Isolation, structure, and radiochemical synthesis of 3,6-dimethyl-4-hydroxy-2-pyrone.

A metabolite from P. stipitatum, C7H803, was deduced to have the structure 3,6-dimethyl-4-hydroxy-2pyrone from its spectral properties, and from conversion into its corresponding pyridone. This structure was fully confirmed by synthesis starting from ethyl acetoacetate and methylmalonic acid, a route which allows for the preparation of various isotopic species. The microscale radiochemical synthesis of this metabolite is exemplified by the preparation of 3,6-dimethyl-4-hydroxy-2-pyrone-3-C14. The biogenesis of this oligoketide and its possible relationship to tropolone biosynthesis are discussed. he biogenesis of secondary aromatic metabolites T produced by the higher fungi has now been firmly established to result from the acetate-polymalonate p a t h ~ a y . ~ In contrast to what has recently been found in the case of the closely related biosynthetic pathway to avian or mammalian fatty acids,4 fungal aromatic formation is still predicted to involve the obligatory participation of poly-P-ketide intermedia te~ .~' In spite of the fact that many organic analogies exemplify8-l0 the conversion of poly-/I-ketides into aromatic derivatives, these compounds have yet to be detected in living systems presumably because of inherent instability. Equally disappointing have been attempts5 to demonstrate incorporation of labeled lower polyketides into fungal metabolites. We wish to record the finding of a methylated oligoketide which may be a prototype of enzyme-bound higher polyketides. involving characterization of the metabolites elaborated by a strain (NRRL 1006) of Penicillium stipifatum, several new compounds were isolated following chromatography on activated alumina of ethereal extracts from the fermentation beer.13 One of these (la) [mp 212-214"; XI : " 288 mp (E 8300), unchanged by acid or base; A:, ": 3.72, 5.99, 6.08, 6.31 p ; [ a ] ~ 0; pK = 5.05; neut equiv 139; nmr (CDC13(CD3)2SO) T = 4.05, 5.06, 7.85, and 8.25 ppm, relative intensity 1 : 1 : 3 : 3 l 4 In continuation of a (1) A preliminary report has been published: P. E. Brenneisen, T. E. (2) Geigy and Co., Basel, Switzerland. (3) The extensive literature which deals with this subject has most recently been comprehensively reviewed by J. H. Richards and J. B. Hendrickson, "The Biosynthesis of Steroids, Terpenes and Acetogenins," W. A. Benjamin, Inc., New York, N. Y., 1964. (4) J. D. Brodie, G. Wasson, and J. W. Porter, J . Biol. Chem., 239, 1346 (1964). ( 5 ) A. 9. Birch, Proc. Chem. Soc., 3 (1962). (6) F. Lynen and M. Tada, Angew. Chem., 73, 513 (1961). (7) J. F. Grove, Fortschr. Chem. Org. Naturstoffe, 22 , 253 (1964). (8) A. J . Birch, D. W. Cameron, and R. W. Rickards, J. Chem. Soc., (9) J. R. Bethell and P. Maitland, ibid., 3751 (1962). (10) T. Money, I. H. Qureshi, G. B. Webster, and A. I. Scott, J. Am. Chem. SOC., 87, 3004 (1965). (11) S. W. Tanenbaum, E. W. Bassett, and M. Kaplan, Arch. Biochem. Biophys., 81, 169 (1959). (12) P. V. Divekar, P. E. Brenneisen, and S. W. Tanenbaum, Biochim. Biophys. Acta, 50, 588 (1961). (13) Tabulation of these metabolites has been presented elsewhere: S. W. Tanenbaum, "Biogenesis of Antibiotic Substances," Z. Vanek and Z. Hostalek, Ed., Academic Press Inc., New York, N. Y., 1965, p 143. (14) We are indebted to Dr. D. P. Hollis, Varian Associates, for n m spectra obtained at 60 Mc using tetramethylsilane = 10 as reference. Acker, and S. W. Tanenhaum, J . Am. Chem. Soc., 86, 1264 (1964). 4395 (1960). (Anal. Calcd for C7H803: C, 59.99; H, 5.75. Found: C, 59.91; H, 5.79)] appeared to be closely related in structure to triacetic lactone (lg) or to dehydroacetic acid (If), and for the foregoing biogenetic considerations was chosen for further characterization. In order to demonstrate that l a was not an artifact resulting from the isolation procedure, control chromatograms were run using identical developing solvents with activated alumina columns. In these experiments, the presence of l a was not detected. To preclude the possibility that l a arose from facile rearrangement of a closely related metabolite on activated alumina, the following method for its isolation was developed in parallel. Crude stipitatic acid and its tropolone congeners were obtained from the ether extract of the fermentation beer. After crystallization from methanol, the filtrates were concentrated and