The emergence of pathogenic bacteria resistant to many current antibiotics is a major public health concern and one of particular importance in clinical settings (nosocomial infections). Restocking the armamentarium of antibacterial agents, especially broad-spectrum antibiotics such as the tetracyclines, promises to be one of the most effective means to combat infectious disease, from hospital-acquired Gram-positive and Gram-negative pathogens to unforeseen and evolving microbial threats. To date, all commercial tetracycline antibiotics have been prepared by fermentation-semisynthesis, which is inherently limited. Our laboratory has developed the first highly practical synthetic route to the tetracycline antibiotics. More than 2,000 novel tetracyclines, molecules with structural modifications that would not have been feasible using a semi-synthetic approach, have been prepared using this technology, many of them potent antibiotics active in broad panels of organisms resistant to classical tetracyclines, as well as other antibiotics. Ongoing research in our laboratory and in conjunction with Tetraphase Pharmaceuticals aims to multiplicatively expand this number, potentially to tens of thousands of compounds, by variation of positions in portions of the tetracycline scaffold that have never been modified before. Preliminary data shows that compounds with such modifications retain or enhance antibacterial activity. A number of active fully synthetic tetracycline scaffolds have been identified thus far, including penta- and hetero-cyclines as well as position specific variants, each representing novel classes of antibiotics. These fully synthetic routes have been designed to ensure that any new antibiotics identified can be scaled economically to prepare multikilogram amounts, as will be required to pursue clinical investigations and, ultimately, large-scale commercial manufacturing.


The strategy for the discovery of new tetracyclines had not varied since the discovery of the first tetracycline (chlortetracycline) more than 60 years ago, which is to say semi-synthetic transformations of complex fermentation products. The human semi-synthetic evolution of the tetracyclines is marked by specific, impactful discoveries that led to the production of new antibiotics. The first enabling advance in tetracycline semisynthesis was achieved by Pfizer scientists: reductive removal of the C6-hydroxyl group of the natural products tetracycline and oxytetracycline.1 The important and now generic antibiotics doxycycline and minocycline followed as a consequence, the latter arising from the additional discovery that electrophilic aromatic substitution at C7 becomes possible when the more stable 6-deoxytetracyclines are used as substrates.2 Decades later, a team of Wyeth scientists synthesized 7,9-disubstituted tetracycline derivatives, leading to the discovery of the antibiotic tigecycline.3




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 MichaelClaisen 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 (1020 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.

Figure 1. A. Tetracycline bound to the 30S subunit of the bacterial ribosome.9 B. Structure of (–)-tetracycline with generalized structure activity relationships.


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(2) (a) Spencer, J. L.; Hlavka, J. J.; Petisi, J.; Krazinski, H. M.; Boothe, J. H. 6-Deoxytetracyclines. V.1a 7,9-Disubstituted Products. 
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