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Austrocortilutein stereoisomers from Australian fungi of the genus Dermocybe
CO3 l-9422/92$5.00+ 0.00 Q 1992PergamonPressplc
Phytochemlstry, Vol 31,No. 3, PP.947 951,1992 Printedin Great Bntain.
AUSTROCORTILUTEIN STEREOISOMERS FROM AUSTRALIAN OF THE GENUS DERMOCYBE*
FUNGI
MELVYNGILL, ALBERTOGIM~NEZ, AKHIL G. JHINGRANand ASFIA QURESHI School of Chemistry, The University of Melbourne, Parkville, Victoria, Australia 3052 (Received 8 July 1991)
Key Word Index-Dermocybe chemistry.
spp.; Agaricales; pigments; tetrahydroanthraquinones; austrocortilutein; stereo-
Abstract-(lR,3R)-Austrocortilutein, (lR,3S)-austrocortilutein and (S)-1-deoxyaustrocortilutein have been isolated from Australian toadstools belonging to the genus Dermocybe; all four stereoisomers of austrocortilutein (1,2,3,4tetrahydro-1,3,8-trihydroxy-6-methoxy-3-methyl-9,l~anthra~n~ione) and both enantiomers of l-deoxyaustrocortilutein are now known as natural products.
INTRODUCTION
RESULTSANDDISCUSSION
(lS,3S)-Austrocortilutein (l), its (R)-1-deoxy derivative 2 and its (lS,3R) diastereoisomer 3 are the pigments responsible for the yellow colours in the fruit bodies of the Australasian toadstool Dermocybe splendida [l-3]. The tetrahydroanthraquinones 1 and 3 and their red cometabolites 5 and 6 are active at low concentrations against a range of Gram-positive and Gram-negative bacteria and fungi [S], and are becoming increasingly important in the taxonomy of Dermocybe [4-61. The absolute configuration of the major quinones 1 and 5 from D. splendida was established by chemical conversion via their respective l-deoxy derivatives 2 and 6 to the (R) lactone 7 [7] with which direct chiroptical comparison was made [3]. Alternatively, the (lS,3R) configuration of the minor yellow quinone 3 rested on the evident antipodal relationship between its hydrogenolysis product 4 {[LY]~’+ 73” (CHCI,)} and the (R) quinone 2 {[a];‘- 78” (CHCI,)} [3]. We report here the isolation from within a small group of toadstools related to D. splendida of three new tetrahydroanthraquinones of the austrocortilutein type. Two of these pigments are the enantiomers 9 and 12 of the known quinones 1 and 3, respectively, while the third is the antipode 4 of the known pigment 2. Consequently, all four possible stereochemical modifications of austrocortilutein (1,2,3,4-tetrahydro-1,3,8-trihydroxy-6-methoxy-3methyl-9,10-anthracenedione) are now known as natural products.
*Part 25 in the series ‘Pigments of Fungi’. For Part 24 see ref. [13]. ?‘These fungi are placed as new taxa in Dermocybe(Watling, R., personal communication; May, T., in [S]). They are referred to here by the accession numbers under which voucher spccimens are held in the Herbarium of the Royal Botanic Garden, Edinburgh, U.K.
We have found several yellow and yellow-brown species of Dermocybe toadstools in S. E. Victoria and Tasmania which, while lacking red pigments of the types 5 and 6, contain quinones of the austrocortilutein type as the major colouring matters [8]. Three of these species, WAT20934, WAT21568 and WAT 21567,t are discussed further here. Fruit bodies of WAT 20934 have a brown, umbonate cap supported by a slender, brown-yellow stipe that tapers to bright yellow basal mycelium. In young specimens the yellow gills are protected by an intense yellow evanescent cortina. Solvent extraction of whole, fresh fruit bodies gave a golden yellow solution. The orange residue obtained after evaporation of the solvent was partitioned between ethyl acetate and water and the components of the organic phase were purified by preparative TLC. The two major orange zones [R, 0.22 and 0.29 (toluene-ethyl formate-formic acid, 50:49: l)] were separated from several minor constituents (Experimental) and purified individually by further chromatography. The contents of the more mobile zone crystallized as orange needles, mp 183-187”, [cr]~z-61” (EtOH), in a yield corresponding to 9 x lo-*% of the fresh weight of the fungus. Mass spectroscopic and combustion analysis data led to the molecular formula C,,H,,06, which, together with the UV, IR and ‘HNMR data (below), were immediately suggestive of an austrocortilutein structure. In particular, the aliphatic proton region of the ‘H NMR spectrum (Fig. la) was indistinguishable from that recorded for (lS,3S)-austrocortilutein (1) from D. splendida [3], thus providing unequivocal proof of the presence of a cis-1,3-dihydroxy-3_methyltetrahydroaromatic ring in the new pigment. In accord with this conclusion the new quinone reacted smoothly with 2,2dimethoxypropane to yield a cyclic acetonide derivative, mp 194-198”, [a];‘- 54” (CHCI,). Comparison of the specific rotation of this orange pigment and its acetonide (uide supra) with data reported for (l&3@austrocortilutein (1) {[a];‘+ 62” (EtOH)} [l]
941
M. GILL et al.
948
Me0
Me0
Me0
5 R=OH 6 R=H
3 R=OH 4 R=H
1 R=OH
2 R=H
J--h U’
0
0
R’ R*
7 R’ = CMf.Z02Me, R* = Me 8 R’ = Me. R2 = CH#$@e
OH
0
OH
13
12
its acetonide derivative f[a];40+60” (CHCI,)) [3] suggested that the new natural product and its acetonide must possess the (lR,3R) absolute stereochemistry depicted in formulae 9 and 10,respectively. This conclusion was verified by a series of degradation reactions that linked (lR,3R)-austrocortilutein (9) with the (S) lactone 8 [7]. Thus, catalytic hydrogenolysis of 9 gave the deoxy derivative 4, mp 205-209”, [a]i”+69” (CHCI,), which upon oxidation with ruthenium tetraoxide [9] and subsequent este~fi~tion gave the lactone 8, which was purified by HPLC. The structure of the chiral lactone 8 was confirmed by spectroscopy but the limited quantity available (ca 1 mg) prevented reliable measurement of the specific rotation. Consequently, the (S) absolute stereochemistry of 8 was determined by ‘H NMR experiments using tris[3~heptafluoropropylhydroxymethylene)-( +)-camphoratol-europium (HI) [Eufhfc),] as chiral shift reagent [lo]. It follows that the dextrorotatory I-deoxy derivative 4 and its laevorotatory progenitor 9 must, therefore, possess (S) and (lR,3R) stereochemistries, respectively. This is the first report of ~lR,3R)-austro~orti~utein (9) as a natural product. The less mobile orange pigment (4 x IO-‘%) from WAT20934, mp 162-165”, [a];’ -1-262” (CHCl,), was identified as (lS,3R)-austrocortiiutein (3) from the spectroscopic data and by direct comparison with material isolated from D. sp~en~idu [3]. Solvent extraction of the yellow fruit bodies of WAT21568 gave an orange extract. Preparative TLC and
effected preliminary separation of an anthraquinone fraction [Rf 0.82 (toluene~thyl formate-formic acid, 50:49:1)-J from a group of less mobile, less abundant preanthraquinones (Rr 0.54-0.22 in the same solvent system). Crystallization of the contents of the more mobile zone gave 6-methylxanthopurpurin-3-O-methyl ether (11) (0.14%), which was identified by comparison with authentic material [l $1. Further chromatography involving the preanthraquinone fraction gave (lS,3R )austroco~iiutein (3) (4 x 10m2%) and (tR,3R)austrocortilutein (9) (7.5 x 1O-2%), identical with materials isolated from WAT20934, together with a third, minor quinone (2 x 10w3%), ClbHtb05 (mass spec.), mp 205-209”, [a]k2 t-63” (CHCI,). This pigment, a new natural product, was identified as (S)-l-deoxyaustrocortilutein (4) by direct comparison with a sample obtained by hydrogenolysis of (lR,3R)-austrocortilutein (9) (uide supra).
It is notable from a stereochemical viewpoint that whereas Dermocybe splendida produces the (lS,3S)- and (lS,3R)-austrocortiluteins, pigments epimeric at the tertiary chiral centre (C-3), WAT20934 and WAT21568 produce the novel (lR,3R) diastereoisomer 9 of austrocortilutein together with its (lS,3R) benzyhc {i.e. C-l) epimer. Fruit bodies of WAT21567 resemble the two species described above by virtue of the predominant brown, orange and orange-yellow tones. Extraction of WAT21567 gave a golden yellow extract which by using
Austrocortilutein stereoisomers from fungi
949
3.30. s 1.43,s
\
2.99, cld, 19.9& 2.2Hz
14.6.2.2 & 1.9Hz 2.37, dd, 19.9 & I.9 Hz 1.78, dd. 14.6 & 5.1 Hz
1.73, dd, 13.6 & 9.2 Hz
2.53, d&
2.30, ddd,
br d, 19.4 Hz
(b) Fig. 1. ‘H NMR data for the protons in the tetrahydroaromatic
TLC could be seen to contain two major orange pigments [Rf 0.22and 0.29 (toluene~thyl fo~at~fo~ic acid, 50:49: l)] and a third, minor and less mobile orangeyellow zone (R,0.15). The components of this polar zone have been dealt with in detail elsewhere [12, 131. Preparative TLC afforded the major pigment (R,0.29; 6.7 x lo-‘%) as orange needles, mp 183--187”, [a]:’ +60 (EtOH), which was identified as (lS,3S)-austrocortilutein (1) by direct comparison with authentic material [l, 31. Its more polar counterpart (R, 0.22; 2x 10m2%), mp 162-165”, [x]gz-262” (CHCI,), was assigned the molecular formula C,,Hi60, from mass spectroscopic and combustion analysis data. It was identified as the last remaining austrocortilutein stereoisomer 12 from the spectroscopic data and by chemical correlation with the known (all-deoxyaustroco~ilutein (2) [2, 33. Thus, the mass, UV, IR and ‘H NMR spectra of 12 clearly indicated
rings in (a) 9 and (b) 12.
the familiar austrocortilutein nucleus. Comparison of that part of the ‘HNMR spectrum between 1.7 and 5.2 ppm (Fig. lb) with data previously reported for (lS,3R)-austrocortilutein (3) [3] firmly established that the hydroxy groups at C-l and C-3 in the new quinone 12 must be truns disposed. The (lR,3S) absolute stereochemistry for the new natural product 12 was evident from comparison of its specific rotation (vide supra) with data reported for (lS,3R)austrocortilutein (3) ([a]F +288” (CHCI,)} and was confirmed by hydrogenolysis of 12 which furnished the deoxy derivative 2, [x]$’ -70” (CHCl,), of known (R) absolute configuration [3]. The isolation of (lR,3S)-austrocortilutein (12) from WAT 21567 brings to four the number of stereoisomers of this tetrahydroan~raquinone that have now been isolated,
each
in homochiral
form,
from
within
a small
M. GILL et al.
950
group of related Basidiomycotina. Again, WAT 21567, like WAT 20934 and WAT 21568, produces austrocortiluteins epimeric at the benzylic (C-l) centre but does so, remarkably, by producing both pigments in the opposite enantiomeric series to that found in its close relatives. We are aware of no other example of the occurrence of all four stereoisomeric modifications of a natural product containing two chiral centres within such a closely related group
of organisms.
EXPERIMENTAL ‘H NMR: 399.65 MHz; 13C NMR: 100.40 MHz, CDCI, with TMS as int. standard; EIMS: 70 eV (probe); prep. TLC: Merck Kieselgel60 GF,s, layers (0.1 x 20 x 20 cm) on glass plates using toluene-HCO,Et-HCO,H (50:49: 1) as eluant. Voucher specimens of the fungi discussed here are lodged at the Herbarium of the Royal Botanic Garden, Edinburg, U.K., under the accession numbers quoted in the text. Elemental analyses were performed by Chemical and Micro Analytical Services Pty Ltd, North Essendon, Victoria. Isolation of pigments from WAT20934. Fruit bodies were collected along the Hop Island track in State Forest close to Marysville, Victoria in June and July 1988. Fresh toadstools (35 g) were finely chopped and soaked in EtOH (500 ml) for 3 hr. The yellow extract was evapd under red. pres. and the orange residue (0.7 g) was partitioned between EtOAc (3 x 100 ml) and H,O (1OOml). The organic phase was coned and the residue (0.2 g) was purified by prep. TLC to afford, in order of increasing polarity: 6-methylxanthopurpurin-3-O-methyl ether (11) (R,0.82) (0.2 mg, 6 x 10m4% fr. wt), yellow needles (from CHCI,-EtOH at -2o”), mp 183-185” (ref. [ll] 186-187”), identical with an authentic sample [ll], l-deoxyaustrocortilutein (2 or 4) (RI 0.54)(0.1 mg, 3 x 10-4% fr. wt) and torosachrysone (13) (R, 0.35) (0.1 mg, 3 x 10-4% fr. wt). both identified by TLC comparison with authentic samples [2, 141, (lR,3R)austrocortilutein (9) (RI 0.29) (32 mg, 9 x 10m2% fr. wt). orange needles (from CHCl-EtOH), mp 183-187” (Found: C, 63.0; H, 5.3. C 16H 160 6 requires C. 63.15; H, 5.3%); [a];‘-61’ (EtOH; c 0.24); CD I::;H-CHC” nm (As): 260 (-2.56), 264 (O.O), 282 (+ 2.08), 320 (O.O), 350 (-0.24), 400 (0.0); ‘H NMR: Fig. la and 63.88 (3H, s, OMe), 6.58 (lH, d, J=2.6 Hz, H-7), 7.09 (lH, d, J h2.6 Hz, H-S), 12.19 (lH, s, OH-8); identical in all other respects with 1 [3], and (lS,3R)-austrocortilutein (3) (R, 0.22) (15 mg, 4x lo-‘% fr. wt), orange needles (from EtOAc-petrol), mp 162-165” (ref. [3] 162-164”), [~(];‘+262(CHCl~;c0.21);CD 1~‘$H-cHC’3 nm (As): 230 (- 1.1 l), 24.5 (O.O),250 (+0.52), 263 (O.O), 294 (-0.96), 400 (0.0); identical in all other respects with an authentic sample [3J. (lR,3R)-Austrocortilutein acetonide (IO). (lR,3R)-Austrocortilutein (9) (8 mg) in CH,Cl, (1.5 ml) containing 2,2-dlmethoxypropane (3 ml) and para-toluenesulphonic acid (1 mg) was stirred at room temp. for 16hr. The mixture was diluted with CHCI, (10 ml) and washed with dil. aq. NaHCO, (1 M, 20 ml). The organic phase was coned and purified by prep. TLC to give the acetonide 10 (7 mg, 77%), yellow needles (from EtOAcpetrol), mp 194-198” (Found. [M]’ 344.1258. C,,H,,O, requires 344.1259); [a] A2- 54” (CHCI,; ~0.2); otherwise indistinguishable from the acetonide derivative of 1 [l, 31. Isolation of pigments from WAT21568. Frmt bodies were
*Prolonged storage during transportation to Melbourne may account wholly or in part for the high concentration of the anthraqumone 11 present in the extracts.
collected in the Mount Field National Park, Tasmania in March 1987. Fresh toadstools (146 g) were finely chopped and immersed in EtOH (1 1)for 10 days.* The orange extract was evapd and the residue (2.2 g) was partitioned between EtOAc (3 x 200 ml) and H,O (200 ml), The organic phase was coned and the residue (0.9 g) was purified by prep. TLC to afford, in order of increasing polarity; 6-methylxanthopurpurin-3-O-methyl ether (11) (208mg, 0.14% fr. wt), (S)-1-deoxyaustrocortilutein (4) (R/ 0.54) (3 mg, 2 x 10e3% fr. wt), orange needles (from MeOH at -2o”), mp 205-209” (Found, [M]’ 288.0997. C,,H,,O, reqmres 288.0997); [a];’ +63” (CHCI,: ~0.18); CD ~.~$‘“‘“‘c” nm (As): 270 (+0.28), 280 (O.O), 300 (-0.22). 345 (0.0). 380 (+005), 400 (O.Ok identical in all other respects with its antipode 2 [2. 31, (lR,3R)-austrocortilutein (9) (109mg. 7.5 x lo-‘% fr. wt), and (lS,3R)-austrocortilutein (3) (58 mg, 4 x lo-‘% fr. wt). Isolation of pigments from w.47’21567 Fruit bodies were collected along the Dickinson track in State Forest near Marysville. Victoria during June 1985. Fresh toadstools (48 g) were finely chopped and soaked in EtOH (500 ml) for 3 hr. The orange extract was evapd and the residue (1.2 g) was partitioned between EtOAc (3 x 100 ml) and H,O (100ml). The organic phase was coned and the residue (0.3 g) was punfied by prep. TLC to afford, in order of Increasing polarity. (lS,3S)austrocortilutein (1) (R, 0.29) (32 mg, 6.7 x 10 ‘% fr. wt), orange needles (from CHCI,-EtOH), mp 183-187” (ref. [3] 183-185”); [a];’ f60” (EtOH; ~0.26); CD ,?~~~H+cHC’~ nm (As): 230 (+ 1.23), 2.50 (O.O),284 (- 1.48). 400 (0.0); otherwise identical with an authentic specimen [S], (lR,3S)-austrocortilutem (12) (R,0.22) (10 mg, 2 x lo-‘% fr. wt), orange needles (from EtOAc-petrol), mp 162-165. (Found: C. 63.0; H, 5 4. C,,H,,O, requires C, 63.15; H. 5.3%); [a];‘-262” (CHCI,; ~0.18); CD 1.~:~H+CWC’3nm (As): 235 (+0.8X), 245 (0.0). 255 (-0.37). 265 (0.0). 288 (+0.74), 330 (O.O), 355 (-0.29). 400 (0.0): ‘H NMR Fig. lb and 63.90 (3H. s, OMe), 5.17 (1H. m. H-l), 6.61 (IH, d, J =2.6 Hz. H-7), 7.14(1H,d. J=2.6 Hz, H-5), 12.13 (lH, s. OH-8), identical in all other respects with its antipode 3 [3], and a yellow zone(R,0.15)(4.5 mg) [12,13J. The quinones2(or 4)and 11,and the dihydroanthracenone 13 were observed m the chromatograms but were not isolated. Hydrogenolysrs of (1R,3R)-austrocortilutem (9). (lR,3R)Austrocortilutein (9) (10 mg) m MeOH (3 ml) was exposed to H, in the presence of Pd-C (lo%, 1.5 mg) until uptake of H, was complete (ca 1 hr). The catalyst was filtered off and washed with MeOH (10 ml) and the filtrate evapd under red. pres. The residue was purified by prep. TLC to afford (S)-l-deoxyaustrocortdutein (4) (R,0.54) (8.2 mg, 76%), orange needles (from MeOH at -2o”), mp 205-209” (Found: [M]’ 288.0994. Calc. for C,,H,,05: 288.0997); [a]:* +69’ (CHCI,, ~0.18); identical in all other respects with material isolated from WAT21568. Sinularly, hydrogenolysrs of (lS,3R)-austrocortilutein (3) (1.5mg) from WAT20934 gave 4 (0.9mg, 63%); [a]i2+70” (CHCI,; c 0.13). Hydrogenolysis of (lR,3S)-austrocortilutein (12) (1.8 mg) under the same conditions gave (R)-1-deoxyaustrocortilutein (2) (1 mg, 59%), orange needles (from MeOH at -20”). mp 205-209” (ref. [3] 206-212”) (F ound: [Ml+ 288.0997. Calc. for C,,H,,O,: 288.0997); [a];’ -70” (CHCl,; ~0.10); CD ~~$‘H+CHC” nm (A&): 270 (-0.28), 280 (0.0). 300 (+0.22). 345 (O.O),380 (-0.05), 400 (0.0); identical in all other respects with an authentic sample [2, 31. Oxidation of(S)-l-deoxyaustrocortilutein (4) A mixture of the quinone 4 (6.1 mg, 0.02 mmol), NaIO, (222 mg) and a trace. of RuCI,.3H,O was stirred vigorously in a heterogeneous mixture of Ccl, (2 ml), MeCN (2 ml) and H,O (3 ml) at room temp. for 24 hr. After this time iso-PrOH (2 ml) was added followed by an excess of BaCl,. The mixture was stirred vigorously for 30 min,
951
Austrocortilutein stereoisomers from fungi filtered, and the filtrate was extracted with CH,Cl, (3 x 10 ml), acidified and exhaustively extracted (24 hr) with Et,O. The organic phases were combined and evapd. The residue was dissolved in Et,0 (1 ml) and an excess of CH,N, was added. After 30 min the excess CH,N, was destroyed with HOAc and the solvent was evapd. HPLC [Ultrasphere-Si, 5~~ 1 x 25 cm, EtOAc-hexane (3:2)] gave the (S) lactone 8 (1.1 mg, 18%) as an oil. The absolute configuration was determined by ‘H NMR as follows: to the lactone 8 (0.95 mg) in CDCI, (0.5 ml) in an NMR tube was added an aliquot (40~1) of a solution of Eu(hfc), (150 mg) in CDCI, (1 ml). At that point the OMe and C-Me signals appeared at 63.94 and 1.75, respectively, shifted from 63.70 and 1.51 in the spectrum of 8 alone, but neither signal was resolved. Addition of a soln of the (R) lactone 7 (200 pg) [7] in CDCl, (100 ~1) gave rise to additional signals at 63.95 (OMe) and 1.76 (C-Me). Acknowledgements-We are indebted to Dr R. Watling (Royal Botanic Garden, Edinburgh) for lodging voucher specimens and Mr T. May (National Herbarium of Victoria) for invaluable help on the location, and advice on the identification, of fungi. We thank Mr R.W. Rickards, F.A.A. (ANU) for providing facilities for recording CD spectra. The Australian Research Council provided generous financial support and A.G. is grateful for a Melbourne University Postgraduate Research Award. Thanks are extended to the Department of Conservation, Forests and Lands for permission to collect fungi in National Parks under their jurisdiction.
REFERENCES 1. Gill, M. and Strauch, R. J. (1985) Tetrahedron Letters 26, 2593. 2. Gill, M. and Smrdel, A. F. (1987) Phytochemistry 26, 2999. 3. Gill, M., Smrdel, A. F., Strauch, R. J. and Begley, M. J. (1990) J. Chem. Sot., Perkin Trans. I 1583. 4. Gill, M. and Steglich, W. (1987) Prog. Chem. Org. Nat. Prods 51, 125. 5. Keller, G., Moser, M., Horak, E. and Steglich, W. (1987) Sydowia 40, 169. 6. Horak, E. (1987) Sydowia 40, 81. 7. Gill, M. and Smrdel, A. F. (1990) Tetrahedron: Asymmetry 1, 453. 8. Gimknez, A. (1990) Ph.D. Thesis. The University of Melbourne. 9. Carlsen, P. H. J., Katsuki, T., Martin, V. S. and Sharpless, K. B. (1981) J. Org. Chem. 46, 3936. 10. Gill, M.,Gim&ez, A., Jhingran, A. G. and Palfreyman, A. R. (1990) Tetrahedron: Asymmetry 1, 621. 11. Archard, M. A., Gill, M. and Strauch, R. J. (1985) Phytochemistry 24, 2755. 12. Burns, C. J., Gill, M. and Gimknez, A. (1989) Tetrahedron Letters 30, 7269. 13. Burns, C. J., Gill, M. and Gimbnez, A. (1992) Aust. J. Chem. (in press). 14. Gill, M., Gimknez, A., Jhingran, A. G. and Smrdel, A. F. (1989) Phytochemistry 28, 2647.
Phytochemlstry, Vol 31,No. 3, PP.947 951,1992 Printedin Great Bntain.
AUSTROCORTILUTEIN STEREOISOMERS FROM AUSTRALIAN OF THE GENUS DERMOCYBE*
FUNGI
MELVYNGILL, ALBERTOGIM~NEZ, AKHIL G. JHINGRANand ASFIA QURESHI School of Chemistry, The University of Melbourne, Parkville, Victoria, Australia 3052 (Received 8 July 1991)
Key Word Index-Dermocybe chemistry.
spp.; Agaricales; pigments; tetrahydroanthraquinones; austrocortilutein; stereo-
Abstract-(lR,3R)-Austrocortilutein, (lR,3S)-austrocortilutein and (S)-1-deoxyaustrocortilutein have been isolated from Australian toadstools belonging to the genus Dermocybe; all four stereoisomers of austrocortilutein (1,2,3,4tetrahydro-1,3,8-trihydroxy-6-methoxy-3-methyl-9,l~anthra~n~ione) and both enantiomers of l-deoxyaustrocortilutein are now known as natural products.
INTRODUCTION
RESULTSANDDISCUSSION
(lS,3S)-Austrocortilutein (l), its (R)-1-deoxy derivative 2 and its (lS,3R) diastereoisomer 3 are the pigments responsible for the yellow colours in the fruit bodies of the Australasian toadstool Dermocybe splendida [l-3]. The tetrahydroanthraquinones 1 and 3 and their red cometabolites 5 and 6 are active at low concentrations against a range of Gram-positive and Gram-negative bacteria and fungi [S], and are becoming increasingly important in the taxonomy of Dermocybe [4-61. The absolute configuration of the major quinones 1 and 5 from D. splendida was established by chemical conversion via their respective l-deoxy derivatives 2 and 6 to the (R) lactone 7 [7] with which direct chiroptical comparison was made [3]. Alternatively, the (lS,3R) configuration of the minor yellow quinone 3 rested on the evident antipodal relationship between its hydrogenolysis product 4 {[LY]~’+ 73” (CHCI,)} and the (R) quinone 2 {[a];‘- 78” (CHCI,)} [3]. We report here the isolation from within a small group of toadstools related to D. splendida of three new tetrahydroanthraquinones of the austrocortilutein type. Two of these pigments are the enantiomers 9 and 12 of the known quinones 1 and 3, respectively, while the third is the antipode 4 of the known pigment 2. Consequently, all four possible stereochemical modifications of austrocortilutein (1,2,3,4-tetrahydro-1,3,8-trihydroxy-6-methoxy-3methyl-9,10-anthracenedione) are now known as natural products.
*Part 25 in the series ‘Pigments of Fungi’. For Part 24 see ref. [13]. ?‘These fungi are placed as new taxa in Dermocybe(Watling, R., personal communication; May, T., in [S]). They are referred to here by the accession numbers under which voucher spccimens are held in the Herbarium of the Royal Botanic Garden, Edinburgh, U.K.
We have found several yellow and yellow-brown species of Dermocybe toadstools in S. E. Victoria and Tasmania which, while lacking red pigments of the types 5 and 6, contain quinones of the austrocortilutein type as the major colouring matters [8]. Three of these species, WAT20934, WAT21568 and WAT 21567,t are discussed further here. Fruit bodies of WAT 20934 have a brown, umbonate cap supported by a slender, brown-yellow stipe that tapers to bright yellow basal mycelium. In young specimens the yellow gills are protected by an intense yellow evanescent cortina. Solvent extraction of whole, fresh fruit bodies gave a golden yellow solution. The orange residue obtained after evaporation of the solvent was partitioned between ethyl acetate and water and the components of the organic phase were purified by preparative TLC. The two major orange zones [R, 0.22 and 0.29 (toluene-ethyl formate-formic acid, 50:49: l)] were separated from several minor constituents (Experimental) and purified individually by further chromatography. The contents of the more mobile zone crystallized as orange needles, mp 183-187”, [cr]~z-61” (EtOH), in a yield corresponding to 9 x lo-*% of the fresh weight of the fungus. Mass spectroscopic and combustion analysis data led to the molecular formula C,,H,,06, which, together with the UV, IR and ‘HNMR data (below), were immediately suggestive of an austrocortilutein structure. In particular, the aliphatic proton region of the ‘H NMR spectrum (Fig. la) was indistinguishable from that recorded for (lS,3S)-austrocortilutein (1) from D. splendida [3], thus providing unequivocal proof of the presence of a cis-1,3-dihydroxy-3_methyltetrahydroaromatic ring in the new pigment. In accord with this conclusion the new quinone reacted smoothly with 2,2dimethoxypropane to yield a cyclic acetonide derivative, mp 194-198”, [a];‘- 54” (CHCI,). Comparison of the specific rotation of this orange pigment and its acetonide (uide supra) with data reported for (l&3@austrocortilutein (1) {[a];‘+ 62” (EtOH)} [l]
941
M. GILL et al.
948
Me0
Me0
Me0
5 R=OH 6 R=H
3 R=OH 4 R=H
1 R=OH
2 R=H
J--h U’
0
0
R’ R*
7 R’ = CMf.Z02Me, R* = Me 8 R’ = Me. R2 = CH#$@e
OH
0
OH
13
12
its acetonide derivative f[a];40+60” (CHCI,)) [3] suggested that the new natural product and its acetonide must possess the (lR,3R) absolute stereochemistry depicted in formulae 9 and 10,respectively. This conclusion was verified by a series of degradation reactions that linked (lR,3R)-austrocortilutein (9) with the (S) lactone 8 [7]. Thus, catalytic hydrogenolysis of 9 gave the deoxy derivative 4, mp 205-209”, [a]i”+69” (CHCI,), which upon oxidation with ruthenium tetraoxide [9] and subsequent este~fi~tion gave the lactone 8, which was purified by HPLC. The structure of the chiral lactone 8 was confirmed by spectroscopy but the limited quantity available (ca 1 mg) prevented reliable measurement of the specific rotation. Consequently, the (S) absolute stereochemistry of 8 was determined by ‘H NMR experiments using tris[3~heptafluoropropylhydroxymethylene)-( +)-camphoratol-europium (HI) [Eufhfc),] as chiral shift reagent [lo]. It follows that the dextrorotatory I-deoxy derivative 4 and its laevorotatory progenitor 9 must, therefore, possess (S) and (lR,3R) stereochemistries, respectively. This is the first report of ~lR,3R)-austro~orti~utein (9) as a natural product. The less mobile orange pigment (4 x IO-‘%) from WAT20934, mp 162-165”, [a];’ -1-262” (CHCl,), was identified as (lS,3R)-austrocortiiutein (3) from the spectroscopic data and by direct comparison with material isolated from D. sp~en~idu [3]. Solvent extraction of the yellow fruit bodies of WAT21568 gave an orange extract. Preparative TLC and
effected preliminary separation of an anthraquinone fraction [Rf 0.82 (toluene~thyl formate-formic acid, 50:49:1)-J from a group of less mobile, less abundant preanthraquinones (Rr 0.54-0.22 in the same solvent system). Crystallization of the contents of the more mobile zone gave 6-methylxanthopurpurin-3-O-methyl ether (11) (0.14%), which was identified by comparison with authentic material [l $1. Further chromatography involving the preanthraquinone fraction gave (lS,3R )austroco~iiutein (3) (4 x 10m2%) and (tR,3R)austrocortilutein (9) (7.5 x 1O-2%), identical with materials isolated from WAT20934, together with a third, minor quinone (2 x 10w3%), ClbHtb05 (mass spec.), mp 205-209”, [a]k2 t-63” (CHCI,). This pigment, a new natural product, was identified as (S)-l-deoxyaustrocortilutein (4) by direct comparison with a sample obtained by hydrogenolysis of (lR,3R)-austrocortilutein (9) (uide supra).
It is notable from a stereochemical viewpoint that whereas Dermocybe splendida produces the (lS,3S)- and (lS,3R)-austrocortiluteins, pigments epimeric at the tertiary chiral centre (C-3), WAT20934 and WAT21568 produce the novel (lR,3R) diastereoisomer 9 of austrocortilutein together with its (lS,3R) benzyhc {i.e. C-l) epimer. Fruit bodies of WAT21567 resemble the two species described above by virtue of the predominant brown, orange and orange-yellow tones. Extraction of WAT21567 gave a golden yellow extract which by using
Austrocortilutein stereoisomers from fungi
949
3.30. s 1.43,s
\
2.99, cld, 19.9& 2.2Hz
14.6.2.2 & 1.9Hz 2.37, dd, 19.9 & I.9 Hz 1.78, dd. 14.6 & 5.1 Hz
1.73, dd, 13.6 & 9.2 Hz
2.53, d&
2.30, ddd,
br d, 19.4 Hz
(b) Fig. 1. ‘H NMR data for the protons in the tetrahydroaromatic
TLC could be seen to contain two major orange pigments [Rf 0.22and 0.29 (toluene~thyl fo~at~fo~ic acid, 50:49: l)] and a third, minor and less mobile orangeyellow zone (R,0.15). The components of this polar zone have been dealt with in detail elsewhere [12, 131. Preparative TLC afforded the major pigment (R,0.29; 6.7 x lo-‘%) as orange needles, mp 183--187”, [a]:’ +60 (EtOH), which was identified as (lS,3S)-austrocortilutein (1) by direct comparison with authentic material [l, 31. Its more polar counterpart (R, 0.22; 2x 10m2%), mp 162-165”, [x]gz-262” (CHCI,), was assigned the molecular formula C,,Hi60, from mass spectroscopic and combustion analysis data. It was identified as the last remaining austrocortilutein stereoisomer 12 from the spectroscopic data and by chemical correlation with the known (all-deoxyaustroco~ilutein (2) [2, 33. Thus, the mass, UV, IR and ‘H NMR spectra of 12 clearly indicated
rings in (a) 9 and (b) 12.
the familiar austrocortilutein nucleus. Comparison of that part of the ‘HNMR spectrum between 1.7 and 5.2 ppm (Fig. lb) with data previously reported for (lS,3R)-austrocortilutein (3) [3] firmly established that the hydroxy groups at C-l and C-3 in the new quinone 12 must be truns disposed. The (lR,3S) absolute stereochemistry for the new natural product 12 was evident from comparison of its specific rotation (vide supra) with data reported for (lS,3R)austrocortilutein (3) ([a]F +288” (CHCI,)} and was confirmed by hydrogenolysis of 12 which furnished the deoxy derivative 2, [x]$’ -70” (CHCl,), of known (R) absolute configuration [3]. The isolation of (lR,3S)-austrocortilutein (12) from WAT 21567 brings to four the number of stereoisomers of this tetrahydroan~raquinone that have now been isolated,
each
in homochiral
form,
from
within
a small
M. GILL et al.
950
group of related Basidiomycotina. Again, WAT 21567, like WAT 20934 and WAT 21568, produces austrocortiluteins epimeric at the benzylic (C-l) centre but does so, remarkably, by producing both pigments in the opposite enantiomeric series to that found in its close relatives. We are aware of no other example of the occurrence of all four stereoisomeric modifications of a natural product containing two chiral centres within such a closely related group
of organisms.
EXPERIMENTAL ‘H NMR: 399.65 MHz; 13C NMR: 100.40 MHz, CDCI, with TMS as int. standard; EIMS: 70 eV (probe); prep. TLC: Merck Kieselgel60 GF,s, layers (0.1 x 20 x 20 cm) on glass plates using toluene-HCO,Et-HCO,H (50:49: 1) as eluant. Voucher specimens of the fungi discussed here are lodged at the Herbarium of the Royal Botanic Garden, Edinburg, U.K., under the accession numbers quoted in the text. Elemental analyses were performed by Chemical and Micro Analytical Services Pty Ltd, North Essendon, Victoria. Isolation of pigments from WAT20934. Fruit bodies were collected along the Hop Island track in State Forest close to Marysville, Victoria in June and July 1988. Fresh toadstools (35 g) were finely chopped and soaked in EtOH (500 ml) for 3 hr. The yellow extract was evapd under red. pres. and the orange residue (0.7 g) was partitioned between EtOAc (3 x 100 ml) and H,O (1OOml). The organic phase was coned and the residue (0.2 g) was purified by prep. TLC to afford, in order of increasing polarity: 6-methylxanthopurpurin-3-O-methyl ether (11) (R,0.82) (0.2 mg, 6 x 10m4% fr. wt), yellow needles (from CHCI,-EtOH at -2o”), mp 183-185” (ref. [ll] 186-187”), identical with an authentic sample [ll], l-deoxyaustrocortilutein (2 or 4) (RI 0.54)(0.1 mg, 3 x 10-4% fr. wt) and torosachrysone (13) (R, 0.35) (0.1 mg, 3 x 10-4% fr. wt). both identified by TLC comparison with authentic samples [2, 141, (lR,3R)austrocortilutein (9) (RI 0.29) (32 mg, 9 x 10m2% fr. wt). orange needles (from CHCl-EtOH), mp 183-187” (Found: C, 63.0; H, 5.3. C 16H 160 6 requires C. 63.15; H, 5.3%); [a];‘-61’ (EtOH; c 0.24); CD I::;H-CHC” nm (As): 260 (-2.56), 264 (O.O), 282 (+ 2.08), 320 (O.O), 350 (-0.24), 400 (0.0); ‘H NMR: Fig. la and 63.88 (3H, s, OMe), 6.58 (lH, d, J=2.6 Hz, H-7), 7.09 (lH, d, J h2.6 Hz, H-S), 12.19 (lH, s, OH-8); identical in all other respects with 1 [3], and (lS,3R)-austrocortilutein (3) (R, 0.22) (15 mg, 4x lo-‘% fr. wt), orange needles (from EtOAc-petrol), mp 162-165” (ref. [3] 162-164”), [~(];‘+262(CHCl~;c0.21);CD 1~‘$H-cHC’3 nm (As): 230 (- 1.1 l), 24.5 (O.O),250 (+0.52), 263 (O.O), 294 (-0.96), 400 (0.0); identical in all other respects with an authentic sample [3J. (lR,3R)-Austrocortilutein acetonide (IO). (lR,3R)-Austrocortilutein (9) (8 mg) in CH,Cl, (1.5 ml) containing 2,2-dlmethoxypropane (3 ml) and para-toluenesulphonic acid (1 mg) was stirred at room temp. for 16hr. The mixture was diluted with CHCI, (10 ml) and washed with dil. aq. NaHCO, (1 M, 20 ml). The organic phase was coned and purified by prep. TLC to give the acetonide 10 (7 mg, 77%), yellow needles (from EtOAcpetrol), mp 194-198” (Found. [M]’ 344.1258. C,,H,,O, requires 344.1259); [a] A2- 54” (CHCI,; ~0.2); otherwise indistinguishable from the acetonide derivative of 1 [l, 31. Isolation of pigments from WAT21568. Frmt bodies were
*Prolonged storage during transportation to Melbourne may account wholly or in part for the high concentration of the anthraqumone 11 present in the extracts.
collected in the Mount Field National Park, Tasmania in March 1987. Fresh toadstools (146 g) were finely chopped and immersed in EtOH (1 1)for 10 days.* The orange extract was evapd and the residue (2.2 g) was partitioned between EtOAc (3 x 200 ml) and H,O (200 ml), The organic phase was coned and the residue (0.9 g) was purified by prep. TLC to afford, in order of increasing polarity; 6-methylxanthopurpurin-3-O-methyl ether (11) (208mg, 0.14% fr. wt), (S)-1-deoxyaustrocortilutein (4) (R/ 0.54) (3 mg, 2 x 10e3% fr. wt), orange needles (from MeOH at -2o”), mp 205-209” (Found, [M]’ 288.0997. C,,H,,O, reqmres 288.0997); [a];’ +63” (CHCI,: ~0.18); CD ~.~$‘“‘“‘c” nm (As): 270 (+0.28), 280 (O.O), 300 (-0.22). 345 (0.0). 380 (+005), 400 (O.Ok identical in all other respects with its antipode 2 [2. 31, (lR,3R)-austrocortilutein (9) (109mg. 7.5 x lo-‘% fr. wt), and (lS,3R)-austrocortilutein (3) (58 mg, 4 x lo-‘% fr. wt). Isolation of pigments from w.47’21567 Fruit bodies were collected along the Dickinson track in State Forest near Marysville. Victoria during June 1985. Fresh toadstools (48 g) were finely chopped and soaked in EtOH (500 ml) for 3 hr. The orange extract was evapd and the residue (1.2 g) was partitioned between EtOAc (3 x 100 ml) and H,O (100ml). The organic phase was coned and the residue (0.3 g) was punfied by prep. TLC to afford, in order of Increasing polarity. (lS,3S)austrocortilutein (1) (R, 0.29) (32 mg, 6.7 x 10 ‘% fr. wt), orange needles (from CHCI,-EtOH), mp 183-187” (ref. [3] 183-185”); [a];’ f60” (EtOH; ~0.26); CD ,?~~~H+cHC’~ nm (As): 230 (+ 1.23), 2.50 (O.O),284 (- 1.48). 400 (0.0); otherwise identical with an authentic specimen [S], (lR,3S)-austrocortilutem (12) (R,0.22) (10 mg, 2 x lo-‘% fr. wt), orange needles (from EtOAc-petrol), mp 162-165. (Found: C. 63.0; H, 5 4. C,,H,,O, requires C, 63.15; H. 5.3%); [a];‘-262” (CHCI,; ~0.18); CD 1.~:~H+CWC’3nm (As): 235 (+0.8X), 245 (0.0). 255 (-0.37). 265 (0.0). 288 (+0.74), 330 (O.O), 355 (-0.29). 400 (0.0): ‘H NMR Fig. lb and 63.90 (3H. s, OMe), 5.17 (1H. m. H-l), 6.61 (IH, d, J =2.6 Hz. H-7), 7.14(1H,d. J=2.6 Hz, H-5), 12.13 (lH, s. OH-8), identical in all other respects with its antipode 3 [3], and a yellow zone(R,0.15)(4.5 mg) [12,13J. The quinones2(or 4)and 11,and the dihydroanthracenone 13 were observed m the chromatograms but were not isolated. Hydrogenolysrs of (1R,3R)-austrocortilutem (9). (lR,3R)Austrocortilutein (9) (10 mg) m MeOH (3 ml) was exposed to H, in the presence of Pd-C (lo%, 1.5 mg) until uptake of H, was complete (ca 1 hr). The catalyst was filtered off and washed with MeOH (10 ml) and the filtrate evapd under red. pres. The residue was purified by prep. TLC to afford (S)-l-deoxyaustrocortdutein (4) (R,0.54) (8.2 mg, 76%), orange needles (from MeOH at -2o”), mp 205-209” (Found: [M]’ 288.0994. Calc. for C,,H,,05: 288.0997); [a]:* +69’ (CHCI,, ~0.18); identical in all other respects with material isolated from WAT21568. Sinularly, hydrogenolysrs of (lS,3R)-austrocortilutein (3) (1.5mg) from WAT20934 gave 4 (0.9mg, 63%); [a]i2+70” (CHCI,; c 0.13). Hydrogenolysis of (lR,3S)-austrocortilutein (12) (1.8 mg) under the same conditions gave (R)-1-deoxyaustrocortilutein (2) (1 mg, 59%), orange needles (from MeOH at -20”). mp 205-209” (ref. [3] 206-212”) (F ound: [Ml+ 288.0997. Calc. for C,,H,,O,: 288.0997); [a];’ -70” (CHCl,; ~0.10); CD ~~$‘H+CHC” nm (A&): 270 (-0.28), 280 (0.0). 300 (+0.22). 345 (O.O),380 (-0.05), 400 (0.0); identical in all other respects with an authentic sample [2, 31. Oxidation of(S)-l-deoxyaustrocortilutein (4) A mixture of the quinone 4 (6.1 mg, 0.02 mmol), NaIO, (222 mg) and a trace. of RuCI,.3H,O was stirred vigorously in a heterogeneous mixture of Ccl, (2 ml), MeCN (2 ml) and H,O (3 ml) at room temp. for 24 hr. After this time iso-PrOH (2 ml) was added followed by an excess of BaCl,. The mixture was stirred vigorously for 30 min,
951
Austrocortilutein stereoisomers from fungi filtered, and the filtrate was extracted with CH,Cl, (3 x 10 ml), acidified and exhaustively extracted (24 hr) with Et,O. The organic phases were combined and evapd. The residue was dissolved in Et,0 (1 ml) and an excess of CH,N, was added. After 30 min the excess CH,N, was destroyed with HOAc and the solvent was evapd. HPLC [Ultrasphere-Si, 5~~ 1 x 25 cm, EtOAc-hexane (3:2)] gave the (S) lactone 8 (1.1 mg, 18%) as an oil. The absolute configuration was determined by ‘H NMR as follows: to the lactone 8 (0.95 mg) in CDCI, (0.5 ml) in an NMR tube was added an aliquot (40~1) of a solution of Eu(hfc), (150 mg) in CDCI, (1 ml). At that point the OMe and C-Me signals appeared at 63.94 and 1.75, respectively, shifted from 63.70 and 1.51 in the spectrum of 8 alone, but neither signal was resolved. Addition of a soln of the (R) lactone 7 (200 pg) [7] in CDCl, (100 ~1) gave rise to additional signals at 63.95 (OMe) and 1.76 (C-Me). Acknowledgements-We are indebted to Dr R. Watling (Royal Botanic Garden, Edinburgh) for lodging voucher specimens and Mr T. May (National Herbarium of Victoria) for invaluable help on the location, and advice on the identification, of fungi. We thank Mr R.W. Rickards, F.A.A. (ANU) for providing facilities for recording CD spectra. The Australian Research Council provided generous financial support and A.G. is grateful for a Melbourne University Postgraduate Research Award. Thanks are extended to the Department of Conservation, Forests and Lands for permission to collect fungi in National Parks under their jurisdiction.
REFERENCES 1. Gill, M. and Strauch, R. J. (1985) Tetrahedron Letters 26, 2593. 2. Gill, M. and Smrdel, A. F. (1987) Phytochemistry 26, 2999. 3. Gill, M., Smrdel, A. F., Strauch, R. J. and Begley, M. J. (1990) J. Chem. Sot., Perkin Trans. I 1583. 4. Gill, M. and Steglich, W. (1987) Prog. Chem. Org. Nat. Prods 51, 125. 5. Keller, G., Moser, M., Horak, E. and Steglich, W. (1987) Sydowia 40, 169. 6. Horak, E. (1987) Sydowia 40, 81. 7. Gill, M. and Smrdel, A. F. (1990) Tetrahedron: Asymmetry 1, 453. 8. Gimknez, A. (1990) Ph.D. Thesis. The University of Melbourne. 9. Carlsen, P. H. J., Katsuki, T., Martin, V. S. and Sharpless, K. B. (1981) J. Org. Chem. 46, 3936. 10. Gill, M.,Gim&ez, A., Jhingran, A. G. and Palfreyman, A. R. (1990) Tetrahedron: Asymmetry 1, 621. 11. Archard, M. A., Gill, M. and Strauch, R. J. (1985) Phytochemistry 24, 2755. 12. Burns, C. J., Gill, M. and Gimknez, A. (1989) Tetrahedron Letters 30, 7269. 13. Burns, C. J., Gill, M. and Gimbnez, A. (1992) Aust. J. Chem. (in press). 14. Gill, M., Gimknez, A., Jhingran, A. G. and Smrdel, A. F. (1989) Phytochemistry 28, 2647.