zyxwv zyxwvu zy HELVETICA CHIMICA ACTA- Vol. 72 (1989) 1455 zyxw 162. New Chromonocoumarin (= 6H,7H-[ l]Benzopyrano[4,3-b][l]benzopyran6,7-dione) Derivatives from Polygulu fruticosa BERG. by Ermindo R. Di Paoloa), Matthias 0.Hamburgera),Helen Stoeckli-Evansb),Colin Rogersc), and Kurt Hostettmanna)* ') Institut de Pharmacognosie et Phytochimie, Ecole de Pharmacie, Universite de Lausanne, 2, rue Vuillermet, CH-1005 Lausanne b, Institut de Chimie, UniversitC de Neuchitel, 51, avenue de Bellevaux, CH-2000 Neuchitel ") Department of Chemistry, University of Durban-Westville, Durban 4000, Republic of South Africa zyxwvuts (18.VIII.89) Three chromonocoumarins ( = 6H,7H-[l]benzopyrano[4.3-6][l]benzopyran-6,7-diones)1-3 have been isolated from the leaves and the root bark of Polygala fruticosa BERG.(syn. Polygala oppositifolia L.; Polygalaceae). The structure of frutinone A (1) was established by X-ray diffraction analysis. The structures of the previously undescribed compounds frutinone B (2) and C (3) were deduced by spectroscopic methods (EI-MS, UV, IR, 'Hand I3C-NMR, including NOE and COSY) in comparison with 1. Chromonocoumarins 1-3 are the first representatives of a new type of naturally occurring compounds. Frutinone A shows strong fungicidal activity against Cladosporium cucumerinum. Introduction. - The genus Polygala (Polygalaceae) comprises ca. 500 species distributed over the temperate, subtropical, and tropical regions of all continents, with exception of Australia [l]. Polygala senega (vernacular name: snakeroot) is the source of the still officinal drug Senegae radix, which is used as an expectorant for its high saponin content. Previous phytochemical investigations of the genus have shown the presence of numerous oleanene-type saponins [2-41, lignans [S] [6], coumarins [7] [8], xanthones [9], hydroxycinnamoyl esters [lo], flavonol glycosides [ 111, and methyl salicylate [ 121. Among these, several biologically active compounds have been found, such as saponins which are inhibitors of CAMPphosphodiesterase [3], antitumor lignans [6], a fungicidal coumarin [8], and xanthones with potential inhibitory activity on the monoamino oxidases A and B ~31. Contrary to the European, American, and Asian Polygala species, the African representatives of this genus have not been investigated phytochemically. Therefore, as a part of our ongoing search for novel biologically active compounds from African medicinal plants, we undertook a study of Polygala fruticosa BERG., a little shrub growing in the Cape Province and Natal (South Africa) [14]. The roots have been used by the Zulus as a remedy against dropsy, scrofula, and to produce profuse perspiration and as an ingredient in a decoction taken for treatment of tuberculosis [15]. In a preliminary screening, the CH,Cl, extract of the leaves of P . fruticosa exhibited strong activity against the plant pathogenic fungus Cladosporiurn cucurnerinurn in a TLC bioassay [16]. The present paper deals with the isolation and structure determination of three chromonocoumarins ( = 6H,7H-[ l]benzopyrano[4,3-b][l]benzopyran-6,7-diones) 1-3, the first representatives of a new type of naturally occurring compounds. 1456 zyxwvu zyxw zyxwvutsrq HELVETICA &MICA 0 ACTA- Vol. 72 (1989) 0 Results. -The leaves and the root bark of P.fruticosa were extracted successivelywith petroleum ether, CH,CI,, and MeOH (see Exper. Part). The fungicidal CH,CI, extract from the leaves was submitted to column chromatography on silica gel with petroleum ether/AcOEt mixtures of increasing polarity. Subsequent recrystallization yielded chromonocoumarins 1 and 2 which we name frutinone A and B, respectively. Column chromatography of the methanolic root extract on silica gel with CHCl,/MeOH/H,O mixtures as mobile phase yielded an enriched fraction of frutinone C(3) which was purified by filtration on Sephadex-LH-20 (MeOH). The structure of frutinone A (1) was deduced from UV, IR, ‘H- and 13C-NMR,and MS data as being 6H,7H-[ l]benzopyrano[4,3-b][llbenzopyran-6J-dione. zyxwv zyxw zyxwvu zyxw zyxw In the ELMS of 1, the molecularion appeared at m/z 264 (CI6H8O4).T h e IR spectrum indicated the presence of lactone C=O (1745 cm-I) and a$-unsaturated C=O (1640 m-’)groups. The U V spectrum (MeOH) showed absorption maxima at 229,261, and 296 and a shoulder at 3 18 nm.No shift was observed upon addition of NaOMc or AICI, indicating the absence of free phenolic OH groups [17]. The ‘H-NMR spectrum (Table 1) displayed 8 zyxwv Table 1. ‘H-NMR Data of 1-3 md3a 17 H-C(I) H-C(2) H-C(3) H-C(4) H-C(8) H-C(9) H-C(1O) H-C(I 1) CH3O-C(4) H-C(1) H-C(2) H-C(3) H-C(4) H-C(8) H-C(9) H-C(I0) CH,O-C(I 1) ”) ”) ’) IN CDCI,. In CDCI,/CD,OD 10 :1. In (D,)DMSO. 8.20 (ddd, J = 0.5, 1.7,g.O) 7.46(&,J=l.l,7.4,8.0) 7.75 (ddd, J = 1.7, 7.4,8.3) 7.38(ddd,J=00.5,1.1,8.3) 8.32 (ddd, J = 0.5, 1.7,S.O) 7.49 (&, J = 1.2, 7.1,g.O) 7.79(ddd,J= 1.7,7.1,8.3) 7.63 (ddd, J =0.5, 1.2,8.3) 2”) 7.79 (dd,J = 1.6, 8.0) 7.40 (dd,J = 8.0, 8.2) 7.29 (dd, J = 1.6, 8.2) 8.33 (&id, J = 0.5, 1.7,S.O) 7.53 (M,J = 1.2,7.1,8.0) 7.81(ddd,J=1.7,7.1,8.5) 7.65 ( d d , J = 0.5, 1.2,8.5) 4.00 (s) 39 3aa) 8.29 (ddd, J = 0.5, 1.7, 7.9) 7.59 (&, J = 1.2,7.4,7.9) 7.89 (M,J = 1.7,7.4,8.3) 7.52(&,J=00.5, 1.2,8.3) 7.87 (dd, J = 1.6,7.9) 7.50 (dd, J = 8.0,S.l) 7.83 (dd, J = 1.6, 8.1) 8.28 (did, J = 0.5, 1.7,7.9) 7.45 (M,J = 1.2, 7.4,7.9) 7.75 (ddd,J = 1.7,7.4,8.3) 7.40 (ddd, J = 0.5, 1.2,8.3) 7.90 (dd, J = 1.6, 8.0) 7.42 (dd,J = 8.0, 8.1) 7.28 (dd, J = 1.6, 8.1) 4.07 (s) zyx zyxwv zyxwvut zyxwv zyxwv HELVETICA CHIMICA ACTA- Vol. 72 (1989) 1457 Table 2. "C-NMR Data of 1-3 124.2 124.8 135.6 117.4 154.2 156.0 105.0 173.0 124.4 126.8 126.6 134.8 117.8 154.3 164.9 113.2 114.9d) 124.7 116.gd) 147.4 144.2 156.0 104.5 173.3 124.2 126.79 126.63 135.0 117.8 154.4 165.3 113.8 56.3 124.4') 125.0') 135.8 116.9 153.8 155.8 104.6 172.6 125.1 119.6 126.19 127.19) 146.5 142.6 164.5 113.5 zyxwvutsrq In CDCI,. CDC13/CD30D1O:l. ') In (DdDMSO. d-g) Values may be interchanged. ") b, aromatic protons in the region 7.38-8.32 ppm. Analysis of the COSY and of a resolutionenhanced 'H-NMR spectrum revealed that the signals were attributable to 2 four-spin systems of orthodisubstituted aromatic rings. The "C-NMR spectrum (Table 2) displayed 16 signals belonging to 2 C=O groups, 3 0-bearing aromatic C-atoms, 3 olefinic or aromatic quaternary C-atoms and 8 protonated aromatic C-atoms. The unsaturated carbonyl group observed in the IR spectrum, the deshielded aromatic proton at 8.32 ppm @eri to a C=O), and the I3C-NMR data indicated that one half of the molecule was a ychromone moiety. The NMR spectral data were in good agreement with data reported for the chromone moiety of flavone [18]. The structural elements of the other part of the molecule (one ortho-disubstituted aromatic ring and one lactone C=O) suggested a mumarin or isocoumarin (4 possible isomeric structures). In a comparison of the observed 'H- and I3C-NMR chemical shifts with values calculated [I91for these structures, formula 1was found to fit best with the experimental data. Further indication was the presence of a second low-field signal at 8.20 ppm (H-C(1)). zyxwvu The structure of compound 1was finally confirmed by X-ray diffraction analysis (see the Fig. and Exper. Part). In crystalline state, the molecule was found to be planar. To accomplish this, the structure exhibits large deformations in the bond angles involving the two central pyranone rings. The bond distances indicate considerable charge delocalization in the central part of the molecule. The two carbonyl 0-atoms are separated by 2.839 A, little more than the sum of their van der Waals radii (2.8 A). On considering that molecule 1 is made up of a flavonoid and a coumarin moiety, an analysis of these structures in the Cambridge Structural Data Base [20] [21] indicates that the bond lengths and angles observed for 1 are normal within experimental error (see Table 3 in Exper. Part). No 'H- and I3C-NMRdata have been previously reported for chromonocoumarins. Unambiguous assignment of the I3C-NMR resonances of 1was, therefore, essential for the structure elucidation of the chromonocoumarins 2 and 3. Protonated C-atoms of 1 were first assigned with a 2D one-bond heteronuclear correlation experiment (HETCOR) [22], while the signals of the quaternary C-atoms could be attributed via long-range 1458 zyxw zyxwv zyxw HELVETICA CHIMICA ACTA- Vol. 72 (1989) P zyxwvut zyxwvutsr zy zyxwvutsrqp zyxwvu o(7) O(6) Figure. View of 1 showing the crystallographic atomic numbering scheme and the vibrational ellipsoids (50% probability level) connectivities in a HETCOR experiment with delay settings optimized for JcccH= 7 Hz. Cross peaks were observed for C(7) and H-C(8), C(7a) and H-C(9), C(4a) and H-C(3), and C(12b) and H-C(2). Additional long-range correlations are listed in the Exper. Part. The structures of frutinone B (2) and C(3) were deduced from spectroscopic data by comparison with 1 and from data of the monomethyl ether 3a obtained from 3 by methylation with (CH,),SO, (see Exper. Part). Thus, 2 and 3 are 4-methoxy- and 1 l-hydroxy-6H,7H-[ I]benzopyrano[4,3-b][l]benzopyran-6,7-dione, respectively. zyxwvu The EI-MS spectrum of 2 exhibited a molecular ion at m/z 294 (C,,Hlo05), 30 amu higher, than that of 1. The IR spectrum presented also absorption band of 2 C=O groups at 1735 and 1650 cm-I. The UV spectrum (MeOH) showing two major maxima at 247 and 303 nm, remained unchanged upon addition of NaOMe or AIC1,. 'H- and I3C-NMR signals at 4.00 (3H, s) and 56.3 ppm, respectively, were indicative of an aromatic M e0 group. Its position of attachment at C(4) was readily apparent from the 'H-NMR spectrum (Table 1 ) . While the 4 protons of the y-cbromone moiety were quasi identical with those of 1, signals for 3 vicinal aromatic protons were present on the aromatic ring of the coumarin part. Compared to 1, H-C(1) was shifted upfield ( A S = -0.41 ppm) and appeared at 7.79 ppm as a dd. The signal attributable to H-C(3) was also shielded (AS = -0.46 ppm), while H-C(2), meta to the substituent, remained almost unchanged. The ',C-NMR spectrum of 2 was in accordance with MeO-C(4) (see Table 2). In comparison with 1, upfield shifts of the signals for C(l) (1 14.9 ppm; AS = -9.3 pprn), C(3) (116.8 ppm; AS = -18.8 ppm), and C(4a) (144.2 ppm; A S = -10.0 ppm) were observed, C(4) (147.4 ppm) was deshielded by 30 ppm, whereas the signals attributable to C(2), C( 12b), and the other C-atoms remained unaffected. Final confirmation was obtained by NOE difference spectroscopy. Irradiation of the Me0 signal at 4.00ppm resulted in an enhancement of the dd at 7.29 ppm previously assigned to H-C(3). The EI-MS of 3, showed a molecular ion at m / z 280 (C,,H,O,), 16 amu higher than that of 1. In addition to the 2 C=O bands, the IR spectrum indicated the presence of an OH group (3500 cm-I). The UV spectrum (MeOH) displayed a major maximum at 267 nm and a shoulder at 316 nm. Identical UV spectra were obtained upon addition of NaOAc or AICI,, while bathochromic shifts to 303 and 378 nm were observed in strongly basic solution (MeOH + NaOMe). Consequently, the phenolic group was not attached to C(8) and C(10) [17]. The presence of the aromatic OH group of 3 was confirmed by its methylation yielding the monomethyl ether 3a (C17H1005). Compounds 2 and 3a were different as a comparison of the data (UV, IR, 'H-NMR, and m.p.) and TLC analysis revealed. The attachment of the Me0 group of 3a at C(11) was deduced from the 'H-NMR spectrum (Table I ) . When compared to 1, the chemical shifts of the 4 aromatic protons on the coumarin moiety of 3a were almost identical. Three vicinal aromatic protons remained on the chromone moiety. While the resonances of the protons zyxwv zy zyxwvut zyxwvut HELVETICA CHIMICA ACTA- Vol. 72 (1989) 1459 ortho (H-C(1O)) and para (H-C(8)) to the M e 0 group appeared upfield ( A S = -0.51 and -0.42 ppm, resp.), H-C(9) was almost unaffected. The substition pattern was corroborated by a NOE difference experiment. An enhancement of H-C(10) (7.28 ppm, dd) was observed upon irradiation of the M e 0 group at 4.07 ppm. The 13C-NMRdata of 3 confirmed the substitution at C(11). In accordance with chemical-shift rules [19], the signals of C(8), C(lO), and C(l1a) appeared at 119.6 (AS = -7.2 ppm), 127.1 (AS = -7.7 ppm) and 142.6 pprn (AS = 11.7 ppm), respectively, while the substituted C(11) was observed at 146.5ppm (AS = f28.7 ppm). Discussion. - Three chromonocoumarins 1-3 have been isolated from Polygala fruticosa BERG.Although this type of compounds has been synthesized, the occurrence in nature of chromonocoumarins has not been reported. The frutinones 1-3 bear two peri C=O groups on the two central rings, a rarity in natural products. This structural feature explains the large deformations in the bond angles of the two central pyranone rings, as observed by X-ray diffraction analysis of 1. The biogenesis of the chromonocoumarins is probably best considered in analogy to the 6-keto dehydrorotenoids, with a 2’-methoxyflavone as precursor instead of a 2’methoxyisoflavone [23]. After ring closure to the chromanochromanone, oxidation to a 6a-hydroxy compound, followed by a subsequent dehydration and oxidation at C(6) would lead to the 6H,7H-[ l]benzopyrano[4,3-b][ l]benzopyran-6,7-dione. A particular feature of compounds 1-3 is the lack or uncommon position of substituents on the benzo rings. No definite answer can be presently given to the question as whether the chromonocoumarins can be considered as genuine compounds or artifacts. For 6-keto dehydrorotenoids, their natural occurrence has not yet been firmly established, as they may be artifacts formed by photo-oxidation of dehydrorotenoids [23]. We are currently pursuing the isolation and structure elucidation of additional phenolic constituents in the search of possible biogenetic precursors. As mentioned above, molecules containing a chromonocoumarin nucleus have been synthesized [2427]. Frutinone A (1) has been obtained via three different synthetic routes [24-261. The scarcity of spectral data, however, precluded any comparison with the natural compound. Good bacteriostatic activity against S. aureus, B. subtilis, and B. coli has been reported for several halogenated chromonocoumarins [25]. Compounds 1-3 and 3a were tested against Cladosporium cucumerinum, a plant pathogenic fungus, using a TLC bioassay [16]. Frutinone A (1) was active, and the mininum quantity required to show antifungal activity in the test was 0.25 pg. Interestingly, the presence of a M e 0 group in position 4 and substitution by a M e 0 or an OH moiety at C(l1) rendered compounds 2,3, and 3a inactive. This finding suggests a certain degree of selectivity in the mode of action of 1. Further biological testing of 1-3 is presently underway. zyxw The Swiss National Science Foundation provided financial support for this work. H . St.-E. wishes to thank the Swiss National Science Foundation for an equipment grant (No. 2.372-0.84). Experimental Part General. TLC: silica gel precoated A1 sheets (Merck) ; RP-8 precoated glass plates (HPTLC; Merck); detection at 254 and 366 nm. Low-pressure liquid chromatography (LPLC): Lobar Diol(4&63 pm; 27 x 2.5 cm i.d.; Merck, Darmstadt), equipped with Duramat-80 pump (Chemie and Filter, Regensdorf). Purity of compounds 1-3 and 3a was checked on TLC and HPLC with a ,u-Bondapuk C-18column (10 pm; 30 cm x 3.9 mm i d .; Waters) and a Spectra Physics 8700 pump (San Jos6, USA); the chromatograms at 210 and 254 nm and the UVjVIS spectra 1460 zyxwv zyxwvut zyx zy zyx zyxwvu zyxwv zyxw zyxw HELVETICA CHIMICA ACTA Vol. 72 (1989) - were recorded with a photodiode array detector H P 1040A, an HP-85B computer, and an H P 74704 plotter (Hewlett-Packard). M.p.: Mettler FP 80/82 hot stage apparatus; uncorrected. UV (A, (log 8 ) ) : Vuriun D M S IUOSspectrophotometer; in MeOH before and after addition of shift reagents according to [17]. IR: Philips PU 9716 spectrophotometer. 'H- and I3C-NMR: Varian VXR-200 equipped with a switchable 5-mm probe at 200 and 50.1 MHz, resp.; chemical shifts in S (ppm) rel. to TMS as internal standard; 'H-NMR with 0.1 Hz digital were extracted after suitable resolution enhancement. COSY spectra: 128 x 512 K data resolution; accurate J(H,H) sets, after zero filling to 512 x 512 K complex data points, pseudo-echo weighting in both dimensions was used. NOE difference spectra: at 30" with a presaturation of 3 s (128 transients). A 1 Hz line broadening function was applied to the difference. FID prior to Fourier transformation. Multiplicities of the I3C signals were determined by DEPTexperiments. 2D HETCOR spectrum of 1: at 35", concentration 1.7. lo-' M in CDCI,; spectral width 333 Hz in F1 and 5630 Hz in F2, data matrix of 64 x 1024 datapoints with 1024 transients per increment; after zero-filling to 128 x 2048 K, pseudo-echo weighting was applied in both dimensions prior to Fourier transformation; the long-range HETCOR experiment was performed with delays optimized for JcccH = 7 Hz. EI-MS: Nermag R 10-10 spectrometer Plant Material. Polygalafruticosa BERG.was collected in Silverglen Nature Reserve, Durban, South Africa, in June 1988. A voucher specimen is deposited at the Herbarium, University of Durban-Westville (South Africa). Exlruction and Isolation. Powdered leaves (60 g) and root bark (90 g) of P . fruticosa were extracted at r.t. successively with petroleum ether, CH2C12,and MeOH. A part (3.5 g) of the CH2C12extract of the leaves (4.5 g) was fractionated by column chromatography (4.5 x 60 cm) on silica gel 60 ( 4 0 4 3 pm, Merck) into 9 fractions (I-IX) ; using steps-gradient petroleum ether/AcOEt 1 :I -2 :3 +2 : 5 , followed by AcOEt. Recrystallisation of Fraction VI (298 mg) and Fraction VII (728 mg) from CH2C12/AcOEt5:1 provided 1 (90 and 31 1 mg, resp.). Recrystallisation ofFraction IX(331 mg)fromCH2CI2/MeOH5:2gave2(29mg). Furtheramountsof1(19mg)and2(15mg) were obtained by LPLC of Fraction VIII (244 mg) on Diol with CHCl,/petrolenm ether 1 : 1. After elimination of 10.0 g of a white precipitate (sucrose), a portion (17.9 g) of the MeOH extract of the root bark (29.0 g) was submitted to column chromatography (5.0 x 80 cm) on silica gel (40-63 km, Merck) using step-gradient CHC13/MeOH/H2080:20:2 +60 :40: 10 (monitoring by TLC) to afford 9 fractions (I-ZX). Compound 3 (40 mg) was isolated from Fraction V (840 mg) by filtration on Sephadex-LH-20 with MeOH as eluent. 6H,?H-(I]Benzop)~ran0[4,3-b]/l]benzopyran-6,7-dione ( = Frutinone A ; 1). White prisms from CH2CI,/ AcOEt 5 : l . M.p. 235-236" ([24]: 233"; [25]: 245"; [26]: 236"). TLC (SO2, CHCI,/MeOH 95:5): R, 0.47, dark fluorescence at 366 nm. HPTLC (RP-8, MeOH/H207 :3): R,0.49. UV (MeOH): 229 (4.18), 261 (4.28), 296 (4.08), 318 (sh, 3.98; [24]: 231 (4.21), 263 (4.49), 298 (4.11), 320 (4.00)). UV (MeOH+NaOMe): unchanged. UV (MeOH + AICI,): unchanged. IR (KBr): 3050, 1745, 1640, 1610, 1540, 1410, 1105,900,870,755 ([24]: 1727, 1648, 1620, 1548). 'H-NMR: Table 1. 13C-NMR: Table 2. Heteronuclear connectivities (long-range HETCOR, ,J(C,H) = 7): C(l)/H-C(3), C(2)/H-C(4), C(3)/H-C(1), C(4)/H-C(2), C(4a)/H-C(3), C(6)/-, C(6a)/-, C(7)/ H-C(8), C(7a)/H-C(9), C(7a)/H-C(Il), C(8)/H-C(10), C(9)/H-C(1 I), C(IO)/H-C(8), C(Il)/H-C(9), C(l la)/ H-C(IO), C(12a)/H-C(1), C(12b)/H-C(2), C(12b)/H-C(4). EI-MS: 264 (100, M+'),236 (59), 208 (22), 116 (43, 104 (19), 92 (23), 88 (62), 76 (19). X-Ray Analysis ofl. Suitable crystals, in the form of transparent plates, were grown from MeCN. Crystal data: CI6H8O4,M , = 264.2, space group Pnam, u = 22.630(9), b = 7.892(1), c = 6.484(1) A, V = 1158.0 A3, F(000) = 544, Z = 4, D , = 1.516 gem-,, MoKcc, A = 0.71073 A, p = 0.10 mm-'. A crystal of dimensions 0.44 x 0.38 x 0.02 mm was used for data collection. Preliminary Weissenberg and precession photographs indicated the crystals to be orthorhombic, space group Pna2 or Pnam. Intensity data, with index limits h 0 to 24, k 0 to 8, I 0 to 6 and Om,, = 22.5", were measured on a Stoe Siemens AED2 four-circle diffractometer (graphitemonochromated MoKa radiation), o/O scan mode, on-line profile fitting [28] [29]. There was a 3% intensity variation for 3 standard reflections measured every h. Of the 649 unique reflections measured, 436 were considered observed (F, > 30(F0)).Cell parameters were from & w values of 14 reflections and their equivalents in the range 7" < 2 8 < 31". No absorption or extinction corrections were applied. The E statistics clearly indicated a centrosymmetric system which was confirmed by the successful least-squares refinement. The structure was solved by direct methods using the program SHELXS-86 [30]. The program SHELX-76 [31] was used for all further calculations. In the final cycles of least-squares refinement the benzene H-atoms were included in idealized positions and treated as 'riding atoms' with an overall isotropic thermal parameter (refined value 0.0823). Weighted anisotropic full-matrix least-squares refinement for 436 reflections converged at R = 0.073, R, = 0.064; w-' = a2(Fn) + O.O0132(F2). Average parameter shift/e.s.d. < 0.008. Heights in final difference map pmax= 0.43, pmin= -0.3 1 e.k3. The rather high R factor is probably due to the fact that the crystal did not diffract significantly zyxwv zyxwvutsrqp zyxwvutsrq zyx HELVETICA CHIMICA ACTA- Vol. 72 (1989) 1461 Table 3. Bond Distances (A; average error 0.014(3) A) and Angles (";average error 1.1(2)")for 1 ~~ ~ C( 1)-C(2) C(l)-C(12b) C(2)-C(3) C(3)-C(4) C(4)-C(4a) C(4a)-O(5) C(4a)-C( 12b) 0(5)-C(6) C(6)-0(6) C(6)-C(6a) C(6a)-C(7) C(6a)-C( 12a) C(2)-C( 1)-C( 12b) C( i)-c(2)-c(3) C(2)-C(3)-C(4) C(3)-C(4)-C(4a) C(4)-C(4a)-0(5) C(4)-C(4a)-C( 12b) 0(5)-C(4a)-C( 12b) C(4a)-0(5)-C(6) 0(5)-C(6)-C(6a) 0(5)-C(6)-0(6) 0(6)-C(6)-C(6a) C(6)-C(6a)-C(7) C(6)-C(6a)-C( 12a) C(7)-C(6a)-C( 12a) C(6a)-C(7)-C(7) C(6a)-C(7)-C(7a) 0(7)-C(7)-C(7a) 1.383 1.374 1.391 1.387 1.392 1.358 1.366 1.394 1.211 1.448 1.460 1.379 C(7)-0(7) C(7)-C(7a) C(7a)-C(8) C(7a)-C(1 la) C@-C(9) C(9)-C( 10) C(l0)-C(11) C(1l)-C(l la) C( 1la)-O( 12) O( 12)-C( 12a) C( 12a)-C( 12b) O(6). ..0(7) 1.231 1.487 1.385 1.340 1.362 1.407 1.398 1.393 1.403 1.337 1.442 2.839 120.5 119.6 120.5 118.0 114.8 122.0 123.1 121.9 117.9 115.0 127.1 122.4 118.0 119.6 124.2 114.8 121.0 C(7)-C(7a)-C(8) C(7)-C(?a)-C(l la) C(X)-C(7a)-C( 1la) C(7a) -C(X)-C(9) C(X)-C(9)-C( 10) c(9)-c(lo)-c(ll) C(l0)-C(l1)-C(1 la) C(7a)-C(1 la)-C(11) C(7a)-C(1 la)-O(12) C( 1 1)-C(l la)-O( 12) C( 1la)-O( 12)-C( 12a) C(6a)-C(12a)-O( 12) C(6a)-C( 12a)-C( 12b) 0(12)-C(12a)-C( 12b) C( 1)-C( 12b)-C(4a) C( 1)-C( 12b)-C( 12a) C(4d)-C( 12b)-C( 12a) 120.0 120.2 119.8 119.0 120.7 120.8 115.3 124.5 122.9 112.7 118.6 123.9 122.9 113.2 119.4 124.5 116.1 zyxwv zyxwvutsr zyxw zyxw beyond 40"in 2 0 , hence the reflections/parameters ratio is poor (ca. 4). Atomic scattering factors were taken from [32]. Bond distances and angles are given in Table 3. The crystallographic numbering scheme is apparent from the Figure, prepared using ORTEP-I1 [33]. Final positional and equivalent isotropic thermal parameters and supplementary material are available from H . St.-E. and deposited with the CCDC. 4-Methoxy-6H,7H-(l]benzopyrano[4,3b][l]benzopyran-6,7-dione ( = Frutinone B ; 2). White needles from CH,Cl,/MeOH 5:2. M.p. 279-280". TLC (SO,, CHCl,/MeOH 95:5): R, 0.43, blue fluorescence at 366 nm. HPTLC (RP-8, MeOH/H,O 8 :2): R, 0.56. UV (MeOH): 247 (4.34), 303 (4.22). UV (MeOH + NaOMe): unchanged. UV (MeOH + AlC1,): unchanged. IR (KBr): 2850-3100, 1755, 1650, 1620, 1555, 1470, 1410, 1280,890, 770. 'H-NMR: Table 1. ',C-NMR: Table 2. EI-MS: 294 (88, M " ) , 265 (42), 251 (51), 237 (14), 210 (13), 208 (lo), 139 (26), 131 (95), 103 (55), 92 (79), 77 (loo), 76 (43), 75 (99). 11-Hydroxy-6H.7H-[ l]benzopyrano[4,3- b][l]benzopyran-6.7-dione ( = Frutinone C; 3). White amorphous solid. M.p. 240-250" (dec.). TLC (SO2, CHC13/MeOH/H2065: 35 :5): R,0.45, yellow fluorescence at 366 nm. UV (MeOH): 267 (4.33), 316 (sh, 4.07). UV (MeOH + NaOMe): 303 (4.03), 378 (3.57). UV (MeOH + NdOAc): unchanged. UV (MeOH AlCl,): unchanged. IR (KBr): 3500, 1720, 1600, 1410, 1250, 1050, 980, 760, 720. 'H-NMR: Table 1. ',C-NMR: Table 2. EI-MS: 280 (100, M " ) , 265 (5), 224 (X), 145 (16), 139 (16), 121 (18), 116 (19), 107 (13), 88 (24), 56 (27), 41 (18). + ll-Methoxy-6H,7H-[l]benzopyrano[4.3-b][l]benzopyran-6,7-dione (3a). Methylation was carried out by stirring under reflux for 1 h 10 mg of 3 in 10 ml of dried acetone in the presence of K,CO, (0.7 g) and (CH,),SO, (0.3 ml). The mixture was filtered and the residue washed several times with acetone. The product was purified by column chromatography on silica gel with CHCl,/MeOH 99:l and recrystallised from MeCN giving 3a as white needles. M.p. 259-260". TLC (SO,, CHCl,/MeOH 95: 5): Rf0.45, white fluorescence at 366 nm. UV (MeOH): 273 (4.31), 315 (sh, 3.98). UV (MeOH NaOMe): unchanged. UV (MeOH AlCl,): unchanged. IR (KBr): 2800- + + 1462 zyxwv zyxwvu zyxwv zy zyxwvuts zyxwvu HELVETICA CHIMICA ACTA Vol. 72 (1989) ~ 3000, 1755, 1610, 1555, 1495, 1420, 1280, 1110,935,715. ‘H-NMR: Table 1. ELMS: 294 (100, M ” ) , 279 (6), 265 ( I l ) , 251 (8), 223 (7), 149 (7), 116 (lo), 88 (12), 85 (16), 83 (23), 43 (16). REFERENCES [l] A. Engler, ‘Syllabus der Pflanzenfamilien’, 12. Aufl., Borntrager Verlag, Berlin, 1964, Vol. 2, p. 275. [2] S. Sakuma, J. Shoji, Chem. Pharm. Bull. 1981, 30, 810. 131 T. Nikaido, T. Ohmoto, H. Saitoh, U. Sankawa, S. Sakuma, J. Shoji, Chem. Pharm. Bull. 1982,30,2020. [4] M. Hamburger, K. 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