Constituents of the higher fungi Part XV. 3-(3,4-Dihydroxyphenyl)-2,7,8-trihydroxydibenzofuran-1,4-dione, a precursor of thelephoric acid from the fungusSuillus grevillei(klotsch) sing. Boletus elegans(schum. per Fries)

摘要

1975 351Constituents of the Higher Fungi Part XV. 3-(3,4-Dihydroxyphenyl)-2,7,8-trihydroxydibenzof uran-1 ,dime, a Precursor of Thelephoric Acidfrom the Fungus Sur'//usgrevillei (Klotsch) Sing. Boletus elegans (Schum.per Fries)By Raymond L. Edwards and Melvyn Gill, Scfiool of Chemistry, University of Bradford, Bradford BD7 1 DP3-(3,4-Dihydroxyphenyl)-2,7,8-trihydroxydibenzofuran-l,4-dione has been isolated from t h e fungus Sui//usgrevillei. Solutions of this compound are unstable and yield thelephoric acid.IN Part XIV we described the isolation of thelephoricacid (I) and seven new pigments from the larch boleteSuillus grevillei. Structures were assigned to three of0(I1these compounds (El1, B,, and A) and we now report theassignment of the structure (11) to the fourth compoundCompound C, (C18H,00,J m.p.300°, M+ 354) was ob-tained as dark red needles which form brown soiutions inacetone and in alcohols. The presence of five hydroxy-groups was established by the formation of a penta-acetate, and the quinonoid nature of the molecule wasshown by its reversible reduction and oxidation withsodium dithionite and air arid the formation of a leuco-hepta-acetate. The remaining, unreactive oxygen atommust be involved in an ether bridge since the lH n.m.r.spectrum shows absorption only in the aromatic region.0 0(C,> *..-a Rl0(137)(Y)(PI1( = I R ' = R 2 = R 3 z O H I R ' = 4 jR' = R 2 = R 3 = R 4 = HR' = R 4 = H I R ' z R3=OHR' = R 2 z R3 E R ~ = O HThe molecular formula and polyhydroxyiated quinon-oid nature of C, suggested that it was a hydroxyphenyl-benzoquinone.However, the U.V. spectrum of theleuco-acetate (Amx 257, 289, and 312sh nm) differs sig-nificantly from those of atromentin leuco-acetate (249nm) and polyporic acid leuco-acetate (245 nm). Also,1 Part XIV, R. L. Edwards and M. Gill, J.C.S. Pevkin I, 1973,1921.C, is unstable in alkali: in aqueous sodium hydroxide ablue colouration is produced which fades within 10 s togreen and then yellow, and in ammonium hydroxide thetransient blue colour fades to a more stable green showinglong-wavelength absorption a t 658 nm. Polyporic acid(IV) and atromentin (V) are relatively stable in alkali.Solutions of C, are unstable and slowly deposit thele-phoric acid (I), identified from its i.r.and U.V. spectra andby the formation of a tetra-acetate and a leuco-hexa-acetate. This instability is particularly marked inacetone solution. The similarity between the chromo-phores of C, and thelephoric acid is illustrated in theTable.U.V. maxima (nm; log E in parentheses) of the pigmeatC, and thelephoric acidSolventAbsolute ethan~lloo/, EtOH10 EtOH +2 drops dilutea1 ka1.i0.1 N-NaOHPyridinePigment C,259 (4.391, 301 14-44),340infl(4-01), 445(3.84)364 (3.97), 300 (4.00)(NH40H) 270 (4-12),316 (4*11), 658(3.84)(3.77)342 (3-96), 394hfl306 (4.24), 331infl(3*78), 446 (3.55)Thelephoric acid264 (4.27), 30.5 (4*30),483 (3.86)271 (3*48), 326 (3.61)(NaOH) 274 (3-85),334 (3*95), 690(3.69)344 (446), 394infl(4013)~ >700311 (4.53), 396infl(3*58), 493 (4.06)Although the formation of the acetate derivativesproves the presence of a quinone ring and five acidichydroxy-groups it does not distinguish between the twopossible structures (11) and (111).Either could con-ceivably be a direct precursor of thelephoric acid, andstructure (11) has recently been suggested as a possibleintermediate in the formation of this compound.2Polyporic acid (IV) and atromentin (V) are relativelystable in alkali but the stability of the related poly-hydroxylated compounds has not been studied. In orderto make a direct comparison with C,, variegatin (VI) (thehypothetical quinone corresponding to variegatic acid)and leucomelone (VII) have been synthesised.2,5-Dihydroxy-3,6-bis(hydroxyphenyl) benzoquinones areusually prepared from 2,5-~blorobenzoquinone byarylation, demethylation, and replacement of halogen byhydroxyl (by treatment with hot sodium hydroxidesolutjm). The synthesis of Leucomelone by this sequencehas been ~laimed,~ a d this would imply that the corn-pound is stable in alkaline solution. However, efforts byother workers to prepare leucomelone by reductivedemethylation of 3,4,4-trimethoxypolyporic acid anda J. Gripenberg, Tahedvun Letters, 1974, 619.M. Akagi, J. Phavm. SOC. Japan, 1942, $2, 202.G. J. Benett and N. Uri, J . Chem. SOC., 1962, 2763352 J.C.S. Perkin Ioxidation of the resulting quinol in alkaline solution gavean amorphous product.Also, a demethylation withhydrogen bromide gave a product yielding an unstableacetate of m.p. (196---200") significantly different fromthat (226-227") reported earlier.3For our synthesis, the acid labile methoxymethylgroup was chosen as the hydroxy-protecting group.Diazotised 3,4-bis (methoxymethoxy) aniline was coupledwith 2,5-dichlorobenzoquinone to yield the diarylatedquinone; this was treated with alkali to remove thehalogen and then demethoxymethylated with dilute acidto yield variegatin. Similarly sequential monoarylationof 2,5-dic hlor o benzoquinone with diazot ised 4- (met hoxy-methoxy)aniline and then with diazotised 3,4-bis-(1nethoxymethoxy)aniline gave the unsymmetrical di-arylated product, which on treatment with alkali anddemethoxymethylation gave leucomelone.In boththese syntheses the use of alkali after removal of theprotecting groups was unnecessary, and during the finaldemethoxymethylation the crystalline quinones wereformed quantitatively during I min.Variegatin (VI) and leucomelone (VII) are both un-stable in dilute alkali. In dilute aqueous sodiumhydroxide, variegatin produces a blue colouration whichConclusive proof of structure (11) was obtained fromthe mass spectrum, which shows features in common withthose of variegatin and leucomelone. All three com-pounds exhibit abundant M+ and M+ - CO peaks.Both variegatin and C, exhibit a peak at m/e 177, whichcan be represented as the fragment (a) from the 'di-hydroxyphenyl' side of the quinone.This would beexpected to fragment to yield the ions (b) m/e 149 and(c) m/e 121; both are found in variegatin and C,. Thefragment of m/e 177 is absent from the spectrum ofleucomelone but the ions (b) and (c) do appear. Mono-hydroxy-analogues of these fragments appear only in thespectrum of leucomelone. A compound with structure(111) would be expected to produce the phenylacetyleneion (d) as a major fragment. This type of fragmentationoccurs in 2,5-di-(p-tolyl)-l,4-benzoquinone, which showsmajor ions at m/e 288 (M+), 273 (M - CH,), 245(M+ - CH, - CO), and 116 (e). Although a peak ispresent at m/e 150 in the spectrum of C,, which couldcorrespond to (d), this is weaker than that of the mle 149ion, and could arise from a simple fragmentation of thebenzoquinone nucleus to give (f) ; this is supported by thepresence of this peak in the spectra of variegatin andleucomelone and, in the case of leucomelone, a mono-fades to yellow within 15 s; in ammonium hydroxide theblue colour fades to a more stable green (A*= 660 nm).Leucomelone produces a blue colouration which fades toa more stable green with both these reagents.Com-pound C, resembles variegatin in these colour reactions.In the i.r. spectrum C, shows strong hydroxy-absorp-tion and complex bands in the carbonyl region. Thecompound tenaciously holds solvent of crystallisation andremoval of this causes marked changes in intensity ofbands at 1692, 1650, and 1640 cm-1. In particular thatat 1692 cm-l almost disappears after vacuum drying athydroxy-analogue at mle 136.Thelephoric acid is a widespread metabolite of lichensand fungi and the ease with which it can be extractedfrom some of these sources by either acetone or alcoholshas been noted by several workers.Zopf reported awine-red alcoholic extract from Thelephora species andseveral authors have used acetone as the extractingsolvent .6-8 Asahina and Shibata used acetone to extractthelephoric acid from Lobaria retigera and pyridine toextract the same pigment from Thelephora palmata.KoglQ noted that the compound could only be readilyc fO+room temperature for 3 days. Bands near 1640 and 1650cm-l would be expected for a compound of structure (11),whereas structure (111) would be expected to absorb at1680 (non-chelated C=O) and 1640 cm-l (C=O in sameenvironment as in thelephoric acid).The possibilitythat cyclisation might be taking place during drying wasruled out by the preparation of the penta-acetate and theleuco-hepta-acetate from the dried material.W. Zopf, Bot. Z., 1889, 8, 69.6 J. Gripenberg, Acta Chem. Scand., 1968, 12, 1411.7 G. Sullivan, L. R. Brady, and V. E. Tyler, Uoydia, 1967, 80,84.extracted from wet Thelephora species by pyridine. Theinsolubility of thelephoric acid in most solvents and thisvariation in the ease of extraction from different sourcescombined with our observation that C, readily changes inacetone solution suggests that precursors similar to, orthe same as, the compound described in this paper areresponsible for the apparent initial solubility of this com-pound in organic solvents.The initial difficulties in* Y. Asahina and S. Shibata, Ber., 1939, 72, 1631.F. Kbgl, H. Erxleben, and L. Janecke, Annalen, 1930, 482,1061975structure determination and purification also suggest theprobable presence of mixtures.1° It is probable that C,is the colouring matter in the cap of Suillus grevillei var.badius and is the precursor of the thelephoric acidisolated by us from this species.EXPERIMENTALM.p.s were determined on a Kofler hot-stage apparatus,i.r. spectra on a Perkin-Elmer 237 spectrophotometer, U.V.spectra on a Unicam SP 800 spectrophotometer, lH n.m.r.spectra on a JEOL JNM-MH-100 spectrometer (tetramethyl-silane as internal standard), and mass spectra on an A.E.I.M S 9 spectrometer.All thin layer (t.l.c,), preparative layer(p.l.c.), and column chromatography was carried out onMerck Kieselgel PF256 + 366.Isolation of 3-(3,4-Dihydroxyphenyl)-2,7,8-trihyd~oxydi-benzofuran-l,4-dione (C,) (11) .-The pigment was extractedand isolated as described in Part X1V.l Fractions 79-85deposited dark red needles (38 mg), m.p. >300° Found: C,60.6; H, 2.9 (after drying under vacuum a t room temp.for 48 h) ; Mf, 354.037624. C18Hlo08 requires C, 61-0; H,2.85 ; M, 354.0375591 ; vmxa 3490, 3320, 2640, 2530, 1650,and 1640 cm-1; m/e 356 (16), 355 (25), 354 (loo), 353 (ll),352 (39), 326 (8), 177 (7), 176 (5), 168 (5), 149 (5), 150 (4), 121(5), and 120 (5); 7 (CD,),CO 2.2-3.8 (10H).Thelephoric Acid from Comfiound C,.-A solution of thepigment (10 mg) in cold acetone (0.3 ml) was set aside for3 days.The brown solution slowly deposited crystals ofthelephoric acid (5 mg), which were filtered off and washedwith acetone and alcohol (Found: Mf, 352.021009. Calc.for C18H,0,: M , 352.021910); vmx. 1630 and 1605 cm-l;lmx. (pyridine) 493 nm (log E 4-02).Acetylation of Compound C,.-A mixture of C, (10 mg),acetic anhydride (0.4 ml), and pyridine (1 drop) was set asideat room temperature. After 48 h the yellow solution wasfiltered and the filtrate poured into water. Recrystallis-ation of the solid from benzene-light petroleum (b.p. 80-100") gave 2,7,8-triacetoxy-3- (3,4-diacetoxyphenyZ)dibenzo-furapt-l,4-dione (4 mg) as bright yellow needles, m.p.137-142" (Found : M f , 564-093684. C2,,H,o01, requires M,564.090684) ; vmx, (CHC1,) 1780, 1680, 1603, 1583, and 1374cm-l; m/e 564, 522, 480, 438, 396, 354, 337, 326, 177, 149,and 121; LX. 249, 274sh, and 350 nm.Reductive A cetylation of C, A cetute.-A mixture of C,acetate (5 mg), acetic anhydride (1 ml), anhydrous sodiumacetate (0.1 mg), and zinc dust (10 mg) was refluxed for 30min and then poured into water. 1,2,4,7,8-Penlu-acetoxy-3-(3,4-diacetoxyphenyl)dibenzofu~an (4.2 mg) was filtered offand crystallised from benzene-light petroleum (b.p. 80-100") asrods, m.p. 177' (Found: M+, 650.128480. C3,H2,O15requires M , 650.127153) ; v- (CHC1,) 1780, 1509, and 1375cm-l; LX 227, 257,289, and 310sh nm (log E 4.56,4-18,4.37,and 4-01>; m/e 650, 608, 566, 524, 482, 440, 398, 356, 337,326, 177, and 149.3,4-Bis(methoxymethoxy) -1-nitrobenzene.-A mixture of 4-nitrocatechol(lO3 g), chloromethyl methyl ether (160 g ) , andanhydrous potassium carbonate (360 g) in anhydrousacetone (1.2 1) was stirred and refluxed for 48 h.The mix-ture was filtered, the residue was well washed with acetone,and the filtrate was evaporated under reduced pressure. Asolution of the oily residue in ether was shaken with aqueoussodium hydroxide (3 x 50 ml; 2 ~ ) , then water, dried(Na,S04), and evaporated. Crystallisation of the residuefrom aqueous ethanol and recrystallisation from methanolgave 3,4-bis(methoxymethoxy)-l-nitrobenzene (126 g ) as paleyellow needles, n1.p.59-63" (Found: C, 49.5; H, 5.4; N,5.8. C1,Hl,N06 requires C, 49.4; H, 5-35; N, 5.8).3,4-Bis(methoxymethoxy)aniline.-A solution of 3,kbis-(methoxymethoxy)- l-nitrobenzene (146 g) in ethanol (600ml) was added dropwise to a stirred suspension of palladisedcharcoal (3 g; 10) in aqueous sodium borohydride (55 g in1500 ml) cooled to 5'. Nitrogen was passed through themixture during the addition, Stirring was continued for 1 hand after filtration the alcohol was evaporated off under re-duced pressure. The mixture was acidified with acetic acidand extracted with ether, and the extract washed with colddilute hydrochloric acid. The base was liberated withalkali and re-extracted into ether. Evaporation, and dis-tillation of the product in uucuo gave the aniline (82 g), b.p.146-148" a t 0.7 mmHg.Crystallisation from cyclohexanegave the needles, m.p. 52-56' (Found: C, 56.5; H, 7.0;N, 6-5. CloH,NO, requires C, 56.3; H, 7.0; N, 6.6).3-3,4-Bis(methoxymethoxy)~heny~-2,5-dichloro- 1,4-benzo-quitzone.-Sodium nitrite (2.3 g) in water (10 ml) was addeddropwise a t 5" to a solution of 3,4-bis(methoxymethoxy)-aniline (6 g, 0-03 mol) in concentrated hydrochloric acid-water (1 : 1 ; 14 ml). Sodium acetate (7 g ) was added andthe mixture poured into a solution of 2,5-dichlorobenzo-quinone (5.3 g, 0-03 mol) in ethanol (250 ml) and ether (250ml) at 10". The dark red solution was set aside overnightand then evaporated to low bulk under reduced pressure.The mixture was extracted with ether, the extract evapor-ated, and the residue adsorbed on silica gel (20 g) fromacetone.The dry gel was placed on a column of silica gel(70 x 4 cm), which was developed with benzene-acetic acid(95 : 5 ) . The product from the first intense red band to beeluted yielded red plates (620 mg), m.p. 130-132" (frommethanol) (Found: c , 51.4; H, 3.9; c1, 19.1. C16H14C120,requires C, 51.5; H, 3.75; C1, 19.0); vmx. 1677, 1667, and1600 cm-1.The product from the second red band gave 3,64-3,4-bis (methoxymethoxy)phenylJ 2,5-dichloro- 1,4-benzoquinone(155 me) as bronze plates (from ethanol), m.p. 163-165"(Found: C, 54.7; H, 4.4; C1, 12.4. C,6H,6C12010 requiresC, 54.8; H, 4.6; C1, 12.5); vmx. 1674 and 1601 cm-1.Similarly prepared from 2,5-dichlorobenzoquinone ( 19.5 g )and diazotised Pmethoxyrnethoxyphenylaniline (15.3 g) was2,5-dichloro-3-4- (methoxymethoxy)fihenyll- 1,4-benzoquinone(7.2 g) as orange-red rods (from ethanol), m.p.110-112"(Found: C, 54.0; H, 3.4; Cl, 22.6. Cl,Hl0Cl2O4 requiresC, 53.7; H, 3.2; C1, 22.7); vmX 1672, 1665, and 1604cm-1.3-3,4-Bis (methoxymethoxy)phenylJ-2,5-dichloro-6-4-(metk-oxymethoxy)-phertyll- 1,4-benzoquinone.- 3,4-Bis (methoxy-methoxy)aniline (9-8 g) in hydrochloric acid (32 ml; 50)was diazotised with sodium nitrite (6 g) in water (30 ml) at6". Sodium acetate (20 g) was added and the mixturepoured into a solution of 2,5-dichloro-3-4-(methoxy-methoxy)phenyl-1,4-benzoquinone (6.26 g) in ethanol (600ml) and ether (180 ml) at 10". The mixture was stirred for3 h during which time the solution became dark brown anddeposited a red solid.Crystallisation from acetic acidyielded the product (1-18 g) as bronze Pplates, m.p. 147-150'(Found: C, 56.7; €3, 4.3; C1, 14.0. C,,H2,Cl2O, requiresC, 56.6; H, 4.3; C1, 13.95); vmaX 1676, 1603, 1580, and1504 cm-l.3,s-Di- 3,4-bis (methoxymethoxy)phenyl-J -2, 5-dihydroxy- 1,4-benzoquinone.-A suspension of 3,6-di-3,4-bis(methoxy-methoxy)phenyl-2,5-dichloro-l,4-benzoquinone (100 mg) inlo G. Read and L. C. Vining, Canad. J. Chem., 1969, 87, 1442354methanol (1.5 ml) and aqueous sodium hydroxide (1.5 ml;2N) was warmed on a water-bath for 30 min. Acidificationof the cold purple solution gave a brown precipitate whichwas filtered off and crystallised from dioxan to yield theproduct (70 mg) as yellow needles, m.p.149-153' (Found:C, 58.4; H, 5-2. C2sH28012 requires C , 58.65; H, 5.3);vmX 1646, 1626, 1599, 1580, and 1513 cm-1.3- 3,C-Bis (methoxymethoxy)pheny ZJ -2,B-dihydvoxy- 6- 4(methoxymethoxy)phe~~~- 1,4-benzoquinone, similarly pre-pared from the corresponding dichloro-compound, wasobtained as orange needles (from dioxan), m.p. 120'(Found: C, 60.8; H, 5.0. C2#1, requires C, 61.0; H,54); vw 1630, 1619, 1600, and 1510 cm-1.Variegatzn (VI) and Leucomelone ( V I I ) .-A mixture of2,5-dihydroxy-3,6-bis(methoxymethoxy)phenyl- 1,kbenzo-quinone (35 mg), acetic acid (1.5 ml), and sulphuric acid (1drop: 2N) was boiled for 1 min. The solution deposited acrystalline brown solid.Recrystallisation from pyridinegave 2,B-bis- (3,4-dihydroxyphenyZ) -3,6-dihydroxy- 1,4-benzo-quinune (variegatin) (1 9 me;) as red rods which turned brownon heatiirlg in vacuo; m.p. > 320' (Found: C, 60.7; H, 3.55.C18H1208 requires C, 60.7; H, 3.4); v,, 3490, 1640sh,1629, 1620, 1605, and 1514 cm-l; A,mx. 216sh, 270, and 282nrn (log E 4.31, 4.16, and 4.21); A,, (10 ethanol) 285 and354sh nm (log e 3.85 and 2-90); Amx. (10 ethanol + 2drops ~N-NH,OH) 250sh, 331, and 660 nm (log E 3.87, 3.94,and 3-83) ; Amma (0-1N-NaOH) 250sh, 332, and 384 nm (log E:4.42, 4.43, and 4.32); m/e 356 (looyo), 328 (751, 255 {20), 177(20), 161 (49), 150 (80), 140 (49), 123 (64), 122 (44), and121 (72).Similarly, hydrolysis of 3-3,4-bis(methoxyinethoxy)-phenyl-2,5-dihydroxy-6-~4-(methoxynietlioxy)phenyl- 1,4-benzoquinone (100 mg) gave leucomelone (36 mg) as orangeplates (from dioxan), m.p.>320" (Found: C, 63.3; H , 3.4.Calc. for C18H,,0,: C, 63.55; H, 3.55) ; v,. 3400, 1637sh,1625, 1602, and 1510 cm-1; A,,,. 271, 282, and 386 nm (log EJ.C.S. Perkin I4.42, 4-41, and 3.29) ; A,,, ( lOyo ethanol) 281 and 345sh nm(log E '4.19 and 3.26); I.-. (10 ethanol + 2 drops 2 ~ -NH40H) 295 and 664 nm (log E 4.14 and 4.79); LX (0.1s-NaOH) 260, 326, 382, and 676 nm (log E 4-06, 4.07, 3-82, and3.33); m/e 340 (looyo), 312 (47), 161 (43), 160 (40), 149 (19),145 (22), 134 (43), 133 (26), 123 (47), 122 (22), 121 (39), and105 (41).Leucomelone Penta-acetate and Variegatin Hexa-acetate.-A mixture of leucomelone (50 mg), acetic anhydride (0.4 ml),and sulphuric acid ( 1 drop) was heated on a water-bath for16 min, cooled, and poured into water. The product (42 mg)crystallised from acetic acid to yield lemon-yellow needles ofleucomelone penta-acetate, m.p. 174-182" (decomp.) (lit.,4196-200"; lit.,3 226-227") (Found: C, 61.3; H, 4.3.Calc. for C.2: C, 61.1; H, 4-00/,); wmK 1773, 1671,1613, and 1499 cm-l.Similarly, variegatin gave variegatin hexa-acetate as yellowneedles (from acetic anhydride), m.p. 184-180" (decornp.)(Found: C, 58.8; H, 4-0. C,H2,01, requires C, 59.2; H,3.9); vmx. 1780, 1680, 1620, 1610, and 1503 cm-l.Leucomelone Hepta-acetate and Variegatin Octa-acetate.-Amixture of leucomelone (25 mg), acetic anhydride (3 ml),anhydrous sodium acetate (10 mg), and zinc dust (50 mg)was heated under reflux for 10 min. The mixture wascooled, filtered, and poured into water. Crystallisation ofthe solid from ethanol gave needles of leucomelone hepta-acetate (19 ing), m.p. 218" (after softening a t 205") (lit.,3204-205") (Found: C, 60.1; H, 4.3. Calc. for C,2H2801a:C , 60.4; H, 4.4); urn=. 1775 cm-l.Similarly, variegatin gave variegatin octu-acetate asneedles (from acetic acid), m.p. 242-244" (Found: C, 58.6;H, 4.25. C,,H,,O1, requires C, 58.8; H, 4.3); v " ~ , ~ .1775 cm-l.We thank the S.R.C. for a research grant (to M. G.).4/1886 Received, 16th September, 1974