Murakami, Yoshito

Ohashi, Takashi

Takaishi, Yoshihisa

Tomimatsu, Toshiaki

Kyoto University Research Information Repository

Title Investigation of the Constituents of Inonotus mikadoiAuthor(s)Takaishi, Yoshihisa; Ohashi, Takashi; Murakami, Yoshito;Tomimatsu, ToshiakiCitationBulletin of the Institute for Chemical Research, KyotoUniversity (1987), 65(3): 134-140Issue Date1987-09-24URL http://hdl.handle.net/2433/77192RightType Departmental Bulletin PaperTextversionpublisherKyoto UniversityBull. Inst. Chem. Res., Kyoto Univ., Vol. 65, No. 3, 1987      Investigation of the Constituents of Inonotus mikadoi              Yoshihisa  TAKAISHI*, Takashi OHASHI*, Yoshito MURAKAMI*                            and Toshiaki ToMIMATSU*                                 Received June 1, 1987    Investigation of the constituents of Inonotus mikadoi led to isolation and characterization of ergosterol peroxide (7), cerevisterol (8) and a cerebroside (11).      KEY WORDS: Basidiomycetes/ Inonotus mikadoi/ Steroid/ Cerebroside/    Basidiomycetes are the natural sources rich in biologically active substances. In our studies on the biologically active components of Basidiomycetes of Gasteromycetes, we have isolated three new lanostane derivatives 1, 2 and 3 from Astraeus hygrometricusll. Antitumor lanostane-type triterpene, inotodiol (4), was isolated from Inonotus obliquus (Mucronoporaceae) along with 5 and 6.2,3) Several polysaccharides which have an antitumor activity were isolated from I. cuticularis,4l I. kanekirae5l and I. sciurinus.sl We report here the investigation of the constituents of I. mikadoi.    The methanol extract from the fresh fruit bodies of I. mikadoi was partitioned with water and ethyl acetate. Ethyl acetate extract was separated by silica gel and Sephadex LH-20 column chromatography .to give compounds 7, 8 and 11.    Compound 7, mp 181-182°C, [4+57.7° (CHC13), showed absorption at 3400(OH) cm-1 in the IR spectrum. Its 1H NMR spectrum showed the presence of four secondary                                                   • o HO    SOHO 1:R=rHH4 2:R =<.~H                       3:R = 0 AcO  O •,• p ,/AcO   56 *fj'±AA j%flj6A : Faculty of Pharmaceutical Science, The University of   Tokushima, 1-78 Shomachi, Tokushima 770. ( 134 )                      Investigation of the Constituents of Inonotus mikadoi                 oal      HORO OH                                                  OH  7  OR                    28 8:  R=H                21 22 26 9:R=Ac 121820 2321 25                                             27                   1911616                             4 15                                 7 AcO                        10                           +Hm/z 180 ------------------- 548(11a),576(11b)276 1-H20                                                                   294 6°          CH2OH1 OHCH3 chain A HO 4'  • 07/.1 1 1 1                            4 6 8 10 12 14 16 18           HO 3" 2. OH                                  NH H O 6               OHchain B 11a: n=1 11b: n=3 methyls [6 0.82 (d, J=6.8 Hz), 0.83 (d, J=6.8 Hz), 0.91 (d, J=7.0 Hz) and 1.00 (d, J=6.6 Hz)] and two tertiary methyls [8 8.01 and 0.88 (each s)]. The signal at 8 3.96 (1H, m) was assigned to the proton on carbon bearing the hydroxy group. The signals at 8 5.17 (2H, m), 6.24 (1H, d, J=8.5 Hz) and 6.50 (1H, d, J=8.5 Hz) were assigned to the protons on the double bond. The 13C NMR (Table 1) spectrum of 7 showed the presence of four signals [o 119.6 (d), 130.7 (tl), 132.2 (d) and 135.5 (d)] assigned to olefinic carbons, three signals [8 66.3 (d), 82.1 (s) 'and 82.7 (s)] assigned to carbons bearing an oxygen atom, six methyls, seven methylenes, six methines and two quaternary carbons.    The EIMS of 7 exhibited a molecular ion at m/z 428. These spectral data agreed with a molecular formula for 7 of C28H4403, which was confirmed by HRMS. On the basis of above results, it was concluded that compound 7 was based on the ergosterol skeleton found in Basidiomycetes. In the EIMS, 7 gave a characteristic fragment ion peaks at 7//z 410 [M— H2O], 396 [M-02] and 303 [M-side chain (C9H17)], among which the peak at mlz 396 was characteristic of ergosterol peroxide!) Compound 7 was identified from the spectral data and direct comparison with synthetic compound derived from ergosterol.$)    Compound 8, mp 253-255°C, [a]D-74.0° (CHC13), showed absorption at 3400(OH) ( 135 )                       Y. TAKAISHI, T. OHASHI, Y. MURAKAMI and T.  TOMIMATSU.                   Table 1. The 13C NMR data of compounds7, 8 and 10.     Carbons 7 810 1 30.1 t33.8 t 36.8t          2 32.6 t 32.5 t 27.4 t          366.3 d67.6 d73.4 d         441.2 t41.8 t 33.8t          5 82.7 s 76.1 s 40.0 d           6 130.7 d 74.2 d 29.5 t           7 119.6 d I20.4 d 117.2 d          8 82.1 s 141.6 s 139.4 s          9 44.6 d 43.8 d 49.3 d          10 38.0 s 38.0 s 34.1 s          1123.4 t22.4 t 21.4t          1228.6 t 40.0t 39.3t         1343.6 s43.8 s43.2 s          14 55.9 d 55.2 d 55.0 d          15 23.4t23.4 t 22.9t          16 29.4t28.4 t 28.3t          17 56.2 d 56.2 d 55.9 d          18 12.8 q 12.5 q 12.0 q          19 18.1 q 18.7 q 12.8 q          20 39.9 d 40.8 d 40.5 d          21 19.9 q 19.8 q 21.1 q           22 135.5 d 136.2 d 135.5 d            23 132.2 d 132.1 d 131.9 d          24 42.8 d 43.1 d 43.0 d          25 33.1 d 33.3 d 33.2 d          26 20.6 q 20.1 q 19.6 q          27 20.9 q 20.1 q 19.6 q          2817.6 q17.8 q17.9 q cm-1 in the IR spectrum. Its IH NMR spectrum (in C5D5N) showed the presence of four secondary methyls [8 0.87, 0.89, 0.97 and 1.07 (each 3H, d, J=6.8 Hz)] and two tertiary methyls [6 0.67, 1.53 (each 3H, s)]. The signal at 6 4.83 (1H, ddt, J=11.5 and 5.5 Hz) was assigned to the proton on the carbon atom bearing the oxygen atom, which was coupled with the signal at 6 3.03 (1H, dd, J=13.2 and 11.5 Hz). Thus, the partial structure I for compound 8 was deduced. The presence of the partial structure II was indicated from the signal at 64.34 (1H, d, J=5.1 Hz) coupled with that at 8 5.74 (1H, d, J=5.1 Hz). Also, the IH NMR spectrum of 8 showed the presence of two olefinic protons [8 5.32 (2H, m)]. The 13C NMR    H H HH H    I I III / -C C-C-III--- C------C------C~ I    H 0- H0-   III ( 136 )                      Investigation of the Constituents of  Inonotus mikadoi spectrum (Table 1) of 8 indicated the presence of four signals [6120.4 (d), 132.1 (d), 136.2 (d) and 141.6 (s)] assigned to olefinic carbons, three signals [8 67.6 (d), 74.2 (d) and 76.1 (s)] assigned to carbons bearing oxygen atom, seven methyls, six methines and two quaternary carbons. The molecular formula. C28H4603, of compound 8 was determined by the high resolution mass spectrum on the peaks at m/z 412 (M-H20), 394 (M-2H20), and 376 (M-3H20). Acetylation of 8 gave diacetate 9 indicating the presence of two secondary hydroxy groups and a tertiary hydroxy group in 8 in the molecule. Thus, the molecular formula of compound 8 was assigned to C28H4603. The peaks at m/z 412, 394 and 376 were shown to M-H2O, M-2H20 and M-3H20, respectively, in which assignment were confirmed by HRMS. These fact indicated that compound 8 was based on the ergosterol skeleton.    The MS of compound 8 showed a peak at m/z 251 [M-side chain (C9H17)-3H20], which indicated the presence of one double bond on side chain. The partial structures I and II were placed in the ring portion of ergosterol skeleton.    The comparison of 13C NMR spectra of compound 8 and 5,6-dihydro ergosterol acetate (10)9) indicated almost same chemical shifts on the ring C, D and side chain. The partial structure I and II could be placed on the ring A and B. The 1H NMR signal at 8 4.83 was appeared down field than usual proton on carbon bearing a hydroxy group in the ergosterol skeleton. This phenomenon was explained the effect of neighboring oxygen function. From this reason, the secondary hydroxy group in the partial structure I and tertiary hydroxy group were placed on C-3 and C-5, respectively. Remaining partial structure II was put on the ring B from the 13C NMR data. Thus the structure of 8 was represented as ergosta 7,22-diene-3,5,6-triol, which was known compound named as cerevistero1.101 The spectral data of compound 8 was good agreement with literature data of cerevisterol.    Compound 11, mp 180-182°C, [a]D+18.7° (EtOH), showed absorption at 3360(OH), 1640 and 1530(CONH) cm-1 in the IR spectrum. Its 111 NMR spectrum (in C5D5N) showed the presence of three methyls [o 0.88 (3HX2, brt), 1.63 (3H, s)] and many methylenes [61.27 (ca. 42H), 2.20 (2H, brt) and 2.17 (4H, brt)]. The signal at 61.63 was attributed to a methyl on double bond from the chemical shift. The signals at 8 5.99 (2H, m) and S 8.37 (1 H, d, J= 7.8 Hz) were assigned to the proton on double bond and amide proton, respectively. The                       Table 2. The 13C NMR data of compound 11.      CH3CH2CHC 14.2X2 22.8X2 54.3135.5         16.0 25.771.2175.5                28.172.1                 28.272.3 29.5X3 74.6 30.0X10 77.9X2 32.1X2 105.0                   32.9124.0                   35.4131.5                   39.9132.2                                62.4                                69.7                                          •  (137)                       Y.  TAKAISHI, T. OHASHI, Y. MURAKAMI and T. TOMIMATSU complex signals between 8 3.8-5.0 were estimated to protons of carbohydrate. These ill NMR data suggested that 11 was a sphingolipid. The 13C NMR spectrum (Table 2) showed the presence of three methyls, many methylenes, eleven methines and two quaternary carbons. The signals at 8 105.6 (s), 74.6 (d), 77.9 (d), 71.2 (d), 77.9 (d) and 62.4 (t) were assigned to glucopyranose carbons (C"-1-C"-6) from the comparison of reported data." The signal at 8 175.5 was assigned to amide carbon. The signals at 5124.0 (d), 131.5 (d), 132.2 (d) and 135.5 (s) were assigned to olefinic carbons. The remaining low field signals at 569.7 (t), 72.1 (t) and 72.3 (d) were attributed to carbons bearing oxygen atom.    From the above evidence, the partial structure of compound 11 was shown below. CH2OH      Hu ------O O--CH=CH--CH-=C--C-NH-  OH H\CHs HO ----~H H OH        CH2CH3X2 -CH2-O- -CH-0- X2 -(CH2)n Recently, Kawai et al. isolated a cerebroside [(4E, 8E)-N-n-2'-hydroxypalmitoyl-1-O-,6-o-glucopyranosyl-9-methyl-4,8-sphingodienine] from Schizophyllum commune, which has a fruiting-inducing activety on the same fungus.12) 1H NMR data of compound 11 was almost same with literature data. The FABMS of compound 11 showed peaks at m/z 750 [M/(727)+ +, 778 [Mjj(755)+Na]+, 576 [Mjj-glucose]+, 548 [Mj-glucose]+, 294, 276 and 180 [glucose]+. These results indicated that compound 11 was mixture of [1 la (M1=727), llb (M11=755)] due to chain B (11a=palmitoyl, 11b=stearoyl). Further purification of compound 11 is progress.    The isolation of this type cerebroside from Basidiomycetes is the second example, the compound 11 may be related to fruiting-inducing activity for I. mikadoi. The study on this point is underway.                            EXPERIMENTAL     M.p.s. were taken on Yanagimoto melting point apparatus and are uncorrected. IR spectra were recorded on Hitachi type 215 spectrometer for KBr disks. 1H and 13C NMR spectra were taken with JEOL JMN FX 200 spectrometers for solution in deuterio-chloroform or d5pyridine. Tetramethylsilane was used as internal standard and chemical shifts were given in 8 (p.p.m.) value. Mass spectra were determined with JEOL JMS D-300 spectrometer. Optical rotations were measured with Union PM-201 polarimeter. Kiesel gel 60 (70-230 mesh or 230-400 mesh, Merck) and Sephadex LH-20 (Pharmacia) were used for chromatogra-phy, and precoated silica gel plates F254 (0.25 mm and 0.5 mm in thickness) were used for TLC.    Material. The fresh fruitbody of I. mikadoi were collected from the Kainan, Tokushima, Japan in July 1984. ( 138 )                               Investigation of the Constituents of Inonotus mikadoi            Isolation of compounds 7, 8 and 11. Fresh fruit bodies (1.8 kg) were cut and extracted with        hot  McOH (20 1) at three times. The McOH solution was evaporated to dryness (136 g),         dissolved in H2O and extracted with EtOAc. The EtOAc extract was evaporated under red.        pres. to give a residue (24.5 g). The residue was chromatographed on silica gel (600 g) column        and eluted succesively with hexane-CHC13 and CHC13-MeOH to afford 15 fractions. Fr. 5        (3.2 g) containing compound 7 was chromatographed on silica gel column and eluted with hexane-EtOAc (2 : 1) to afford fr. 5-4 (1.10 g), which was crystallized from McOH to give        compound 7 (481 mg). Fr. 9 (0.25 g) containing compound 8 was filtrated, the filtrate was        concentrated to give a residue (48 mg), which was purified by using preparative TLC (CHC13—        McOH=9:1) and crystallized from McOH to give compound 8 (26 mg). Fr. 11 (1.65 g)        containing compound 11 was chromatographed on silica gel column and eluted with CHC13—        McOH—H20 (88:12 : 1) to give Fr. 11-2 (510 mg), which was crystallized from McOH to give        compound 11 (346 mg).           Compound 7, colorless needles, mp 181-182°C, [4+57.7° (c=0.1, CHC13), IR 4,1X cm—I: 3350(OH), 1650, 1610, 1450, 1380, 1360, 1060, 1030, 960; EI—MS m/z (rel. int.): 428 [M]+ (2), 410 [M—H2O]+ (6), 396 [M—O2]+ (19), 251 (25), 69 (100); HR—MS m/z: 428.3305 [M]+ for C28H4403, required 428.3291; Ill 8 (CDC13): 0.81 (3H, s), 0.82 (3H, d, J=6.8        Hz), 0.83 (3H, d, J=6.8 Hz), 0.88 (3H, s), 0.91 (3H, d, J=7.0 Hz), 1.00 (3H, d, J=6.6 Hz),        3.96 (1H, m, 3—H), 5.17 (2H, m, 26—H and 27—H), 6.24 and 6.50 (each 1H, d, J=8.5 Hz, 6—H        and 7—H); 13C NMR (CDC13): Table 1.           Compound 8, colorless needles, mp 253-255°C, [4-74.0° (c=0.2, CHC13), IR vmBx cm—t: 3400(OH), 1610, 1430, 1360, 1240, 1000, 940, 920; EI—MS m/z (rel. int.): 412 [M—       H201+ (43), 394 [M-2H2O]+ (52), 376 [M-3H2O]+ (53), 251 [C19H23]+(100); HR—MS m/z:        412.3340 for C28114402, required 412.3341, 394.3215 for C28H420, required 394.3236, 376.3124        for C28H40, required 376.3130; IH NMR 8 (C5D5N): 0.67 (3H, s), 0.87, 0.97, 1.07 and 1.26        (each 3H, d, J=6.8 Hz), 1.53 (3H, s), 3.03 (IH, dd, J=13.2 and 11.5 Hz, 4—H), 4.34 (1H, d, J=5.1 Hz, 6—H), 4.83 (1H, m, 3—H), 5.74 (1H, d, J=5.1 Hz, 7—H); 13C NMR (C5D5N): Table 1.           Compound 11, colorless needles, mp 180-182°C, [4+18.7° (c=0.1, EtOH), IR vmBx cm-1: 3360(OH), 1640 and 1530(CONH), 1460, 1080; EI—MS m/z (rel. int.): 276 [Ci8H46N]+       (63), 258 (32), 180 [glu]+ (9), FAB—MS m/z (rel. int.): 778 [Mjj+Na]+ (15), 750 [Mj+Na]+       (20), 576 [Mjj-glu+H]+ (12), 548 [Mi-g1u+H]+ (21), 294 [C18H49NO]+ (8), 276 [C18H47N]+ (15); 1H NMR 8 (C5D5N): 0.88 (3HX2, brt, 18—H3 and 16'—H3), 1.27 (ca. 42H, 11—H2--17—H2i 3'—H2--15'—H2), 1.63 (3H, s, 19—H3), 2.02 (2H, brt, 6—H2), 2.17 (2HX2, brt, 7—H2i 10—H2), 4.92 (1H, d, J=7.6 Hz, anomeric H), 5.99 (2H, m, 5-H, 8—H), 8.37 (1H, d, J=        7.8 Hz, CONH); 13C NMR (C5D5N): Table 2.            Ergosterol peroxide from ergosterol. A solution of ergosterol (50 mg) in CHC13 (50 ml)         was stirred at 25°C under sun light for 5 hr, the reaction mixture was evaporated to give a         residue, which was chromatographed on silica gel column and eluated with hexane-EtOAc (4:1) to give ergosterol peroxide, mp 182-183°C, EI—MS m/z: 428 [M]+. This compound        was identifide with compound 7 by direct comparison (TLC, IR, EI—MS and 1H NMR).           Acetylation of compound 8. A solution of 8 (10 mg) in pyridine (0.5 ml) and Ac20 (0.5         ml) was kept at room temperature for 12 hr., the reaction mixture was work up in usual way to        give a residue, which was crystallized from McOH to give a needles (6.4 mg), compound 9, 1H ( 139 )                                    Y. TAKAISHI, T. OHASHI, Y. MURAKAMI and T. TOMIMATSU         NMR  8  (CDC13): 0.82 (3H, d, J=7.0 Hz, 26-H3), 0.83 (3H, d, J=7.0 Hz, 27-H3), 0.92 (3H, d,         J=7.0 Hz, 28-H3), 1.04 (3H, d, J=7.0 Hz, 21-H3), 1.08 (3H, s, 19-H3), 1.58 (3H, s, 18-H3),         4.91 (1H, d, J=3.5 Hz, 6-H), 5.12 (1H, m, 3-H), 5.26 (1H, m, 7-H), 2.02 and 2.06 (each 3H,         s, COCH3).                                    REFERENCES (1) Y. Takaishi, Y. Murakami, T. Ohashi, K. Nakano, K. Murakami and T. Tomimatsu, Phytochemistry,             26, 2341 (1987). (2) R. S. Ludwiczak and U. Wrzeciono, Roczniki Chem., 36, 497 (1962). (3) F. R. Hirtzbach and G. Ourisson, Tetrahedron, 28, 2259 (1972). (4) M. Takatsu, M. Tabuchi, S. Saburo, T. Minami and S. Otani, Japan Kokai Pt., 75, 12, 296. (5) Aginomoto Co. Inc., Morishita Pharmaceutical Co. Ltd., Fr. Demanda, 2360310. (6) S. Ohtsuka, S. Ueno, C. Yoshikuni, F. Hirose, Y. Ohmura, T. Wada, T. Fuji and E. Takahashi, U. S.                A. Pt., 4051314. ( 7) E. P. Serebryakov, A. V. Simolin, V. F. Kucherov and B. V. Rosynov, Tetrahedron, 26, 5215 (1970);               J. Seibl, Helu. Chim. Acta., 50, 263 (1967). (8) S. C. Pakrashi, B. Achari and P. C. Majumdar, Indian J. Chem., 13,755 (1975). (9) T. Matsumoto, T. Shigemoto and I. Itoh, Phytochemistry, 22, 1300 (1983).           (10) Y. Sugimoto, T. Yamada and N. Ikekawa, Chem. Pharm. Bull., 33, 3129 (1985).           (11) R. Kasai, M. Okihara, J. Asakawa, K. Mizutani and O. Tanaka, Tetrahedron, 35, 1427 (1979).          (12) G. Kawai and Y. Ikeda, Biochim. Biophys. Ada., 754, 243 (1983); G. Kawai, The 13 rd Symposium               on Phytochemistry, Nagoya, p. 26 (1987). (140)

Investigation of the Constituents of Inonotus mikadoi

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Investigation of the Constituents of Inonotus mikadoi

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