Baran Lab T h e T e t r a c y c l i n e s D. W. Lin Cl HO Me NMe2 OH NMe2 The tetracycline family of antibiotics H H H H OH OH The natural products H N NH2 Cl HO Me NMe2 HO Me OH NMe2 H H H H OH OH OH OH OH O HO O O OH OH O HO O O clomocycline methacycline (rondomycin) NH2 NH2 (megaclor) 1963 1965 OH OH OH O HO O O OH O HO O O Me OH NMe2 NMe2 NMe2 chlortetracycline oxytetracycline H H H H OH OH (aureomycin) (terramycin) 1948 1949 NH2 NH2 HO Me NMe2 Cl HO NMe2 OH OH H H H H OH O HO O O OH O HO O O OH OH doxycycline minocycline NH NH (vibramycin) (minocin) 2 2 1967 1972 OH OH OH O HO O O OH O HO O O NMe2 NMe2 H H tetracycline demethylchlortetracycline OH (achromycin) (declomycin) O H 1953 1957 Me N NH2 N H OH Me Me OH O HO O O Semisynthetic derivatives on the market t-butylglycylamidominocycline (tigilcycline) 1993 HO Me OH NMe2 HO Me OH NMe2 H H H H (Phase III clinical trials in progress) OH OH H H Cl HO Me OH NMe2 N N 5 7 6 5a 4a 4 3 OH OH OH 8 OH O HO O O N OH O HO O O HN D C B A 2 9 NH2 4 12a rolitetracycline limecycline 10 11 12 1 (reverin) (tetralysal) H2N CO2H OH O HO O O 1958 1961 Notation I. Chopra, M. Roberts. Microbiol. Mol. Biol. Rev. 2001, 65, 232. 1 Baran Lab T h e T e t r a c y c l i n e s D. W. Lin Discovery and The Dawn of Semisynthetic Antibiotics HO Me OH NMe2 H H OH NH2 OH OH O HO O O Cl HO Me NMe2 H H OH terramycin NH2 OH OH O HO O O aureomycin Bayer Pharmaceuticals Benjamin Duggar University of Missouri The first tetracycline antibiotic discovered, aureomycin was isolated in 1948 from a Missouri The Nobel Prize Committee soil sample by Lederle R. B. Woodward Laboratories. The Lederle team was led by Benjamin Duggar - a About the same time as the Lederle discovery of aureomycin, Pfizer was consultant who was a 73-year-old scouring the globe for new antibiotics. Soil samples were collected from emeritus professor of botany who jungles, deserts, mountaintops, and oceans. But ultimately terramycin was had recently retired from the isolated in 1949... from a soil sample collected on the grounds of a factory in University of Missouri! As Jie Terre Haute, Indiana, owned by Pfizer! Jack Li cracks, "Your greatest discovery could happen in your retirement." From the beginning, terramycin was a molecule enveloped in controversy. It was the subject of the first mass-marketing campaign by a modern About Lederle Labs: pharmaceutical company. Pfizer advertised the drug heavily in medical Lederle Labs was founded in journals, eventually spending twice as much on marketing as it did to discover 1902 in an old farmhouse on the and develop terramycin. Still, it turned Pfizer - then a small company - into a Pearl River in New York. pharmaceutical giant. Aureomycin was one of many lifesaving products developed by Pfizer and R.B. Woodward collaborated to determine the structure of Lederle, including vaccines for terramycin, succeeding for the most part in 1952 (JACS 1953, 75, 5455). The polio and smallpox. It is now a stereochemistry at C was revised after X-ray crystallography and NMR part of Wyeth Pharmaceuticals. 4a studies in the 1960's (JACS 1965, 87, 134; JACS 1963, 85, 851). Ebay Ad for aureomycin as additive in cattle feed J. J. Li. "A History of Drugs and Their Discoverers." Pfizer Intranet Magazine. March-April 2004. 2 Baran Lab T h e T e t r a c y c l i n e s D. W. Lin Big Pharma Behaving Badly: In 1955, Conover Now, Back to Actual Science discovered that hydrogenolysis of aureomycin gives a deschloro product that is just as active as the original Biosynthesis and Biological Activity: Tetracyclines are polyketide antibiotics, product. This proved for the first time that chemically- biosynthesized in a fashion similar to that of fatty acids, erythromycin and a host of modified antibiotics could have biological activity. Within a other antibiotics. Tetracyclines are produced naturally by Streptomyces few years, a number of semisynthetic tetracyclines had aureofaciens (T. Nakano, et al. Biosci. Biotechnol. Biochem. 2004, 68, 1345.). entered the market, and now most antibiotic discoveries are of novel active derivatives of older compounds. Tetracyclines bind to the bacterial ribosome, preventing the binding of aminoacyl- tRNA to the ribosomal A site. This prevents bacterial protein translation (I. Chopra, Conover's discovery, however, provoked further controversy M. Roberts. Microbiol. Mol Biol. Rev. 2001, 65, 232.). for tetracycline. Pfizer became embroiled in a patent dispute with American Cyanamid, which owned the rights to The Challenge to Synthetic Chemists: Muxfeldt and colleagues outline the basic aureomycin (the starting material for Conover's procedure obstacles to achieving a total synthesis of any of the natural tetracyclines: to make tetracycline). Pfizer and American Cyanamid eventually settled the dispute out of court when they Stereochemical Complexity. There are up to five contiguous asymmetric centers realized that neither company held truly exclusive rights to (terramycin) which must be established. the drug, and agreed to cooperate on selling the drug in order to drive off competitors trying to enter the tetracycline Chemical Sensitivity. For the 6-methyl-6-hydroxy tetracyclines, mild acid rapidly market. At one point, Pfizer employed a private decective catalyzes dehydration, ketalization and a retro-aldol to produce the lactone below. to tap the phones of Bristol-Meyers, a competitor seeking to Mildly basic conditions results in deprotonation of the C5 and C6 hydroxyls, enter the tetracycline market! Bristol-Meyers agreed to About.com initiating a cascade of events which leads to decomposition of the molecule. overlook this brazen act in exchange for a share of the Finally, the C4 stereocenter is readily epimerized upon exposure to acetic acid or Lloyd Conover aqueous buffers. tetracycline market. Eventually five companies colluded in Pfizer order to maintain artificially high prices for tetracycline. However, the Federal Trade Commission stepped in after several years, finding Pfizer and company guilty of patent HO Me OH NMe2 Me NMe2 fraud and anti-trust violations, and broke up the monopoly. H H H OH OH Legal issues aside, for this discovery Lloyd Conover is now in the American acid Inventors' Hall of Fame, alongside Thomas Edison and the Wright brothers. O NH2 NH2 U.S. Federal Trade Commission, "Anticipating the 21st Century: Competition OH OH Policy in the New High-Tech, Global Marketplace". OH O HO O O OH OH O O O base M. Mintz. "Golden Ox of Antitrust." The Nation 14 April 1969, Vol. 208, Issue 15. acid pp. 467-468. O Cl HO Me NMe2 H HO Me NMe2 HO Me OH NMe2 H H H H H H OH OH OH COOH H2, Pd/C HO Me NH2 MeOH/ NH2 NH2 COOH dioxane OH OH OH OH O HO O O OH O HO O O OH O HO O O Conover, L.H. 1955. U.S. Patent No. 2,699,054. H. Muxfeldt, et al. J. Am. Chem. Soc. 1979, 101, 689. 3 Baran Lab T h e T e t r a c y c l i n e s D. W. Lin Woodward's First Total Synthesis of a Biologically-Active Tetracycline, 6- O O Demethyl-6-deoxytetracycline. Cl Cl MeO OMe CO Me L.H. Conover, et al. J. Am. Chem. Soc. 1962, 84, 3222. (Initial communication) CO Me (2 eq.) 2 R.B. Woodward. Pure Appl. Chem. 1963, 6, 561. (A personal account) 2 J.J. Korst, et al. J. Am. Chem. Soc. 1968, 90, 439. (Full article) then NaH (4 eq.), OH (i) O then MeOH (1 eq.), rt --> 80 oC OMe O OH OMe O OMe O O O 45% NaH CO2Me intermolecular condensation outcompetes intramolecular! DMF OMe OMe (ii) O OMe Triton-B MeOH is essential to suppressing the kinetically Cl dioxane-MeOH Cl OMe o favorable intramolecular condensation and OMe OMe O 50-70 C permitting the intermolecular condensation with NaH CO2Me DMF 88% the oxalate prior to formation of the desired 55% tricycle. In the absence of MeOH, Woodward observed formation of the intramolecular product OH O O first, followed by condensation onto the oxalate OMe OH O CO2Me (i) AcOH/ to form the five-membered ring shown: CO2Me CO2Me H2SO4 Cl O (ii) H2SO4 Cl CO Me CO Me 2 MeOH/CHCl3 2 CO2Me n OMe 44% OMe AcOH/HCl H CO2 Bu (i) H2, 200 psi (i) NaOH H2O Mg(OMe)2 OH o O Pd/C CO2Me H2O 90 C toluene o AcOH, 30 oC 100 C OMe O OH 73% OMe O OH reflux 52% (ii) H SO (ii) I , AcOH; 2 4 CO2Me 2 MeOH/CHCl Cl CO nBu 3 then Cl2 in AcOH 2 Cl NMe2 93% OMe (iii) HF, neat o H H (i) NHMe2, -10 C; 63% over 3 steps o n (ii) NaBH4, -70 C CO2 Bu Cl Cl H SO O 69% CO2H 2 4 CO Me OH MeOH/CHCl 2 3 OMe O OH OMe O OH reflux 66% The thermodynamically more favorable diastereomer is formed OMe O OMe O exclusively in this step, with the carboxyamino substituent assuming an equatorial position and thus establishing the cis relationship of Chlorination blocks the para the bridgehead hydrogens.