, –3.4; HRMS–ESI (m/z): [M+H]+ calcd for C42H56N3O10Si, 790.3730; found, 790.3736. 206 5-( )-Methoxyminocycline (86). Concentrated aqueous hydrofluoric acid solution (48 wt%, 3.0 mL) was added to a solution of the Michael–Claisen cyclization product 83 (472 mg, 0.597 mmol, 1 equiv) in acetonitrile (4.5 mL) in a polypropylene reaction vessel at 23 °C. The reaction solution was stirred vigorously at 23 °C for 14 h, then was poured into water (150 mL) containing dipotassium hydrogenphosphate trihydrate (30 g). The resulting mixture was extracted with ethyl acetate (150 mL, then 2 x 100 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording an orange-brown solid. Methanol (4.0 mL) and dioxane (4.0 mL) were added to the crude product. Palladium black (25.4 mg, 0.239 mmol, 0.4 equiv) was added in one portion to the resulting solution at 23 °C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The reaction mixture was stirred at 23 °C for 75 min, then was filtered through a plug of Celite. The filtrate was concentrated. The crude product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, 2 batches, injection volume: 8.0 mL (6.0 mL 0.1% trifluoroacetic acid in water, 2.0 mL acetonitrile), gradient elution with 20 40% B over 40 min, flow rate: 15 mL/min]. Fractions eluting at 4–9 min were 207 collected and concentrated, affording 5-( )-methoxyminocycline trifluoroacetate (86) as a yellow solid (359 mg, 100%, 2 steps). 1 H NMR (600 MHz, CD3OD, trifluoroacetate) 7.83 (d, 1H, J = 9.4 Hz), 7.03 (dd, 1H, J = 9.2, 0.7 Hz), 4.25 (d, 1H, J = 1.2 Hz), 3.67 (s, 3H), 3.62 (dd, 1H, J = 15.2, 4.4 Hz), 3.22 (dd, 1H, J = 11.4, 9.1 Hz), 3.16 (s, 6H), 3.07–3.02 (m, 1H), 2.99 (dd, 1H, J = 11.4, 9.3 Hz), 2.94 (s, 6H), 2.66 (dd, 1H, J = 14.5, 14.4 Hz); HRMS–ESI (m/z): [M+H]+ calcd for C24H30N3O8, 488.2027; found, 488.2052. 208 9-Nitro-5-( )-methoxyminocycline (87). Potassium nitrate (46.6 mg, 0.461 mmol, 1.1 equiv) was added in one portion to a cooled orange solution of 5-( )methoxyminocycline trifluoroacetate (86, 252 mg, 0.419 mmol, 1 equiv) in concentrated sulfuric acid (2.0 mL) at 0 °C. The reaction mixture was stirred at this temperature for 2 h, then was added portionwise and dropwise over 5 min to ice-cold diethyl ether (20 mL), leading to formation of a yellow precipitate. The resulting suspension was transferred using a wide-bore pipette to a Celite pad contained in a sintered glass funnel. The Celite pad was washed thoroughly with ice-cold diethyl ether (100 mL). The crude nitration product was then eluted with methanol (100 mL). The orange-yellow filtrate from elution with methanol was concentrated. The crude product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, 2 batches, injection volume: 8.0 mL (6.0 mL 0.1% trifluoroacetic acid in water, 2.0 mL acetonitrile), gradient elution with 20 40% B over 40 min, flow rate: 15 mL/min]. Fractions eluting at 18-28 min were collected and concentrated, affording 9-nitro-5-( )-methoxyminocycline trifluoroacetate (87) as a brownish yellow solid (171 mg, 63%). 1 H NMR (600 MHz, CD3OD, trifluoroacetate) 8.01 (s, 1H), 4.26 (d, 1H, J = 1.2 Hz), 3.80 (dd, 1H, J = 15.8, 4.5 Hz), 3.69 (s, 3H), 3.27 (dd, 1H, J = 11.6, 9.1 Hz), 2.99–2.92 209 (m, 2H), 2.94 (brs, 6H), 2.73 (s, 6H), 2.55 (dd, 1H, J = 15.7, 13.6 Hz); HRMS–ESI (m/z): [M+H]+ calcd for C24H29N4O10, 533.1878; found, 533.1881. 210 5-( )-Methoxytigecycline (89). Palladium black (14.8 mg, 0.139 mmol, 0.4 equiv) was added in one portion to a solution of 9-nitro-5-( )-methoxyminocycline trifluoroacetate (87, 225 mg, 0.348 mmol, 1 equiv) in methanol (8.0 mL) at 23 °C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The reaction mixture was stirred at 23 °C for 1 ½ h, then was filtered through a plug of Celite. The filtrate was concentrated. The crude aniline product (88) was then divided into 8 equal portions (0.0435 mmol each, assuming 100% yield for nitro reduction) and used in diversifying steps without further purification. 2-(tert-Butylamino)acetyl chloride hydrochloride (16.2 mg, 0.087 mmol, 2.0 equiv) was added in one portion to a solution of crude 9-amino-5-( )-methoxyminocycline trifluoroacetate (0.0435 mmol, 1 equiv) in N,N-dimethylformamide (200 µL) and acetonitrile (200 µL) at 23 °C. The reaction mixture was stirred at this temperature for 2 h, whereupon 0.1% aqueous trifluoroacetic acid solution was added (6.0 mL). The resulting crude product solution was filtered and then purified by preparative HPLC by loading directly onto an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting 21-24 min were collected and concentrated, affording 5-( )-methoxytigecycline bistrifluoroacetate 211 (89) as a yellow solid (18.0 mg, 49% over 2 steps from 9-nitro-5-( )methoxyminocycline). 1 H NMR (600 MHz, CD3OD, bistrifluoroacetate) 8.48 (s, 1H), 4.26 (d, 1H, J = 1.2 Hz), 4.09 (s, 2H), 3.71 (dd, 1H, J = 15.2, 4.5 Hz), 3.68 (s, 3H), 3.23 (dd, 1H, J = 11.6, 8.9 Hz), 3.00–2.94 (m, 2H), 2.94 (brs, 6H), 2.88 (s, 6H), 2.51 (dd, 1H, J = 15.2, 13.6 Hz), 1.43 (s, 9H); HRMS–ESI (m/z): [M+H]+ calcd for C30H42N5O9, 616.2977; found, 616.2969. 212 9-(N,N-dimethylglycylamido)-5-( )-methoxyminocycline (90). 2-(Dimethylamino)acetyl chloride hydrochloride (13.7 mg, 0.087 mmol, 2.0 equiv) was added in one portion to a solution of crude 9-amino-5-( )-methoxyminocycline trifluoroacetate (88, 0.0435 mmol, 1 equiv) in N,N-dimethylformamide (200 µL) and acetonitrile (200 µL) at 23 °C. The reaction mixture was stirred at this temperature for 3 h, whereupon 0.1% aqueous trifluoroacetic acid solution was added (6.0 mL). The resulting crude product solution was filtered and then purified by preparative HPLC by loading directly onto an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting 18-20 min were collected and concentrated, affording 9-(N,N-dimethylglycylamido)-5-( )-methoxyminocycline bistrifluoroacetate (90) as a yellow solid (15.0 mg, 42% over 2 steps from 9-nitro-5-( )methoxyminocycline). 1 H NMR (600 MHz, CD3OD, bistrifluoroacetate) 8.49 (s, 1H), 4.25 (d, 1H, J = 1.0 Hz), 4.25 (s, 2H), 3.71–3.68 (m, 1H), 3.68 (s, 3H), 3.23 (dd, 1H, J = 11.4, 9.1 Hz), 3.02 (s, 6H), 3.02–2.94 (m, 2H), 2.94 (s, 6H), 2.92 (s, 6H), 2.53 (dd, 1H, J = 15.1, 13.6 Hz); HRMS–ESI (m/z): [M+H]+ calcd for C28H38N5O9, 588.2664; found, 588.2663. 213 CH3 N(CH3)2 O N(CH3)2 H H OH H2N NH2 OH O HO O O H 88 O O Br Br O N then NH N H N(CH3)2 H O DMF CH3CN (3:1) ; CH3 N(CH3)2 H OH NH2 OH O HO O O H 91 O 9-(Pyrollidinoglycylamido)-5-( )-methoxyminocycline (91). Bromoacetyl bromide (4.5 µL, 0.052 mmol, 1.2 equiv) was added dropwise to a mixture of crude 9-amino-5-( )methoxyminocycline trifluoroacetate (88, 0.0435 mmol, 1 equiv) and sodium carbonate (23 mg, 0.22 mmol, 5.0 equiv) in N,N-dimethylformamide (300 µL) and acetonitrile (100 µL) at 23 °C. The resulting mixture was stirred at this temperature for 15 min, whereupon pyrrolidine (36.0 µL, 0.435 mmol, 10.0 equiv) was added. The reaction mixture was stirred at 23 °C for a further 2 h, then 0.1% aqueous trifluoroacetic acid solution was added (6.0 mL). The resulting crude product solution was filtered and then purified by preparative HPLC by loading directly onto an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting 20–23 min were collected and concentrated, providing 9(pyrrolidino)acetamido-5-( )-methoxyminocycline bistrifluoroacetate (91) as a yellow solid (14.5 mg, 40% yield over 2 steps from 9-nitro-5-( )-methoxyminocycline). 1 H NMR (600 MHz, CD3OD, bistrifluoroacetate) 8.56 (s, 1H), 4.34 (s, 2H), 4.25 (d, 1H, J = 1.2 Hz), 3.80 (brs, 2H), 3.68 (s, 3H), 3.69–3.65 (m, 1H), 3.24–3.18 (brm, 2H), 3.23 (dd, 1H, J = 11.5, 9.0 Hz), 3.03–2.94 (m, 2H), 3.00 (s, 6H), 2.94 (s, 6H), 2.57 (dd, 1H, J = 15.1, 13.6 Hz), 2.18 (brs, 2H), 2.09 (brs, 2H); HRMS–ESI (m/z): [M+H]+ calcd for C30H40N5O9, 614.2821; found, 614.2823. 214 Michael–Claisen cyclization product 92. A freshly prepared solution of lithium diisopropylamide (1.0 M, 3.45 mL, 3.45 mmol, 3.0 equiv) was added dropwise via syringe to a solution of phenyl ester 81 (1.19 g, 3.45 mmol, 3.0 equiv) and TMEDA (1.04 mL, 6.89 mmol, 6.0 equiv) in tetrahydrofuran (40 mL) at –78 °C, forming a dark red solution. After stirring at –78 °C for 45 min, a solution of -( )-fluoro AB enone 72 (575 mg, 1.15 mmol, 1 equiv) in tetrahydrofuran (5 mL) was added dropwise via syringe to the reaction solution. The resulting mixture was allowed to warm slowly to –10 °C over 100 min, then was partitioned between aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 125 mL) and dichloromethane (125 mL). The phases were separated and the aqueous phase was further extracted with dichloromethane (2 x 50 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated, affording a brown oil. The product was purified by flash-column chromatography (8% ethyl acetate– hexanes), providing the Michael–Claisen cyclization product 92 as a greenish-yellow solid (523 mg, 61%). Rf = 0.48 (25% ethyl acetate–hexanes); 1H NMR (500 MHz, CDCl3) 15.64 (s, 1H), 7.52–7.50 (m, 2H), 7.41–7.35 (m, 3H), 7.25 (dd, 1H, J = 8.3, 8.3 Hz), 7.07 (dd, 1H, J = 9.0, 4.5 Hz), 5.38 (AB quartet, 2H), 5.03 (ddd, 1H, J = 47.8, 3.5, 1.0 Hz), 3.67 (dd, 1H, J 215 = 15.6, 5.4 Hz), 3.62 (d, 1H, J = 11.2 Hz), 3.56–3.45 (m, 1H), 2.93 (ddd, 1H, J = 22.0, 10.8, 1.0 Hz), 2.52 (s, 6H), 2.48 (dd, 1H, J = 15.6, 15.6 Hz), 1.53 (s, 9H), 0.83 (s, 9H), 0.24 (s, 3H), 0.09 (s, 3H); 13C NMR (125 MHz, CDCl3) 188.0, 186.0, 180.1 (d, J = 4.7 Hz), 178.1, 167.4, 156.6 (d, J = 244.4 Hz), 151.6, 146.1 (d, J = 2.7 Hz), 134.9, 128.6, 128.5, 128.5, 127.8 (d, J = 19.2 Hz), 123.6 (d, J = 8.2 Hz), 123.5 (d, J = 3.7 Hz), 120.6 (d, J = 23.8 Hz), 108.4, 104.0 (d, J = 8.2 Hz), 92.4 (d, J = 180.3 Hz), 84.2, 81.9, 72.7, 59.6 (d, J = 10.1 Hz), 52.5 (d, J = 20.1 Hz), 41.9, 36.8 (d, J = 28.4 Hz), 27.9, 27.6, 25.9, 19.0, –2.4, –3.4; 19F NMR (300 MHz, CDCl3) –115.1, –159.8; FTIR (neat film), 1763 (w), 1721 (m), 1614 (w), 1508 (m), 1452 (m), 1144 (m), 733 (s) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C39H47F2N2O9Si, 753.3013; found, 753.3023. 216 7-Fluoro-5-( )-fluorosancycline (93). Concentrated aqueous hydrofluoric acid solution (48 wt%, 3.0 mL) was added to a solution of the Michael–Claisen cyclization product 92 (416 mg, 0.553 mmol, 1 equiv) in acetonitrile (4.5 mL) in a polypropylene reaction vessel at 23 °C. The reaction solution was stirred vigorously at 23 °C for 16 h, then was poured into water (150 mL) containing dipotassium hydrogenphosphate trihydrate (30 g). The resulting mixture was extracted with ethyl acetate (150 mL, then 2 x 50 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. Methanol (4.0 mL) and dioxane (4.0 mL) were added to the crude product. Palladium black (23.5 mg, 0.221 mmol, 0.4 equiv) was added in one portion to the resulting solution at 23 °C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The reaction mixture was stirred at 23 °C for 1 h, then was filtered through a plug of Celite. The filtrate was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, 2 batches, injection volume: 8.0 mL (6.0 mL 0.1% trifluoroacetic acid in water, 2.0 mL acetonitrile), gradient elution with 20 40% B over 40 min, flow rate: 15 mL/min]. Fractions eluting at 15–25 min were collected and concentrated, affording 7fluoro-5-( )-fluorosancycline trifluoroacetate (93) as a yellow solid (310 mg, 99%, two steps). 217 1 H NMR (600 MHz, CD3OD, trifluoroacetate) 7.32 (dd, 1H, J = 9.1, 8.9 Hz), 6.85 (dd, 1H, J = 9.2, 4.2 Hz), 4.57 (ddd, 1H, J = 48.8, 11.8, 9.0 Hz), 4.56 (s, 1H), 3.49 (dd, 1H, J = 15.7, 4.7 Hz), 3.32 (dd, 1H, J = 11.9, 5.5 Hz), 3.28–3.21 (m, 1H), 2.93 (brs, 6H), 2.48 (dd, 1H, J = 14.8, 14.4 Hz); HRMS–ESI (m/z): [M+H]+ calcd for C21H21F2N2O7, 451.1311; found, 451.1312. 218 Michael–Claisen cyclization product 97. A freshly prepared solution of lithium diisopropylamide (1.0 M, 2.31 mL, 2.31 mmol, 3.1 equiv) was added dropwise via syringe to a solution of phenyl ester 31 (808 mg, 2.23 mmol, 3.0 equiv) and TMEDA (670 µL, 4.47 mmol, 6.0 equiv) in tetrahydrofuran (20 mL) at –78 °C, forming a dark red solution. After stirring at –78 °C for 40 min, a solution of -( )-azido-substituted AB enone 73 (390 mg, 0.745 mmol, 1 equiv) in tetrahydrofuran (4.0 mL) was added dropwise via syringe to the reaction solution. The resulting mixture was stirred at –78 °C for 10 min, then was allowed to warm slowly to –10 °C over 100 min. Aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 50 mL) was added and the resulting mixture was allowed to warm to 23 °C. Dichloromethane (50 mL) was added to the crude product mixture and the phases were separated. The aqueous phase was further extracted with dichloromethane (50 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The product was purified first by flash-column chromatography (12% ethyl acetate–hexanes), then by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: water, Solvent B: methanol, 3 batches, gradient elution with 95 100% B over 50 min, flow rate: 15 mL/min]. 219 Fractions eluting at 16–22 min were collected and concentrated, providing the Michael– Claisen cyclization product 97 as a yellow solid (294 mg, 50%). Rf = 0.28 (20% ethyl acetate–hexanes); 1H NMR (500 MHz, CDCl3) 15.82 (s, 1H), 7.53 (d, 2H, J = 6.8 Hz), 7.45 (d, 2H, J = 6.8 Hz), 7.42-7.29 (m, 6H), 7.22 (d, 1H, J = 8.8 Hz), 6.89 (d, 1H, J = 8.8 Hz), 5.40 (s, 2H), 5.16 (AB quartet, 2H), 4.14 (d, 1H, J = 5.9 Hz), 3.80 (dd, 1H, J = 15.6, 5.9 Hz), 3.74 (d, 1H, J = 10.7 Hz), 3.07-3.01 (m, 1H), 2.82 (d, 1H, J = 11.7 Hz), 2.65 (s, 6H), 2.55 (s, 6H), 2.26 (app t, 1H, J = 15.6, 14.7 Hz), 0.86 (s, 9H), 0.28 (s, 3H), 0.08 (s, 3H); 13 C NMR (125 MHz, CDCl3) 187.3, 186.7, 180.6, 180.1, 167.5, 154.9, 145.1, 136.9, 136.5, 135.0, 128.5, 128.5, 128.5, 128.4, 127.7, 126.8, 125.3, 119.7, 114.0, 108.6, 105.1, 82.6, 72.6, 71.4, 62.7, 61.6, 52.5, 44.8, 41.9, 36.7, 32.3, 26.0, 19.1, -2.3, -3.1; FTIR (neat film), 2097 (s), 1722 (m), 1611 (m), 1512 (s), 1250 (s), 833 (s) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C43H51N6O7Si, 791.3583; found, 791.3605. 220 tert-Butyl carbamate 98. A solution of trimethylphosphine in tetrahydrofuran (1.0 M, 50 µL, 0.050 mmol, 1.4 equiv) was added dropwise to a solution of the Michael–Claisen cyclization product 97 (27.9 mg, 0.035 mmol, 1 equiv) in tetrahydrofuran (2.5 mL) and water (2.5 µL) at 23 °C. The resulting mixture was stirred at this temperature for 90 min, whereupon an additional portion of trimethylphosphine solution (10 µL, 0.010 mmol, 0.3 equiv) was added. After stirring at 23 °C for a further 30 min, the reaction mixture was concentrated. The crude iminophosphorane product was dissolved in tetrahydrofuran (0.5 mL) and water (0.25 mL), and aqueous sodium hydroxide solution (2.0 M, 0.25 mL) was added dropwise to the resulting solution. The reaction mixture was stirred at 23 °C for 1 h, whereupon aqueous hydrochloric acid solution (2.0 M, 0.25 mL) was added. Saturated aqueous sodium bicarbonate solution (10 mL) and ethyl acetate (10 ml) were added in sequence and the phases were separated. The aqueous phase was extracted with ethyl acetate (10 mL). The organic extracts were combined and the combined solution was dried. The dried solution as filtered and the filtrate was concentrated. The crude Staudinger reduction product was dissolved in dichloromethane (1.0 mL), and triethylamine (9.8 µL, 0.071 mmol, 2.0 equiv) and di-tert-butyl dicarbonate (12.3 µL, 0.053 mmol, 1.5 equiv) were added in sequence. The reaction mixture was stirred at 23 °C for 9 h, then was concentrated. The crude product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, 221 Solvent A: water, Solvent B: methanol, gradient elution with 95 100% B over 50 min, flow rate: 15 mL/min]. Fractions eluting at 14–18 min were collected and concentrated, affording tert-butyl carbamate 98 as a yellow solid (18.4 mg, 60%). 1 H NMR (500 MHz, CDCl3) 16.28, 7.51 (d, 2H, J = 7.3 Hz), 7.47 (d, 2H, J = 7.3 Hz), 7.41-7.29 (m, 6H), 7.19 (d, 1H, J = 8.8 Hz), 6.87 (d, 1H, J = 8.8 Hz), 5.38 (s, 2H), 5.18 (AB quartet, 2H), 4.55 (d, 1H, J = 9.8 Hz), 3.95 (dd, 1H, J = 15.7, 4.9 Hz), 3.94 (d, 1H, J = 10.8 Hz), 2.83-2.79 (m, 1H), 2.67-2.63 (m, 1H), 2.63 (s, 6H), 2.52 (s, 6H), 2.35 (appt, 1H, J = 15.1, 15.1 Hz), 1.41 (s, 9H), 0.93 (s, 9H), 0.36 (s, 3H), 0.08 (s, 3H); 13 C NMR (125 MHz, CDCl3) 186.5, 184.4, 181.2, 181.1, 167.4, 155.2, 154.8, 145.3, 138.0, 136.9, 135.0, 128.5, 128.5, 128.5, 128.4, 127.7, 126.8, 125.2, 120.6, 113.6, 108.4, 106.7, 83.1, 79.1, 72.6, 71.3, 60.8, 52.7, 48.5, 44.7, 41.8, 40.2, 33.2, 28.3, 26.3, 19.1, -2.5, -2.6; FTIR (neat film), 1717 (s), 1611 (w), 1512 (m), 1474 (m), 1171 (m) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C48H61N4O9Si, 865.4202; found, 865.4246. 222 5-Aminominocycline (99). Palladium black (23.6 mg, 0.222 mmol, 2.0 equiv) was added in one portion to a solution of tert-butyl carbamate 98 (96 mg, 0.111 mmol, 1 equiv) in methanol–dioxane (4 mL, 1:1 mixture) at 23 °C. An atmosphere of hydrogen was introduced by briefly evacuating the flask, then flushing with pure hydrogen (1 atm). The reaction mixture was stirred at 23 °C for 2 ½ h, then was filtered through a plug of Celite. The filtrate was concentrated, affording an orange solid. Concentrated aqueous hydrofluoric acid (48 wt%, 0.8 mL) was added to a solution of the crude product in acetonitrile (1.6 mL) in a polypropylene reaction vessel at 23 °C. The reaction mixture was stirred vigorously at 23 °C for 12 h. Excess hydrofluoric acid was quenched by the careful addition of methoxytrimethylsilane (6.0 mL). The resulting mixture was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 30–35 min were collected and concentrated, affording C5-aminominocycline trifluoroacetate (99) as a yellow solid (65 mg, 100%, two steps). 1 H NMR (600 MHz, CD3OD, trifluoroacetate) 7.90 (d, 1H, J = 9.2 Hz), 7.07 (d, 1H, J = 9.2 Hz), 4.10 (d, 1H, J = 8.6 Hz), 3.96 (brs, 1H), 3.81 (dd, 1H, J = 15.4, 4.4 Hz), 3.29- 223 3.26 (m, 1H), 3.27 (s, 6H), 3.09 (brs, 1H), 2.92 (s, 6H), 2.92-2.90 (m, 1H); HRMS–ESI (m/z): [M+H]+ calcd for C23H29N4O7, 473.2031; found, 473.2052. 224 5-N-Acetylaminominocycline (100). N,N-Diisopropylethylamine (6.0 µL, 0.034 mmol, 5.0 equiv) and acetyl chloride (1.9 µL, 0.027 mmol, 4.0 equiv) were added sequentially to solution of 5-aminominocycline trifluoroacetate (99, 4.0 mg, 0.0068 mmol, 1 equiv) in methanol (200 µL) at 0 °C. The resulting solution was allowed to warm to 23 °C over 5 min. The reaction mixture was stirred at this temperature for 40 min, then was concentrated. The product was purified by preparative HPLC on an Agilent Prep C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, Solvent A: 0.1% trifluoroacetic acid in water, Solvent B: acetonitrile, injection volume: 5.0 mL (4.0 mL 0.1% trifluoroacetic acid in water, 1.0 mL acetonitrile), gradient elution with 5 40% B over 50 min, flow rate: 7.5 mL/min]. Fractions eluting at 24-27 min were collected and concentrated, affording 5-N-acetylaminominocycline trifluoroacetate (100) as a yellow solid (3.1 mg, 73%). 1 H NMR (600 MHz, CD3OD, trifluoroacetate) 7.69 (d, 1H, J = 9.2 Hz), 6.97 (d, 1H, J = 9.2 Hz), 4.00 (brs, 1H), 3.92 (s, 1H), 3.41 (dd, 1H, J = 15.4, 4.1 Hz), 3.03-2.92 (m, 2H), 2.98 (s, 6H), 2.95 (s, 6H), 2.38 (appt, 1H, J = 14.8, 14.3 Hz), 2.13 (s, 3H); HRMS–ESI (m/z): [M+H]+ calcd for C25H31N4O8, 515.2136; found 515.2128. 225 5-Aminomethylminocycline precursor 102. Michael–Claisen cyclization product 101 (815 mg, 0.938 mmol, 1 equiv) was dissolved in 1,2-dichloroethane (10 mL) and argon was bubbled through the resulting solution for 2 min. The solution was then transferred to a round–bottomed flask containing tetrakis(triphenylphosphine)palladium (108 mg, 0.094 mmol, 0.1 equiv) and 1,3-dimethylbarbituric acid (878 mg, 5.63 mmol, 6.0 equiv). The yellow homogeneous reaction solution was heated to 35 °C. After stirring at this temperature for 80 min, the reaction solution was allowed to cool to 23 °C. The cooled solution was diluted with ethyl acetate (50 mL) and the resulting solution was poured into saturated aqueous sodium bicarbonate solution (50 mL). The phases were separated and the aqueous phase was further extracted with ethyl acetate (2 x 50 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude product was purified by flash-column chromatography (1.5% methanol–dichloromethane, grading to 5%), affording amine 102 as a golden brown solid (571 mg, 77%). Rf = 0.26 (5% methanol–dichloromethane); 1H NMR (500 MHz, CDCl3) 7.50 (d, 2H, J = 6.8 Hz), 7.39–7.32 (m, 3H), 7.23 (d, 1H, J = 8.8 Hz), 7.01 (d, 1H, J = 8.3 Hz), 5.37 (AB quartet, 2H), 3.71 (d, 1H, J = 10.7 Hz), 3.60 (dd, 1H, J = 15.4, 5.2 Hz), 3.09–3.00 (m, 2H), 2.77 (d, 1H, J = 10.3 Hz), 2.75–2.70 (m, 1H), 2.66 (s, 6H), 2.50 (s, 6H), 2.29 226 (dd, 1H, J = 15.2, 15.1 Hz), 2.22–2.18 (m, 1H), 1.52 (s, 9H), 0.87 (s, 9H), 0.24 (s, 3H), – 0.03 (s, 3H); 13 C NMR (125 MHz, CDCl3) 187.3, 187.2, 181.0, 180.1, 167.3, 152.0, 149.4, 145.1, 136.3, 135.0, 128.4, 128.4, 128.3, 124.1, 123.2, 122.1, 108.5, 107.4, 83.6, 83.5, 72.4, 62.6, 53.4, 48.4, 47.7, 44.4, 43.0, 41.8, 33.7, 33.5, 27.6, 26.4, 19.1, –2.5, –2.6; HRMS–ESI (m/z): [M+H]+ calcd for C42H57N4O9Si, 789.3889; found, 789.3920. 227 X-Ray Crystallography (Michael–Claisen cyclization product 105): A crystal mounted on a diffractometer was collected data at 100 K. The intensities of the reflections were collected by means of a Bruker APEX II CCD along with the D8 Diffractometer (30 KeV, = 0.413280 Å), and equipped with an Oxford Cryosystems nitrogen open flow apparatus. The collection method involved 0.5 scans in Phi at -5 in 2 . Data integration down to 0.82 Å resolution was carried out using SAINT V7.46 A (Bruker diffractometer, 2009) with reflection spot size optimisation. Absorption corrections were made with the program SADABS (Bruker diffractometer, 2009). The structure was solved by the direct methods procedure and refined by least-squares methods again F2 using SHELXS-97 and SHELXL-97 (Sheldrick, 2008). Non-hydrogen atoms were refined anisotropically, and hydrogen atoms were allowed to ride on the respective atoms. Crystal data as well as details of data collection and refinement are summarized in Table 3.10, geometric parameters are shown in Tables 3.11, and hydrogen-bond parameters are listed in Table 3.12. The Ortep plots produced with SHELXL-97 program, and the other drawings were produced with Accelrys DS Visualizer 2.0 (Accelrys, 2007). Acknowledgement: We thank Dr. Yu-Sheng Chen at ChemMatCARS, APS, for his assistance with single-crystal data. ChemMatCARS Sector 15 is principally supported by the National Science Foundation/Department of Energy under grant number NSF/CHE0822838. Use of the Advanced Photon Source was supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DEAC02-06CH11357. 228 Table 3.10. Experimental details IV-PMW-117 Crystal data Chemical formula Mr Crystal system, space group Temperature (K) a, b, c (Å) , , (°) V (Å3) Z Radiation type (mm-1) Crystal size (mm) Data collection Diffractometer Absorption correction Tmin, Tmax Bruker D8 goniometer with CCD area detector diffractometer Multi-scan SADABS 0.998, 0.999 C90H110N6O18Si2 1620.02 Triclinic, P1 100 8.5526 (8), 13.1550 (12), 20.6010 (19) 106.209 (2), 90.871 (2), 105.015 (2) 2140.1 (3) 1 Synchrotron, 0.07 0.03 × 0.03 × 0.02 = 0.41328 Å No. of measured, independent and 34033, 12277, 9207 observed [I > 2 (I)] reflections Rint Refinement R[F2 > 2 (F2)], wR(F2), S No. of reflections No. of parameters No. of restraints H-atom treatment max, min 0.042 0.096, 0.211, 1.10 12277 1169 499 H-atom parameters constrained 0.94, -0.51 Flack H D (1983), Acta Cryst. A39, 876-881 -0.4 (12) (e Å-3) Absolute structure Flack parameter 229 Computer programs: APEX2 v2009.3.0 (Bruker-AXS, 2009), SAINT 7.46A (Bruker-AXS, 2009), SHELXS97 (Sheldrick, 2008), SHELXL97 (Sheldrick, 2008), Bruker SHELXTL (Sheldrick, 2008). Table 3.11. Geometric parameters (Å, º) O1—C1 O1—N1 O2—C22 O2—C4 O4—C26 O4—C11 O5—C13 O6—C15 O6—H6 O7—C16 O7—Si1B O7—Si1 O8—C17 O9—C19 O9—C39B O9—C39 N1—C19 N2—C2 N2—C20 N2—C21 N3—C25 N3—C25B N3—C8 N3—C24B N3—C24 C1—C18 C1—C2 1.369 (7) 1.422 (9) 1.398 (9) 1.443 (8) 1.305 (11) 1.346 (10) 1.288 (9) 1.286 (9) 0.8400 1.419 (9) 1.655 (9) 1.674 (9) 1.179 (8) 1.290 (9) 1.518 (18) 1.520 (9) 1.304 (10) 1.451 (9) 1.453 (10) 1.467 (10) 1.292 (14) 1.35 (2) 1.403 (12) 1.42 (2) 1.453 (14) 1.313 (10) 1.459 (10) O16—H16 O17—C66 O17—Si2B O17—Si2 O18—C67 O19—C69 O19—C89 O19—C89B N11—C69 N12—C58 N12—C74 N12—C75 C51—C68 C51—C52 C52—N13 C52—C53 C52—H52 C53—C54 C53—C66 C53—H53 C54—C55 C54—H54 C55—C64 C55—C56 C55—H55 C56—C57 C56—H56A 0.8400 1.418 (10) 1.651 (10) 1.723 (8) 1.200 (9) 1.315 (10) 1.530 (16) 1.550 (12) 1.256 (10) 1.420 (11) 1.460 (12) 1.467 (13) 1.282 (11) 1.512 (11) 1.409 (11) 1.580 (9) 1.0000 1.509 (11) 1.524 (11) 1.0000 1.523 (9) 1.0000 1.462 (10) 1.529 (11) 1.0000 1.511 (10) 0.9900 230 Table 3.11. (Continued) C2—C3 C2—H2 C3—C16 C3—C4 C3—H3 C4—C5 C4—H4 C5—C14 C5—C6 C5—H5 C6—C7 C6—H6A C6—H6B C7—C8 C7—C12 C8—C9 C9—C10 C9—H9 C10—C11 C10—H10 C11—C12 C12—C13 C13—C14 C14—C15 C15—C16 C16—C17 C17—C18 C18—C19 C20—H20A C20—H20B C20—H20C 1.566 (8) 1.0000 1.513 (10) 1.541 (9) 1.0000 1.512 (9) 1.0000 1.523 (10) 1.543 (9) 1.0000 1.500 (9) 0.9900 0.9900 1.392 (11) 1.396 (11) 1.367 (11) 1.364 (12) 0.9500 1.367 (11) 0.9500 1.403 (9) 1.455 (10) 1.412 (9) 1.340 (10) 1.537 (9) 1.520 (10) 1.491 (9) 1.434 (10) 0.9800 0.9800 0.9800 C56—H56B C57—C62 C57—C58 N13—C70 N13—C71 C58—C59 C59—C60 C59—H59 C60—C61 C60—H60 C61—O14 C61—C62 C62—C63 C63—C64 C64—C65 C65—C66 C66—C67 C67—C68 C68—C69 C70—H70A C70—H70B C70—H70C C71—H71A C71—H71B C71—H71C C72—O13 C72—H72A C72—H72B O13—C73 C73—H73A C73—H73B 0.9900 1.353 (11) 1.416 (11) 1.449 (10) 1.462 (11) 1.424 (12) 1.359 (13) 0.9500 1.389 (12) 0.9500 1.335 (10) 1.424 (10) 1.487 (11) 1.420 (9) 1.390 (10) 1.520 (9) 1.559 (11) 1.471 (10) 1.439 (10) 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 1.366 (12) 0.9900 0.9900 1.439 (12) 0.9800 0.9800 231 Table 3.11. (Continued) C21—H21A C21—H21B C21—H21C C22—O3 C22—H22A C22—H22B O3—C23 C23—H23A C23—H23B C23—H23C C24—H24A C24—H24B C24—H24C C25—H25A C25—H25B C25—H25C C24B—H24D C24B—H24E C24B—H24F C25B—H25D C25B—H25E C25B—H25F C26—C27 C26—H26A C26—H26B C27—C28 C27—C32 C28—C29 C28—H28 C29—C30 C29—H29 C30—C31 0.9800 0.9800 0.9800 1.347 (10) 0.9900 0.9900 1.382 (12) 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 1.508 (10) 0.9900 0.9900 1.3900 1.3900 1.3900 0.9500 1.3900 0.9500 1.3900 C73—H73C C72B—O13B C72B—H72C C72B—H72D O13B—C73B C73B—H73D C73B—H73E C73B—H73F C74—H74A C74—H74B C74—H74C C75—H75A C75—H75B C75—H75C O14—C76 C76—C77 C76—C77B C76—H76A C76—H76B C76—H76C C76—H76D C77—C78 C77—C82 C78—C79 C78—H78 C79—C80 C79—H79 C80—C81 C80—H80 C81—C82 C81—H81 C82—H82 0.9800 1.36 (2) 0.9900 0.9900 1.44 (2) 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 1.449 (10) 1.471 (11) 1.475 (18) 0.9900 0.9900 0.9900 0.9900 1.3900 1.3900 1.3900 0.9500 1.3900 0.9500 1.3900 0.9500 1.3900 0.9500 0.9500 232 Table 3.11. (Continued) C30—H30 C31—C32 C31—H31 C32—H32 Si1—C35 Si1—C33 Si1—C34 C33—H33A C33—H33B C33—H33C C34—H34A C34—H34B C34—H34C C35—C38 C35—C37 C35—C36 C36—H36A C36—H36B C36—H36C C37—H37A C37—H37B C37—H37C C38—H38A C38—H38B C38—H38C Si1B—C35B Si1B—C34B Si1B—C33B Si1B—C38B C34B—H34D C34B—H34E C34B—H34F 0.9500 1.3900 0.9500 0.9500 1.875 (17) 1.918 (19) 1.951 (15) 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 1.38 (2) 1.44 (2) 1.53 (2) 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 1.908 (19) 1.935 (19) 1.965 (18) 2.33 (2) 0.9800 0.9800 0.9800 C77B—C78B C77B—C82B C78B—C79B C78B—H78A C79B—C80B C79B—H79A C80B—C81B C80B—H80A C81B—C82B C81B—H81A C82B—H82A Si2—C85 Si2—C83 Si2—C84 C83—H83A C83—H83B C83—H83C C84—H84A C84—H84B C84—H84C C85—C88 C85—C86 C85—C87 C86—H86A C86—H86B C86—H86C C87—H87A C87—H87B C87—H87C C88—H88A C88—H88B C88—H88C 1.3900 1.3900 1.3900 0.9500 1.3900 0.9500 1.3900 0.9500 1.3900 0.9500 0.9500 1.794 (16) 1.94 (2) 1.942 (15) 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 1.476 (19) 1.490 (19) 1.69 (2) 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 233 Table 3.11. (Continued) C33B—H33D C33B—H33E C33B—H33F C35B—C38B C35B—C37B C35B—C36B C36B—H36D C36B—H36E C36B—H36F C37B—H37D C37B—H37E C37B—H37F C38B—H38D C38B—H38E C38B—H38F C39—C40 C39—H39A C39—H39B C40—C41 C40—C45 C41—C42 C41—H41 C42—C43 C42—H42 C43—C44 C43—H43 C44—C45 C44—H44 C45—H45 C39B—C40B C39B—H39C C39B—H39D 0.9800 0.9800 0.9800 1.38 (2) 1.40 (2) 1.53 (2) 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 1.425 (12) 0.9900 0.9900 1.3900 1.3900 1.3900 0.9500 1.3900 0.9500 1.3900 0.9500 1.3900 0.9500 0.9500 1.434 (19) 0.9900 0.9900 Si2B—C85B Si2B—C84B Si2B—C83B C83B—H83D C83B—H83E C83B—H83F C84B—H84D C84B—H84E C84B—H84F C85B—C86B C85B—C88B C85B—C87B C86B—H86D C86B—H86E C86B—H86F C87B—H87D C87B—H87E C87B—H87F C88B—H88D C88B—H88E C88B—H88F C89—C90 C89—H89A C89—H89B C90—C91 C90—C95 C91—C92 C91—H91 C92—C93 C92—H92 C93—C94 C93—H93 1.786 (19) 1.918 (19) 2.07 (2) 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 1.45 (2) 1.50 (2) 1.70 (3) 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 0.9800 1.453 (19) 0.9900 0.9900 1.3900 1.3900 1.3900 0.9500 1.3900 0.9500 1.3900 0.9500 234 Table 3.11. (Continued) C40B—C41B C40B—C45B C41B—C42B C41B—H41A C42B—C43B C42B—H42A C43B—C44B C43B—H43A C44B—C45B C44B—H44A C45B—H45A O11—C51 O11—N11 O12—C54 O12—C72 O12—C72B O15—C63 O16—C65 1.3900 1.3900 1.3900 0.9500 1.3900 0.9500 1.3900 0.9500 1.3900 0.9500 0.9500 1.363 (8) 1.409 (9) 1.431 (10) 1.453 (10) 1.47 (2) 1.267 (9) 1.287 (9) C94—C95 C94—H94 C95—H95 C89B—C90B C89B—H89C C89B—H89D C90B—C91B C90B—C95B C91B—C92B C91B—H91A C92B—C93B C92B—H92A C93B—C94B C93B—H93A C94B—C95B C94B—H94A C95B—H95A 1.3900 0.9500 0.9500 1.463 (18) 0.9900 0.9900 1.3900 1.3900 1.3900 0.9500 1.3900 0.9500 1.3900 0.9500 1.3900 0.9500 0.9500 C1—O1—N1 C22—O2—C4 C26—O4—C11 C15—O6—H6 C16—O7—Si1B C16—O7—Si1 C19—O9—C39B C19—O9—C39 C19—N1—O1 C2—N2—C20 C2—N2—C21 C20—N2—C21 C25—N3—C8 107.8 (5) 114.6 (7) 113.4 (7) 109.5 142.6 (6) 131.4 (6) 119.7 (12) 115.5 (7) 105.7 (5) 114.3 (6) 112.5 (7) 112.4 (6) 111.7 (11) C58—N12—C75 C74—N12—C75 C68—C51—O11 C68—C51—C52 O11—C51—C52 N13—C52—C51 N13—C52—C53 C51—C52—C53 N13—C52—H52 C51—C52—H52 C53—C52—H52 C54—C53—C66 C54—C53—C52 110.3 (8) 107.7 (9) 111.1 (7) 131.6 (6) 116.8 (7) 121.5 (7) 112.2 (6) 106.2 (6) 105.2 105.2 105.2 112.0 (6) 109.6 (6) 235 Table 3.11. (Continued) C25B—N3—C8 C25—N3—C24B C25B—N3—C24B C8—N3—C24B C25—N3—C24 C25B—N3—C24 C8—N3—C24 C18—C1—O1 C18—C1—C2 O1—C1—C2 N2—C2—C1 N2—C2—C3 C1—C2—C3 N2—C2—H2 C1—C2—H2 C3—C2—H2 C16—C3—C4 C16—C3—C2 C4—C3—C2 C16—C3—H3 C4—C3—H3 C2—C3—H3 O2—C4—C5 O2—C4—C3 C5—C4—C3 O2—C4—H4 C5—C4—H4 C3—C4—H4 C4—C5—C14 C4—C5—C6 C14—C5—C6 C4—C5—H5 114 (2) 124 (2) 111 (3) 124 (2) 117.2 (11) 81 (3) 112.6 (9) 109.8 (6) 129.9 (6) 120.0 (6) 118.3 (6) 112.0 (5) 109.7 (6) 105.2 105.2 105.2 110.9 (6) 115.9 (6) 108.4 (6) 107.1 107.1 107.1 112.8 (6) 107.1 (5) 110.7 (6) 108.7 108.7 108.7 111.6 (6) 112.3 (5) 108.0 (5) 108.3 C66—C53—C52 C54—C53—H53 C66—C53—H53 C52—C53—H53 O12—C54—C53 O12—C54—C55 C53—C54—C55 O12—C54—H54 C53—C54—H54 C55—C54—H54 C64—C55—C54 C64—C55—C56 C54—C55—C56 C64—C55—H55 C54—C55—H55 C56—C55—H55 C57—C56—C55 C57—C56—H56A C55—C56—H56A C57—C56—H56B C55—C56—H56B H56A—C56—H56B C62—C57—C58 C62—C57—C56 C58—C57—C56 C52—N13—C70 C52—N13—C71 C70—N13—C71 C57—C58—N12 C57—C58—C59 N12—C58—C59 C60—C59—C58 117.5 (6) 105.6 105.6 105.6 106.8 (6) 110.3 (6) 112.1 (6) 109.2 109.2 109.2 112.8 (6) 108.7 (6) 113.7 (6) 107.1 107.1 107.1 107.6 (7) 110.2 110.2 110.2 110.2 108.5 122.9 (7) 119.4 (7) 117.7 (8) 114.0 (7) 113.0 (7) 110.5 (7) 121.1 (7) 115.0 (9) 123.6 (8) 122.3 (8) 236 Table 3.11. (Continued) C14—C5—H5 C6—C5—H5 C7—C6—C5 C7—C6—H6A C5—C6—H6A C7—C6—H6B C5—C6—H6B H6A—C6—H6B C8—C7—C12 C8—C7—C6 C12—C7—C6 C9—C8—C7 C9—C8—N3 C7—C8—N3 C10—C9—C8 C10—C9—H9 C8—C9—H9 C9—C10—C11 C9—C10—H10 C11—C10—H10 O4—C11—C10 O4—C11—C12 C10—C11—C12 C7—C12—C11 C7—C12—C13 C11—C12—C13 O5—C13—C14 O5—C13—C12 C14—C13—C12 C15—C14—C13 C15—C14—C5 C13—C14—C5 108.3 108.3 109.9 (6) 109.7 109.7 109.7 109.7 108.2 120.3 (7) 119.8 (7) 119.8 (6) 119.3 (9) 121.0 (8) 119.7 (7) 120.8 (8) 119.6 119.6 121.1 (8) 119.4 119.4 120.5 (7) 119.9 (7) 119.5 (8) 118.7 (7) 117.7 (6) 123.5 (8) 119.8 (6) 119.6 (6) 120.6 (7) 120.5 (7) 122.7 (6) 116.5 (6) C60—C59—H59 C58—C59—H59 C59—C60—C61 C59—C60—H60 C61—C60—H60 O14—C61—C60 O14—C61—C62 C60—C61—C62 C57—C62—C61 C57—C62—C63 C61—C62—C63 O15—C63—C64 O15—C63—C62 C64—C63—C62 C65—C64—C63 C65—C64—C55 C63—C64—C55 O16—C65—C64 O16—C65—C66 C64—C65—C66 O17—C66—C65 O17—C66—C53 C65—C66—C53 O17—C66—C67 C65—C66—C67 C53—C66—C67 O18—C67—C68 O18—C67—C66 C68—C67—C66 C51—C68—C69 C51—C68—C67 C69—C68—C67 118.8 118.8 121.5 (8) 119.2 119.2 123.3 (7) 119.2 (7) 117.6 (9) 120.3 (8) 119.6 (6) 120.0 (8) 122.7 (7) 121.4 (6) 115.9 (7) 116.8 (7) 123.9 (6) 119.2 (7) 124.0 (6) 114.3 (6) 121.7 (7) 109.1 (6) 109.6 (6) 111.5 (6) 105.1 (6) 110.1 (6) 111.3 (6) 125.4 (7) 125.1 (6) 109.3 (7) 104.4 (7) 121.7 (7) 133.9 (8) 237 Table 3.11. (Continued) O6—C15—C14 O6—C15—C16 C14—C15—C16 O7—C16—C3 O7—C16—C17 C3—C16—C17 O7—C16—C15 C3—C16—C15 C17—C16—C15 O8—C17—C18 O8—C17—C16 C18—C17—C16 C1—C18—C19 C1—C18—C17 C19—C18—C17 O9—C19—N1 O9—C19—C18 N1—C19—C18 N2—C20—H20A N2—C20—H20B H20A—C20—H20B N2—C20—H20C H20A—C20—H20C H20B—C20—H20C N2—C21—H21A N2—C21—H21B H21A—C21—H21B N2—C21—H21C H21A—C21—H21C H21B—C21—H21C O3—C22—O2 O3—C22—H22A 122.7 (6) 114.0 (6) 123.3 (7) 107.4 (6) 106.0 (6) 114.4 (6) 108.3 (6) 111.0 (6) 109.5 (6) 123.9 (6) 126.8 (6) 109.3 (6) 106.0 (6) 121.1 (6) 132.8 (7) 124.1 (7) 125.3 (7) 110.6 (7) 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 114.7 (7) 108.6 N11—C69—O19 N11—C69—C68 O19—C69—C68 N13—C70—H70A N13—C70—H70B H70A—C70—H70B N13—C70—H70C H70A—C70—H70C H70B—C70—H70C N13—C71—H71A N13—C71—H71B H71A—C71—H71B N13—C71—H71C H71A—C71—H71C H71B—C71—H71C O13—C72—O12 O13—C72—H72A O12—C72—H72A O13—C72—H72B O12—C72—H72B H72A—C72—H72B C72—O13—C73 O13—C73—H73A O13—C73—H73B H73A—C73—H73B O13—C73—H73C H73A—C73—H73C H73B—C73—H73C O13B—C72B—O12 O13B—C72B—H72C O12—C72B—H72C O13B—C72B—H72D 125.0 (7) 111.4 (8) 123.4 (7) 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 112.9 (8) 109.0 109.0 109.0 109.0 107.8 112.0 (10) 109.5 109.5 109.5 109.5 109.5 109.5 110 (3) 109.7 109.7 109.7 238 Table 3.11. (Continued) O2—C22—H22A O3—C22—H22B O2—C22—H22B H22A—C22—H22B C22—O3—C23 O3—C23—H23A O3—C23—H23B H23A—C23—H23B O3—C23—H23C H23A—C23—H23C H23B—C23—H23C N3—C24—H24A N3—C24—H24B H24A—C24—H24B N3—C24—H24C H24A—C24—H24C H24B—C24—H24C N3—C25—H25A N3—C25—H25B H25A—C25—H25B N3—C25—H25C H25A—C25—H25C H25B—C25—H25C N3—C24B—H24D N3—C24B—H24E H24D—C24B—H24E N3—C24B—H24F H24D—C24B—H24F H24E—C24B—H24F N3—C25B—H25D N3—C25B—H25E H25D—C25B—H25E 108.6 108.6 108.6 107.6 112.1 (9) 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 O12—C72B—H72D H72C—C72B—H72D C72B—O13B—C73B O13B—C73B—H73D O13B—C73B—H73E H73D—C73B—H73E O13B—C73B—H73F H73D—C73B—H73F H73E—C73B—H73F N12—C74—H74A N12—C74—H74B H74A—C74—H74B N12—C74—H74C H74A—C74—H74C H74B—C74—H74C N12—C75—H75A N12—C75—H75B H75A—C75—H75B N12—C75—H75C H75A—C75—H75C H75B—C75—H75C C61—O14—C76 O14—C76—C77 O14—C76—C77B O14—C76—H76A C77—C76—H76A C77B—C76—H76A O14—C76—H76B C77—C76—H76B C77B—C76—H76B H76A—C76—H76B O14—C76—H76C 109.7 108.2 117 (4) 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 120.1 (6) 109.3 (7) 108.6 (11) 109.8 109.8 103.9 109.8 109.8 116.2 108.3 110.0 239 Table 3.11. (Continued) N3—C25B—H25F H25D—C25B—H25F H25E—C25B—H25F O4—C26—C27 O4—C26—H26A C27—C26—H26A O4—C26—H26B C27—C26—H26B H26A—C26—H26B C28—C27—C32 C28—C27—C26 C32—C27—C26 C27—C28—C29 C27—C28—H28 C29—C28—H28 C28—C29—C30 C28—C29—H29 C30—C29—H29 C31—C30—C29 C31—C30—H30 C29—C30—H30 C30—C31—C32 C30—C31—H31 C32—C31—H31 C31—C32—C27 C31—C32—H32 C27—C32—H32 O7—Si1—C35 O7—Si1—C33 C35—Si1—C33 O7—Si1—C34 C35—Si1—C34 109.5 109.5 109.5 108.8 (8) 109.9 109.9 109.9 109.9 108.3 120.0 121.6 (5) 118.2 (6) 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 104.6 (7) 118.2 (7) 115.3 (9) 107.0 (7) 106.0 (8) C77—C76—H76C C77B—C76—H76C H76B—C76—H76C O14—C76—H76D C77—C76—H76D C77B—C76—H76D H76A—C76—H76D H76C—C76—H76D C78—C77—C82 C78—C77—C76 C82—C77—C76 C77—C78—C79 C77—C78—H78 C79—C78—H78 C80—C79—C78 C80—C79—H79 C78—C79—H79 C79—C80—C81 C79—C80—H80 C81—C80—H80 C82—C81—C80 C82—C81—H81 C80—C81—H81 C81—C82—C77 C81—C82—H82 C77—C82—H82 C78B—C77B—C82B C78B—C77B—C76 C82B—C77B—C76 C77B—C78B—C79B C77B—C78B—H78A C79B—C78B—H78A 115.6 110.0 102.1 110.0 103.4 110.0 114.3 108.3 120.0 120.5 (6) 119.1 (6) 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 125.3 (16) 105.4 (16) 120.0 120.0 120.0 240 Table 3.11. (Continued) C33—Si1—C34 Si1—C33—H33A Si1—C33—H33B H33A—C33—H33B Si1—C33—H33C H33A—C33—H33C H33B—C33—H33C Si1—C34—H34A Si1—C34—H34B H34A—C34—H34B Si1—C34—H34C H34A—C34—H34C H34B—C34—H34C C38—C35—C37 C38—C35—C36 C37—C35—C36 C38—C35—Si1 C37—C35—Si1 C36—C35—Si1 C35—C36—H36A C35—C36—H36B H36A—C36—H36B C35—C36—H36C H36A—C36—H36C H36B—C36—H36C C35—C37—H37A C35—C37—H37B H37A—C37—H37B C35—C37—H37C H37A—C37—H37C H37B—C37—H37C C35—C38—H38A 104.8 (9) 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 113.1 (17) 105.9 (15) 113.0 (15) 104.3 (14) 111.9 (13) 108.1 (13) 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 C78B—C79B—C80B C78B—C79B—H79A C80B—C79B—H79A C81B—C80B—C79B C81B—C80B—H80A C79B—C80B—H80A C82B—C81B—C80B C82B—C81B—H81A C80B—C81B—H81A C81B—C82B—C77B C81B—C82B—H82A C77B—C82B—H82A O17—Si2—C85 O17—Si2—C83 C85—Si2—C83 O17—Si2—C84 C85—Si2—C84 C83—Si2—C84 Si2—C83—H83A Si2—C83—H83B H83A—C83—H83B Si2—C83—H83C H83A—C83—H83C H83B—C83—H83C Si2—C84—H84A Si2—C84—H84B H84A—C84—H84B Si2—C84—H84C H84A—C84—H84C H84B—C84—H84C C88—C85—C86 C88—C85—C87 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 108.6 (6) 115.6 (7) 100.4 (8) 107.2 (7) 106.8 (8) 117.4 (9) 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 118.5 (14) 101.3 (14) 241 Table 3.11. (Continued) C35—C38—H38B H38A—C38—H38B C35—C38—H38C H38A—C38—H38C H38B—C38—H38C O7—Si1B—C35B O7—Si1B—C34B C35B—Si1B—C34B O7—Si1B—C33B C35B—Si1B—C33B C34B—Si1B—C33B O7—Si1B—C38B C34B—Si1B—C38B C33B—Si1B—C38B Si1B—C34B—H34D Si1B—C34B—H34E H34D—C34B—H34E Si1B—C34B—H34F H34D—C34B—H34F H34E—C34B—H34F Si1B—C33B—H33D Si1B—C33B—H33E H33D—C33B—H33E Si1B—C33B—H33F H33D—C33B—H33F H33E—C33B—H33F C38B—C35B—C37B C38B—C35B—C36B C37B—C35B—C36B C38B—C35B—Si1B C37B—C35B—Si1B C36B—C35B—Si1B 109.5 109.5 109.5 109.5 109.5 122.1 (7) 111.3 (8) 114.7 (10) 101.9 (8) 100.2 (9) 102.8 (11) 101.7 (7) 102.3 (10) 136.1 (9) 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 113.7 (19) 104.2 (17) 108.5 (15) 89.0 (13) 129.7 (16) 108.2 (14) C86—C85—C87 C88—C85—Si2 C86—C85—Si2 C87—C85—Si2 C85—C86—H86A C85—C86—H86B H86A—C86—H86B C85—C86—H86C H86A—C86—H86C H86B—C86—H86C C85—C87—H87A C85—C87—H87B H87A—C87—H87B C85—C87—H87C H87A—C87—H87C H87B—C87—H87C C85—C88—H88A C85—C88—H88B H88A—C88—H88B C85—C88—H88C H88A—C88—H88C H88B—C88—H88C O17—Si2B—C85B O17—Si2B—C84B C85B—Si2B—C84B O17—Si2B—C83B C85B—Si2B—C83B C84B—Si2B—C83B Si2B—C83B—H83D Si2B—C83B—H83E H83D—C83B—H83E Si2B—C83B—H83F 105.7 (14) 111.9 (12) 112.2 (12) 105.6 (11) 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 124.3 (8) 116.1 (9) 118.0 (11) 90.7 (9) 87.8 (11) 104.5 (12) 109.5 109.5 109.5 109.5 242 Table 3.11. (Continued) C35B—C36B—H36D C35B—C36B—H36E H36D—C36B—H36E C35B—C36B—H36F H36D—C36B—H36F H36E—C36B—H36F C35B—C37B—H37D C35B—C37B—H37E H37D—C37B—H37E C35B—C37B—H37F H37D—C37B—H37F H37E—C37B—H37F C35B—C38B—Si1B C35B—C38B—H38D Si1B—C38B—H38D C35B—C38B—H38E Si1B—C38B—H38E H38D—C38B—H38E C35B—C38B—H38F Si1B—C38B—H38F H38D—C38B—H38F H38E—C38B—H38F C40—C39—O9 C40—C39—H39A O9—C39—H39A C40—C39—H39B O9—C39—H39B H39A—C39—H39B C41—C40—C45 C41—C40—C39 C45—C40—C39 C40—C41—C42 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 54.9 (11) 109.5 82.3 109.5 163.7 109.5 109.5 75.4 109.5 109.5 109.0 (8) 109.9 109.9 109.9 109.9 108.3 120.0 118.1 (9) 121.9 (9) 120.0 H83D—C83B—H83F H83E—C83B—H83F Si2B—C84B—H84D Si2B—C84B—H84E H84D—C84B—H84E Si2B—C84B—H84F H84D—C84B—H84F H84E—C84B—H84F C86B—C85B—C88B C86B—C85B—C87B C88B—C85B—C87B C86B—C85B—Si2B C88B—C85B—Si2B C87B—C85B—Si2B C85B—C86B—H86D C85B—C86B—H86E H86D—C86B—H86E C85B—C86B—H86F H86D—C86B—H86F H86E—C86B—H86F C85B—C87B—H87D C85B—C87B—H87E H87D—C87B—H87E C85B—C87B—H87F H87D—C87B—H87F H87E—C87B—H87F C85B—C88B—H88D C85B—C88B—H88E H88D—C88B—H88E C85B—C88B—H88F H88D—C88B—H88F H88E—C88B—H88F 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 114.4 (18) 114 (2) 96.2 (17) 126.4 (16) 105.2 (16) 95.4 (14) 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 243 Table 3.11. (Continued) C40—C41—H41 C42—C41—H41 C41—C42—C43 C41—C42—H42 C43—C42—H42 C44—C43—C42 C44—C43—H43 C42—C43—H43 C43—C44—C45 C43—C44—H44 C45—C44—H44 C44—C45—C40 C44—C45—H45 C40—C45—H45 C40B—C39B—O9 C40B—C39B—H39C O9—C39B—H39C C40B—C39B—H39D O9—C39B—H39D H39C—C39B—H39D C41B—C40B—C45B C41B—C40B—C39B C45B—C40B—C39B C42B—C41B—C40B C42B—C41B—H41A C40B—C41B—H41A C41B—C42B—C43B C41B—C42B—H42A C43B—C42B—H42A C42B—C43B—C44B C42B—C43B—H43A C44B—C43B—H43A 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 107 (2) 110.3 110.3 110.3 110.3 108.5 120.00 (7) 129 (2) 110 (3) 120.0 120.0 120.0 120.0 120.0 120.0 120.00 (6) 120.0 120.0 C90—C89—O19 C90—C89—H89A O19—C89—H89A C90—C89—H89B O19—C89—H89B H89A—C89—H89B C91—C90—C95 C91—C90—C89 C95—C90—C89 C92—C91—C90 C92—C91—H91 C90—C91—H91 C91—C92—C93 C91—C92—H92 C93—C92—H92 C94—C93—C92 C94—C93—H93 C92—C93—H93 C93—C94—C95 C93—C94—H94 C95—C94—H94 C94—C95—C90 C94—C95—H95 C90—C95—H95 C90B—C89B—O19 C90B—C89B—H89C O19—C89B—H89C C90B—C89B—H89D O19—C89B—H89D H89C—C89B—H89D C91B—C90B—C95B C91B—C90B—C89B 97.5 (13) 112.3 112.3 112.3 112.3 109.9 120.0 104 (2) 136 (2) 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 111.0 (13) 109.4 109.4 109.4 109.4 108.0 120.0 125.8 (14) 244 Table 3.11. (Continued) C45B—C44B—C43B C45B—C44B—H44A C43B—C44B—H44A C44B—C45B—C40B C44B—C45B—H45A C40B—C45B—H45A C51—O11—N11 C54—O12—C72 C54—O12—C72B C65—O16—H16 C66—O17—Si2B C66—O17—Si2 C69—O19—C89 C69—O19—C89B C69—N11—O11 C58—N12—C74 120.0 120.0 120.0 120.0 120.0 120.0 106.4 (6) 113.1 (7) 139 (3) 109.5 135.8 (7) 135.7 (7) 108.4 (12) 117.7 (9) 106.4 (7) 118.4 (8) C95B—C90B—C89B C92B—C91B—C90B C92B—C91B—H91A C90B—C91B—H91A C93B—C92B—C91B C93B—C92B—H92A C91B—C92B—H92A C92B—C93B—C94B C92B—C93B—H93A C94B—C93B—H93A C93B—C94B—C95B C93B—C94B—H94A C95B—C94B—H94A C94B—C95B—C90B C94B—C95B—H95A C90B—C95B—H95A 114.2 (14) 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 120.0 C1—O1—N1—C19 N1—O1—C1—C18 N1—O1—C1—C2 C20—N2—C2—C1 C21—N2—C2—C1 C20—N2—C2—C3 C21—N2—C2—C3 C18—C1—C2—N2 O1—C1—C2—N2 C18—C1—C2—C3 O1—C1—C2—C3 N2—C2—C3—C16 C1—C2—C3—C16 N2—C2—C3—C4 C1—C2—C3—C4 -0.3 (8) 2.1 (9) 175.8 (7) -77.7 (9) 52.2 (8) 153.2 (7) -76.9 (7) -143.3 (8) 44.4 (10) -13.1 (12) 174.6 (6) 118.9 (7) -14.6 (9) -115.7 (7) 110.8 (7) O11—C51—C52—N13 C68—C51—C52—C53 O11—C51—C52—C53 N13—C52—C53—C54 C51—C52—C53—C54 N13—C52—C53—C66 C51—C52—C53—C66 C72—O12—C54—C53 C72B—O12—C54—C53 C72—O12—C54—C55 C72B—O12—C54—C55 C66—C53—C54—O12 C52—C53—C54—O12 C66—C53—C54—C55 C52—C53—C54—C55 47.7 (11) -10.7 (14) 177.5 (7) -114.7 (8) 110.3 (8) 115.9 (8) -19.0 (10) 135.4 (7) 156 (3) -102.5 (8) -82 (3) 61.1 (7) -71.2 (8) -59.9 (8) 167.9 (7) 245 Table 3.11. (Continued) C22—O2—C4—C5 C22—O2—C4—C3 C16—C3—C4—O2 C2—C3—C4—O2 C16—C3—C4—C5 C2—C3—C4—C5 O2—C4—C5—C14 C3—C4—C5—C14 O2—C4—C5—C6 C3—C4—C5—C6 C4—C5—C6—C7 C14—C5—C6—C7 C5—C6—C7—C8 C5—C6—C7—C12 C12—C7—C8—C9 C6—C7—C8—C9 C12—C7—C8—N3 C6—C7—C8—N3 C25—N3—C8—C9 C25B—N3—C8—C9 C24B—N3—C8—C9 C24—N3—C8—C9 C25—N3—C8—C7 C25B—N3—C8—C7 C24B—N3—C8—C7 C24—N3—C8—C7 C7—C8—C9—C10 N3—C8—C9—C10 C8—C9—C10—C11 C26—O4—C11—C10 C26—O4—C11—C12 C9—C10—C11—O4 -101.7 (7) 136.3 (6) 59.9 (7) -68.3 (7) -63.4 (7) 168.3 (6) -72.7 (7) 47.2 (8) 48.8 (8) 168.7 (6) 179.4 (6) -57.0 (7) -143.2 (8) 33.9 (10) -1.3 (15) 175.7 (9) 176.6 (9) -6.3 (14) -84.1 (13) -40 (3) 101 (3) 50.2 (15) 98.0 (13) 142 (3) -77 (3) O12—C54—C55—C64 C53—C54—C55—C64 O12—C54—C55—C56 C53—C54—C55—C56 C64—C55—C56—C57 C54—C55—C56—C57 C55—C56—C57—C62 C55—C56—C57—C58 C51—C52—N13—C70 C53—C52—N13—C70 C51—C52—N13—C71 C53—C52—N13—C71 C62—C57—C58—N12 C56—C57—C58—N12 C62—C57—C58—C59 C56—C57—C58—C59 C74—N12—C58—C57 C75—N12—C58—C57 C74—N12—C58—C59 C75—N12—C58—C59 C57—C58—C59—C60 N12—C58—C59—C60 C58—C59—C60—C61 C59—C60—C61—O14 C59—C60—C61—C62 -75.7 (8) 43.2 (9) 48.6 (9) 167.5 (7) -59.4 (8) 174.1 (7) 37.3 (10) -142.4 (8) -78.2 (10) 154.7 (7) 49.1 (9) -78.0 (8) 178.9 (8) -1.5 (13) -7.7 (13) 172.0 (8) 155.2 (9) -80.2 (10) -17.6 (14) 106.9 (10) 3.0 (14) 176.2 (9) 1.0 (15) 179.4 (9) -0.7 (13) 8.3 (13) -171.3 (7) -175.4 (8) 4.9 (12) 176.0 (8) -3.8 (12) -0.2 (12) -127.7 (11) C58—C57—C62—C61 -2.1 (16) C56—C57—C62—C61 -180.0 (11) C58—C57—C62—C63 5.2 (17) 90.5 (11) -87.5 (10) 177.2 (9) C56—C57—C62—C63 O14—C61—C62—C57 C60—C61—C62—C57 O14—C61—C62—C63 246 Table 3.11. (Continued) C9—C10—C11—C12 C8—C7—C12—C11 C6—C7—C12—C11 C8—C7—C12—C13 C6—C7—C12—C13 O4—C11—C12—C7 C10—C11—C12—C7 O4—C11—C12—C13 C10—C11—C12—C13 C7—C12—C13—O5 C11—C12—C13—O5 C7—C12—C13—C14 C11—C12—C13—C14 O5—C13—C14—C15 C12—C13—C14—C15 O5—C13—C14—C5 C12—C13—C14—C5 C4—C5—C14—C15 C6—C5—C14—C15 C4—C5—C14—C13 C6—C5—C14—C13 C13—C14—C15—O6 C5—C14—C15—O6 C13—C14—C15—C16 C5—C14—C15—C16 Si1B—O7—C16—C3 Si1—O7—C16—C3 Si1B—O7—C16—C17 Si1—O7—C16—C17 Si1B—O7—C16—C15 Si1—O7—C16—C15 C4—C3—C16—O7 -4.8 (16) 1.7 (13) -175.4 (7) -175.8 (8) 7.1 (11) 179.4 (8) 1.4 (13) -3.3 (13) 178.7 (9) 154.7 (7) -22.6 (12) -24.4 (11) 158.2 (8) 3.6 (11) -177.3 (7) 177.9 (6) -2.9 (10) -18.7 (9) -142.7 (7) 167.0 (6) 43.1 (8) -3.4 (11) -177.5 (7) 177.7 (6) 3.7 (11) -152.6 (8) 159.0 (6) 84.7 (10) 36.3 (8) -32.7 (12) -81.1 (8) 164.5 (5) C60—C61—C62—C63 C57—C62—C63—O15 C61—C62—C63—O15 C57—C62—C63—C64 C61—C62—C63—C64 O15—C63—C64—C65 C62—C63—C64—C65 O15—C63—C64—C55 C62—C63—C64—C55 C54—C55—C64—C65 C56—C55—C64—C65 C54—C55—C64—C63 C56—C55—C64—C63 C63—C64—C65—O16 C55—C64—C65—O16 C63—C64—C65—C66 C55—C64—C65—C66 Si2B—O17—C66—C65 Si2—O17—C66—C65 Si2B—O17—C66—C53 Si2—O17—C66—C53 Si2B—O17—C66—C67 Si2—O17—C66—C67 O16—C65—C66—O17 C64—C65—C66—O17 O16—C65—C66—C53 C64—C65—C66—C53 O16—C65—C66—C67 C64—C65—C66—C67 C54—C53—C66—O17 C52—C53—C66—O17 C54—C53—C66—C65 179.9 (7) 154.4 (8) -29.3 (11) -25.2 (11) 151.1 (7) 4.0 (11) -176.4 (7) 179.3 (7) -1.2 (10) -14.8 (11) -141.8 (7) 170.3 (7) 43.3 (9) -5.5 (11) 179.4 (7) 176.7 (7) 1.7 (12) -31.1 (11) -81.4 (8) -153.4 (8) 156.3 (6) 86.9 (9) 36.6 (9) 44.3 (9) -137.7 (8) 165.4 (7) -16.6 (10) -70.5 (8) 107.4 (8) 165.7 (6) -66.0 (8) 44.9 (8) 247 Table 3.11. (Continued) C2—C3—C16—O7 C4—C3—C16—C17 C2—C3—C16—C17 C4—C3—C16—C15 C2—C3—C16—C15 O6—C15—C16—O7 C14—C15—C16—O7 O6—C15—C16—C3 C14—C15—C16—C3 O6—C15—C16—C17 C14—C15—C16—C17 O7—C16—C17—O8 C3—C16—C17—O8 C15—C16—C17—O8 O7—C16—C17—C18 C3—C16—C17—C18 C15—C16—C17—C18 O1—C1—C18—C19 C2—C1—C18—C19 O1—C1—C18—C17 C2—C1—C18—C17 O8—C17—C18—C1 C16—C17—C18—C1 O8—C17—C18—C19 C16—C17—C18—C19 C39B—O9—C19—N1 C39—O9—C19—N1 C39B—O9—C19—C18 C39—O9—C19—C18 O1—N1—C19—O9 O1—N1—C19—C18 C1—C18—C19—O9 -71.4 (7) -78.2 (7) 45.9 (8) 46.3 (7) 170.4 (6) 45.5 (9) -135.5 (7) 163.2 (6) -17.9 (10) -69.6 (8) 109.3 (8) -109.7 (9) 132.2 (8) 6.9 (12) 69.0 (8) -49.2 (8) -174.5 (7) -2.8 (9) -175.8 (8) -179.1 (7) 7.9 (13) -157.3 (8) 24.0 (10) 27.6 (14) -151.1 (8) 18 (2) -5.4 (13) C52—C53—C66—C65 C54—C53—C66—C67 C52—C53—C66—C67 O17—C66—C67—O18 C65—C66—C67—O18 C53—C66—C67—O18 O17—C66—C67—C68 C65—C66—C67—C68 C53—C66—C67—C68 O11—C51—C68—C69 C52—C51—C68—C69 O11—C51—C68—C67 C52—C51—C68—C67 O18—C67—C68—C51 C66—C67—C68—C51 O18—C67—C68—C69 C66—C67—C68—C69 O11—N11—C69—O19 O11—N11—C69—C68 C89—O19—C69—N11 C89B—O19—C69—N11 C89—O19—C69—C68 C89B—O19—C69—C68 C51—C68—C69—N11 C67—C68—C69—N11 C51—C68—C69—O19 C67—C68—C69—O19 173.1 (7) -78.5 (7) 49.7 (9) -108.5 (8) 8.8 (11) 133.0 (8) 67.2 (7) -175.5 (6) -51.3 (8) -4.1 (10) -176.2 (10) 178.5 (7) 6.3 (16) -158.2 (9) 26.1 (11) 25.2 (15) -150.5 (10) -179.9 (9) -4.2 (11) 6 (2) -13.7 (17) -169.1 (17) 171.1 (12) 5.3 (12) -177.7 (9) -178.9 (9) -1.9 (17) -80.9 (9) 126 (4) -79.7 (11) -87 (4) -47 (3) -163.1 (19) C54—O12—C72—O13 173.7 (9) 177.9 (8) -1.4 (9) -176.6 (8) C72B—O12—C72—O13 O12—C72—O13—C73 C54—O12—C72B—O13B C72—O12—C72B—O13B 248 Table 3.11. (Continued) C17—C18—C19—O9 C1—C18—C19—N1 C17—C18—C19—N1 C4—O2—C22—O3 O2—C22—O3—C23 C11—O4—C26—C27 O4—C26—C27—C28 O4—C26—C27—C32 C32—C27—C28—C29 C26—C27—C28—C29 C27—C28—C29—C30 C28—C29—C30—C31 C29—C30—C31—C32 C30—C31—C32—C27 C28—C27—C32—C31 C26—C27—C32—C31 C16—O7—Si1—C35 Si1B—O7—Si1—C35 C16—O7—Si1—C33 Si1B—O7—Si1—C33 C16—O7—Si1—C34 Si1B—O7—Si1—C34 O7—Si1—C35—C38 C33—Si1—C35—C38 C34—Si1—C35—C38 O7—Si1—C35—C37 C33—Si1—C35—C37 C34—Si1—C35—C37 O7—Si1—C35—C36 C33—Si1—C35—C36 C34—Si1—C35—C36 C16—O7—Si1B—C35B -0.9 (15) 2.7 (10) 178.3 (8) -78.6 (9) -79.8 (10) -178.9 (6) 104.0 (8) -71.8 (8) 0.0 -175.7 (8) 0.0 0.0 0.0 0.0 0.0 175.9 (7) 135.3 (8) 9.4 (8) 5.5 (10) -120.4 (9) -112.5 (9) 121.6 (10) -62.0 (15) 69.6 (16) O12—C72B—O13B—C73B C60—C61—O14—C76 C62—C61—O14—C76 C61—O14—C76—C77 C61—O14—C76—C77B O14—C76—C77—C78 C77B—C76—C77—C78 O14—C76—C77—C82 C77B—C76—C77—C82 C82—C77—C78—C79 C76—C77—C78—C79 C77—C78—C79—C80 C78—C79—C80—C81 C79—C80—C81—C82 C80—C81—C82—C77 C78—C77—C82—C81 C76—C77—C82—C81 O14—C76—C77B—C78B C77—C76—C77B—C78B O14—C76—C77B—C82B C77—C76—C77B—C82B -99 (6) 35.5 (12) -144.4 (8) 162.2 (7) 154.7 (14) -49.8 (10) 36 (9) 137.4 (6) -137 (10) 0.0 -172.7 (8) 0.0 0.0 0.0 0.0 0.0 172.8 (8) 11 (2) -86 (9) 156.6 (13) 60 (9) C82B—C77B—C78B—C79B 0.0 C76—C77B—C78B—C79B 142 (2) C77B—C78B—C79B—C80B 0.0 -174.9 (15) C78B—C79B—C80B—C81B 0.0 60.6 (13) C79B—C80B—C81B—C82B 0.0 -167.9 (12) C80B—C81B—C82B—C77B 0.0 -52.4 (15) C78B—C77B—C82B—C81B 0.0 -148 (2) 136.8 (9) 28.0 (9) 24.9 (10) -174.4 (11) C76—C77B—C82B—C81B -42.8 (14) 72.7 (14) -77.6 (12) C66—O17—Si2—C85 Si2B—O17—Si2—C85 C66—O17—Si2—C83 249 Table 3.11. (Continued) Si1—O7—Si1B—C35B C16—O7—Si1B—C34B Si1—O7—Si1B—C34B C16—O7—Si1B—C33B Si1—O7—Si1B—C33B C16—O7—Si1B—C38B Si1—O7—Si1B—C38B O7—Si1B—C35B—C38B C34B—Si1B—C35B—C38B C33B—Si1B—C35B—C38B O7—Si1B—C35B—C37B C34B—Si1B—C35B—C37B C33B—Si1B—C35B—C37B C38B—Si1B—C35B—C37B O7—Si1B—C35B—C36B C34B—Si1B—C35B—C36B C33B—Si1B—C35B—C36B C38B—Si1B—C35B—C36B C37B—C35B—C38B—Si1B C36B—C35B—C38B—Si1B O7—Si1B—C38B—C35B C34B—Si1B—C38B—C35B C33B—Si1B—C38B—C35B C19—O9—C39—C40 C39B—O9—C39—C40 O9—C39—C40—C41 O9—C39—C40—C45 C45—C40—C41—C42 C39—C40—C41—C42 C40—C41—C42—C43 C41—C42—C43—C44 C42—C43—C44—C45 13.0 (8) 63.2 (14) 153.8 (12) 172.1 (11) -97.2 (9) -45.2 (11) 45.5 (7) 63.0 (14) -76.6 (16) 174.1 (13) Si2B—O17—Si2—C83 C66—O17—Si2—C84 Si2B—O17—Si2—C84 O17—Si2—C85—C88 C83—Si2—C85—C88 C84—Si2—C85—C88 O17—Si2—C85—C86 C83—Si2—C85—C86 C84—Si2—C85—C86 O17—Si2—C85—C87 -83.9 (9) -108.2 (10) 143.0 (11) -49.4 (15) 72.3 (15) -164.7 (14) 174.6 (12) -63.7 (14) 59.3 (16) 59.9 (10) -178.4 (10) -55.4 (12) -103.4 (13) 4.9 (11) 62.3 (16) 170.6 (16) 168.8 (11) -82.9 (10) -175 (2) 20 (3) -86 (2) -38 (2) 156.8 (18) 51.5 (18) 60.2 (16) -105.3 (16) 149.5 (15) -146 (2) -25 (4) -100 (2) 83 (3) 0.0 -176.5 (17) C83—Si2—C85—C87 44 (2) -65 (2) 120 (2) -41.6 (16) 178.8 (14) 69.5 (15) C84—Si2—C85—C87 C66—O17—Si2B—C85B Si2—O17—Si2B—C85B C66—O17—Si2B—C84B Si2—O17—Si2B—C84B C66—O17—Si2B—C83B -104.6 (18) Si2—O17—Si2B—C83B -133.6 (19) O17—Si2B—C85B—C86B 108.5 (15) C84B—Si2B—C85B—C86B -129.6 (12) C83B—Si2B—C85B—C86B 115.3 (14) -8.4 (18) -169.5 (8) 84 (4) 97.5 (10) -85.5 (10) 0.0 177.1 (8) 0.0 0.0 0.0 O17—Si2B—C85B—C88B C84B—Si2B—C85B—C88B C83B—Si2B—C85B—C88B O17—Si2B—C85B—C87B C84B—Si2B—C85B—C87B C83B—Si2B—C85B—C87B C69—O19—C89—C90 C89B—O19—C89—C90 O19—C89—C90—C91 O19—C89—C90—C95 C95—C90—C91—C92 250 Table 3.11. (Continued) C43—C44—C45—C40 C41—C40—C45—C44 C39—C40—C45—C44 C19—O9—C39B—C40B C39—O9—C39B—C40B O9—C39B—C40B—C41B O9—C39B—C40B—C45B 0.0 0.0 -177.0 (8) -163 (2) -79 (4) 42 (4) -129 (2) C89—C90—C91—C92 C90—C91—C92—C93 C91—C92—C93—C94 C92—C93—C94—C95 C93—C94—C95—C90 C91—C90—C95—C94 C89—C90—C95—C94 C69—O19—C89B—C90B C89—O19—C89B—C90B O19—C89B—C90B—C91B O19—C89B—C90B—C95B -177.7 (15) 0.0 0.0 0.0 0.0 0.0 177 (2) -178.1 (14) 115 (7) -97.3 (14) 85.1 (17) C45B—C40B—C41B—C42B 0.0 C39B—C40B—C41B—C42B -170 (3) C40B—C41B—C42B—C43B 0.0 C41B—C42B—C43B—C44B 0.0 C42B—C43B—C44B—C45B 0.0 C43B—C44B—C45B—C40B 0.0 C41B—C40B—C45B—C44B 0.0 C39B—C40B—C45B—C44B 172 (2) C51—O11—N11—C69 N11—O11—C51—C68 N11—O11—C51—C52 C68—C51—C52—N13 1.7 (10) 1.8 (10) 175.3 (8) C95B—C90B—C91B—C92B 0.0 C89B—C90B—C91B—C92B -177.5 (14) C90B—C91B—C92B—C93B 0.0 C91B—C92B—C93B—C94B 0.0 C92B—C93B—C94B—C95B 0.0 C93B—C94B—C95B—C90B 0.0 C91B—C90B—C95B—C94B 0.0 -140.4 (10) C89B—C90B—C95B—C94B 177.8 (12) Table 3.12. Hydrogen-bond parameters D—H···A O6—H6···O5 D—H (Å) 0.84 H···A (Å) 1.78 1.96 D···A (Å) 2.458 (6) 2.474 (7) D—H···A (°) 136.3 118.9 O16—H16···O15 0.84 251 Figure 3.4a Figure 3.4b Figure 3.4. Perspective views showing 50% probability displacement (the H atoms that rides on C atoms and the disorder parts have been omitted). 252 b c Figure 3.5. Three-dimensional supramolecular architecture viewed along the a-axis direction. 253 Catalog of Spectra H N(CH 3) 2 O N TBSO O OTBS 68 OBn H N(CH 3) 2 O N TBSO O OTBS 68 OBn 254 I H N(CH 3 )2 O N O O OTBS 70 OBn I H N(CH 3 )2 O N O O OTBS 70 OBn 255 OH N(CH 3)2 H O N O O OTBS 71 OBn OH N(CH 3)2 H O N O O OTBS 71 OBn 256 F H N(CH 3 )2 O N O O OTBS 72 OBn F H N(CH 3 )2 O N O O OTBS 72 OBn 257 N3 H N(CH 3 )2 O N O O OTBS 73 OBn N3 H N(CH 3 )2 O N O O OTBS 73 OBn 258 OH N(CH 3)2 H O N O O OTBS 74 OBn OH N(CH 3)2 H O N O O OTBS 74 OBn 259 CH3 O H N(CH 3) 2 O N O O OTBS 76 OBn CH3O H N(CH 3) 2 O N O O OTBS 76 OBn 260 OCH3 O O H N(CH3 )2 O N O O OTBS 75 OBn OCH3 O O H N(CH3 )2 O N O O OTBS 75 OBn 261 N H N(CH3 )2 O N O O OTBS 80 OBn N(CH 3) 2 H F H N(CH 3)2 O N BocO O HO 82 O OTBS OBn 262 N(CH 3) 2 H F H N(CH 3)2 O N BocO O HO 82 O OTBS OBn N(CH 3) 2 H F H N(CH3)2 OH NH2 OH O O HO H O 107 O 263 N(CH 3) 2 H CH 3 N(CH 3)2 O H O N O OTBS OBn BocO O HO 83 N(CH 3) 2 H CH 3 N(CH 3)2 O H O N O OTBS OBn BocO O HO 83 264 N(CH 3) 2 H O CH 3 N(CH3 )2 H OH NH2 OH O O HO H O 86 O N(CH 3)2 H O CH 3 N(CH3 )2 H OH NH2 O2 N OH O HO O O H 87 O 265 N(CH 3)2 H N O N H H O CH 3 N(CH3)2 H OH NH2 t-Bu OH O HO O O H 89 O N(CH3 )2 CH 3 O CH3 N N H OH O H O CH3 N(CH3 )2 H OH NH 2 HO O O H 90 O 266 N(CH 3) 2 O N N H H O CH 3 N(CH 3) 2 H OH NH 2 OH O HO O O H 91 O F H F H N(CH 3)2 O N BocO O HO 92 O OTBS OBn 267 F H F H N(CH 3)2 O N BocO O HO 92 O OTBS OBn F H F H N(CH3)2 OH NH2 OH O O HO H O 93 O 268 F O (CH 3) 2N N H H F H N(CH3 )2 OH NH 2 OH O HO O O H 95 O F O N N H H F H N(CH3 )2 OH NH2 OH O HO O O H 96 O 269 N(CH 3)2 H N3 H N(CH3 )2 O N BnO O HO 97 O OTBS OBn N(CH 3)2 H N3 H N(CH3 )2 O N BnO O HO 97 O OTBS OBn 270 Boc N(CH 3 )2 NH N(CH 3) 2 H H O N BnO O HO 98 O OTBS OBn Boc N(CH 3 )2 NH N(CH 3) 2 H H O N BnO O HO 98 O OTBS OBn 271 N(CH 3) 2 H NH2 N(CH3 )2 H OH NH2 OH O O HO H O 99 O H3 C O N(CH3 )2 N(CH 3) 2 HN H H OH NH2 OH O O HO H O 100 O 272 N N(CH 3) 2 H H N(CH 3)2 O N BocO O HO O OTBS 101 OBn N(CH 3) 2 H NH 2 N(CH 3)2 H O N O OTBS 102 OBn BocO O HO 273 N(CH 3) 2 H NH 2 N(CH 3)2 H O N O OTBS 102 OBn BocO O HO N(CH 3) 2 H NH 2 N(CH3 )2 H OH NH2 OH O HO O O H 103 O 274 N(CH 3) 2 H OH N(CH3 )2 H OH NH2 OH O O HO H O 106 O 275 Chapter 4 Progress Toward the Synthesis of 5-Hetero-Tetracyclines 276 Introduction Gram-negative bacterial cells are bounded by two permeability barriers: (1) the cytoplasmic membrane, which is permeable to uncharged, lipophilic molecules; and (2) the outer membrane, which has a different constitution and is significantly less permeable to lipophilic molecules.97 Tetracyclines, like most antibacterials, penetrate the outer membrane of Gram-negative cells predominantly by passing through aqueous channels provided by porin proteins imbedded in the outer membrane. Antibacterial agents with activity against Gram-negative bacteria tend to have higher relative polar surface area (see Chapter 1 for discussion) and lower mean molecular mass than antibacterials which are only active against Gram-positive organisms. These requirements for Gram-negative activity are believed to be driven by the properties of porin proteins. OmpF is one of the porin proteins found in the outer membrane of Escherichia coli. The X-ray crystal structure of OmpF revealed a trimer of identical subunits, each consisting of a -barrel with 16 transmembrane -strands.98 At its most constricted (at about half the height of the barrel) the pore narrows to an ellipse of cross-section 11 x 7 Å. Pore diameter increases abruptly to 22 x 15 Å beyond the constriction zone (which is 97 98 Nikaido, H. Microbio. Mol. Biol. Rev. 2003, 67, 593-656. Cowan, S. W.; Schirmer, T.; Rummel, G.; Steiert, M.; Ghosh, R.; Pauptit, R. A.; Jansonius, J. N.; Rosenbusch, J. P. Nature 1992, 358, 727–733. 277 approximately 9 Å long). The constriction region of OmpK36, a porin found in Klebsiella pneumoniae, was found to be almost exactly the same size as that of OmpF.99 In theory, the introduction of new hydrogen bond-forming substituents on the tetracycline scaffold could make the displacement of water molecules associated with passage through porin channels less disfavored. Any structural decoration would (to lesser or greater extents) increase overall size of the molecule in one or more dimensions, which in the case of larger groups could disrupt movement of the small molecule through the constricted regions of porin proteins. We were led to consider the possibility that we could increase the polarity of tetracyclines without increasing size (and thereby disrupting movement through porin channels) by incorporating a heteroatom into the polyketide-derived carbocyclic scaffold. The incorporation of a heteroatom (oxygen or nitrogen) at position 5 on the tetracycline scaffold would add a hydrogen bonding substituent and increase the polar surface area of these antibacterial small molecules without significantly increasing molecular weight or size. In theory, this could make the displacement of water molecules associated with passage through porin channels less disfavored, thus making penetration of Gram-negative cells more efficient. For these reasons, we chose to consider the preparation of fully synthetic 5-oxo- and 5-aza-tetracyclines. Dutzler, R.; Rummel, G.; Alberti, S.; Hernández-Allés, S.; Phale, P. S.; Rosenbusch, J. P.; Benedi, V. J.; Schirmer, T. Structure 1999, 7, 425–434. 99 278 Unlike the - and -substituted AB precursors to fully synthetic 5a- and 5- substituted tetracyclines described in preceding chapters, it would not be possible to prepare “5-hetero” AB precursors directly from the AB enone 10. We instead considered synthesizing these modified AB components by adaptation of our third-generation synthesis of the AB enone 10.100 The third-generation synthesis comprises a five-step synthetic sequence from two starting materials of near equal complexity, as measured by the number of steps required to prepare each starting material. Asymmetry is transferred from the B-ring precursor 121 to C4 and C4a via a highly diastereoselective Michael– Claisen cyclization reaction with the sodium enolate of isoxazole precursor 120 (Scheme 4.1).101 Michael addition proceeds by addition to the sterically more accessible face of cyclohexenone 121 (along a pseudoaxial trajectory) and with complete control of relative stereochemistry at C4 and C4a. 100 101 Kummer, D. A.; Li, D.; Dion, A.; Myers, A. G. Chem. Sci. 2011, 2, 1710–1718. Stork reported the first synthesis of isoxazole ester 120 while developing the 5-benzyloxyisoxazole function as a protective group for the A ring of tetracyclines, an innovation that was critical to the development of the Myers tetracycline platform. For original work, see: (a) Stork, G.; Hagedorn, A. A. J. Am. Chem. Soc. 1978, 100, 3609–3611. (b) Stork, G.; LaClair, J. J.; Spargo, P.; Nargund, R. P.; Totah, N. J. Am. Chem. Soc. 1996, 118, 5304–5305. For an optimized protocol for the synthesis of isoxazole 120, see: Kummer, D. A. (2011) II. A Practical, Convergent Route to the Key Precursor to the Tetracycline Antibiotics. Ph.D. Thesis, Harvard University. 279 Scheme 4.1. Diastereoselective Michael–Claisen cyclization of components 120 and 121. Michael–Claisen cycloadduct 122 was transformed into the AB enone 10 by the following sequence (Scheme 4.2): (1) expulsion of cyclopentadiene from 122 by retroDiels–Alder fragmentation, (2) C12a-hydroxylation of 123 with 3-(4-nitrophenyl)-2(phenylsulfonyl)-oxaziridine (119),102 (3) C4-epimerization upon heating a solution of 124 in tetrahydrofuran–methanol with sodium dihydrogen phosphate, and (4) protection of the C12a-hydroxyl group of 125 as a tert-butyldimethylsilyl ether. The relative stereochemical outcome of the A-ring-forming cyclization described above did not match the stereochemistry of tetracyclines at C4, but this result proved advantageous as the dimethylamino group of intermediate 123 directs the approach of oxaziridine 119 to the sterically more accessible lower face, providing hydroxylation product 124 with the required stereochemistry at C12a. 102 Vishwakarma, L. C.; Stringer, O. D.; Davis, F. A. Organic Syntheses 1993, Coll. Vol. 8, 546. 280 Scheme 4.2. Synthesis of AB enone 10 from Michael–Claisen cycloadduct 122. It was well known prior to this work that various conditions could be used to effect epimerization at C4 of tetracyclines. Lederle scientists observed in 1955 that tetracycline is converted to a mixture of C4 stereoisomers in 1M sodium dihydrogen phosphate–methanol (2:1, 25 °C).103 It has also been demonstrated that a mixture of C4 epimers of sancycline (6-deoxy-6-demethyltetracycline) can be isomerized to provide predominantly the natural stereoisomer by treatment with calcium (II) chloride in a water-butanol mixture containing ethanolamine (pH 8.5, reflux).104 103 (a) Doerschuk, A. P.; Bitler, B. A.; McCormick, J. R. D. J. Am. Chem. Soc. 1955, 77, 4687. (b) McCormick, J. R. D.; Fox, S. M.; Smith, L. L.; Bitler, B. A.; Reichenthal, J.; Origoni, V. E.; Muller, W. H.; Winterbottom, R.; Doerschuk, A. P. J. Am. Chem. Soc. 1957, 79, 2849. M. M. Noseworthy. U.S. Patent 3,009,956, November 21, 1961. 104 281 Retrosynthetic Strategy and Background We envisioned preparing 5-hetero-tetracyclines using an iterative Michael– Claisen strategy, a conceptual approach that had been successful in the third-generation synthesis of the AB enone 10. 5-Hetero-tetracyclines could be formed by cyclization reactions of D-ring precursors with 5-hetero AB precursors (represented by structure 126, where X is oxygen or protected nitrogen; Scheme 4.3). 5-Hetero AB precursors could in turn be accessed via A-ring-forming Michael–Claisen cyclizations of isoxazole ester anions and heterocyclic B-ring precursors. Dihydro-4-pyranone 127 was selected as a potential precursor to the B ring of 5-oxo-tetracyclines. There is literature precedent for a diastereoselective conjugate addition reaction of the enantiomer of 127 with a thiol nucleophile, with addition occurring from the face opposite the isopropylidene substituent.105 An analogous stereochemical outcome in the cyclization we envisioned would provide the correct stereochemistry at C4a. Numerous challenges were anticipated in transforming the Michael–Claisen product 128 into AB precursor 129, including removal of the B-ring isopropylidene substituent, stereoselective C12a-hydroxylation and C4-epimerization. Efforts toward the synthesis of 5-oxo AB enone 129 are described below. 105 Witczak, Z. J.; Lorchak, D.; Nguyen, N. Carbohydrates Res. 2007, 342, 1929–1933. 282 Scheme 4.3. Retrosynthesis of 5-hetero-tetracyclines: an iterative Michael–Claisen cyclization strategy. There are a few examples in the literature that provide precedent for the Michael– Claisen cyclizations proposed in Scheme 4.3 above. Hauser described a regiospecific annulation reaction of the sulfone-stabilized phthalide anion derived from 130 with benzopyranone 131, affording Michael–Claisen cyclization product 132 in 27% yield (eq 1, Scheme 4.4).106 Tatsuta reported an efficient coupling reaction involving phthalide 133 and oxocyclic enone 134 (eq 2); oxidation of the cyclization product afforded quinone 135 in 64% yield over two steps.107 This is the only example of which we are aware of an 106 107 Hauser, F. M.; Hewawasam, P.; Baghdanov, V. M. J. Org. Chem. 1988, 53, 223-224. Tatsuta, K.; Tanaka, Y.; Kojima, M.; Ikegami, H. Chem. Lett. 2002, 14–15. 283 electrophile of this type (dihydro-4-pyranone) undergoing a successful Michael–Claisen reaction. There is also some precedent for cyclizations involving , -unsaturated lactone Michael acceptors (see eq 3 for an example).108 Scheme 4.4. Literature examples of Michael–Claisen and Michael–Dieckmann reaction sequences involving oxocyclic enones: benzopyranone, dihydro-4-pyranone and unsaturated lactone Michael acceptors. , - There are two examples in the literature of Michael–Claisen reactions sequences of benzylic anions and azacyclic enone electrophiles.109 In both examples the Michael 108 Examples of Michael–Claisen reactions involving , -unsaturated lactones as Michael acceptors: (a) Tatsuta, K.; Yamazaki, T.; Mase, T.; Yoshimoto, T. Tetrahedron Lett. 1998, 39, 1771–1772. (b) Braukmuller, S.; Brückner, R. Eur. J. Org. Chem. 2006, 2110–2118. (a) Alvarez, M.; Ajana, W.; Lopez-Calahorra, F.; Joule, J. A. J. Chem. Soc. Perkin Trans. 1 1994, 917919. (b) Ajana, W.; Lopez-Calahorra, F.; Joule, J. A.; Alvarez, M. Tetrahedron 1997, 53, 341-356. 109 284 acceptor is N-methoxycarbamoyl-4-quinolone (137). Reaction of 137 with the dithianestabilized benzylic anion derived from methyl ester 136 afforded Michael–Claisen product 138 in 64% yield (eq 1, Scheme 4.5). In addition, quinolone 137 was found to undergo a Michael–Claisen reaction with the phthalide anion of pyridine N-oxide 139, providing the product 140 following post-cyclization transformations (eq 2). Similar cyclization reactions of 4-pyridone and dihydro-4-pyridone electrophiles have not been reported as far as we are aware, but these azacyclic enones have been shown to undergo conjugate addition reactions with organometallic reagents.110,111 Scheme 4.5. Literature examples of Michael–Claisen cyclizations of benzylic anions and N-methoxycarbamoyl-4-quinolone (137). Examples of conjugate addition reactions of N-carbamoyl-4-pyridones: Dieter, R. K.; Guo, F. J. Org. Chem. 2009, 74, 3843–3848. Examples of conjugate addition reactions of a dihydro-4-pyridone electrophile: (a) Ye, X. M. et al. Bioorg. Med. Chem. Lett. 2010, 20, 2195–2199. (b) Jagt, R. B. C.; de Vries, J. G.; Feringa, B. L.; Minnaard, A. J. Org. Lett. 2005, 7, 2433–2435. 111 110 285 Results Before targeting 5-hetero AB precursors it was first necessary to investigate the feasibility of constructing the C ring of 5-hetero-tetracyclines by Michael–Claisen cyclization. The results of model systems are presented in Scheme 4.6 below. Addition of N-benzyloxycarbamoyl-dihydro-4-pyridone 141 (1 equiv)112 to a solution of the anion formed by LDA deprotonation of D-ring precursor 81 (2 equiv) in the presence of TMEDA (4 equiv) at –78 °C, followed by warming of the reaction mixture to –10 °C provided the desired Michael–Claisen product 142 in 64% yield after purification by flash-column chromatography. As far as we are aware, this is the first example of a Michael–Claisen reaction with a dihydro-4-pyridone as the conjugate acceptor. This reaction was also successful in the absence of TMEDA, although product 142 was formed in slightly lower yield. The analogous cyclization reaction of Nbenzyloxycarbamoyl-quinolone 143 was extremely efficient without TMEDA additive, affording Michael–Claisen cycloadduct 144 in 85% yield following purification.113 The feasibility our synthetic plan to access 5-hetero-tetracyclines using Michael–Claisen 112 N-Benzyloxycarbamoyl-dihydro-4-pyridone 141 was prepared from 4-piperidone monohydrate hydrochloride by treatment with benzyl chloroformate (1 equiv) and sodium hydroxide (2 equiv) followed by oxidation of the acylation product, N-benzyloxy-4-piperidone, with o-iodoxybenzoic acid (IBX) in the presence of 4-methylmorpholine N-oxide. For oxidation conditions, see: Nicolaou, K. C.; Gray, D. L. F.; Montagnon, T.; Harrison, S. T. Angew. Chem. Int. Ed. 2002, 41, 996–1000. N-Benzyloxycarbamoyl-4-quinolone 143 was prepared from 2’-nitroacetophenone according to procedures detailed in the following literature precedents: (a) Tois, J.; Vahermo, M.; Koskinen, A. Tetrahedron Lett. 2005, 46, 735–737. (b) Shintani, R.; Yamagami, T.; Kimura, T.; Hayashi, T. Org. Lett. 2005, 7, 5317–5319. 113 286 chemistry was further affirmed by a successful cyclization reaction of D-ring precursor 81 with dihydro-4-pyranone 145,114 providing the desired product 146 in 39% yield. Scheme 4.6. Michael–Claisen cyclization reactions of D-ring precursor 81 with dihydro4-pyridone, 4-quinolone and dihydro-4-pyranone electrophiles. Once the feasibility of constructing the C ring of 5-hetero-tetracyclines using Michael–Claisen cyclizations had been established, synthetic approaches to 5-hetero AB precursors could be considered and developed. Heterocyclic enone 127 was identified as a possible precursor to the B ring of 5-oxo AB enone 129 (see Scheme 4.3 above). This potential B-ring precursor (127) was prepared as a single enantiomer via an efficient For the preparation of dihydro-4-pyranone 145, see: (a) Paquette, L. A.; Oplinger, J. A. Tetrahedron 1989, 45, 107–124. (b) Smith, A. B.; Fukui, M. J. Am. Chem. Soc. 1987, 109, 1269–1272. 114 287 three-step sequence adapted from literature precedent (Scheme 4.7). D-(–)-Arabinose (147) was converted into the corresponding bis-O-isopropylidene derivative 148 upon prolonged stirring with a catalytic quantity of tetrabutylammonium tribromide in dry acetone (80% yield, 2 d, 12-g batch).115 Treatment of bis-O-isopropylidene 148 with an excess of lithium diisopropylamide afforded allylic alcohol 149 as the product of a lithiation–elimination sequence.116 In a significant improvement upon a literature procedure, oxidation of allylic alcohol 149 was achieved rapidly and efficiently with tetrapropylammonium perruthenate (10 mol %) and N-methylmorpholine N-oxide (NMO, 1.5 equiv), providing the potential B–ring precursor 127 in 75% yield.117 Scheme 4.7. Synthesis of heterocyclic enone 127, a possible B-ring precursor. 115 116 117 Khan, A. T.; Khan, M. M.; Adhikary, A. Carbohydrates Res. 2011, 346, 673–677. Klemer, A.; Jung, G. Chem. Ber. 1981, 114, 1192–1195. Ley, S. V.; Norman, J.; Griffith, W. P.; Marsden, S. P. Synthesis 1994, 7, 639–666. 288 The next challenge was to construct the A ring of 5-oxo-tetracyclines by a Michael–Claisen reaction of B-ring precursor 127 with isoxazole ester anions known from prior research to be effective nucleophiles in analogous A-ring-forming cyclizations. Attempted cyclization of 127 with the anion derived from methyl ester isoxazole 120 was not successful. The major product of this cyclization attempt was compound 152 (Scheme 4.8). A possible mechanism for the formation of 152 is presented below. This product could be formed by Michael addition of the isoxazole ester anion (to form enolate 150) followed by sequential B-ring opening, expulsion of acetone (to give alkoxide 151), tautomerization and cyclization to form the B ring. No Michael– Claisen products were observed in this reaction, indicating that the energy barrier for Claisen cyclization is higher than that for B-ring opening of enolate intermediate 150. Scheme 4.8. Unsuccessful A-ring cyclization attempt with isoxazole methyl ester 120. 289 It was hoped that this problem could be overcome by increasing the electrophilicity of the isoxazole ester. Addition of B-ring precursor 127 (1 equiv) to a solution of the anion formed by NaHMDS deprotonation of the phenyl ester isoxazole 153 at –78 °C, followed by warming of the reaction mixture to –15 °C and stirring at this temperature for 2 ½ h afforded the Michael–Claisen product 128 in 40% yield as a single diastereomer after purification by flash-column chromatography and rp-HPLC (Scheme 4.9 below). NMR analysis revealed trace amounts of by-products in the crude product mixture, but no diastereomers of 128 were isolated following purification. The stereochemical outcome of the cyclization is homologous with the outcome of the A-ringforming cyclization reaction in the third-generation synthesis of the AB enone 10.100 Scheme 4.9. Synthesis of trimethylsilyl ether 155 via sequential diastereoselective reactions: Michael–Claisen coupling of isoxazole phenyl ester 153 and B-ring precursor 127, followed by C12a-hydroxylation. 290 With Michael–Claisen product 128 in hand, we next sought to introduce hydroxylation at C12a using optimized conditions that were developed for the thirdgeneration synthesis of the AB enone 10. It was not known whether the isopropylidene substituent would block approach of the electrophilic oxidant from the lower face, potentially eroding the stereoselectivity observed in the third-generation synthesis. Fortuitously, addition of lithium tert-butoxide to a THF solution of 128 and 3-(4nitrophenyl)-2-(phenylsulfonyl)-oxaziridine (119)102 at –40 °C followed by warming of the reaction mixture to –5 °C afforded the desired C12a-hydroxylated compound 154 as the only major product (Scheme 4.9 above). Partial purification of 154 followed by protection of the C12a-hydroxyl group as a trimethylsilyl ether by treatment with 1(trimethylsilyl)imidazole (5 equiv) at 0 °C afforded compound 155 in 43% yield over two steps. The relative stereochemical outcomes of all reactions in this synthetic sequence were confirmed by an X-ray crystal structure of C12a-hydroxylation product 154 (Figure 4.1). Figure 4.1. X-ray crystal structure of C12a-hydroxylation product 154. 291 Two significant challenges remained in order to convert trimethylsilyl ether 155 (or the C12a-hydroxy compound 154) into an AB precursor to 5-oxo-tetracyclines: (1) inversion of C4 stereochemistry, and (2) removal of the B-ring “chiral auxiliary” and formation of an enone. Initial investigations indicate that C4-epimerization may be an intractable problem in this system. Heating of a solution of trimethylsilyl ether 155 in aqueous sodium dihydrogen phosphate, methanol and tetrahydrofuran at 60 °C quickly led to formation of a dark red reaction mixture. Following work-up and purification it was found that the starting material had been cleanly converted to a red solid which was characterized as diketone 156 (70% yield, Scheme 4.10). Interestingly, graduate researcher Fan Liu observed formation of the same decomposition product (156, 92% yield) from piperidone 157 (a possible precursor to 5-aza-tetracyclines) under identical conditions. Scheme 4.10. Attempts to achieve C4-epimerization led to decomposition. 292 Attempts to achieve C4-epimerization using both acidic and basic conditions have led to the formation of diketone 156. The highly distinctive dark red color of 156 has been observed upon formation of even small amounts of this product in various reaction mixtures. Treatment of substrates 154 and 155 with bases such as DBU and phosphazene P4-t-Bu in various solvents with different reaction temperatures either provided recovered starting material or led to decomposition. An attempt to form an extended C1–C4 enolate by treatment of a THF solution of 155 with an excess of NaHMDS at –78 °C afforded recovered starting material. The characteristic dark red color of diketone 156 was also observed upon prolonged exposure of 12a-hydroxylation product 154 to silica gel, and upon attempts to purify this compound by reverse-phase HPLC (methanol–water solvent system). A possible mechanism for the formation of 156 from various precursors to 5hetero AB enones is presented in Scheme 4.11. N(CH3)2 O N O O OP OBn O OH OP X N(CH3)2 O N OBn X H H N(CH3)2 O N O O 156 OBn PO [O] N(CH3)2 O N OH OBn XH N(CH3)2 O N O O OP OBn Scheme 4.11. Possible mechanism for the formation of decomposition product 156. 293 Despite these failures, we also sought to find conditions for removal of the acetonide functionality and formation of a B-ring enone (Scheme 4.12 below). Addition of boron trichloride to a solution of acetonide 155 in dichloromethane at –78 °C followed by warming to 0 °C provided diol 158 (29%) and enol 159 (32%). Treatment of enol 159 with trifluoromethanesulfonic anhydride (Tf2O, 1.5 equiv) and 2,6-lutidine afforded enone triflate 160 in 56% yield following purification by flash-column chromatography. Diol 158 could also be converted to 160 using an excess of Tf2O (2.5 equiv) and 2,6lutidine (5 equiv). A dark red color was observed quickly following purification of enone triflate 160, and complete decomposition to diketone 156 occurred upon standing overnight at 23 °C under an inert atmosphere. An attempt to achieve palladium-catalyzed reduction of triflate 160 immediately following purification also led to decomposition. Scheme 4.12. Synthesis of enone triflate 160. 294 Conclusion The chemical innovations described herein have established the viability of an iterative Michael–Claisen strategy for the synthesis of 5-hetero-tetracyclines. The substrate scope of the Michael–Claisen cyclization reaction has been expanded to include new heterocyclic enone electrophiles such as dihydro-4-pyridones. 295 Experimental Section Michael–Claisen cyclization product 142. A freshly prepared solution of lithium diisopropylamide in tetrahydrofuran (1.0 M, 418 µL, 0.418 mmol, 2.1 equiv) was added dropwise via syringe to a solution of phenyl ester 81 (138 mg, 0.398 mmol, 2.0 equiv) and TMEDA (120 µL, 0.796 mmol, 4.0 equiv) in tetrahydrofuran (2.5 mL) at –78 °C, forming a bright red solution. After stirring at –78 °C for 45 min, a solution of Nbenzyloxycarbamoyl-2,3-dihydro-4-pyridone 141 (46 mg, 0.199 mmol, 1 equiv) in tetrahydrofuran (0.5 ml) was added to the reaction solution dropwise via syringe. The resulting mixture was stirred at –78 °C for 10 min, then was allowed to warm slowly to – 10 °C over 50 min. Aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 25 mL) was then added to the reaction solution and the resulting mixture was allowed to warm to 23 °C. The product mixture was extracted with ethyl acetate (20 mL, then 10 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The product was purified by flash-column chromatography (12% ethyl acetate–hexanes), providing Michael–Claisen cyclization product 142 as a pale yellow solid (60 mg, 62%). Rf = 0.19 (15% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 15.32 (s, 1H), 7.38-7.33 (m, 5H), 7.24 (app t, 1H, J = 8.8 Hz), 7.08 (dd, 1H, J = 8.8, 3.9 Hz), 5.20 (AB 296 quartet, 2H), 4.88 (brd, 1H, J = 9.8 Hz), 4.41 (brs, 1H), 3.46 (brd, 1H, J = 13.7 Hz), 3.11 (app t, 1H, J = 12.7, 11.7 Hz), 2.61 (app t, 2H, J = 14.6, 13.7 Hz), 2.38 (brd, 1H, J = 17.6 Hz), 1.58 (s, 9H); 13C NMR (125 MHz, CDCl3) 184.2, 180.8, 157.3 (d, J = 245.3 Hz), 154.8, 151.5, 146.5 (d, J = 2.7 Hz), 136.1, 128.6, 128.2, 128.1, 127.7 (d, J = 19.2 Hz), 125.1 (d, J = 3.7 Hz), 123.4 (d, J = 8.2 Hz), 120.4 (d, J = 23.8 Hz), 110.6, 105.9, 84.0, 67.6, 48.9, 37.4, 31.9, 27.7; FTIR (neat film), 1761 (m), 1701 (m), 1225 (s), 1144 (s), 733 (s) cm–1; HRMS–ESI (m/z): [M+Na]+ calcd for C26H26FNNaO7, 506.1591; found, 506.1549. 297 F CH3 CO2Ph BocO 81 O N OBn LDA –78 O 143 –10 °C F O N OBn BocO O HO 144 Michael–Claisen cyclization product 144. A freshly prepared solution of lithium diisopropylamide in tetrahydrofuran (1.0 M, 421 µL, 0.421 mmol, 2.1 equiv) was added dropwise via syringe to a solution of phenyl ester 81 (139 mg, 0.401 mmol, 2.0 equiv) in tetrahydrofuran (2.5 mL) at –78 °C, forming a bright red solution. After stirring at –78 °C for 35 min, a solution of N-benzyloxycarbamoyl-quinolone 143 (56 mg, 0.201 mmol, 1 equiv) in tetrahydrofuran (0.5 ml) was added to the reaction solution dropwise via syringe. The resulting mixture was stirred at –78 °C for 10 min, then was allowed to warm slowly to –10 °C over 75 min. Aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 25 mL) was then added to the reaction solution and the resulting mixture was allowed to warm to 23 °C. The product mixture was extracted with ethyl acetate (2 × 30 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The product was purified by flash-column chromatography (8% ethyl acetate–hexanes), providing Michael–Claisen cyclization product 144 as an orangeyellow solid (91 mg, 85%). Rf = 0.37 (15% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 14.59 (s, 1H), 7.86 (d, 1H, J = 7.8 Hz), 7.74 (d, 1H, J = 8.8 Hz), 7.42-7.33 (m, 6H), 7.24 (app t, 1H, J = 8.8, 7.8 Hz), 7.14 (app t, 1H, J = 8.8, 7.8 Hz), 7.09 (dd, 1H, J = 8.8, 3.9 Hz), 5.51 (dd, 1H, J = 298 12.7, 3.9 Hz), 5.30 (AB quartet, 2H), 3.50 (dd, 1H, J = 14.6, 3.9 Hz), 2.92 (app t, 1H, J = 14.6, 13.6 Hz), 1.60 (s, 9H); 13C NMR (125 MHz, CDCl3) 182.8, 166.8, 157.3 (d, J = 245.3 Hz), 154.4, 151.5, 146.3 (d, J = 2.7 Hz), 138.1, 135.4, 132.8, 128.6, 128.4, 128.2, 126.6 (d, J = 19.2 Hz), 125.6 (d, J = 3.7 Hz), 124.8, 124.2, 123.8, 123.6 (d, J = 8.2 Hz), 121.8, 120.7 (d, J = 24.7 Hz), 107.0, 84.1, 68.4, 53.3, 29.6, 27.7; FTIR (neat film), 1759 (m), 1713 (m), 1285 (s), 1265 (s), 1225 (s), 1140 (s) cm–1; HRMS–ESI (m/z): [M+Na]+ calcd for C30H26FNNaO7, 554.1586; found, 554.1571. 299 Michael–Claisen cyclization product 146. A freshly prepared solution of lithium diisopropylamide in tetrahydrofuran (1.0 M, 171 µL, 0.171 mmol, 2.1 equiv) was added dropwise via syringe to a solution of phenyl ester 81 (56.5 mg, 0.163 mmol, 2.0 equiv) and TMEDA (49.2 µL, 0.326 mmol, 4.0 equiv) in tetrahydrofuran (1.25 mL) at –78 °C, forming a bright red solution. After stirring at –78 °C for 30 min, a solution of 2,3dihydro-4-pyranone 145 (8 mg, 0.082 mmol, 1 equiv) in tetrahydrofuran (0.25 ml) was added to the reaction solution dropwise via syringe. The resulting mixture was stirred at – 78 °C for 5 min, then was allowed to warm slowly to –10 °C over 55 min. Aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 15 mL) was then added to the reaction solution and the resulting mixture was allowed to warm to 23 °C. The product mixture was extracted with dichloromethane (2 × 15 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The product was purified by flashcolumn chromatography (9% ethyl acetate–hexanes, grading to 12%), providing Michael–Claisen cyclization product 146 as a pale orange-yellow solid (11 mg, 39%). Rf = 0.29 (15% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 15.37 (s, 1H), 7.22 (app t, 1H, J = 8.8, 7.8 Hz), 7.05 (dd, 1H, J = 8.8, 4.9 Hz), 4.56 (dd, 1H, J = 12.7, 4.8 Hz), 4.21 (dd, 1H, J = 11.7, 7.8 Hz), 3.82 (app td, 1H, J = 11.7, 3.9 Hz), 3.43 (dd, 1H, J = 15.6, 5.9 Hz), 2.85-2.78 (m, 1H), 2.62 (app t, 1H, J = 14.7, 13.7 Hz), 2.35 (dd, 1H, J = 300 18.6, 3.9 Hz), 1.57 (s, 9H); 13 C NMR (125 MHz, CDCl3) 182.4, 181.3, 157.1 (d, J = 245.3), 151.5, 146.1 (d, J = 2.7 Hz), 126.4 (d, J = 19.2 Hz), 125.1 (d, J = 3.7 Hz), 123.3 (d, J = 8.3 Hz), 120.2 (d, J = 23.8 Hz), 108.5, 84.0, 70.5, 63.5, 31.5, 27.9 (d, J = 2.7 Hz), 27.7; FTIR (neat film), 1759 (m), 1614 (w), 1279 (m), 1225 (s), 1144 (s) cm–1; HRMS– ESI (m/z): [M+H]+ calcd for C18H19FNaO6, 373.1063; found, 373.1025. 301 HO HO O OH OH 147 n-Bu4NBr3 O O O O O O 148 Bis-O-isopropylidene 148.118 Tetrabutylammonium tribromide (1.29 g, 2.66 mmol, 0.04 equiv) was added in one portion to a white suspension of D-(–)-arabinose (147, 10.0 g, 66.6 mmol, 1 equiv) in dry acetone (250 mL, dried over anhydrous calcium sulfate) at 23 °C. The resulting mixture was stirred at 23 °C for 2 d, whereupon triethylamine (1.0 mL) was added carefully. The yellow product solution was concentrated. The crude product was purified by flash-column chromatography (2% acetone–hexanes, grading to 5%), affording bis-O-isopropylidene 148 as a white solid (12.3 g, 80%). 1H NMR data for 148 closely matched that reported in the literature.119 118 This procedure is adapted from that of Khan et al.: Khan, A. T.; Khan, M. M.; Adhikary, A. Carbohydrates Res. 2011, 346, 673–677. Pedatella, S.; Guaragna, A.; D’Alonzo, D.; De Nisco, M.; Palumbo, G. Synthesis 2006, 2, 305–308. 119 302 Allylic alcohol 149.120 A round-bottomed flask containing a solution of bis-Oisopropylidene 148 (13.8 g, 59.9 mmol, 1 equiv) in tetrahydrofuran (500 mL) was placed in a cooling bath containing an ice–water mixture. A commercial solution of lithium diisopropylamide (2.0 M in tetrahydrofuran–heptane–ethylbenzene, 102 mL, 204 mmol, 3.4 equiv) was added carefully via cannula over 20 min to the cooled starting material solution. The resulting mixture was stirred for 30 min, whereupon the cooling bath was removed. The reaction mixture was allowed to warm to 23 °C. After stirring at this temperature for 6 h, the reaction flask was placed in a cooling water bath and water (400 mL) was added carefully. The cooling bath was removed and the product mixture was poured into a separatory funnel containing chloroform (400 mL). The phases were separated and the aqueous phase was further extracted with chloroform (400 mL, then 200 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The product was purified by flash-column chromatography (20% ethyl acetate–hexanes, grading to 25%), providing allylic alcohol 149 as a pale yellow solid (5.05 g, 49%). This procedure is adapted from that of Klemer et al.: Klemer, A.; Jung, G. Chem. Ber. 1981, 114, 1192– 1195. 120 303 Rf = 0.22 (30% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 6.34 (d, 1H, J = 6.3 Hz), 5.44 (d, 1H, J = 2.9 Hz), 4.96-4.93 (m, 1H), 4.20-4.18 (m, 2H), 2.33 (d, 1H, J = 4.9 Hz), 1.45 (s, 3H), 1.39 (s, 3H); 13 C NMR (125 MHz, CDCl3) 143.2, 111.0, 99.0, 93.4, 77.9, 60.3, 27.7, 25.8; FTIR (neat film), 3431 (br), 1651 (m), 1227 (s), 1076 (s), 1022 (s) cm–1. 304 Dihydro-4-pyranone 127. Tetrapropylammonium perruthenate (343 mg, 0.976 mmol, 0.1 equiv) was added portionwise over 5 min to a cooled mixture (ice–water cooling bath) of allylic alcohol 149 (1.68 g, 9.76 mmol, 1 equiv), N-methylmorpholine N-oxide (1.72 g, 14.6 mmol, 1.5 equiv) and powdered 4Å molecular sieves in anhydrous dichloromethane (25 mL). The cooling bath was removed and the reaction mixture was allowed to warm to 23 °C. After stirring at this temperature for 20 min, the reaction mixture was filtered through a thick pad of silica gel, washing through with ethyl acetate. The filtrate was concentrated. The crude product was purified by flash-column chromatography (20% ethyl acetate–hexanes), affording dihydro-4-pyranone 127 as a very pale yellow solid (1.25 g, 75%). Rf = 0.26 (30% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 7.25 (d, 1H, J = 6.0 Hz), 5.88 (d, 1H, J = 3.7 Hz), 5.43 (d, 1H, J = 6.4 Hz), 4.19 (d, 1H, J = 2.7 Hz), 1.52 (s, 3H), 1.43 (s, 3H); 13C NMR (125 MHz, CDCl3) 186.3, 160.7, 113.3, 103.8, 101.0, 76.7, 27.4, 25.7; FTIR (neat film), 1678 (s), 1599 (s), 1223 (s), 1032 (s) cm–1. 305 Methyl ester 152. A 1.0 M solution of sodium bis(trimethylsilyl)amide in tetrahydrofuran (197 µL, 0.197 mmol, 2.1 equiv) was added dropwise via syringe to a solution of methyl 3-benzyloxy-5-(dimethylaminomethyl)isoxazole-4-carboxylate 120 (54.6 mg, 0.188 mmol, 2.0 equiv) in tetrahydrofuran (1.5 mL) at –78 °C (dry ice–acetone bath). The resulting yellow solution was stirred at this temperature for 5 min, then was allowed to warm to –20 °C (dry ice–acetonitrile bath). After stirring at –20 °C for 30 min, the reaction flask was placed in a dry ice–acetone bath at –78 °C. After stirring at this temperature for a further 5 min, a solution of B-ring precursor 127 (16.0 mg, 0.094 mmol, 1 equiv) in tetrahydrofuran (0.3 mL) was added slowly to the isoxazole anion solution at –78 °C. The reaction mixture was stirred at this temperature for 5 min, then was allowed to warm slowly to 23 °C over 70 min. Aqueous potassium phosphate buffer solution (pH 7.0, 0.2 M, 10 mL) and dichloromethane (10 mL) were added in sequence and the phases were separated. The aqueous phase was extracted with dichloromethane (10 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The product was purified by flash-column chromatography (25% ethyl acetate–hexanes, grading to 30%), affording methyl ester 152 as a yellow solid (14.0 mg, 37%). 306 Rf = 0.09 (30% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 7.49 (d, 2H, J = 7.8 Hz), 7.42-7.35 (m, 3H), 7.20 (s, 1H), 5.36 (s, 2H), 5.34 (brs, 1H), 4.94-4.89 (m, 1H), 4.80 (d, 1H, J = 9.8 Hz), 3.86 (s, 3H), 2.98 (dd, 1H, J = 17.6, 3.5 Hz), 2.74 (dd, 1H, J = 17.6, 14.7 Hz), 2.26 (s, 6H); 13C NMR (125 MHz, CDCl3) 187.5, 174.2, 168.8, 161.6, 145.0, 136.7, 135.4, 128.6, 128.4, 127.8, 104.3, 76.8, 72.0, 62.3, 51.9, 42.1, 38.2; FTIR (neat film), 1717 (m), 1674 (w), 1634 (w), 1613 (m), 1508 (m), 1173 (s), 1113 (s), 733 (s) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C19H23N2O6, 403.1500; found, 403.1524. 307 N(CH3)2 O O O 127 O PhO O 153 O N OBn –78 –15 °C NaHMDS O O O H N(CH3)2 O N O HO 128 OBn Michael–Claisen cyclization product 128. A 1.0 M solution of sodium bis(trimethylsilyl)amide in tetrahydrofuran (12.1 mL, 12.1 mmol, 2.05 equiv) was added dropwise via syringe to a solution of phenyl 3-benzyloxy-5- (dimethylaminomethyl)isoxazole-4-carboxylate 153 (4.14 g, 11.8 mmol, 2.0 equiv) in tetrahydrofuran (100 mL) at –78 °C (dry ice–acetone bath). The resulting brownish yellow solution was stirred at this temperature for 5 min, then was allowed to warm to – 30 °C (dry ice–acetonitrile bath). After stirring at –30 °C for 40 min, the reaction flask was placed in a dry ice–acetone bath at –78 °C. After stirring at this temperature for a further 5 min, a solution of B-ring precursor 127 (1.00 g, 5.88 mmol, 1 equiv) in tetrahydrofuran (7.0 mL) was added slowly to the orange isoxazole anion solution at –78 °C. The reaction mixture was stirred at this temperature for 5 min, then was allowed to warm slowly to –15 °C over 80 min. After stirring at –15 °C for a further 2 h, saturated aqueous ammonium chloride solution (30 mL) was added. The cooling bath was removed and the reaction flask was allowed to warm to 23 °C. Water (150 mL) and ethyl acetate (200 mL) were added and the phases were separated. The aqueous phase was extracted with ethyl acetate (200 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The product was purified first by flash-column chromatography (18% acetone–hexanes, grading to 24%), then by preparative HPLC on an Agilent Prep 308 C18 column [10 µm, 250 x 21.2 mm, UV detection at 350 nm, solvent A: water, solvent B: methanol, gradient elution with 70–90% B over 50 min, flow rate: 15 mL/min, 5 batches]. Fractions eluting 14–20 min were collected and concentrated, providing the Michael–Claisen cyclization product 128 as a yellow solid (1.00 g, 40%). Rf = 0.15 (25% acetone–hexanes); 1H NMR (500 MHz, CDCl3) 13.62 (s, 1H), 7.49 (d, 2H, J = 7.8 Hz), 7.40–7.34 (m, 3H), 5.96 (d, 1H, J = 4.9 Hz), 5.37 (s, 2H), 5.10 (d, 1H, J = 6.8 Hz), 4.54 (d, 1H, J = 4.9 Hz), 4.22 (d, 1H, J = 7.8 Hz), 2.32 (s, 6H), 1.42 (s, 6H); 13 C NMR (125 MHz, CDCl3) 182.3, 175.5, 167.3, 167.2, 134.8, 128.6, 128.6, 128.3, 109.5, 108.9, 106.5, 98.4, 72.6, 68.7, 64.6, 58.1, 42.2, 27.0, 26.8; FTIR (neat film), 2932 (w), 1699 (s), 1649 (s), 1510 (s), 1250 (s), 1125 (s), 836 (s) cm–1; HRMS–ESI (m/z): [M+H]+ calcd for C22H24N2O7, 429.1662; found, 429.1656. 309 Trimethylsilyl ether 155. A commercial solution of lithium tert-butoxide in tetrahydrofuran (1.0 M, 305 µL, 0.305 mmol, 0.3 equiv) was added dropwise via syringe to a stirred suspension of 3-(4-nitrophenyl)-2-(phenylsulfonyl)-oxaziridine102 (404 mg, 1.32 mmol, 1.3 equiv) and the Michael–Claisen cyclization product 128 (435 mg, 1.02 mmol, 1 equiv) in tetrahydrofuran (6.0 mL) at –40 °C. The resulting mixture was allowed to warm slowly to –5 °C over 30 min. After stirring at –5 °C for 1 h, saturated aqueous sodium bicarbonate solution (40 mL) was added and the product was extracted with ethyl acetate (2 x 40 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The product was partially purified by flash-column chromatography (30% ethyl acetate–hexanes), affording impure tertiary alcohol 154 (mass of impure product = 395 mg). The impure hydroxylation product 154 was dissolved in dichloromethane (5.0 mL) and the resulting solution was cooled to 0 °C. 1-(Trimethylsilyl)imidazole (652 µL, 4.44 mmol, 5.0 equiv) was added dropwise to the cooled solution of hydroxylation product 154. After stirring at 0 °C for 1 h, the reaction mixture was diluted with dichloromethane (10 mL) and saturated aqueous sodium bicarbonate solution (10 mL) was added dropwise over 5 min with an ice–water cooling bath. The resulting mixture was allowed to warm to room temperature whereupon dichloromethane (10 mL) and 310 water (10 mL) were added. The phases were separated and the aqueous phase was extracted with dichloromethane (20 mL). The organic extracts were combined and the combined solution was dried over anhydrous sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The crude product was purified by flash-column chromatography (12% ethyl acetate–hexanes, grading to 15%), providing trimethylsilyl ether 155 as a pale yellow solid (223 mg, 43% over two steps). Rf = 0.18 (15% ethyl acetate-hexanes); 1H NMR (500 MHz, CDCl3) 7.46 (d, 2H, J = 7.3 Hz), 7.38-7.32 (m, 3H), 5.74 (d, 1H, J = 5.0 Hz), 5.37 (s, 2H), 4.79 (d, 1H, J = 2.7 Hz), 4.48 (d, 1H, J = 5.5 Hz), 4.45 (d, 1H, J = 3.2 Hz), 2.62 (s, 6H), 1.43 (s, 3H), 1.41 (s, 3H), 0.08 (s, 9H); 13 C NMR (125 MHz, CDCl3) 200.9, 186.6, 179.4, 168.0, 134.8, 128.5, 128.5, 128.1, 110.0, 105.1, 99.4, 86.4, 77.4, 76.5, 72.4, 59.2, 43.3, 27.4, 26.9, 1.7; FTIR (neat film), 1753 (m), 1703 (m), 1512 (s), 1157 (s), 1072 (s), 849 (s) cm–1; HRMS– ESI (m/z): [M+H]+ calcd for C25H33N2O8Si, 517.2001; found, 517.2022. 311 X-Ray Crystallography (C12a-hydroxylation product 154): A crystal mounted on a diffractometer was collected data at 180 K. The intensities of the reflections were collected by means of a Bruker APEX II DUO CCD diffractometer (CuK radiation, =1.54178 Å), and equipped with an Oxford Cryosystems nitrogen flow apparatus. The collection method involved 1.0 scans in at 30 , 55 , 80 and 115 in 2 . Data integration down to 0.84 Å resolution was carried out using SAINT V7.46 A with reflection spot size optimization.121 Absorption corrections were made with the program SADABS.121 The structure was solved by the direct methods procedure and refined by least-squares methods again F2 using SHELXS-97 and SHELXL-97.122 Non-hydrogen atoms were refined anisotropically, and hydrogen atoms were allowed to ride on the respective atoms. Crystal data as well as details of data collection and refinement are summarized in Table 4.1, geometric parameters are shown in Table 4.2, and hydrogenbond parameters are listed in Table 4.3. The Ortep plots produced with SHELXL-97 program, and the other drawings were produced with Accelrys DS Visualizer 2.0.123 121 122 123 Bruker AXS APEX II, Bruker AXS, Madison, Wisconsin, 2009. G. M. Sheldrick, Acta Cryst. 2008, A64, 112-122. Accelrys DS Visualizer v2.0.1, Accelrys Software. Inc., 2007. 312 Table 4.1. Experimental details V-PMW-526 Crystal data Chemical formula Mr Crystal system, space group Temperature (K) a, b, c (Å) (°) V (Å3) Z Radiation type µ (mm-1) Crystal size (mm) Data collection Diffractometer Absorption correction Bruker D8 goniometer with CCD area detector diffractometer Multi-scan SADABS C22H24N2O8 444.43 Monoclinic, P21 100 9.5161 (2), 17.6610 (3), 13.0462 (2) 107.255 (1) 2093.91 (7) 4 Cu K 0.91 0.03 × 0.01 × 0.01 313 Table 4.1. (Continued) Tmin, Tmax 0.973, 0.991 No. of measured, independent 33387, 6774, 6441 and observed [I > 2 (I)] reflections Rint Refinement R[F2 > 2 (F2)], wR(F2), S No. of reflections No. of parameters No. of restraints H-atom treatment max, 0.038 0.028, 0.066, 1.05 6774 593 1 H atoms treated by a mixture of independent and constrained refinement 0.17, -0.15 Flack H D (1983), Acta Cryst. A39, 876-881 0.02 (10) min (e Å-3) Absolute structure Flack parameter Computer programs: APEX2 v2009.3.0 (Bruker-AXS, 2009), SAINT 7.46A (BrukerAXS, 2009), SHELXS97 (Sheldrick, 2008), SHELXL97 (Sheldrick, 2008), Bruker SHELXTL (Sheldrick, 2008). 314 Table 4.2. Geometric parameters (Å, º) C1—N1 C1—O1 C1—C2 C2—C11 C2—C3 C3—O2 C3—C4 C4—O3 C4—C5 C4—C9 C5—O4 C5—C6 C6—O5 C6—C8 C6—H6 1.307 (2) 1.328 (2) 1.429 (3) 1.360 (3) 1.447 (3) 1.217 (2) 1.549 (3) 1.408 (2) 1.521 (2) 1.542 (2) 1.202 (2) 1.527 (3) 1.407 (2) 1.531 (2) 1.0000 C31—N3 C31—O11 C31—C32 C32—C41 C32—C33 C33—O12 C33—C34 C34—O13 C34—C35 C34—C39 C35—O14 C35—C36 C36—O15 C36—C38 C36—H36 1.315 (2) 1.328 (2) 1.423 (3) 1.351 (3) 1.460 (2) 1.213 (2) 1.546 (3) 1.407 (2) 1.526 (3) 1.551 (2) 1.205 (2) 1.519 (3) 1.404 (2) 1.523 (2) 1.0000 315 Table 4.2. (Continued) C7—O5 C7—O6 C7—C20 C7—C19 C8—O7 C8—O6 C8—H8 C9—O7 C9—C10 C9—H9 C10—N2 C10—C11 C10—H10 C11—O8 C12—O1 C12—C13 1.434 (2) 1.446 (2) 1.505 (3) 1.511 (3) 1.394 (2) 1.416 (2) 1.0000 1.429 (2) 1.550 (2) 1.0000 1.464 (2) 1.501 (3) 1.0000 1.330 (2) 1.456 (2) 1.502 (3) C37—O15 C37—O16 C37—C49 C37—C50 C38—O17 C38—O16 C38—H38 C39—O17 C39—C40 C39—H39 C40—N4 C40—C41 C40—H40 C41—O18 C42—O11 C42—C43 1.442 (2) 1.446 (2) 1.501 (3) 1.514 (3) 1.398 (2) 1.423 (2) 1.0000 1.425 (2) 1.549 (2) 1.0000 1.472 (2) 1.494 (3) 1.0000 1.333 (2) 1.457 (2) 1.501 (3) 316 Table 4.2. (Continued) C12—H12A C12—H12B C13—C18 C13—C14 C14—C15 C14—H14 C15—C16 C15—H15 C16—C17 C16—H16 C17—C18 C17—H17 C18—H18 C19—H19A C19—H19B C19—H19C 0.9900 0.9900 1.380 (3) 1.394 (3) 1.390 (3) 0.9500 1.367 (4) 0.9500 1.372 (3) 0.9500 1.387 (3) 0.9500 0.9500 0.9800 0.9800 0.9800 C42—H42A C42—H42B C43—C44 C43—C48 C44—C45 C44—H44 C45—C46 C45—H45 C46—C47 C46—H46 C47—C48 C47—H47 C48—H48 C49—H49A C49—H49B C49—H49C 0.9900 0.9900 1.393 (3) 1.397 (3) 1.390 (3) 0.9500 1.375 (3) 0.9500 1.389 (3) 0.9500 1.382 (3) 0.9500 0.9500 0.9800 0.9800 0.9800 317 Table 4.2. (Continued) C20—H20A C20—H20B C20—H20C C21—N2 C21—H21A C21—H21B C21—H21C C22—N2 C22—H22A C22—H22B C22—H22C N1—O8 O3—H3 0.9800 0.9800 0.9800 1.458 (3) 0.9800 0.9800 0.9800 1.466 (3) 0.9800 0.9800 0.9800 1.448 (2) 0.90 (3) C50—H50A C50—H50B C50—H50C C51—N4 C51—H51A C51—H51B C51—H51C C52—N4 C52—H52A C52—H52B C52—H52C N3—O18 O13—H13 0.9800 0.9800 0.9800 1.468 (2) 0.9800 0.9800 0.9800 1.476 (3) 0.9800 0.9800 0.9800 1.4343 (19) 0.87 (3) N1—C1—O1 N1—C1—C2 125.05 (17) 112.66 (17) N3—C31—O11 N3—C31—C32 123.85 (16) 112.13 (16) 318 Table 4.2. (Continued) O1—C1—C2 C11—C2—C1 C11—C2—C3 C1—C2—C3 O2—C3—C2 O2—C3—C4 C2—C3—C4 O3—C4—C5 O3—C4—C9 C5—C4—C9 O3—C4—C3 C5—C4—C3 C9—C4—C3 O4—C5—C4 O4—C5—C6 C4—C5—C6 122.21 (16) 103.57 (16) 123.97 (18) 132.43 (17) 125.31 (18) 121.30 (17) 113.37 (15) 112.00 (14) 110.37 (14) 108.76 (14) 107.42 (15) 107.85 (15) 110.41 (14) 121.55 (17) 121.48 (16) 116.96 (15) O11—C31—C32 C41—C32—C31 C41—C32—C33 C31—C32—C33 O12—C33—C32 O12—C33—C34 C32—C33—C34 O13—C34—C35 O13—C34—C33 C35—C34—C33 O13—C34—C39 C35—C34—C39 C33—C34—C39 O14—C35—C36 O14—C35—C34 C36—C35—C34 123.94 (16) 104.02 (15) 122.80 (16) 133.18 (16) 125.09 (17) 121.96 (16) 112.84 (15) 111.76 (14) 106.97 (14) 108.32 (15) 109.47 (14) 108.37 (14) 111.98 (14) 121.70 (17) 120.63 (17) 117.67 (14) 319 Table 4.2. (Continued) O5—C6—C5 O5—C6—C8 C5—C6—C8 O5—C6—H6 C5—C6—H6 C8—C6—H6 O5—C7—O6 O5—C7—C20 O6—C7—C20 O5—C7—C19 O6—C7—C19 C20—C7—C19 O7—C8—O6 O7—C8—C6 O6—C8—C6 O7—C8—H8 112.59 (15) 103.25 (14) 113.72 (14) 109.0 109.0 109.0 105.33 (14) 108.47 (16) 109.71 (16) 111.26 (16) 108.58 (16) 113.20 (19) 111.22 (14) 115.42 (15) 103.12 (15) 108.9 O15—C36—C35 O15—C36—C38 C35—C36—C38 O15—C36—H36 C35—C36—H36 C38—C36—H36 O15—C37—O16 O15—C37—C49 O16—C37—C49 O15—C37—C50 O16—C37—C50 C49—C37—C50 O17—C38—O16 O17—C38—C36 O16—C38—C36 O17—C38—H38 112.00 (14) 103.00 (14) 112.57 (15) 109.7 109.7 109.7 105.64 (13) 111.96 (15) 108.52 (16) 107.28 (16) 110.39 (15) 112.81 (17) 111.08 (14) 115.21 (15) 102.20 (14) 109.4 320 Table 4.2. (Continued) O6—C8—H8 C6—C8—H8 O7—C9—C4 O7—C9—C10 C4—C9—C10 O7—C9—H9 C4—C9—H9 C10—C9—H9 N2—C10—C11 N2—C10—C9 C11—C10—C9 N2—C10—H10 C11—C10—H10 C9—C10—H10 O8—C11—C2 O8—C11—C10 108.9 108.9 107.08 (14) 106.36 (14) 113.55 (14) 109.9 109.9 109.9 113.77 (15) 116.78 (15) 106.17 (14) 106.5 106.5 106.5 110.96 (17) 123.03 (16) O16—C38—H38 C36—C38—H38 O17—C39—C40 O17—C39—C34 C40—C39—C34 O17—C39—H39 C40—C39—H39 C34—C39—H39 N4—C40—C41 N4—C40—C39 C41—C40—C39 N4—C40—H40 C41—C40—H40 C39—C40—H40 O18—C41—C32 O18—C41—C40 109.4 109.4 106.40 (13) 108.99 (14) 113.84 (14) 109.2 109.2 109.2 112.22 (14) 117.08 (15) 107.96 (14) 106.3 106.3 106.3 110.79 (16) 121.42 (15) 321 Table 4.2. (Continued) C2—C11—C10 O1—C12—C13 O1—C12—H12A C13—C12—H12A O1—C12—H12B C13—C12—H12B H12A—C12—H12B C18—C13—C14 C18—C13—C12 C14—C13—C12 C15—C14—C13 C15—C14—H14 C13—C14—H14 C16—C15—C14 C16—C15—H15 C14—C15—H15 125.95 (16) 108.31 (15) 110.0 110.0 110.0 110.0 108.4 118.0 (2) 119.89 (17) 122.12 (19) 120.4 (2) 119.8 119.8 120.5 (2) 119.7 119.7 C32—C41—C40 O11—C42—C43 O11—C42—H42A C43—C42—H42A O11—C42—H42B C43—C42—H42B H42A—C42—H42B C44—C43—C48 C44—C43—C42 C48—C43—C42 C45—C44—C43 C45—C44—H44 C43—C44—H44 C46—C45—C44 C46—C45—H45 C44—C45—H45 127.66 (16) 109.34 (14) 109.8 109.8 109.8 109.8 108.3 118.93 (18) 122.94 (17) 118.09 (16) 119.91 (18) 120.0 120.0 120.73 (18) 119.6 119.6 322 Table 4.2. (Continued) C15—C16—C17 C15—C16—H16 C17—C16—H16 C16—C17—C18 C16—C17—H17 C18—C17—H17 C13—C18—C17 C13—C18—H18 C17—C18—H18 C7—C19—H19A C7—C19—H19B H19A—C19—H19B C7—C19—H19C H19A—C19—H19C H19B—C19—H19C C7—C20—H20A 119.7 (2) 120.2 120.2 120.1 (2) 119.9 119.9 121.22 (18) 119.4 119.4 109.5 109.5 109.5 109.5 109.5 109.5 109.5 C45—C46—C47 C45—C46—H46 C47—C46—H46 C48—C47—C46 C48—C47—H47 C46—C47—H47 C47—C48—C43 C47—C48—H48 C43—C48—H48 C37—C49—H49A C37—C49—H49B H49A—C49—H49B C37—C49—H49C H49A—C49—H49C H49B—C49—H49C C37—C50—H50A 119.86 (19) 120.1 120.1 119.89 (19) 120.1 120.1 120.66 (18) 119.7 119.7 109.5 109.5 109.5 109.5 109.5 109.5 109.5 323 Table 4.2. (Continued) C7—C20—H20B H20A—C20—H20B C7—C20—H20C H20A—C20—H20C H20B—C20—H20C N2—C21—H21A N2—C21—H21B H21A—C21—H21B N2—C21—H21C H21A—C21—H21C H21B—C21—H21C N2—C22—H22A N2—C22—H22B H22A—C22—H22B N2—C22—H22C H22A—C22—H22C 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 C37—C50—H50B H50A—C50—H50B C37—C50—H50C H50A—C50—H50C H50B—C50—H50C N4—C51—H51A N4—C51—H51B H51A—C51—H51B N4—C51—H51C H51A—C51—H51C H51B—C51—H51C N4—C52—H52A N4—C52—H52B H52A—C52—H52B N4—C52—H52C H52A—C52—H52C 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 109.5 324 Table 4.2. (Continued) H22B—C22—H22C C1—N1—O8 C21—N2—C10 C21—N2—C22 C10—N2—C22 C1—O1—C12 C4—O3—H3 C6—O5—C7 C8—O6—C7 C8—O7—C9 C11—O8—N1 109.5 104.29 (14) 111.45 (15) 109.98 (16) 117.13 (14) 116.77 (15) 109.9 (18) 108.05 (13) 109.74 (13) 115.50 (14) 108.48 (13) H52B—C52—H52C C31—N3—O18 C51—N4—C40 C51—N4—C52 C40—N4—C52 C31—O11—C42 C34—O13—H13 C36—O15—C37 C38—O16—C37 C38—O17—C39 C41—O18—N3 109.5 104.38 (13) 111.14 (14) 110.08 (15) 116.75 (14) 115.91 (14) 106.9 (17) 108.05 (13) 108.24 (13) 115.65 (13) 108.61 (13) N1—C1—C2—C11 O1—C1—C2—C11 N1—C1—C2—C3 O1—C1—C2—C3 2.3 (2) -174.62 (17) -175.66 (19) 7.5 (3) N3—C31—C32—C41 O11—C31—C32—C41 N3—C31—C32—C33 O11—C31—C32—C33 3.0 (2) -173.92 (16) -176.46 (18) 6.6 (3) 325 Table 4.2. (Continued) C11—C2—C3—O2 C1—C2—C3—O2 C11—C2—C3—C4 C1—C2—C3—C4 O2—C3—C4—O3 C2—C3—C4—O3 O2—C3—C4—C5 C2—C3—C4—C5 O2—C3—C4—C9 C2—C3—C4—C9 O3—C4—C5—O4 C9—C4—C5—O4 C3—C4—C5—O4 O3—C4—C5—C6 C9—C4—C5—C6 C3—C4—C5—C6 -168.86 (19) 8.7 (3) 12.8 (3) -169.64 (19) -95.7 (2) 82.68 (18) 25.2 (2) -156.42 (15) 143.88 (18) -37.7 (2) 14.3 (2) 136.56 (18) -103.7 (2) -166.24 (15) -44.0 (2) 75.77 (19) C41—C32—C33—O12 C31—C32—C33—O12 C41—C32—C33—C34 C31—C32—C33—C34 -166.79 (18) 12.6 (3) 17.0 (2) -163.66 (18) O12—C33—C34—O13 -96.5 (2) C32—C33—C34—O13 O12—C33—C34—C35 C32—C33—C34—C35 O12—C33—C34—C39 C32—C33—C34—C39 79.89 (17) 24.1 (2) -159.49 (15) 143.59 (17) -40.0 (2) O13—C34—C35—O14 15.6 (2) C33—C34—C35—O14 C39—C34—C35—O14 O13—C34—C35—C36 C33—C34—C35—C36 C39—C34—C35—C36 -102.0 (2) 136.33 (18) -164.94 (15) 77.47 (19) -44.2 (2) 326 Table 4.2. (Continued) O4—C5—C6—O5 C4—C5—C6—O5 O4—C5—C6—C8 C4—C5—C6—C8 O5—C6—C8—O7 C5—C6—C8—O7 O5—C6—C8—O6 C5—C6—C8—O6 O3—C4—C9—O7 C5—C4—C9—O7 C3—C4—C9—O7 O3—C4—C9—C10 C5—C4—C9—C10 C3—C4—C9—C10 O7—C9—C10—N2 C4—C9—C10—N2 -35.2 (2) 145.32 (15) -152.22 (17) 28.3 (2) -151.86 (15) -29.5 (2) -30.37 (17) 91.94 (17) -176.43 (14) 60.32 (18) -57.82 (18) -59.34 (19) 177.42 (15) 59.28 (19) -60.33 (19) -177.85 (15) O14—C35—C36—O15 -31.4 (2) C34—C35—C36—O15 O14—C35—C36—C38 C34—C35—C36—C38 149.19 (15) -146.88 (18) 33.7 (2) O15—C36—C38—O17 -156.30 (15) C35—C36—C38—O17 -35.5 (2) O15—C36—C38—O16 -35.74 (17) C35—C36—C38—O16 85.08 (17) O13—C34—C39—O17 178.49 (14) C35—C34—C39—O17 C33—C34—C39—O17 O13—C34—C39—C40 C35—C34—C39—C40 C33—C34—C39—C40 O17—C39—C40—N4 C34—C39—C40—N4 56.37 (18) -63.05 (18) -62.92 (19) 174.96 (14) 55.5 (2) -50.47 (19) -170.53 (15) 327 Table 4.2. (Continued) O7—C9—C10—C11 C4—C9—C10—C11 C1—C2—C11—O8 C3—C2—C11—O8 C1—C2—C11—C10 C3—C2—C11—C10 N2—C10—C11—O8 C9—C10—C11—O8 N2—C10—C11—C2 C9—C10—C11—C2 O1—C12—C13—C18 O1—C12—C13—C14 C18—C13—C14—C15 C12—C13—C14—C15 C13—C14—C15—C16 C14—C15—C16—C17 67.73 (17) -49.79 (19) -1.9 (2) 176.28 (17) 175.38 (17) -6.5 (3) -29.2 (2) -158.99 (16) 153.90 (18) 24.1 (2) 143.90 (18) -36.4 (2) 1.1 (3) -178.61 (19) 0.0 (3) -0.6 (3) O17—C39—C40—C41 C34—C39—C40—C41 C31—C32—C41—O18 C33—C32—C41—O18 C31—C32—C41—C40 C33—C32—C41—C40 N4—C40—C41—O18 C39—C40—C41—O18 N4—C40—C41—C32 C39—C40—C41—C32 O11—C42—C43—C44 O11—C42—C43—C48 C48—C43—C44—C45 C42—C43—C44—C45 C43—C44—C45—C46 C44—C45—C46—C47 77.28 (17) -42.77 (19) -2.6 (2) 176.91 (16) 173.18 (17) -7.3 (3) -34.4 (2) -164.85 (15) 150.24 (17) 19.7 (2) -10.8 (2) 171.70 (16) 1.0 (3) -176.51 (18) -0.1 (3) -0.9 (3) 328 Table 4.2. (Continued) C15—C16—C17—C18 C14—C13—C18—C17 C12—C13—C18—C17 C16—C17—C18—C13 O1—C1—N1—O8 C2—C1—N1—O8 C11—C10—N2—C21 C9—C10—N2—C21 C11—C10—N2—C22 C9—C10—N2—C22 N1—C1—O1—C12 C2—C1—O1—C12 C13—C12—O1—C1 C5—C6—O5—C7 C8—C6—O5—C7 O6—C7—O5—C6 0.1 (3) -1.6 (3) 178.11 (19) 1.0 (3) 175.07 (17) -1.7 (2) 176.40 (15) -59.3 (2) -55.7 (2) 68.5 (2) -4.1 (3) 172.35 (17) -148.03 (16) -91.18 (16) 31.89 (18) -21.19 (19) C45—C46—C47—C48 C46—C47—C48—C43 C44—C43—C48—C47 C42—C43—C48—C47 O11—C31—N3—O18 C32—C31—N3—O18 C41—C40—N4—C51 C39—C40—N4—C51 C41—C40—N4—C52 C39—C40—N4—C52 N3—C31—O11—C42 C32—C31—O11—C42 C43—C42—O11—C31 C35—C36—O15—C37 C38—C36—O15—C37 0.9 (3) 0.0 (3) -0.9 (3) 176.68 (18) 174.81 (16) -2.11 (19) -179.95 (15) -54.3 (2) -52.6 (2) 73.1 (2) -8.9 (3) 167.68 (16) -144.68 (16) -90.26 (17) 30.95 (18) O16—C37—O15—C36 -14.48 (18) 329 Table 4.2. (Continued) C20—C7—O5—C6 C19—C7—O5—C6 O7—C8—O6—C7 C6—C8—O6—C7 O5—C7—O6—C8 C20—C7—O6—C8 C19—C7—O6—C8 O6—C8—O7—C9 C6—C8—O7—C9 C4—C9—O7—C8 C10—C9—O7—C8 C2—C11—O8—N1 C10—C11—O8—N1 C1—N1—O8—C11 -138.59 (16) 96.28 (19) 142.43 (15) 18.15 (18) 0.52 (19) 117.08 (17) -118.74 (17) -65.49 (19) 51.5 (2) -67.14 (18) 171.14 (14) 0.9 (2) -176.38 (16) 0.48 (19) C49—C37—O15—C36 C50—C37—O15—C36 103.47 (17) -132.25 (15) O17—C38—O16—C37 150.94 (14) C36—C38—O16—C37 27.55 (17) O15—C37—O16—C38 -9.60 (18) C49—C37—O16—C38 C50—C37—O16—C38 -129.83 (16) 106.06 (18) O16—C38—O17—C39 -62.13 (19) C36—C38—O17—C39 C40—C39—O17—C38 C34—C39—O17—C38 C32—C41—O18—N3 C40—C41—O18—N3 C31—N3—O18—C41 53.4 (2) 172.73 (14) -64.12 (18) 1.48 (19) -174.63 (15) 0.43 (18) 330 Table 4.3. Hydrogen-bond parameters D—H···A O3—H3···N4i D—H (Å) 0.90 (3) H···A (Å) 2.03 (3) 2.12 (3) D···A (Å) 2.893 (2) 2.915 (2) D—H···A (°) 161 (3) 151 (2) O13—H13···N2ii 0.87 (3) Symmetry code(s): (i) x+1, y, z; (ii) x-1, y, z-1. Figure 4.1a 331 Figure 4.1b Figure 4.1. Perspective views showing 50% probability displacement. c b Figure 4.2. Three-dimensional supramolecular architecture viewed along the a-axis direction. 332 Catalog of Spectra F O N OBn BocO O 142 HO F O N OBn BocO O 142 HO 333 F O N OBn BocO O HO 144 F O N OBn BocO O HO 144 334 F O BocO O 146 HO F O BocO O 146 HO 335 O O O OH 149 O O O OH 149 336 O O O O 127 O O O O 127 337 N(CH 3) 2 O HO O O 152 O N OBn OCH3 N(CH 3) 2 O HO O O 152 O N OBn OCH3 338 O O O H N(CH3)2 O N O HO 128 OBn O O O H N(CH3)2 O N O HO 128 OBn 339 O O O H N(CH3 )2 O N O O O H 154 OBn O O O H N(CH3 )2 O N O O O H 154 OBn 340 O O O H N(CH3 )2 O N O O OTMS 155 OBn O O O H N(CH3 )2 O N O O OTMS 155 OBn 341 N(CH 3 )2 O N O O OBn 156 N(CH 3)2 O N O O OBn 156 342