Fungal Transformation of the Antifungal Isoflavone Luteone Satoshi Tahara, Shiro Nakahara, Junya Mizutani and John L

Home , Formononetin, Luteone (isoflavone), Rotenoid

Agric. Biol. Chem., 48 (6), 1471 ~ 1477, 1984 1471

Fungal Transformation of the Antifungal Isoflavone Luteone Satoshi Tahara, Shiro Nakahara, Junya Mizutani and John L. Ingham* Department of Agricultural Chemistry, Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo 060, Japan *Phytochemical Unit, Department of Botany, University of Reading, Whiteknights, Reading RG6 2AS, England Received November 4, 1983

An antifungal isoflavone, luteone [5,7,2/,4/-tetrahydroxy-6-(3,3-diniethylallyl)isoflavone] is metabolised by cultures of Aspergillus flavus and Botrytis cinerea into 2",3"-dihydro-3"- hydroxyluteone (AF- 1), 2//,3 '/-dihydrodihydroxyluteone, a dihydrofuranoisoflavone (BG-1) and a dihydropyranoisoflavone. The structures of the metabolites were elucidated by physico-chemical and chemical procedures. The major metabolites, AF-1 and BC-1 are much less toxic than luteone against Cladosporium herbarum. The possible metabolic pathways are briefly discussed.

SJ^^'-Tetrahydroxy^-^B-dimethyl- demethylating or reducing the molecule.6'7* allyl)isoflavone (luteone, 1) was first isolated In the case of 5,7,2',4'-tetrahydroxy-8-(3,3- from the young fruits of Lupinus luteus L. dimethylallyl)isoflavanone (kievitone) and 3,9- (yellow lupin) and found to be strongly dihydroxy- 1 0-(3, 3-dimethylallyl)pterocarpan antifungal.1) Considerable quantities of 1 and (phaseollidin), it was found that metabolism the related fungitoxin 5,7,4'-trihydroxy-6- by Fusalium solani f. sp. phaseoli involved a (3,3-dimethylallyl)isoflavone (wighteone) also straightforward hydration of the sidechain to occur on the surface of lupin Ieaves2'3) where, yield kievitone hydrate8) and phaseollidin hy- in conjunction with other isoflavones [e.g. drate,9* respectively. Both these modification 5,7,4'-trihydroxyisoflavone (genistein) and products were much less antifungal than either its 2'-hydroxy analogue3)], they may offer kievitone or phaseollidin.8'9* some degree of protection against potential We have recently isolated several fungal fungal pathogens. It has long been recognized, metabolites of luteone (1) by the incubation of however, that some fungi have the ability to this lupin-derived isoflavone with cultures of metabolise and detoxify isoflavonoid com- Aspergillus flavus and Botrytis cinerea. The pounds,^ and in certain cases this process spectroscopic and chemical evidence to show seems to be closely linked with pathogen- that four of the metabolites have structures 2 icity.4'5) Because 1 may function as a pre- (luteone hydrate), 3,4 and 5a (in Scheme 1) are infectional antifungal agent (prohibitin) on presented in this paper. leaves of L. luteus and other Lupinus species,3) it would be useful to determine if this isofla- MATERIALS AND METHODS vonoid was also susceptible to detoxification by micro-organisms. Previous studies have de- Substrate. Luteone (1) used in this experiment was monstrated that several Fusarium fungi me- isolated from L. albus and L. luteus as previously.3* MS m/z (%): 355 (M++l, 22), 354 (M+, 88), 339 (6.9), 312 tabolise simple isoflavones such as biochanin (6.9), 311 (M+ -43, 100), 300 (5.6), 299 (M+ -55, 80), 298 A (5,7-dihydroxy-4/-methoxyisoflavone) and (5.5), 165 (ll), 134 (5.1). XH-NMR <5^.one-d6 (100 MHz): formononetin (7-hydroxy-4'-methoxyisofla- 1.65 and 1.78 (each 3H, two br s, 4"- and 5"-H3), 3.37 (2H, vone) by first hydroxylating, methylating, brd,.7=7.3Hz, 1"-H2), 5.28 (1H, brt, J= 7.3Hz, 2"-H), 1472 S. Tahara et al.

6.44 (1H, dd, 7=8.9 and 2.4Hz, 5'-H), 6.48 (1H, incom- circular zone (13 ~ 14mmi.d.) of the test material adsorb- pleted, 3'-H), 6.53 (1H, s, 8-H), 7.12(1H,d,.7=8.9Hz, 6'- ed homogeneously. After the acetone had evaporated, a H), 8.14 (1H, s, 2-H), 13.05 (s, 5-OH). spore suspension of Cladosporium herbarum Fr. AHU 9262 in a mediumwas sprayed onto the plate and in- Epoxyluteone tetraacetate (IAc). The prenyl sidechain of cubated in a moist atmosphere for 2~3 days at 25°C luteone tetraacetate1* was oxidized to the corresponding according to Homans and Fuchs.n) epoxide according to Gupta et al.10) ra-Chloroperbenzoic acid (26mg) was added to a chilled (ice-bath) solution of Properties of metabolites. AF-1 (2): Colorless needles luteone tetraacetate (42mg) in CHC13 (4ml) and the from acetone,.mp 224~226°C. UV, MS and XH-NMR reaction mixture was stirred at 15°C for 6 hr. After diluting data are shown in Table I. to 25 ml with CHC13,the mixture was washed successively AF-2 (3). MS m/z(%): 388 (M+, 16), 370 (3.9), 330 (14), with 5% aqueous NaHCO3and brine. The organic layer 329 (97), 300 (25), 299 (100), 298 (4.5), 167 (4.7), 165 (39), was dried over Na2SO4and concentrated to near dryness 134 (5.0). UV l^?Hnm: 264, 293sh; +NaOMe, 276, in vacuo. The concentrate was subjected to preparative 340sh; +A1C13, 274, 315, 373; +NaOAc, 272, 338 TLC (PTLC) in benzene-EtOAc (4: 1) to isolate IAc as a (the addition of solid boric acid regenerated the MeOH colorless oil (23mg). MS m/z (%): 538 (M+, 0.9), 496 (5.9) spectrum). ^-NMR ^Sone"d6 (100 MHz): 1.26 and 1.28 454, (47), 412 (33), 395 (8.3), 379 (6.5), 371 (8.1), 370 (100), (each 3H, both s, 4"- and 5"-H3), 2.62 (1H, dd, J= 14 and 354 (6.7), 353 (10), 352(9.8), 312 (29), 311 (16), 177 (8.4), 9.8Hz, 1"-Ha), 3.25 (1H, dd, 7=14 and 2.0Hz, 1"-Hb), 134 (6.7), 59 (56). ^-NMR ^S0"6"'6 (100 MHz): 1.21 and 3.65 (1H, dd, J= 9.8 and 2.0Hz, 2"-H), 6.44(1H, dd,/= 1.38 (each 3H, both s, 4"- and 5"-H3), 2.ll, 2.28, 2.35 and 10 and 2.4Hz, 5 -H), 6.49 (1H, incomplete d, 3'-H), 6.49 2.38 (each 3H, 4xs, 4xCH3CO), 2.62~3.04 (3H, m, 1"- (1H, s, 8-H), 7.13 (1H, d, J=10Hz, 6'-H), 8.15 (1H, s, 2- H2 and 2"-H), 7.08 (1H, d, /=2.2Hz, 3 -H), 7.09 (1H, dd, H), 13.23 (s, 5-OH). .7=8.9 and 2.2Hz, 5 -H), 7.41 (1H, d,.7=8.9Hz, 6'-H), BC-1 (4). Pale yellow needles from MeOH, mp 7.43 (1H, s, 8-H), 8.16 (1H, s, 2-H). 229~231.5°C. [a£3-10.6° (c=0.094, MeOH). UV, MS and XH-NMRdata are shown in Table I. Metabolic experiments. Cultures of Aspergillus flavus BC-2 (5a). MS m/z (%): 371 (M++l, 16), 370 (M+, AHU7049 and Botrytis cinerea AHU9424 were grown in 100), 300 (4.0), 299 (58), 298 (12), 165 (35), 134 (31). UV a liquid medium consisting of glucose (5 g), peptone (1 g), A^Hnm: 227sh, 263, 292sh; +NaOMe, 247, 262, 279; yeast extract (0.1 g) and H2O(100ml). The medium was +AICI3, 230sh, 273, 316, 372; +NaOAc, 263, 292sh. 1H- dispensed into 500 ml shaking flasks (100 ml/flask), steri- NMR ^Sone"d6 (100MHz): 1.34 and 1.40 (each 3H, both lized (120°C for 12min) and then inoculated with 1 ml of s, 4"- and 5"-H3), 2.62 (1H, dd, /=17 and 7.1 Hz, 1"-HJ, mycelial suspension of the appropriate fungus. The cul- 2.98 (1H, dd, /=17 and 5.4Hz, r'-Hb), 3.87 (1H, dd, /= tures were placed on a reciprocal shaker (lOOrpm) and 7.1 and 5.4Hz, 2"-H), 6.40 (1H, s, 8-H), 6.44 (1H, incubated (25°C) for 4 days, at which point a solution of 1 incomplete dd, 5'-ft), 6.48 (1H, incomplete d, 3'-H), 7.13 in ethanol (5mg/ml) was added to give a final luteone (1H, d,.7=8.8Hz, 6'-H), 8.16 (1H, s, 2-H), 13.13 (s, 5- concentration of about 50 /ig/ml. After a further 3 days OH). incubation, an equal volumeof acetone wasaddedto each Tetraacetyl BC-2 (5b). The tetraacetate (5b) was formed flask, the contents immediately filtered by suction, and the by treating BC-2 (5a) with a mixture of acetic anhydride- filtrate concentrated in vacuo (30°C) to remove the ace- pyridine (1 : 1). MS m/z (%): 538 (M+, 4.1), 496 (9.2), 437 tone. The concentrate was then acidified to pH 3 (HC1), (ll), 436 (84), 422 (10), 421 (93), 394 (15), 380 (9.9), 379 shaken ( x 3) with ethyl acetate, and the combined ethyl (100), 352 (7.8), 337 (27), 203 (13), 165 (6.0), 134 (7.1). acetate fractions were washed first with 5% aqueous ^å NMR <5xMsone'd6 (100 MHz): 1.46 (6H, br s, 4"- and NaHCO3and then with a saturated solution of NaCl. 5//-H3), 2.7-3.0 (2H, m, 1"-H2), 5.14 (1H, t, 7=4.6Hz, Evaporation of the ethyl acetate gave a residue from which 2"-H), 6.87 (1H, s, 8-H), 7.07 (1H, d, /=2.2Hz, 3/-H), the A. flavus- or B. cinerea-derived metabolites of 1 (AF- 7.08 (1H, dd, 7=9.0 and 2.2Hz, 5 -H), 7.39 (1H, d, J= 1 and AF-2 or BC-1, BC-2 and BC-3) were isolated 9,0 Hz, 6'-H), 8.07 (1H, s, 2-H). Four acetyl methyl groups by PTLCusing pre-coated silica gel plates (Merck, Silica were found at S 2.02, 2.10, 2.28 and 2.31 as four singlets Gel 60 F-254, 0.25 or 0.5mm thickness) and the fol- (each 3H). The two methyl groups (4//- and 5//-H3) were lowing solvent systems: CM=CHCl3-MeOH, 50: 2-3; not equivalent in CDC13 [100MHz, 1.35 and 1.40 (each CAAm= CHCl3-acetone-conc. ammonia water (28%), 3H, both s)]. 70 : 60 : 1; PEAa=pentane-diethyl ether-glacial acetic BC-3 (2): MS m/z (%): 388 (M+, 25), 370 (4.7), 330 (19), acid, 70:30:6. 329 (96), 300 (30), 299 (100), 298 (4.5), 167 (8.6), 165 (38), 134 (9.3). The metabolite BC-3 was indistinguishable Assay of antifungal activities. The specified amount of from AF-2 on TLC and UV. pure isolate was dissolved in acetone. The solution (25 p\) was spotted on a thin layer plate (Merck, Silica Gel 60 F- 254, 0.25mmthickness) by a micro syringe to give a Fungal Transformation of Luteone 1473

RESULTS AND DISCUSSION there were no MS fragments at m/z 329 (M+ -43) or m/z 317 (M+ -55) characteristic Aspergillus flavus metabolites of a 3,3-dimethylallyl substituent (cf. data for Cultures of A. flavus were found to contain 1, lupisoflavone, lupalbigenin and wighte- two metabolites (AF-1 and AF-2) of luteone one1>3)), and fungal modification of 1 was, (1) with Rfvalues of 0.ll and 0.08, respec- therefore, considered to have occurred by tively, in the CMsystem. A small quantity of hydration of the alkenyl sidechain. unchanged 1 (Rf 0.19 in CM) was also re- This possibility was confirmed by the XH- covered from the medium. About 50mg of 1 NMRspectrum of AF-1 (determined at 500 yielded 25.8mg of AF-1, but only 1.1mg of MHz in acetone-d6) which differed from that AF-2. Like 1, the metabolite AF-1 gave a of 1 (see Table I and Materials) in two strong purple/blue color on chromatograms important respects. Firstly, signals due to the treated with Gibbs reagent. High resolution non-equivalent methyls of the luteone side- MSdeduced the molecular formula C20H20O7 chain ((51.65 and 1.78) were replaced by a (observed, 372. 120; calculated, 372. 121) which sharp 6-proton singlet at S 1.26. Secondly, the differs by 18 mass units (H2O) from that of 1 methylene (1 "-H2) and olefinic (2"-H) protons (M+354). Signals at m/z 165 (main A-ring of1, which appeared as a doublet (S 3.37) and fragment) and m/z 134 (main B-ring frag- as a broad triplet (S 5.28) respectively, were ment) were commonto the MSof both AF-1 replaced by two high-field methylene multi- and 1, but the metabolite also gave intense plets (<5 1.71 and 2.79). These changes in fragments corresponding to M+-H2O (m/z AF-1 relative to luteone mirror exactly those 354), M+-(H2O+C3H7) (m/z 311) and reported in the formation of kievitone hy- M+ -(H2O+C4H7) (m/z 299). Significantly, drate from kievitone8) and phaseollidin hy-

Table I. Properties of Major Fungal Metabolites of Luteone

AF-1 BC-1

Molecular formula (HR-MS) C20H20O7 C20H18O7 MSm/z(%) . 372(M+, 5.5), 354(19), 371 (15), 370(M+, 100) 311 (16), 300 (6.7), 299 337 (26), 312 (51), 311 (73), (100), 298 (17), 165 (36), 179 (14), 177 (14), 150 134 (8.2), 123 (5.4), 69 (8.3), 134 (13), 59 (51) (8.4), 55 (7.6) UV AjJ^nm 265, 293sh 263, 289sh (loge) (4.47) (4.15) (4.49) (4.24) +NaOMe 277, 300sh, 336 280, 295sh +A1C13 274, 315, 367 271, 318, 363 +NaOAc 267, 292sh, ca. 340sh 263, 289sh ^-NMR S acetone-d6 500MHz /=Hz 2-H 8.15 s 8.18 s 5-OH 13.05 s 12.96 s 8-H 6.52 s 6.43 s 3 -H 6.49 d 7=2.4 6.49 d 7=2.4 5 -H 6.44 dd /=7.9, 2.4 6.45 dd /=7.9, 2.4 6'-H .7.12 d /=7.9 7.13 d /=7.9 1^-Ha l 3.16 dd /=16, 9.8 r'-Hb j 3.21 dd /=16, 7.9 2/r-Ha ) t _t 4.86 dd.7=9.8, 7.9 2"-Hb J L71 m 4--H3 1 1.25 s 5"-H3 'J 1.30 s 1474 S. Tahara et al.

æf Q |_| I I I I. I *y^n y=0H 2 OH^ iH 'O HO^=>^OH

m/z 165 m/z 177 m/z 134 m/z 59

æf å '

O y. ^ å MO O

0H ° ^J'-nu 0H ° J^Anu

m/z 311 m/z 299 Fig. 1. Mass Fragments Mentioned in the Text.

shifted bathochromically by the addition of AICI3 (C-5-OH), and NaOAc (C-7-OH).12) As hohV|^kA\ \^JuHOv| ha-V-^^V with AF-1, the intact luteone nucleus was confirmed by comparing XH-NMRdata of 59 89 AF-2 with those of 1 (three protons on the B- a** r*b r*e ring and two protons of C-2 and C-8); therefore, AF-2 arose by dihydroxylation of the 3,3- dimethylallyl sidechain. Although several part structures (e.g. a and b) are possible for AF-2, the MSfragments at m/z 329 and 299 tend to favor a. This estimation was validated by 1H- d e^ NMRdetection of two methyl groups on the carbinol carbon (<5 1.26 and 1.28) and three Fig. 2. Partial Structures Mentioned in the Text. HA OH protons showing an AMXsystem (-C-C-, drate from phaseollidin.9) They clearly indi- HMHx cate that a tertiary alcohol derivative of 1, /AM=14Hz, yAX=9.8Hz, /MX=2.0Hz). The which we proposed to name luteone hy- whole structure of AF-2 (3) was thus eluci- drate (2), had been produced by Markov- dated as depicted in Scheme 1. nikov addition of H2O to the C=C bond of the 3,3-dimethylallyl sidechain. As shown Botrytis cinerea metabolites of luteone in Table I, the aromatic (8-, 3'-, 5'- and 6'- Luteone (1) was transformed by B. cinerea H) and heterocyclic (2-H) proton signals of into three metabolites designated BC- 1 (Rf0. 19 luteone hydrate closely correspond with in CM), BC-2 (#0.15 in CM) and BC-3 (Rf those given by luteone itself, thereby exclud- 0.08 in CM). Although BC-1 and 1 ran to- ing any possibility of modification elsewhere gether in the CM system, they were easily in the molecule. separated by silica gel PTLCusing PEAa (1, The second A. flavus metabolite (AF-2; Rf #0.23; BC-1, Rf0.07). About 50mg of1 were 0.08 in CM)showed the molecular ion at mjz metabolized by the fungus to yield 23mg of 388 (16%) in addition to the MSfragments at BC-1, 4.38mg of BC-2 and 2.26mg of BC-3, m/z 370 (M+-H2O, 3.9), 329 (M+-59, 97), respectively. Metabolite BC-1 was laevor- 299 (M+-89,100), 165 (main A-ring frag- otatory and gave a purple/blue color with ment, 39) and 134 (main B-ring fragment, 5.0). Gibbs reagent. High resolution MS indicated The methanolic UVmaximumof AF-2 was the molecular formula to be C20H18O7 (ob- Fungal Transformation of Luteone 1475 served, M+ 370.103; calculated, 370.105), and l-methyl)ethyldihydrofuran attachment.16* major associated fragments were evident at Major MSfragments were also observed at m/z337, 312, 311 (M+-59), 177, 134and 59. m/z 177 (A-ring with the C-5-OH group and Anion at m/z 165 corresponding to an A-ring alkyl sidechain; cf. MS data for erythrinin derived fragment of 1, was not observed in the C15)) and m/z 134 (B-ring with OH groups at MS ofBC-1. C-2' and C-4'), and these presumably arise In addition to the expected heterocyclic (2- by RDA (retro-Diels-Alder) fission of the H) and aromatic ring (8-, 3'-, 5'- and 6/-H) heterocyclic ring C following the loss of protons, the ^-NMRspectrum (500 MHz; C3H7Ofrom the molecular ion to give m/z acetone-d6) of BC-1 also contained signals 311. The above data indicate that BC-1 has entirely consistent with the presence of a structure 4, and is thus the 2/-hydroxy ( 1 -hydroxy- 1 -methyl)ethyl-dihydrofuran sub- analogue of erythrinin C. Compound BC-1 stituent (c). Thus, apart from two non- has recently been isolated from the uninfected equivalent C-methyl singlets (5 1.25 and 1.30; roots of L. albus and named lupinisoflavone each 3H), an isolated ABXsystem of aliphatic B.20) It is also knownto occur with erythri- proton signals (5 3.16, 1H, dd, /=16 and nin C and wighteone in the fungus-inoculated 9.8Hz, ABX; 3.21, 1H, dd, /=16 and 7.9Hz, twigs of the fig tree, Ficus carica (Mora- ABX; 4.86, 1H, dd, /=9.8 and 7.9Hz, ABX) ceae).21) was attributable to the methylene (1 "-H2) and The second Botrytis metabolite (BC-2) gave methine (2"-H) protons of a dihydrofuran M+ 370 (100%) and prominent ions at m/z 299 ring (c). Since the 263nm UV(MeOH) max- (M+ -71, 58%), 165 (an A-ring derived frag- imumof BC-1 shifted bathochromically with ment, 35%) and 134 (a B-ring derived frag- A1C13 (8nm; C-5-OH group) but not with ment, 31%). The methanolic UVmaximum NaOAc, the sidechain was considered to oc- (263"nm) of BC-2 was shifted bathochromi- cupy the linear position shown in 4.12) Lastly, cally by lOnm upon the addition ofA1C13 (C- the presence of a non-aromatic tertiary OH 5-OH), but was completely unaffected by group at C-3" was deduced from both 1H- NaOAc. Like 1, AF-1 and BC-1, metabolite NMR and MS data. Thus, chemical shift BC-2 afforded a purple/blue color on TLC values for the gem-dimethyl groups (5 1.25 and plates treated with Gibbs reagent. 1.30) closely resembled those reported for dal- In the ^-NMRspectrum (100 MHz; ace- panol13) (6 1.22 and 1.34 in CDC13) whereas tone-d6), aliphatic proton signals were observ- corresponding signals given by the model com- ed at 5 1.34 and 1.40 (each 3H, both s, 2.x pound dihydrorotenone were evident as a 6H CH3), 2.62 (1H, dd, /=17 and 7.1Hz, ABX, doublet at a considerably high-field position (8 >CHAHB), 2.98 (1H, dd, J=ll and 5.4Hz, 0.92).14) It should be noted, incidentally, that ABX, >CHAHB) and 3.87 (1H, dd, 7=7.1 whilst the sidechain methyls of erythrinin C15) and 5.4Hz, ABX, 7CHJ. Otherproton signals are equivalent, appearing at 5.1.34 (s, 6H), were similar to those reported for BC-1 (Table they are clearly non-equivalent in several com- I). From the XH-NMRdata, partial structures pounds (e.g. the rotenoid dehydrodalpanol,13) d and e were equally plausible for BC-2 al- the coumarins columbianetin and marme- though d is favored over the epoxy arrange- sin16jl7) and the alkaloids choisyine18) and ment e because of the lack of a bathochromic platidesmine19)) other than dalpanol and BC- UVshift with NaOAc.12) Also, the loss of 71 1. Significantly, the MS of BC-1 exhibited mass units from the molecular ion, as in the prominent fragments at m/z 311 (M+-59) MSof BC-2, is typical of compounds (e.g. the and m/z 59 (C3H7O), both of which are con- coumarins lomatin and decursinol16)) with a sidered to be characteristic of compounds hydroxy-dimethyldihydropyran substituent. (e.g. columbianetin, marmesin, choisyine and Partial structure e was firmly eliminated when erythrinin C15~18)) possessing a (1-hydroxy- the acetate derivative of BC-2 (BC-2 tetra- 1476 S. Tahara et at.

5 H HO3^OH H H° 0H i 2(AF-1)

\ o oo^-ior h a ,,o u" I R R H I -. R=H 3 (AF-2,BC-3) IAc= R=Ac ^

H H0 °H R R°

4(BC-1) 5a:R=H(BC-2) 5b:R=Ac

Scheme 1. Structure of Luteone (1), Its Metabolites (2, 3, 4, 5a) and a Possible Intermediate (I). The metabolites are arranged to illustrate their possible derivation from luteone (1). AFand BCmean Aspergillus flavus and Botrytis cinerea, respectively. acetate, 5b) was found to be chromatographi- Metabolic aspect of luteone in fungi cally (silica gel TLC) different from epoxy- The major fungal metabolites (2~4, 5a) luteone tetraacetate (IAc in Scheme 1) pre- isolated during the course of the present study pared by the oxidation of luteone tetraacetate are depicted in Scheme 1 where they are ar- with ra-chloroperbenzoic acid10) (Rf values in ranged to illustrate their possible derivation benzene-EtOAc/4 : 1 : 5b, 0.24; IAc, 0.22). Fur- from luteone (1). Structure 4 is particularly thermore, acetylation of BC-2 resulted in a interesting because it possesses a dihydrofuran pronounced lower field shift of the Hxsignal ring identical with, or very similar to, that (A 1.27ppm) which permitted the assignment found in many rotenoid derivatives23* and of this proton to a carbon atome bearing a denoted as ring E. In fact, the biosynthesis of secondary OH group.16) The Hx proton of amorphigenin24'25) is thought to involve ep- psoralenol (S 3.85 in CDC13) exhibits an al- oxidation at the olefinic bond of a 3,3- most identical lower field shift to 6 5.13 (A dimethylallyl-substituted precursor, although 1.28 ppm) upon acetylation of the correspond- as yet the epoxide itself has not been found in ing OHgroup.22) Together with the other data Nature. Subsequent steps via dalpanol (with already presented, this fact fully supports the a sidechain as in BC-1) and rotenone afford view that BC-2 has structure 5a. The MSpeaks amorphigenin. An epoxide intermediate (I in at m/z 165 and 134 could therefore be derived Scheme 1) may likewise be formed during the by RDAfission of the fragment (m/z 299) transformation of l->4 by B. cinerea, and if resulting from the loss of C4H7O from the this is the case, luteone metabolism could molecular ion. provide a useful model for the mechanistic The third B. cinerea metabolite BC-3 was investigation of E-ring formation in rotenone confirmed to be identical with AF-2 by spec- biosynthesis. Alternatively, B. cinerea may troscopic (MS, UV) and chromatographic convert 1 directly, or perhaps indirectly by (TLC in CM, CAAmand PEAa) procedures. epoxidation, to the glycol 3 (Botrytis metab- Fungal Transformation of Luteone 1477 olite BC-3 and Aspergillus metabolite AF-2) 343 -405. 6) U. Willeke and W. Barz, Z. Naturforsch., 37C, 861 which could then be easily cyclized to give 4 (1982). 7) K. M. Weltring, K. Mackenbrock and W. Barz, Z. The antifungalandactivities of5a.compound2 and Naturforsch., 37C, 570 (1982). 4 were compared with that of luteone (1) using 8) P. J. Kuhn, D. A. Smith and D. F. Ewing, Phytochemistry, 16, 296 (1977). the thin-layer plate (silica gel: layer thickness, 9) D. A. Smith, P. J. Kuhn, J. A. Bailey and R. S. 0.25mm) bioassay procedure described by Burden, Phytochemistry, 19, 1673 (1980). Homansand Fuchs.n) Whentested against the 10) B. K. Gupta, G. K. Gupta, K. L. Dhar and C. K. growth of Cladosporium herbarum, spots of 2 Atal, Phytochemistry, 19, 2232 (1980). and 4 gave only weak inhibition zones at ll) A. L. Homans andA. Fuchs, /. Chromatogr., 51, 327 applied levels of 28 ~ 56 fig/cm2. In direct con- (1970). 12) T J. Mabry, K. R. Markham and M. B. Thomas, trast, the growth of C. herbarum was com- "The Systematic Indentification of Flavonoids," pletely inhibited by 1 at levels of 7 ~ 14 jug/cm2. Springer, Berlin, 1970, p. 169. It is clear, therefore, that fungal conversion of 13) D. Adinarayana, M. Radhakrishniah, J. R. Rao, R. 1 into 2 and 4 (and presumably 3 and 5a) Campbell and L. Crombie, J. Chem. Soc. (C), 29 results in a significant detoxification of this (1971). 14) L. Crombie and J. W. Lown, /. Chem. Soc, 775 naturally occurring antifungal agent. (1962). 15) V. H. Deshpande, A. D. Pendse and R. Pendse, Acknowledgments. Wethank Professor S. Takao for his kind supply of the fungal strains, Mr. M. Ikura and Indian J. Chem., 15B, 205 (1977). Miss R. Kato for NMR,and Miss Y. Atsuta for MS 16) R. D. H. Murray, M. Sutcliffe and H. McCabe, Tetrahedron, 27, 4901 (1971). analyses. Financial support (to S. T.) by a Grant-in-Aid 17) W. Steck, Can. J. Chem., 49, 1197 (1971). for Scientific Research (No. 585601 14) from the Ministry of Education, Science and Culture of Japan is also grate- 18) S. R. Johns, J. A. Lamberton and A. A. Sioumis, fully acknowledged. Aust. J. Chem., 20, 1975 (1967). 19) S. R. Johns andJ. A. Lamberton, Aust. J. 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