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J Basic Microbiol 39 (1999) –6, 345 –349 (Hans-Knöll-Institute for Natural Products Research, Beutenbergstrasse 11, D-07745 Jena, Germany and The Centre for Mycological Research of National Hanoi University, College of Natural Sciences – VNU, 90 Nguyen Trai Str., Dong Da – Hanoi, Vietnam) New fasciculol-type triterpene compounds from Hypholoma fasciculare PETER KLEINWÄCHTER, UDO LUHMANN, BRIGITTE SCHLEGEL, STEFAN HEINZE, ALBERT HÄRTL, TRINH TAM KIET1 and UDO GRÄFE Dedicated to Prof Dr WOLFGANG STEGLICH on the occasion of his 65th birthday (Received 27 July 1999/Accepted 27 August 1999) New fasciculol-type triterpene compounds 1, and were discovered in extracts of the mushroom Hypholoma fasciculare by LC-NMR and LC-MS methods Isolation and structure elucidation are reported Fasciculol and fasciculic acid production of H fasciculare was compared with that of H sublateritium and H capnoides and inhibited moderately 3D-hydroxysteroid dehydrogenase (IC50: 14.0 µM) Hypholoma species (formerly Naematoloma) belong to the Euro-Asian-North-American group of Basidiomycota Fasciculols and fasciculic acids were described as lanosterol-type sterol metabolites from Hypholoma fasciculare which is a rich source of triterpenoid homologues and congenors (Fig 1) Nine derivatives of the basic lanostane skeleton have been reported which are discernible by the side chain at either C-2 or C-3 (R1 or R2 = 3Dhydroxy-3E-methylglutaric acid (Y); 3D-hydroxy-3E-methylglutaryl-glycine or its methylester (X) and hydroxyl substituents at C-12 (R3) and/or C-21 (TURNER and ALDRICH 1982, DE BERNARDI et al 1981, SUZUKI et al 1983, IKEDA et al 1977 a, 1977 b, 1977 c) Previous interest in these compounds focused on their toxicity (KIKUCHI et al 1979), activity as calmodulin antagonists (TAKAHASHI et al 1989, KUBO et al 1985, NOZOE et al 1993) and plant growth inhibitors (IKEDA et al 1977 a –c) In the course of a screening for new fungal metabolites we investigated the extract of H fasciculare by HPLC-coupled NMR spectroscopy and electrospray mass spectrometry Comparison of the mass spectrometric and 1H NMR data suggested the occurrence of the three hithero not reported fasciculol-type compounds 1, and (fasciculic acids D, E and F; Fig 1) Here we describe their isolation, structure and biological activity Materials and methods Mp uncorr.; NMR spectra were recorded on 300 MHz and 500 MHz instruments (BRUKER AVANCE DPX 300 and BRUKER AVANCE DRX 500); HRFAB-MS on an AMD 402 instrument (AMD Intectra, Harpstedt near Bremen, Germany), electrospray-MS (ESI-MS) on a triple quadrupole instrument Quattro (VG BIOTECH, Altrincham, England) and FINNIGAN LCQ and MAT 95 XL equipments; optical rotation on a Propol polarimeter (KERNCHEN Optics, Seelze, Germany), IR spectra on SHIMADZU FT IR 470 spectrometer, UV-VIS spectra on a BECKMAN DU 640 scanning instrument (BECKMAN, USA) Assay of 3D-hydroxysteroid dehydrogenase: The NADPH-dependent reduction of 5E-dihydrocortison (PENNING 1985) by 3D-hydroxysteroid dehydrogenase from Pseudomonas testosteroni 346 P KLEINWÄCHTER et al (SIGMA) was measured in presence of increasing concentrations of 1, and The standard assay of the enzyme contained 840 µl distilled water, 100 µl phosphate buffer (pH 6.0, 12 mM), 20 µl NADPH (10 µg), 10 µl cortison (18 µg), 10 µl of a solution of fasciculols in methanol and 10 µl of a solution of rat liver cytosolic fraction ( Ô àg) The assay was started by the addition of the liver cytosole fraction and the decrease in optical density at 340 nm was recorded The inhibitory activity was estimated by comparison of controls (without the addition of fasciculols) and assays run with standards such as ibuprofene with the assay containing the samples 1, and as was described (PENNING 1985) Results and discussion Fruit bodies of Hypholoma fasciculare, H sublateritium and H capnoides were collected in the forestal region of Hummelshain/Neustadt (Thuringia, Germany) 2.5 kg of wet fruit bodies were extracted threetimes by l ethyl acetate The extract was dried over Na2SO4 and evaporated to dryness The residue obtained from the extract of H fasciculare was subjected to the top of a column (silica gel 60, Ô mm, MERCK, cm u 40 cm) and elution occurred stepwise by each column volumes of CHCl3 and CHCl3 –MeOH (95 : 1, : 1, : v/v) The eluate fractions were checked by TLC (silica gel 60 aluminium sheets, MERCK, CHCl3/MeOH : v/v) New fasciculic acids 1, and (Fig 1) were visualized by blueish staining with 3% vanillin in conc H2SO4 (Rf: 0.75, 0.7 and 0.13, respectively, CHCl3/MeOH : v/v) Final purification was achieved by preparative HPLC (Spherisorb RP18 ODS-2; 2.5 u 25 cm, µm, 10 ml/min, 210 nm, gradient water to 83% acetonitrile, 15 min, subsequently isocratic run with 83% acetonitrile) The purity of the fractions thus obtained was checked by analytical HPLC (GROM-SIL RP18 ODS-0 AB, u 250 mm, µm, 0.5 ml/min, 210 nm, acetonitrile/H2O 83 : 17, 0.1% TFA) In addition to 1, and (for the physico-chemical data see below) the known fasciculols C, E, F, D and fasciculic acids B and C were isolated from the fruiting bodies of Hypholoma fasciculare The structures of 1, and as new triterpene structures were settled by mass spectrometry (electrospray-MS (ESI-MS), high-resolution fast atom bombardment MS (HRFAB-MS), Fig Structures of new triterpenes fasciculic acid D (1), fasciculic acid E (2) and fasciculic acid F (3) New fasciculol-type triterpene compounds from Hypholoma fasciculare 347 1D- and 2D-NMR experiments (1H, 13C, COSY, DEPT, HSQC, HMBC)) For their detection amongst the other fasciculol and fasciculic acid components LC-MS and LC-NMR experiments were particularly helpful Thus in the H NMR spectra of and the methoxyl proton signal was missing which is a characteristic of the fasciculols D and F But otherwise the other 1H NMR data of and were nearly identical The third metabolite (3) was detected due to the upfield chemical shifts and the coupling pattern of the H-12 and H-21 protons which were caused by the presence of hydroxyl substituents ESI-MS of (m/z 653 [M + H]+) suggested that there is a 3D-hydroxy-3E-methylglutaryl substituent at O-3E-position (see Fig 1) Physico-chemical properties of new fasciculic acids D, E and F Fasciculic acid D (1): colorless solid, mp 124 –126 °C (uncorrected); [D]D25 + 11.5 (c = 0.5 mg/ml, MeOH), ESI-MS: m/z 710 [M + H]+, 732 [M + Na]+, HRESI-MS: m/z 732.4427 (calcd 4309 for C38H63NO11Na; intensity 10%), m/z 305.1 (100%), m/z 220.6 (60%) IRQKBr cm–1: 3415, 2965, 1721, 1653, 1542, 1452, 1375, 1199, 1076, 1037, UV OMeOH nm) max max 1H NMR (CD OD): G 1.23 (H -1, m); 2.10 (H -1, m), 5.02 (H-2, td, log H : 205 (83.71) A B 10.8, 4.3 Hz), 3.20 (H-3, d, 10.8 Hz), 1.20 (H-5, br), 1.56 (HA-6, m), 1.74 (HB-6, m), 2.09 (2 H-7, m), 1.98 (HA-11, br), 2.70 (HB-11, br), 4.01 (H-12, dd, 9.1, 2.3 Hz), 1.21 (HA-15, m), 1.74 (HB-15, m), 1.44 (HA-16, m), 2.08 (HB-16, m), 2.42 (H-17, q, 9.4 Hz), 0.63 (3 H-18, s), 1.10 (3 H-19, s), 1.38 (H-20, m), 3.69 (HA-21, d, 11.6 Hz), 3.79 (HB-21, d, 11.6 Hz), 1.44 (HA-22, m), 1.65 (HB-22, m), 1.42 (HA-23, m), 1.55 (HB-23, m), 3.25 (H-24, dd, 10.2, 2.0 Hz), 1.13 (3 H-26, s), 1.16 (3 H-27, s), 1.06 (3 H-28, s), 1.06 (3 H-29, s), 0.88 (3 H-30, s), 2.65 (HA-2c, d, 14.2 Hz), 2.69 (HB-2c, d, 14.2 Hz), 1.39 (3 H-4c, s), 2.57 (HA-5c, d, 14.2 Hz), 2.61 (HB-5c, d, 14.2 Hz), 3.91 (HA-7c, d, 17.9 Hz), 3.95 (HB-7c, d, 17.9 Hz), 13C NMR (CD OD): G 41.75 (C-1), 74.43 (C-2), 80.88 (C-3), 40.62 (C-4), 51.84 (C-5), 19.30 (C-6), 27.52 (C-7), 136.68 (C-8), 133.89 (C-9), 39.31 (C-10), 33.12 (C-11), 74.30 (C-12), 51.08 (C-13), 51.19 (C-14), 32.82 (C-15), 28.83 (C-16), 39.21 (C-17), 17.35 (C-18), 20.11 (C-19), 44.23 (C-20), 61.95 (C-21), 28.04 (C-22), 29.37 (C-23), 79.43 (C-24), 73.84 (C-25), 24.94 (C-26), 25.67 (C-27), 24.16 (C-28), 28.94 (C-29), 17.15 (C-30), 172.71 (C-1c), 47.07 (C-2c), 71.48 (C-3c), 27.98 (C-4c), 47.22 (C-5c), 174.04 (C-6c), 41.75 (C-7c), 172.94 (C-8c) Fasciculic acid E (2): colorless solid, mp Ô& (uncorrected); [D]D25 + 15.4 (c = 0.4 mg/ml, MeOH), ESI-MS: m/z 694 [M + H]+, 716 (M + Na]+, HRESI-MS: m/z 716.4618 (calcd 4870 for C38H63NO10Na; intensity 10%), m/z 304.0 (20%), m/z 220.6 cm–1: 3410, 2955, 1726, 1679, 1525, 1459, 1438, 1378, 1343, 1251, 1232, (100%) IRQKBr max nm (log H): 205 (3.72) 1H NMR (CD3OD): G 1.25 (HA-1, m), 1138, 1109, 1027 UV OMeOH max 2.09 (HB-1, m), 5.02 (H-2, m), 3.21 (H-3, d, 10.7 Hz), 1.21 (H-5, br), 1.57 (HA-6, m), 1.74 (HB-6, m), 2.10 (2 H-7, m), 2.02 (HA-11, br), 2.62 (HB-11, br), 4.00 (H-12, dd, 7.7, 1.5 Hz), 1.16 (HA-15, m), 1.68 (H-15, m), 1.38 (HA-16, m), 2.04 (HB-16, m), 2.22 (H-17, q, 9.2 Hz), 0.65 (3 H-18, s), 1.10 (3 H-19, s), 1.43 (H-20, m), 1.03 (3 H-21, d, 6.3 Hz), 1.34 (HA-22, m), 1.52 (HB-22, m), 1.39 (HA-23, m), 1.53 (HB-23, m), 3.23 (H-24, dd, 10.6, 2.0 Hz), 1.13 (3 H-26, s), 1.16 (3 H-27, s), 1.09 (3 H-28, s), 1.06 (3 H-29, s), 0.88 (3 H-30, s), 2.65 (HA-2c, d, 14.2 Hz), 2.69 (HB-2c, d, 14.2 Hz), 1.39 (H-4c, s), 2.57 (HA-5c, d, 14.2 Hz), 2.61 (HB-5c, d, 14.2 Hz), 3.91 (HA-7c, d, 17.9 Hz), 3.95 (HB-7c, d, 17.9 Hz) 13C NMR (CD OD): G 42.03 (C-1), 74.51 (C-2), 80.88 (C-3), 40.65 (C-4), 51.78 (C-5), 19.33 (C-6), 27.42 (C-7), 136.77 (C-8), 133.56 (C-9), 39.25 (C-10), 34.53 (C-11), 73.57 (C-12), 50.61 (C-13), 50.74 (C-14), 33.22 (C-15), 28.98 (C-16), 44.10 (C-17), 16.98 (C-18), 20.25 (C-19), 37.57 (C-20), 18.00 (C-21), 34.39 (C-22), 29.02 (C-23), 79.78 (C-24), 73.91 (C-25), 25.0 (C-26), 25.62 (C-27), 25.26 (C-28), 28.96 (C-29), 17.17 (C-30), 172.71 (C-1c), 348 P KLEINWÄCHTER et al 47.09 (C-2c), 71.48 (C-3c), 27.96 (C-4c), 47.22 (C-5c), 174.04 (C-6c), 41.74 (C-7c), 172.95 (C-8c) Fasciculic acid F (3): colorless solid, mp Ô C (uncorrected); [D]D25 14.7 (c = 0.3 mg/ml, MeOH); HRESI-MS: m/z 651.4116 ([M + H]–, calcd 4108 for C36H59O10) cm–1: 3420, 2945, 1698, 1677, 1454, 1425, 1375, 1200, 1138, 1083, 1033 UV OMeOH nm IRQKBr max max (log H): 207 (3.78) H NMR (CD3OD): G 1.23 (HA-1, m), 2.10 (HB-1, m), 5.01 (H-2, td, 10.8, 4.3 Hz), 3.19 (H-3, d, 10.8 Hz), 1.20 (H-5, br), 1.57 (HA-6, m), 1.74 (HB-6, m), 2.09 (2HA-7, m), 1.96 (HA-11, br), 2.70 (HB-11, br), 4.00 (H-12, dd, 8.9, 2.5 Hz), 1.21 (HA-15, m), 1.75 (HB-15, m), 2.08 (HA-16, m), 1.44 (HB-16, m), 2.42 (H-17, q, 9.6 Hz), 0.63 (3 H18, s), 1.10 (3 H-19, s), 1.38 (H-20, m), 3.69 (HA-21, d, 11.7 Hz), 3.79 (HB-21, d, 11.7 Hz), 1.43 (HA-22, m), 1.65 (HB-22, m), 1.42 (8 HA-23, m), 1.55 (HB-23, m), 3.25 (H-24, dd, 10.1, 1.9 Hz), 1.13 (3 H-26, s), 1.16 (3 H-27, s), 1.06 (3 H-28, s), 1.05 (3 H-29, s), 0.88 (3 H-30, s), 2.71 (2 H-2c, s), 1.40 (3 H-4c, s), 2.64 (HA-5c, d, 15.3 Hz), 2.68 (HB-5c, d, 15.3 Hz) 13C NMR (CD OD) G 41.83 (C-1), 74.31 (C-2), 80.93 (C-3), 40.65 (C-4), 51.85 (C-5), 19.30 (C-6), 27.53 (C-7), 136.68 (C-8), 133.90 (C-9), 39.30 (C-10), 33.09 (C-11), 74.31 (C-12), 51.08 (C-13), 51.20 (C-14), 32.82 (C-15), 28.83 (C-16), 39.22 (C-17), 17.35 (C-18), 20.11 (H-19), 44.22 (C-20), 61.94 (C-21), 28.04 (C-22), 29.38 (C-23), 79.43 (C-24), 73.84 (C-25), 24.95 (C-26), 25.67 (C-27), 24.17 (C-28), 28.95 (C-29), 17.16 (C-30), 172.70 (C-1c), 46.71 (C-2c), 70.87 (C-3c), 27.90 (C-4c), 45.87 (C-5c), 174.87 (C-6c) Biological activities of 1, and Low antimicrobial activity of 1, and was established against some Gram-positive bacteria such as Bacillus subtilis ATCC 6633 (MIC > 500 µg/ml) but not against Gram-negative bacteria and fungi However, 1, and inhibited moderately the 3D-hydroxysteroid dehydrogenase (PENNING 1985) with IC50 = 14 µM Occurrence of 1, and in related mushrooms such as Hypholoma sublateritium and Hypholoma capnoides: Though H sublateritium ist capable of producing fasciculols B, C, D and F there was no evidence suggesting the presence of 1, and in this organism H capnoides was incapable of producing fasciculols The new metabolites 1, and (fasciculic acids D, E and F) enlarge the spectrum of the known fasciculol-type sterol compounds from Hypholoma species Their occurrence in H fasciculare was disclosed in addition to the hithero known closely related compounds such as fasciculols $Ô) DQG IDVFLFXOLF DFLGV $Ô& TURNER and ALDRICH 1982, TAKAHASHI et al 1989) However, sensitive analytical methods such as LC-NMR and MS were necessary to discover the presence of these new minor components amongst the bulk of recurrent metabolites The optical rotations of 1, and were similar to those reported for fasciculol D, fasciculol F and fasciculic acid B suggesting that there is an identical stereochemistry Acknowledgements We gratefully acknowledge support of this work by FCI Frankfurt a.M and DLR Bonn (project VIE 008/97), Germany The skillful technical assistance of Mrs H HEINECKE, A PERNER, R KOCH and C KARKOWSKI is appreciated References DE BERNARDI, M., MELLERIO, G., VIDARI, G., VITA-FINZI, P., FRONZA, G., KOCOR, M and PYREK, J S., 1981 Fungal Metabolites IX Triterpenes from Naematoloma sublateritium J Nat Prod., 44, 351 –356 New fasciculol-type triterpene compounds from Hypholoma fasciculare 349 IKEDA, M., SATO, Y., IZAWA, M., SASSA, T and MIURA, Y., 1977 a Isolation and structure of fasciculol A, a new plant growth inhibitor from Naematoloma fasciculare Agric Biol Chem., 41, 1539 –1541 IKEDA, M., WATANABE, H., AYAKAWA, A., SATO, K., SASSA, T and MIURA, Y., 1977 b Structures of fasciculol B and its depsipeptide, new biologically active substances from Naematoloma fasciculare Agric Biol Chem., 41, 1543 –1545 IKEDA, M., NIWA, G., TOHYAMA, K., SASSA, T and MIURA, Y., 1977 c Structures of fasciculol C and its depsipeptide, new biologically active substances from Naematoloma fasciculare Agric Biol Chem., 41, 1803 –1805 KIKUCHI, T., KANAOKA, M., HANAGAKI, S and KADOTA, S., 1979 Syntheses of 24,25-dideoxyfasciculol A and fasciculol A, constituent of Naematoloma fasciculare (FR.) KARST, a poisonous bitter mushroom Chem Letters (Japan), 1979, 1495 KUBO, I., MATSUMOTO, A., KOZUOKA, M and WOOD, W F., 1985 Calmodulin inhibitors from the bitter mushroom Naematoloma fasciculare (FR.) KARST and absolute configuration of fasciculols Chem Pharm Bull., 33, 3821 –3825 NOZOE, S., TAKAHASHI, A and OHTA, T., 1993 Chirality of the 3-hydroxy-3-methylglutaric acid moiety of fasciculic acid A, a calmodulin antagonist isolated from Naematoloma fasciculare Chem Pharm Bull., 41, 1738 –1742 PENNING, T M., 1985 Inhibition of 5E-dihydrocortisone reduction in rat liver cytosol: a rapid spectrophotometric screen for nonsteroidal anti-inflammatory drug potency J Pharmaceut Sci., 74, 651 –654 SUZUKI, K., FUJIMOTO, H and YAMAZAKI, M., 1983 The toxic principles of Naematoloma fasciculare Chem Pharm Bull., 31, 2176 –2178 TAKAHASHI, A., KUSANO, G., OHTA, T., OHIZUMI, Y and NOZOE, S., 1989 Fasciculic acids A, B and C as calmodulin antagonists from the mushroom Neamatoloma fasciculare Chem Pharm Bull., 37, 3247 TURNER, W B and ALDRICH, D C., 1983 Fungal Metabolites II Academic Press, New York, London, pp 320 –321 Mailing address: Prof Dr UDO GRÄFE, Hans-Knöll-Institut für Naturstoff-Forschung, Beutenbergstr 11, 07745 Jena, Germany 350 P KLEINWÄCHTER et al ... –356 New fasciculol-type triterpene compounds from Hypholoma fasciculare 349 IKEDA, M., SATO, Y., IZAWA, M., SASSA, T and MIURA, Y., 1977 a Isolation and structure of fasciculol A, a new plant... fast atom bombardment MS (HRFAB-MS), Fig Structures of new triterpenes fasciculic acid D (1), fasciculic acid E (2) and fasciculic acid F (3) New fasciculol-type triterpene compounds from Hypholoma. .. physico-chemical data see below) the known fasciculols C, E, F, D and fasciculic acids B and C were isolated from the fruiting bodies of Hypholoma fasciculare The structures of 1, and as new triterpene
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