554 J.C.S. Perkin IThe Biosynthesis of Fungal Metabolites. Part VIP Production andB iosy nt h esis of 4,7- D i met hoxy- 5 -met h y I co u ma r i n i n Aspergillus varie-colorBy Kuldip K. Chexal, Christopher Fouweather, and John S. E. Holker,‘ Robert Robinson Laboratories,4.7-Dimethoxy-5-methylcoumarin (I) (4.7-dimethoxy-5-methylchrornen-2-one) is shown to be a metabolite ofAspergi//us variecolor, strain IMi 53749. Incorporation of [2-14C]acetate Into this natural product shows thatit is derived on the P-ketide pathway. A synthesis of compound (I) is reported.University of Liverpool, P.O. Box 147, Liverpool 96L 3BXDURING an investigation 1 into the xanthone and di-benzoxepin metabolites of a number of variant strains ofA . variecolor, a biogenetically unrelated metabolite,C12H1204, was isolated from strain I M I 53749.Initiallythis was produced in relatively large quantities togetherwith arugosin C 1,2 and minor amounts of arugosins Aand B.193 In later cultures it was formed in decreasingquantities and finally production ceased altogether. Atthe same time the amount of arugosin C also decreasedand was replaced by a mixture of ~hamixanthone,l,~ epi-isoshamixanthone,l 25-0-methylarugosin A,l arugosinD,l and sterigmat~cystin.l*~ Fortunately in the earlystages of the investigation we were able to produce asufficient quantity of the new metabolite for identific-ation and to carry out a study of the incorporation of[2-l4Chce t at e.The lH n.m.r. spectrum of the new compound showedPart VI, K. K.Chexal, J. S. E. Holker, and T. J. Simpson,J. A. Ballantine, V. Ferrito, C . H. Hassall, and M. L. Jen-J . A. Ballantine, D. J. Francis, C . H. Hassall, and J. L. C .4 K. K. Chexal, C. Fouweather, J. S. E. Holker, and T. J.preceding paper.kins, J.C.S. Perkin I, 1973, 1826.Wright, J . Chem. SOC. (C), 1970,1176.Simpson, J.C.S. Perkin I, 1974, 1684.two meta-coupled aromatic protons [T 3.34 and 3-42(J 3 Hz)], a singlet vinyl proton (T 4-48) two methoxy-groups (7 6.10 and 6-20), and an aromatic methyl sub-stituent (z 7.41). The U.V. spectrum [A,, 226,288,306,and 317 nm (E 90o0, 11,600, 14,700, and 12,700)] wasclosely similar to that of kotanin (IV), a recently charac-terised bicoumarin which occurs with the correspondingdemethyl compound (V) in the related organism A .cZavatzcs.6 It therefore seemed likely that the new com-pound was the corresponding 4,7-dimethoxy-5-methyl-coumarin (I).This structure was readily confinned by synthesis.4’,6’-Dimethoxy-2’-methylacetophenone (VI),’ wasmonodemethylated with boron trichloride, a convenientreagent for the specific demethylation of ortho-methoxy-carbonyl con-ipounds,8 to give the 6‘-hydroxy-compound6 E.Bullock, J. C. Roberts, and J. G. Underwood, J . Chem.SOC., 1962, 4179.6 G. Buchi, D. H. Klaubert, R. C. Shank, S. M. Weinreb, andG. N. Wogan, J . Org. Chein., 1971,56,1143; G. Buchi, Y. Kitaura,S. S. Yuan, H. E. Wright, J. Clardy, A. L. Demain, T. Glinsukon,N. Hunt, and G. N. Wogan, J . Amer. Chem. SOC., 1973,95, 6423.7 K.Hoesch, Ber., 1916, 48, 1122. * F. M. Dean, J. Goodchild, L. E. Houghton, J. -4. Martin,R. B. Morton, B. Parton, A. W. Price, and N. Somvichien, Tetra-hedron Letters, 1966, 41631975 555(VII). Treatment of this with ethyl chloroformate gavethe ethoxycarbonyl derivative (VIII), which was con-verted into 4-hydroxy-7-methoxy-6-methylcoumarin (11)with potassium t-butoxide under the same conditions asthose used in the synthesis of kotanin.6 Methylation of4-hydroxycoumarins with dimethyl sulphate gives onlyR 2 0 W oMe OR'Me OMeRO qoOMe Me ( I I I I R 1 = R 2 = HMeoooR C O M eMe( = I R z M e(JLU1 R = H(Pml R : C O z E tthe corresponding 4-methoxycoumarins, whereas use ofdiazomethane gives both this type of compound and theisomeric 2-metho~ychromone.~ The 4-hydroxycou-marin (11) was methylated with dimethyl sulphate to give4,7-dimethoxy-5-methylcoumarin (I), identical with thenatural product.Since the completion of this work, sideriii, a metaboliteof Sideritis canariensis Ait.and S. romana L. (Labiatae),originally thought to be 6,7-dimethoxy-4-methylcou-rnarir~,~ has been shown to be 4,7-dimethoxy-5-methyl-coumarin.1° This compound has been synthesised byinethylation of the corresponding 4,6-dihydroxy-com-pound (111), obtained by condensation of orcinol withmalonic acid.Formally, the metabolite (I) could be considered to bederived biogenetically from acetate and malonate on the(3-ketide pathway with introduction of the two O-methylgroups from the C, pool, or on the shikimate-prephenatepathway with introduction of the C-methyl group fromthe C, pool.In fungi, the operation of the P-ketide path-way seemed more likely but it has been pointed out thatin the cyclisation of a poly-p-ketide intermediate the un-cyclised residues from the methyl ends of the chains arenever shorter than the residues from the carbonyl ends.llThus, a p-ketide origin for compound (I) would requirean exceptional cyclisation of the pentaketide inter-mediate. Hence, the biogenetic origin of this compoundin A . variecolor was worthy of investigation.[2-14C]Acetate was incorporated by A . variecolor intothe metabolite (I) with 0.75% overall efficiency. Kuhn-Roth oxidation of the labelled metabolite gave aceticA.G. GonzQles, 13. M. Fraga, JI. G. Hernandez, arid J. G.Luis, Phytochcm., 1972, 11, 2116.acid, which was isolated and counted as the p-bromo-phenylacyl ester. This contained 25.8% of the totalactivity of the starting material. Even incorporation ofacetate into a pentaketide-derived compound would re-quire 20% activity in the acetic acid. However, sincethis degradation involves the isolation of the startingacetate it is perhaps not surprising that it has a highactivity, especially as this particular feeding experi-ment involved rather large amounts of acetate precursor.It would have been desirable to investigate the distribu-tion of label in other parts of the molecule but lack ofmaterial precluded any further degradations. However,we feel that the result justifies the conclusion that thefungal metabolite is derived on the p-ketide pathway.It would be interesting to see if siderin is produced onthe same pathway in higher plants.EXPERIMENTALUnless otherwise stated, i.r.spectra were measured witha Perkin-Elmer 257 instrument for KBr discs, U.V. spectrawith a Unicam SP 800 instrument for solutions in ethanol,and 1H n.m.r. spectra with a Varian HA-100 instrument forsolutions in deuteriochloroform containing tetramethylsilaneas internal standard. Radioactivity measurements weremade by liquid scintillation counting [Packard 3003 TricarbScintillation Spectrometer and Butyl-PBD (CIBA) scintil-lator solution]. Counting efficiencies were determined with[l*C]hexadecane as internal standard.3f.p.s were deter-mined with a Kofler hot-stage instrument.4,7-Dimethoxy-5-methylcouinarin (I) from A. variecolor(IMI 53749).-This organism was grown in flat vessels eachcontaining Czapek-Dox medium (500 ml) as previouslydescribed . I s 4 The dried mycelium was exhaustively ex-tracted with light petroleum (b.p. 60-80") and the solutionevaporated. The residue (1.1 g 1-1) was dissolved in theminimum volume of hot methanol and the crude productseparated on cooling. Recrystallisation from hot methanolgave 4,7-dimethoxy-5-methylcoumarin (I) (4,7-dimethoxy-5-methylchromen-2-one) as needles (100 mg l-l), m.p. 194-195" (lit.,lO 194-195"), vmX. 1725, 1625, and 1612 cm-l(Found: C, 65.3; H, 5.5. Calc. for C12H120,: C, 65.4; H,5.5%).The methanolic mother liquors were used in theisolations of the xanthones and dibenzoxepins previouslydescribed.l6'- Hyd~oxy-4'-snethoxy-2'-nzethyEacetophenone (VII) .-4',6'-Dimethoxy-2'-methylacetophenone (VI) , m.p. 45-47"(lit.,' 48") (1 g ) , in dichloromethane (20 ml) was stirred at 0"with a large excess of boron trichloride for 5 min and themixture was then quenched in water (30 ml). The di-chloromethane layer was separated, washed with water(2 x 10 ml), dried (MgSO,), and evaporated to give a darkbrown solid. This was crystallised from hexane giving longneedles of the monomethyl ether (VII) (0.8 g), m.p. 78-79"(lit.,' 79').4-Hydroxy-7-nzethoxy-5-unethyZcou~~zarin (11) .-Compound(VII) (0.7 g) and ethyl chloroformate (2.8 g) in pyridine (3ml) were heated a t 85" for 5 h under nitrogen.The cooledsolution was poured into 2~-hydrochloric acid (20 ml) andthe product isolated in chloroform (2 x 50 ml). Evapor-ation left the crude carbonate (VIII) (0.9 g), which was10 P. Venturella, A. Bellino, and F. Piozzi, Tetrahedron Letters,1974, 979.11 W. B. Turner, ' Fungal Metabolites,' Academic Press,London, 1971, p. 198556dissolved in a solution of potassium t-butoxide in t-butylalcohol (80 ml) [from potassium (1.0 g)] and heated underreflux for 4 h. After cooling, the solution was poured into2~-hydrochloric acid (200 ml) and the resultant yellow solidcollected. Crystallised from methanol, 4-hydroxy-7-meth-oxy-5-methylcournarin (11) (4-hydroxy-7-methoxy-5-methyl-chromen-2-one) formed needles (0.5 g), m.p.264-265",vlmX 1690 and 1612 cm-l, A,, 234, 289, 309, and 319 nm(E 8850, 10,800, 18,800, and 10,900), T (CDCl,-CF,CO,H)3-14 (ArH, d, J 3.5 Hz), 3-20 (ArH, d, J 3-5 Hz), 3.70 (3-H,s), 6.10 (OMe, s), and 7-24 (5-Me, s) (Found: C, 63.9; H, 4.8.C,,Hl0O4 requires C, 64.1; H, 4.9%).4,7-Dimethoxy-5-methyZcoumari.~ (I) .-Prepared from com-pound (11) (410 mg) with dimethyl sulphate (700 mg) andanhydrous potassium carbonate (1 g) in 1,2-dimethoxy-ethane ( 100 ml) ,4,7-dimethoxy-5-methylcoumarin separatedfrom methanol in needles (250 mg), m.p. and mixed m.p.with the natural product, 194-195". The samples hadidentical i.r., u.v., and n.m.r. spectra.[14C]-4,7-Dirnethoxy-4-methyZcournarin.-A . varieco Zov,IMI 53749, was grown as previously described, except thatJ.C.S. Perkin Ion the seventh day sodium [2-14C]acetate (100 mg; 0.50pCi) in water was added to each of 5 flasks (500 ml culturefluid in each) under aseptic conditions. After a further 21days the cultures were harvested and [14C]-4,7-dimethoxy-4-methylcoumarin was isolated as previously described. Thecompound was purified by crystallisation from methanol toconstant radioactivity (12 mg, and 3-73 x lo-, pCi per pan).The overall efficiency of incorporation was thus 0.75%.[14q -p-BromophenacyZ A cetate .-[1*C]-4,7-Dimethoxy-4-methylcoumarin (52 mg; 3.73 x pCi mmol-l) was sub-jected to Kuhn-Roth oxidation and the resultant (14Claceticacid was converted into its p-bromophenacyl ester, whichwas purified by preparative t.1.c. on silica gel GF (Merck),followed by crystallisation from light petroleum (b.p. 60-80") to constant radioactivity (9 nig; 9.62 x yCimmol-l) ; m.p. 82-83".We thank Dr. W. B. Turner, Imperial Chemical In-dustries Ltd., Pharmaceutical Division, for discussions.[4/1945 Received, 23rd September, 1974
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