Our laboratory has shown that the key “AB enone” can be transformed into tetracycline antibiotics using a sequence of as few as three chemical steps.4 The first and key step of the sequence forms the C-ring of the tetracyclines by a Michael–Claisen cyclization reaction, which has since proven to be a general means for constructing tetracycline analogs widely variant in the left-hand or D-ring portion of the molecule. After cyclization, deprotection affords fully synthetic tetracycline antibiotics. An essential step in the deprotection sequence is hydrogenolysis of the benzyloxyisoxazole protective group, which Stork and Hagedorn had developed for the purpose of masking the reactive functional groups of the A-ring of tetracyclines.5 This synthetic sequence has enabled the synthesis of large numbers of tetracycline analogs, many of which exhibit activity against Gram-positive and Gram-negative bacteria, including tetracycline-resistant strains. 8-Fluoro sancycline, 6-phenyl sancycline, a D-ring pyrazole, a pentacycline, and a 6-aryl heteropentacycline exemplify novel structures synthesized using this strategy, structures which would have been difficult, if not impossible, to prepare using conventional “semi-synthetic” pathways.
The first-generation synthetic approach to the AB enone proceeded in 11 steps from benzoic acid and achieved the goal of providing gram amounts of this substance for coupling with D-ring precursors. C-ring construction and deprotection then provided testable quantities (10–20 mg) of tetracycline antibiotics that would have been difficult if not impossible to prepare before.4a,6 More than 50 different tetracyclines and tetracycline analogs were synthesized as a result of this initial effort.4b
Further attempts to scale the first-generation synthetic route to the AB enone were discontinued upon the innovation of a second-generation route, which has provided a scalable synthetic route to this substance. The second-generation route to the AB enone, like the first, is enantioselective, but is a more convergent process. It begins with the coupling of readily available 3-methoxyfurfural and the optically active organolithium reagent shown below to provide an intermediate alcohol (depicted), which in four steps is transformed to the key AB enone (40 grams of AB enone were prepared in a single batch in an early demonstration of the route).7Scientists at Tetraphase Pharmaceuticals, a company founded to commercialize the development of fully synthetic tetracyclines, have developed the synthetic route to allow for economical production of the AB enone and tetracyclines derived from it on multi-kilogram scales.
Recent chemical innovations have allowed us to synthesize new positional variants of the AB Enone at C4, C4a, C5, and C5a (positions indicated above), and thereby the correspondingly numbered positions of fully synthetic tetracyclines. This has been made possible by the development of a third-generation synthetic approach to the AB enone that is more amenable to substitutional variations, as well as the discovery of new transformations of the AB enone that provide highly diversifiable intermediates. These innovations, combined with modifications to positions C6, C7, C8, C9, and C10, enabled by prior work, should allow for multiplicative expansion of the pool of tetracycline antibiotics. Compounds synthesized have been evaluated against panels of >16 different bacterial cell lines (including Staphylococcus, Streptococcus, Haemophilus, Acinetobacter, Klebsiella, Pseudomonas, as well as others), many of which are multiply resistant to known antibiotics, including tetracycline. Tetracyclines with substitutions at C4, C4a, C5, C5a, and C6, heretofore inaccessible due to the limitations of semi-synthesis, hold promise to be novel and potent antibiotics.
More than fifty years of empirical results based on semi-synthetic modification8 of the tetracyclines and (more recently) two crystal structures of tetracycline bound to the bacterial ribosome suggest that a broad array of substitutions upon the tetracycline scaffold may be feasible without impairing (and perhaps enhancing) ribosomal binding.9 The bacterial ribosome is a time-proven molecular target, both time in the modern scientific era (60 years of human chemical evolution) and, more significantly, in the evolutionary history of life (more than a billion years of bacterial evolution). Targeting the bacterial ribosome continues to promise perhaps the highest probability of success in the search for new antibacterial agents relative to any other single target.
X-ray crystal structures of tetracycline bound to the bacterial ribosome have revealed that the highly oxygenated periphery and A-ring vinylogous hydroxamic acid of tetracycline provide the primary binding contacts with the 30S subunit of the ribosome (Figure 1A).9 Positions C4, C4a, C5, C5a, and C6 are all found on the opposite side of the molecule, in the so-called “modifiable” or hydrophobic region (Figure 1B). Of these positions, C4a and C5a have never been modified, while only a limited number of C4, C5 and C6 substitutional variants have been accessible by semi-synthetic methods.8 Improved binding to the bacterial ribosome is not the sole objective of our research, though certainly this is one goal. It is well appreciated that effective antibiotics must navigate complex processes of absorption, bacterial cell penetration (different for Gram-positive and Gram-negative pathogens), and bacterial resistance (such as inducible pumps, as well as many other mechanisms of resistance) while at the same time avoiding problems associated with metabolism and toxicity.
(2) (a) Spencer, J. L.; Hlavka, J. J.; Petisi, J.; Krazinski, H. M.; Boothe, J. H. 6-Deoxytetracyclines. V.1a 7,9-Disubstituted Products. J. Med. Chem. 1963, 6, 405–407. (b) Martell, M. J.; Boothe, J. H. The 6-Deoxytetracyclines. VII. Alkylated Aminotetracyclines Possessing Unique Antibacterial Activity. J. Med. Chem. 1967, 10, 44–46. (c) Church, R. F. R.; Schaub, R. E.; Weiss, M. J. Synthesis of 7-dimethylamino-6-demethyl-6-deoxytetracycline (minocycline) via 9-nitro-6-demethyl-6-deoxytetracycline. J. Org. Chem. 1971, 36, 723–725. (d) Zambrano, R. T. U.S. Patent 3,483,251, Dec 9, 1969.
(3) (a) Sum, P. E.; Lee, V. J.; Testa, R. T.; Hlavka, J. J.; Ellestad, G. A.; Bloom, J. D.; Gluzman, Y.; Tally, F. P. Glycylcyclines. 1. A new generation of potent antibacterial agents through modification of 9-aminotetracyclines. J. Med. Chem. 1994, 37, 184–188. (b) Sum, P.-E.; Petersen, P. Synthesis and structure-activity relationship of novel glycylcycline derivatives leading to the discovery of GAR-936. Bioorg. Med. Chem. Lett. 1999, 9, 1459–1462.
(4) (a) Charest, M. G.; Lerner, C. D.; Brubaker, J. D.; Siegel, D. R.; Myers, A. G. A Convergent Enantioselective Route to Structurally Diverse 6-Deoxytetracycline Antibiotics. Science 2005, 308, 395–398. (b) Sun, C.; Wang, Q.; Brubaker, J. D.; Wright, P. M.; Lerner, C. D.; Noson, K.; Charest, M.; Siegel, D. R.; Wang, Y.-M.; Myers, A. G. A Robust Platform for the Synthesis of New Tetracycline Antibiotics. J. Am. Chem. Soc. 2008, 130, 17913–17927.
(5) Stork, G.; Hagedorn, A. A. 3-Benzyloxyisoxazole System in Construction of Tetracyclines. J. Am. Chem. Soc. 1978, 100, 3609–3611.
(6) Myers, A. G.; Siegel, D. R.; Buzard, D. J.; Charest, M. G. Synthesis of a Broad Array of Highly Functionalized, Enantiomerically Pure Cyclohexanecarboxylic Acid Derivatives by Microbial Dihydroxylation of Benzoic Acid and Subsequent Oxidative and Rearrangement Reactions. Org. Lett. 2001, 3, 2923–2926.
(7) Brubaker, J. D.; Myers, A. G. A Practical, Enantioselective Synthetic Route to a Key Precursor to the Tetracycline Antibiotics. Org. Lett. 2007, 9, 3523–3525.
(8) (a) Rogalski, W. In The Tetracyclines; Hlavka, J.J., Boothe, J. H. Eds.; Handbook of Experimental Pharmacology Vol. 78; Springer-Verlag:Berlin, 1985; 179–316. (b) Nelson, M. L. InTetracyclines in Biology, Chemistry and Medicine; Nelson, M., Hillen, W., Greenwald, R. A., Eds.; Birkhauser:Boston, 2001; 3–63.
(9) (a) Brodersen, D. E.; Clemons, W. M.; Carter, A. P.; Morgan-Warren, R. J.; Wimberly, B. T.; Ramakrishnan, V. The Structural Basis for the Action of the Antibiotics Tetracycline, Pactamycin, and Hygromycin B on the 30S Ribosomal Subunit. Cell 2000, 103, 1143–1154. (b) Pioletti, M.; Schlunzen, F.; Harms, J.; Zarivach, R.; Gluhmann, M.; Avila, H.; Bashan, A.; Bartels, H.; Auerbach, T.; Jacobi, C.; Hartsch, T.; Yonath, A.; Franceschi, F. Crystal Structures of Complexes of the Small Ribosomal Subunit with Tetracycline, Edeine and IF3. EMBO J. 2001, 20, 1829–1839.