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Handbook of Secondary Fungal Metabolites VOLUME I

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Handbook of Secondary Fungal Metabolites VOLUME

I

RICHARD J. COLE

Albany, Georgia

MILBRA A. SCHWEIKERT National Peanut Research Laboratory Dawson, Georgia

ACADEMIC PRESS An imprint of Elsevier Science Amsterdam Boston London New York Oxford Paris San Diego San Francisco Singapore Sydney Tokyo

Cover images: Photography by Dr. Bruce Horn and design by Brian E. Cole. The cover is a collage of various fungi, some presented in pure culture and others in their natural forms, i.e. mushrooms, which are the easily recognizable fruiting structures of some fungi, superimposed on these fungi are the chemical structures of some representative secondary fungal metabolites. Academic Press Rapid Manuscript Reproduction This book is printed on acid-free paper. @ Copyright

92003, Elsevier Science (USA).

All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier's Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail: [email protected]. You may also complete your request on-line via the Elsevier Science homepage (http://elsevier.com), by selecting "Customer Support" and then "Obtaining Permissions." A c a d e m i c Press An imprint of Elsevier Science 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.academicpress.com A c a d e m i c Press 84 Theobald's Road, London WC1X 8RR, UK http://www.academicpress.com Library of Congress Catalog Card Number: 2003103019 International International International International

Standard Standard Standard Standard

Book Book Book Book

Number: Number: Number: Number:

0-12-179460-1 0-12-179461-X 0-12-179462-8 0-12-179463-6

(Set) (Volume 1) (Volume 2) (Volume 3)

PRINTED IN THE UNITED STATES OF AMERICA 03 04 05 06 07 8 7 6 5 4 3 2 1

Contents

Preface I ix Acknowledgments I xi

Indole Alkaloids / 1 Diketopiperazines / 145

I~l

ChaetoglobosinslCytochalasins /

I~1

Aflavinines and Related Indoles / 355

I ~ l Tryptoquivalines /

377

I(~ I Penitrems/Lolitrems / 411

245

vi

[~J

Contents

Paspaline and Related Metabolites / 441 J a n , h , t r e m s , ,83

] ~ I Miscellaneous Indole Metabolites /

493

I~~l Loline Alkaloids / 525 l~i

Aflatoxins / 545

i~l

Versicolorins / 571

I~ ~1 Sterigmatocystin and Related Metabolites I 595 l~ ~i Chokols ! 613 I~ ~] Enaminomycins and Related Metabolites /

629

l~ ~1 Boviquinones and Related Metabolites / 651 I~ ~1 Fusicoccins I 665

Contents

vii

I~ ~1 Altenuene and Related Metabolites / 695 I~ ~] Viridin and Related Metabolites / 715 I~(~

Cercosporin and Related Metabolites /

I~J

Cyathanes / 751

I~1

Alliacolide and Related Metabolites / 797

~~

Botrydial and Related Metabolites /

813

I~~1 Herbarin and Related Metabolites /

837

I~~[ Miscellaneous Metabolites I Secondary Metabofite Index I 989 Molecular Formula Index I 995 Molecular Weight Index I 999 Fungal/Plant Source Index I 1003

897

729


Preface

The "Handbook of Secondary Fungal Metabolites" is presented in three volumes and is comprehensive to the extent that all major groups of secondary fungal metabolites are included. The format is similar to that presented in the "Handbook of Toxic Fungal Metabolites" with the major exception that actual spectra are not included; however, spectral data are included where available. Also included in these volumes are the methods used by the authors to isolate and purify metabolites. Another major difference is that the appropriate references are presented with each metabolite, negating the need to turn to the end of each group to find the appropriate references. Each volume contains four indexes: secondary metabolite index, molecular formula index, molecular weight index, and fungal/plant source index. In a few instances, plant sources are included when the metabolites are closely related to fungal metabolites or the source of precursors may be fungal; i.e., the baccharins, which are found in extracts from Baccharis megapotamica. These metabolites are closely related to the macrocyclic trichothecenes found in extracts of fungi such as Myrothecium spp. and Stachybotrys spp. Also, metabolites from the fungal symbiont of lichens are sometimes presented. To aid in the interpretation of NMR data, the numbering system presented in the literature is included for the major representative fungal metabolite and, at times, for several related metabolites. Fungal sources are given as reported in the original references. It is recognized that the taxonomy in several cases has been revised, perhaps more than once. It is beyond the scope of these volumes to deal with what is "currently accepted taxonomy" because this is a dynamic science that, in many cases, is as yet undefined. The "Handbook" has been divided into sections, and the placement of metabolites is based on chemical relationships. One section of each volume contains a miscellaneous section to accommodate metabolites difficult to place into one of the sections. The miscellaneous section of Volume III contains some metabolites related to those that appear in Volumes I and II. This occurred when related metabolites were discovered after Volumes I and II were completed.

ix

x

Preface

It is hoped that this compilation of data on secondary fungal metabolites will aid investigators in the identification of known or related fungal metabolites. Because fungal metabolites represent a wide diversity of chemical species, these volumes will be useful to scientists interested in correlations of structural features with various spectral and biological characteristics. The known biological activity of metabolites is presented, which may aid in future studies related to the identification of new uses for fungal metabolites. Richard J. Cole Milbra A. Schweikert

Acknowledgments

The authors thank the following investigators for their assistance in producing the "Handbook of Secondary Fungal Metabolites." Their contributions made this compilation of data on fungal metabolites possible. Wayne L. Bryden

James K. Porter

University of Queensland Gotton, Queensland 4343 Australia

Toxicology and Mycotoxin Research Unit USDA-ARS Athens, Georgia

Brian E. Cole

Images, Inc. Snellville, Georgia Horace G. Cutler

Department of Pharmacology Mercer University Atlanta, Georgia Jens C. Frisvad

Department of Biotechnology Technical University of Denmark DK-2800 Lyngby Denmark Bruce Horn

USDA-ARS National Panut Research Laboratory Dawson, Georgia

Ronaid T. Riley

Toxicology and Mycotoxin Research Unit USDA-ARS Athens, Georgia Victor S. Sobolev

USDA-ARS National Peanut Research Laboratory Dawson, Georgia Bruce B. Jarvis

Department of Chemistry and Biochemistry University of Maryland College Park, Maryland

William Norred Toxicology and Mycotoxin Research Unit USDA-ARS Athens, Georgia

xi


Indole Alkaloids

3-(3-Indolyl)propane-1,2,3-triol 3-(3,3-Diindolyl)propane-1,2-diol 4-(3-Indolyl)butane-l,2,3-triol N-Methyl-4-dimethylallyltryptophan Lysergic Acid Ergine; Lysergic acid amide 8-Hydroxyergine Erginine; Isolysergic acid amide 8-Hydroxyerginine Lysergol Lysergene Lysergine Ergonovine; Ergometrine; Ergobasine Ergonovinine; Ergometrinine; Ergobasinine Agroclavine 6, 7-seco-Agroclavine Dihydroagroclavine Festuclavine Elymoclavine Elymoclavine-O-B-fructofuranoside Elymoclavine-O-B-fructofuranosyl-(2-1)-O-B-Dfructofuranoside Chanoclavine-I; Chanoclavine Isochanoclavine-I Chanoclavine-II N-Demethylchanoclavine-II; Norchanoclavine II Setoclavine Isosetoclavine Costaclavine Pyroclavine Molliclavine Penniclavine Cycloclavine Ergotamine Ergotaminine 8-Hydroxyergotamine Ergosine Ergosinine Ergostine Ergostinine Ergonine Ergovaline Ergoptine Ergocomine

2

1.

Indole Alkaloids

Ergocominine O- 12'-Methylergoeomine Ergocristine Ergocristinine Ergosecaline Ergosecalinine Ergobalansine Ergobalansinine a-Ergocryptine O- 12'-Methyl-a-ergocryptine 13-Ergocryptine 5'-epimer of 13-Ergocryptine 13-Ergocryptam 13,13-Ergoannam Ergobutine Ergobutyrine Rugulovasine A 8-Chlororugulovasine A Rugulovasine B 8-ChlororugulovasineB Fumigaclavine A; 9~-Acetoxy-6,8a-dimethylergoline Roquefortine A; IsofumigaclavineA; 9-Acetoxy-6,8-dimethylergoline Fumigaclavine B; 9-Hydroxy-6,8-dimethylergoline Roquefortine B; IsofumigaclavineB; 9-Hydroxy-6,8-dimethylergoline Fumigaclavine C; 2-Dimethylallyl-9-acetoxy-6,8-dimethylergoline

1. Indole Alkaloids

3

Common/Systematic Name 3-(3-Indolyl)propane- 1,2,3-triol Molecular Formula/Molecular Weight CllH13NO3; M W = 207.08954

cH2oH OH

Io.

General Characteristics Red-violet color reaction with p-dimethylaminocinnamaldehyde. Fungal Source Balansia epichlod. Isolation/Purification Purification was achieved by column chromatography on Porapak Q and preparative TLC on silica gel GF254[TLC developing systems were chloroform-methanol (80:20, v/v) and benzene-dimethylformamide (86.5:13.5)]. Biological Activity Toxic to fertile Leghorn chicken eggs: 23~g/egg = 80%; 68~g/egg = 100% mortality. Spectral Data UV:

~,~ff" 220(log e=4.95), 273(4.00), 280(4.02), and 289nm (3.95). IR:

(KBr) 1550, 1420, 1410, 1335, 1065, 1050, 740, and 780cm"1. Mass Spectrum: 207.08, 189.07, 188.06, 186.05, 172.07, 171.06, 170.05, 160.07, 159.06, 146.05, 145.05, 144.08, 144.04, 142.06, 130.06, 118.06, 117.05, 116.05, 103.05, 91.05, 90.04, and 89.03m/e. Reference J. K. Porter, C. W. Bacon, J. D. Robbins, D. S. Himmelsbach, and H. C. Higman; Indole Alkaloids from Balansia epichlo~ (Weese); J. Agric. Chem., Vol. 25, pp. 88-93 (1977).

4

1.

Indole Alkaloids

Common/Systematic Name 3-(3,3-Diindolyl)propane-l,2-diol Molecular Formula/Molecular Weight C19H18N202; ~

= 306.13683

CH20H I CHOH

~!LNH~ ~NH/L~ General Characteristics Red-violet color reaction with p-dimethylaminocinnamaldehyde. Fungal Source Balansia epichlo~. Isolation/Purification Purification was achieved by column chromatography on Porapak Q and preparative TLC on silica gel GF254 [TLC developing systems were chloroform-methanol (80:20, v/v) and benzene-dimethylformamide (86.5 13.5, v/v)]. Biological Activity Toxic to fertile Leghorn chicken eggs: 20l.tg/egg = 20%; 60lag/egg = 55% mortality; 99ktg/egg = 100% mortality. Spectral Data UV:

~

MeOH max

221(1og e=4.88), 275(3.97), 282(4.01), and 291nm (3.96).

IR:

(KBr) 1550, 1410, 1335, 1080, 1050, and 780cm1. Mass Spectrum: 306.1368(M+), 272.1326, 270.1145, 258.1132, 257.1049, 256.0993, 245.1069, 218.0958, 217.0887, 188.0671, 171.0675, 170.06, 160.07, 159.06, 144.04, 142.06, 130.06, 118.06, 117.05, 116.05, 103.05, 91.05, 90.04, and 89.03role. Reference J. K. Porter, C. W. Bacon, J. D. Robbins, D. S. Himmelsbach, and H. C. Higman; Indole Alkaloids from Balansia epichlo~ (Weese); J. Agric. Chem., Vol. 25, pp. 88-93(1977).

1. Indole Alkaloids

5

Common/Systematic Name 4-(3-Indolyl)butane- 1,2,3-triol Molecular Formula/Molecular Weight C12H15NO3, M W = 221.10519

OH

{~}LNHI ~ H CH20 H General Characteristics Red-violet color reaction with p-dimethylaminocinnamaldehyde. Fungal Source Balansia epichlos Isolation/Purification Purification was achieved by column chromatography on Porapak Q and preparative TLC on silica gel GF254[TLC developing systems were chloroform-methanol (80:20, v/v) and benzene-dimethylformamide (86.5:13.5, v/v)]. Biological Activity Toxic to fertile Leghorn chicken eggs: 57~g/egg = 53% mortality; 113l.tg/egg = 100% mortality. Spectral Data UV:

~

MeOH max

221(log e=4.65), 272(3.78), 279(3.8), and 288nm (3.73).

IR:

(KBr) 1550, 1410, 1340 1080, 1030, and 780cmq. Mass Spectrum: 221.10(M+), 203.09, 201.08, 189.07, 188.07, 186.05, 172.07, 171.06, 170.06, 160.07, 159.06, 146.05, 145.05, 144.08, 144.04, 142.06, 130.06, 118.06, 117.05, 116.05, 103.05, 91.04, 91.05, 90.04, and 89.08role. Reference J. K. Porter, C. W. Bacon, J. D. Robbins, D. S. Himmelsbach, and H. C. Higrnan; Indole Alkaloids from Balansia epichlo3 (Weese), J. Agile. Chem., Vol. 25, pp. 88-93(1977).

6

1.

Indole Alkaloids

Common/Systematic Name N-Methyl-4-dimethylallyltryptophan Molecular Formula/Molecular Weight C]THz2NzO2; MW' = 286.16813

Me Me

/

CO2H HMe

NH General Characteristics N-Methyl-4-dimethylallyltryptophan crystallized from methanol as needles; mp., 232~ Fungal Sourc.e

Clavicepsfusiformis.

Isolation/Purification

Clavicepsfusiformis was grown aerobically in submerged cultures in both shaken flasks and stirred fermenters. When alkaloid production began, anaerobic conditions were imposed and the cultures stood for three days. Clavine alkaloids were extracted with chloroform at alkaline pH and then the amphoteric metabolites extracted with n-butanol at neutral pH. The butanol extract, which contained considerable quantities of chanoclavines and other oxygenated clavine alkaloids, was chromatographed on silica gel with chloroform/methanol/ammonia as the eluant.

Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV: ~, m=M~" 274, 280, and 295nm.

1. Indole Alkaloids

7

IR~

(KBr) 3580, 3250(broad) 1640, 1400, and 770cm1. 1H NMR: (CDaCOOD) inter alia 8.64(s, 6H), 7.64(s, 3H), 5.06(t, 1H, J=-7.0Hz), and 6.3-7.0ppm (complex, 4H).

Mass Spectrum: 286, 198, 156, 155, and 154m/e. The fragmentation under electron-impact was very similar to bis-seco-dehydrocyclopiazonic acid with allylic cleavage of the amino acid side chain giving the ion ofm/e 198, followed by cyclization to a series oftricyclic ions m/e 156, 155, and 154 with elimination of a C-3 unit. Cyclization of this type is only possible if the two side chains are located in the peri-position of the indole nucleus, Le. at positions 3 and 4. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or l:l, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4: l, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References K. D. Barrow and F. R. Quigley, Ergot Alkaloids HI : The Isolation of N-Methyl.4dimethylallyltryptophan from Clavicepsfusiformis; Tetrahedron Letters, pp. 4269-4270 (1975). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978).

J. K. Porter, Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

8

1.

Indole Alkaloids

Common/Systematic Name Lysergic acid Molecular Formula/Molecular Weight CI6H16N202; ~

= 268.12118

H,, COOH '~N--Me

NH General Characteristics Hexagonal scales, plates from water (associated with one or two moles water); mp., 240~ (dec.); [tt]D2~ +40 ~ (C=0.5, in pyridine); pKa=3.44/pI~,=7.68. Moderately soluble in pyridine; sparingly soluble in water and neutral organic solvents; soluble in NaOR NH4OH, Na2CO3, and HCI solutions; and slightly soluble in dilute H2SO4. Methyl ester derivative, thin leaflets from benzene; mp., 168 ~C. Fungal Source Sclerotia and saprophytic culture of Claviceps purpurea. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data IR:

See A. Hofmann, 1964. 13C NMR:

(CDCI3) (methyl lysergate) C-2, 118.2; C-3, 110.2; C-4, 26.9; C-5, 62.6; C-7, 54.6; C8, 41.8; C-9, 117.6; C-10, 136.0; C-11,127.6; C-12, 112.0; C-13, 122.9; C-14, 109.4; C-15, 133.7; C-16, 125.9; C-17, 172.4; Me, 51.9; and NMe, 43.4ppm.

1. Indole Alkaloids

9

Mass Spectrum: LREIMS: 268(M+, 100%), 224, 221,207, 192, 180, 167,and 154role. References N. J. Bach, H. E. Boaz, E. C. Kornfeld, C-J Chang, H. G. Floss, E. W. Hagaman, and E. Wenkert; Nuclear Magnetic Resonance Spectral Analysis of the Ergot Alkaloids; J. Org. Chem., Vol. 39, pp. 1272-1276(1974). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkom Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964.

10

1.

Indole Alkaloids

Common/Systematic Name Ergine; Lysergic acid amide Molecular Formula/Molecular Weight CI6HI7N30; M ~ = 267.13716

0 II

H2N--C. ,H N--Me

\

H

General Characteristics Crystallized from acetone as massive colorless prisms; m.p. 196~ [a]D 20 q" 414 ~ [a]54612~+ 520 ~ (c--1.0, in CHCI3); pK = 6.2 (in 80% methylcellosolve); blue color with Keller's reagent. Fungal Source Ergot of Clavicepspurpurea and Paspalum distichum L. (also isolated from seeds of Rivea corymbosa (L.) and Ipomoea tricolor; Convolvulaceae). Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some ergopeptine alkaloids are used routinely in medical practice. Central American Indians used seeds ofRivea corymbosa and Ipomoea tricolor as a magic drug called "Ololiuqui". Spectral Data IR:

See A. Hofmann, 1964. UV:

UV spectrum identical to that of lysergic acid or isolysergic acid.

1. Indole Alkaloids

11

Mass Spectrum: LREIMS: 267(M+, 100%), 249, 224, 221,207, 192, 180, 167, and 154role. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70% alkaline methanol. This technique works well for ergopeptide alkaloids. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964.

12

1.

Indole Alkaloids

Common/Systematic Name 8-Hydroxyergine Molecular Formula/Molecular Weight C16HITN302; MW = 283.13208

0II C~NH2 HO""'~~8 7~N_Me 13 14

I 3 d

2

Fungal Source

Clcn,iceps paspali (strain MG-6).

Isolation/Purification The strain C. paspali MG-6 was isolated from the grass Paspalum dilatatum in the vicinity of Rome. Alkaloids were separated by adsorption on bentonite (Flieger et al., 1989b). A crude alkaloid mixture was chromatographed on Kieselgel 60 F254, Merck preparative TLC plates and eluted with chloroform-isopropyl alcohol-ammonia (90:10:0.036, v/v/v); Rf values of 8-hydroxyergine and 8-hydroxyerginine were 0.50 and 0.91, respectively. Prepurified alkaloids were chromatographed on a Separon SGX C~s column (Tessek, Czechoslovakia) particle size 71.tm. The mobile phase consisted of (A) MeOH-H20-NH3 (90:10:0.036, v/v/v) and (B) MeOH-H20-NH3 (20:80:0.036, v/v/v). The column was equilibrated with 4% A in B and subsequently eluted with a linear gradient up to 54% A in B. Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic comlSonents). The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medicine.

1.

Indole Alkaloids

13

Spectral Data 1H NMR: (CDaOD) H=2, 6.984(J2,4a=l.8Hz); H-4a, 2.749; H-4b, 3.569(J2,4b=>0,J~,4b-= 14.5Hz); H-5, 3.265(J4~,5=11.8Hz, J4b,5=5.9Hz); H-7a, 2.936(Js~m,=-I1.7Hz, Js~,9=l.0I-Iz); H=7b, 2.965; H-9, 6.358(J4b,9=>0, J5,9=2.1Hz); H=12, 7.193(Jla, la--7.4I-Iz, .J12,14=0.7I-Iz);H-13, 7.107(J~a,14=7.9Hz); H14, 7.231; and N-Me, 2.590ppm. 13C NMR: (CDaOD) C-2, 120.66; C-3, 110.52; C-4, 27.15; C-5, 64.20; C-6, 62.7 1; C-7, 73.84; C-8, 121.00; C-9, 139.91; C-10, 128.13; C-11,113.43; C-12, 123.93; C-13, 111.95; C-14, 136.02; C-15, 128.13; C-16, 177.92; and N-Me, 43.75ppm.

Mass Spectrum: EIMS: 283(M+, C16H17N302,61%), 266(C16H16N30, 27), 265(C16HIsN30, 37), 248(C16H12N20, 59), 240(CIA-II2N202, 86), 223(Cl,d-IiEN202, 86), 221(C~5HlaN2, 42), 206(C14HloN2, 19), 195(CIaH9NO, 36), 194(CIaHsNO, 32), 181(CI3HIIN, 26), 180(ClaHloN, 26), 167(C12HaN, 83), and 154(C~HsN, 100). TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References M. Flieger, R. Linhartova, P. Sedmera, J. Zima, P. Sajdl, J. Stuchlik, and L. Cvak; New Alkaloids ofClavicepspaspali; J. Nat. Prod., Vol. 52, pp. 1003-1007 (1989a). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

14

1.

Indole Alkaloids

Common/Systematic Name Erginine; Isolysergic acid amide Molecular Formula/Molecular Weight CIrHITN30; MW = 267.13716

O II H2N--C,,

H. N--Me

NH General Characteristics Crystallized from methanol as solvated prisms; mp., 132-134~ 608 ~ (C=0.5, in pyridine); pK=6.1 (in 80% methylcellosolve).

[tt]D2~+ 480 ~ [tt]54612~+

Fungal Source Ergot and saprophytic culture of Clavicepspurpurea. Epimers are not considered as naturally occurring, but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base. Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some ergopeptine alkaloids are used routinely in medical practice. Central American Indians used seeds of Rivea corymbosa and Ipomoea tricolor as a magic drug called "Ololiuqui". Spectral Data _

IR:

See A. Hofmann, 1964.

1.

Indole Alkaloids

15

TLC Purification Silica gel plates developed with methylene chloride-isopropyi alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70% alkaline methanol. This technique works well for ergopeptide alkaloids. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

16

1.

Indole Alkaloids

Common/Systematic Name 8-Hydroxyerginine Molecular Formula/Molecular Weight C]6H]7N302; MW = 283.13208 O II

C_--NH2 HOy8 "' 7~N--Me

Fungal Source Claviceps paspali MG-6. Epimers are not considered as naturally occurring but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base. Isolation/Purification The strain C. paspali MG-6 was isolated from the grass Paspalum dilatatum in the vicinity of Rome. Alkaloids were separated by adsorption on bentonite (Flieger et al., 1989b). A crude alkaloid mixture was chromatographed on Kieselgel 60 F254,Merck preparative TLC plates, and eluted with chloroform-isopropyl alcohol-ammonia (90:10:0.036, v/v/v); Rf values of 8-hydroxyergine and 8-hydroxyerginine were 0.50 and 0.91, respectively. Prepurified alkaloids were chromatographed on a Separon SGX C18 column (Tessek, Czechoslovakia) of particle size 71~. The mobile phase consisted of (A) MeOH-H20-NH3 (90:10:0.036, v/v/v)and (B) MeOH-H20-NH3 (20:80:0.036, v/v/v). The column was equilibrated with 4% A in B and subsequently eluted with a linear gradient up to 54% A in B. Spectral Data ]H NMR: (CDsOD) H-2, 6.974(J2,4a=l.6Hz); H-4a, 2.649; H-4b, 3.606(J2,4b=>0, dna,4b=-14.6Hz); H-5, 3.150(J4a,5=l 1.5Hz, Jnb,5=5.9Hz); H-7a, 3.080(J7~,Tb=-I1.3Hz, J7a,9=l.5Hz); H-Tb, 2.626; H-9, 6.268(Jab,9=0.8Hz, Js,9=2.2Hz); H- 12, 7.118(J]2,13=7.0Hz, J]2,14=l.THz); H- 13, 7.090(Jx3,14=7.2Hz); H- 14, 7.219ppm; and N-Me, 2.614ppm.

1.

Indole Alkaloids

17

13CNMR: (CD3OD) C-2, 120.53; C-3, 110.55; C-4, 28.27; C-5, 64.11; C-6, 63.06; C-7, 71.87; C8, 124.30; C-9, 139.13; C-10, 128.04; C-11, 113.15; C-12, 123.96; C-13, 111.72; C14, 135.95; C-15, 127.99; C-16, 179.59; and N-Me, 43.39ppm. Mass Spectrum: EIMS: 283(M§ C16H17N302, 100), 266(C16H16N30, 14), 265(C16H15N30, 29), 248(C16H12N20, 35), 240(C14H12N202, 93), 223(C14H12N202, 31), 221(C15I-I13N2,42), 206(C14H1oN2, 12), 195(C13HgN,60), 194(CI3HsN, 61), 181(C13HllN, 50), 180(C13HloN, 20), 167(C12H9N,94), and 154(C1lI-lsN, 96%). Reference M. Flieger, R. Linhartova, P. Sedmera, J. Zima, P. Sajdl, J. Stuchlfk, and L. Cvak; New Alkaloids of Clawcepspaspali; J. Nat. Prod.; Vol. 52, pp. 1003-1007(1989a).

18

1.

Indole Alkaloids

Common/Systematic Name Lysergol Molecular Formula/Molecular Weight C16HIsN20, MW' = 254.14191

HOH2C. ,H

.~'~N--Me

General Characteristics Colorless prisms from ethanol; mp., 245~ (uncorr. decomp.); as plates and prisms; mp., 253-255~ (dec.); [a]D 18 +49 ~ (C=0.2, in pyridine); [a]D2~ +54 ~ [a]5~sl2~ +87 ~ (c=0.3, in pyridine). Gave a light purple and purplish blue colors with van Urk's and Allport-Cocking's reagents, respectively. Sublimes at high vacuum at 1800C; pK=6.6 (in 80% aqueous methylcellosolve). Soluble in 350 parts of boiling methanol or 100 parts boiling ethanol, sparingly soluble in chloroform or water. Fungal Source Saprophytic cultures of Elymus-type ergot fungus. Isolation/Purification Purified either by countercurrent distribution or by column chromatography using Hyflo Super Cell treated with a buffer solution (Mcllvaine). Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV: ~, M.o. 225, 242, and 312nm. max

1. Indole Alkaloids

19

IR; See A. Hofmann, 1964. Mass Spectrum: LREIMS: 254(M+, 100%), 235, 223,221,207, 205, 193, 192, 180, 167, and 154role. References M. Abe, S. Yamatodani, T. Yamano, and M. Kusumoto; Isolation of Lysergol, Lysergene and Lysergine from the Saprophytic Cultures of Ergot Fungi; Agile. Biol. Chem. [Tokyo], Vol. 25, pp. 594-595(1961). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964.

20

1. Indole Alkaloids

Common/Systematic Name Lysergene Molecular Formula/Molecular Weight Cl6Hl6N2, M W = 236.13135

CH2 N--Me

NH General Characteristics Colorless needles or prisms from ethyl acetate; mp., 244~ (uncorr. decomp.); colorless needles or prisms from methanol, 247-249~ (dec.); [a]o TM +461 (c=0.2, in pyridine); [a]Dz~ +504 ~ (C=0.4, in pyridine). Sparingly soluble in most organic solvents, moderately soluble in chloroform or pyridine. Gave a yellowish-green color with both van Urk's and Allport-Cocking's reagents, respectively. Fungal Source Saprophytic cultures of Elymus-type ergot fungus. Isolation/Purification Purified either by countercurrent distribution or by column chromatography using Hyflo Super Cell treated with a buffer solution (Mcllvaine). Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UW:

m~H 243, 263, and 335nm. IR:


1.

Indole Alkaloids

21

References M. Abe, S. Yamatodani, T. Yamano, and M. Kusumoto; Isolation ofLysergol, Lysergene and Lysergine from the Saprophytic Cultures of Ergot Fungi; Agile. Biol. Chem. Vol., pp. 594-595(1961). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964.

22

1. Indole Alkaloids

Common/Systematic Name Lysergine Molecular Formula/Molecular Weight CI6HlsN2; M W = 238.14700

Me

,H

N--Me

'

Nit

General Characteristics Colorless prisms from ethyl acetate; mp., 275~ (uncorr. decomp.); prisms from methanol, ethanol, or ethyl acetate, 286-289~ (dec.); [a]DTM = +70 ~ (c=0.2, in pyridine), [a]D2~ +65 ~ (C=0.5, in pyridine); sparingly soluble in methanol, ethanol, and ethyl acetate. Fungal Source Ergot or saprophytic cultures ofAgropyrum sp. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV~

Very similar to lysergic acid. IR~

See A. Hofmann, 1964. References Abe, S. Yamatodani, T. Yamano, and M. Kusumoto; Isolation ofLysergol, Lysergene and Lysergine from the Saprophytic Cultures of Ergot Fungi; Agric. Biol. Chem. [Tokyo], Vol. 25, pp. 594-595(1961).

1.

Indole Alkaloids

23

B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook 0fExperimental Pharmacology; Springer-Verlag, New York (1978) A. Hofmann; Die Mutterkom Alkaloide, Enke Verlag, Stuttgart, 218 pp., 1964.

24

1.

Indole Alkaloids

Common/Systematic Name Ergonovine; Ergometrine; Ergobasine Molecular Formul~olecu!ar Weight C19H23N302; M W = 325.17903

0 II C

CH20H I

NH~C ~H

...)___.

Hi,

e

N--Me

NH General Characteristics Tetrahedra from ethyl acetate; fine needles from benzene; tendency to form solvated crystals; mp., 162~ (nonsolvated, mp., 212~ dec.); [a]D2~+ 90~ in water); -16 ~ (c=l.0, in pyridine); [aid 2~ + 41~ [a]546~2~ + 60 ~ (c=l.0, in alcohol); pK = 6.8. Freely soluble in lower alcohols, ethyl acetate, and acetone; more soluble in water than other principal alkaloids of ergot; slightly soluble in chloroform. Fungal Source

Claviceps purpurea, Balansia epichlo~, B. henningsiana, and B. claviceps.

Isolation/Purification/Analysis Extract with either aqueous tartaric or lactic acid solution, partition chromatography with chloroform or methylene chloride at appropriate pH, column clean-up procedures using either silica, alumina or ion exchange resin, and identification and analysis using a combination of co-chromatography using TLC and/or HPLC with UV or fluorescence detection. Mass spectrometry is quite useful for identification, analysis and quantitation. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central neivous system effects (bulbomedullary and mesodiencephalic components). MLD (IV) in rabbits was 7.5mg/kg.

1.

Indole Alkaloids

25

Spectral Data IR~

See A. Hofmann, 1964. UV~

Identical to that of lysergic acid or isolysergic acid. ~3CNMR: (DMSO-d6) C-2, 119.1; C-3, 108.9; C-4, 26.8; C-5, 62.6; C-7, 55.5; C-8, 42.8; C-9, 120.1; C-10, 135.0; C-11, 127.4; C-12, 111.0; C-13, 122.4; C-14, 109.0; C-15, 133.7; C-16, 125.8; C-17, 171.2; Me, 17.4; NCH, 46.4; OCH2, 64.4; and N-Me, 43.4ppm. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1) followed by rechromatography in chloroform-methanol (9:1 or 4:1) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography: a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70% alkaline methanol. This technique works well for ergopeptide alkaloids. References N. J. Bach, H. E. Boaz, E. C. Kornfeld, C-J Chang, H. G. Floss, E. W. Hagaman, and E. Wenkert; Nuclear Magnetic Resonance Spectral Analysis of the Ergot Alkaloids; J. Org. chem., Vol. 39, pp. 1272-1276(1974). C. W. Bacon, J. K. Porter, and J. D. Robbins; Lysergic Acid Amide Derivatives from

Balansia epichlo3 and Balansia claviceps (Clavicipitaceae); J. Nat. Prod., 42:309-314 (1979). C. W. Bacon, J. K. Porter, and J. D. Robbins; Laboratory Production of Ergot Alkaloids by Species ofBalansia; J. Gen. Microbiol., 113: 119-126(1979).

26

1.

Indole Alkaloids

C. W. Bacon, J. K. Porter, and J. D. Robbins; Ergot Alkaloids Biosynthesis by Isolates of

Balansia epichlo~ and Balansia henningsiana; Can. J. Bot., 59:2534-2538(1981).

C. W. Bacon, J. K. Porter, J. D. Robbins, and D. Betowski; Ergot Alkaloids Identification in Clavicipitaceae Systemic Fungi of Pasture Grass; J. Agric. Food Chem., 29:653-657 (1981). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. W. A. Jacobs and L. C. Craig; On an Alkaloid from Ergot; Science, Vol. 82, pp. 16-17 (1935). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

1. Indole Alkaloids

27

Common/Systematic Name Ergonovinine; Ergometrinine; Ergobasinine Molecular Formula/Molecular Weight C19H23N302, ~

O II C

= 325.17903

CH2OH I NH~C ~H

H

e N--Me

"

NH

General Characteristics Forms large colorless prisms from acetone; mp., 196~ (dec.); [a]D2~+ 414 ~ [a]5~l 2~ + 520 ~ (c=l.0, in CHCI3); pK=6.2 (in 80% methylcellosolve); blue color with Keller's reagent. Fungal Source

Claviceps purpurea.

Epimers are not considered as naturally occurring, but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base. Isolation/Purification/Analysis Extract with either aqueous tartaric or lactic acid solution, partition chromatography with chloroform or methylene chloride at appropriate pH, column clean-up procedures using either silica, alumina or ion exchange resin, and identification and analysis using a combination of co-chromatography using TLC and/or HPLC with UV or fluorescence detection. Mass spectrometry is quite useful for identification, analysis and quantitation. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism and adrenergic blockage) and central nervous system effects (bulbomeduUary and mesodiencephalic components). MLD (IV) in rabbits was 7.5mg/kg.

28

1. Indole Alkaloids

Spectral Data IR-

See A. Hofmann, 1964. UV:

Identical to that of lysergic acid or isolysergic acid. 13C N M R :

(DMSO-d6) C-2, 119.0; C-3, 108.9; C-4, 26.9; C-5, 62.0; C-7, 54.0; C-8, ca. 42.2; C9, 119.0; C-10, 136.1; C-11,127.6; C-12, 111.0; C-13, 122.1; C-14, 109.8; C-15, 133.7; C-16, 125.7; C-17, 172.1; Me, 17.2; NCH, 46.2; OCH2, 64.3; and N-Me, 43.6ppm. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NHs (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70% alkaline methanol. This technique works well for ergopeptide alkaloids. References N. J. Bach, ,. H. E. Boaz, E. C. Kornfeld, C-J Chang, H. G. Floss, E. W. Hagaman, and E. Wenkert; Nuclear Magnetic Resonance Spectral Analysis of the Ergot Alkaloids; J. Org. chem., Vol. 39, pp. 1272-1276(1974). C. W. Bacon, J. K. Porter, and J. D. Robbins; Lysergic Acid Amide Derivatives from Balansia epichlo3and Balansia claviceps (Clavicipitaceae); J. Nat. Prod., 42:309-314 (1979). C. W. Bacon, J. K. Porter, and J. D. Robbins; Laboratory Production of Ergot Alkaloids

1. Indole Alkaloids

29

by Species ofBalansia; J. Gen. Microbiol., 113:119-126(1979). C. W. Bacon, J. K. Porter, and J. D. Robbins; Ergot Alkaloids Biosynthesis by Isolates of

Balansia epichlo~and Balansia henningsiana; Can. J. Bot., 59: 2534-2538(1981).

C. W. Bacon, J. K. Porter, J. D. Robbins, and D. Betowski; Ergot Alkaloids Identification in Clavicipitaceae Systemic Fungi of Pasture Grass; J. Agile. Food Chem., 29:653-657 (1981). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacolo~_; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. W. A. Jacobs and L. C. Craig; On an Alkaloid from Ergot; Science, Vol. 82, pp. 16-17 (1935). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

30

1. Indole Alkaloids

Common/Systematic Name Agroclavine Molecular Formula/Molecular Weight C16HIgN2; M~W = 238.14700 Me N--Me ll~

NH

General Characteristics Colorless needles from acetone; mp., 205-206~ sublimed under high vacuum between 110-130~ [tt]D2~-155 ~ (C=0.9 in CHCIs); [a]D2~ -182 ~ (C=0.5 in pyridine); pK=6.8 (in 80% aqueous methylcellosolve). Violet/blue color with Keller's reagent. Fungal Source First found from sclerotia and cultures ofAgropyrum semicostatum Nees and A. ciliare Fr. Also found in Pennisetum typhoideum. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV~ )~0o.

225, 284 and 293nm (log e=4.47, 3.88 and 3.81, respectively).

IR~ See A. Hofmann, 1964. IH NMR: (pyridine-ds) H-6, 2.38(3H, s); H-7, 2.73(1H, d); H-4, 2.90(1H, ddd); H-7', 3.18(1H, d); H-4', 2.51(1H, ddd); H-5, 3.36(1H, dd); H-10, 3.89(1H, m); 7-CHs, 1.68(3H, s);

1.

Indole Alkaloids

31

H-9, 6.30(1H, m); aromatic-H, 7.1-7.4(4H, m); and H-l, 11.43ppm (1H, s). (Note: Possible incorrect assignment of the 44, 413, and 5 hydrogens). (CDCI3) H-4a, 2.78(dd, ,/=15, 12Hz); H-413, 3.3 l(dd, J=15, 4Hz); H-5, 2.52(ddd, J=12, 9.5 and 4Hz); H-7a, 3.24(d, J=17Hz); H-713, 2.93(dd, broad signal, J=17, 4Hz); H-9a and 13, 6.18(s, broad signal); H- 10, 3.74(dd, broad signal, J=9.5, 4Hz); H- 17, 1.77(s); and N-Me, 2.49ppm (s). ~3CNMR: (pyridine-ds) C-2, 118.3; C-3, 111.2; C-4, 26.4; C-5, 63.6; C-7, 60.2; C-8, 131.9; C-9, 119.4; C-10, 40.8; C-11,131.9; C-12, 112.0; C-13, 122.0; C-14, 108.4; C-15, 134.0; C-16, 126.6; C-17, 19.9; and N-Me, 40.2ppm. Mass Spectrum: LREIMS: 238(M+, 52%), 237(100), 167(17), and 154role (16). References M. Abe, T. Yamano, Y. Kozu, and M. Kusumoto; Isolation of Further Two Water-soluble Ergot Alkaloids; J. Age. Chem. Soc., Vol. 28, pp. 501-510(1954). N. J. Bach, H. E. Boaz, E. C. Cornfield, C-J Chang, H. G. Floss, E. W. Hegemon, and E. Wenkert; Nuclear Magnetic Resonance Spectral Analysis of the Ergot Alkaloids; J. Org. Chem., Vol. 39, pp. 1272-1276(1974). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology, Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. R. G. Mrtek, H. L. Crespi, G. Norman, M. I. Blake, and J. J. Katz; Biosynthesis of Clavine Alkaloids: Proton Magnetic Resonance Studies; Phytochemistry, Vol. 7, pp. 1535-1541(1968). A. Stoll, A. Brack, H. Kobel, A. Hofmann, and R. Brunner; Die Alkaloide eines Mutterkornpilzes von Pennisetum typhoideum rich. und deren Bildung in Saprophytischer Kultur; Helvetica Chimica Acta, Vol. 37, pp. 1815-1825(1954).

32

1. Indole Alkaloids

Common/SystematicName

6,7-seco-Agroelavine

Molevular F0rmula/Moleeular Weight C16H2oN2;MW = 240.16265

......

HMe

General Characteristics Crystals; mp., 126-129~ subl., gives a blue color with Allport and Cocking's reagent. Fungal Source v

Claviceps purpurea, strain AA-218, Balansia epichlo~ B. strangukms, and Epichlo~ trphina.

Isolation/Purification Purified by HPLC followed by PLC using 1% conc. ammonia, 5% Meg)H, and 94% chloroform. TLC using silica gel with same solvent system. Soeetral Data _

UV~

m,x~~a 225, 283, and 293nm.

(CHCI3): 3480(indole NH), 3320(aliphatic NH), 1605, and 1445cm"~(C=C). ~H NMR: (CDCI3): 1.85(s, 6H); 2.53(s, with hyperfme splitting, 3H); 2.4(br s, 1H); 2.6-3.5(m, 4H); 3.75-4.15(m, 1H); 5.0-5.3(d, 1H (C-10-H)); 6.6-7.3(m, 4H); and 8.1-8.5ppm (br s, 1H(indole)). Mass Spectrum: EIMS: 240, 225, 208, 197, 184, 168, and 155m/e. References C. HorweH and J. P. Verge; Isolation and Identification of 6,7-seco-Agroclavine l~om Claviceps trurpurea, Phytoehemistry, Vol. 18, p. 519 (1979).

1. Indole Alkaloids

J. K. Porter, C. W. Bacon, J. D. Robbins, and D. Betowski; Ergot Alkaloid Identification in Clavicipitaceae Systemic Fungi of Pasture Grasses; J. Agric. Food Chem., 29:653-657 (1981).

33

34

1.

Indole Alkaloids

Common/Systematic Name Dihydroagroclavine Molecular Formula/Molecular Weight C16H20N2; ~

= 240.16265

Me

J

"

~N--Me

NH

General Characteristics Crystals as long needles from toluene, benzene, ether, chloroform, ethyl acetate, acetone, methanol, ethanol or pyridine; mp., 242~ (dec.); [a]D2~ - 69 ~ [tt]5~ 2~ - 83 o (C=0.5, in CHCI3); [et]D~ - 111 ~ [~]54612~ - 129 ~ (c=0.5, in pyridine). Insoluble in toluene, benzene and ether; readily soluble in chloroform, ethyl acetate, acetone, methanol, ethanol and pyridine. Succinate derivative, C16H20N2. 0.5 C4H604, crystals as prisms from water; mp., 213~ (dec.); [t~]D~7 - 87 ~ (C=0.13, in pyridine). Fungal Source Ergot and saprophytic culture of Phalaris and Agropyrum sp. Spectral Data Mass Data: Found: C 79.94, H 8.34, N 11.64 (calcd. for CI6H20N2:C 79.95, H 8.39, N 11.66). Reference M:Abe and S. Yamatodani; Isolation of Further Two Water Soluble Ergot Alkaloids; J. Agr. Chem. Soc., Vol. 28, p. 501 (1954).

1. Indole Alkaloids

35

Common/Systematic Name Festuclavine Molecular Formula/Molecular Weight C16I-I20N2; MW Me

-~

240.16265

H

N--Me

NH General Characteristics Crystals (long needles) from methanol; mp., 238-2390C (dec.); 242-244~ (dec.); [a]u 15 -98 ~ (c=0.3, pyridine); [a]D 20 -70 ~ [a]546120 -83 ~ (c=0.5, in CHCI3); [a]D z0 -110 ~ [a]546120 -128~ pK=7.4 (in 80% aqueous methylcellosolve); positive for van Urk's reaction; insoluble in petroleum ether, sparingly soluble in ethyl acetate, moderately soluble in benzene and chloroform and readily soluble in acetone, methanol, ethanol, and pyridine. Fungal Source Penicillium chermesinum (PC 106-1), Agropyrum type ergot fungus parasitic on Agropyrum semicostatum, Trisetum bifidum Ohwi, Festuca rubra L., etc. growing in Japan. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV:

~

MeOH max

224(log e=4.54), 275(3.81), and 281nm (3.84).

IR;


36

1.

Indole Alkaloids

1H NMR: (CDCI3) H-4a, 2.68(dd, J=-15.0, 11.5Hz); H-4p, 3.39(dd, J=15.0, 4.5Hz); H-5, 2.10(ddd, J=l 1.5, 9.5, 4.5Hz); H-7tt, 2.95(d[broad], J=-I 1.0Hz); H-713, 1.87(t, J=l 1.0Hz); H-8, 2.01(ddd, J=-12, 11, 6.5Hz); H-9a, 2.63(dd, J=12.0, 3.5Hz); H-9[i, 1.08(q, J=12.0Hz); H-10, 2.97(ddd, J=-12.0, 9.5, 3.5Hz); H-17, 0.99(d, J=6.5Hz); and N-Me, 2.45ppm (s). 13C NMR: (CDC13) C-2, 117.7; C-3, 110.5; C-4, 26.6; C-5, 66.7; C-7, 65.0; C-8, 30.2; C-9, 36.2; C-10, 40.4; C-11, 132.7"; C-12, 112.0; C-13, 122.0; C-14, 108.3; C-15, 133.1; C-16, 125.9; C-17, 19.3; and N-Me, 42.7ppm.

* Assignment may be reversed. Mass Spectrum: LREIMS: 240(M+, 100%), 197, 182, 167, 154, and 144m/e. References N. J. Bach, H. E. Boaz, E. C. Kornfeld, C-J Chang, H. G. Floss, E. W. Haganmn, and E. Wenkert; Nuclear Magnetic Resonanc~ Spectral Analysis of the Ergot Alkaloids; J. Org. Chem., Vol. 39, pp. 1272-1276(1974). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook

of Experimental Pharmacology; Springer-Vedag, New York (1978). A. Hofinann; Die Mutterkom Alkaloide, Enke Verlag, Stuttgart, 218 pp., 1964. S. Ohmomo, T. Sato, T. Utagawa, and M. Abe; Isolation of Festuelavine and Three New Indole Alkaloids, Roquefortine A, B and C from the Cultures ofPenicillium roqueforti; Agr. Biol. Chem., Vol. 39, pp. 1333-1334(1975).

1.

Indole Alkaloids

37

Common/Systematic Name Elymoclavine Molecular Formula/Molecular Weight C16HlgN20; M ' W = 254.14191

CH20H s LH 12 13

General Characteristics Crystallized as prisms from methanol; mp., 245-249~ [a]D 20 -152 ~ (c=0.9, in pyridine), [a]D2~-111 ~ (C=0.1, in EtOH); pK=6.7 (in 80% aqueous methylcellosolve); violet-blue color with Keller's reagent. Fungal Source Saprophytic culture of ergot fungus Claviceps sp. SD 58 (ATCC 26019), Pennisetum typhoideum sclerotia and saprophytic cultures, and Elymus mollis. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV:

~

MeOH max

227, 283, and 293nm (log e=4.31, 3.84, and 3.76, respectively).

See A. Hofmann, 1964. ~H NMR: (pyridine-ds) H-6, 2.41(3H, s); H-7, 3.05(IH, d); H-4, 2.92(IH, ddd); H-7', 3.61(IH,

d); H-4', 2.59(IH, ddd); H-5, 3.35(IH, dd); H-10, 3.94(IH, m); H-17, 4.36(21-I); 17-

38

1.

Indole Alkaloids

OH, 4.75(1H, s); H-9, 6.71(1H, m); aromatic-H, 7.1-7.4(4H, m); and H-l, 11.40ppm(1H, s). (CD3OD) H-2, 6.922; H-4a, 2.798; H-413, 3.357; H-5, 2.622; H7a, 3.026; H-713, 3.444; H-9, 6.464; H-10, 3.798; H-12, 6.967; H-13, 7.121; H-14, 7.191; H-17upfield, 4.106; H-17downfield, 4.141; and N-Me, 2.523ppm. ~3C NMR: (CD3OD) C-2, 119.09; C-3, 111.45; C-4, 27.17; C-5, 64.76; N(6)-Me, 41.15; C-7, 57.20; C-8, 134.37; C-9, 121.55; C-10, 40.98; C-11, 131.74; C-12, 112.83; C-13, 123.12; C-14, 109.60; C-15, 136.12; C-16, 126.88; and C-17, 65.06ppm. Mass Spectrum: LREIMS: 254(M+, 52%), 253(100), 237(23), 167(36), and 154m/e (30). References N. J. Bach, H. E. Boaz, E. C. Komfeld, C-J Chang, H. G. Floss, E. W. Hagaman, and E. Wenkert; Nuclear Magnetic Resonance Spectral Analysis of the Ergot Alkaloids; J. Org. Chem., Vol. 39, pp. 1272-1276(1974). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacolo~; Springer-Verlag, New York (1978). M. Flieger, N. F. Zelenkova, P. Sedmera, V. Kren, J. Novak, V. Rylko, P. Sajdl, and Z. l~eh~i6ek, Ergot Alkaloid Glycosides from Saprophytic Cultures of Claviceps, I. Elymoclavine Fructosides; J. Natural Products, Vol. 52, pp. 506-510(1989). A. Hofmann; Die Mutterkom Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. R. G. Mrtek, H. L. Crespi, G. Norman, M. I. Blake, and J. J. Katz; Biosynthesis of Clavine Alkaloids: Proton Magnetic Resonance Studies; Phytochemistry, Vol. 7, pp. 1535-1541(1968). A. Stoll, A. Brack, H. Kobel, A. Hofmann, and R. Brunner; Die Alkaloide eines Mutterkornpilzes von Pennisetum typhoideum rich. und deren Bildung in Saprophytischer Kultur; Helvetica Chimica Acta, Vol. 37, pp. 1815-1825(1954).

1.

Indole Alkaloids

39

Common/Systematic Name Elymoclavine-O-13-fructofuranoside Molecular Formula/Molecular Weight C22H28N206, MW = 416.19474

17 HOH2C~ O ~ 2,/O--0 H2 5'H~H, HHO H~~ CH2OH 6'

OH

H

8

,..

N--Me

//' NH

Fungal Source Saprophytic culture of ergot fungus Claviceps sp. SD 58 (ATCC 26019) and 88 EP; evidence suggested that this alkaloid formed from elymoclavine and the sucrose in the medium by the action of invertase present in the fungal mycelium. Isolation/Purification Alkaloids were separated from the culture broth (pH adjusted to 7.5 with concentrated NH3) by adsorption on bentonite (Lachema, Brno, Czechoslovakia) and desorbed with MeOH and the crude alkaloid solution was concentrated to a final volume of 10ml under low pressure conditions. The MeOH solution was loaded on a Separon SGX ClS column and eluted with MeOH-H20-concentrated NH3 (30:70:0.34, v/v/v). The column effluent was monitored by UV (288nm). The first alkaloid fraction contained a mixture of all elymoclavine fructosides. The mixture of elymoclavine fructosides was repeatedly loaded on Separon SGX Cl8 column and eluted with the above-mentioned mixture. A base line separation of all fructosides was reached. The Separon SGX C~8 column with the same mobile phase was also used for checking purity. Column effluent was monitored by UV at 224nm. Spectral Data IH NMR: (CD3OD) 6.937, H-2; 2.797, H-4tt; 3.379, H-413; 2.691, H-5; 3.174, H-7a; 3.631, H713; 6.553, H-9; 3.854, H-10; 6.950, H-12; 7.163, H-13; 7.143, H-14; 4.112, H-17u; 4.327, H-17d; 2.588, N-Me; 3.590, H-l'u; 3.717, H-I'd; 4.154, H-3'; 4.008, H-4'; 3.779, H-5'; 3.628, H-6'u; and 3.734ppm, H-6'd.

40

1.

Indole Alkaloids

13CNMR: (CD3OD) C-2, 119.98; C-3, 111.58; C-4, 27.56; C-5, 65.87; N(6)-Me, 41.02; C-7, 58.13; C-8, 134.63; C-9, 123.34; C-10, 41.65; C-11,131.91; C-12, 113.51; C-13, 123.74; C-14, 110.35; C-15, 135.61; C-16, 127.74; C-17, 65.01; C-I', 62.41; C-2', 105.79; C-3', 78.85; C-4', 77.26; C-5', 83.82; and C-6', 64.88ppm. Mass Spectrum: CIMS: (NH3) 417(33%), 416(22), 254(23), 253(30), 237(100), 236(85), 223(9), 207(6), 167(9), 154(6), and 127m/e (1). References M. Flieger, N. F. Zelenkova, P. Sedmera, V. Kren, J. Novak, V. Rylko, P. Sajdl, and Z. l~eh~i~ek; Ergot Alkaloid Glycosides from Saprophytic Cultures of Claviceps, I. Elymoclavine Fructosides; J. Natural Products, Vol. 52, pp. 506-510(1989). H. G. Floss, H. Gunter, U. Mothes, and I. Becker. Z.; Isolierung von Elymocalvin-O-13fruktosid aus Kulturen des Mutterkompilzes; Naturforsch,. Vol. 22b, pp. 399-402(1967).

1. Indole Alkaloids

41

Common/Systematic Name Elymoelavine-O-13-fruetofuranosyl-(2-,l)-O-~-D-ffuetofuranoside Molecular Formula/Molecular Weight C28H35N2Oll, ~ = 578.24756 17

I'"

OH

a"l

H

OH

Fungal Source Saprophytic culture of ergot fungus Claviceps sp. SD 58 (ATCC 26019) and 88 EP. Isolati0n/Purification Alkaloids were separated from the culture broth (pH adjusted to 7.5 with concentrated NH3) by adsorption on bentonite (Lachema, Bmo, Czechoslovakia) and desorbed with MeOH and the crude alkaloid solution concentrated to a final volume of 10ml under low pressure conditions. The MeOH solution was loaded on a Separon SGX Cls column and eluted with MeOH-H20-eoneentrated NH3 (30:70:0.34, v/v/v). The column effluent was monitored by UV (288nm). The first alkaloid fraction contained a mixture of all elymoclavine fruetosides. The mixture of elymoclavine fruetosides was repeatedly loaded on Separon SGX C18 column and eluted with the above-mentioned mixture. A base line separation of all fructosides was reached. The Separon SGX C~8 column with the same mobile phase was also used for checking purity. Column effluent was monitored by UV at 224nm. Spectral Data 1H NMR: (CI)3OD) 6.949, H-2; 2.841, H-4a; 3.404, H-4~; 2.797, H-5; 3.230, H-7a; 3.676, H7~; 6.557,1-I-9; 3.851, H-10; 6.966, H-12; 7.073, H-13; 7.151,1-1-14; 4.131, H-17u; 4.328, H-17d; 2.648, N-Me; 3.577, H-l'u; 3.649, H-l'd; 4.165, H-3'; 4.012, H..4'; N. D., H-5'; 3.627, H-6'u; and N. D., H-6'd.

42

1.

Indole Alkaloids

~3CNMR:

(CD3OD)C-2, 120.07; C-3, 111.30; C-4, 27.38; C-5, 65.82; N(6)-Me, 40.81; C-7,

58.02; C-8, 134.13; C-9, 123.62; C-10, 41.43; C-11,131.58; C-12, 113.58; C-13, 123.76; C-14, 110.44; C-15, 135.60; C-16, 127.67; C-17, 64.81; C-I', 62.54; C-2', 105.52; C-3', 79.21; C-4', 77.06; C-5', 83.80; C-6', 64.77; C-I", 62.85; C-2", 105.00; C-3", 80.01; C-4", 76.50; C-5", 83.67; and C-6", 63.91ppm. Mass Spectrum: CIMS: (NH3) 579(25%), 578(8), 416(25), 254(60), 253(45), 237(100), 236(93), 223(7), 207(3), 167(9), 154(10), and 127m/e (24). Reference M. Flieger, N. F. Zelenkova, P. Sedmera, V. I~en, J. Novfik, V. Rylko, P. Sajdi, and Z. l~ehfi~ek; Ergot Alkaloid Glycosides From Saprophytic Cultures of Claviceps, I. Elymoclavine Fructosides; J. Natural Products, Vol. 52, pp. 506-510(1989).

1. Indole Alkaloids

43

Common/Systematic Name Chanoclavine-I; Chanoclavine Molecular Formula/Molecular Weight CI6H20N20; ~ = 256.15756

CH20H Me

Me

General Characteristics Prisms and polyhedral crystals from acetone/methanol; mp., 220-222~ (dec.), [ a ] D 20 240~ [tt]54612~ 294 ~ (c=l.0, in pyridine); [tt]D2~- 205 ~ (C=0.75, in alcohol); pI~,=5.80; pK=8.2 (in aqueous methylcellosolve); violet-blue color with Keller's or van Urk's reagents. N-acetyl derivative crystallized as massive prisms; mp., 226-227 ~ C (dec.); [tt]D2~ - 180 ~ (C=0.5, in pyridine). Fungal Source Saprophytic culture of ergot fungus isolated from a tropical millet (Pennisetum typhoideum). Ergots of Elymus sp., Phragmites sp., Phalaris sp., Agropyrum sp., Balansia epichlo~ B. strangulans, B. claviceps, B. henningsiana, and Acremonium

coenophialum.

Biological Activity All natural ergot alkaloids possess to a greater or lesser degreel biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV: ,~ McOH max

225, 284, and 293nm (log c=4,44, 3.82, and 3.76, respectively).

IR: 1600-1650cm "~ (characteristic of indole); see A. Hofmann, 1964.

44

1. Indole Alkaloids

Mass Spectrum: 256(M+), 237, 183(100%), 182, 167, 168, 154, and 155m/e. References W. Achlin, T. Fehr, and D. Arigoni; The Stereoehemistry of Chanoelavine-I and Isochanoielavine-I; Chemical Communications, pp. 799-800(1966). C. W. Bacon, J. K. Porter, and J. D. Robbins; Lysergic Acid Amide Derivatives from

Balansia epichlo~and Balansia claviceps (Clavicipitaceae); J. Nat. Prod., 42:309-314 (1979). C. W. Bacon, J. K. Porter, and J. D. Robbins; Laboratory Production of Ergot Alkaloids by Species of Balansia; J. Cren. Mierobiol., 113: 119-126(1979). C. W. Bacon, J. K. Porter, and J. D. Robbins; Ergot Alkaloids Biosynthesis by Isolates of

Balansia epichlo~ and Balansia henningsiana; Can. J. Bot., 59: 2534-2538(1981).

C. W. Bacon, J. K. Porter, J. D. Robbins, and D. Betowski; Ergot Alkaloids Identitication in Clavieipitaeeae Systemic Fungi of Pasture Grass; J. Agile. Food Chem., 29:653-657 (1981). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkom Alkaloide; Enke Verlag, Stuttgart 218 pp., 1964. A. Hofinam~ R. Brunner, H. Kobel, and A. Braek; Neue Alkaloide aus der saprophytischen Kultur des Mutterkornpilzes von Pennisetum typhoideum Rich.; Helvetica Chimica Aeta, Vol. XI, pp. 1358-1373(1957) P. C. Lyons, R. D. Plattner, and C. W. Bacon; Occurance of Peptide and Clavine Ergot Alkaloids in Tall Fescue Grass; Science, 232: 487-489(1986). D. Stauffacher and H. Tscherter; Isomere des Chanoclavins aus Clavicepspurpurea (Ft.) Tul.; Helvetica Chimica Acta, Vol. 47, pp. 2186-2194(1964).

1. Indole Alkaloids

45

Common/Systematic Name Isochanoclavine-I Molecular Formula/Molecular Weight C16H20N20; ~

= 272.15248

Me

~ - - - - C H20H Me

NH General Characteristics Crystals; mp., 190~ [a]D -208 ~ (in pyridine); rods from isopropanol; mp., 1810C,; [a]D2~ -216 ~ (C=0.5, in pyridine); blue color with van Urk's reagent; violet-blue with Keller's reagent. Fungal Source Saprophytic culture of ergot fungus, Claviceps purpurea (Fr.). Isolation/Purification The alkaloid was purified by aluminum oxide column chromatography (Act. III) eluted with chloroform/0.75% MeOH. The crude alkaloid fraction was crystallized from acetone followed by isopropanol to give rods, mp., 181 ~ sublimed under high vacuum at 170~ Spectral Data UV:

/~

MeOH max

222, 281,275(sh), and 291nm (log c=4.5, 3.89, 3.86, and 3.82, respectively).

IR.:

See Stauffacher and Tscherter, 1964. ~H NMR: See Stauffacher and Tscherter, 1964. References W. Achlin, T. Fehr, and D. Arigoni; The Stereochemistry of Chanoclavine-I and Isochanoclavine-I; Chemical Communications, pp. 799-800(1966).

46

1.

Indole Alkaloids

D. Stauffacher and H. Tscherter; Isomere des chanoclavins aus Claviceps purpurea (Fr.) Tul. Helvetica Chimica Acta, Vol. 47, pp. 2186-2194(1964).

1. Indole Alkaloids

47

Common/Systematic Name Chanoclavine-II Molecular Formula/Molecular Weight C16H20N20; MW = 256.15756 CH2OH Me Me

'~

NH

General Characteristics Prisms from acetone; mp., 174 ~ [a]D 20 -332 ~ (c--0.5, in pyridine); violet-blue color with Keller's and blue with van Urk's reagents. HCl.salt crystals from alcohol; mp., 247~ [a]D2~ -271 ~ (C=0.5, in 50% alcohol). N-Acetyl derivative, large crystalline prisms from methanol; mp., 203 ~ [a]D2~-455 ~ (C=0.54, in pyridine). Fungal Souree Saprophytic culture of ergot fungus, Clawcepspurpurea(FR.). Spectral Data UV:

~.Mm~H 222, 281, and 291nm (log c=4,50, 3.89, and 3.82, respectively); shoulders at ~,=x 275(Iog c=3.86), 245, and 289nm. IR:

(Nujol) N-aeetyl derivative: 1610em"l, (N-C)CH3. 1H NMR: N-acetyl derivative: 4.82(1H, dd, J=-10 and 4Hz, H-10); 5.53(1H, octet, J=4, 5, and 11Hz, H-5); and 2.67-3.61ppm (2H, AB part of an ABX system, J~=14Hz, J~c=l 1Hz, Jsx=SHz, H-4). N-aeetyl derivative, 4.28ppm (2H, s, allyl-CH2-O group). Reference D. Stauffacher and H. Tseherter, Isomere des Chanoclavins aus Clavicepspurpurea (Fr.) Tul.; Helvetica Chimica Acta, Vol. 47, pp. 2186-2194(1964).

48

1.

Indole Alkaloids

Common/Systematic Name N-Demethylchanoclavine-II; Norchanoclavine II Molecular Formula/Molecular Weight C15HIsN20; MW = 242.14191

CH2OH Me 2

NH General Characteristics Gray color, turning blue with Ehrlich's reagent. Fungal Source

Claviceps sp. (strain SD 58).

Isolation/Purification Culture filtrates were made alkaline to pH 11 with ammonia and extracted several times with chloroform or chloroform-isopropanol (3:1, v/v). The extracts were combined and evaporated to dryness in a vacuum. The residue was dissolved in 2% aqueous succinic acid, the solution washed 3 times with methylene chloride, made alkaline with ammonia to pH 11 and extracted with methylene chloride. This alkaloid extract was dried over anhydrous sodium sulfate, concentrated in a vacuum and left in the refrigerator overnight. The solution was filtered through a fine sintered glass funnel to remove the crystallized elymoclavine, which was washed with 3ml cold methylene chloride. The filtrate and washings were then passed throughan alumina column(Brockmann activity II-III) suspended in methylene chloride. The column was eluted with methylene chloride containing 2% methanol until no more isochanoclavine-I could be detected in the eluate. These fractions contained agroclavine, elymoclavine and isochanoclavine-I, chanoclavine-II and chanoclavine-I. The elution was continued with methylene chloride containing 10% methanol to give two more fractions. The first contained the chanoclavine-I, some chanoclavine-II and N-demethylchanoclavine-II and the following fraction contained mainly N-demethylchanoclavine-II. These two fractions were evaporated and streaked, respectively, on silica gel G plates. The plates were developed twice in acetone-ethyl acetate-N,N-dimethylfohnamide (5:5:1, v/v/v) system. The band containing N-demethylchanoclavine-II was scraped off and the alkaloid was eluted from the gel. This material was rechromatographed in chloroform-methanol (9:1, v/v) in an ammonia atmosphere to yield a chromatographically homogeneous material.

1.

Indole Alkaloids

49

Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. Spectral Data

UV;

~bMmax~H 223,274, 283, jand 294nm. Mass Spectrum: HREIMS: 242.1430 (calcd for CIsHIsN20, 242.1419). The mass spectrum also showed strong peaks at 154, 156, 167, 169, 182, 194, 209, and 223m/e. The M + ion peak (242re~e) was the base peak (100%). Reference J. M. Cassady, C. I. Abou-chaar, and H. G. Floss; Ergot Alkaloids. Isolation of N-Demethylchanoclavine-II from Claviceps Strain SD 58 and the Role of Demethylchanoclavines in Ergoline Biosynthesis; Lloydia, Vol. 36, pp. 390-396(1973).

50

1.

Indole Alkaloids

Common/Systematic Name Setoclavine Molecular Formula/Molecular Weight C16HlsN20, lk4Vr = 254.14191 Me

OH N--Me

NH General Characteristics Prisms from methanol-acetone; m.p. 229-234~ (dec.); [a]Dz~+ 174~ [a]546120 + 232 ~ (c=l.1, in pyridine); [a]Dz~+ 165 ~ (C=0.3, in alcohol); pK=6.4 (in 80% aqueous methylcellosolve). Fungal Source Saprophytic culture of ergot fungus isolated from a tropical millet (Pennisetum typhoideum); Elymus mollis, Agropyrum semicostatum, Trisetum bifidum, and Festuca

rubra.

Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV~ ~b MeOH max

243 and 313nm (log e=4.38 and 4.04).

IR:

See A. Hofmann, 1964. Mass Spectrum: 254(M+), 236, 235, 234, 219, 211, 196, 181,168, and 154m/e (100%).

1. Indole Alkaloids

51

References B. Berde and H. O. Scheld (eds.), Ergot Alkaloids and Related Compounds, In Handbook of Experimental Pharmacology, Springer-Verlag, New York (1978). A. Hofmann, Die Mutterkom Alkaloide, Enke Verlag, Stuttgart, 218 pp, 1964. A. Hofinann, R. Brunner, H. Kobel, and A. Brack, Neue Alkaloide aus der saprophytischen Kultur des Mutterkompilzes von Pennisetum typhoideum Rich., Helvetica Chimica Acta, Vol. XI, pp. 1358-1373(1957).

52

1. Indole Alkaloids

Common/Systematic Name Isosetoclavine Molecular Formula/Molecular Weight CI6HIsN20, MW = 254.14191 HO

Me

N--Me

NH General Characteristics Large polyhedral crystals from methanol; m.p. 234-237~ [a]u 2~+ 107 ~ [a]s~il 2~ + 147 ~ (c=0.5, in pyridine); [a]D2~+ 129 ~ (C=0.4, in alcohol); pK=5.9 (in 80% aqueous methylcellosolve). Soluble in 70 parts boiling methanol, 60 parts boiling acetone or 160 parts boiling chloroform. Responses to various color reactions were similar to setoclavine. Hydrochloride crystallized as rosettes from methanol diluted with acetone, did not melt at temperatures up to 300~ Fungal Source Saprophytic culture of ergot fungus isolated from a tropical millet (Pennisetum

typhoideum).

Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV~

~

MeOH max

242 and 317nm (log e=4.42 and 4.10).

IR~ See A. Hofmann, 1964.

1. Indole Alkaloids

53

References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmar~logy_; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkom Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. A. Hofmann, R. Brunner, H. Kobel, and A. Brack; Neue Alkaloide aus der saprophytischen Kultur des Mutterkompilzes yon Pennisetum typhoideum Rich.; Helvetica Chimiea Aeta, Vol. XI, pp. 1358-1373(1957).

54

1.

Indole Alkaloids

Common/Systematic Name Costaclavine Molecular Formula/Molecular Weight C16H2oN2, M W = 240.16265

Me H ~:---~/N--Me

NH General Characteristics Crystals (prisms) from ether-acetone, acetone, methanol or ethanol; mp., 182-184 ~ (dec.); [a]D2~+ 44 ~ [a]546~2~+ 59 ~ (c=0.2 in pyridine); positive for van Urk's reaction; insoluble in petroleum ether, sparingly soluble in ethyl acetate, moderately soluble in benzene and chloroform and readily soluble in acetone, methanol, ethanol, and pyridine. Fungal Source Penicillium chermesmum (PC 106-1), Agropyrum type ergot fungus parasitic on Agropyrum semicostatum Nees., Trisetum bifidum Ohwi, and Festuca rubra L., etc. growing in Japan. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Dat.a. UV:

~

MeOH max

225,275, 282, and 292nm.

IR:


1.

Indole Alkaloids

55

References M. Abe, S. Yamatodani, T. Yamono, and M. Kusumoto; Bull. Agr. Chem. Soc. (Japan), Vol. 20, pp. 59-60(1956). S. L. Agurell; Costaclavine from Penicillium chermesinum; Experientia, Vol. 20, pp. 2526(1964). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Vedag, New York (1978). A. Hofmann; Die Mutterkom Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964.

56

1.

Indole Alkaloids

Common/Systematic Name Pyroclavine Molecular Formula/Molecular Weight C16H20N2; M W -- 2 4 0 . 1 6 2 6 5

••/N--Me H

Me

NH

General Characteristics Crystals (needles) from ethyl acetate, methanol or benzene, mp., 204~ (uncorr.); [a]D2~ - 90 ~ [a]54~ 2~- 105 ~ (c=0.2 in pyridine); gave a deep blue color with Allport and Cockings's reagent and coned, sulfuric acid; insoluble in petroleum ether; moderately soluble in ethyl acetate and benzene; readily soluble in chloroform, acetone, methanol, ethanol and pyridine. Color reactions were identical to festuclavine. Fungal Source

Agropyrum-type ergot fungus parasitic on Agropyrum semicostatum, Trisetum bifidum, Festuca rubra L., etc. growing in Japan.

Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV~

~

MeOH max

225, 275, 282, and 292nm.

IR~


1.

Indole Alkaloids

57

References M. Abe, S. Yamatodani, T. Yamono, and M. Kusumoto; Bull. Agr. Chem. Soc. (Japan), Vol. 20, pp 59-60(1956). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964.

58

1.

Indole Alkaloids

Common/Systematic Name Molliclavine Molecular Formula/Molecular Weight C16HIsN202; M W = 270.13683

CH2OH HO

N--Me

General Characteristics Crystallized as prisms from methanol or acetone; mp., 253~ (dec.); [a]D 17 "+"3 0 ~ [~]546117 -k- 42 ~ (c=0.2, in pyridine); green color with Keller's and van Urk's reagent. Soluble in methanol and acetone; sparingly soluble in benzene and chloroform; and very soluble in water. Fungal Source Sclerotia and saprophytic cultures ofElymus mollis. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV: ~, M,o. 226, 287, and 294nm. max

IR:

See A. Hofmann, 1964. References Abe and S. Yamatodani; Isolation of Further Two Water Soluble Ergot Alkaloids; J. Agric. Chem. Soc. Japan, Vol. 28, p. 501 (1954).

1. Indole Alkaloids

59

A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. B. Berde and H. O. Seheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology, Springer-Verlag, New York (1978).

60

1.

Indole Alkaloids

Common/Systematic Name Penniclavine Molecular Formula/Molecular Weight CI6HIsN202;

MW

-

270.13683

HOH2C. ,OH .~-"~N--Me

~"

H

General Characteristics Crystals from methanol or acetone; mp., 222-225~ [a]D2~ +151 ~ [a]s~il2~ +201 o (c=0.5, in pyridine); pK=6.4 (in 80% aqueous methylcellosolve). Fungal Source Sclerotia and cultures of Pennisetum typhoideum. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomeduUary and mesodiencephalic components). Spectral Data UV; MeOH ~ max

240 and 315nm (log e=4.29 and 3.93, respectively).

IR~ See A. Hofmann, 1964. IH NMR: (pyridine-ds) H-6, 2.46(3H, s); H-7, 2.79(1H, d); H-4, 2.73(1H, ddd); H-7', 3.17(1H, dd); H-4', 3.20(1H, ddd); H-5, 3.53('1H, dd); 17-CH2, 4.00(2H, s); 17-OH, 5.26.2(2H); H-9, 6.85(1H, m); aromatic-H, 7.1-7.4(4H, m); and H-l, 11.43ppm (1H, s).

1. Indole Alkaloids

61

Mass Spectrum: LREIMS: 270(M+, 93%), 243(23), 234(72), 219(98), 208(60), 196(57), 192(43), 181(57), 167(32), and 154m/e (100). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook 0fExperimental Pharmacoloffy; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkom Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. R. G. Mrtek, H. L. Crespi, G. Norman, M. I. Blake, and J. J. Katz; Biosynthesis o f Clavine Alkaloids: Proton Magnetic Resonance Studies; Phytochemistry, Vol. 7, pp. 1535-1541(1968). A. Stoll, A. Brack, H. Kobel, A. Hofmann, and R. Brunner; Die Alkaloide eines Mutterkornpilzes von Pennisetum typhoideum rich. und deren Bildung in Saprophytiseher Kultur; Helvetica Chimica Acta, Vol. 37, pp. 1815-1825(1954).

62

1. Indole Alkaloids

Common/Systematic Name Cycloclavine Molecular Formula/Molecular Weight C16HIsN2; MW = 238.14700 Me

~N--Me

9

NH

General Characteristics Colorless needles from methanol; mp 164-165 ~ C; [a]D 22 + 40 ~ (c=0.175, in pyridine); [a]54622 + 58~ in pyridine); gave a reddish purple color, turning to bluish-purple, with Allport-Cocking's reagent; an orange color with Dragendorff reagent. Sparingly soluble in benzene; moderately soluble in acetone, methanol, ethanol, chloroform, and ethyl and butyl acetate; readily soluble in pyridine; almost insoluble in cold water; readily soluble in dilute acids. It yielded no isomer on treatment with acid or alkali. Fungal Source

Aspergillusjaponicus.

Isolation/Purification Mycelia extracted with 0.1N H2504, combined with the culture filtrate, pH adjusted to 10 and the mixture repeatedly extracted with ethyl acetate. The alkaloid residue purified with silica gel column chromatography eluting with chloroform-methanol (955, v/v). Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV: ~ EtOH max

227(1og 6=19.6), 276(3.66), 282(3.84), and 293nm (2.66).

1.

Indole Alkaloids

63

IR~ (KBr) 3400(indole NH) and 3150cm"~. IH NMR: (CDCI3) 0.45 and 1.62ppm (2H, AB system, cyclopropyl-CH2); 1.69ppm (3H, s, CCH3); 2.35ppm (3H, s, N-CH3); 2.4-3.3ppm (5H, m, -CH-N- or-CH-C=C-); 6.77.3ppm (4H, m, indole protons); and 8.2ppm (1H, s, indole NH).

Mass Data: 238(M+), 237(base peak), 167, and 154m/e; anal. found; C, 80.24; H, 7.72; N, 11.77; calcd, for Cl6HlsN2: C, 80.63; H, 7.61; N, 11.75%. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994). S. Ohmomo, T. Sato, T. Utagawa, and M. Abe; Isolation of Festuclavine and Three New Indole Alkaloids, Roquefortine A, B and C from the Cultures of Pellicillium roqueforti; Agr. Biol. Chem., Vol. 39, pp. 1333-1334(1975).

64

1.

Indole Alkaloids

Common/Systematic Name Ergotamine Molecular Formula/Molecular Weight C33H35NsOs; MW = 581.26382

o

Me

II

N H .......... "~' " ~ ' O " ~ N :/

C

" I.... .

OHI

,\

o _- -1

'

/ --

NI . i I ==O

General Characteristics Elongated prisms from benzene; mp., 212-214 ~ (dee.); [a]D2~ 160", [a]5~sl2~ -192" (c--0.5, in CHCI3); [a]Dz~ -12.7 ~ [a]5~ 2~ -8.6 ~ (C----0.5,in pyridine). Becomes solventfree only after prolonged heating at high vacuum; darkens and decomposes on exposure to air, heat, and light. Very hygroscopic; pK = 5.6 (in 80% aqueous methyleellosolve); blue color with Keller's reagent. Soluble in 70 parts methanol, 150 parts acetone, 300 parts ethanol; freely soluble in chloroform, pyridine, and glacial acetic acid; slightly soluble in benzene; almost insoluble in water and petroleum ether. Fungal Source

Claviceps purpurea.

Isolation/Purification/Analysis Extract with either aqueous tartaric or lactic acid solution, partition chromatography with chloroform or methylene chloride at appropriate pH, column clean-up procedures using either silica, alumina or ion exchange resin, and identification and analysis using a combination of co-chromatography using TLC and/or HPLC with UV or fluorescence detection. Mass spectrometry is quite useful for identification, analysis, and quantitation. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergie blockage), and central nervous system effects (bulbomedullary and mesodiencephalie components). LDs0 in rats 62mg/kg (IV).

1. Indole Alkaloids

65

Spectral Data (See A. Hofmann, 1964). ~H NMR: (CDCIa) H-I, 8.14(s); H-2, 6.91(m); H-4a, 2.79(m, ,/=14.2, 11.9Hz); H-4b, 3.32(,/=14.2, 5.0Hz); H-5, 3.73(m, 3=11.9, 5.0Hz); 6-N-Me, 2.61(s); H-7a, 2.96(`/=11.9, 3.9Hz); H-7b, 2.78(dd, 3--11.9, 3.4Hz); H-8b, 3.18(,/=-5.5, 3.9, 3.4Hz); H-9, 6.34(J=5.5Hz); 8-NH, 9.04(s); H-12-14, 7.0-7.5(m); H-2'Me, 1.51(s); H-5', 4.69; OH, 6.97(s); and 7'-NH, not obs. 13C NMR: (DMSO-d6) C-2, 119.4; C-3, 108.8; C-4, 26.6; C-5, 62.4; C-7, 55.1; C-8, 42.5; C-9, 118.3; C-10, 136.0; C-11, 127.1; C-12, 111.0; C-13, 122.2; C-14, 110.2; C-15, 133.8; C-16, 125.9; C-17, 174.3; and N-Me, 43.4ppm. TLC Data Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70% alkaline methanol. This technique works well for ergopeptide alkaloids. References N. J. Bach, H. E. Boa.z, E. C. Kornfeld, C-J Chang, H. G. Floss, E. W. Hagaman, and E. Wenkert; Nuclear Magnetic Resonance Spectral Analysis of the Ergot Alkaloids; J. Org. Chem., Vol. 39, pp. 1272-1276(1974). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. R. G. Mrtek, H. L. Crespi, G. Norman, M. I. Blake, and J. J. Katz; Biosynthesis of Clavine Alkaloids: Proton Magnetic Resonance Studies; Phytochemistry, Vol. 7, pp. 1535-1541(1968).

66

1. Indole Alkaloids

J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994). J. K. Porter and D. Betowski; Chemical-ionization Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 29, pp. 651-653(1981). R. G. Powell, R. D. Plattner, S. G. Yates, K. Clay, and A. Leuchtmann; Ergobalansine, A New Ergot-Type Peptide Alkaloid Isolated from Cenchrus echinatus (Sandbur Grass) Infected with Balansia obtecta, and Produced in Liquid Cultures ofB. obtecta and B. cyperi; Journal of Natural Products, Vol. 53, pp. 1272-1279(1990).

1. Indole Alkaloids

67

Common/Systematic Name Ergotaminine Molecular Formula/Molecular Weight C33H35NsOs; MW' = 579.24817

o

Me

II C

NH..

H -

OHI

.......... ,, N ,T,,,.O/~ --_ l 1~ i

o=1

'

/

J

o

NH

General Characteristics Crystallizes solvent-free as thin rhombic plates from methanol; m.p. 241-243 ~ (dec.); [tt]o 2~+ 369 ~ (c=0.5, in CHCI3); [tt]D2~+ 397 ~ (C=0.5, in pyridine). Soluble in about 1000 parts boiling ethanol, 1500 parts boiling methanol; fairly soluble in chloroform, pyridine, and glacial acetic acid; pK = 5.6 (in 80% aqueous methylcellosolve); blue color with Keller's reagent; does not form salts. Fungal Source

Claviceps purpurea.

Epimers are not considered as naturally occurring, but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base. Isolation/Purification/Analysis Extract with either aqueous tartaric or lactic acid solution, partition chromatography with chloroform or methylene chloride at appropriate pH, column clean-up procedures using either silica, alumina or ion exchange resin, and identification and analysis using a combination of co-chromatography using TLC and/or HPLC with UV or fluorescence detection. Mass spectrometry is quite useful for identification, analysis, and-~-uantitation. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components).

68

1. Indole Alkaloids

Spectral Data IR:

(See A. Hofmann, 1964.)

~H NMR: (CDCI3) H-I, 8.00(s); H-2, 6.91(m); H-4a, 3.59(m, J=15.3, 4.9Hz); H-4b, 2.62(J=15.3, 10.4Hz); H-5, 3.23(m, J=10.4, 4.9Hz); 6-N-Me, 2.61(s); H-7a, 3.13(J=11.7, 1.7Hz); H-7b, 2.76(dd, J=l 1.7, 3.8Hz); H-8b, 3.07(J=6.3Hz); H-9, 6.25(J=6.3Hz); 8-NH, 9.83(s); H-Z'Me, 1.49(s); H-5', 4.61; OH, 6.94(s); and 7'-NH, not obs. ~3CNMR: (DMSO-d6) C-2, 119.7; C-3, 109.0; C-4, 26.9; C-5, 61.7; C-7, 53.0; C-8, 41.8; C-9, 118.1; C-10, 137.1; C-11,127.9; C-12, 111.4; C-13, 122.4; C-14, 110.3; C-15, 133.8; C-16, 126.1; C-17, 175.3; and N-Me, 42.5ppm. References N. J. Bach, H. E. Boaz, E. C. Kornfeld, C-J Chang, H. G. Floss, E. W. Hagaman, and E. Wenkert; Nuclear Magnetic Resonance Spectral Analysis of the Ergot Alkaloids; J. Org. Chem., Vol. 39, pp. 1272-1276(1974). B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. R. G. Mrtek, H. L. Crespi, G. Norman, M. I. Blake, and J. J. Katz; Biosynthesis of Clavine Alkaloids: Proton Magnetic Resonance Studies; Phytochemistry, Vol. 7, pp. 1535-1541(1968). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994). R. G. Powell, R. D. Plattner, S. G. Yates, K. Clay, and A. Leuchtmann; Ergobalansine, A New Ergot-Type Peptide Alkaloid Isolated from Cenchrus echinatus (Sandbu~Grass) Infected with Balansia obtecta, and Produced in Liquid Cultures ofB. obtecta and B. cyperi; Journal of Natural Products, Vol. 53, pp. 1272-1279(1990).

1. Indole Alkaloids

69

Common/Systematic Name 8-Hydroxyergotamine Molecular Formula/Molecular Weight C33HasNsO6, M W = 5 9 7 . 2 5 8 7 3 13'

M e ~, O H I

.o oo .,oj ,

I

9'

8'

NN General Characteristics Crystals from hot ethyl acetate and chloroform were obtained; mp., 197~ [tt]D2~ +14" (C=1%, in pyridine); [a]2546.12~+32.9 (c=1%, in pyridine). The compound has good solubility in dioxane, pyridine, and dimethyl sulfoxide;is less soluble in methanol and chloroform. It gave positive van Urk's and Keller's tests. Fungal Source Natural ergot (Claviceps purpurea). Isolation/Purification Ground ergot from the field culture containing mainly ergotamine was extracted with a mixture of ether and ethanol (1:1, v/v) at room temperature. The extracted alkaloids were treated with 1% tartaric acid. After neutralization by aqueous ammonia to pH 7.5, crude alkaloid mixture was obtained. The peptide alkaloids were then transformed to their levorotatory forms by reaction with 1N sulfuric acid in absolute ethanol and acetic acid. The bases were prepared from the mixture of crude crystalline sulfates by aqueous sodium hydrocarbonate and extracted with ether. Ergotamine was removed by crystallization. Mother liquors were concentrated in vacuo at 40~ giving the bases. Ergotamine, ergocristine and ergostine were transformed into their poorly soluble dextrorotatory forms by boiling eight hours in twenty-fold excess of methanol and their crystals were filtered off alter cooling. The residue in the mother liquors was subjected again to the same operation. It was further separated by chromatography on silica gel using chloroform-ethanol, 99:1, v/v. The new alkaloid was rechromatographed on silica gel using chloroform-benzene, 4:1, v/v. The product was crystallized from hot ethyl acetate and then from hot chloroform.

70

1. Indole Alkaloids

Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components).

Spectral Data UV: 3,~2~

261(1oge=3.95) and 381nm (3.57).

13C NM~: (DMSO-d6) Peptide part- C-2', 86.8 s; C-3', 166.9 s; C-5', 57.2 d; C-6', 165.3 s; C-8', 46.8 t; C-9', 22.6 t; C-10', 26.8 t; C-1 l', 64.8 d; C-12', 103.7 s; C-13', 24.8 q; C-14', 39.4 d; C-15', 139.7 s; C-16', C-20', 130.7 d; C-17', C-19', 128.6 d; and C-18', 128.5ppm d. Ergoline part- C-2, 120.6 d; C-3, 109.5 s; C-4, 26.8 t; C-5, 62.4 d; C-7, 62.9 t; C-8, 72.7 s; C-9, 121.5 d; C-10, 139.0 s; C-1 l, 127.1 s; C-12, 112.3 d, C-13, 123.2 d; C-14, 111.5 d; C-15, 134.8 s; C-16, 127.1 s; C-17, 176.6 s; and N-CH3, 43.8ppm q. Mass Data: 314(30%, C17HlgN204), 283(13, CI6HI7N302), 265(C16HIsN30), 244(C14I-'I16N202), 240(21, C~4H~2N202), 196(10, C~3HIEN2), 167(22, C~2HgN), 154(36, C~IHsN), 153(78, CTH9N202), 125(53, C6HgN20), 120(7, CsH~oN), 91(50, C7H7), 70(100, C4HsN), and 43m/e (535, CzH30); elemental analyses found: 66.88% C, 6.0% H, 11.7% N; for C33H35N506 calcd 66.32% C, 5.90% H, 11.72% N. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds, In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Krajicek, B. Trtik, J. Sp~i~il,P. Sedmera, J. Vokoun, and Z. Reh~t~,ek; 8Hydroxyergotamine, a New Ergot Alkaloid; Czechoslov. Chem. Commun. Vol. 44, pp. 2255-2260(1979).

1. Indole Alkaloids

71

Common/Systematic Name Ergosine Molecular Formula/Molecular Weight C3oH37NsOs; MW' = 547.27947

H,,

o II C "'

Me la 17

NH .........

.~~a~'N'N--Me H

0----~ a

0

OH, 11'

l

N s..'.,~J==O

/ _

H

/Me

CH2CH \Me

"

Nit

General Characteristics Prisms from ethyl acetate; mp., 228~ (dec.); [a]D2~- 161 o (c=l.0 ' in CHCI3), [tt]D2~-8 ~ (C=I.0, in pyridine). Soluble in chloroform; fairly soluble in methanol and acetone; sparingly soluble in ethyl acetate and benzene; pK = 5.5 (in 80% aqueous methylcellosolve); blue color with Keller's reagent; does not form salts. Fungal Source

Claviceps purpurea.

Isolation/Purification/Analysis Extract with either aqueous tartaric or lactic acid solution, partition chromatography with chloroform or methylene chloride at appropriate pH, column clean-up procedures using either silica, alumina or ion exchange resin, and identification and analysis using a combination of co-chromatography using TLC and/or HPLC with UV or fluorescence detection. Mass spectrometry is quite useful for identification, analysis, and quantitation. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components).

72

1.

Indole Alkaloids

Spectral Data IR:

(See A. Hofmann, 1964.) Mass Spectrum: CIMS: (isobutane) 268, 281, and 21 lm/e; LREIMS: 267,196, 154, 125, 86, 70 (100%), 44, and 43m/e. TLC Data Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or l:l, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4: l, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70% alkaline methanol. This technique works well for ergopeptide alkaloids. References B. Berde and H. O. Seheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann, Die Mutterkom Alkaloide, Enke Verlag, Stuttgart, 218 pp., 1964. J. K. Porter, Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock, J. Animal Science, Vol. 73, pp. 871-880(1994). R. G. Powell, R. D. Plattner, S. G. Yates, K. Clay, and/L Leuchtmann, Ergobalansine, A New Ergot-Type Peptide Alkaloid Isolated from C e n c ~ echinatus (Sandbur Grass) Infected with Bakmsia obtecta, and Produced in Liquid Cultures ofB. obtecta and B. cyperi; Journal of Natural Products, Vol. 53, pp. 1272-1279(1990).

1.

Indole Alkaloids

73

Common/Systematic Name Ergosinine Molecular Formula/Molecular Weight CaoHaTNsOs; MW = 547.27947

0n C H :- 17

Me OH, , 18 , , , ,/, 1~" ~ / . ~11' .,J NH......... O~N~s'i~"-O N--Me 3' ~ . Me

r YIH' "

H'CH2CH

\Me

General Characteristics Prisms from ethyl acetate, 90% alcohol, aqueous acetone or benzene; mp., 228~ (dec.); [a]D 20 + 420 ~ (c=l.0, in CHCI3), [a]546120 + 522 ~ (c=l.0, in CHCI3), [a]D 20 + 3 8 0 ~ (c=l.0, in acetone). Very readily soluble in chloroform; readily soluble in acetone; less soluble in ethyl acetate; very sparingly soluble in methanol; almost insoluble in water; pK = 5.5 (in 80% aqueous methylcellosolve); blue color with Keller's reagent; some amorphous salts have been prepared, but only with difficulty. Fungal Source Claviceps purpurea. Epimers are not considered as naturally occurring, but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base. Isolation/Purification/Analysis Extract with either aqueous tartaric or lactic acid solution, partition chromatography with chloroform or methylene chloride at appropriate pH, column clean-up procedures using either silica, alumina or ion exchange resin, identification and analysis using a combination of co-chromatography using TLC and/or HPLC with UV or fluorescence detection. Mass spectrometry is quite useful for identification, analysis and quantitation. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and

74

1.

Indole Alkaloids

adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data IR~ (See A. Hofmann, 1964.) TLC Data Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group, and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70% alkaline methanol. This technique works well for ergopeptide alkaloids. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

1. Indole Alkaloids

75

CommordSystematic Name Ergostine Molecular Formula/Molecular Weight C34H37NsOs; M W - 5 9 5 . 2 7 9 4 7

Me

,

H,,

o C

....

I CI-12 O H ,

L

,7

~,

'

NH............. , ~ 0 s

N--Me

3'

,

o.i-

Y

N T~-i N~s'jJ===O '-_

.--.

NH

General Characteristics Prisms from acetone or ethyl acetate; mp., 211-212~ (dec.); [a]D 2~ - 169 ~ [a]5,s~ 2~ 203~ in CHCI3); [ a i D 20 - 38 ~ [ a ] 5 4 6 1 2 0 - 39~ in pyridine). Moderately soluble in acetone; slightly soluble in chloroform and ethanol; sparingly soluble in benzene and ethyl acetate. Blue color with Keller's reagent changing to blue-green in approximately 15 seconds. Fungal Source

Claviceps purpurea strain D-3-18.

Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some are used routinely in medicine. Soectral Data IR:

(See Hofmann, 1964.)

76

1.

Indole Alkaloids

Mass Spectrum: 328, 267, 244, 154, 153, 125, 91, 70(100%), and 57m/e. CIMS: (isobutane) 268(74%), 329(100), and 245m/e (47). TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology_; Springer-Verlag, New York (1978). M. Flieger, P. Sedmera, J. Vokoun, Z. l~ehfi~ek, J. Stuchlik, Z. Malinka, L. Cvak, and P. Harazim; New Alkaloids from a Saprophytic Culture of Claviceps purpurea; J. Natural Products, Vol. 47, pp. 970-976(1984). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994). J. K. Porter and D. Betowski; Chemical-Ionization Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 29, pp. 651-653(1981).

1. Indole Alkaloids

77

Common/Systematic Name Ergostinine Molecular Formula/Molecular Weight C34I-I37N505; MW = 595.27947 Me I

CH2 OH,

o

II

C H : 17

18

NH ......... O===i3,

0

-

11'

Ns . . ~ = = = O

~~'~N--Me

"

NH

General Characteristics Prisms from methanol; mp., 215-216~ (dec.); [a]D 20 + 357 ~ [a]s4612~+ 4460(c=1.0, in CHCI3); [a]D2~ + 429 ~ [a]54612~+ 538~ in pyridine). Slightly soluble in chloroform and sparingly soluble in benzene. Blue color with Keller's reagent changing to blue-green in 2-3 minutes. Fungal Source

Claviceps purpurea strain D-3-18. Epimers are not considered as naturally occurring, but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base.

Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some are used routinely in medicine. Spectral Data IR.: (See Hofmann, 1964.)

78

1.

Indole Alkaloids

TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacolo~r Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide, Enke Verlag, Stuttgart, 218 pp., 1964. J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

1.

Indole Alkaloids

79

Common/Systematic Name Ergonine Molecular Formula/Molecular Weight C30H37NsOs; MW = 547.27947 Me

H,,

I

0 C

CH20HI

/

NH

|l,

N, "

~

I

o

Me NH General Characteristics Crystals from EtOH/diisopropylether; mp., 206-207~ (synthetic ergonine; mp., 207208~ Fungal Source Claviceps purpurea. Isolation/Purification The mother liquor of the ergokryptine-ergocornine producing strain of Claviceps purpurea consisting of ergosine (90%) and ergonine (10%) was chromatographed on alumina (activity II) atter removing the ergosine as the di-p-toluyl-L-tartaric acid salt (twice). Elution with dichloromethane/0.3% methanol followed by crystallization from ethanol/diisopropylether yielded purified ergonine. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components).

80

1.

Indole Alkaloids

Spectral Data UV: ~

Dichlommethzne max

238(1og 6=4.31) and 307.5nm (3.95).

IR:

(dichloromethane) 3460, 1728, 1668(sh), and 1649cmq. 1H NMR:

(CDCI3) 9.05(1H, s, N1-H); 8.20(1H, s, CONH); 7.0-7.3(3 + 1H, m, aromatic H, OH); 6.90(1H, s, C2-H); 6.20-6.5(1H, m, C9-H); 4.40(1H, d, J=5Hz, C5'-H); 1.54(--16H, m), 2.6(3H, s, N6-CH3); 1.15(3 + 3H, d, J=THz, CH(CH3)2; and 0.91(3H, t, J=THz, CH2-CHs). Mass Spectrum: (Field desorption) Found 547re~e;calcd for C30H37N505 547m/e. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). R. Brunner, P. L. Stutz, H. Tschekter, and P. A. Stadler; Isolation of ergovaline, ergoptine, and ergonine, new alkaloids of the peptide type, from ergot sclerotia ; Can. J. Chem., Vol. 57, pp. 1638-1641(1979).

1. IndoleAlkaloids

81

Common/Systematic Name Ergovaline Molecular Formula/Molecular Weight C29H3sNsOs; MW = 533.26382

o

Me

II

C

OHI

/

N H ......

H,. . . .

0

~N--Me

~

~

0

H-

'--_ CHiMe

I

Me

NH General Characteristics Crystallized from ethyl acetate; mp., 207-208~ (synthetic ergovaline; mp., 207-208"C; [t~]D2~- 172 ~ (C=0.5, in CHCI3). Fungal Source Claviceps purpurea (strain No. 235), Epichlo~ typhina and Acremonium coenophialum infected fescue (Festuca arundinacea Schreb.). Isolation/Purification Sclerotia of Claviceps purpurea were ground and defatted by extraction with petroleum ether. The dark violet residue was extracted with a mixture of 70% acetone and 30% water containing 5% tartaric acid. The extracts were concentrated in vacuo at 50~ The acidic solution was rendered alkaline by addition of Na2CO3, extracted with ethyl acetate, washed with water, dried 0Nla2SO4), and evaporated at 50~ m vacuo. Chromatography of the crude extract on alumina, activity II followed by chromatography on DS-O Camag, activity I and crystallization from ethyl acetate gave purified ergovaline. Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components).

82

1.

Indole Alkaloids

Spectral Data UV:

~,m~O~.....~e

238(1oge=4.3) and 307nm (3.95).

IR:

(dichloromethane) 3466, 1727, 1665(sh), and 1650cm~. 'H NMR: (DMSO) 10.8(1H, s, N1-H); 9.37(1H, s, CONH); 7.0-7.3(4H, m, aromatic H); 6.75(1H, d, J-2Hz, OH); 6.33(1H, s, C9-H); 4.31(1H, d, J=5Hz, C5'-H); 2.61(3H, s, N6-CH3); 1.7-3.9(~14H, m); 1.58(3H, s, C2'-CH3); and 1.08ppm (6H, d, J=7H~ CH(CH3)2). Mass Spectrum: HRIMS: found 533.2602m/e; calcd for C29H35NsO5 533.2638; CIMS (isobutane): 534(M § + 1, 8), 268(100), 267(74), and 197m/e (12%). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology_, Springer-Verlag, New York (1978). R. Brunner, P. L. Stutz, H. Tschekter, and P. A. Stadler; Isolation of Ergovaline, Ergoptine, and Ergonine, New Alkaloids of the Peptide Type, from Ergot Sclerotia; Can. J. Chem., Vol. 57, pp. 1638-1641(1979). A. Hofmann; Die Mutterkorn Alkaloide; Elke Verlag, Stuttgart, 218 pp., 1964. J. K. Porter, C. W. Bacon, J. D. Robbins, and D. Betowski; Ergot Alkaloid Identification in Clavicipitaceae Systemic Fungi of Pasture Grass; J. Agric. Food Chem., Vol. 29, pp. 653-657(1981). J. K. Porter and D. Betowski; Chemical Ionization Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 29, pp. 651-653(1981). M. R. Siegel, G. C. M. Latch, L. B. Bush, F. F. Fannin, D. D. Rowan, B. A. Tapper, C. W. Bacon, and M. C. Johnson; Fungal Endophyte-infected Grasses: Alkaloid Accumulation and Aphid Response; J. Chem. Ecol., 16:3301-3315(1990).

1.

Indole A l k a l o i d s

83

Common/Systematic Name Ergoptine Molecular Formula/Molecular Weight CalH3aNsOs; MW = 561.29512 Me

I

o

c�89

"

C

NH .....

/

~

~N--Me

i"~l i |,,

~

H

~

I CH

M/ \ Me /

NH

General Characteristics Crystals from acetone/water (7:3), mp., 198-199~ (synthetic ergoptine; mp., 199200~ [a]D2~-188 ~ (C=0.8, in CHCI3). Fungal Source

Claviceps purpurea.

Isolation/Purification The mother liquor from ergokaTptine-ergocomine production with a total alkaloid content of about 80% (determined colorimetrically with van Urk's reagent) was chromatographed on alumina (activity II). Elution with chloroform/0.3% methanol led to a first fraction, which consisted primarily of ergokryptine and ergocomine. A second fraction was obtained by elution with chloroform/0.6% methanol which contained about 25% ergoptine. This fraction was further enriched by chromatography on alumina (activity H). The first fraction obtained on elution with chloroform/0.3% methanol contained kryptine and ergocornine. Elution with chloroform/0.6% methanol gave ergoptine (80% purity). Ergoptine was further purified by conversion into a salt with di-p-toluyl-L-tartaric acid. The product was crystallized and cleaved by partition between chloroform Na2COs giving an amorphous resin which was again converted into the salt with di-p-toluyl-L-tartaric acid. The salt obtained, mp 177-178 ~ was cleaved by partition in chloroform/2 N Na2COs and afforded ergoptine with a 95% purity. Further purification of the base by silica gel column chromatography followed by crystallization from acetone/water (7:3, v/v) yielded homogeneous ergoptine.

84

1.

Indole Alkaloids

Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). v

Soeetral Data UV:

238(1og e=4.34) and 308nm (3.98). IR:

(dichloromethane) 3470, 3150-3300, 1732, 1671(sh), and 1653em"1. 1H M R : (CDCI3) 9.58(1H, s, N1-H); 8.17(1H, s, CONH); 7.1-7.3(3 + 1H, m, aromatic I-I, OH); 6.99(1H, s, C2-I-I); 6.34-6.5(1H, m, C9-H); 4.58(1H, t, J----6I-Iz,C5'-H); 2.8-4(-~9H, m, C4-H, C5-H, C7-H, C8-H, C8'-H, and C11'-H); 2.68(3H, s C6-CI-I3); 1.7-2.4(--9H, m, C2'-CH2, C5'-CH2, C9'-CH2, C10'-CH2 and side chain CI-I); and 1.9-2.2ppm (9H, side chain CH3). Mass Spectrum: HRIMS" Found 561.2875m/e; ceded for C31H39N~Os,561.2951. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92: 8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook ofExperim, entfl Pharmacoloffy, Springer-Verlag, New York (1978).

1.

Indole Alkaloids

85

R. Brunner, P. L. Stutz, H. Tschekter, and P. A. Stadler; Isolation of ergovaline, ergoptine, and ergonine, new alkaloids of the peptide type, from ergot sclerotia, Can. J. Chem., Vol. 57, pp. 1638-1641(1979). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

86

1.

Indole Alkaloids

Common/Systematic Name Ergocornine Molecular Formula/Molecular Weight C31H39NsOs" M W = 561.29512

M e \ /Me CH _OH

o II

C

,NH.......

HIIll,

0

~ N - - M e

Nil General Characteristics Solvated polyhedra from methanol; mp., 182-184~ (dec.); [a]D2~ -188", [a]s~l 2~ 224~ in CHCI3); [a]D2~ -111 ~ [tg]546120 -129 ~ (c=l.0, in pyridine); pK = 5.5 (in 80% methylcellosolve); blue color changing to green with Keller's reagent. Soluble in acetone, chloroform, and ethyl acetate; slightly soluble in ethyl and methyl alcohol; nearly insoluble in water. Fungal Source Sclerotia of Claviceps purpurea. Isolation/Purification/Analysis Extract with either aqueous tartaric or lactic acid solution, partition chromatography with chloroform or methylene chloride at appropriate pH, column clean-up procedures using either silica, alumina or ion exchange resin, and identification and analysis using a combination of co-chromatography using TLC and/or HPLC with UV or fluorescence detection. Mass spectrometry is quite useful for identification, analysis, and quantitation. Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects

1.

Indole Alkaloids

87

(bulbomedullary and mesodiencephalic components). LDs0 IV in rabbits 1.17mg/kg.

Spectral Data IR:

(See A. Hofmann, 1964.) Mass Spectrum: CIMS: (isobutane) 562(M+ + 1, 14%), 268(100), 297(72), and 197m/e (11). TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70% alkaline methanol. This technique works well for ergopeptide alkaloids. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology_; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. R. D. Plattner, S. H. Yates, and J. K. Porter; Quadrupole Mass Spectrometry/Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 31, pp. 785-789 (1983). J. K. Porter and D. Betowski; Chemical Ionization Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agile. Food Chem., Vol. 29, pp. 651-653(1981).

88

1. Indole Alkaloids

Common/Systematic Name Ergocorninine Molecular Formula/Molecular Weight C31H39N505; M W "- 561.29512

M e \ /Me CH

o H

II C

NH ....

\

H

N--

_OH O

Me/`` Me

NH

General Characteristics Crystallizes solvent-free, prisms from alcohol; mp., 228~ (dec.); [tt]D2~+ 404 ~ [a]54612~ + 504 ~ (c=l.0, in CHCI3); [t~]D2~+ 488 ~ [a]546120 "~"624 ~ (c= 1.0, in pyridine); pK = 4.8 (in 80% methylcellosolve); blue color changing to green with Keller's reagent. Soluble in 15 parts boiling ethanol, 25 parts boiling methanol, 30 parts boiling benzene, 30 parts boiling ethyl acetate; freely soluble in acetone and chloroform; nearly insoluble in water. Does not seem to form salts. Fungal Source Sclerotia of Clavicepspurpurea. Epimers are not considered as naturally occurring, but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base. Isolation/Purification/Analysis Extract with either aqueous tartaric or lactic acid solution, partition chromatography with chloroform ot methylene chloride at appropriate pH, column clean-up procedures using either silica, alumina or ion exchange resin, and identification, and analysis using a combination of co-chromatography using TLC and/or HPLC with UV or fluorescence detection. Mass spectrometry is quite useful for identification, analysis, and quantitation. Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser

1.

Indole Alkaloids

89

degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some ergopeptine alkaloids are used routinely in medicine. LDs0 IV in rabbits 1.17mg/kg. Spectral Data. IR; See A. Hofmann, 1964. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70% alkaline methanol. This technique works well for ergopeptide alkaloids. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide, Enke Verlag, Stuttgart, 218 pp., 1964. R. D. Plattner, S. H. Yates, and J. K. Porter; Quadrupole Mass Spectrometry/Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 31, pp. 785-789 (1983). J. K. Porter and D. Betowski; Chemical Ionization Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 29, pp. 651-653(1981).

90

1. Indole Alkaloids

.Common/Systematic Name O- 12'-Methylergocomine Molecular Formula/Molecular Weight C32H41NsOs, ~ = 575.31077 Me

Me

,3[

,c\ H/

0 II

~7C

H,, .... 8

18

Me_O

I

]

I ~ 0 . ~ i_--~ 1 ~11' ~ j " ~ I_ " N " ~ I H I O~ 3 N ~' 6 0

NH ...............' - r 2 ' Me

16

e)2

Fungal Source

Clavicepspurpurea Strain 231 FI.

Isolation/Purification Crude alkaloids were obtained by extraction of fungal cultures with CH2CI2-MeOH (80:20, v/v) followed by precipitation with petroleum ether; the crude alkaloid extract was chromatographed by Extrelut (Merck) column chromatography eluted with CH2C12. The crude alkaloids thus obtained were chromatographed on a Silica gel 60 (Merck) column eluted with CH2CI2-MeOH (98:2, v/v); the column was monitored by UV and TLC. Final purification was by preparative TLC in a cold room in the dark. TLC was performed on Silica gel F254, plates (Merck) using the following solvent systems: CH2C12-isopropyl alcohol (92:8, v/v) or CH2CI2-MeOH (90:10, v/v). The alkaloids were detected by examination under UV light at 254 and 366 nm while some were sprayed with N,N-p-dimethylaminobenzaldehyde. The various bands were scraped off and eluted with CH2CI2-MeOH (1:1, v/v). Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater of lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Soectral Data IH NMR: (CDCI3) 8.07(1H, s, NH-1); 2.66 (3H, s, N-CH3), 6.40(1H, dd, J=-2.0, 5.9Hz, H-9); 9.17 (1H, CONH); 3.31 (3H, s, OCH3); and 4.51ppm (1H, d, J--4.1Hz, H-5').

1.

Indole Alkaloids

91

~3CNMR (CDC13) C-2, 119.0"; C-3, 110.8; C-4, 26.6; C-5, 59.4; N(6)-CH3, 44.1; C-7, 49.2; C-8, 41.3; C-9, (119.2); C-10, 138.8; C-11, 129.8; C-12, 112.0; C-13, 123.4; C-14, 110.0; C-15, 133.8; C-16, 126.2; 8-CONH, 173.9; C-2', 89.2; C-3', 164.7"*, C-5', 60.7; C-6', 168.7"*; C-8', 45.8; C-9', 22.1"**; C-10', 22.2***; C-11', 61.4; C-12' , 106.8; 12'-OCH3, 49.2; (2')-C, 35.7; (2')-CH-(CH3)2, 16.0, 16.7; (5')-C, 32.2; and (5')-C-C-(CH3)2, 19.5, 19.2ppm. *, **, and *** assignments may be reversed. Mass Spectrum: EIMS: 575(M+), 543,446, 418, 347, 319, 304, 221, 196, 154, and 70m/e (100%). Reference N. Crespi-Perellino, M. Ballabio, B. Gioia, and A. Minghetti; Two Unusual Ergopeptines Produced by a Saprophytic Culture of Claviceps purpurea; J. Natural Products, Vol. 50, pp. 1065-1074(1987).

92

1. Indole Alkaloids

Common/Systematic Name Ergocristine Molecular F0rmula/Molecular,Weight C35H39N505; MW 609.29512 -

-

Me

\/

Me

CH

o II

C

�9N H ......

OH,

.

'0

Nit General Characteristics Orthorhombic crystals from benzene with 2 molecules benzene of crystallization; the solvent-free base melts at 155-157~ (dec.); [a]D 2~ -183 ~ [a]5,s] 2~-217 ~ (c=1.0, in CHCI3); [a]D2~ -108 ~ [a]~] 2~ -125 ~ (C=I.0, in pyridine); pg = 5.5 (in 80% methylcellosolve); blue color slowly changing to olive-green with Keller's reagent. Very soluble in ethyl and methyl alcohol, acetone, chloroform, and ethyl acetate; slightly soluble in ether; practically insoluble in water and petroleum ether. Fungal Source

Claviceps purpurea.

Isolation/Purification/Analysis Extract with either aqueous tartaric or lactic acid solution, partition chromatography with chloroform or methylene chloride at appropriate pH, column clean-up procedures using either silica, alumina or ion exchange resin, and identification, and analysis using a combination of co-chromatography using TLC and/or I-IPLC with UV or fluorescence detection. Mass spectrometry is quite useful for identification, analysis, and quantitation. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components).

1.

Indole Alkaloids

93

Spectral Data See A. Hofmann, 1964. ~3CNMR: (CDCI3) C-2, 119.2; C-3, 110.6; C-4, 30.9; C-5, 59.3; 6-NMe, 40.9; C-7, 48.2, C-8, 44.3; C-9, 118.8; C-10, 138.9; C-II, 129.6; C-12, 111.9; C-13, 123.3; C-14, 110.2; C15, 133.8; C-16, 125.9; 17-CONH, 176.2; C-2', 89.9; C-Y, 165.7"; C-5', 56.8; C-6', 165.4"; C-8', 46.1; C-9' 21.7; C-10', 22.4; C-I 1', 64.3; and C-12', 103.7ppm. * Assignment may be reversed. Mass Spectrmn: LREIMS: 342(10OA), 267(11), 244(29), 154(19), 153(73), 125(81), 91(64), and 70m/e (100); CIMS: (isobutane) 268(100%), 343(55), and 245m/e (47). TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). HPLC Data Extract with alkaline methanol, filter, followed by direct HPLC analysis with fluorescence detection; mobile phase, either 60% or 70OAalkaline methanol. This technique works well for ergopeptide alkaloids. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkom Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. R. D. Plattner, S. H. Yates, and J. K. Porter; Quadrupole Mass Spectrometry/Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 31, pp. 785-

94

1.

Indole Alkaloids

789(1983). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994). J. K. Porter and D. Betowski; Chemical Ionization Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 29, pp. 651-653(1981). R. G. Powell, R. D. Plattner, S. G. Yates, K. Clay, and A. Leuchtmann; Ergobalansine, A New Ergot-Type Peptide Alkaloid Isolated From Cenchrus echinatus (Sandbur Grass) Infected With Balansia obtecta, and Produced in Liquid Cultures ofB. obtecta and B. cyperi; Journal of Natural Products, Vol. 53, pp. 1272-1279(1990).

1. Indole Alkaloids

95

Common/Systematic Name Ergocristinine Molecular Formula/Molecular Weight C35H39NsOs; ~

= 609.29512 Me

CH

o

II C H, -

Me

\/

la

17

0

NH .........

0-"-33,

N--Me

OH,

'

-

I 11'

Ns , , , , , ~ ~ O

.

//__,

c

/

,\

/

General Characteristics Crystallizes solvent-free as long prisms from absolute alcohol solution; mp., 226~ (dec.); [aiD 20 -I- 366 ~ [a]546120 d- 460 ~ (c=0.7, in CHCI3); laiD 20 -t- 462 ~ [a]546120 -I- 582 ~ (c=l.0, in pyridine); pK = 5.0 (in 80% methylcellosolve); blue color slowly changing to olivegreen with Keller's reagent. Much less soluble than ergocristine; apparently does not form salts. Fungal Source

Clavicepspurpurea strain D-3-18. Epimers are not considered as naturally occurring, but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base.

Isolation/Purification via TLC Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25)

96

1.

b) c) d) e)

Indole Alkaloids

Chloroform-methanol (90:10; 80:20; 90:10 in saturated NHs atmosphere). Chloroform-methanol-NH3 (94:5:1). Chloroform-ethylamine (90:10). Benzene-dimethylformamide (86.5:13.5).

Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data IR~

See A. Hofmann, 1964. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimenta! Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

1. Indole Alkaloids

97

Common/Systematic Name Ergosecaline Molecular Formula/Molecular Weight C24H28N404; MW = 436.21106

?o Me

O II C

NH,,,......

i,,

~=0

Me z

O==~N/CH~CH N--Me

H

Me

NH Fungal Source Claviceps purpurea strain D-3-18. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some are used routinely in medical practice. Spectral Data IR:

See Hofmann, 1964. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica usifig methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25).

98

1.

Indole Alkaloids

b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

1.

Indole Alkaloids

99

Common/Systematic Name Ergosecalinine Molecular Formula/Molecular Weight_ C24H2gN404; M W -- 436.21106 Me

O

C H =

NH,,,,.....,.."~ ~ = 0 /Me O==a,,.N/CH--CH ~N--Me H Me

NH General Characteristics Prisms from ethyl acetate; mp., 217~ (dec.); [a], ~8+ 298 ~ [tt]546~18 + 375 ~ (c=l.0, in CHCI3); [aiD Is + 417 ~ [a]s46~Is + 512 ~ (C=I.0, in pyridine); blue color with AllportCocking's reagent. Readily soluble in acetone and methanol; moderately soluble in ethyl acetate; sparingly soluble in benzene and chloroform. Fungal Source Sclerotia and saprophytic culture of Claviceps purpurea. Epimers are not considered as naturally occurring, but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some are used routinely in medicine. Spectral Data IR:

See Hofmann, 1964.

100

1. Indole Alkaloids

TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharm.acolo~; Springer-Verlag, New York (1978). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

1.

Indole Alkaloids

101

Common/Systematic Name Ergobalansine Molecular Formula/Molecular Weight CEgH35NsOs; MW = 521.26382

0 17~

~3' Me Me 0H/ 0 - : 11,

18

H,....

\

o 6 N--Me

/

"--_

H - / - - ---CH2 CH M e / Me NH

General Characteristics Ergobalansine was obtained as a white solid. Fungal Source Cenchrus echinatus infected with Balansia obtecta and Cyperus virens infected with B. cyperi. Isolation/Purification Aerial parts of C echinatus infected with B. obtecta were ground in a Wiley mill fitted with a 1/4 inch screen and extracted with 95% EtOH for 24 h. The extraction process was repeated three additional times and combined EtOH extracts were evaporated to a volume of 6 liters on a rotary evaporator below 42~ An aliquot was evaporated to dryness and a portion of the concentrate was coated onto silica gel. The material was added to the top of a silica gel column packed in methylene chloride and the column was eluted with CH2C12, 5%, 10%, and 25% MeOH in CH2C12. Fractions were analyzed by TLC and recombined into fractions on the basis of similarity of TLC patterns. The remaining extract was processed by dividing into 6 portions and repeating the chromatographic procedure 6 additional times. Like materials were combined and all 8 fractions were examined for the presence of ergot-peptide alkaloids. An alkaloid-enriched fraction was prepared by solvent partitioning the entire sample in EtOAc and aqueous 2% tartaric acid. The aqueous layer was partitioned a second time with EtOAc. The aqueous layer was adjusted to pH 9 by slow addition of concentrated NI-hOH. The resulting basic solution was extracted with EtOAc, and the EtOAc extract yielded an alkaloidal material. Analysis of the alkaloid-enriched fractions by reversed-phase I-IPLC revealed two highly fluorescent

102

1.

Indole Alkaloids

compounds. Final purification of ergobalansine and ergobalansinine was achieved by preparative TLC. Purified ergobalansine and ergobalansinine readily epimerized to an approximate 1:1 equilibrium mixture on standing in solutions containing MeOH, particularly at elevated temperatures. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some are used routinely in medicine. Spectral Data IH NMR: (CDCI3) H-l, 7.99(s); H-2, 6.93(m); H-4a, 2.82(m, J=14.3, 12.2Hz); H-4b, 3.32(m, J=14.3, 5.0Hz); H-5, 3.79(m, J=12.2, 5.0Hz); 6-NMe, 2.64(s); H-7a, 2.98(dd, ,/=-12.0, 3.7Hz); H-7b, 2.88(dd, J=12.0, 2.8Hz); H-8a, 3.18(m, J=5.6, 3.7, 2.8Hz); H-9, 6.35(dd, J=5.6Hz); 8-NH, 9.26(s); H-12-14, 7.18(m); H-2'Me, 1.52(s); H-5', 4.52(dd); 5'-CH2, 1.94(m); 5'-CH2EH, 1.94(m); 5'-CH2CH(CH3)2, 1.05(d), 1.00(d); H-11', 3.56(m); 11'-CH3, 1.34(d); OH, 5.84(s); and 7'-NH, 6.78ppm(bs). ~3CNMR: (CDCI3) C-2, 119.0; C-3, 110.8; C-4, 22.1; C-5, 59.6; 6-NMe, 41.1; C-7, 48.9; C-8, 44.1; C-9, 118.4; C-10, 139.2; C-11,129.5; C-12, 112.0; C-13, 123.4; C-14, 110.0; C15, 133.8; C-16, 126.2; 17-CONH, 175.7; C-2', 85.4; C-3', 166.3"; C-5', 53.2; C-6', 169.1"; C-8', not obs.; C-9' not obs.; C-10', not obs.; C-11', 57.0; C-12', 102.7; T-Me, 24.6; 5'-CH2, 43.5; 5'-CH2CH, 24.7; 5'-(Me)2, 21.9, 22.8; and 11'-CH3, 14.7ppm. "Assignment may be reversed. Mass Data: EIMS: [M]§ 521(3%), 337(6), 267(100), 224(30), 221(52), 207(49), 196(21), 184(38), 180(29), 167(19), 154(22), 141(17), 128(54), 113(21), 44(48), and 43(59); CIMS: (isobutane) MH+ 522(6%), 504(2), 268(93), 255(100), and 185(12); negative ion CIMS: (isobutane) [M] 521(36), and 254(100). The MS-MS daughter spectrum of 254m/e (negative ion CIMS) was: 254(27), 211(7), 183(100), 126(2), 113(2), and 86(4). Found for [AH]§ 268.1422 (C16HlsN30 requires 268.1450); [BH] § 255.1362(C12H19N20; requires 255.1345). TLC Data Analytical and preparative TLC were carried out on Silica gel 60-F254plates (E. Merck) developed with chloroform-methanol (9:1, v/v).

1.

Indole Alkaloids

103

HPLC Data Preparative HPLC utilized a Rainin Dynamax-60/~ silica column with isocratic elution using methylene chloride-methanol (93:7, v/v). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). R. G. Powell, R. D. Plattner, S. G. Yates, K. Clay, and A. Leuchtmann; Ergobalansine, A New Ergot-Type Peptide Alkaloid Isolated From Cenchrus echinatus (Sandbur Grass) Infected with Balansia obtecta, and Produced in Liquid Cultures ofB. obtecta and B. cyperi; Journal of Natural Products, Vol. 53, pp. 1272-1279(1990).

104

1. Indole Alkaloids

Common/Systematic Name Ergobalansinine Molecular Formula/Molecular Weight C28H35N505; MW = 521.26382

17~

13' Me

18

| O

Me OH=

:

11'

......

,~

~N--Me

12

//'-____ _

H-

---CH2

I

CH Me/

~Me

NH

General Characteristics Ergobalansinine was obtained as a white solid; ergobalansine and ergobalansinine readily epimerized to an approximate 1:1 equilibrium mixture on standing in solutions containing MeOH. Fungal Source Cenchrus echinatus infected with Balansia obtecta and Cyperus virens infected with B. cyperi. Epimers are not considered as naturally occurring, but as products formed during extraction and purification; epimerization at C-8 occurs in either acid or base. Isolation/Purification Aerial parts of C. echinatus infected with B. obtecta were ground in a Wiley mill fitted with a ~Ainch screen and extracted with 95% EtOH 24 h. The extraction process was repeated three additional times and combined EtOH extracts were evaporated to a volume of 6 liters, below 42 ~ on a rotary evaporator. An aliquot was evaporated to dryness and a portion of the concentrate was coated onto silica gel. The material was added to the top of a silica gel column packed in methylene chloride and the column was eluted with CH2C12, 5%, 10%, and 25% MeOH in CH2C12. Fractions were analyzed by TLC and recombined into fractions on the basis of similarity of TLC patterns. The remaining extract was processed by dividing into 6 portions and repeating the chromatographic procedure 6 additional times. Like materials were combined and all 8 fractions were examined for the presence of ergot-peptide alkaloids. An alkaloid-enriched fraction was prepared by solvent partitioning the entire sample in EtOAc and aqueous 2% tartaric acid. The aqueous layer was partitioned a second time with EtOAc. The aqueous layer

1. Indole Alkaloids

105

was partitioned a second time with EtOAc. The aqueous layer was adjusted to pH 9 by slow addition of concentrated Nt-hOH. The resulting basic solution was extracted with EtOAc, and the EtOAc extract yielded an alkaloidal material. Analysis of the alkaloid-enriched fractions by reversed-phase I-IPLC revealed two highly fluorescent compounds. Final purification of ergobalansine and ergobalansinine was achieved by preparative TLC. Purified ergobalansine and ergobalansinine readily epimerized to an approximate 1:1 equilibrium mixture on standing in solutions containing MeOR particularly at elevated temperatures. Spectral Data IH NMR: (CDCI3) H-I, 8.05(s); H-2, 6.90(m); H-4a, 3.59(m, J=14.4, 5.3Hz); H-4b, 2.64(m, J=14.4, 12.0Hz); H-5, 3.20(m, ,/=12.0, 5.3Hz); 6-NMe, 2.61(s); H-7a, 3.12(J=l 1.9Hz); H-To, 2.75(dd, J=l 1.9, 3.5Hz); H-8b, 3.06(m, J=6.3Hz); H-9, 6.49(dd, J=6.3Hz); 8-NI-I, 9.85(s); H-12-14, 7.15(m); H-2'Me, 1.50(s); H-5 ~ 4.42(dd); 5'-CH2, 1.90(m); 5'-CH2CH, 1.90(m); 5'-CH2CH(CH3)2, 0.96(d), 0.95(d); H11', 3.51(m); 11'-CH3, 1.32(d); OH, 6.01(s); and 7'-NH, 6.77ppm (brs). 13C N M R :

(CDCI3) C-2, 118.4; C-3, 110.1; C-4, 27.7; C-5, 62.6; 6-NMe, 43.6; C-7, 54.5; C-8, 43.0; C-9, 117.6; C-10, 137.5; C-11,127.5; C-12, 112.8; C-13, 123.3; C-14, 110.2; C15, 133.9; C-16, 126.2; 17-CONH, 176.0; C-2', 85.4; C-3', 165.7~ C-5', 53.0; C-6', 169.1*; C-8', not obs.; C-9' not obs.; C-10', not obs.; C-11', 56.9; C-12', 102.5; T-Me, 24.5; 5'-CH2, 43.4; 5'-CH2CH, 24.6; 5'-(Me)2, 22.0, 22.7; and 11'-CH3, 14.7ppm. ,

Assignments may be reversed.

Mass Data: EIMS: [M] § 521(3%), 337(4), 267(64), 221(43), 207(47), 196(25), 184(36), 180(34), 167(22), 154(33), 141(25), 128(96), 113(33), 44(89), and 43(100); CIMS: (isobutane) MIT 522(8%), 504(2), 268(97), 255(100), and 185(13); negative ion CIMS (isobutane) ([M] 42), and 254(100). Found for [AH]§ 268.1412 (Cl6HlsN30 requires 268.1450); [BH]§ 255.1362 (C~2H~9N20;requires 255.1345). TLC Data Analytical and preparative TLC were carried out on silica gel 60-F254plates (E. Merck) developed with chloroform-methanol (9:1). HPLC Data Preparative HPLC utilized a Rainin Dynamax-60A silica column with isocratic elution using methylene chloride-methanol (93:7, v/v).

106

1. Indole Alkaloids

References R. G. Powell, R. D. Plattner, S. G. Yates, K. Clay, and A. Leuchtmann; Ergobalansine, A New Ergot-Type Peptide Alkaloid Isolated From Cenchrus echinatus (Sandbur Grass) Infected With Balansia obtecta, and Produced in Liquid Cultures orB. obtecta and B. cyperi; Journal of Natural Products, Vol. 53, pp. 1272-1279(1990). B. Berde and H. O. Scheld (eds.), Ergot Alkaloids and Related Compounds, In Handbook of Experimental Pharmacolowr Springer-Verlag, New York (1978). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock, J. Animal Science, Vol. 73, pp. 871-880(1994).

1. Indole Alkaloids

107

Common/Systematic Name ct-Ergocryptine Molecular Formula/Molecular Weight C32H41NsOs" M W = 575.31077

Me

0 17 II C

~/ /

\/

Me

13,CH OH r----q la L O . ~ . . ~ ' _J NH.............'1~' ~ i.!.I "?2'

1

II /

Me/

'

kMe

19'

Fungal Source Claviceps purpurea strain D-3-18. Isolation/Purification The fungal mycelium was wetted and extracted with Et20-EtOH (9:1, v/v). Alkaloids were extracted into a 1% tartaric acid solution and precipitated by adjusting the pH to 7.5. They were collected by filtration and dried. The crude alkaloid mixture was defatted with petroleum ether; the resulting alkaloid mixture was chromatographed on a silica gel column eluted with CHCI3. The alkaloids were further purified by HPLC on a semipreparative column: MicroPak NH2, particle size 101.tM; mobile phase, Et20-EtOH (22:3, v/v); flow rate, 220ml/h; UV detection at 240nm. Purity was monitored on an analytical HPLC column: Perkin-Elmer NH2; particle size 10btM; mobile phase, hexane-Et20-EtOH (41:41:8, v/v/v); flow rate 0.6ml/min; detection with UV at 240nm. Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components).

108

1. Indole Alkaloids

Spectral Data IR: See A. Hofinann, 1964. 1H NMR: (CDCh) NH(1), 8.37(s); H-2, 6.94(s); H-9, 6.37(dd, J--6.4, 1.SHz); N-CH3(6), 2.63(s); N-H(18), 9.86(s); H-5', 4.520, J=6.4Hz); C-2' side chain methyls, 1.03(d, 6.1Hz), 0.91(d, 6.1Hz); and C-5' side chain methyls, 1.02ppm (d, J=-7.3Hz).

~3CNMR: (CDCI3) C-2, 119.2; C-3, 110.6; C-4, 26.5; C-5, 64.5; C-7, 48.1; C-8, 40.9; C-9, 118.8; C-10, 139.2; C-11,129.6; C-12, 111.9; C-13, 123.3; C-14, 110.1; C-15, 133.9; C-16, 126.3; C-17, 176.3; N-Me, 44.3; C-2', 89.7; C-3', 165.8; C-5', 53.3; C-6', 166.2; C-8', 46.0; C-9' 21.6; C-10', 22.2; C-11', 59.3; C-12', 103.4; C-lY, 34.3; C-14', 15.3"; C-15', 16.9"; C-16', 43.5; C-17', 25.1; C-18', 22.1"; and C-19', 22.6"ppm. *, **Assignments may be reversed. Mass Spectrum: No M + observed in the mass spectrum; 308(C16I-I24N204,3%), 267(C~6I-I~7N30,22); 210(C~H18N202, 14), 209(C~H~TN202, 33), 154(CTH~oN202, 53), 125(C6H9N20, 8), 86(C~I12N, 6), 71(C4H70, 61), and 70role (C~J-IsN, 100). CIMS: (isobutane) Results suggested that major fragments for the cyclol alkaloids are due to cleavage of the bond joining the tricyclic peptide moiety with the lysergic acid amide portion of the molecule. (M+ + 1)+ was not observed in the CI spectrum; however, daughter ions 268(100%), 309 (30), and 211(3) were observed. Quadrupole MS/MS: (CI with isobutane) 576[(M+H)§ 35%], 268(AW, 100), 309(BH+, 64), and 211(CI-F, 7); Negative CIMS, 575(M', 65%), 266[(A-H)', 0.1], 308(B, 100), and 209[(C-H), 0.1 ]. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel clwomatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 7525). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere).

1.

Indole Alkaloids

109

c) Chloroform-methanol-NH3 (94"5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References M. Flieger, P. Sedmera, J. Vokoun, Z. l~eh~i~ek, I. Stuchlik, Z. Malinka, L. Cvak, and P. Harazim, New Alkaloids from a Saprophytic Culture of Clavicepspurpurea; J. Natural Products, Vol. 47, pp. 970-976(1984). A. Hofmann, Die Mutterkom Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. R. D. Plattner, S. G. Yates, and J. K. Porter, Quadrupole Mass Spectrometry/Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 31, pp. 785789(1983). J. K. Porter and D. Betowski, Chemical Ionization Mass Spectrometry of Ergot Cyclol

Alkaloids, J. Agric. Food Chem., Vol. 29, pp. 650-653(1981). R. G. Powell, R. D. Plattner, S. G. Yates, K. Clay, and A. Leuchtmann; Ergobalansine, A New Ergot-Type Peptide Alkaloid Isolated from Cenchrus echina~s (Sandbur Grass) Infected With Balansia obtecta, and Produced in Liquid Cultures ofB. obtecta and B. cyperi; Journal of Natural Products, Vol. 53, pp. 1272-1279(1990).

110

1. Indole Alkaloids

Common/Systematic Name O- 12'-Methyl-ct-ergokryptine Molecular Formula/Molecular Weight C33H43NsOs; MW = 589.32642 Me \ / Me

! Y8

H,,

13,0H

,=

C

NH .............. 'B'

~

OMe

ho." ~

...

~ 0--'43'

6 N--Me

-

i

l

'

1, "N n

N5 ~ O

..J i

16'

/"--_ 16'

Fungal Source

Clavicepspurpurea strain 231 FI.

Isolation/Purification Crude alkaloids were obtained by extraction of fungal cultures with CH2CI2-MeOH (80:20) followed by precipitation with petroleum ether; the crude alkaloid extract was chromatographed by Extrelut (Merck) column chromatography eluted with CH2C12. The crude alkaloids thus obtained were chromatographed on a Silica gel 60 (Merck) column eluted with CH2CI2-MeOH (98:2, v/v); the column was monitored by UV and TLC. Final purification was by preparative TLC in a cold room in the dark. TLC was performed on Silica gel F254plates (Merck) using the following solvent systems: CH2Cl2-isopropyl alcohol (92:8, v/v) or CH2CI2-MeOH (90:10, v/v). The alkaloids were detected by examination under UV light at 254 and 366nm while some were sprayed with N,N-p-dimethylaminobenzaldehyde. The various bands were scraped off and eluted with CH2CI2-MeOH (1:1, v/v). Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components).

1.

Indole Alkaloids

111

Spectral Data IH NIV[R: (CDCI3) NH(1), 8.09(s); N(6)-CH3, 2.66; H-9, 6.41(J=1.8, 5.5Hz); (8)-CONH, 9.16; OCH3-12', 3.24; and H-5', 4.62ppm (,/=3.8, 10.8Hz). 13CNMR: (CDCI3) C-2, 119.0"; C-3, 110.3; C-4, 26.4; C-5, 59.5; N(6)-CH3, 44.6; C-7, 48.4; C8, 41.3; C-9, 119.2"; C-10, 138.8; C-11,129.8; C-12, 112.1; C-13, 123.5; C-14, 110.0; C-15, 133.8; C-16, 126.2; 8-CONH, 174.0; C-2', 89.2; C-3', 165.7"*; C-5', 53.1; C-6', 167.4"*; C-8', 46.1; C-9', 21.9"**; C-10', 22.0"**; C-11', 61.9; C-12', 106.4; 12'-OCH3, 48.8; (2')-C, 35.9; (2')-CH-(CH3)2, 16.0, 16.8; (5')-C, 42.2; (5')-C-C, 25.0; and (5')-CC-(CH3)2, 22.1, 21.4ppm.

*, **, and *** Assignments may be reversed. Mass Spectrum: EIMS: 589(M+), 557, 432, 347, 319, 304, 221,196, 154, and 70m/e (100%). Reference N. Crespi-Perellino, M. Ballabio, B. Gioia, and A. Minghetti; Two Unusual Ergopeptines Produced by a Saprophytic Culture of Claviceps purpurea; J. Natural Products, Vol. 50, pp. 1065-1074(1987).

112

1.

Indole Alkaloids

Common/Systematic Name 13-Ergocryptine Molecular Formula/Molecular Weight C32H41NsOs; M'W = 575.31077 Me

_

,o C

H,,I

Y s /.

' /

\ /

Me

13'CH

,=

NH .............,~,

~

OH,

Lo.i. I-

0----43,

6 N--Me

"~

I,,.1..]r) I"

"~_I'N

J

,

I H 16,

Ns...~O /-

16'

Me/ CH2--Me

'~ '"

'"

General Characteristics Prisms from methanol solution (solvated with MeOH); mp., 210-212~ (dec.); [a]o 2~ 1910, [~]546120 - 228 ~ (c=l.0, in CHCI3); [~]D2~- 117 ~ [et]5~ 2~ - 138 ~ (C=I.0, in pyridine); pK = 5.5 (in 80% methylcellosolve); blue color slowly changing to olive-green with Keller's reagent. Fungal Source


Isolation/Purification The fungal mycelium was wetted and extracted with Et20-EtOH (9:1, v/v). Alkaloids were extracted into a 1% tartaric acid solution and precipitated by adjusting the pH to 7.5. They were collected by filtration and dried. The crude alkaloid mixture was defatted with petroleum ether; the resulting alkaloid mixture was chromatographed on a silica gel column eluted with CHCI3. The alkaloids were further purified by HPLC on a semipreparative column: MicroPak NH2, particle size 101.tm; mobile phase, Et20-EtOH (22:3, v/v); flow rate, 220ml/h; UV detection at 240nm. Purity was monitored on an analytical HPLC column: Perkin-Elmer NH2; particle size 101~m; mobile phase, hexane-Et20-EtOH (41:41:8, v/v/v); flow rate 0.6ml/min; detection with UV at 240nm. Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser

1.

Indole Alkaloids

113

degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). LDs0 IV in rabbits, 1.05mg/kg. Spectral Data IR:

See A. Hofmann, 1964. IH NMR: (CDCI3) NH(1), 8.21(s); H-2, 6.92(s); H-9, 6.35(dd, J-6.1, 1.4Hz); N-CH3(6), 2.63(s); N-H(18), 9.88(s); H-5', 4.49(d, J=2.5Hz); C-2' side chain methyls, 1.113(d, 6.1Hz), 0.87(d, 6.1Hz); and C-5' side chain methyls, 1.04(d, J=6.8Hz, and 0.94ppm (t, J=7.3Hz). 13C N M R :

(CDC13) C-2, 119.1; C-3, 110.7; C-4, 26.6; C-5, 64.0; C-7, 48.0; C-8, 40.9; C-9, 119.2; C-10, 139.2; C-11, 129.7; C-12, 112.0; C-13, 123.3; C-14, 110.1; C-15, 133.8; C-16, 126.3; 17-CONH, 176.3; N-Me, 44.3; C-2', 89.5; C-3', 164.6; C-5', 59.7; C-6', 166.6; C-8', 45.9; C-9' 21.3; C-10', 22.2; C-11', 59.2; C-12', 106.3; C-13', 34.3; C-14', 15.3"; C-15', 17.0~ C-16', 39.4; C-IT, 27.9; C-18', 12.6; and C-19', 16.6ppm. ,

Assignments may be reversed.

Mass Spectrum: No M + observed in the mass spectrum; 308(C16H24N204, 2%), 267(C16HI7N30, 47); 210(CllHlsN202, 8), 209(CI1HITN202, 22), 154(C7H10N202, 90), 125(C6I-I9N20, 17), 86(CsH12N, 10%), 71(C4H70, 44), and 70m/e (C4I-IsN, 100). CIMS (isobutane) Results suggested that major fragments for the cyclol alkaloids are due to cleavage of the bond joining the tricyclic peptide moiety with the lysergic acid amide portion of the molecule. (M+ + 1)+ was not observed in the CI spectrum; however, daughter ions 268(100%), 309(53), and 211(4) were observed. Quadrupole MS/MS: (CI with isobutane) 576[(M+H) +, 52%], 268(AW, 100), 309(BIT, 67), and 211(CIT, 8); Negative ElMS 575(M', 37%), 266[(A-H)', 0.1], 308(B-, 100), and 209[(CH), 0.2]. TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates

114

1.

Indole Alkaloids

ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook 0.f Experimental Pharmacology; Springer-Verlag, New York (1978). M. FUieger, P. Sedmera, J. Vokoun, Z. l~ehd~,ek, J. Stuchlik, Z. Malinka, L. Cvak, and P. Harazim; New Alkaloids from a Saprophytic Culture of Claviceps purpurea; J. Natural Products, Vol. 47, pp. 970-976(1984). A. Hofmann; Die Mutterkom Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. R. D. Plattner, S. G. Yates, and J. K. Porter; Quadrupole Mass Spectrometry/Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 31, pp. 785789(1983). J. K. Porter and D. Betowski; Chemical Ionization Mass Spectrometry of Ergot Cyclol Alkaloids; J. Agric. Food Chem., Vol. 29, pp. 650-653(1981). R. G. Powell, R. D. Plattner, S. G. Yates, K. Clay, and A. Leuchtmann; Ergobalansine, A New Ergot-Type Peptide Alkaloid Isolated From Cenchrus echinatus (Sandbur Grass) Infected With Balansia obtecta, and Produced in Liquid Cultures ofB. obtecta and B. cyperi; Journal of Natural Products, Vol. 53, pp. 1272-1279(1990).

1. Indole Alkaloids

115

Common/Systematic Name 5'-epimer of p-Ergocryptine Molecular Formula/Molecular Weight C32H41N5Os, ~ = 575.31077 Me

\/

Me

CH

O II

_OH

H

ill,,

H2CHC

H

NH Fungal Source


Isolation/Purification The fungal mycelium was wetted and extracted with Et20-EtOH (9:1, v/v). Alkaloids were extracted into a 1% tartaric acid solution and precipitated by adjusting the pH to 7.5. They were collected by filtration and dried. The crude alkaloid mixture was defatted with petroleum ether, the resulting alkaloid mixture was chromatographed on a silica gel column that was eluted with CHCI3. The alkaloids were further purified by HPLC on a semi-preparative column: MicroPak NH2, particle size 10btm, mobile phase, EhO-EtOH (22:3, v/v), flow rate, 220ml/h, UV detection at 240nm. Purity was monitored on an analytical HPLC column: Perkin-Elmer NH2; particle size 1Own; mobile phase, hexane-Et20-EtOH (41:41:8, v/v/v); flow rate 0.6ml/min; detection with UV at 240nm. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some are used routinely in medicine. Spectral Data ~H NMR:

116

1.

Indole Alkaloids

(CDCI3) NH(1), 8.47(s); H-2, 6.94(s); H-9, 6.36(dd, J=5.5, 1.3Hz); N-CH3(6), 2.65(s); N-H(18), 9.84(s); H-5', 4.53(d, J=3.1Hz); C-2' side chain methyls, 1.03(d, 6.1Hz), 0.89(d, 6.1Hz); and C-5' side chain methyls, 1.14(d, J--6.8Hz), 0.96ppm (t, J=-7.3Hz). ~3CNMR: (CDCI3) C-2, 119.2; C-3, 110.5; C-4, 26.7; C-5, 64.1; C-7, 48.2; C-8, 40.9; C-9, 118.9; C-10, 139.0; C-11, 129.6; C-12, 111.9; C-13, 123.2; C-14, 110.2; C-15, 133.9, C-16, 126.3; C-17, 176.1; N-Me, 44.3; C-2', 89.5; C-3', 165.0; C-5', 60.4; C-6', 167.1; C-8', 46.0; C-9' 21.7; C-10', 22.1; C-11', 59.3; C-12', 103.7; C-13', 34.3; C-14', 15.3"; C-15', 16.9"; C-16', 39.8; C-17', 26.3; C-18', 12.5; and C-19', 16.6ppm. * Assignment may be reversed. Mass Spectrum: No M + observed in the mass spectrum; 308(C16H24N204, 1%), 267(Ct6H17N30, 15); 210(CnHlsN202, 8), 209(C~IHl~N202, 22), 154(C~H~oN202,67), 125(C6HgN20, 12), 86(CsH12N, 8), 71 (C4H70, 47), and 70m/e (C4I-IsN, 100). TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds, In Handbook 0.f Experimental Pharmacology; Springer-Verlag, New York (1978). M. Flieger, P. Sedmera, J. Vokoun, Z. l~ehfi6ek, J. Stuchlik, Z. Malinka, L. Cvak, and P. Harazim; New Alkaloids from a Saprophytic Culture of Clavicepspurpurea; J. Natural Products, Vol. 47, pp. 970-976(1984). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-881(1994).

1. Indole Alkaloids

117

Common/Systematic Name 13-Ergocryptam Molecular Formula/Molecular Weight C32H41NsO4; M W = 559.15855

M e \ /Me 13' CH

o

...........

H,,,I 17 /~ -

,,Vc.o J.

I 0----~3, N--Me

I H I N ~ O /-

.,

Me

,

Nil Fungal Source


Isolation/Purification The fungal mycelium was wetted and extracted with Et20-EtOH (9:1, v/v). Alkaloids were extracted, into a 1% tartaric acid solution and precipitated by adjusting the pH to 7.5. They were collected by filtration and dried. The crude alkaloid mixture was defatted with petroleum ether; the resulting alkaloid mixture was chromatographed on a silica gel column eluted with CHCI3. The alkaloids were further purified by I-IPLC on a semipreparative column: MicroPak NH2, particle size 101.tm; mobile phase, Et20-EtOH (22:3, v/v); flow rate, 220ml/h; UV detection at 240nm. Purity was monitored on an analytical HPLC column: Perkin-Elmer NH2; particle size 101.tm; mobile phase, hexane-Et20-EtOH (41:41:8, v/v/v); flow rate 0.6ml/min; detection with UV at 240nm. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some are used routinely in medicine.

118

1.

Indole Alkaloids

Spectral Data 1H ~ : (CDCI3) NH(1), 8.30(s); H-2, 6.92(s); H-9, 6.46(dd, ,/--4.3, 1.2Hz); N-CH3(6), 2.67(s); N-H(18), 8.22(d, J=9.2Hz); H-2', 5.83(dd, :=-9.2, 3.1Hz); H-5', 4.96(d, d=9.2Hz); HI I', 4.36(t, d=7.8Hz); C-2' side chain methyls, 0.75(d, 6.7Hz), 0.94(d, 6.7Hz); and C-5' side chain methyls, 0.98(d, J=6.7Hz), and 0.93ppm (t, ,/=7.3Hz). 13C NMPx: (CDC13) C-2, 118.9; C-3, 110.9; C-4, 29.6; C-5, 63.2; C-7, 50.6; C-8, 41.7; C-9, 119.2; C-10, 137.8; C-11,129.4; C-12, 111.8; C-13, 123.2; C-14, 109.9; C-15, 133.9; C-16, 126.3; C-17, 174.4; N-Me, 44.3; C-2', 58.1; C-3', 173.6; C-5', 60.0; C-6', 165.6; C-8', 45.5; C-9' 22.9; C-10', 23.3; C-11', 60.4; C-12', 170.0; C-13', 30.3; C-14', 16.1"; C-15', 20.1*; C-16', 37.2; C-17', 25.7; C-18', 10.9; and C-19', 15.Sppm. " Assignments may be reversed. Mass Spectrum: M + observed in the mass spectrum, 559(1%), 349(C21H23N302, 2), 321 (C2oH23N30, 4), 221(C~sH~3N2, 9), 154(CTH10N202, 100), 125(C6HgN20, 26), 86(CsH~2N, 13), and 70role (CaHsN, 87). TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde arid H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). M. Flieger, P. Sedmera, J. Vokoun, Z. l~eh~i6ek,J. Stuchlik, Z. Malinka, L. Cvak, and P. Harazim; New Alkaloids from a Saprophytic Culture of Claviceps purpurea; J. Natural Products, Vol. 47, pp. 970-976(1984).

1.

Indole Alkaloids

119

J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

120

1.

Indole Alkaloids

Common/Systematic Name 13,13-Ergoannam Molecular Formula/Molecular Weight C33H43NsO4; MW = 573.33151

M e \ /CH2Me 13, CH

oII

I

'"

3'

N H

,,

"

J

18 O . . ........... , , c .

c

O CH~CH2Me I

Me

NH

Fungal Source Claviceps purpurea strain D-3-18. Isolation/Purification The fungal mycelium was wetted and extracted with Et20-EtOH (9:1, v/v). Alkaloids were extracted into a 1% tartaric acid solution and precipitated by adjusting the pH to 7.5. They were collected by filtration and dried. The crude alkaloid mixture was defatted with petroleum ether; the resulting alkaloid mixture was chromatographed on a silica gel column eluted with CHCI3. The alkaloids were further purified by HPLC on a semipreparative column: MicroPak NH2, particle size 101,tm; mobile phase, Et20-EtOH (22:3, v/v); flow rate, 220ml/h; UV detection at 240nm. Purity was monitored on an analytical HPLC column: Perkin-Elmer NH2; particle size 101.tm; mobile phase, hexane-Et20-EtOH (41:41:8, v/v/v); flow rate 0.6ml/min; detection with UV at 240nm. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Some are used routinely in medicine.

1.

Indole Alkaloids

121

Spectral Data 1H NMR: (CDCI3) NH(I), 8.00(s); H-2, 6.92(s); H-9, 6.46(dd, J=4.4, 1.5Hz); N-CH3(6), 2.67(s); N-H(18), 8.26(d, J=9.8Hz); H-2', 5.94(dd, J=9.8, 2.4Hz); H-5 ~ 4.94(d, J=9.3Hz); H11', 4.36(t, J=7.6Hz); C-2' side chain methyls, 0.70(d, 6.8Hz), 0.90(d, 7.3Hz); and C-5' side chain methyls, 1.06(d, J=6.8Hz); 0.92ppm (t, J=7.3Hz). 13C NMR: (CDCI3) C-2, 118.8; C-3, 111.1; C-4, 29.8; C-5, 63.3; C-7, 50.4; C-8, 41.7; C-9, 119.8; C-10, 137.9; C-11,129.7; C-12, 111.8; C-13, 123.3; C-14, 109.8 C-15, 133.9; C-16, 126.3; C-17, 174.7; N-Me, 44.4; C-2', 56.5; C-3', 173.6; C-5', 60.0; C-6', 165.6; C-8', 45.5; C-9' 22.9; C-10 ~ 23.1; C-11', 60.3; C-IT, 170.0; C-13', 36.8; C-14', 27.4; C-15', 11.7; C-16', 13.6; C-IT, 37.3; C-18', 25.7; C-19', 10.9, and C-20', 15.5ppm. Mass Spectrum: M* observed in the mass spectrum, 573(4%), 363(C22H25NaO2, 7), 335(C21H25N30, 12), 221(C~sH13N2, 21), 154(CTH~oN202,91), 125(C6H9N20, 22), 86(CsH~2N, 12), and 70m/e (CaHsN, 100). TLC Purification Silica gel plates developed with methylene chloride-isopropyl alcohol (92:8, v/v) three times which results in the separation of alkaloids into the isolysergic acid group, the ergotoxine group, the ergoxine group and a mixture of the ergotamine and clavine groups. Extraction of the alkaloids from the silica using methanol-chloroform (1:4 or 1:1, v/v) followed by rechromatography in chloroform-methanol (9:1 or 4:1, v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines). TLC Solvent systems for silica gel chromatography (v/v): a) Methylene chloride-isopropyl alcohol (92:8; 90:10; 75:25). b) Chloroform-methanol (90:10; 80:20; 90:10 in saturated NH3 atmosphere). c) Chloroform-methanol-NH3 (94:5:1). d) Chloroform-ethylamine (90:10). e) Benzene-dimethylformamide (86.5:13.5). References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacolowy; Springer-Verlag, New York (1978). M. Flieger, P. Sedmera, J. Vokoun, Z. l~eh~i~ek,J. Stuchlik, Z. Malinka, L. Cvak, and P. Harazim; New Alkaloids from a Saprophytic Culture of Claviceps purpurea; J. Natural Products, Vol. 47, pp. 970-976(1984). J. K. Porter; Analysis of Endophyte Toxins: Fescue and Other Grasses Toxic to Livestock; J. Animal Science, Vol. 73, pp. 871-880(1994).

122

1.

Indole Alkaloids

Common/Systematic Name Ergobutine Molecular Formula/Molecular Weight C29HasNsOs; MW = 533.26382 Me I

13' CI-12 _OH I

o

I n--- J

,7~

H , , ~ ,~ 12

"J

1

NH ............ ,,,l~z" ' ~ l ' N / II_ H 16'

O---a3, s/N--Me

,

Ns,,,,,5,~O / ' ""-.1..6'

H-

---cN I Me

NH Fungal Source C l a v i c e p s p u r p u r e a strain 231 FI.

Isolation/Purification The whole broth culture was acidified to pH 2.5 with solid tartaric acid, homogenized, and centrifuged. The sediment was extracted twice with an aqueous solution of tartaric acid. The pooled supernatants were adjusted to pH 9-10 with 5N NaOH and extracted three times with chloroform-methanol (1:1, v/v). The combined extracts were dried over anhydrous Na2SO4 and concentrated in vacuo. The alkaloids were precipitated with petroleum ether, washed with the same solvent and dried m vacuo. The solid material was subjected to partial purification by dissolving it into 15ml of methanol and 15ml of an aqueous solution of 5% tartaric acid (w/v). The solution was brought to 600ml with water, and aliquots of 20ml were adsorbed onto 30 Extrelut (Merck) columns. Each column was percolated with chloroform, and the combined eluates were concentrated; the alkaloids were precipitated with petroleum ether, and the recovered material was washed and dried as above. Thin-layer chromatography was performed with silica gel 60 F254 (Merck). The crude extract was applied atter being dissolved in chloroform-methanol (1:1, v/v). Solvent systems were: chloroform-isopropanol (92:8, v/v); chloroform-methanol (90:10, v/v); and chloroform-methanol-cone, ammonia (90:10:1, v/v/v). The alkaloids were detected under UV light at 254 and 366nm and/or detected by spraying with N,N-p-dimethylaminobenzaldehyde. The alkaloids were scraped and eluted from the silica gel with chloroform-methanol (11 v/v).

1.

Indole Alkaloids

123

Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV: ~ MeOH max

310nm typical for lysergic acid derivatives.

Mass Spectrum: Field desorption mass spectrum 533(M+) and 267m/e (fragment ion for lysergamide); EIMS: 533(M+), 351,267, 181, 154, and 70role (100%). Reference M. L. Bianchi, N. C. Perellino, B. Gioia, and A. Minghetti; Production by Claviceps purpurea of Two New Peptide Ergot Alkaloids Belonging to a New Series Containing aAminobutyric Acid; J. Natural Products, Vol. 45, pp. 191-196(1982).

124

1.

Indole Alkaloids

Common/Systematic Name Ergobutyrine v

Molecular Formula/Molecular Weightht C3oH37NsOs, M W = 547.27947

Me

\/

Me

13' C H

o

II

la

17C

NH ............ ,,

O--L, '

'"

N--Me 12

I

OH f

O

-

11' 0

N '--. is' Me

NH

Fungal Source Claviceps purpurea strain 231 FI.

Isolation/Purification The whole broth culture was acidified to pH 2.5 with solid tartaric acid, homogenized, and centrifuged. The sediment was extracted twice with an aqueous solution of tartaric acid. The pooled supernatants were adjusted to pH 9-10 with 5 N NaOH and extracted three times with chloroform-methanol (1:1, v/v). The combined extracts were dried over anhydrous Na2SOa and concentrated in vacuo. The alkaloids were precipitated with petroleum ether, washed with the same solvent and dried m vacuo. The solid material was subjected to partial purification by dissolving it into 15 ml of methanol and 15 ml of an aqueous solution of 5% tartaric acid (w/v). The solution was brought to 600ml with water, and aliquots of 20ml were adsorbed onto 30 Extrelut (Merck) columns. Each column was percolated with chloroform, and the combined eluates were concentrated; the alkaloids were precipitated with petroleum ether, and the recovered material was washed and dried as above. Thin-layer chromatography was performed with silica gel 60 F254 (Merck). The,crude extract was applied after being dissolved in chloroform-methanol (1:1, v/v). Solvent systems were: chloroform-isopropanol (92:8, v/v); chloroform-methanol (90:10, v/v); and chloroform-methanol-conc, ammonia (90:10:1, v/v/v). The alkaloids were detected under UV light at 254 and 366nm and/or detected by spraying with N,N-p-dimethylarninobenzaldehyde. The alkaloids were scraped and eluted from the silica gel with chloroform-methanol (1:1, v/v).

1.

Indole Alkaloids

125

Biological Activity The ergopeptine alkaloids produce a wide range of biological activities and some are used routinely in medical practice. All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components). Spectral Data UV; ~MeOH max

310nm typical for lysergic acid derivatives.

Mass Spectrum: Field desorption mass spectrum 547(M +) and 267m/e (fragment ion for lysergamide); EIMS: 547(M+), 365, 280, 267, 181, 154, and 70role (100%). Reference M. L. Bianchi, N. C. Perellino, B. Gioia, and A. Minghetti; Production by Claviceps purpurea of Two New Peptide Ergot Alkaloids Belonging to a New Series Containing aAminobutyric Acid; J. Natural Products, Vol. 45, pp. 191-196(1982).

126

1. IndoleAlkaloids

C0mmon/Systematic Name Rugulovasine A Molecular Formula/Molecular Weight C16HI6N202; MW = 268.12118 16

Me~

~O

17

[[ .'5OI .,,,,,NHMe

8

General Characteristics Crystallizes as large, clear cubes from chloroform; rap., 138~ (dec.); [a]43622 -3.0 ~ (c=l.0, in pyridine); [~]D22 0.0. Rugulovasine A can be interconverted with its diastereomer, rugulovasine B upon warming in polar solvents. Fungal Source Penicillium islandicum, P. concavo-rugulosum, P. verruculosum, P. rubrum, and P. biforme. Isolation/Purification See 8-chlororugulovasine A. Biological Activity Rugulovasine A exhibited hypotensive action on chloralose-urethanized cats; the minimum effective dose was 0.2-0.5mg/kg. Spectral Data UV: ~,E~

224(e=23,400), 277(5,000), 288(6,000), and 295nm (6,000).

~3C NMR: C-2, 119.6 d; C-3, 109.8 s; C-4, 126.4 s*; C-5, 128.2 s*; C-6, 114.8 d; C-7, 122.8 d; C-8. 111.4 d; C-9, 133.9 s; C-10, 25.4 t; C-11, 63.7 d; C-12, 88.5 s; C-13, 150.6 d; C-14, 129.2 s; C-15, 174.3 s;'C-16, 10.8 q; and C-17, 3S.lppm q. * Assignments could be reversed.

1. Indole Alkaloids

127

TLC Data Silica gel PF254; Solvent: chloroform-acetone-methanol, 93:7:5, v/v/v; Re: 0.22. Detection: rose-colored spot after spraying with 50% ethanolic H2SO4 followed immediately by spraying with ninhydrin and heating at 100 ~ for 5-10 min.

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 537 (1981). R. J. Cole, J. W. Kirksey, J. Clardy, N. Eichman, S. M. Weinreb, P. Singh, and D. Kim; Structures of rugulovasine-A and rugulovasine-B and 8-chlororugulovasine-A and B; Tet. Lett., pp. 3849-3852(1976). R. J. Cole, J. W. Kirksey, H. G. Cutler, D. M. Wilson, and G. Morgan-Jones; Two toxic indole alkaloids from Penicillium islandicum; Can. J. Microbiol., Vol. 22, pp. 741-744 (1976).

128

1.

Indole Alkaloids

Common/Systematic Name 8-Chlororugulovasine A Molecular Formula/Molecular Weight C16HIsN202CI; MW = 302.08221 16

Me~

~,O 17

I "/

,NHMe

]: CI Fungal Source Penicillium islandicum. Isolation/Purification Rugulovasines A & B and chlororugulovasines A & B were extracted from fungal cultures with ethyl acetate in a Waring Blender after the pH was adjusted to 10.5 with aqueous sodium carbonate. The ethyl acetate fraction was separated from the aqueous fraction and dried over anhydrous sodium sulfate. After filtering, the crude extract was evaporated to dryness under vacuum and chromatographed on a silica gel column (70-230 mesh). The column was packed as a slurry in ethyl acetate and the extract was applied to the column in ethyl acetate solution. Toxins were eluted from the column by a linear gradient of ethyl acetate to acetone. 8-Chlororugulovasine A can be interconverted with its diastereomer, chlororugulovasine B upon warming in polar solvents. Biological Activity LDs0 in day old cockerels dosed orally, 75-125mg/kg. Spectral Dat___~a UV~

~

EtOH max

225(e=21,400), 280(sh)(4,600), 292(5,200), and 298nm (5,400).

1H NMR: H-l, 8.60; H-2, 7.00; H-6, 6.77(`/=7.6); H-7, 7.11(J=7.6); H-10, 3.30; H-11, 3.30; NH-1, 1.43; H-13, 7.35(./=1.5); H-16, 2.02(,/=1.5); and H-17, 2.43ppm.

1.

Indole Alkaloids

129

13C NMR: C-2, 120.6 d; C-3, 109.9 s; C-4, 124.8 s; C-5, 127.4 s; C-6, 116.0 d; C-7, 122.3 d; C-8, 116.5 s; C-9, 131.1 s*; C-10, 24.3 t; C-11, 63.6 d; C-12, 87.4 s; C-13, 150.2 d; C-14, 129.8 s*; C-15, 173.5 s; C-16, 10.8 q; and C-17, 34.8ppm q. * Assignments could be reversed. Mass Spectrum: HREIMS mass spectrum showed 302.0800m/e (M+). The presence of a chlorine atom was supported by the expected isotope ratios (relative intensity) 302.0800 = 86.5 ; 304.0778 = 25. Base peak was at 259.064 lm/e. TLC Data Silica gel PF254, chloroform-acetone-methanol, 93:7:5, v/v/v; Re: 0.27. Detection: rose-colored spot atter spraying with 50% ethanolic H2SO4 followed immediately by spraying with ninhydrin and heating at 100 ~ for 5-10 min. References R. J. Cole, J. W. Kirksey, H. G. Cutler, D. M. Wilson, and G. Morgan-Jones; Two Toxic Indole Alkaloids from Penicillium islandicum; Can. J. Microbiol., Vol. 22, pp. 741-744(1976). R. J. Cole, J. W. Kirksey, J. Clardy, N. Eichman, S. M. Weinreb, P. Singh, and D. Kim; Structures of Rugulovasine-A and Rugulovasine-B and 8-Chlororugulovasine-A and B; Tet. Lett., pp. 3849-3852(1976). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 528 (1981).

130

1. Indole Alkaloids

Common/Systematic Name Rugulovasine B Molecular Formula/Molecular Weight C16H16N202; M W -- 268.12118 16

Me"-r~-~O II ~(~

%,.,,,~

17 NHMe

8

General Characteristics Colorless resinous oil; [a]43622 +1.4 ~ (c=l.0, in pyridine); [a]D 22 0.0. Fungal Source Penicillium islandicum, P. concavo-rugulosum, P. rubrum, and P. biforme. Isolation/Purification See 8-chlororugulovasine A. Biological Activity Rugulovasine B exhibited hypotensive action in experimental animals; in chloralose-urethanized cats, the minimum effective dose was 0.025-0.05mg/kg. Spectral Data UV:

~,mE~H

224(e=14,500), 277(4,800), 288(5,400), and 295nm (5,200).

1H NMR: H-I, 8.68; H-2, 7.00; H-6, 6.83; H-7, 7.2: H-8, 7.2; H-10, 3.25; H-11, 3.25; NH-1, 1.70~ H-13, 6.98; H-16, 2.02; and H-17, 2.43ppm. 13C NMR: C-2, 120.0 d; C-3, 108.4 s; C-4, 125.8 s*;C-5, 126.1 s*; C-6, 114.4 d; C-7, 122.7 d; C-8, 111.2 d; C-9, 133.9 s; C-10, 24.3 t; C-11, 63.6 d; C-12, 88.2 s; C-13, 150.9 d; C-14, 129.2 s; C-15, 174.0 s; C-16, 10.7 q; and C-17, 34.7ppm q. " Assignments could be reversed.

1.

Indole Alkaloids

131

TLC Data Silica gel PF254; solvent chloroform-acetone-methanol, 93:7:5, v/v/v; Re: 0.34. Detection: rose-colored spot after spraying with 50% ethanolic H2SO4 followed immediately by spraying with ninhydrin and heating at 100 ~ for 5-10 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 540 (1981). R. J. Cole, J. W. Kirksey, J. Clardy, N. Eichman, S. M. Weinreb, P. Singh, and D. Kim; Structures of Rugulovasine-A and Rugulovasine-B and 8-Chlororugulovasine-A and B; Tet. Lett., pp. 3849-3852(1976). R. J. Cole, J. W. Kirksey, H. G. Cutler, D. M. Wilson, and G. Morgan-Jones; Two Toxic Indole Alkaloids from Penicillium islandicum; Can. J. Microbiol., Vol. 22, pp. 741-744 (1976).

132

1.

Indole Alkaloids

Common/Systematic Name 8-Chlororugulovasine B Molecular Formula/Molecular Weight CI6HlsN202CI; M W

302.08221

=

16

M e ~ II

O 17 ~l

NHMe

k. ,,,,O / 4

3

CI Fungal Source

Penicillium islandicum.

Isolation/Purification See 8-chlororugulovasine A Biological Activity LDs0 of chlororugulovasine B in preliminary studies showed it to be as toxic as chlororugulovasine A in day-old cockerels dosed orally (75-125mg/kg). Spectral Data UV: ~ mEtOH ax

224(e=22,400), 278(4,700), 288(5,100), and 295nm (5,400).

~H NMR: H-l, 8.89; H-2, 7.09; H-6, 6.73(,/--7.6); H-7, 7.05(J=7.6); H-10, 3.07; H-11, 3.07; NH-1, 1.61; H-13, 6.78(J=1.5); H-16, 2.01(J=1.5); and H-17, 2.44ppm. 13C NMR: C-2, 120.1 d; C-3, 110.6 s; C-4, 124.9 s; C-5, 127.5 s; C-6, 115.8 d; C-7, 122.0 d; C-8, 116.5 s; C-9, 130.9 s*; C-10, 25.1 t; C-11, 63.0 d; C-12, 87.8 s; C-13, 149.9 d; C-14, 129.3 s*; C-15, 173.8 s;.C-16, 10.7 q; and C-17, 34.9ppm q. * Assignments could be reversed.

1.

Indole Alkaloids

133

TLC Data Silica gel PF2s4; solvent: chloroform-acetone-methanol, 73:7:5, v/v/v; Re 0.44. Detection: rose-colored spot after spraying with 50% ethanolic H2SO4 followed immediately with ninhydrin spraying and heating at 100 ~ for 5-10 min. References R. J. Cole, J. W. Kirksey, J. Clardy, N. Eichman, S. M. Weinreb, P. SingJt, and D. Kim; Structures of Rugulovasine-A and gugulovasine-B and 8-Chlororugulovasine-A and B; Tet. Lett., pp. 3849-3852(1976). R. J. Cole, J. W. Kirksey, H. G. Cutler, D. M. Wilson, and G. Morgan-Jones; Two Toxic Indole Alkaloids from Penicillium islandicum; Can. J. Microbiol., Vol. 22, pp. 741-744 (1976). R.J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabo!ites; Academic Press, New York, p. 532 (1981).

134

1. Indole Alkaloids

Common/Systematic Name Fumigaclavine A 9-Acetoxy-6, 8-dimethylergoline Molecular Formula/Molecular Weight CIgH22N202; M W = 298.16813 Me

0

H

H

'--

"~

Nil

General Characteristics Colorless needles; mp., 84-~176 (crystals from aqueous MeOH); [tt]5~s~ -56.7 ~ (pyridine); intense blue color with AUport and Cocking's reagent. Hydrochloride: prisms from EtOH; mp., 3040-305 ~ (dec.), [tt]~24622, -56.7 ~ (c=1.5, in MeOH) Fungal Source

Aspergillusfumigatus.

Isolation/Purification Extracted with chloroform, filtered, and dried over anhydrous sodium sulfate. The crude chloroform extract was concentrated under vacuum, redissolved in ethyl acetate, and partitioned three times between an aqueous solution adjusted to pH 2.0 with HCI. The acidic phases were combined, made basic (pH 10.0) with sodium carbonate, and partitioned three times with chloroform. The chloroform fractions were pooled and washed three times with distilled water. The extract was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The resulting concentrate was chromatographed on a column containing neutral alumina that was deactivated to activity grade IV. The column was packed as a slurry in benzene, the sample was applied in benzene solution, and the column was eluted with benzene. This yielded fumagaclavines A and C in pure form after crystallization from methanol solution. Biological Activity All natural ergot alkaloids possess to a greater or lesser degree, biological effects that can be classified as direct peripheral effects on smooth muscle (uterine contraction and vasoconstriction), indirect peripheral effects (humoral, serotonin antagonism, and adrenergic blockage), and central nervous system effects (bulbomedullary and mesodiencephalic components).

1. Indole Alkaloids

135

Spectral Data UV: M,K)H ~ m~

225, 275, 282, and 293nm (e max notreported).

IR~

See Hofmann, 1964 ~H NMR: 1-NH, 7.94; H-2, 6.86; H-9, 5.70; H-12, 7.12; H-13, 6.76; H-14, 7.16; H-17, 2.47; H18, 1.35(d=7.0); and H-20, 1.90ppm. 13C NMR: C-2, 117.7 d; C-3, 111.6 s; C-4, 26.8 t; C-5, 61.9 d; C-7, 57.9 t; C-8 33.2 d; C-9, 71.6 d; C-10, 39.8 d; C-11, 129.9 s; C-12, 112.9 d; C-13, 123.2 d; C-14, 108.3 d; C-15, 133.6 s; C-16, 126.8 s; C-17, 43.6 q; C-18, 16.8 q; C-19, 170.8 s; and C-20, 21.2ppm q. TLC Data Silica gel G-HR; toluene-ethyl acetate-formic acid, 5:4:1 v/v/v; Re: 0.05. Detection: purple spot after spraying with 50% ethanolic H:SO4 and heating at 100~ for 5-10 min. References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). R. J. Cole, J. W. Kirksey, J. W. Domer, D. M. Wilson, J. C. Johnson, Jr., A. N. Johnson, D. M. Bedel, J. P. Springer, K. K. Chexal, J. C. Clardy, and R. H. Cox; Mycotoxins Produced by Aspergillusfumigatus Species Isolated From Molded Silage; Agile. Food Chem., Vol. 25, pp. 826-830(1977). A. Hofmann; Die Mutterkorn Alkaloide; Enke Verlag, Stuttgart, 218 pp., 1964. J. F. Spilsbury and S. Wilkinson; J. Chem. Soc. p. 2085 (1961).

136

1. Indole Alkaloids

Common/Systematic Name Roquefortine A; Isofumigaclavine A 9-Acetoxy-6, 8-dimethylergoline Molecular Formula/Molecular Weight CIgH22N202; M W = 298.16831 Me H M e C O 0 .......

N--Me

NH

General Characteristics Crystals from benzene; mp., 190~176

[tg]D22 -54.1 ~

(c=0.67, in CHCI3).

Fungal Source Penicillium roqueforti. Biological Activity Roquefortine A exhibited a variety of weak pharmacological actions, such as muscle relaxant, antidepressant, local anesthetic, etc., in experimental animals. Its LDs0 in mice dosed IP was 340m~kg. Spectral Data UWz

~,mM~" 226(e=46,300), 277(sh)(6,000), 283(6,500), and 293nm (5,600). IH NMR: 1-NH, 10.76i H-2, 7.07; H-9, 5.06; H-12, 6.70(J=6.7); H-13, 7.07; H-14, 7.25(J=8.5); H-17, 2.40; H-18, 0.95(J=4.0); and H-20, 2.28ppm. 13C N M R :

C-2, 119.1 d; C-3, 110.7 s; C-4,28.4 t; C-5, 62.3 d; C-7, 55.4 t; C-8, 37.5 d; C-9, 79.6 d; C-10, 38.5 d; C-11, 131.5 s; C-12, 113.9 d; C-13, 122.8 d; C-14, 110.0 d; C-15, 134.2 s; C-16, 113.9 s; C-17, 45.9 q; C-18, 16.4 q; C-19, 171.5 s; and C-20, 22.2ppm q.

1.

Indole Alkaloids

137

TLC Data Silica gel F 1500/LS 254 (Schleicher and Schuell); solvent: chloroform-methanol-28% ammonium hydroxide, 85 15"1 v/v/v; Re 0.62. Detection: a mauve spot after spraying with 50% H2SO4 and heating at about 100~ for 10 min. References R. J. Cole and R. H. Cox; Handbook of Toxic F unga.! Metabolites; Academic Press, New York, p. 549 (1981). P. M. Scott, J. Polonsky, and M. A. Merrien; Configuration of the 3,12- Double Bond of Roquefortine; J. Agrie. Food Chem. Vol. 27, p. 201 (1979).

138

1.

Indole Alkaloids

Common/Systematic Name Fumigaclavine B 9-Hydroxy-6,8-dimethylergoline Molecular Formula/Molecular Weight CI6H2oN20; M W -- 2 5 6 . 1 5 7 5 6

Me

H

General Characteristics Needles from aqueous methanol; 244-245~ (at 260~ the melt solidified and remelted at 265-267~ [a]546~22 -6.3 ~ (c=1.2, in MeOH); -113 ~ (c=0.6, in pyridine). Fungal Source

Aspergillusfumigatus and Rhizopus arrhizus.

Spectral Data UV~

225(e=30,900), 275(6,200), 282(6,600), and 293nm (5,200). IR~

(See A. Hofmann, 1964). ~H NMR: 1-NH, 8.04; H-2, 6.96; H-9, 4.54; H-11, 6.87; H-12, 7.18; H-13, 7.21; H-17, 2.43; and H- 18, 1.30ppm. 13C NMR: C-2, 117.6 d; C-3, 111.6 s; C-4, 26.2 t; C-5, 60.9 d; C-7, 57.4 t; C-8, 34.0 d; C-9, 69.0 d; C-10, 41.6 d; C-11, 129.5 s; C-12, 112.2 d; C-13, 123.0 d; C-14, 108.7 d; C-15, 133.5 s; C-16, 126.9 s; C-17, 43.4 q; and C-18, 16.9ppm q. TLC Data Silica gel G-HR; toluene-ethyl acetate-formic acid, 5:4:1 v/v/v; Re origin. Detection: purple spot after spraying with 50% ethanolic H2SO4 and heating at 100~ for 5-10 rain.

1. Indole Alkaloids

139

References B. Berde and H. O. Scheld (eds.); Ergot Alkaloids and Related Compounds; In Handbook of Experimental Pharmacology; Springer-Verlag, New York (1978). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 553 (1981). R. J. Cole, J. W. Kirksey, J. W. Dorner, D. M. Wilson, J. C. Johnson, Jr., A. N. Johnson, D. M. Bedel, J. P. Springer, K. K. Chexal, J. C. Clardy, and R. H. Cox; Mycotoxins Produced by Aspergillusfumigatus Species Isolated from Molded Silage; Agric. Food Chem., Voi. 25, pp. 826-830(1977). A. Hofmann; Die Mutterkorn Alkaloide, Enke Verlag, Stuttgart, 218 pp., 1964. J. F. Spilsbury and S. Wilkinson; J. Chem. Soc., p. 2085 (1961).

140

1.

Indole Alkaloids

Common/Systematic Name Roquefortine B; Isofumigaclavine B 9-Hydroxy-6, 8-dimethylergoline Molecular Formula/Molecular Weight CI6H2oN20; MW = 256.15756 18

.o"N-Me Me

General Characteristics Melting point, 278~176 (dec.); colorless needles from methanol; mp., 222~ (760mm Hg); [a]D~s -147~ in pyridine). Fungal Source

Penicillium roqueforti.

Biological Activity The LD~0 of roquefortine B dosed IP to mice was 1000mg/kg. Spectral Data UV: ~mM~176 224(e=44,700), 275(6,200), 282(7,000), and 294nm(6,000); ~ , 225(e=25,100), 277(sh)(4,300), 283(4,600), and 293nm(3,800). TLC Data Silica gel F 1500FLS254 thin-layer sheets; chloroform-methanol-28% ammonium hydroxide, 85:15:1 v/v/v; Rf: 0.31. Detection: a mauve spot after spraying with 50% H2SO4 and heating at about 100~ for 10 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 557 (1981).

1.

Indole Alkaloids

141

P. M. Scott, J. Polonsky, and M. A. Merrien; Configuration of the 3,12-Double Bond of Roquefortine; J. Agric. Food Chem. Vol. 27, p. 201 (1979).

142

1.

Indole Alkaloids

Common/Systematic Name Fumigaclavine C 2-Dimethylallyl-9-acetoxy-6, 8-dimethylergoline Molecular Formula/Molecular Weight C23H3oN202; MW = 366.23073

Mecoo N-Mo

LL. General Characteristics Crystals from ethanol, acetone, or ethyl acetate (prisms), mp 191-193 ~ Needles from methanol; mp., 198~ [a]o 22 -90; [a]546122 -119 ~ (c=l.0, in CHCI3); [a]o 22 -132~ [~]546122 -160 ~ (C-'I.0, in pyridine). Fungal Source

Aspergillusfumigatus.

Isolation/Purification See fumigaclavine A. Biological Activity The LDs0 of fumigaclavine C dosed orally to day-old cockerels was 150mg/kg. Histopathology of surviving cockerels showed vacuolation of the hepatic parenchymal cells that may have been directly related to the toxic action of the toxin or due to anorexia. Spectral Data UV:

M.o. 229, 284, and 292nm (e max not reported); ~,.~M.o. 227(10,200), 283(11,000), and 292nm(9,500).

225(e=34,700),

IH ~ : 1-NH, 7.76; H-9, 5.67; H-12, 7.04; H-13, 6.72; H-14, 7.08; H-17, 2.46; H-18, 1.35; H-20, 1.90; H-22, 1.56; H-23, 1.56; H-24, 6.10; H-25a, 5.05; and H-25b, 5.20ppm.

1.

Indole Alkaloids

143

~3C NMR: C-2, 131.1 s; C-3, 106.2 s; C-4, 28.1 t; C-5, 61.6 d; C-7, 57.8 t; C-8, 33.1 d; C-9, 71.4 d; C-10, 39.4 d; C-11,129.1 s; C-12, 112.8 d; C-13, 122.2 d; C-14, 107.6 d; C-15, 136.6 s; C-16, 128.0 s; C-17, 43.5 q; C-18, 16.7 q; C-19, 170.8 s; C-20, 21.2 q, C-21, 39.1 s; C-22, 27.3 q; C-23, 27.4 q; C-24, 145.7 d; and C-25, 111.Sppm t. TLC Data Silica gel G-HR; toluene-ethyl acetate-formic acid, 5:4:1 v/v/v; Re: 0.14. Detection: blue spot aiter spraying with 50% ethanolic H2SO4 and heating at 100~ for 5-10 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 560-561 (1981). R. J. Cole, J. W. Kirksey, J. W. Dorner, D. M. Wilson, J. C. Johnson, Jr., A. N. Johnson, D. M. Bedel, J. P. Springer, K. K. Chexal, J. C. Clardy, and R. H. Cox; Mycotoxins Produced by Aspergillusfumigatus Species Isolated from Molded Silage; Agile. Food Chem., Vol. 25, pp. 826-830(1977).


Diketopiperazines Roquefortine; Roquefortine C Aurantiamine Viridamine Okaramine A Okaramine B

2

cyclo-L-Phenylalanyl-L-alanine cyclo-L-Prolylglycine L-Propyldiketopiperazine B; L-Homoleucyl-D-proline-lactam L-Propyl-L-tyrosine Isoleucylisoleucyl anhydride Phomamide D-Valyl-L-tryptophan anhydride L-Alanyl-L-tryptophan anhydride

cyclo-(L-Isoleucyl-L-valine ) cyclo-(L-Alanyl-L-proline)

Neoxaline Oxaline Aszonalenin LL-$49013; Acetylaszonalenin Bipolaramide Austamide 12,13-Dihydroaustamide 12,13-Dihydro- 12-hydroxyaustamide Cycloechinulin 12,13-Dehydroprolyl-2-(l', l'-dimethylallyltryptophyl)diketopiperazine Alanyl-2-(1,1-dimethyl-2-propenyl)dehydrotryptophan anhydride 10,20-Dehydro [ 12,13-dehydroprolyl]-2-( 1', 1'-dimethylallyltryptophyl)diketopiperazine Deoxybrevianamide E Brevianamide C Brevianamide D Brevianamide F Eehinulin Preechinulin Neoechinulin Neoechinulin A Neoechinulin B; Cryptoechinulin C; E 10 Neoechinulin C; Cryptoechinulin A; E8 Neoechinulin D Neoechinulin E Isoechinulin A Isoechinulin B Isoechinulin C E-7 Cryptoechinulin G

145

146

2. Diketopiperazines

Cryptoechinulin B; Aurechinulin; E6 Cryptoechinulin D Fumitremorgin A Fumitremorgin B; Lanosulin Fumitremorgin C; SM-Q Verruculogen 15-Acetoxyverruculogen TR-2 Epiamauromine N-Methylepiamauromine Ditryptophenaline Exserohilone 9,10-Dihydroexserohilone Epoxyexserohilone


147

Common/Systematic Name Roquefortine; Roquefortine C 1013-(Dimethyl-2-propenyl)-3-(imidazol-4-ylmethylene)-5 a, 1013,11,11atetrahydro-2H-pyrazino [ 1',2': 1,5]pyrrolo[2,3-b]indole- 1,4-(3H,6H)-dione Molecular Formula/Molecular Weight C22H23NsO2; M W = 389.18518 20

16

F,7 O General Characteristics Colorless needles of the solvate from methanol-water; mp., 195-200~ (dec.); [a]o 22 -703 ~ (c=1.0, in CHCI3). Fungal Source Penicillium roqueforti, P. notatum, P. oxaficum, P. commune, P. corymbiferum, P. expansum, and P. urticae. Isolation/Purification Fungal cultures were filtered and the mycelium macerated with acetone in a Waring blender. The acetone solution was evaporated and the aqueous residue partitioned between dichloromethane and water. The residue from the dichloromethane solution was partitioned between n-hexane and 90% methanol. The 90% methanol solution was evaporated and the aqueous residue once again partitioned between dichloromethane and water. The crude extract obtained from the dichloromethane solution was percolated through a short silica-gel column with benzene-acetone (4:1, v/v). Residual material on the column was eluted with benzene-acetone (1:4, v/v) to give the fraction containing crude roquefortine. This material was purified by percolation through an aluminum oxide (activity II-III) column with chloroform-methanol (9:1, v/v) to yield, after crystallization from aqueous methanol, crystalline roquefortine. Biological Activity The LDs0 (IP, male mice) was 15-20mg/kg. Doses of 50-100mg/kg caused prostration and an atonic posture. The prostration was interrupted for a few seconds by opisthotonoid seizures which could be brought about by noise or a tap on the cage. Death followed within a few hours. At doses of 10mg/kg, convulsions were replaced by simple contractions and the state of prostration by equilibrium disorders during movement.

148


Spectral Data UV~

EtOH

~max

209(e= 29,500), 240(16,200), and 328nm (27,000).

IR~

(CHCI3) 3430, 3380, 3190, 1685, 1665, and 1608cm"~. IH NMR: (CDC13) H-5a, 5.71; H-7-10, 6.58-7.30; H-11, 2.56; H-1 la, 4.00; H-12, 6.48; H-15, 7.73; H-17, 7.20; H-19, 6.12; H-20, 5.10; H-21, 1.06; and H-22, 1.17pm. ~3C NMR: (CDCI3) C-I, 166.2 s; C-3, 123.7 s; C-4, 159.8 s; C-5a, 78.9 d; C-6a, 151.9 s; C-7, 110.3 d; C-8, 129.8 d; C-9, 118.9 d; C-10, 125.6 d; C-10a, 129.4 s; C-10b, 62.1 d; C11,38.0 t; C-1 la, 59.4 d; C-12, 109.6 d; C-13, 125.6 d; C-15, 137.3 d; C-17, 134.5 d; C-18, 41.7 s; C-19, 144.9 d; C-20, 114.2 t; C-21, 23.0 q; and C-22, 23.3ppm q. Mass Spectrum: LREIMS: 389(M+), 320(base peak), 198, 192, 157, 130, and 108m/e. TLC Data TLC: silica gel F 1500/LS254; chloroform-methanol-28% ammonium hydroxide, 90:10:1 v/v/v; Rf: 0.46. Detection: a blue spot after spraying with 50% H2SO4 and heating at 110~ for 10 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp.564 (1981). A. E. de Jesus, P. S. Steyn, F. R. van Heerden, R. Vleggaar, P. L. Wessels, and W. E. Hull; Tremorgenic Mycotoxins from Penicillium crustosum: Isolation ofPenitrems A-F and the Structure Elucidation and Absolute Configuration of Penitrem A; J. Chem. Soc. Perkin Trans. I, pp. 1847-1856(1983). P. M. Scott; Roquefortine, In Mycotoxins-Production, Isolation, Separation, and Purification; V. Betina (ed.) Elsevier Science Publishers B.V., Amsterdam (1984). P. M. Scott, M. A. Merrien, and J. Polonsky; Roquefortine and Isofumigaclavine A, Metabolites from Penicillium roqueforti; Experientia, Vol. 32, pp. 140-141(1976). P. M. Scott, M. A. Merrien, and J. Polonsky; Roquefortine and Isofumigaclavine A, Metabolites from Penicillium roqueforti; Annales De La Nutrition et De L'Alimentation, Vol. 31, pp. 693-698(1977). P. M. Scott, J. Polonsky, and M. A. Merrien; Configuration of the 3, 12 Double Bond of Roquefortine; J. Agric, Food Chem. Vol. 27, p. 201 (1979).


149

Common/Systematic Name Aurantiamine (Isomeric with viridamine) Molecular Formula/Molecular Weight C16H22N402; ~

= 302.17428

~

Me

"

H

1

12

NH

Me

0

General Characteristics Crystals from ethyl acetate; mp., 238-239~

[a]D23 -116 ~ (C=0.5, in MeOH).

Fungal Source Aurantiamine was produced by the majority of strains ofPenicillium aurantiogriseum var. aurantiogriseum and var. neoechinulatum. P. aurantiogriseum Diercks var. aurantiogriseum is the most common of all fungi in cereals and it is considered highly toxigenic. Isolation/Purification The fungal mat was extracted with CHCI3-MeOH (2:1, v/v). The extract was taken to dryness and the residue chromatographed on a silica gel column eluted with tolueneEtOAc (1:1 to 1:4, v/v). The fractions containing aurantiamine by TLC were pooled and taken to dryness, dissolved in hot EtOAc and filtered through activated charcoal. Crystals were formed on cooling and partial evaporation of the solvent. This procedure was repeated to give pure aurantiamine. Spectral Data UV:

~m~x (H20/MeCN with CF3CO2H) 230(40) and 320nm (100%). IH NIVIR:

(CDCI3) 1;2.16(exch., brs, H-12); 10.0(exch., brs, H-I); 7.57(brs, H-2); 6.96(s, H-6); 6.81(exch., brd, J=2Hz, H-9); 6.04[dd, 3--10.8 and 17.1Hz (X part of ABX pattern) H-17]; 5.15(2H, AB part of ABX pattern, CH2-18); 4.08(dd, ,/=2 and 3Hz, H-10); 2.47(dhept, J=3 and 7Hz, H-13); 1.49(6H, s, Me-19 and Me-20); and 10.9 and 0.95ppm (each 3H, d, J=7.0Hz, Me-14 and Me-15). 13C NMR: (CDCI3) C-2, 132.6 Dd; C-4, 132.1 Sdd; C-5, 136.7 Sm; C-6, 105.3 D; C-7, 123.5 Sd;

150


C-8, 160.8 Sm; C-10, 61.1 Dm; C-11,165.1 Stm; C-13, 33.0 Dm; C-14, 18.6 Qm; C15, 15.8 Qm; C-16, 37.5 Sm; C-17, 144.6 Dm; C-18, 113.1 DDs; and C-19/20, 27.9 Qm. (CD3OD) C-2,133.0 Ds ; C-4,130.5 Sdd ; C-5, 137.0 Sm; C-6, 105.3 D; C-7, 122.5 S; C-8, 160.6 Sdd; C-10, 60.0 Dm; C-11,165.5 Sddd; C-13, 33.1 Dm; C-14, 16.8 Qm; C-15, 14.8 Qm; C-16, 36.8 Sm; C-17, 144.4 Dm; C-18, 110.9 DDs; and C19/20, 26.5 Qm. * Capital letters refer to directly bonded protons and small letters to long-range couplings. Mass Spectrum: EIMS: 302(M+, 50%), 287(15), 260(28), 231(35), 203(100), and 188m/e. Reference T. O. Larsen, J. C. Frisvad, and S. R. Jensen; Aurantiamine, a Diketopiperazine from Two Varieties ofPenicillium aurantiogriseum; Phytochemistry, Vol. 31, pp. 1613-1615 (1992).


151

Common/Systematic Name Viridamine 2-(3',3'-DimethylaUyl)-6,7-didehydrocyclo-L-prolylhistdyl Molecular Formula/Molecular Weight C16H22N402; M W -" 302.17428

Me

,

HN---~

O

O

3N

Me General Characteristics A colorless glass;

[ a ] D 20 -

95,0 ~ (c=l.0, in CHCI3).

Fungal Source Penicillium viridicatum (strain no. CSIR 354). Isolation/Purification Viridamine was extracted from the dried moldy maize meal with 1:1 chloroform-methanol. An equal volume of water was added to the extract and the chloroform phase was collected. The aqueous phase was re-extracted with chloroform, the chloroform extracts were combined and the solvent was removed under reduced pressure. The residue was dissolved in a minimum of chloroform and diethyl ether was added. Precipitated material was removed by filtration and the diethyl ether-chloroform solution was repeatedly extracted with ice cold 2N hydrochloric acid. The hydrochloric acid extract was neutralized with sodium carbonate and extracted with chloroform. The chloroform was washed with 5N ammonia solution and water. The chloroform solution was dried over anhydrous Na2SO4 and evaporated under reduced pressure to yield a basic fraction which was chromatographed over alumina. The column was developed in chloroform-benzene (3:1, v/v) and chloroform. Elution with chloroform-methanol (95:5, v/v) gave viridamine as a colorless glass which could not be induced to crystallize. Spectral Data UV: MeOH

315(e=33,795), shii~ing to 300(28,280), and 338nm (39,610) on the addition of HCl and NaOH, resp.

~j'~max

152


IR~ (CHCI3) 3460, 3420, 3140-3340, 1680, and 1645 cm"~. IH NMR: (CDI3) 11.88(s, 12-a); 10.1(s, l-H); 7.22(d, ,]=2.0nz, 9-n); 7.05(s, 5-n); 6.63(s, 6H); 5.32(t, J=7Hz, 17-H); 4.04(t, J=2 and 4Iq~ 10-H); 3.43(d, J=-7Hz, 16-1-12);2.35(m, J13,14and Jl3,1s=7Hz, Jl3,10=4Hz); 1.71 and 1.65(2xs, 19-and 20-H3); 1.05 and 0.94ppm (2xd, ,]14,13and Jls, 13=7Hz; 14- and 15-H3). (DMSO-d6) 12.14(s, 12-H); 11.56(s, I-H); 8.21(d, J=2Hz, 9-H); 7.31(s, 5-H); 6.45(s, 6-H); 5.33(t, J=7Hz, 17-H); 3.91(q, J=4 and 2Hz, 10-H); 3.41(d, J=7Hz, 16-1-12); 2.14(m, J13,14and Jls,13=7Hz, Jl3,1o=4Hz, 13-H); 1.68(s, 19-and 20-H3); 0.93 and 0.83ppm (2xd, J14,13and Jls, la=7Hz, 14- and 15-H3). Mass Spectrum: LREIMS: 302(49%), 287(4), 274(3), 259(3), 258(4), 231(11), 203(12), 176(13), 175(100), 174(5), 120(13), 119(24), 79(5), and 69m/e (5); HREIMS: found: M +, 302.1701; calc. for CI6H22N402, 302.1743. Reference C. W. Holzapfel and J. J. Marsh; Isolation and Structure of Viridamine, A New Nitrogenous Metabolite of Penicillium viridicatum; S. Afr. J. Chem., Vol. XXX, pp. 197204(1977).


153

Common/Systematic Name Okaramine A Molecular Formula/Molecular Weight C32H32N403; MW' = 520.24744

11

.~,,.11,'

10' r ~

-~.

11b'/~~ ,1~

.

l

91

3"N'~_

General Characteristics Pale green needles from toluene; mp., 210-212~

[aid 30 +101 (c--0.09, in MeOH).

Fungal Source Penicillium simplicissimum (AK-40) -" P. brasilianum.

Isolation/Purification The acetone extract of okara (the insoluble residue of whole soybean) fermented with this fungal strain was concentrated and partitioned against ethyl acetate. Column chromatography of the ethyl acetate extract resulted in the isolation of okaramine A. Biological Activity Okaramine A caused about 90% of the test insects to die within 24hr at a dose of 30ppm in the diet when applied to the third instar larvae ofBombyx mori. Spectral Data UV:

~

MeOH

229(e=29,900), 255(21,100). 284(17,400), and 371 nm (19,400).

max

(KBr) 3460. 3300, 1610, 1480, 1375, and 742cm"~. ~H M R : Spectrum indicated the presence of the following partial structures: four tertiary methyls [1.52(3H, s), 1.65(3H, s), 1.69(3H s), 1.70(3H, s)];-CH2CH-[2.55(1H, dd,

154


J=13.5, 9.6Hz); 3.1 l(1H, dd, J=13.5, 5.9); 4.54(1H, dd, J=9.6, 5.9)]; a vinyl [5.04(1H, dd,`/=l 1.0, 1.1); 5.17(1H, dd, J=17.8, 1.1); 6.11(1H, dd,`/=17.8, 11.0)]; an isolated methine [5.52(1H, s)], a cis substituted ene [5.60(1H, d, J=7.9), 5.87(1H, d, J=7.9)]; a 2,3-disubstituted indole [7.08-7.20(2H, m); 7.38(1H, dd, `/=7.2, 1.0); 7.2 1H, dd, J=6.5, 2.6); 10.64(1H, br, s)]; a 1,2-disubstituted benzene ring [6.75(1H, td, ,/=7.2, 1.0); 6.90(1H, dd, `/=8.4, 1.0); 7.05(1H, ddd, `/=8.4, 7.2, 1.5); 7.72(1H, dd, ,/=7.2, 1.5)]; an olefinic methine [7.68(1H, s)], and a hydroxyl [4.71(1H, s)]. Mass Spectrum: The molecular formula of okaramine A was established to be C32H32N403,by high-resolution mass spectral data (found, 520.2477; caled., 520.2457). Reference S. Murao, H. Ayashi, K. Takiuchi, and M. Arai; Okaramine A, a Novel Indole Alkaloid with Insecticidal Activity, from Penicillium simplicissimum AK-40; Agrie. Biol. Chem.,Vol. 52, pp. 885-886(1988).


155

Common/Systematic Name Okaramine B Molecular Formula/Molecular Weight C33H34N4Os; M W = 5 6 6 . 2 5 2 9 2 14

,,,,!.0. 12,,,,!,!

N

4

O\ I~ OHm3OH 10'

11a'

Me

11

j3'N-"%_o

~ N

A General Characteristics Crystallized from methanol to afford okaramine B as pale yellow needles; mp., 295-298"C (dec.); [a]D2~+570 ~ (C=0.24, pyridine). Fungal Source Penicillium simplicissimum (AK-40) -, P. brasilianum.

Isolation/Purification The cultures were extracted with acetone, partitioned with ethyl acetate and chromatographed on a column containing Wakogel C-200 eluted with various concentrations of hexane in acetone. The active fractions eluted with 40 and 60% acetone-hexane. The active fractions were concentrated and chromatographed on alumina eluted with various concentrations of ethyl acetate-hexane. The 60% and 80% ethyl acetate fractions contained okaramine B which was crystallized from methanol as pale yellow needles. Biological Activity Insecticidal activity against third instar larvae of silkworm at 0.3ppm, 100% of the larvae were killed within 24hr. Spectral Data El"V:

~

MeOH max

233(e=27,000), 288 (12,900), and 375nm (17,900).

156


IR:

(KBr) 3400, 3320, 1670, 1610, 1465, 1360, and 750cm-1. IH M R : (DMSO-d6) 8-3, 4.19 s; 8-4, 7.38 d (2'=7.8); H-5, 7.01 t (2"=7.8);8-6, 7.20 t (2"=7.8); H-7, 6.72 d (2"=7.8); H-11, 3.10 q (,/=7.3); H-12, 1.23 d (/=7.3); H-13, 1.65 s; H-14, 0.86 s; H-I', 7.39 s; H-4', 5.90 d (2'=8.3); H-5', 5.67 d (,/=8.3); H-7', 11.42 br s; H-8', 7.41 d (2"=4.4); H-9', 7.14 m; H-10' 7.13 m; H-11', 7.59 dd (2"=5.4, 2.8); H-13', 1.58 s; H-14', 1.66 s; 2-OH, 6.42 s; 3-OMe, 3.73 s; and 3a-OH, 5.26ppm s. 13C NMR: (DMSO-d6) C-2, 86.0; C-3, 82.7; C-3a, C-3b, 126.4; C-4, 125.2; C-5, 123.0; C-6, 129.4; C-7, 117.8; C-7a, 140.1; C-8a, 93.6; C-9, 164.1; C-10, 62.3; C-I 1, 40.9; C-12, 11.1; C-13, 25.2; C-14, 26.3; C-I', 112.1; C-2', 150.3; C-4', 139.5; C-5', 122.3; C-6', 36.2; 6a', 148.1; C-7a', 134.2; C-8', 113.8; C-9', 121.9; C-10', 120.8; C-11', 116.7, C-1 la', 129.8; C-1 lb', 104.6; C-12', 162.4; C-13', 28.1; C-14' 27.3; and 3-OMe, 60.1ppm.

Reference H. Hayashi, K. Takiuchi, S. Murao, and M. Arai; Okaramine B and Insecticidal Indole Alkaloid, Produced by Penicillium simplicissimum AK-40; Agrie. Biol. Chem., Vol. 52, pp. 2131-2133(1988).


157

Common/Systematic Name cyclo-L-Phenylalanyl-L-alarfine Molecular Formula/Molecular Weight C12HI4N202; M W = 218.10553

O

"~

"Me

O General Characteristics Crystals from methanol; mp., 272-274~ + 63 ~(c=0.95, in acetic acid).

285~ (sealed tube); sublimed at 240~

[a]D~9

Fungal Source Entoloma haastii (fruiting bodies). Spectral Data UV: /~MeOH

max

204 and 285nm (weak).

IR:

(KBr) 3340, 3200, 1660, 1600, 1500, 750, 700, 1340, 1245, 1212, 1192, 1160, 970, 923, and 780cm"1. Mass Data: HREIMS: 318.1060m/e; found: C, 65.9, 66.1; H, 6.8, 6.9; N, 11.1, 12.1; calcd for CI2HI4N202: C, 66.0; H, 6.4; N, 12.9%. Reference E. P. White; 2,5-Dioxopiperazines from the Fungal Genera Entoloma and Fusarium; New Zealand J. of Science, Vol. 15, pp. 178-181(1972).

158


Common/Systematic Name cyclo-L-Prolyl glycine Molecular Formu.la/M01ecul.arWeight CTHIoN202; ~

= 154.07423

O

O General Characteristics Crystals from ethanol-ethyl acetate; mp., 202-204~

[ a ] D 20 -

88~

in water).

Fungal Source Fusarium equiseti, and F. scirpi (CMI 112503). Isolation/Purification Cultures were extracted with chloroform, reduced to dryness, and material soluble in hot ether concentrated to yield heavy crystals. These were washed with acetone and recrystallized from ethanol-ethyl acetate. Spectral D.ata UV: ~MeOH

max

202nm

IR:

(Nujol) 3150, 3160, 3200, 1675, 1640, 1295, 1270, 1110, 1005, 900, 890, 790, and 775cm~. Mass Analysis: LREIMS: 154m/e; found: C, 54.3; H, 6.7; N, 18.2; calcd for c7nl0N202, C, 54.5; H, 6.5; N, 18.1%. Reference E. P. White; 2,5-Dioxopiperazines from the Fungal Genera Entoloma and Fusarium; New Zealand J. of Science, Vol. 15, pp. 178-181 (1972).


159

Common/Systematic Name Prolyldiketopiperazine B; L-Homoleucyl-D-proline laetam Molecular Formula/Molecular Weight CI2H2oN202, M W = 224.15248

O

Me2CH(CH2)2~~L~ N 0 Fungal Source Sclerotia and saprophytic cultures of Clawceps sp. Spectral Data Mass Spectrum: LREIMS: 224(M+), 223, 195, 180, 153(100%), 99, 69, 43, and 28m/e. Reference S. Ohmomo and M. Abe; On a New Prolyldiketopiperazine Produced by Ergot Fungi; Nippon Nogei Kagaku Kaishi, Vol. 50, pp. 37-40(1976).

160


Common/Systematic Name L-Propyl-L-tyrosine Molecular Formula/Molecular Weight CI4HI6N203; MW - 260.11609

NH./L'CH2~ OH General Characteristics Crystals from benzene (contained benzene of crystallization); mp., 127~ atter drying over P2Os for 24hrs. at 70~ mp., 154~ [a]D25 - 58.60(C=0.5, in MeOH). Fungal Source

Fusarium nivale Fn-2-B.

Isolation/Purification The metabolite was adsorbed onto activated carbon directly from the culture broth. The metabolite was eluted from the carbon with methanol, precipitated with the addition of chloroform, and purified with silica gel column chromatography by elution with chloroform-methanol (1 O:1, v/v) followed by crystallization from benzene solution. Spectral Data ~

MeOH max

205(e=9,620), 228(5,850), and 280nm (1,040).

IR:

(KBr) 3300, 3200, 3005, 2945, 2855, 1660, 1635, 1602, 1590, 1510, 1450, 1440, 1340, 1320, 1210, 1180, 1010, 870, and 805cm"i. 1H NMR: (CDCI3) 1.60; 2.30(2H, dd, J=72 and 11Hz); 2.02(2H, quintet); 3.32(2H, dd, J=43 and 15Hz); 3.70(2H); 4.32(1H); 4.68(1H, t); and 7.05ppm (4H, aromatic, dd, ,/=39 and 9Hz). Mass Data: Found: C, 71.27; H, 7.55; N, 8.27; calcd for CI4HI6N203oC6H6: C, 70.92, H, 6.39; N, 8.31; found: C, 65.68; H, 6.29; N, 9.37; calcd for C14I-I16N203: C, 64.74; H, 6.14; N, 10.75%.


161

Reference T. Tatsuno, M. Sato, Y. Kubota, and H. Tsunoda; Recherches Toxicologiques des substances Metaboliques du Fusarium nivale. VIII. La Quatrieme Substance Metabolique de F. nivale; Chem. Pharm. Bull., Vol. 19, pp. 1498-1500(9171).

162

2.

Diketopiperazines

Common/Systematic Name Isoleucylisoleucyl anhydride Molecular Formula/Molecular Weight C 12H22N202; MW' = 226.16813

H2C/Me O NH

I

CH Me

Me~CH~NH~ O I Me"~CH2 Fungal Source

Ustilago cynodontis, a plant pathogenic microorganism isolated from Bermuda grass, and Beauveria bassiana.

Isolation/Purification Fungal cultures were extracted with dichloromethane after the removal of the mycelium by centrifugation. After evaporation of the dichloromethane, a small amount of ether was added to afford the white crystals of isoleucylisoleucyl anhydride. The metabolite sublimed at 253 ~ Spectral Data IR;

(Nujol) 3200 and 1660cm"! for N-H and amide carbonyl groups. Mass Data: 226(M+), 170(M + - C4I-I8), 113(M+ - C~t-I~7), 86, 69, and 57re~e; found: C, 63.29; H, 10.25; N, 12.32; calcd, for C~2Hz2N202: C, 63.68; H, 9.80; N, 12.38%. References J. F. Grove and M. Pople; Nitrogen-Containing Minor Metabolic Products of Beauveria bassiana; Phytochem., Vol. 20, pp. 815-816(1981). Y. Yamada, S. Sawada, and H. Okada; Production of Isoleucylisoleucyl Anhydride by Ustilago cynodontis; J. Ferment. Technol., Vol. 52, pp. 143-145(1974).


163

Common/Systematic Name Phomamide Molecular Formula/Molecular Weight CITH22N204; M W = 318.15796

s

O'OH2

HN~0 CH20H

General Characteristics White prisms from ethyl acetate; mp., 213-215 ~

[a]D 20 - 7 6 ~

(MeOH).

Fungal Source

Phoma lingam.

Isolation/Purification The fungal cultures were extracted with ethyl acetate and submitted to silica gel column chromatography using a gradient of methanol in ethyl acetate as eluant; the more polar fraction was combined and a second chromatography eluting with chloroform-ethyl acetate-methanol (12:4:1, v/v) afforded a crude product (amorphous). The final purification was achieved on a Sephadex LH 20 column using methanol for elution while monitoring by TLC (Re 0.50; ethyl acetate-methanol, 4:1, v/v; Schleicher-Schiill F254 SiO2 films). The pure phomamide was obtained after crystallization from ethyl acetate as white prisms. Biological Activity Biological activity not reported, but the metabolite is related to the sirodesmin group of antibiotics. Spectral Data UV:

~

MeOH max

209(e=12,400), 229(13,500), and 277nm (1,450).

(KBr) 3500, 3320, 3200, 1680, 1650, 1620, 1585, 1515, and 825cm4. CD: (MeOH) 217(-10.4), 227(-0.2), and 275nm (+l).

164


~H N M R : [(CD3)2SO] 1.69(3H, s, 17-H3); 1.73(3H, s, 16-H); 2.91(IH, m, A part of A~B~X~ system, ,/=4.5 and 14I-Iz,8-Ha); 2.91(IH, m, A part of A2B2X2 system, ,/=6 and I IHz, 7-Ha); 3.01(I H, dd, B part of A~BIX~ system,`/=6.I and 14Hz, 8-I-Ib);3.35(IH, m, B part of A2B2X2 system, J=3 and 11Hz, 7-Hb); 3.67 (I H, m, X part of A2B2X2 system, ,/=6 and 3Hz, 6-1-1);3.99(IH, m, X part of AzB~X~ system, ,/=4.5 and 6.IHz, 3-H); 4.48(2H, d, ,/=6.8Hz, 13-H2); 4.89(IH, t, exchangeable with D20, 7-OH); 5.41(IH, m, 14-H); 6.95(4H, m, aromatic);and 7.90 and 7.92ppm (2H, m, exchangeable with D20, I- and 4-NH). 13C NMR: (CD3)2SO 17.9(q, C-17); 25.3(q, C-16); 38.9(t, C-8); 55.6(d, C-3 or-6); 57.1(d, C-3 or-6); 63.1(t, C-7); 64.2(t, C-13); 114.3(d, C-11 and-11'); 120.1(d, C-1 and C-10'); 128.3(s, C-9); 130.9(d, C-14); 136.7(s, C-15); 157.2(s, C-12); 165.8(s, C-2 or-5); and 166.7ppm (s, C-2 or C-5, C-carbonyl atoms). Mass Data: 318(M+, 2%), 250(30), 144(95), 107(97), and 69m/e (100); found: C, 64.3; H, 7; N, 8.5; O, 20.2; calcd, for CiTH22N204: C, 64.1; H, 7; N, 8.8; O, 20.1%. Reference J-P Ferezou, A. Quesneau-Thierry, M. Barbier, A. Kollmann, and J-F Bousquet; Structure and Synthesis ofPhomamide, A New Piperazine-2,5-dione Related to the Sirodesmins, Isolated from the Culture Medium ofPhoma lingam Tode; J. C. S. Perkin I, pp. 113115(1980).


165

Common/Systematic Name D-Valyl-L-tryptophan anhydride Molecular Formula/Molecular Weight C16H19N302; M W = 285.14773

~~ L .... ~I NH

.~ ,~ Me_/..

0"" NH

"CH Me

General Characteristics Rhombic crystals changed to long needles upon heating between 235-255"C; melted between 277-279~ [a]D +78 ~ (C=0.16, in acetic acid). Fungal Source Aspergillus chevalieri = Eurotium chevalieri. Isolation/Purification Isolated by silica gel column chromatography eluted with 1.5% methanol in chloroform. Partially purified fractions were combined and rechromatographed as above; D-valyl-Ltryptophan anhydride containing fractions crystallized as rhombic crystals. Spectral Data UV:

~

EtOH m~

290(~=5,800), 281(6,900), 274.5(6,500), and 220nm (43,400).

IR:

(KBr) 1660cm"~indicated amide functionality. ~H NMR: Spectrum revealed a six proton doublet of doublets at 0.85 and 0.88ppm (derived from proline) that was coupled to a single proton centered at 2.23ppm (m). A one proton doublet appeared at 3.23ppm. Mass Spectrum: LREIMS: 285 and 130m/e(lO0%). HREIMS indicated that 285 was CI6HIgN302 (M+), and 130m/e was CgI-IsN. Reference R. D. Stipanovic, H.W. Schroeder, and H. Hein, Jr.; Identification of D-Valyl-Ltryptophan Anhydride from Aspergillus chevalieri; Lloydia, Vol. 39, pp. 158-159(1976).

166


Common/Systematic Name L-Alanyl-L-tryptophan anhydride Molecular Formula/Molecular Weight C1~-I15N302; MW = 257.11643 O

HNg"Me

I I/"Y )..NH O

General Characteristics Colorless needles; mp., 290-292~ (decomp.); [a]D 25 -F 36 ~ (c=0.5, in EtOH); positive to Ehrlich's reagent, negative to ninhydrin reagent. Treatment with 6N HCI afforded Lalanine and L-tryptophan. Fungal Source

Aspergillus chevalieri, IFO 4090.

Isolation/Purification The fungus was grown as a surface culture for 3 weeks at 24~ on potato extract medium. The culture filtrates were stirred with activated charcoal and the adsorbed metabolites were eluted with acetone. The acetone extracts were chromatographed on a column of silicic acid and eluted with benzene-acetone (4:1, v/v) which gave L-alanyl-2-(1,1-dimethylallyl)-L-tryptophan anhydride. Elution with benzene-acetone (2:1, v/v) gave L-alanyl-L-tryptophan anhydride. Spectral Data UV: Zm~x 220(e=38,000), 273(6,100), 280(6,300), and 290nm (5,800). IR:

3420(NH), 1675(C=O, amide), and 1665cm~ (C=O, amide). IH NMR: The spectrum revealed: a doublet at 0.48ppm (3H, doublet, J=7Hz) due to the alanyl methyl protons which showed coupling with a proton at 3.63ppm (1H, quartet of doublets, J=7 and 2Hz); two doublets of doublets at 3.05ppm (1H, doublet of doublets, ,/--15 and 5Hz) and 3.22ppm (1H, doublet of doublets, J=15 and 3Hz) assigned to the allylic methylene protons which showed coupling with a proton at 4.12ppm (1H, multiplet); a signal at 10.86ppm (IH, broad) assigned to the -NH group proton in the


167

indole ring system. This proton coupled with a proton at 7.09ppm (1H, doublet, J=3Hz) because of a proton at position 2 in the indole ring system. Protons at 7.90ppm (1H, broad) and 7.95ppm (1H, broad) assigned to -NH groups in the dioxopiperazine ring system showed coupling with protons at 3.63 and 4.12ppm, respectively. These three protons at 7.90, 7.95, and 10.86ppm disappeared on treatment with deuterium oxide. Signals of the four aromatic protons were observed at 6.8-7.8ppm (4H, multiplet). Mass Data: LREIMS: 257(M+); found: C, 65.00; H, 5.79; N, 16.18; calcd, for cl4nlsN302: C, 65.35; H, 5.88; N, 16.33% Reference T. Hamasaki, K. Nagayama, and Y. Hatsuda; A New Metabolite, L-Alanyl-L-tryptophan Anhydride from Aspergillus chevalier/; Agr. Biol. Chem., Vol. 40, p. 2487(1976).

168


Common/Systematic Name

cyclo-(L-Isoleucyl-L-valine)

Molecular Formula/Molecular Weight Clln2oN202; M W = 212.15248 Me

0

NH

M e ~ N H ~ O

.,i- Me "

Me General Characteristics Sublimed without melting at 250~

[a]D22 -400(c=0.908, in EtOH)

Fungal Source

Beauveria bassiana.

Isolation/Purification The metabolite was purified with silica gel column chromatography by elution with benzene-ethyl acetate (9:1, v/v) followed by sublimation at 250~ Spectral Data IR~

(Nujol) 3195, 3050, and 1642 cm"~for N-H and amide carbonyl groups. Mass Spectrum: LREIMS: 212(M+), 197(-CH3), 183(-C2H5), 170, 156(100%), 141, 127, 114, 113(100), 86, 85, 72, 69, and 57m/e. References S. Eriksen and I. S. Fagerson; Mass Spectra of Some Cyclic Dipeptides (2,5Diketopiperazines); J. Agile. Food Chem., Vol. 24, pp. 1242-1243(1976). J. F. Grove and M. Pople; Nitrogen-Containing Minor Metabolic Products of Beauveria bassiana; Phytochem., Vol. 20, pp. 815-816(1981).


169

Common/Systematic Name cyclo-(L-Alanyl-L-proline) Molecular Formula/Molecular Weight CsHI2N202; ~

0

NH

= 168.08988

Me

General Characteristics Crystals from methanol; mp., 162-166~

[ a i D 22 -

85~

in EtOH)

Fungal Source Beauveria bassiana. Isolation/Purification The metabolite was purified with silica gel column chromatography by elution with benzene-ethyl acetate (9:1, v/v) followed by crystallization from methanol. Spectral Data IR:

(Nujol) 3280, and 1657cm"l for N-H and amide carbonyl groups. Mass Spectrum: HREIMS: 168.0904m/e (M+); CsHI2N202 requires 168.0898. Reference J. F. Grove and M. Pople; Nitrogen-Containing Minor Metabolic Products of Beauveria bassiana; Phytochem., Vol. 20, pp. 815-816(1981).

170


Common/Systematic Name Neoxaline Molecular Formula/Molecular Weight C23H25NsO4; M W - 4 3 5 . 1 9 0 6 5 23

18

MeO

0

NH

General Characteristics Colorless needles from benzene; mp., 202~ (dec.); laiD 24 -16.3~(c=l.0, in CHCI3). Fungal Source Penicillium sp. Fg-234 and Aspergillusjaponicus Fg-551. Spectral Data UV:

~ 9

MeOH max

330(e=29,560) and 237nm (17,620).

IR:

3500(OH), 3425(CONH), 3200(NH, imidazole), 1710, and 1685cm~ (NC=O). CD: (c=0.0009, in EtOH) - 430(372nm) (negative maximum), + 1,200(339nm) (positive maximum), + 800(335.5nm) (negative maximum), + 1,200(328nm) (positive maximum), + 800(311nm) (negative maximum), + 4,800(290nm) (positive maximum), + 4,750(285nm) (negative maximum), + 8,970(267nm) (positive maximum), 11,200(245.5nm) (negative maximum), and + 5,600(223nm) (positive maximum). -

1H NMR: (acetone-d6) H-4, 7.58(dd, ,/=8.0, 2.014_z);H-5, 7.02(dt, ,/--2.0, 8.0Hz); H-6, 7.27(dt, ,/=2.0, 8.0Hz); H-7, 6.91(dd, J=2.0, 8.0Hz); H-8, 2.44(t, J=12.0Hz), 2.23(dd, ,/--12.0, 6.0Hz); H-9, 4.60(dd, J=12.0, 6.0Hz); n-15, 8.29(s); n-18, 7.79(s); H-20, 7.3 l(s); H22, 6.12(dd, J=18.0, 10.0Hz); H-23, 5.11(d, J=18Hz), ca. 4.97; 1-OMe, 3.73(s); 21OMe2, 1.3 l(s), 1.3 l(s); NH, 8.98(br s), 2.32(s); and OH, ca. 1.3 lppm.


171

13C NMR:

C-2, 100.6(s); C-3, 53.3(s); C-3a, 128.2(s); C-4, 124.8(d); C-5, 123.6(d); C-6, 128.9(d); C-7, l ll.5(d); C-7a, 145.7(s); C-8, 40.8(t); C-9, 66.4(d); C-10, 171.65(s); C-12, 122.3(s); C-13, 165.6(s); C-15, 110.3(d); C-16, 125.8(s); C-18, 136.8(d); C-20, 134.4(d); C-21, 43.5(s); C-22, 144.5(d); C-23, 114.1(t); 1-OMe, 65.2(q); and 21-Me2, 24.7(q), 24.5ppm (q). Mass Data: HREIMS: 435.191m/e (M+) (calcd. 435.190); anal calcd, for C23H25N504;C, 63.43%; H, 5.79; N, 16.08; found: 63.23; H, 5.70; N, 15.90. Reference Y. Konda, M. Onda, A. Hirano, and S. Omura; Oxaline and Neoxaline, Chem. Pharm. Bull., Vol. 28, pp. 2987-2993(1980).

172


Common/Systematic Name Oxaline Molecular Formula/Molecular Weight C24H25NsO4; M W = 4 4 7 . 1 9 0 6 5

22•,

OMe

L MeO

~0

~NH

General Characteristics Colorless prisms from acetone; rap., 230-232~

[aiD 22

-45 ~ (c=0.3, in MeOH).

Fungal Source Penicillium sp. Fg-234, P. oxalicum M-555, and Aspergillusjaponicus Fg-551.

Isolation/Purification The culture broth including mycelium was adjusted to pH 10.0 with aqueous ammonia and extracted with butyl acetate. The extract was concentrated in vacuo and the precipitate removed by filtration. The filtrate, which was dried over Na2SO4 and concentrated m vacuo, yielded pale brownish crystals which were recrystallized from acetone to yield colorless prisms. Biological Activity Biologically inactive. Spectral Data UV~

~

MeOH max

345(e=25,200) and 228nm (21,300).

IRz

(CHCI3) 3420(CONH), 3180(NH), 1710, and 1680cm q (NC=O). CD: (c=0.001, in MeOH) - 24,400(344nm) (negative maximum), + 38,200(273nm) (positive maximum), + 34,300(260nm) (negative maximum), + 42,00(247nm) (positive maximum), and - 98,300 (223nm) (negative maximum).


173

IH NMR:

(CDCI3) H-4, 7.57(d, J=8.0Hz); H-5, 7.06(t, J=8.0Hz); H-6, 7.27(t, J=g.0Hz); H-7, 6.95(d, J=8.0Hz); H-S, 5.12(s); H-15, 8.32(s); H-18, 7.42(s); H-20, 7.02(s); H-22, 6.09(dd, ,/=18.0, 10.0Hz); H-23, 5.06(d, J=18Hz), 5.02(d, J=10.0Hz); 1-OMe, 3.70(s); 9-OMe, 3.62(s); 21-Me2, 1.29.(s), 1.25(s); NH, 12.76(s); and 9.59ppm (s). 13CNMR:

C-2, 101.7(s); C-3, 52.5(s); C-3a, 126.1(s); C-4, 124.7(d); C-5, 123.2(d); C-6, 128.5(d); C-7, l12.0(d); C-7a, 146.8(s); C-8, 106.9(d); C-9, 146.4(s); C-10, 157.5(s); C-12, 123.2(s); C-13, 166.3(s); C-15, 109.6(d); C-16, 126.1(s); C-IS, 136.4(d), C-20, 133.8(d); C-21, 42.5(s); C-22, 142.8(d); C-23, 113.9(t); 1-OMe, 65.2(q); 9-OMe, 55.7(q); and El-Me2, 24.1(q), 23.7ppm (q). Reference Y. Konda, M. Onda, A. Hirano, and S. Omura; Oxaline and Neoxaline, Chem. Pharm. Bull., Vol. 28, pp. 2987-2993(1980).

174


Common/Systematic Name Aszonalenin Molecular Formula/Molecular Weight C23H23N302; MW = 3 73.17903 26

24 2 2 ~

25

General Characteristics Colorless needles from chloroform-methanol; mp., 244-247~ CHCI3); positive to Ehrlich's reagent.

[aiD 20 q-

53 ~ (c=1.31, in

Fungal Source Aspergillus zonatus IFO 8817. Isolation/Purification The fungus was stationarily cultured at 24 ~ for 21 days in malt extract medium. The acetone extract from the dry mycelial mats was chromatographed on a silica gel column eluted with benzene-acetone (19:1, v/v). After elution ofLL-S49013 , aszonalenin was obtained as a crude substance, which was rechromatographed over silica gel eluted with benzene-ethyl acetate (19:1, v/v) to give colorless crystals of aszonalenin. Biological Activity Application of aszonalenin at a concentration of 50~g/ml apparently induced the abnormal cleavage of sea urchin embryos. Spectral Data UV:

~

EtOH max

210(e=44,700), 233(sh, 25,100), and 290nm (5,050).

IR~ (KBr) 3400(NH), 1700(C-O, amide), 1640(C-O, amide), 1620(C=C), and 1578cmq (aromatic).


175

~H NMR: (CDCI3) ~H NMR spectrum was very similar to that ofLL-S490g. A signal at 2.59ppm due to methyl protons of an acetyl group in the spectrum of LL-S49013 was not observed in the NMR spectrum of aszonalenin; an additional signal at ca. 7.0ppm assignable to -NH- was present in the spectrum of aszonalenin. Signals from a 1,1dimethyl-2-propenyl group were observed at 1.08, 1.16, 5.04, 5.07, and 6.10ppm; a signal at 8.77ppm was assigned to an amide proton; and signals between 6.55-7.92ppm were assigned to eight aromatic protons.

13C NMR: (CDCI3) Signals at 24.2(q) and 171.7(s) due to an acetyl group in spectrum of LL$49013 were not observed in this case. Signals from a 1,1-dimethyl-2-propenyl group were observed at 22.6, 22.9, 41.7, 114.3, and 144.1ppm; C-10, 33.6; C-11, 57.5; C-12, 170.2; C-16, 168.0; twelve signals between 119.3-141.8, aromatic carbons; 82.0, C-2; and 61.1 ppm, C-3. Mass Spectrum: LREIMS: 373(M+), 304(M + - 69, base peak), and 130m/e (due to indoline-3-methylene ion). Reference Y. Kimura, T. Hamasaki, and H. Nakajima; Structure of Aszonalenin, a New Metabolite ofAspergillus zonatus; Tetrahedron Letters, Vol. 23, pp. 225-228(1982).

176


Common/Systematic Name LL-$49013; Acetylaszonalenin Molecular Formula/Molecular Weight C2sH25N3Oa; MW = 415.18959

4

O

General Characteristics Crystals from ethyl acetate-benzene; mp., 238-240~

[aiD 20 +

425 ~ (c=0.20, in MeOH).

Fungal Source

Aspergillus sp.

Isolation/Purification The whole mash from a fermentation was extracted with an equal volume of ethyl acetate at pH 5.0. The extract was concentrated to dryness and the residue was partitioned between methanol and heptane to remove fatty material. Evaporation to dryness of the methanol portion gave a crude residue. This residue was chromatographed over a silica gel column (acid washed) packed in methylene chloride. A gradient elution between 0.5% methanol-methylene chloride and 3% methanol-methylene chloride provided cladosporin after evaporation of the solvent and crystallization from ethyl acetate-benzene. Further elution, removal of the solvent, and crystallization from ethyl acetate gave LL-$49013. Spectral Data UV~ ~

MeOH max

210(e=61,000), 245(22,000), and 284nm (sh, 3,940).

IR~ (KBr) 3300(NH), 1689(C=O, amide), and 1647cmq (C=O, amide). IH NMR: (CDCI3) 1.02 and 1.21(3H, s); 2.60(3H, s); 2.46(q, J~=14, Jp~= 8.0Hz); 3.42(q, J~=14, JBx=8.5Hz); 3.90(t, J=8 Hz); 5.16(m, AB ofvinylidene); 5.92 (q, Jvo,~=18, Jcis=9.5Hz, X ofvinylidene); 6.00(1H, s); 6.83-8.17(8H, m); and 8.45ppm (1H, S).


177

Mass Data: HREIMS: 415.18919 (calcd.for C25H25N303, 415.18959). Reference G. A. Ellestad, P. Mirando, and M. P. Kunstmann; Structure of the Metabolite LL-S49013 from an Unidentified Aspergillus species; J. Org. Chem., Voi. 38, pp. 4204-4205(1973).

178


Common/Systematic Name Bipolaramide Molecular Formula/Molecular Weight ClsH14N204; MW' = 322.09536

6

4

0

OH

O2 OH

0

General Characteristics Crystallized from acetone as colorless needle-shaped crystals; mp., 296-297~ 210 ~ (C=1.0, in acetone).

[a]D -

Funsal Source

Bipolaris sorokiniana (IMI 115076); a toxigenic strain isolated in the Karroo from an indigenous weed Tribulis terrestris.

Spectral Data UV:

~,~"

218, 260, and 294nm (log e=4.65, 4.17, and 3.88, respectively).

IR:

(KBr) 1645(amide CO) and 1610cm"l. IH NMR:

(acetone-d6) 7.15(1H, t, J=7.5Hz); 6.87(1H, dd, J=7.5, 2.8Hz); 6.80(1H, dd, J=7.5, 2.8Hz); 5.53(1H, t, J=9.8Hz); 3.78(1H, dd, J=17.3, 9.8Hz); 3.51(1H, dd, J=17.3, 9.8Hz); and 11.40ppm (D20-exchangeable hydroxy group). ~3C NMR: [(CD3)zCO] 164.5(s); 145.1(s); 132.4(d); 127.5(d); 126.3(d); 116.4(d); 115.4(d); 61.4(d); and 30.3ppm (t). These data indicated the presence of three contiguous aromatic protons and of an ABX aliphatic system. The vicinal coupling constants (J=9.8Hz) observed for the ABX system are in accordance with the dihedral angular relationship obtained from X-ray crystallography: 149.8 and 31.6 ~ for H(3)-C(3)-C(4)H(4A) and H(3)-C(3)-C(4)-H(4B), respectively. Mass Spectrum: 322(M +) and 16 lm/e.


179

Reference C. M. Maes, P. S. Steyn, P. H. van Rooyen, and C. J. Rabieb; The Structure of Bipolaramide, a Novel Dioxopiperazine from Bipolaris sorokiniana; J. Chem. Soc. Chem. Commun., pp. 350-351(1982).

180


Common/Systematic Name Austamide Molecular Formula/Molecular Weight C21H21N303; M W -- 363.15829

O

:

O

19

General Characteristics A yellow amorphous compound or homogeneous powder;

l a i D 20 + 1 5 2 ~

(c=l, in EtOH).

Funsal Source Aspergillus ustus (C.S.I.R. 1128; NRRL 5856). Isolation/Purification The dried Aspergillus ustus cultures were extracted with CHCI3-MeOH and the solvent removed under reduced pressure which yielded homogenous crystalline material. This represented the main toxic component of the fungal culture. The latter in CHCI3 was extracted twice with water. Evaporation of the CHCI3 yielded material which was partitioned between 90% MeOH and hexane, yielding toxic material in the MeOH layer. This toxic material was further purified by chromatography on formamide-impregnated cellulose powder. The cellulose column was developed with a mixture of hexane and benzene. The toxic material was further separated on A1203 (activity III). Elution with mixtures of benzene and CHCI3 gave pure austamide and a mixture of austamide and 12,13-dehydroaustamide. Biological Activity Austamide was one of the active components in extracts ofA. ustus (cultured on maize) causing acute toxicosis in day old ducklings. Spectral Data UV~

~,~m~ H 234(r 256(117), 268 sh (1,096), 282(8,709), and 392nm (2,691) (unchanged upon addition of acid or base). IR:

(CHCI3) 3420, 1700, 1680, and 1650cm "l.


181

CD: (in MeOH) 390(+2.0), 285(+1.8), and 234nm (20-25). ~3CNMR: 23.6, 26.1, 28.0, 41.9, 42.0, 45.5, 62.5, 70.7, 111.9, 118.7, 120.3, 120.4, 124.4, 125.4, 128.3, 132.2, 137.3, 154.7, 160.1, 161.1, and 200.8ppm.

TLC Data Silica gel; chloroform-methanol, 97:3 v/v; Re not reported. Detection: not reported. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp.484 (1981). P. S. Steyn; The Structures of Five Diketopiperazines from Aspergillus ustus; Tetrahedron, Vol. 29, pp. 107-120(1973).

182



12,13-Dihydroaustamide

Molecular Formula/Molecular Weight C21H23N303; MW = 365.17394

4

0

O~N~~2 13 ]17

T,,,, H

General Characteristics Crystallized from acetone; mp., 235-238~

[a]D 22 + 5 5 ~

(c=l.1, in CHCI3).

Fungal Source Aspergillus ustus (C.S.I.R. 1128; NRRL 5856) and Penicillium italicum. Isolation/Purification The dried moldy maize culture was extracted with CHCI3-MeOH and the solvent removed under reduced pressure which yielded homogenous crystalline material which represented the main toxic components of the fungal culture. The latter in CHCI3 was twice extracted with water. Evaporation of the CHCI3 yielded material which was partitioned between 90% MeOH and hexane, yielding toxic material in the MeOH layer. This toxic material was further purified by chromatography on formamide-impregnated cellulose powder. The cellulose column was developed with a mixture of hexane and benzene. The toxic material was further separated on A1203 (activity III). Elution with mixtures of benzene and CHCI3 gave a mixture of austamide and 12,13-dehydroaustamide. The latter metabolites were separated by repeated preparatory silica gel TLC developed in CHCI3-MeOH (97:3, v/v) yielding pure 12,13-dehydroaustamide. Biological Activity Toxic to ducklings. Spectral Data UV:

~

EtOH max

238(1og c=4.49), 256(sh)(4.13), and 390nm (3.52).

IR;

(CHCI3) 3420, 3335, 3,000, 1670, and 1620cm"l.


183

~H NMR: The NMR spectrum of 12,13-dihydroaustamide when compared with that of austamide showed the absence of the olefinic triplet at 3.74z and the newly formed proton at C12 at 5.82z. Concurrently the peaks comprising the proline part became more complex. Mass Spectrum: HREIMS: 365.1750(M+, C21H23N303 requires 365.1739), 192.1268 (CIIHI6N20 requires 192.1262), and 70.0656m/e (C~HsN requires 70.0656). Reference P. S. Steyn; The Structures of Five Diketopiperazines from Aspergillus ustus; Tetrahedron, Vol. 29, pp. 107-120(1973).

184


Common/Systematic Name 12,13-Dihydro- 12-hydroxyaustamide 2S, 9S, 12R- 12,13-Dihydro- 12-hydroxyaustamide Molecular Formula/Molecular Weight C21H23N304; M W -- 381.16886

oxy

4

0

H,,,.I

"

I....'OH

General Characteristics Crystals from MeOH; mp., 164-165~ Fungal Source

Aspergillus ustus.

Isolation/Purification Isolated from A. ustus fermented corn and separated by chromatography on formamideimpregnated cellulose powder; further purified by extensive column chromatography and TLC on silica gel and aluminum oxide; crystallized from methanol. Biological Activity Toxic to ducklings. Spectral Data UV: ,~ McOH max

231,255, and 390nm (log e = 4.48, 4.04, and 3.47, respectively).

CD: ,~ MeOH max

420(0), 374(-2.0), 355(0), 341(+2.35), 3140, 305(-0.34), and 290 (0).

IR: (CHCI3) 3430-3350, 1670, and 1618cm"~. IH N]VIR: (CDCI3) Spectrum showed 2 singlets at 8.48(3H) and 9.16z (3H) which were assigned to the two geminal methyl groups; the cis-olefirfic protons appeared as an AB-pattern


185

at 3.29 and 4.98Z(JAa=10Hz); protons at C-8 and C-9 appeared as an ABX system, HA being I-ls~q, 7.13z(q, JAa=15Hz, JBx=12Hz); a complex pattern centered around 6.361: was assigned to the methylene protons adjacent to the proline N-atom; the remaining 4 methylene protons which comprised the proline ring appeared as an unresolved multiplet between 7.6 and 8.1z; the proton at C-12 resonated between 5.80-5.90z; and the splitting pattern of the aromatic region was identical to that of dihydroaustamide. Mass Spectrum: HR IMS: 381.1685(M+, C21Hz3N304 requires 381.1688) and 363m/e for C~zH~4N202. Reference P. S. Steyn and R. Vleggaar; 12,13-Dihydro-12-hydroxyaustamide, A New Dioxopiperazine from Aspergillus ustus; Phytochemistry, Vol. 15, pp. 355-356(1976).

186


Common/Systematic Name Cycloechinulin Molecular Formula/Molecular Weight C2oH21N303, MW = 352.15829 22 Me H N ~ .... ,H

4

r"

\17

MeO-/~~~8 NI~I /eMe 7 2o General Characteristics Cycloechinulin was isolated as a yellow solid; [~]D -23.3 ~ (c=0.06g/dl, in CHCIa). Fungal Source Sclerotia ofAspergillus ochraceus (NRRL 3519). Isolation/Purification Intact sclerotia of A. ochraceus were extracted at room temperature with hexane, then CHCI3. After removal of the solvent in vacuo, the hexane extract was subjected to reversed-phase HPLC [MeOH-H20 (9:1, v/v), 2ml/min} to give N-methylepiamauromine. The CHCI3 extract, after removal of the solvent in vacuo, was fractionated through a column of Sephadex LH-20 (25-1001,t) using CH2Cl2-hexane (1:1, v/v), then CH2CI2-MeOH (1:1, v/v) as eluents. The fractions obtained with CH2Cl2-hexane (1:1, v/v) were combined, concentrated, and purified by HPLC as above to give epiamauromine and N-methylepiamauromine. The fractions eluted with CH2CI2-MeOH (1:1, v/v) were likewise combined, concentrated, rechromatographed on Sephadex LH-20 using CHCI3-MeOH (2:1, v/v), and purified by reversed-phase HPLC to yield cycloechinulin. Biological Activity Caused moderate reduction in weight gain in assays against the lepidopteran crop pest Helicoverpa zea.

Spectral Data UV:

~

MeOH max

214(1og e=4.3), 228(4.2), 267(4.0), 300(4.1), and 377nm (3.9).


187

IH N]VIR: (CDCI3) H-1,8.39, br s;H-4, 7.63, d(J=8.7);H-5, 6.84, dd(J=2.2, 8.7);H-7, 6.81, d(J=2.1); H-10, 7.57, s;H- 13, 6.37, br s; H-14, 4.13, dq(J=2.3, 6.9);H-17, 5.82, d(J=8.2); H-18, 5.94, d(J=8.2);H-20, 1.68, s;H-21, 1.67, s;H-22, 1.51, d(J=6.9); and H-23, 3.81ppm, s. 13CNMR: (CDCI3) C-2, 145.8; C-3, 105.7; C-4, 118.7; C-5, 110.9; C-6, 157.2; C-7, 94.8; C-8, 134.0; C-9, 124.4; C-10, 115.4; C-11,125.0; C-12, 165.6; C-14, 51.1; C-15, 167.2; C-17, 122.4; C-18, 139.7; C-19, 36.0; C-20, 27.2; C-21, 27.0; C-22, 18.4; and C-23, 55.8ppm. Mass Spectrum: EIMS: [M]§ 351(77), 336(100), 308(20), 296(26), 293(37), 280(93), 265(34), 252(46), 251(47), 237(94), 225(82), 222(31), and 197m/e (64); HRFABMS found 352.1671; calcd for C20H21N303+H 352.1661. HPLC Data Beckman Ultrasphere 51.tm(10mm x 25cm) C~8 reversed-phase column was used on all HPLC separations; HPLC retention time for cycloechinulin was 7.5 min. Reference F. S. de Guzman, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; New Diketopiperazine Metabolites from the Sclerotia of Aspergillus ochraceus; Journal of Natural Products, Vol. 55, pp. 931-939(1992).

188


Common/Systematic Name 12,13-Dehydroprolyl-2-(l', l'-dimethylallyltryptophyl) diketopiperazine Molecular Formula/Molecular Weight C21H23N302; MW = 349.17903

4 ~ N _ \1o~/OST/ 7

20/19

General Characteristics Homogenous powder;

l a i D 22 - 3 8 ~

(c=1.3, in CHCI3).

Fungal Source Aspergillus ustus (C.S.I.R. 1128; NRRL 5856) and Penicillium italicum. Isolation/Purification The dried moldy corn was extracted with CHCIa-MeOH and the solvent removed under reduced pressure which yielded homogenous crystalline material which represented the main toxic components of the fungal culture. The latter in CHCI3 was twice extracted with water. Evaporation of the CHCI3 yielded material which was partitioned between 90% MeOH and hexane, yielding toxic material in the MeOH layer. This toxic material was further purified by chromatography on formamide-impregnated cellulose powder. The cellulose column was developed with a mixture of hexane and benzene. The toxic material was further separated on A1203 (activity III). Elution with benzene gave a mixture of compounds. The latter material was separated by silica gel TLC developed in CHCI3MeOH (98:2, v/v) yielding pure 10,20-dehydro[ 12,13-dehydropropyl]-2-(l', l'-dimethylallyltryptophyl)diketopiperazine. Elution of the foregoing A1203 column with CHCI3benzene (6:4, v/v) gave pure 12,13-dehydroprolyl-2-(l', 1'- dimethylallyltryptophyl)diketopiperazine as a homogenous powder. Biological Activity Toxic to ducklings. Spectral Data UV:

223(1og e=4.54), 268(sh)(4.03), 283(4.00), and 292nm (3.89).


189

IR;

(CHCI3) 3482, 3460, 3380, 1670, and 1650cm"~. ~H NMR: The NMR spectra of deoxybrevianamide E and 12,13-dehydroprolyl-2-(1 ', 1'-dimethyl allyltryptophyl)diketopiperazine had many features in common, the most striking difference being due to protons of the proline ring. These protons appeared as an A2M2X pattern, represented by a triplet at 6.0~ (2H, J=-9Hz, C-15 CH2), a sextet at 7.311: (2H, ,/=3, 9, 9I-~ C-14 CH2), and a triplet at 3.94~ (1H, J-=-3I-~ C-13 CH). Mass Spectrum: HREIMS: 349.1720(M +, C21H23N302 requires 349.1708). References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 453 (1981). P. S. Steyn; The Structures of Five Diketopiperazines from Aspergillus ustus; Tetrahedron, Vol. 29, pp. 107-120(1973).

190


Common/Systematic Name Alanyl-2-(1,1-dimethyl-2-propenyl)dehydrotryptophan anhydride Molecular Formula/Molecular Weight CwH21N302; MW = 323.16338 O

[~N

NHMe

General Characteristics Crystals; mp., 262-264 ~C. Fungal Source Aspergillus ruber -, Eurotium rubrum isolated from beet pulp.

Isolation/Purification Mycelia was extracted with ethyl acetate. The ethyl acetate-soluble/neutral fraction was chromatographed on a silica gel column using benzene-ethyl acetate. The benzene-40% ethyl acetate eluant gave two indole metabolites which were separated with preparative thin-layer chromatography developed with benzene-ethyl acetate (1:1, v/v). Biological Activity Growth retardant for silkworm larvae (Bombyx mori L.). Spectral Data UV: 1' MeOH ^ ....

224(E=31,600), 283 (8,300), 290(8,050), and 338nm (9,340).

IR;

(Nujol) 1675 and 1633cm"1. ~H NMR: (DMSO-dr) 11.03(IH, s, -NH-, indole nucleus), 8.57(1H, s, -NH-CO-), 8.3 I(1H, s,-NH-CO-), 7.44-7.00(4H, m), 6.92(1H, s), 6.10(1H, dd, J=9.9Hz and 17.8Hz -CH=CH2), 5.02(1H, dd, J=9.9Hz and 1.1Hz; 1H, dd, J=17.8Hz and 1.1Hz, -CH=CH2), 4.17(1H, q, J=7.0Hz, =CH-CH3), 1.48(6H, s, (CH3)2C=), and 1.38ppm (3H, d, J=7.0Hz, =CH-CH3).


191

Mass Spectrum: 323.160 lm/e (M +) and 254(M + - C5H9); calcdfor C19H2~N302, 323.1631. Reference H. Nagasawa, A. Isogal, K. Ikeda, S. Sato, S. Murakoshi, A. Suzuki, and S. Tamura; Isolation and Structure Elucidation of a New Indole Metabolite from Aspergillus ruber; Agr. Biol. Chem., Vol. 39, pp. 1901-1902(1975).

192


Co.mmon/Systematic Name 10,20-Dehydro [ 12,13-dehydroprolyl]-2-( 1', 1'-dimethylallyltryptophyl)diketopiperazine Molecular Formula/Molecular Weight C21H21N302;

MW

-- 3 4 9 . 1 6 3 3 8

13

O 8 4

I

7 Fungal Source v Aspergillus ustus (C.S.I.R. 1128, NRRL 5856) and Penicillium italicum. Isolation/Purification Dried moldy maize was extracted with CHCI3-MeOH and the solvent removed under reduced pressure which yielded homogenous crystalline material which represented the main toxic components of the fungal culture. The latter in CHCI3 was twice extracted with water. Evaporation of the CHCI3 yielded material which was partitioned between 90% MeOH and hexane, yielding toxic material in the MeOH layer. This toxic material was further purified by chromatography on formamide-impregnated cellulose powder. The cellulose column was developed with a mixture of hexane and benzene. The toxic material was further separated on A1203 (activity HI). Elution with benzene gave a mixture of compounds. The latter material was separated by silica gel TLC developed in CHCI3MeOH (98:2, v/v) yielding pure 10,20-dehydro[ 12,13-dehydropropyl]-2-(l',l'-dimethylallyltryptophyl)diketopiperazine. Elution of the foregoing A1203 column with CHCl3benzene (6:4, v/v) gave pure 12,13-dehydroprolyl-2-(1 ', 1'-dimethylallyltryptophyl)diketopiperazine as a homogenous powder. Biologic~ Activity Toxic to ducklings. Spectral Data UV"

~ EtOH max

224(1og e=4.44), 272(sh)(3.93), 284(3.84), and 292nm (3.71).


193

IR; (CHCI3) 3485(sharp peak), 3350(weak broad band), 3,000, 1675, and 1650cm "l. 1H The NMR spectrum showed the presence of an indole NH-proton 1.70% (s) and four contiguous aromatic protons 2.4-3.1% (m). The two 3-proton singlets at 8.39 and 8.66% were assigned to the gem-dimethyl group and the 2-proton singlet at 4.26% to the olefinic protons at positions 19 and 20. The three protons at C-8 and C-9 appeared as an ABX pattern as quartets: Hx at 5.751: (J=l, 6Hz, C9-H), HA at 6.40(J=1, 15Hz, CS-H, q), and lib at 6.54z (,/=6, 15Hz, C8 H,x.). The small coupling constant (1Hz) between C9-H and C8-I-I~ is consonant with a dihedral angle of close to 90 ~ while JBX 6Hz is consistent with a dihedral angle of close to 135"; the dihedral values are in agreement with those obtained from an inspection of a Dreiding model of 10,20-dehydro [ 12,13-dehydroprolyi]-2-( 1', 1'-dimethylallyl tryptophyl)diketopiperazine. A two-proton triplet at 6.421: was assigned to the protons at C15, a 2-proton sextet at 8.17~ (,/=3, 10, 10Hz) was assigned to the methylene protons at C~4, while the olefinic proton at C~3 resonated as a triplet at 4.58z (J=3Hz). Irradiation at the center of this latter triplet led to the collapse of the sextet to a triplet at 8.17% (J=-I 0Hz). Mass Spectrum: HREIMS: 347.161(M +, C21H21N302requires 347.162). Reference P. S. Steyn; The Structures of Five Diketopiperazines from Aspergillus ustus; Tetrahedron, Vol. 29, pp. 107-120(1973).

194


Common/Systematic Name Deoxybrevianamide E L-Prolyl-2-( 1', 1'-dimethylallyl)-L-tryptophyldiketopiperazine Molecular Formula/Molecular Weight C21H25N302; M W -- 351.19468 O

4

8 ~

N

General Characteristics Homogeneous powder from benzene;

is

[a]D 22

-59 ~

(c=1.2, in CHCI3).

Fungal Source Aspergillus ustus (C.S.I.R. 1128; NRRL 5856) and Penicillium italicum. Biological Activity Toxic to ducklings. Spectral Data UV;

~

EtOH max

225(E=32,300), 275(sh)(7,000), 283(8,100), and 29 lnm (7,000).

IR;

(CHCI3) 3481, 3458, 3366, 1670, and 1665cml. lH N]V[R; Two exchangeable ringlets at 1.25 and 4.28ppm were assigned to NH protons. A multiplet at 2.48-3.05ppm was attributed to the four neighboring aromatic protons. A 6-proton singlet at 8.50 was due to the gem-dimethyl group while the three exocyclic olefinic protons appeared as an AA'X system at 3.90(1H, X part, dax 18.2, Jh~:9Hz, C19-H) and 4.92(2H, AA' part of AA'X system, J,x 18.2, JA~ 9Hz,-C=CH2). The protons at C-8 and C-9 appeared as an ABX system at 5.56(1H, X part of ABX system, Jgx 4.0, JBx 11.0Hz, C9-H, 6.25 and 6.83(2H, AB part of an ABX system, HA(6.25) JAB 15.5, JAX 4.0Hz, and HB(6.83) JAB 15.5, JBx 11.0Hz, 8 CH2). The triplet at 5.95ppm (J=7.0Hz) was assigned to the methine proton at position 12. The protons adjacent to the proline nitrogen resonated as an ill-defined triplet at 6.34ppm; the other four protons comprising the proline ring appeared as an unstructured multiplet between 7.6-8.2ppm.


195

Mass Spectrum: Only one prominent fragment ion at 198role (C14HI6N) from cleavage of the 8,9-bond. TLC Data Silica gel, chloroform-acetone, 9:1 v/v, Rf: 0.75. Detection: blue spot atter spraying with 65% H2SO4 and heating at ca. 120~ for 10 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 453 (1981). P. S. Steyn; The Structures of Five Diketopiperazines from Aspergillus ustus; Tetrahedron, Vol. 29, pp. 107-120(1973).

196


Common/Systematic Name Brevianamide C Molecular Formula/Molecular Weight C21H23N303; MW' = 365.17394

0 0 N

N

Me

General Characteristics Brevianamide C was obtained as an orange glass that could not be induced to crystallize. Fungal Source

Penicillium brevi-compactum.

Isolation/Purification The fungus was cultured on Czapek-Dox broth at 25 ~ Brevianamide C was isolated from the crude pigment mixture remaining after the crystallization ofbrevianamide A by repetitive preparative TLC using 5% methanol in chloroform or 1% ethanol in ether. The various metabolites in descending gf, brevianamide C, D, A, B, and F were scraped from the plates and eluted with methanol. Spectral Data UV~

~m~

234, 259, 277(in0, 300(in0, and 450rim.

IR~

(CHCI3) 3410, 3350, 1710, 1680, and 1615cm"l. ~H NMR: Refer to Birch and Russell (1972) for copy of spectrum. Mass Spectrum: HREIMS: 365.174Ira~e, M +, C21H23N303requires 365.1739; LREIMS" 365(13%), 322(15), 295(100), 177(5), 171(4), and 146m/e (3).


Reference A. J. Birch, and R. A. Russell; Studies in Relation to Biosynthesis-XLIV Structural Elucidations of Brevianamides-B, -C, -D and-F; Tetrahedron, Vol. 28, pp. 2999-3008 (1972).

197

198


Common/Systematic Name Brevianamide D Molecular Formula/Molecular Weight C21H23N303; M W -- 365.17394

General Characteristics Brevianamide D was obtained as a red glass that could not be induced to crystallize. Fungal Source Penicillium brevi-compactum. Isolation/Purification The fungus was cultured on Czapek-Dox broth at 25 ~ Brevianamide C was isolated from the crude pigment mixture remaining after the crystallization of brevianamide A by repetitive preparative TLC using 5% methanol in chloroform or 1% ethanol in ether. The various metabolites in descending Rf, brevianamide C, D, A, B, and F were scraped from the plates and eluted with methanol. Spectral Data UV~

~.,,~x 235, 264, 306, and 470nm. IR~

3440, 3200, 1710(sh), 1680, 1630, and 1615cmq. IH NMR: Refer to Birch and Russell (1972) for copy of spectrum. Mass Spectrum: HREIMS: 365.1738re~e, M +, C21H23N303requires 365.1739; LREIMS: 365(25%), 322(14), 295(100), 177(14), 171(6), 146(3), and 133role (7). Reference A. J. Birch, and R. A. Russell; Studies in Relation to Biosynthesis-XLIV Structural Elucidations ofBrevianamides-B, -C, -D and -F; Tetrahedron, Vol. 28, pp. 2999-3008 (1972).


199

Common/Systematic Name Brevianamide F; L-Tryptophanyl-L-prolyl anhydride Molecular Formula/Molecular Weight CI6HITN302; M W = 283.13208

0 N

HN

General Characteristics Brevianamide F was obtained as white needles from ethanol; mp., 173-175~ with Ehrlich's reagent.

blue color

Fungal Source Penicillium brevi-compactum. Isolation/Purification The fungus was cultured on Czapek-Dox broth at 25 ~C. Brevianamide C was isolated from the crude pigment mixture remaining after the crystallization of brevianamide A by repetitive preparative TLC using 5% methanol in chloroform or 1% ethanol in ether. The various metabolites in descending Re, brevianamide C, D, A, B, and F were scraped from the plates and eluted with methanol. Spectral Data UV:

~,m~x 277(inf), 283, and 292nm. IR:

3280, 1670, 1650(weak), and 1640 cm"~(weak). ~H NMR: (DMSO-dr) 1.2-2. l(4H, methylene multiplet); 2.9-3.5(4H, =CCHAHa, N-CH2CH2); 4.05(1H, broad triplet, NHCHCO, J=7.0Hz); 4.30(1H, triplet, NCHCO, J=6Hz); 6.97.6(4H, rn, aromatic H); 7.98(1H, d, indole NHCH=C, J~rHJCh,=2Hz);7.66(1H, s, NH); and 10.8ppm (1H, b.s., indole NH): Mass Data: LREIMS: 282(9%), 154(8), 130(100), and 83role (9); found: C, 67.8; H, 6.1; N, 14.8 CI6H17N302 requires C, 67.7; H, 5.9; N, 14.7%.

200


Reference A. J. Birch and R. A. Russell; Studies in Relation to Biosynthesis-XLIV Structural Elucidations of Brevianamides-B, -C, -D and-F; Tetrahedron, Vol. 28, pp. 2999-3008 (1972).


201

Common/Systematic Name Echinulin 3{[2•(•••-Dimethy•al•y•)-5,7-bis(3•methy••2•buteny•)ind•••3-y•]methyl}•6•methy•-2•5piperazinedione Molecular Formula/Molecular Weight

C29H39N302;MW- 461.30423

~ ~

28 ~

~

4

10

~11

0

[[

General Characteristics Needles from butanol; mp., 242-243 ~ Soluble in glacial acetic acid, chloroform, pyridine, dioxane; slightly soluble in warm alcohol, butanol, benzene, ethyl ether, acetone, and carbon tetrachloride. Fungal Source Aspergillus amstelodami = Eurotium amstelodami, A. chevalieri = E. chevalieri, and A. echinulatus = E. echmulatum.

Spectral Data

UV: Z ~m~

230(39,810), 279(9,549), and 286nm (9,120).

13C NMR: C-2, 141.1 s; C-3, 104.5 s; C-4, 121.5 d; C-5, 130.4 s; C-6, 114.5 d; C-7, 124.4 s; C-8, 131.8 s; C-9, 130.4 s; C-10, 29.9 t; C-11, 55.2 d; C-12, 167.4 s; C-14, 50.4 d; C-15, 167.8 s; C-17, 20.2 q; C-18, 38.9 s; C-19, 27.9 q; C-20, 27.9 q; C-21, 145.9 d; C-22, 111.2 t; C-23, 30.4 t; C-24, 122.3 d; C-25, 130.4 s; C-26, 25.6 q; C-27, 17.7 q; C-28, 34.3 t; C-29, 123.2 d; C-30, 131.9 s; C-31, 25.6 q; and C-32, 17.7ppm q. TLC Data Silica gel HF2s4; A: ethyl acetate-hexane, 2:1 v/v; B: benzene-ethyl acetate, 1:1 v/v; C: benzene-ethyl acetate, 2:1 v/v. Detection: not reported.

202


Reference R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 480 (1981).


203

Common/Systematic Name Preechinulin (+)-L-Alanyl(1,1-dimethylallyl)tryptophan anhydride Molecular Formula/Molecular Weight C19Hx3N302, ~

= 325.17903

O 4

10 11 I [

General Characteristics Fine white needles from methylene chloride-benzene; mp., 294~176 (C=0.38, in acetic acid); [a]D2~ +22.3 o (in acetic acid).

[a]D 24

+50 ~

Fungal Source Aspergillus amstelodami = Eurotium amstelodami, A. chevalieri = E. chevalieri, and A. repens = E. repens.

Spectral Data UV: EtOH

225(e=32,500), 283(7,750), and 291nm (7,000).

IR:

(KCI) 3380, 3210, 1670, 1460, 993, and 910cmq. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 459 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983). ,

204


Common/Systematic Name Neoechinulin Molecular Formula/Molecular Weight C23H25N303; M ' ~ = 391.18959 O 4

26

~ 25~y

~

10

11

NH

NH

~7

27 22

Fungal Source A s p e r g i l l u s a m s t e l o d a m i = E u r o # u m amstelodami.

Spectral Data UV:

~, ~,O=H 231(C=32,300), 287(13,100), and 420nm (9,700). ~H NMR: H-4,7.11; H-5,6.83; H-7,7.51; H-19,1.57; H-20,1.57; H-21, 6.10; H-22, 5.12; 5.11; H23, 3.42; H-24, 5.38; H-26, 1.75; H-27, 1.75; and NH, 9.59; 11.06; 12.07ppm. 13C N M R :

C-2, 131.1; C-3, 103.4; C-4, 116.3; C-5, 119.2; C-6, 124.0; C-7, 110.8; C-8, 134.6; C9, 123.1; C-10, 120.9; C-11, 145.7; C-12, 152.0; C-14, 157.0; C-15, 160.2; C-18, 38.9; C-19, 27.7; C-20, 27.7; C-21,145.1; C-22, 111.9; C-23, 33.9; C-24, 124.0; C25, 135.7; C-26, 25.4; and C-27, 17.6ppm. TLC Data Silica gel I-'~254. Solvent: A, ethyl acetate-hexane, 2:1 v/v; B, benzene-ethyl acetate, 1:1 v/v; C, benzene-ethyl acetate, 2:1 v/v. Rr: A, 0.75; B, 0.50; C, 0.30. Detection: not reported. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolit.e_s; Academic Press, New York, pp. 463 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

2.

Diketopiperazines

205

Common/Systematic Name Neoechinulin A Molecular Formula/Molecular Weight CI9H21N302; M W = 323.16338

O 4

10 11 [ [ 3

Nil

General Characteristics Ivory crystals from methanol; mp., 264-265~ Fungal Source A s p e r g i l l u s ruber = E u r o t i u m rubrum

and A.

amstelodami = E. amstelodami.

Spectral Data UV~

Z ~m"~

229(e=23,300), 286(11,700), 292(10,400), and 338nm (9,500).

IR~

(KBr) 3360, 2980, 1670, and 1630cm~. IH NMR: H-l, 8.30; H-4, 7.00-7.50; H-5, 7.00-7.50; H-6, 7.00-7.50; H-7, 7.00-7.50; H-10, 6.93; H-13, 8.30; H-14, 4.15; H-16, 10.96; H-17, 1.42; H-19, 1.50; H-20, 1.50; H-21, 6.12; H-23, 5.04; and H-24, 5.06ppm. ~3CNMR: C-2, 144.1; C-3, 103.3; C-4, 118.7; C-5, 120.8; C-6, 119.5; C-7, 110.2; C-8, 135.2; C9, 126.0; C-10, 111.6; C-11, 124.8; C-12, 159.7; C-14, 50.5; C-15, 166.3; C-17, 17.9; C-18, 40.2; C-19, 27.5; C-20, 27.5; C-21,145.2; and C-22, 111.6ppm. TLC Data Silica gel HF254; A: ethyl acetate-hexane, 2:1 v/v; B: benzene-ethyl acetate, 1:1 v/v; C: benzene-ethyl acetate, 2:1 v/v. Re: A, 0.24, B, 0.17, C, 0.09. Detection: not reported. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New

206


York, pp. 469 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

2.

Diketopiperazines

207

Common/Systematic Name Neoechinulin B; Cryptoechinulin C; E 10 Molecular Formula/Molecular Weight C19HI9N302; MW' = 321.14773 O 4

10 11 I I

3

NH

VNH~'8

N 5

17

N General Characteristics Yellow crystals from methanol; mp., 234-236~ Fungal Source Aspergillus amstelodami = Eurotium amstelodami.

Spectral Data UV: Z E~

228(e=28,100), 273(19,000), 284(18,100), and 374nm (10,400).

IR;

(KBr) 3360, 2980, 2925, 1680, and 1645cm~. ~H NMR: H-I, 8.36; H-4-7, 6.70-7.50; H-10, 6.85; H-13, 10.61; H-16, 10.86; H-17, 5.08, 5.34; H-19, 1.50; H-20, 1.50; H-21, 6.07; H-23, 5.07; and H-24, 5.09ppm. TLC Data Silica gel HF254;A: ethyl acetate-hexane, 2:1 v/v; B: benzene-ethyl acetate, 1:1 v/v; C: benzene-ethyl acetate, 2:1 v/v. Re: A, 0.77; B, 0.46; C, 0.26. Detection: not reported. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 471 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

208


Common/Systematic Name Neoechinulin C; E8; Cryptoechinulin A Molecular Formula/Molecular Weight C24H27N302; ]~[W' = 389.21033

O

4

10 11 I I 3

NH

2021~ 6 22

General Characteristics Optically inactive; yellow crystals from light petroleum-benzene, 1"1 v/v; mp., 205-207"C. Fungal Source Aspergillus amstelodami = Eurotium amstelodami.

Spectral Data UV:

231 (e=34,600), 275(22,300), 290(18,600), and 380nm (12,000). IR;

(KBr) 3350, 2970, 2925, 1680, and 1640cmq. IH NMR:

H-l, 8.66; H-4,5,7, 6.80-7.40; H-10, 7.01; H-13, 10.79; H-16, 10.96; H-17, 4.96, 5.27; H-19, 1.48; H-20, 1.48; H-21, 6.07; H-23, 5.06; H-24, 5.08; H-25, 3.40; H-26, 5.35; H-28, 1.76; and H-29, 1.76ppm. 13CNMR: C-2, 131.1; C-3, 102.8; C-4, 112.0; C-5, 118.5; C-6,124.0; C-7, 110.9; C-8, 134.4; C9, 124.0; C-10, 120.9; C-11, 142.2; C-12, 155.7; C-14, 134.8; C-15, 157.1; C-17, 100.0; C-18, 38.6; C-19, 27.5; C-20, 27.5; C-21, 144.1; C-22, 111.7; C-23, 33.9; C24, 124.0; C-25, 135.6; C-26, 25.4; and C-27, 17.6ppm. TLC Data Silica gel I-IF254;A, ethyl acetate-hexane, 2:1 v/v; B, benzene-ethyl acetate, 1"1 v/v; C, benzene-ethyl acetate, 2:1 v/v. Rf: A, 0.80; B, 0.53; C, 0.39. Detection: not reported.


209

R.eferences R.. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 473 (1981). A. Dossena, R. Marchelli, and A. Pochini; Neoechinulin D, a New Isoprenylated Dehydrotryptophyl Metabolite from Aspergillus amstelodami; Experientia, Vol. 31, p. 1249 (1975). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, p. 407 (1983).

210


Common/Systematic Name Neoechinulin D Alanyl-2-(1,1-dimethyl-2-propenyl-6-isopentyl)-dehydrotryptophan anhydride Molecular Formula/Molecular Weight C24H29N302; MW = 391.22598 4

lO

O ]1 NH

20

2021~

L~

6 22

General Characteristics Ivory crystals from methanol; mp., 223-225 ~ Fungal Source Aspergillus amstelodami = Eurotium amstelodami

and A. rubrum = E. t u b e r

(Isolated from beet pulp). Isolation/Purification Neoechinulin D was isolated from the crude ethereal extract of the mycelium by silica gel 60 column chromatography eluted with hexane-ethyl acetate (1:1, v/v). Neoechinulin D rich fractions were combined, concentrated, and further purified by preparatory TLC followed by crystallization from petroleum ether-benzene as ivory crystals; recrystallized from methanol. Biological Activity Growth retardant for silkworm larvae (Bombyx mori L.). Spectral Data UV: ~

EtOH max

231(e=34,600), 265(11,400), 296(10,400), and 345nm (10,900).

IR:

(KBr) 3340, 2940, 1670, and 1630. IH NMR: H-l, 8.32; H-4,5,7, 6.95-7.40; H-10, 7.01; H-13, 10.38; H-14, 4.25; H-16, 10.62; H17, 1.53; H-19, 1.48; H-20, 1.48; H-21, 6.11; H-23, 5.06; H-24, 5.08; H-25, 3.40; H26, 5.33; H-28, 1.72; and H-29, 1.72ppm.


211

Mass Spectrum: 391.2262m/e (M+); calcd for C24H29N302,391.2258. TLC Data Silica gel HF254;A: ethyl acetate-hexane, 21 v/v; B benzene-ethyl acetate, 1"1 v/v; C: benzene-ethyl acetate, 2:1 v/v. Rf: A, 0.37; B, 0.23; C, 0.12. Detection not reported. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 466 (1981). A. Dossena, R. Marchelli, and A. Pochini; Neoechinulin D, a New Isoprenylated Dehydrotryptophanyl Metabolite from Aspergillus amstelodami; Experientia, Vol. 31, p. 1249 (1975). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983). H. Nagasawa, A. Isogal, K. Ikeda, S. Sato, S. Murakoshi, A. Suzuki, and S. Tamura; Isolation and Structure Elucidation of a New Indole Metabolite from Aspergillus ruber; Agr. Biol. Chem., Vol. 39, pp. 1901-1902(1975).

212


Common/Systematic Name_ Neoechinulin E Molecular Formula/Molecular Weight CIgHITN303; ~ = 323.12670 O 4

10

1,1

II

N

H

NH

8 N 2~

k~x

O 22

General Characteristics Orange-red crystals from methanol; mp., 275~ Funsal Source v

Aspergillus amstelodami = Eurotium amstelodami.

Spectral Data UV:

Z Em'~ 228(6=37,100), 281(9,100), and 410nm (8,100). IR:

(KBr) 3330, 3150, 3020, 2840, 1740, 1690, and 1600cmq. IH NMR: H-I, 8.64, H-4-7, 6.90-7.55, H-10, 7.40; H-13, 10.58; H-16, 10.58; H-19, 1.54; H-20, 1.54; H-21, 6.06; H-23, 5.06; and H-24, 5.20ppm. TLC Data Silica gel HF254; A: ethyl acetate-hexane, 2:1 v/v; B: benzene-ethyl acetate, 1:1 v/v; C: benzene-ethyl acetate, 2:1 v/v. Rf: A: 0.51; B: 0.36; C: 0.17. Detection: visible light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 464 (1981). A. Dossena, R. Marchelli, and A. Pochini; Neoechinulin D, a New Isoprenylated Dehydrotryptophyl Metabolite from Aspergillus amstelodami; Experientia, Vol. 31, p. 1249(1975).


W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

213

214


Common/Systematic Name Isoechinulin A Molecular Formula/Molecular Weight C24H29N302; M W ~---3 9 1 . 2 2 5 9 8

23

4

10

I

24

,,

3

Ir , T

NH "

17

7

20 2 1~_2 2 6

Fungal Source

Aspergillus ruber = Eurotium rubrum.

Biological Activity Isoechinulin A inhibited growth of silkworm larvae. Spectral Data UV: EtOH ~m~x

227(e=31,500), 289(8,900), and 34 lnm (9,400).

IR:

(KBr) 3260, 1675, and 1633cmq. ~H NMR: H-I, 10.21; H-4, 7.10; H-6, 6.99; H-7, 7.34; H-10, 7.15; H-13, 7.92; H-14, 4.28; H16, 7.53; H-17, 1.55; H-19, 1.55; H-20, 1.55; H-21, 6.16; H-23, 5.13; H-24, 5.13; H25, 3.40; H-26, 5.38; H-28, 1.71" and H-29, 1.71ppm. 13C N M R :

C-2, 143.9; C-3, 102.8; C-4, 123.2"; C-5, 132.9'*; C-6, 117.9; C-7, 111.1"**;C-8, 134.6; C-9, 126.4; C-10, 112.1"**"C-11,124.3; C-12, 160.3" C-14, 51.6; C-15,165.8; C-17, 20.9; C-18, 39.3; C-19, 27.4; C-20, 27.4; C-21, 144.1; C-22, 113.1; C-23, 34.6; C-24, 124.0"; C-25, 132.0"*; C-26, 25.8" and C-27, 17.9. *, **, *** Assignments may be reversed. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 475 (1981).


W. B. Turner and D. C. Aldridge; Fungal Metabolites I.I; Academic Press, New York, New York, p. 407 (1983).

215

216


Common/Systematic Name Isoechinulin B Molecular Formula/Molecular Weight C24H27N302; M3vV= 389.21033

4

24

10

0 H

I HN J~. NH ~ , 8 " ~ "C H2 ~21

0

22

Fungal Source Aspergillus ruber = Eurotium rubrum.

Spectral Data UV: ~, max MeOH

228(e=27,900), 272(18,700), 285(sh) (16,600), and 370nm (10,400).

IR:

(KBr) 3310, 1678, and 1643cm"l. IH NMR: (CDCI3) H-I, 10.23; H-4, 7.00-7.20; H-6, 6.96; H-7, 7.30; H-10, 7.00-7.20; H-13, 9.80; H-16, 8.08; H-17, 5.00-5.40; H-19, 1.54; H-20, 1.54; H-21, 6.13; H-23, 5.00-5.40; H-24, 5.00-5.40; H-25, 3.38; H-26, 5.00-5.40; H-28, 1.68; and H-29, 1.68ppm. ~3CNMR: C-2, 144.6; C-3, 102.7; C-4, 123.2"; C-5, 133.0"*; C-6, 118.0; C-7, 111.3***;C-8, 134.7; C-9, 126.0; C-10, 113.5"**;C-11,124.0; C-12, 158.5; C-14, 133.6; C-15, 156.1; C-17, 103.0; C-18, 39.2; C-19, 27.4; C-20, 27.4; C-21,144.2; C-23, 34.5; C-24, 124.0"; C-25, 132.0"*; C-26, 25.7; and C-27, 17.8ppm. 9 **

, ,

***

Assignmentsmay be interchanged.

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 476 (1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

2.

Diketopiperazines

217

Common/Systematic Name Isoechinulin C Molecular Formula/Molecular Weight C24H27N303; M W -- 405.20524

.~

~,~

4

10 3

-

~

i

~

0

/

12 NH

.,~1HN

~L,~ 7

"~-NH/~18 ys "~CH2 20 21~22 O

Fungal Source Aspergillus ruber = Eurotium rubrum.

Spectral Data UV:

Meott

229(e=26,700), 272(18,000),286 sh (15,800), and 371nm (9,700).

IR:

(KBr) 3250, 1676, and 1644cm"1. 1H NIVIR:

H-I, 10.21; H-4, 7.02; H-6, 7.02; H-7, 7.31; H-10, 7.07; H-13, 9.73; H-16, 8.12; H17, 5.00; 5.34; H-19, 1.56; H-20, 1.56; H-21, 6.11; H-23, 5.08; H-24, 5.05; H-25, 2.70-3.10; H-26, 2.70-3.10; H-28, 1.22; and H-29, 1.33ppm. ~3CNMR: C-2, 140.2; C-3, 104.5; C-4, 124.5; C-5, 131.2; C-6, 119.9; C-7, 112.8"; C-8, 136.4; C-9, 127.8; C-10, 112.9"; C-11,126.4; C-12, 158.3; C-14, 135.5; C-15, 157.0; C-17, 100.6; C-18, 40.6; C-19, 28.4; C-20, 28.4; C-21,146.2; C-22, 112.9~ C-23, 36.7; C24, 65.9; C-25, 59.4; C-26, 25.5; and C-27, 19.6ppm. "Assignments may be interchanged. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p.477 (1981). W. B. Turner and D. C. Aldri.dge; Fungal Metabolites II; Academic Press, New York, New York, p. 407 (1983).

218


Common/Systematic Name E7 Molecular Formula/Molecular Weight C29H35N302; M ~ = 457.27293

O

H2cHN General Characteristics Crystals from ether; mp., 146-148 ~

Fungal Source Aspergillus amstelodami IFO 6667 = Eurotium amstelodami. Spectral Data IR~

(KBr) 1680, and 1640cm"~. Mass Spectrum: HREIMS: 457.2697(M+), 402, 388, 332, 294, and 69m/e. Reference S. Inoue, J. Murata, N. Takamatsu, H. Nagano, and Y. Kishi; Synthetic Studies on Echinulin and Related Natural Products. V. Isolation, Structure and Synthesis of Echinulin-Neoechinulin Type Alkaloids Isolated from Aspergillus amstelodami; Yakugaku Zasshi, Vol. 97, pp. 576-581(1977).


219

Common/Systematic Name Cryptoechinulin G Molecular Formula/Molecular Weight C29H35N302; M W ' = 4 5 7 . 2 7 2 9 3

24

H 22

General Characteristics Amorphous solid, optically inactive. Fungal Source

Aspergillus ruber = Eurotium rubrum.

Spectral Data ~3CNMR*: C-2, 126.6; C-3, 101.3; C-4, 128.9; C-5, 130.3; C-6, 123.0; C-7, 109.5; C-8, 134.5; C9, 130.2; C-10, 114.6; C-11, 142.2; C-12, 156.6; C-14, 134.6; C-15, 156.1; C-17, 100.4; C-21,145.4; C-22, 111.1; C-24, 124.9; C-25, 130.2; C-29, 124.7; and C-30, 130.2. * Data for saturated carbons not reported. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 468 (1981). G. Gatti; Molecular Structure of Cryptoechinulin G, an Isoprenylated Dehydrotryptophan Metabolite Isolated from Aspergillus ruber; Tetrahedron Lett., p. 2605-2606(1978). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

220


Common/Systematic Name Cryptoechinulin B; Aurechinulin; E6 Molecular Formula/Molecular Weight C43H49N3Os; ~

= 687.36722

3'a

3'

aa a

~T/O H

HO.~ ~.~1~H 0 0 7' ae~ t~, b

S General Characteristics Crystals; mp., 188 ~C. Fungal Source Aspergillus amstelodami -, Eurotium amstelodami. Spectral Data 1H NMR: [(CD3)250] Similar to that of cryptoechinulin D except for the addition of two methyl resonances at 1.7ppm and a -CH2CH= pattern at 3.3 and 5.3ppm.

Mass Spectrum: LREIMS: 687, 618, 481,412, 344, 320, 298, 265, 243,206, and 69m/e. References R. Cardillo, C. Fuganti, D. Ghiringhelli, and P. Grasselli; New Minor Metabolites of Asperg#lus amstelodami; La Chimica E L'Industria, Vol. 57, pp. 678-679(1975). G. Gatti, R. Cardillo, C. Fuganti, and D. GhiringheUi; Structure Determination of Two Extractives from Aspergillus amstelodami by NMR Spectroscopy; J. C. S. Chem. Comm., pp. 435-436(1976).


221

Common/Systematic Name Cryptoechinulin D Molecular Formula/Molecular Weight

C38H41N305;h/IW'= 619.30462 3'0~

3'a 3' ~ , , . . 0 H

HO./~ O ~/~"-' 7' (~HO �9 "

3 ~

....

"NHIg'

o

General Characteristics Crystals; mp., 198 ~C. Fungal Source Aspergillus amstelodami -, Eurotium amstelodami.

Spectral Data ~H NMR: [(CD3)2SO] Five protons that were exchangeable with D20 (11.8; 10.9; 9.2; 8.8, CONH; and 7.8ppm, CONH); 10.9(NH); 6.9-7.5ppm (4 aromatic protons); 1.4(3H); 6.97(3a-H); 6.7(7'H); 3.2 and 5.2ppm (-CH2CH=); 1.6ppm (3H); and 10.2ppm (CHO). 13C NM~:

[(CD3)250] 161.5 and 167.8ppm (two CONH groups); 36.8 (suggests the tt, ttdimethylallyl chain in position 2 of tryptophan); 144.4(C-3b) and 111.7ppm (C-3a); 17.7 and 25.6ppm (methyl resonances); 197.3ppm (CHO). References R. Cardillo, C. Fuganti, D. Ghiringhelli, and P. Grasselli; New Minor Metabolites of Aspergillus amstelodami, La Chimica E L'Industria, Vol. 57, pp. 678-679(1975). G. Gatti, R. Cardillo, C. Fuganti, and D. Ghiringhelli; Structure Determination of Two Extractives from Aspergillus amstelodami by NMR Spectroscopy; J. C. S. Chem. Comm., pp. 435-436(1976).

222


Common/Systematic Name Fumitremorgin A Molecular Formula/Molecular Weight C32H41N307; M W - 5 7 9 . 2 9 4 4 5

0 0 ~ OH

~ 26

" 22

General Characteristics Colorless prisms from methanol; mp., 206-209~ [a]D 10 +61 o (acetone). Soluble in chloroform and ethyl acetate; slightly soluble in methanol and ethanol; unstable to prolonged exposure to light. Fungal Source

Aspergillus fumigatus and A. caespitosus.

Biological Activity Tremorgenic: lmg dosed IP to mice caused perceptible tremors; 5mg dosed IP to mice caused sustained tremors and 70% mortality. Spectral Data UV:

~

EtOH

226(6=31,700), 277(5,300), and 296nm (4,900).

1H NMR: H-4, 6.59; H-5, 6.82; H-7, 7.68; H-10, 5.50; 11 OH, 4.48; H-14, 3.63; H-15, 1.60-2.20; H-16, 1.60-2.20; H-17, 6.13; H-20, 5.06; H-21, 2.40; H-23, 0.99; H-24, 2.00; H-25, 6.62; H-26, 5.02; H-28, 1.71; H-29, 1.71; H-30, 3.84; H-31, 4.71; H-32, 5.60; H-34, 1.81; and H-35, 1.81ppm. TLC Data Silica gel G-HR; A: chloroform-acetone, 93:7, v/v; B: toluene-ethyl acetate-formic acid,


223

5:4:1, v/v/v; Rf: A, 0.30, B, 0.65. Detection: slate gray-blue spot in visible light or mustard-colored spot under UV light; develops immediately alter spraying with 50% ethanolic H2SO4. NOTE: The color response and Rf's ofverruculogen are identical to those of fumitremorgin A in the above conditions. References R. J. Cole and R. H. Cox; Handbook of Toxic Funga_! Metabolites; Academic Press, New York, pp. 357-359(1981). M. Yamazaki, H. Fujimoto, and T. Kawasaki; Tremorgenic Toxins from Aspergillus

fumigatus; Tetrahedron Lett., p. 1241 (1975).

224


Common/Systematic Name Fumitremorgin B; Lanosulin Molecular Formula/Molecular Weight C27H33N3Os; M W -- 479.24202 OH 0 - OH

MeO

N H "'

II r~

0 - -

General Characteristics Colorless needles from methanol; mp., 211-212 oC. Fungal Source

Aspergillus fumigatus, A. caespitosus (NRRL 1929), Penicillium lanosum, and P. piscarium.

Isolation/Purification Cultures were comminuted in 70% aqueous acetone, filtered and extracted with chloroform, adsorbed on a silica gel column and eluted in sequence with benzene and 3% methanol/benzene. The tremorgenic fractions were further purified on a second silica gel column eluted with 1.5% acetone in chloroform. Verruculogen and fumitremorgen B were crystallized from benzene-ethanol solution and finally from methanol solution. Biological Activity Tremorgenic: Fumitremorgin B dosed at lmg/mouse (IP) caused perceptible tremors; at 5mg/mouse (IP), it caused sustained tremors and 70% mortality. Spectral Data UV:

~

MeOH max

228(e=36,400), 278(7,500), and 297nm (8,400).

IR:

(KBr) 3460, 3420, and 1665cm "~. ~H NMR: (CDCI3) H-4, 7.80(J=9.0); H-5, 6.73(J=9.0, 2.0); H-7, 6. 64(J=2. 0); H-10, 5.72;


225

H-14, 3.80; H-15, 2.00; H-16, 2.00; H-17, 4.50; H-20, 5.95; H-21, 4.70; H-23, 1.67; H-24, 1.97; H-25, 4.50; H-26, 4.50; H-28, 1.60; H-29, 1.81; and H-30, 3.82ppm.

13C NMR: (CDCI3) C-2, 130.8 s; C-3, 104.2 s; C-4, 121.1 d; C-5, 104.0 d; C-6, 155.8 s; C-7, 93.7 d; C-8, 137.6 s; C-9, 120.3 s; C-10, 68.8 d; C-I 1, 82.8 d; C-12, 170.0 s; C-14, 48.9 t; C-15, 22.5 t; C-16, 28.9 t; C-17, 58.6 d; C-18, 165.8 s; C-20, 58.2 d; C-21, 122.7 d; C-22, 134.8 s; C-23, 25.5 q; C-24, 18.3 q; C-25, 45.1 t; C-26, 120.0 d; C-27, 134.4 s; C-28, 25.5 q; C-29, 18.3 q; and C-30, 55.6ppm q. References R. J. Cole and R. H. Cox; Handbook of Toxic Fung.a.l_Metabolites..; Academic Press, New York, pp. 360-363(1981). H. W. Schroeder, R. J. Cole, H. Hein, Jr., and J. W. Kirksey; Tremorgenic Mycotoxins from Aspergillus caespitosus; Applied Microbiology, pp. 857-858(1975). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, p. 435 (1983). M. Yamazaki, K. Sasago, and K. Mikyaki; Tremorgenic Toxins from Aspergillus

fumigatus Fres.; J. Chem. Soc., Chem. Commun., p. 408 (1974).

226


Common/Systematic Name Fumitremorgin C (SM-Q) Molecular Formula/Molecular Weight C22H25N303; M W -- 379.18959

O 10

MOO"

v

-NH

15

General Characteristics Crystals from ethyl acetate; mp., 125-130~ Fungal Source

Aspergillusfumigatus isolated from moldy silage suspected of causing disease in beef cattle.

Biological Activity Tremorgenic to day-old cockerels at levels down to 25mg/kg (oral). Spectral Data UV: ~.m,~M~O" 224, 272, and 294nm. IH N]V[R:

(CDCI3) I-NH, 8.32;H-4, 7.48(J=9.0);H-5, 6.86(/=9.0,2.0);H-7, 6.92(,/=2.0);H-17, 4.16; H-20, 4.98(J=9.0);H-21, (6.06)(J=9.0);H-23, 2.07;H-24, 1.72;and H-25, 3.91ppm. 13C N M R :

(CDCI3) C-2, 132.1 s; C-3, 106.2 s; C-4, 118.8 d; C-5, 109.4 d; C-6, 156.4 s; C-7, 95.2; C-8, 136.9 s; C-9, 120.7 s; C-10, 45.4 t; C-11, 51.0 d; C-12, 169.4 s; C-14, 45.4 t; C-15, 23.0 t; C-16, 28.6 t; C-17, 59.2 d; C-18, 165.6 s; C-20, 56.8 d; C-21,124.1 d; C-22, 133.9 s; C-23, 18.1 q; C-24, 25.7 q; and C-25, 55.7ppm, q TLC Data Silica gel G-H~ toluene-ethyl acetate-formic acid, 5:4:1, v/v/v; Rf: 0.55. Detection: bright orange spot develops immediately after spraying with 50% ethanolic H2SO4 and minimal heating.


227

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 364-367(1981). R. J. Cole, J. W. Kirksey, J. W. Dorner, D. M. Wilson, J. Johnson, Jr., D. Bedell, J. P. Springer, K. K. Chexal, and J. Clardy; Ann. Nutr. Alim., Vol. 31, pp. 685-692 (1977).

228


Common/Systematic Name Verruculogen Molecular Formula/Molecular Weight C27H33N307; M W = 511.23185

OH 0 .--- OH '

MeO~,....-.~..f.~N / ~

\~/

N

~

.i-" \ 2~ II

!

,s

0

General Characteristics Colorless plates from benzene-ethanol, 1 1, v/v; mp., 233-235~ (dec.); [a]D -27.7 ~ (in CHCI3). Soluble in benzene, ethyl acetate, and acetone; slightly soluble in ethanol; and very soluble in CHCI3. Verruculogen is unstable to prolonged exposure to light. Fungal Source. Penicillium verruculosum (ATCC 24640; NRRL 5881) --" P. brasilianum, P. paraherquei -, P. brasilianum, P. piscarium -, P. brasilianum, P. janthinellum, P. parilli --"P. graminicola, P. estinogenum --, P. brasilianum, Aspergillus caespitosus (NRKL 1929), and A. fumigatus.

Isolation/Purification Cultures were extracted in hot chloroform. Chloroform extracts were filtered, dried, evaporated to dryness in vacuo. The crude extract was chromatographed on a silica gel (Merck 70-325 mesh) column packed in n-hexane and eluted with n-hexane to remove non-polar triglycerides; the toxin was eluted with ethyl ether. The toxin was purified further on a silica gel column packed in toluene and eluted with toluene followed with a linear gradient from toluene to ethyl acetate. The tremorgen was further purified using a column packed with Florisil and eluted with 5% ethyl acetate in hexane. Active fractions were combined, concentrated m vacuo, and crystallized from benzene-ethanol solution (1:1, v/v). Biological Activity The EDs0 for tremor response in mice was 0.39mg/kg (IP); in day-old cockerels it was 0.33mg/kg (IP). Tremors are sustained at higher levels, intermittent at lower levels, and enhanced by enforced movement. Verruculogen administration produced a decrease in y-aminobutyric acid levels in mouse central nervous system; verruculogen-induced tremor is mediated by a loss of inhibitory GAB A function. The LDs0 of verruculogen to mice was 2.4mg/kg (IP) and 126.7mg/kg (oral); to day-old cockerels, 15.2mg/kg (IP) and 265.5mg/kg (oral).


229

Spectral Data UV: ~, Em ~~

226(E=47,500), 277(11,000), and 295nm (9,750).

CD: AE (in EtOH solution): 290(+0.16) and 265nm (+0.56). 1H NIVIR:

H-4, 7.90(J=9.0); H-5, 6.80(,/=9.0,2.0);H-7, 6.60(,/=2.0);H-10, 5.65; 10-OH, 4.80, H-14, 3.65; H-15, 2.00; H-16, 2.00; H-17, 4.50; H-20, 6.05; H-21, 2.00; H-23, 1.02; H-24, 1.80;H-25, 6.70(J=8.0);H-26, 5.05; H-28, 1.80;H-29, 2.00; and H-30, 3.86ppm. ~3C NMR: C-2, 131.6 s; C-3, 105.7 s; C-4, 121.7 d; C-5, 109.3 d; C-6, 156.4 s; C-7, 93.9 d; C-8, 136.3 s; C-9, 121.1 s; C-10, 68.7 d; C-11, 82.6 s; C-12, 170.7 s; C-14, 51.3 t; C-15, 22.7 t; C-16, 29.1 t; C-17, 58.7 d; C-18, 166.2 s; C-20, 48.9 d; C-21, 45.3 t; C-22, 82.1 s; C-23, 27.1 q; C-24, 25.6 q; C-25, 85.8 q; C-26, 118.7 d; C-27, 142.9 s, C-28, 18.8 q; C-29, 24.2 q; and C-30, 55.7ppm q. TLC Data Silica gel G-HR; A: chloroform-acetone, 93:7, v/v; B: toluene-ethyl acetate-formic acid, 5:4:1, v/v/v. Re: A = 0.30, B = 0.65. Detection: slate gray-blue spot in visible light or mustard-colored spot under UV light; develops immediately atter spraying with 50% ethanolic H2SO4. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolit.e.S.; Academic Press, New York, pp. 368-373(1981). R. J. Cole, J. W. Kirksey, J. H. Moore, B. R. Blankenship, U. L. Diener, and N. D. Davis; Tremorgenic Toxin from Penicillium verruculosum; Applied Microbiology, pp. 248-256 (1972).

230


Common/Systematic Name 15-Acetoxyverruculogen Molecular Formula/Molecular Weight C29H3sN3Og; ~

= 569.23733 OH 0 - OH

i@

N

'l" ,~1

o

o -

~Me

~ ~~~'o_o ._Z_ 0

General Characteristics Melting point, 217-218~ (dec.) Fungal Source P e n i c i l l i u m v e r r u c u l o s u m -, P. brasilianum.

Biological Activity Tremorgenic but activity less than that observed for comparable amount of verruculogen. Spectral Data UV:

~t-~M~~ 227 and 295nm (e max not reported). TLC Data Silica gel G-HR, toluene-ethyl acetate-formic acid, 5:4:1, v/v/v. Rf: 0.66. Detection: slate-gray spot develops immediately atter spraying with 50% ethanolic H2SO4. Reference R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 374-376(1981).


231

Common/Systematic Name TR-2 Molecular Formula/Molecular Weight C22H27N306; M~V = 429.18999

OH

4 5

"

21

0

o ll -

14

~OH

General Characteristics Crystals from benzene-ethyl acetate, 95:5, v/v; mp., 150-152~ Fungal Source Penicillium verruculosum --, P. brasilianum, and Asperg#lus fumigatus Fres. Also,

obtained as one of two products from reductive cleavage of verruculogen. Biological Activity Perceptible tremors in day-old cockerels dosed orally at levels down to 12.5mg/kg. Spectral Data UV:

~

EtOH max

224(e = 37,400), 268(6,830), and 294nm (7,540).

~H NMR: (CDCI3) H-4, 7.60(d-9.0); H-5, 6.60(J=9.0, 3.0); H-7, 6.87; H-10, 5.52; 10-OH, 5.00; 11-OH, 4.20; H-14,3.54; H-15, 1.85; H-16, 1.85; H-17, 4.33; H-20, 5.37; H-21, 1.85; H-23, 1.05; H-24, 1.30; 22-OH, 5.94; and H-25, 3.71ppm. ~3C NMR: (CDC13) C-2, 131.8 s; C-3, 105.1 s; C-4, 121.2 d; C-5, 109.8 d; C-6, 156.6 s; C-7, 95.2; C-8, 137.2 s; C-9, 120.8 s; C-10, 68.8 d; C-11, 83.4 d; C-12, 171.4 s; C-14, 49.9 t; C-15, 22.7 t; C-16, 29.6 t; C-17, 58.9 d; C~ 166.2 s; C-20, 57.4 d; C-21, 45.3 t; C-22, 71.2 s; C-23, 31.9 q; C-24, 29.7 q; and C-25, 55.7ppm q. Mass Spectrum: 429 M + with prominent losses of-15(CH3), 18(OH), and 84m/e (C5H80).

232


TLC Data Silica gel G-HR, toluene-ethyl acetate-formic acid, 5:4:1, v/v/v. Re: 0.46. Detection: light brown fluorescent spot after spraying with 50% ethanolic H2SO4 and heating at 100~ for 5 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 377-381(1981). R. J. Cole and J. W. Kirksey; The Mycotoxin Verruculogen: a 6-O-Methylindole; Agric. Food Chem., Vol. 21, pp. 927-929(1973).


233

Common/Systematic Name Epiamauromine Molecular Formula/Molecular Weight C32H36N402; MW = 508.28383 5//Is

u 0

"2N

H HN,..j."'~.

~

''~"

0 General Characteristics Epiamauromine was isolated as a white solid; mp., 134"C; [a]D -50.0* (C=0.18g/dl, in CHCI3) and [a]D-29.1 ~ (c=0.46g/dl, CHCI3). Fungal Source Sclerotia ofAspergillus ochraceus (NRRL 3519). Isolation/Purification Intact sclerotia ofA. ochraceus were extracted at room temperature with hexane, then CHCI3. After removal of the solvent in vacuo, the hexane extract was subjected to reversed-phase HPLC [MeOH-H20 (9:1, v/v), 2ml/min] to give N-methylepiamauromine. The CHC13 extract, after removal of the solvent in vacuo, was fractionated through a column of Sephadex LH-20 (25-1001.tm) using CH2Cl2-hexane (1:1, v/v), then CH:CI2-MeOH (1:1, v/v) as eluents. The fractions obtained with CH2C12-hexane (1:1, v/v) were combined, concentrated, and purified by HPLC as above to give epiamauromine and N-methylepiamauromine. The fractions eluted with CH2CI2-MeOH (1:1, v/v) were likewise combined, concentrated, rechromatographed on Sephadex LH-20 using CHCIa-MeOH (2:1, v/v), and purified by reversed-phase HPLC to yield cycloechinulin. Biological Activity Caused moderate reduction in weight gain in assays against the lepidopteran crop pest Helieoverpa zea. Spectral Data UV;

~

Mr max

215(1og e=5.0), 243(5.0), and 300nm (4.6).

1H NMR: (CDCI3) H-1,5.36, br s; H-2, 5.30, s; H-4, 7.16(dd, J=-l.2, 7.8); H-5, 6.74(ddd, J=l.2,

234


4.2, 7.2); H-6, 7.08(dt, J=l.2, 7.8); H-7, 6.56(ddd, ,/=0.6, 1.2, 7.8); H-10, 2.45(dd, ,/=9.6, 13.8); 2.75(dd, ,/=9.0, 13.3); H-11, 4.04(dt, ,/=1.8, 9.0); H-15, 5.86(dd, ,/=10.8, 17.4); H-16, 5.06(dd, ,/=1.2, 17.4); 5.09(dd, ,/=0.6, 10.8); H-17, 1.10, s; H-18, 0.93, s; H-I', 4.91, br s; H-2', 5.30, s; H-4', 7.12(dd, ,/=1.2, 7.2); H-5', 6.72(dd, J=l.2, 4.2, 7.2); H-6', 7.06(dt, ,/=1.2, 7.8); H-7', 6.52(dm, ,/=0.6, 1.2, 7.8); H-10', 2.50(dd, ./=6.0, 12.6); 2.35(dd, ./=10.8, 12.6); H-11', 3.90(ddd, ,/=1.8, 6.0, 11.4); H-15', 5.90(dd, ./=10.8, 17.4); H-16', 5.01(dd, ,/=1.2, 17.4); 5.07(dd, ,/=0.6, 10.8); H-17',1.05 s; and H-18', 0.95ppm s. ~3CNMR: (CDCI3) C-2, 79.3; C-3, 62.1; C-4, 125.7; C-5, 118.6; C-6, 128.3; C-7, 108.8; C-8, 148.4; C-9, 131.4; C-10, 36.0; C-11, 60.7; C-12, 166.0; C-14, 41.7; C-15, 143.3; C-16, 14.7; C-17, 22.9; C-18, 22.5; C-2', 77.6; C-3', 61.8; C-4', 125.0; C-5', 118.9; C-6', 128.8; C-7', 109.3; C-8', 150.0; C-9', 129.0; C-10', 35.2; C-11',62.0; C-12', 168.0; C-14', 40.8; C-15', 143.4; C-16', 114.4; C-17', 22.9; and C-18', 22.5ppm. Mass Spectrum: EIMS: [M]§ 508(0.03), 199(61), 158(20), 150(7), and 131m/e (100); HREIMS: found 508.2846; calcd for C32H36N402, 508.2838. HPLC Data Beckman Ultrasphere 5kt (10mm x 25cm) C~s reversed-phase column was used on all HPLC separations; HPLC retention time for epiamauromine was 12.7 min. Reference F. S. de Guzman, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; New Diketopiperazine Metabolites from the Sclerotia ofAspergillus ochraceus; Journal of Natural Products, Vol. 55, pp. 931-939(1992).

2.

Diketopiperazines

235

Common/Systematic Name N-Methylepiamauromine Molecular Formula/Molecular Weight C33H38N402; M W -- 522.29948 ~8

Me

Me

H

M

O

-

"

�9

N

H N

12,

N

,

Me

0 General Characteristics N-Methylepiamauromine was isolated as a white solid; [0g]D -29.1 ~ (c=0.46g/dl, in CHCI3). Fungal Source Sclerotia ofAspergillus ochraceus (NRRL 3519). Isolation/Purification Intact sclerotia of A. ochraceus were extracted at room temperature with hexane, then CHCI3. After removal of the solvent in vacuo, the hexane extract was subjected to reversed-phase HPLC [MeOH-H20 (9:1, v/v), 2ml/min] to give N-methylepiamauromine. The CHCI3 extract, alter removal of the solvent in vacuo, was fractionated through a column of Sephadex LH-20 (25-1001.tm) using CH2Cl2-hexane (1:1, v/v), then CH2CI2-MeOH (1:1, v/v) as eluents. The fractions obtained with CH2Cl2-hexane (1:1, v/v) were combined, concentrated, and purified by HPLC as above to give epiamauromine and N-methylepiamauromine. The fractions eluted with CH2CI2-MeOH (1:1, v/v) were likewise combined, concentrated, rechromatographed on Sephadex LH-20 using CHCI3-MeOH (2:1, v/v), and purified by reversed-phase HPLC to yield cycloechinulin. Biological Activity Caused moderate reduction in weight gain in assays against the lepidopteran crop pest Helieoverpa zea.

Spectral Data UV:

~

MeOH max

214(Iog e=4.4), 244(4.1), and 303nm (3.8).

IR:

(film on NaCI plate) 3380, "1665, and 1605cm"~.

236


IH ~ : (CDCI3) H-I, 5.37, s; H-2, 5.32, S; H-4, 7.16(d, J=7.5); H-5, 6.73(dt, ,/=0.9, 7.5); H-6, 7.07, dd (`/= 14mg/kg IP in mice. Other clinical signs: uncoordinated movement, diarrhea, rough hair coat and hypersensitivity to sound stimuli. Gross clinical signs very similar to those produced by paxilline. Spectral Data UV: ~,~"

232(e=39,800) and 275nm (10,500).

13C N M R :

C-2, 152.1 s; C-3, 117.1 s; C-4, 118.4 d; C-5, 119.5 d; C-6, 120.3 d; C-7, 111.5 d, C-8, 139.9 s; C-9, 125.2; C-10, 51.5 s; C-11, 39.9 s; C-12, 21.1 t; C-13, 26.3 t; C-14, 104.5 s, C-15, 87.9 s; C-16, 197.2 s; C-17, 117.5 d; C-18, 169.9 s, C-19, 77.4 s, C-20, 27.4 t; C-21, 28.2 t; C-22, 48.6 d; C-23, 32.8 t; C-24, 78.6 s; C-25, 23.5 q; C-26, 28.8 q; C-27, 23.0 q; and C-28, 16.3ppm q.

448

7.

Paspaline and Related Metabolites

TLC Data Silica gel G-HR; chloroform-acetone, 93:7, v/v; Re: 0.52. Detection: blue spot after spraying with 50% ethanolic HzSO4 and heating at 100~ for 5 min; fluorescent under UV light. References 1L J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 398-401 ( 1981). R. J. Cole, J. W. Domer, J. P. Springer, and R. H. Cox, Indole Metabolites from a Strain

ofAspergillusflavus; J. Agric. Food Chem., Vol. 29, pp. 293-295(1981).

R. T. Gallagher, J. Finer, J. Clardy, A. Leutwiler, F. Weibel, W. Acklin, and D. Arigoni; Paspalanine, A Tremorgenic Metabolite from Clavicepspaspali Stevens et Hall, Tet. Lett., Vol. 21, pp. 235-238(1980).

7. Paspaline and Related Metabolites

449

Common/Systematic Name Paxilline Molecular Formula/Molecular Weight C27H33NO4, MW = 43 5.24096 4

H

I

21 iOH

I -OH General Characteristics Clear cubes from acetone; mp., 252~ (paxilline crystallized with 1 molecule of acetone H-bonded to the hydrogen of the indole nitrogen). Fungal Sour.ce

Penicillium paxilli, Emericella desertorum, E. foreolata, E. striata, and Acremonium lolii, an endophyte of perennial ryegrass (Lolium perenne L.).

Isolation/Purification The tremorgen was extracted with hot chloroform and the crude extract was chromatographed on a silica gel column; the tremorgen eluted with diethyl ether. The ether fraction was chromatographed on a silica gel column eluted with a linear gradient from toluene to ethyl acetate. Paxilline was crystallized from acetone solution. Biological Activity In day-old cockerels dosed orally: at 25mg/kg (lmg/cockerel) tremors were intermittent; at 100mg/kg (4mg/cockerel) tremors were more sustained. Mortality was observed at higher dosage levels. In mice dosed intraperitoneally, intermittent tremors were observed at levels down to 35mg/kg. Tremors were enhanced when the animal tried to move or was forced to move. Mice were noticeably hypersensitive to sound stimuli. No mortality was observed in mice dosed up to 227mg/kg. Mice continued to feed or attempt to feed. Suspected as one of the tremorgens responsible for the clinical signs of ryegrass staggers. Spectral Data UV: ~b McOH max

230(E=41,500) and 281nm (8,000).

IR: (KBr) 3420, 1650, 1355, 1365, and 740cm "~.

450

7.


CD: AE 335(+ 1.2) and 300nm (+ 3.3). 13C NMR: C-2, 152.1 s; C-3, 114.5 s; C-4, 117.2 d; C-5, 118.4 d; C-6, 118.8 d; C-7, 111.4 d; C-8, 139.3 s; C-9, 124.1 s; C-10, 50.0 s; C-11, 39.9 s; C-12, 20.6 t; C-13, 25.8 t; C-14, 72.2 d; C-15, 83.1 d; C-16, 196.9 s; C-17, 117.9 d; C-18, 169.9 s; C-19, 75.4 s; C-20, 26.7 t; C-21, 28.1 t; C-22, 49.1 d; C-23, 32.4 t; C-24, 70.8 s; C-25, 25.7 qt; C-26, 25.8 q; C-27, 18.6 q; and C-28, 16.2ppm q. Mass Data: 435.2422(M+), 420.2165, 378.2033, 377.1998, 363.1779, and 362.1753role. (Anal. found: C, 75.29; H, 7.68; N, 3.12; calcd for C27H33NO4; C, 74.48; H, 7.58; N, 3.21; O, 14.70). TLC Data Silica gel G-HR; toluene-ethyl acetate-formic acid, 5:4:1 v/v/v; Rf: 0.75. Detection: A dark spot with 50% ethanolic H2SO4 spray; a greenish-gray spot with 3% ethanolic phosphomolybdic acid. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 386-389(1981). R. J. Cole, J. W. Kirksey, and J. M. Wells; The Structure of Paxilline, a Tremorgenic Metabolite of Penicillium paxilli Bainier; Can. J. Microbiol., Vol. 20, pp. 1159-1162(1974). C. O. Miles, A. L. Wilkins, R. T. Gallagher, A. D. Hawkes, S. C. Munday, and N. R. Towers; Synthesis and Tremorgenicity of Paxitriols and Lolitriol: Possible Biosynthetic Precursors of Lolitrem B; J. Agric. Food Chem., Vol. 40, pp. 234-238(1992). J. P. Springer, J. Clardy, J. M. Wells, R. J. Cole, and J. W. Kirksey; The Structure of Paxilline, a Tremorgenic Metabolite ofPenicillium paxilli Bainier; Tet. Lett. pp. 2531-2534(1975).


451

Common/Systematic Name a-Paxitriol Molecular Formula/Molecular Weight C27H35NO4; M W = 437.25661 17

27.,OH

O" ,,,,,,-,~

General Characteristics Colorless solid that decomposed without melting at 250-3000C. Fungal Source A c r e m o n i u m lolii

endophyte on perennial ryegrass.

Isolation/Purification Extracted with dichloromethane and dried over Na2SO4 overnight at 4~ in the dark. Removal of solvent in vacuo afforded a pale yellow oil. Addition of dichloromethane resulted initially in dissolution of the product, followed by precipitation. Atter standing at 4~ for 30 min, solvent was removed by decantation. The off-white crystals were washed with fresh solvent to afford pure 13-paxitriol. The washings were purified by flash chromatography (acetone-dichloromethane, 3:17, v/v) to give pure a-paxitriol (the less mobile diastereoisomer). Biological Activity Nontremorgenic to mice at 100mg/kg; caused lethargy and rough hair coats, and normal activities such as walking, rearing and preening were greatly reduced for several hours. Spectral Data UV:

X^~,a~o

23 l(log e=4.53) and 281nm (3.91).

~H NMR: (CDCla-DMSO) H-5, 2.57; H-6, 2.01; H-7, 4.60; H-9, 3.03; H-1 l, 5.35; H-14, 1.69; H-15, 2.06; H-17, 2.36; H-20, 7.28; H-21, 6.92; H-22, 6.91" H-23, 7.27; H-25, 1.29; H-26, 0.95" H-28, 1.22; and H-29, 1.26ppm. 13C NMR:

(CDCI3-DMSO) C-2, 153.5; C-3, 50.5; C-4, 42.5; C-5, 26.8; C-6, 28.3; C-7, 73.3;

452


C-9, 81.7; C-10, 64.5; C-11,120.2; C-12, 144.5; C-13, 75.8; C-14, 33.5; C-15, 21.2; C-16, 49.7; C-17, 27.1; C-18, 115.1; C-19, 124.7; C-20, 117.6; C-21, 118.4; C-22, 119.1; C-23, 111.7; C-24, 139.9; C-25, 16.3; C-26, 19.5; C-27, 72.4, C-28, 24.2; and C-29, 27.8ppm. Mass Spectrum: 437.2587(M+, 437.2557 for C27HasNO4,55%), 422(47), 419(42), 404(28), 343(15), 328(18), 208(9), 182(100), 169(54), and 230m/e (56). Reference C. O. Miles, A. L. Wilkins, R. T. Gallagher, A. D. Hawkes, S. C. Munday, and N. R. Towers; Synthesis and Tremorgenicity of Paxitriols and Lolitriol: Possible Biosynthetic Precursors of Lolitrem B; J. Agric. Food Chem., Vol. 40, pp. 234-238(1992).

7.


453

Common/Systematic Name 13-Paxitriol Molecular Formula/Molecular Weight C27H35NO4;1VIW= 437.25661 17

(

20

OH 2 ~ ,,,,"

General Characteristics Colorless solid that decomposed without melting at 250-300~ Fungal Source A cremonium lolii

endophyte on perennial ryegrass.

Isolation/Purification Extracted with dichloromethane and dried over Na2SO4 overnight at 4 ~ in the dark. Removal of solvent in vacuo afforded a pale yellow oil. Addition of dichloromethane resulted initially in dissolution of the product, followed by precipitation. ARer standing at 4 ~ for 30 min, solvent was removed by decantation. The off-white crystals were then washed with fresh solvent to afford pure [3-paxitriol. The washings were purified by flash chromatography (acetone-dichloromethane, 3:17, v/v) to give pure tt-paxitriol (the less mobile diastereoisomer). Biological Activity Nontremorgenic to mice at 100mg/kg; caused lethargy and rough hair coats, and normal activities such as walking, rearing and preening were greatly reduced for several hours. Spectral Data UV: ~ Acetonitfil 9 max

23 l(log e=4.58) and 280nm (3.94).

~H NMR: (CsDsN) H-5, 2.61; H-6, 2.19; H-7, 4.61; H-9, 3.06; H-10, 4.19; H-11, 5.64; H-14, 1.69; H-15, 2.03; H-17, 2.39; H-20, 7.33; H-21, 6.96; H-22, 6.94; H-23, 7.32; H-25, 1.33; H-26, 1.04; H-28, 1.29; and H-29, 1.30ppm. IaC NMR: (CsDsN) C-2, 153.4; C-3, 50.5; C-4, 42.4; C-5, 26.8; C-6, 28.3; C-7, 74.1; C-9, 80.4;

454

7.


C-10, 63.3; C-11, 117.5; C-12, 147.9; C-13, 76.2; C-14, 33.7; C-15, 21.2; C-16, 49.8; C-17, 27.1; C-18, 115.2; C-19, 124.7; C-20, 117.6; C-21, 118.5; C-22, 119.2; C-23, 111.8; C-24, 139.9; C-25,16.3; C-26, 19.6; C-27, 72.0; C-28, 26.7; and C-29, 27.1ppm. Mass Spectrum: 437.2558(M § 437.2557 for C27H35NO4,55%), 422(52), 419(15), 343(18), 328(18), 208(17), 182(100), 69(52), and 130m/e (57). Reference C. O. Miles, A. L. Wilkins, R. T. Gallagher, A. D. Hawkes, S. C. Munday and N. R. Towers; Synthesis and Tremorgenicity of Paxitriols and Lolitriol: Possible Biosynthetic Precursors of Lolitrem B; J. Agric. Food Chem., Vol. 40, pp. 234-238(1992).

7.


455

Common/Systematic Name 3-O-Acetoxypaxilline Molecular Formula/Molecular Weight C29H37NOs; ~

8

11

= 479.26717

H

NH

6

i''~ 13

,4 R

H ['OH

General Characteristics Colorless crystalline powder; mp., 162-164 ~C. Fungal Source Penicillium crustosum (strain NHL 6491) isolated from mycelia of contaminated bread

~ i c h had been intended for school lunches in Shinagawa-ku, Tokyo, Japan in 1983. Isolat~ion P. crustosum was cultivated at 25 ~ for 14 days containing Czapek-Dox medium

supplemented with 0.2% yeast extract. The mycelium was extracted with CH2C12; the residue obtained by evaporation of the extract and chromatography on silica gel with benzene-acetone (100:1, v/v) gave ergosterol; with benzene-acetone (50:1, v/v) followed by purification by LPLC using benzene gave penitrems F and B; with benzene-acetone (20:1, v/v) followed by purification by LPLC using benzene-acetone (100:1, v/v) gave penitrems A and E; with benzene-acetone (10:1, v/v) followed by further purification by LPLC [benzene-acetone (50:1, v/v)] gave penitrem D; with benzene acetone (5:1, v/v) followed by repeated purification by LPLC using benzene-acetone (20:1, v/v) and/or hexane-AcOEt-EtOH gave cyclopenin and 3-O-acetoxypaxilline; and with benzene-acetone (3:1, v/v) followed by purification by HPLC using benzene/AcOEt (5:2, v/v) gave dihydrodehydroxypaxilline. Biological Activity Probable biosynthetic precursor for the penitrems and probably tremorgenic; however, tremorgenic activity was not reported. Spectral Data UV:

~

MeOH max

229(1og e=4.74) and 279nm (4.05).

IR:

(KBr) 3500, 3460, 3400(NH, OH), and 1720cm1 (OAc).

456


CD: (c=4.55 x 105, MeOH) [0] 2~ -61,000(232), +4,900(286), and +5,000nm (294). ~H NMR: [(CD3)2CO] 1.00(3H, s, 12C-Me), 1.20(3H, 5, 2'-Me), 1.26(3H, s, 3'-Me). 1.36(3H, s, 12b-Me), 1.70(3H, m), 1.82(1H, dddd, J=13.5, 13.5, 8.5, 5.2), 1.95(1H, ddd, ,/--13.5, 13.5, 4.5), 1.99(3H, s, 3-OCOMe), 2.10(1H, m), 2.39(1H, dd, J=13.0, 10.8, 7B-H), 2.67(1H, dd, ,/=13.0, 6.3, 7a-H), 2.69(1H, ddd, ,]--13.3, 13.3, 4.8), 2.85(1H, m), 3.32(1H, d, J=l.8, 2-H), 3.48(1H, s, OH), 3.49(1H, s, OH), 4.65(1H, br dd, J=10.5, 7.6, 14a-I-I), 5.20(1H, ddd, J=5.8, 1.8, 1.8, 3-H), 5.69(1H, dd, J=5.8, 1.8, 4-I-I), 6.94(2H, m, 9-H, 10-H), 7.28(1H, m, 8-H or 1l-H), 7.32(1H, m, 11-H or 8-H), a~nd 9.85ppm [1H, br s, 12-H (NH)]. 13C NMP~:

[(CD3)2CO]C-2, 82.56; C-3, 66.01; C-4, 115.21; C-4a, 151.29; C-4b, 77.52; C-5, 34.71; C-6, 22.0; C-6a, 50.67; C-7, 30.56; C-7a, 116.71; C-7b, 126.11; C-8, 118.66; C-9, 119.54; C-10, 120.36; C-11,112.45; C-1 la, 141.23; C-12a, 153.84; C-12b, 51.61; 12b-Me, 16.73; C-12c, 43.53; 12-C-Me, 19.93; C-13, 27.87; C-14, 29.13; C-14a, 75.06; C-I', 71.58; C-2', 25.99; C-3', 27.95; MeCOO, 21.28; and MeCOO, 170.69ppm. Mass Spectrum: El-MS" 479.2669(M*, 479.2670 for C29H37NOs,63), 464.2433[(M-Me)+, 464.2433 for C28H34NOs, 100], 182(87), and 130m/e (68). Reference T. Hosoe, K. Nozawa, S. Udagawa, S. Nakajima, and K. Kawai; Structures of New Indoloditerpenes, Possible Biosynthetic Precursors of the Tremorgenic Mycotoxins, Penitrems, from Penicillium crustosum; Chem. Pharm. Bull., Vol. 38, pp. 3473-3475 (1990).


457

Common/Systematic Name 1'-O-Acetylpaxilline Molecular Formula/Molecular Weight C29H35NOs; M W = 477.25152

8

7

H

6

,,

~

~4 R -

R

I~OAc !

3'

General Characteristics Colorless needles from ethyl acetate; mp., 262-265~ (sublim.). Fungal Source Emericella striata, strain 80-NE-22. Isolation/Purification The dried mycelia were extracted with acetone at room temperature. The acetone extract was chromatographed on silica gel with benzene-acetone followed by column chromatography with benzene-ethyl acetate (5:1, v:v). It was further purified by repeated LPLC [hexane-ethyl acetate (10:1, v:v)] to yield l'-O-acetylpaxilline. Biological Activity Tremorgenic. Spectral Data UV: ~EtOH max

23 l(log E=4.62). 280(3.97), and 290nm (sh) (3.92).

IR:

3480, 3400(OH, NH), 1728(OAc), and 1680cm~ (-CO-). CD: e

-80400(244), -1090(268sh), +8260(296), and +3430nm (338).

1H NMR: (CDCI3) 1.02(3H, s, Me); 1.32(3H, s, Me); 1.43(3H, s, Me); 1.50(1H, m); 1.61(1H, s, OH), 1.65(3H, s, Me); 1.55-2.08(6H, m); 2.04(3H, s, OAc); 2.27(1H, m), 2.44(1H, dd J=13.1, 10.9Hz), 2.73(1H, dd J=13.1, 6.1Hz); 2.80(1H, m); 4.77(1H, brt, J=10.0Hz,

458

7.


CH2-CH-O-); 4.79(1H, d J=l.8Hz, (=CH-O-)); 5.78(1H, d J=I.8Hz(=C=CH); 7.07(2H, m); 7.29(1H, m); 7.43(1H, m); and 7.83ppm (1H, brs. NH). 13CNMR: [CDCI3 plus trace (CD3)280] C-2, 80.43; C-3, 195.31; C-4, 120.07; C-4a, 166.21; C-4b, 77.37; C-5, 28.05; C-6, 28.37; C-6a, 49.54; C-7, 34.23; C-7a, 117.49; C-7b, 125.15; C-8, 118.52; C-9, 119.73; C-10, 120.64; C-I 1, 111.52; C-1 la, 139.81; C-12a, 151.79; C-12b, 50.85; C-12c, 43.14; C-13, 20.94; C-14, 27.22; C-14a, 72.94; C-I', 81.89; C-2', 22.84; C-3', 23.86; 12b-Me, 16.21 12c-Me, 19.73" O-CO-Me, 22.31" and O-CO-Me, 170.92ppm. Mass Data: El-MS: 477(100% M~), 462(75 [M-Me]), 417(22 [M+-AcOH], 402(25[M+-AcOH-Me]), 384(20 [M-AcOH-Me-H20]), 182(65), and 130(45); anal calcd for C29H35NOs;C, 72.93: H, 7.39: N, 2.93; found: C, 73.07: H, 7.42: N, 2.81. Reference K. Nozawa, Y. Horie, S. Udagawa, K. Kawai, and M. Yamazaki; Isolation of a New Tremorgenic Indoloditerpene, l'-O-Acetylpaxilline, from Emericella striata and Distribution of Paxilline in Emericella spp.; Chem. Pharm. Bull., Vol. 37, pp. 1387-1389 (1989).


459

Common/Systematic Name Aflatrem; a, a-Dimethylallylpaspalinine Molecular Formula/Molecular Weight C32H39NO4; M W = 5 0 1 . 2 8 7 9 1

29~ ~

H

30

12~~00~24 General Characteristics Colorless needles from ethyl ether-acetone; mp., 222-224 ~ Fungal Source

Aspergillus flavus.

Isolation/Purification Fungal cultures were extracted with chloroform and the crude extract was chromatographed on a silica gel column. Tremorgens eluted with ethyl ether and ethyl acetate. The active fractions were combined and chromatographed on a silica gel column eluted with a linear gradient from benzene to ethyl ether followed by a second gradient from ethyl ether to ethyl acetate. Aflatrem and paspalanine were isolated in the first gradient. Biological Activity Tremorgenic when dosed IP or orally to mice, guinea pigs, and rats. A dose of lmg of partially purified toxin to mice via stomach tube initially resulted in inactivity but animals responded to auditory and tactile stimuli. Tremors became marked when animals attempted to move. Trembling became more pronounced in 1-2 hr after dosing and continued for several hours. Marked improvement was usually evident the day after treatment with the toxin. Spectral Data UV:

MeOH

231(e=27,750), 282(9,050), and 292nm (sh) (7,850).

460


13C NMR: C-2, 151.8 s; C-3, 115.1 s; C-4, 140.6 s; C-5, 115.8 d; C-6, 118.9 d; C-7, 110.6 d; C-8, 139.3 s; C-9, 123.3 s; C-10, 50.5 s; C-11, 39.4 s; C-12, 21.0 t; C-13, 26.4 t; C-14, 104.5 s; C-15, 87.6 d; C-16, 197.0 s; C-17, 116.9 d; C-18, 170.0 s; C-19, 77.6 s; C-20, 28.2 t; C-21, 32.6 t; C-22, 48.0 d; C-23, 33.8 t; C-24, 78.0 s; C-25, 28.8 q; C-26, 22.9 q; C-27, 22.9 q; C-28, 16.0 q; C-29, 111.1 t; C-30, 140.6 d; C-31, 41.3 s; C-32, 29.3 q, and C-33, 29.5ppm q. TLC Data Silica gel G-HR; chloroform-acetone, 93:7 (v/v); Re: 0.65. Detection: blue spot after spraying with H2SO4 followed by 1% dimethyl aminobenzaldehyde (ethanolic) and heating at 100 ~C for 1-2 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 410-413(1981). R. J. Cole, J. W. Dorner, J. P. Springer, and R. H. Cox; Indole Metabolites from a Strain ofAspergiilusflavus; J. Agric. Food Chem., Vol. 29, pp. 293-295(1981). R. T. Gallagher and B. J. Wilson; Aflatrem, the tremorgenic mycotoxin from Aspergillus

flavus; Tet. Lett., Vol. 21, pp. 239-240(1980).

W. B. Turner and D. C. Aldfidge; Fungal Metabolites II, Academic Press, New York, New York, pp. 298-300(1983).


461

Common/Systematic Name 13-Aflatrem Molecular Formula/Molecular Weight C32H39NO4, ~

= 501.28791

23

21

19

17

General Characteristics 13-Aflatrem was obtained as a yellow crystalline solid with the following properties: mp., 188-190~ [a]D +77.9 ~ (C--0.011, in CHCI3). Fungal Source

Aspergillusflavus (NRRL 13462) sclerotia, A. parasiticus (NRRL 6433, 13539), and A. subolivaceus (NRRL 4998).


A. flavus (NRRL 13462) sclerotia were subjected to exhaustive extraction with hexane, and this extract was directly subjected to preparative reversed-phase HPLC [81.tm C~8 column, 2.14x25cm, MeOH:H20 (90:10, v/v) at 8.4ml/min] to afford the following major components: paspalinine (retention time, 17 min), afiatrem (26 min), 13-aflatrem (32 min), nominine (34 min), aflavinine (38.5 min), and paspaline (66 min).

Biological Activity Exhibited significant activity against Helicoverpa zea, causing a 57% reduction in weight gain relative to controls when incorporated into a standard diet at a concentration of 100ppm. Spectral Data UV: Ms ~ max

233(e=18,500), 264(6,300), and 296nm (2,700).

IR:

(CH2CI2) 3572, 3468, 3060, 2971, 2937, 1690, 1614, 1455, 1275, and 894cmq. ~H NMR: (CDCI3) H-l, 7.73, s; H-5, 7.43, d, J=1.5; H-7, 7.13, dd, J=l.5, 8.8; H-8, 7.27, d,

462


,/--8.8; H-12, 2.26, m, 2.69, m; H-13, 1.75, m, 1.82, m; H-15, 4.32, s; H-17, 5.86, s; H-20, 2.73, m, 2.83, m; H-21, 2.45, dd, ,]--12.5, 10.3, 2.73, m; H-22, 2.83, m; H-23, 1.92, m, 1.97, m; H-25, 1.14, s; H-26, 1.42, s; H-27, 1.19, s; H-28, 1.23, s; H-30, 6.12, dd, J=10.5, 17.3; H-31, 5.05, br d, J=10.5, 5.09, br d, J=17.3; and H-32/33, 1.48ppm, br s. ~3CNMR: (CDCI3) C-2, 152.45; C-3, 117.22; C-4, 124.94; C-5, 115.32; C-6, 140.11; C-7, 119.54; C-8, 111.04; C-9, 138.14; C-10, 51.45; C-11, 39.86; C-12, 26.93; C-13, 21.14; C-14, 104.36; C-15, 87.96; C-16, 197.28; C-17, 117.58; C-18, 169.84; C-19, 77.61; C-20, 28.25; C-21, 27.58; C-22, 48.55; C-23, 33.82; C-24, 78.71; C-25, 23.07; C-26, 28.76; C-27, 23.55; C-28, 16.27; C-29, 41.05; C-30, 149.01; C-31, 109.87; and C-32/33, 28.83, 28.76ppm. Mass Spectrum: 501(M+, 20), 4.86(21), 483(18), 468(18), 443(10), 428(16), 425(31), 410(48), 387(17), 372(4), 344(5), 250(31), 237(37), 222(23), 198(35), 182(50), 168(37), and 130m/e (100); HREIMS: found 501.2877; calcd for C32H39NO4, 501.2881. Reference M. R. TePaske, J. B. Gloer, D. T. Wicklow, and P. F. Dowd; Aflavarin and [3-Aflatrem: New Anti-Insectan Metabolites From The Sclerotia ofAspergillusflavus; J. Natural Products, Vol. 55, pp. 1080-1086(1992).


463

Common/Systematic Name Paspalitrem A; 3-Methyl-2-butenylpaspalinine Molecular Formula/Molecular Weight C32H39NO4; M W - 5 0 1 . 2 8 7 9 1

23

H

21

Fungal Source

Claviceps paspali sclerotia (collected from Paspalum dilatatum infected plants) and an endophytic Phomopsis sp. (MF5670) isolated from the living bark of Cavendishia pubescens, a shrub or small tree of dry or exposed slopes and thickets in upland regions of northwest South America. This isolate was from Colombia. A potential ecological role of this metabolite in regard to endophytism of the woody host is possible.

Isolation/Purification Fungal sclerotia were extracted with chloroform; the chloroform extract was partitioned between hexane and 80% aqueous methanol. The aqueous methanol extract was chromatographed on a silica gel column eluted with a linear gradient from benzene to ethyl ether. The active fractions were chromatographed on a silica gel column eluted with a linear gradient from benzene to 10% ethyl ether/benzene. Biological Activity EDs0 in mice dosed IP was 400nm emission. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 29-33(1981). K. Helrich, Ed.; AOAC Official Methods of Analysis, 15th Edition, Vol. II; AOAC, Inc., Arlington, VA, pp. 1199-1205(1990) C. H. Holzapfel, P. S. Steyn, and I. F. H. Purchase; Isolation and Structure of Aflatoxins, M~ and M2; Tet. Lett. p. 2799 (1966).

11.

Aflatoxins

557

Common/Systematic Name Aflatoxin M2 Molecular Formula/Molecular Weight C17H1407; MW = 330.07395 O

O

OH 0~~"~

o,l,o,(Yo,

General Characteristics Crystals from methanol-chloroform; mp., 293 ~ (dec.). Fungal Source

Aspergillus flavus and A. parasiticus.

Biological Activity Aflatoxin M2 was excreted in the urine and milk of animals ingesting mixed aflatoxins. The LDs0 value for aflatoxin M2 was 621.tg/day old duckling compared to 121.tg for aflatoxin B~ in the same assay done simultaneously. Spectral Data UV:

~.~x 221(e=20,000), 264(10,900), and 357nm (21,000). IR:

(CHCI3) 3350, 1760, and 1690cm "l. TLC Data Adsorbosil- 1 silica gel, isopropyl alcohol-acetone-chloroform (5:10:85, v/v/v); Rf, 0.42; detection as blue fluorescence under UV light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 49-50(1981). C. W. Holzapfel, P. S. Steyn, and I. F. H. Purchase; Isolation and Structure of Aflatoxins, M~ and Mz; Tet. Lett., p. 2799 (1966).

558

11.

Aflatoxins

Common/Systematic Name Atlatoxin Pl Molecular Formula/Molecular Weight C16H1oO6; ~

= 298.04774

0

0

0

General Characteristics Pale yellow needles from methanol-benzene-hexane; mp., >320~ in MeOH).

[a]D2~ -574 ~ (C=0.08,

Source Aflatoxin PI was the principal urinary metabolite of aflatoxin B1 in rhesus monkeys. It occurred in the urine in unconjugated (3%), sulfate (10%), and glucuronide (50%) forms. Biological Activity In a mouse bioassay using IP injection, aflatoxin P l showed considerably less toxicity than aflatoxin B1. At a dosage of 100mg/kg, no mortalities were observed; at 150mg/kg there were 2 mortalities in 15 animals; and at 200mg/kg, no mortalities occurred. Atlatoxin B l in the same assay had an LDs0 of 9.5mg/kg. Spectral Data UV:

~..... 226(e=20,400), 267(11,200), 342(14,900), 362(15,400) and 425nm (2,500). References G. Buchi, D. Spitzner, S. Paglialunga, and G. N. Wogan; Life Science, Vol. 13, p. 1145 (1973). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 3-5 (1981). J. Dalezios, G. N. Wogan, and S. M. Weinreb; Science, Vol. 171, p. 584 (1971).

11.

Aflatoxins

559

Common/Systematic Name Aflatoxin D~ Molecular Formula/Molecular Weight C16H1405; M W = 286.08412

0 OH

General Characteristics Sublimed without melting at 2200-290~ in sealed tube crystals decomposed without melting at 255~ Acetate derivative; mp., 155-157~ Fungal Source A major product formed from reacting aflatoxin B~ with ammonium hydroxide at 100~ under pressure. Biological Activity Specific toxicity data not known. It has been reported that the nonfluorescent compounds formed in alkaline solutions are strongly toxic (mixture included aflatoxin D~). Spectral Data UV:

~,~H 227(~=20,792) and 324nm (17,074) [corrected from 227(15,920), and 324nm (12,440)].

IH NMR: H-2, 6.24; H-4, 2.40; H-5, 3.10; H-8, 6.25; H-12, 6.76; H-13, 4.70; H-14, 5.40; H-15, 6.63; and H-16, 3.75ppm. 13C NMR: C-l, 208.6 s; C-2, 131.5 d; C-3, 170.9 s; C-4, 31.9 t; C-5, 34.2 t; C-6, 106.1 s*; C-7, 159.5 s; C-8, 86.6 d; C-9, 158.6 s; C-10, 106.7 s*; C-11,151.3 s, C-12, 111.5 d; C-13, 47.6 d; C-14, 103.1 d; C-15, 144.1 d; and C-16, 55.8ppm q. * Assignment may be reversed.

560

11.

Aflatoxins

TLC Data Silica gel: chloroform-acetone, 95:5, v/v; Re, 0.31. Detection: turns gray-brown after spraying with 10% FeCI3; orange-yellow with 2,4-dinitrophenylhydrazine. Acetate Re, 0.78. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites, Academic Press, New York, pp. 6-9(1981). J. B. Stanley, L. S. Lee, A. F. Cucullu, and I. V. deGruy; J. Agric. Food Chem., Vol. 23, p. 447 (1975).

11.

Aflatoxins

561

Common/Systematic Name Parasiticol; Aflatoxin B3 Molecular Formula/Molecular Weight C16H1406; M W = 302.07904

O O

II I

I

CH20H

'~OJ~O~/'~,OMe General Characteristics Crystals from chloroform; mp., 217~ Fungal Source

Aspergillusflavus, A. parasiticus. Parasiticol appears to be more prominent in older cultures ofA. flavus and A. parasiticus. It was suggested to be the first step in the biological degradation of aflatoxin G~ and was reported as an intermediate in the biodegradation of aflatoxin G~ by Rhizopus spp.

Biological Activity Parasiticol had the same acute toxicity to ducklings as aflatoxin B1, but it had little tendency to cause biliary hyperplasia. It was only 1/100 as toxic as BI in chick embryo studies. Spectral Data UV;

ZmM~H 229(sh) (C=10,000), 253(7,300), 262(7,550), and 326nm (9,350); ~,mx 225(sh) (12,600), 253(6,800), 262(7,400), and 325nm (9,700). 1H NMR: H-2, 5.94; H-6, 6.62; H-10, 6.85 (,/--7.0); H-11, 4.72(,/=7.0, 3.0); H-12, 5.36 (,/--3.0, 3.0); H-13, 6.66; H-14, 3.00; H-15, 3.61; H-16, 3.85; and OH, 4.59ppm. 13C NMR: C-l, 158.6 s*; C-2, 110.8 d; C-3, 154.7 s; C-4, 103.5 s; C-5, 158.8 s*; C-6, 91.0 d; C-7, 160.6 s; C-8, 106.8 s; C-9, 150.7 s; C-10, 112.6 d; C-11, 47.2 d; C-12, 102.0 d; C-13, 145.1 d; C-14, 41.1 t; C-15, 59.9 t; and C-16, 56.4ppm q. * Assignment may be reversed.

562

11.

Aflatoxins

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites.; Academic Press, New York, pp. 10-14(1981). R. J. Cole and J. W. Kirksey; J. Agric. Food Chem., Vol. 19, p. 222 (1971).

11.

Aflatoxins

563

Common/Systematic Name Aflatoxin Q~ Molecular Formula/Molecular Weight C17Hn207; ~ = 328.05830

0

0

0 o.

General Characteristics Colorless needles from methanol-chloroform or hot acetonitrile; mp., 295 ~ (dec.); crystals from chloroform-hexane; mp., 266~ (dec.). Fungal Source Atlatoxin Q~ was a major metabolite of aflatoxin B~ metabolism in monkey, rat, and human liver preparations (in vitro). Biological Activity Aflatoxin Q~ was approximately 18 times less toxic than aflatoxin B~ by the air cell route in the chicken embryo test. No mutagenic activity was detected by the bacterial mutagenesis test, using Salmonella typhimurium TA 1538 with or without microsomal activities. Spectral Data UV: ~MeOH max

366(E=17,500), 267(11,450), and 223nm (19,030); ~, max E~H 365(18,800), 266(11,700), 242(sh) (10,000), and 224nm (20,500). TLC Data Adsorbosil- 1 silica gel, chloroform- acetone-n-hexane, 88:15:20, v/v/v, Rf, 0.23. Detection: intense yellow-green fluorescence under UV light. Silica gel, chloroform-methanol, 20:1, v/v, Rf: 0.25. Detection: yellow fluorescence in 350nm UV light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 20-24(1981). M. S. Masri, W. F. Hadden, R. E. Lundin, and D. P. H. Hsieh; J. Agric. Food Chem., Vol. 22, p. 512 (1974).

564

11.

Aflatoxins

Common/Systematic Name Aflatoxicol A (Ro) Molecular Formula/Molecular Weight C17H1406; M W "- 3 1 4 . 0 7 9 0 4

0

OH

O

General Characteristics Crystals from benzene-n-hexane; mp., 224-226~ Fungal Source Aflatoxicol A (Ro) is produced from the m vitro incubation of aflatoxin B~ with submitochondrial liver fractions from several animal species. It was also produced from biological reduction of aflatoxin BI by Tetrahymenapyriformis, Dactylium dendroides, Rhizopus spp., and other microorganisms. It was reported to occur together with a diastereoisomer (aflatoxicol B). Biological Activity Reportedly 18 times less toxic than aflatoxin B~ in the duckling biliary hyperplasia assay. Spectral Data UV~

~ mEtOH 332(e = 14,100), 261 (10,800), and 254nm (6,790). Fluorescence emission, ax 425nm.

TLC Data Silica gel G-HR (chloroform-acetone, 93:7, v/v); Re, 0.30; detection as blue fluorescence under UV light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 38-40(1981 ). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

11.

Aflatoxins

565

Common/Systematic Name Aflatoxicol B Molecular Formula/Molecular Weight C17H1406; MW = 314.07904 0

OH _

0

General Characteristics Crystals from benzene-n-hexane, decomposed over broad range, starting at 233 ~ Fungal Source Aflatoxicol B was obtained from biological reduction of aflatoxin B~ by Rhizopus spp., Dactylium dendroides, Tetrahymenapyriformis, and other microorganisms. Spectral Data UV:

Z~

325(E=14,100), 261(10,800), and 254nm (6,790).

TLC Data Silica gel G-HR (chloroform-acetone, 93:7, v/v); Re, 0.26; detection as blue fluorescence under UV light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 41-44(1981). W. B. Turner and D. C. Aldridge; Fungal Metabolites II; Academic Press, New York, New York, 631 pp. (1983).

566

11.

Aflatoxins

Common/Systematic Name Aflatoxin B2, Molecular Formula/Molecular Weight C17H1407; M W "- 3 3 0 . 0 7 3 9 5

O

O

H General Characteristics Melting point, 240~ (dec.). Fungal Source

Aspergillusflavus and A. parasiticus. Also biotransformation product of aflatoxin B~ by the liver of some animals and forms spontaneously in acidic conditions.

Biological Activity Aflatoxin B2, was more than 200 times less toxic than aflatoxin BI as measured by the initiation of bile duct proliferation in the standard duckling assay. No acute toxicity was noted in Khaki Cambell ducklings (day-old) at levels up to 12001.tg/duckling. Spectral Data UV:

228(e=17,600), 256(10,300), and 363nm (20,400). ~3C NMR: C-I, 153.9 s; C-2, 117.4 s; C-3, 200.0 s; C-4, 34.6 t; C-5, 28.5 t; C-6, 176.7 s; C-7, 106.9 s; C-8, 160.8 s; C-9, 90.5 d; C-10, 165.3 s; C-11,108.6 s; C-12, 152.0 s; C-13, 113.6 d; C-14, 41.3 d; C-15, 37.2 t; C-16, 99.7 d; and C-17, 56.6ppm q. TLC Data Kieselgel G (chloroform-methanol, 98:2, v/v); Rf, 0.13; detection as blue fluorescence under UV light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 51-54(1981). J. G. Heathcote and J. R. Hibbert; Biochem. Soc. Trans. Vol. 2, p. 301 (1974).

11.

Aflatoxins

567

Common/Systematic Name Aflatoxin G2a Molecular Formula/Molecular Weight Cl7Hl4Os, MW

= 346.06887

O

H

O

.o o o

O

I

O

H

General Characteristics Melting point, 190~ (dec.). Fungal Source

Aspergillusflavus and A. parasiticus. Also biotransformation product of aflatoxin B~ by the liver of some animals and forms spontaneously in acidic conditions.

Biological Activity There were no significant differences in growth and no characteristic liver lesions in day-old Khaki Cambell ducklings dosed up to 1600~g/duckling (LDs0 of aflatoxin BI in same assay was 18.21.tg/duckling). Spectral Data UV:

~b max

MeOH

223(e=18,600), 242(10,100), 262(8,700), and 365nm (18,000).

13C NMR: C-I, 153.9 s; C-2, 111.0 s; C-3, 159.6 s; C-4, 64.1 t; C-5, 28.4 t; C-6, 161.9 s; C-7, 105.3 s; C-8, 161.6 s; C-9, 91.1 d; C-10, 164.9 s; C-11,108.6 s; C-12, 151.6 s; C-13, 113.7 d; C-14, 41.9 d; C-15, 41.9 t; C-16, 91.1 d; and C-17, 56.7ppm q. TLC Data Kieselgel G (chloroform-methanol, 98:2, v/v); Re, 0.10; detection as blue fluorescence under UV light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 55-58(1981). J. G. Heathcote and J. R. Hibbert; Biochem. Soc. Trans. Vol. 2, p. 301 (1974).

568

11.

Aflatoxins

Common/Systematic Name Aflatoxicol O-ethyl ether A Molecular Formula/Molecular Weight C19H1806; MW = 342.11034 0

OCH2Me

0

General Characteristics Colorless crystals from ethanol solution; mp., 198-200 ~ Fungal Source Produced spontaneously from aflatoxicol A during silica gel column chromatography using chloroform (ethanol preservative) as the eluting solvent. Spectral Da.ta UV: ~,Em~H 332(C=14,200), 261(9,660), and 255nm (8,830). TLC Data Silica gel G-HR (chloroform-acetone, 93:7, v/v); Rr, 0.81; detection as blue fluorescence under UV light. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 59-61(1981). R. J. Cole, J. W. Kirksey, and B. R. Blankenship; J. Agric. Food Chem., Vol. 20, p. 11 O0 (1972).

11. Aflatoxins

569

C.ommon/Systematic N.ame Aflatoxicol O-ethyl ether B Molecular Formula/M01ecular Weight ClgHlsO6; MW = 342.11034

O o

I"-

OCH2Me

General Characteristics Colorless crystals from ethanol solution; mp., 194-196 ~C. Fungal Source Produced spontaneously from aflatoxicol B during silica gel column chromatography using chloroform (ethanol preservative) as the eluting solvent. Soectral Data _ UV: ~ , EtOH max

331(e=15,750), 261(12,280), and 255nm (11,170).

TLC Data Silica gel G-HR (chloroform-acetone, 93:7, v/v); Re, 0.73; detection as blue fluorescence under UV light. References R. J. Cole and R. H. Cox; Handboo.k of Toxic Fungal Met abolites; Academic Press, New York, pp. 62-64(1981). R. J. Cole, J. W. Kirksey, and B. R. Blankenship; J. Agric. Food Chem., Vol. 20, p. 1100 (1972).


Versicolorins Versicolorin A Versicolorin B Versicolorin C Averufin Norsolorinic acid Versiconal hemiacetal acetate Versiconol acetate Versiconol Nidurufin Dimethylnidurufin Aversin O-Methylaversin; Tri-O-methylversicolorin B Versicolorone Hydroxyversicolorone

571


12. Versicolorins

573

Common/Systematic Name Versicolorin A Molecular Formula/Molecular Weight C1ffli007; MW = 338.04265

OH

Logo2

0

OH

0

General Characteristics Orange-yellow needles from chloroform-methanol; mp., 303-306~ [ a i D 18 _ 354 ~ (c=0.75, in dioxane). Trimethyl ether; mp., 241 ~ Versicolorin A was soluble in acetone, dioxane, ethyl acetate, ether, and ethanol; sparingly soluble in chloroform and benzene; insoluble in water and aqueous bicarbonate. Versicolorin A is a biosynthetic precursor of the aflatoxins. Fungal source Aspergillus versicolor and A. parasiticus (mutant strain 1- 11-105 wk- 1). Isolation/Purification The mycelium was harvested after 4-7 days growth on a low salts medium. The pigment was obtained by chloroform-methanol (1:1, v/v) extraction of the mycelium in a Soxhlet apparatus, followed by solvent partition with hexane-90% methanol. The 90~ methanol layer contained versieolorin A and was separated by chromatography on silica gel H under pressure (1 kg/cm2). The column was developed with chloroform-methanol (97:3, v/v). Crystallization from chloroform-methanol gave pure versicolorin A. Spectral Data UV: ~ EtOH max

222(c=1og 4.45), 255(4.13), 267(4.26), 290(4.40), 326(3.83), and 450nm

(3.85). IR:

(KBr) 3340, 1679, 1625, and 1610cm ~. 13C N M R :

(CDC13) C-l, 158.6, s; C-2, 119.7, s; C-3, 165.1, s; C-4, 100.9, d, C-5, 108.6, d, C-6, 164.8, s, C-7, 107.6, d, C-8, 163.8, s, C-9, 188.4, s, C-10, 180.1, s; C-11, 43.3, t, C-12, 30.0, t; ,C-13, 66.9, d; C-14, 112.9, d, C-la, 110.4, s; C-4a, 134.2, s, C-5a,

574

12. Versicolorins

134.8, s; and C-8a, 107.9ppm, s.

TLC Data Silica gel (benzene-acetic acid. 95:5, v/v); Re: 0.23; detected as yellow spot in visible fight. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 102 (1981). T. Hamasaki, Y. Hatsuda, N. Terashima, and M. Renbutsu; Studies on the Metabolites of Aspergillus versicolor (Vuillemin) Tiraboschi Part V. Isolation and Structures of Three New Metabolites, Versicoloilns A, B, and C; Agile. Biol. Chem., Vol. 31, pp. 11-17 (1967). G. P. Gorst-Allmart, P. S. Steyn, P. L. Wessels, and D. B. Scott; Carbon-13 Nuclear ~ n e t i c Resonance Assignments and Biosynthesis of Versicolorin A in AspergiUus parasitieus; J. Chem. Soc., Perkin Trans. 1, pp. 961-964(1978).

12. Versicolorins

575

Common/Systematic Name Versicolorin B MolecuLar Formula/Molecular Weight C18H1207; ~

= 340.05830

OH

0

OH

-

la

H. 1-

~,~,lOa~N.~ ~ 2 r~ .Y ~ l a "NH

0

~ -OH8, H , ~ r HN3= \ j l ~j

General Characteristics Fisealin A was recrystallized from dichloromethane-methanol (1:1, v/v) to provide colorless orthorhombic crystals; mp., 242--243*C; [t~]D -169* (C=0.5, in MeOH). Isolation/Purification The fermentation was carried out at 26~ with shaking at 220rpm. Fiscalin production and cell growth peaked after 6 days. The majority of fiscalins was associated with the cell mass. Harvested cells were extracted with ethyl acetate to yield a brown residue. After redissolving in a mixture of methanol and dichloromethane (1:1, v/v), the sample was subjected to flash silica gel column chromatography with stepwise elution using chloroform, chloroform-methanol (1:1, v/v), and methanol. Bioactive eluents were collected from the chloroform-methanol fraction, and dried in vacuo to yield a white powder. After rinsing with methanol, the white solids were redissolved in a mixture of diehloromethane-methanol (1:9, v/v) and then subjected to semi-preparative I-IPLC using a YMC reverse phase column (C18) and isocratic elution with 86% methanol and 14% water at lml/minute. Eluents were collected to yield fiscalin A, B, and C. Fungal Source Neosartoryafischeri isolated from a plant rhizosphere collected near the We Fung Chi Cascade region of Taiwan. Anamorph stage is Aspergillusfischerianus. Biological Activity Inhibited the binding of radiolabeled substance P ligand to the human neurokinin (NK-1) receptor, with Ki value of 5711M.

62

5.

Fiscalins

Soeetral Data _

UV:

~,M~oH 226, 278, and 306nm. max CD: ~,~x 228(-62.1) and 307nm (-9.5Ae). IR:

(KBr) 3378-3279(NH)and 1689em~ (amide). 1H N - ~ : (CDC13 + CD3OD or DMSO-d~) 4.41(1H, H-I); 8.43(1H, NH); 5.31(1H, H-4); 7.99(1H, H-7); 7.26(1H, H-8); 7.55(1H, H-9); 7.43(1H, H-10); 0.94(3H, H-la); 2.85(1H, H-lb); 0.68(3H, H-le); 2.51, 2.68(2h, H-4a); 3.14(11-1,NH-I'); 3.47(1H, H-2'); 0.82(3H, H-2a'); 7.17(1H, H-5'); 6.88(1H, H-6'); 7.07(1H, H-7'); 7.14(1H, H8'); and 4.93ppm (1H, H-9a'). 13CNMR: (CDCI3) 60.4, C1; 172.4, C-3; 54.4, C-4; 163.9, C-6; 122.2, C-6a; 128.4, C-7; 129.1, C-8; 136.8, C-9; 129.2, C-10; 149.4, C-10a; 152.2, C-11a; 20.0, C-la; 31.2, C-lb; 16.5, C-lc; 40.1, C-4a; 62.9, C-2'; 18.0, C-2a'; 177.0, C-3'; 139.1, C-4a'; 117.3, C-3'; 127.5, C-6'; 132.2, C-7'; 126.4, C-8'; 139.2, C-8a'; 76.3, C-9'; and 84.4ppm C-9a'. Mass Spectrum: HREIMS: 473.2032re~e;calculated, 473.2063. Reference S-M. Wong, L. L. Musza, G. C. Kydd, R. Kullnig, A. M. Gillum, and R. Cooper; Fiscalins: New Substance P Inhibitors Produced by The Fungus Neosartorya fischeri, Taxonomy, Fermentation, Structures, and Biological Properties; The Journal of Antibiotics, Vol. 46, pp. 545-553(1993).

5.

Fiscalins

63

Common/Systematic Name Fiscalin B Molecular Formula/Molecular Weight C23H22N402, ~ = 386.17428 H

-

General Characteristics Fiscalin B was obtained as a white amorphous solid. Isolatiort~urification See fiscalin A. Fungal Source

Neosartoryafischeri isolated from a plant rhizosphere collected near the We Fung Chi Cascade region of Taiwan. Anamorph stage is Aspergillusfischerianus.

Biological Activity Inhibited the binding of radiolabeled substance P ligand to the human neurokinin (NK-1) receptor, with Ki value of 157~M. Spectral Data UV: UV suggested the presence of quinazolone and indole chromophores. CD: ~,~x 228(-62.1) and 307nm (-9.5AE). 1H NIV[R: (CDCI3) 2.70(1H, H-l), 5.97(1H, NH); 5.63(1H, H-4); 8.33(1H, H-7), 7.49(1H, H-8); 7.74(1H, H-9); 7.53(1H, H-10); 0.61(3H, H-la); 2.60(1FL n-lb); 0.62(3H, H-lc); 3.69, 3.59(2h, n-4a); 8.21(1H, H-4'); 7.38(1H, n-5'); 6.88(1H, n-6'); 7.08(1H, H-7'); 7.25(1H, n-8'); and 6.57ppm (1H, n-9a').

64

5.

Fiscalins

13CNMR: (CDC13) 58.4, C1,170.2, C-3, 57.1, C-4, 161.6, C-6, 120.7, C-6a, 127.4, C-7, 127.7, C-8, 135.3, C-9, 127.7, C-10, 147.7, C-10a, 151.0, C-11a, 19.0, C-la, 29.8, C-lb, 14.9, C-lc; 27.6, C-4a; 127.8, C-4a'; 119.2, C-3', 120.5, C-6'; 123.1, C-7'; 111.6, C-8'; 136.6, C-8a', 109.7, C-9', and 124.2ppm C-9a'. Mass Spectrum: 387m/e (M + H) + and H R I M S of 386.1743m/e suggesting a molecular formula of C23H2zN4Oz, with two major fragments 257.1158 (C1A-I15N302)and 130.0649m/e

(Cd-hN). Reference S-M. Wong, L. L. Musza, G. C. Kydd, R. Kullnig, A. M. GiUum, and R. Cooper; Fiscalins: New Substance P Inlfibitors Produced by The Fungus Neosartoryafischeri, Taxonomy, Fermentation, Structures, and Biological Properties; The Journal of Antibiotics, Vol. 46, pp. 545-553(1993).

5.

Fiscalins

65

Common/Systematic Name Fiscalin C Molecular Formula/Molecular Weight C27H29NsO4; M W -- 4 8 7 . 2 2 1 9 5

H

-

0 General Characteristics Fiscalin C was obtained as a white amorphous solid. Isolation/Purification See fiscalin A. Fungal Source

Neosartoryafischeri isolated from a plant rhizosphere collected near the We Fung Chi Cascade region of Taiwan. Anamorph stage is Aspergillusfischeriatms.

Biological Activity Inhibited the binding of radiolabeled substance P ligand to the human neurokinin (NK-1) receptor, with Ki value of 68~M. Spectral Data UV;

I.W suggested the presence of quinazolone and indole chromophores. CD" ~.m,~ 228(-44.6) and 307rim (-5.0At).

66

5.

Fiscalins

1HNI~: (CDCI3) 4.56(1H, H-l); 6.12(1H, NH), 5.82(1H, H-4); 8.30(1H, H-7), 7.51(1H, H8); 7.79(1H, H-9), 7.68(1H, n-10); 0.95(3H, H-la); 3.16(1H, U-lb); 1.18(3H, H-lc), 2.47, 2.69(2H, n-4a); 1.39(3H, n-3a'); 1.49(1H, n-4'), 7.51(1H, H-5'), 7.09(1H, n-6'); 7.32(1H, n-7'); 7.32(1H, n-8'), and 5.25ppm (1H, n-9a'). 13CNMR: (CDCI3) 48.6, C-1,170.8, C-3, 51.8, C-4, 161.3, C-6, 120.9, C-6a, 127.7, C-7, 128.0, C-8; 135.5, C-9; 128.1, C-10; 147.5, C-10a, 149.8, C-1 la; 19.8, C-la; 29.0, Clb; 15.6, C-lc; 40.0, C-4a; 65.4, C-2'; 25.4, C-2a', 26.4, C-2b', 175.9, C-Y, 138.0, C4a', 116.3, C-5', 125.8, C-6', 130.8, C-7', 124.7, C-8', 138.5, C-8a', 74.5, C-9', and 78.9ppm C-9a'. Mass Spectrum: HREIMS: 488.2318m/e (M + H) +. Reference S-M. Wong, L. L. Musza, G. C. Kydd, R. KuUnig, A. M. Gillum, and R. Cooper, Fiscalins: New Substance P Inhibitors Produced by The Fungus Neosartoryafischeri, Taxonomy, Fermentation, Structures, and Biological Properties; The Journal of Antibiotics, Vol. 46, pp. 545-553(1993).

Palmarumycins

6

Palmarumycin CP1 Palmarumycin CP2 Palmarumycin CP3 Palmarumycin CP4 Palmarumycin C1 Palmarumycin C2 Palmarumycin C3 Palmarumycin C4 Palmarumycin C5 Palmarumycin C6 Palmarumycin C7 Palmarumycin C8 Palmarumycin Clo Palmarumycin Cll Palmarumycin C12 Palmarumycin C13 Palmarumycin C14 Palmarumycin C15 Palmarumycin C16

67


6. Palmarumycins

69

Common/Systematic Name Palmarumycin CP1 5-Hydroxyspiro [naphthalene- 1(4H),2'-naphtho [ 1,8-de] [ 1,3 ]dioxin]-4-one Molecular Formula/Molecular Weight C2oH1204; M~V = 316.07356 0

d

OH

4'

General Characteristics Light yellow crystals from methylene chloride-petroleum ether; mp., 170~ (dec.); optically inactive. Isolation/Purification The palmarumycins were extracted with chloroform and CP1 and CP3 were isolated in pure form using preparative thin-layer chromatography [silica gel on aluminum foil (Merck)]. Fungal Source Coniothyrium palmarum (internal strain number 2014), an endophytic fungus isolated from Lamium purpureum. Biological Activity Palmarumycin CP1 showed no biological activity against the test fungus Cladosporium cucumerinum. Spectral Data UV~ ~,mM~y'~~ (3,731).

232(10g e = 4,633), 288(3,961), 296(3,966), 330(3,785), and 362nm

IR~ (KBr) 3456(OH), 3057, 1662(C=O), 1611, 1455, 1410, 1379, 1340, 1268(Ar-O), 1236, 1112, 1075, 942, and 744cm "1.

70

6. Palmarumycins

1H NMR: (CDCI3) 12.16(1OH, s); 7.66[1H, pseudo-t (dd), J6,7=8.2Hz, J7,8=7.9Hz, H-7]; 7.58(2H, d, J3,4,,(5,,6,)=8.3Hz, 4'-H and 5'H); 7.4712H, pseudo-t (dd), J3',4;(5"6')- 8.4Hz, Jz3,c6,,7,)=7.5Hz,H-3' and H-6']; 7.46(1H, dd, J6,s=lHz, H-8); 7.14(1H, dd, J6,7=8.4Hz, J6,8=lHz, H-6); 7.02(1H, d, J2,3=10.4Hz, H-2); 6.98(2H, d, J2,,3,(6,,7,)=7.5Hz, H2' and H7'); and 6.36ppm (1H, d, J2,3=10.6Hz, H-3). 13CNMR: (CDC13) 188.8(C-4, s); 161.9(C-5, s); 147.2(C-1', C-8', s); 139.7(C-2, d); 138.8(C-8a, s); 136.6(C-7, d); 134.1(C-4a', s); 129.7(C-3, d); 127.6(C-3', C-6', d); 121.4(C-4', C-5', d); 119.7(C-8, d); 119.4(C-6, d); 113.9(C-4a, s); 113.0(C-8a', s); 109.9(C-2', C-7', d); and 93.0ppm (C- 1, s).

Mass Data: 316(M§ 100%), 288(24, M§ - CO), 287(31, M+ - CO), 209(27), 169(40), and 167m/e (32); HREIMS: obsd 316.0736m/e; calcd for C20H1204, 316.0736; anal C =75.5, H=3.68; calcd C=75.94, H=3.82. Reference K. Krohn, A. Michel, U. Florke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins CP1 -CP4 from Coniothyriumpalmarum: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1093-1097(1994).

6. Palmarumycins

71

Common/Systematic Name Palmarumycin CP2 5-Hydroxy-2,3-dihydrospiro [naphthalene- 1(4H),2'-naphtho [ 1,8-de] [ 1,3 ] dioxin]-4-one _Molecular Formula/Molecular Weight C20H1404; M W = 318.08921 0

O

OH

ii I1'

General Characteristics Crystals; mp., 170~ (dec.); optically inactive. Isolation/Purification The palmarumycins were extracted with ethyl acetate and CP2 was isolated in pure form using repeated column chromatography on silica gel using toluene as eluant. Fungal Source Coniothyrium palmarum (internal strain number 2014), an endophytic fungus isolated from Lamium purpureum. Biological Activity Palmarumycin CP1 showed no biological activity against the test fungus Cladosporium cucumerinum. However, it showed weak activity against Ustilago violacea and Eurotium repens. Spectral Data UV: Z mM~=~176 ~ 285(Iog e = 3.81), 297(4.12), 300(4.11), 315(3.98), and 329nm (3.99). IR: ~

(KBr) 3438(OH), 3056, 1640(C=O), 1609, 1586, 1412, 1381, 1348, 1331, 1237, 1117, 1107, 1074, 920, 885, 819, 804, 795, 758, and 743cm"1.

72

6. Palmarumycins

1H NMR: (CDCI3) 12.43(1OH, s, H-7); 7.62[1H, pseudo-t (dd), J6,7= 8.2Hz, J=7,s7.9Hz, H-7]; 7.54(2H, dd, ffY,4"(5;6')-" 8.3HZ, ff2,,4,(5,,7,)= 0.6Hz, H-4' and H-5'); 7.4612H, pseudo-t (dd), J2,3,(6,,7~=7.3Hz, ff3',4'(5',6~-- 8.3Hg, H-3' and H-6']; 7.45 (1H, dd, Jz, s= 7.6Hz, J6,s = 1.0Hz, H-8); 7.10 (1H, dd, J6,7= 8.3Hz, d6,s=l.OHz, H-6); 6.97(2H, dd, J2,3,(6,z,)=7.3Hz, J2,4,o,,7~ = 0.6Hz, H-2' and H-7' ); 2.85(2H, t, Jz~= 6.5Hz, H-3); and 2.49ppm (2H, t, Jz3=6.5Hz, H-2). 13C NUll: (CDC13) 203.3(C-4, s); 162.5(r s); 147.5(C-1', C-8', s); 140.9(C-8a, s); 137.2(C-7, d); 134.3(C-4a', s); 127.6(C-3', C-6', d); 120.9(C-4', C-5', d); 119.6(C-8, d); 116.7(C-6, d); 115.5(C-4a, s); 109.5(C-2', d); 113.4(C-8a', s); 98.4(C-1, s); 34.1(C-3, t) and 29.5ppm (C-2, t).

Mass Data: ELMS: 318(M§ 100%), 301(M+-OH, 44), 159(36), and 132m/e (26); HR IMS: obsd 318.089m/e; calcd for C20H1404, 318.0892; anal C= 75.21, H= 4.23; calcd C=75.46, H=4.43. Reference K. Krohn, A. Michel, U. Florke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins CP1 CP4 from C o n i o t h y r i u m p a l m a r u m : Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1093-1097(1994). -

6. Palmarumycins

73

Common/Systematic Name Palmarumycin CP3 2S*,4 aS*, 8S*, 8aS*-2,3,8,8a-T etrahydro spiro [2,8-epoxynaphthalene- 1(4H),2'naphtho [ 1,8-de ][ 1,3 ] dioxin]-4,5 (4aH)-dione Molecular Formula/Molecular Weight C20H1405~, ~

0

II1''

= 334.08412

0

~.

General Characteristics Colorless prisms from methylene chloride-petroleum ether; mp., 190~ (dec.); - 108.8 ~ (c=l.0, in CDCls).

[ ~ ] D 20 -"

Isolation/Purification The palmarumycins were extracted 3 times with ethyl acetate. The combined extracts were dried (MgSO4), filtered, and concentrated under reduced pressure. The crude extract was subjected to preparative liquid chromatography four successive times using ethyl acetatemethylene chloride (1:1, v/v); diethyl ether-methylene chloride (5:15, v/v); diethyl ethermethylene chloride (1:10, v/v), and diethyl ether-methylene chloride (2:98, v/v). Fungal Source Coniothyrium palmarum (internal strain number 2014), an endophytic fungus isolated from Lamium purpureum. Biological Activity Palmarumycin CP3 showed biological activity against a variety of bacteria and fungi. Spectral Data ugz Chloroform

1~ (4.06). max

290(log e=4.06), 299(4.34), 307(4.27), 313(4.29), 3.27(4.22), and 381nm

74

6. Palmarumycins

IR~

(KBo 3079, 2982, 2955, 2901, 1728(C=O), 1688(C=O), 1607, 1584, 1414, 1381, 1275(Ar-O), 1252, 1230, 1169, 1153, 1124, 1055, 928, 833, and 770cm1. 1H N1V[R:

(CDCI3) 7.57(1H, d, Jy,6~-8.3Hz, H-5'); 7.56(1H, d, Js,4~-8.3Hz, H-4'); 7.47[1H, pseudo-t (dd), Jzs~7.6Hz, Js,,4,-=8.3Hz,H-3']; 7.45[ 1H, pseudo-t (dd), Jy,6~8.3Hz, J6,z,~-7.8Hz, H-6']; 7.07(1H, dd, Jz, s~7.5Hz, Jz,4~0.6Hz, H-2'; 6.99(1H, dd, J6,7=10.1Hz, Jzs-=5.1Hz, H-7); 6.95(1H, dd, J6,7~lO.1Hz, Jzs-=5.1Hz, H-7); 6.14(1H, d, J6,7=10.1Hz, H-6); 4.97[1H, pseudo-t (dd), Jzs=4.8Hz, Js,sa=4.8Hz, H-8]; 4.55(1H, m, H-2); 3.65(1H, dJ4a,sa=5.4Hz, H-4a); 3.18[1H, pseudo-t (dd), J4a,sa=4.2Hz, Js,s,,=4.9Hz, Jzs~=I.4Hz, H-8a]; 3.04[1H, d, (AB)dd, Js=,s~q=18.7Hz,Jzs~q=2.4Hz, Js~q,4,~=O.7Hz,H-3eq]; and 2.84ppm [ 1H, d (AB)dd, Js,=,s~q=l8.7Hz, Jz3==2.9Hz, J~,4~=l. 1Hz, H-3ax]

13C NMR:

(CDC13) C-4, 200(s); C-5, 189.9(s), C-8', 146.7(s); C-l', 146.5(s); C-7, 143.1(d); C4a', 134.5(s), C-6, 1131.4(d); C-3', 127.7(d); C-6', 127.5(d); C-5', 122.0(d); C-4', 121.5(d); C-8a', 113.9(s); C-2', 110.1(d); C-7', 109.5(d); C-I, 105.6(s); C-2, 76.3(d), C-8, 70. l(d); C-4a, 58.1(d); C-3, 45.6(0; and C-8a, 42.4ppm (d). Mass Data: 334(M+, 100%), 305(8), 263(31), 235(30), 218(18), 212(15), 199(15), 171(14), 115(39), 114(24), 91(18), and 65m/e (24); anal, C=72.05, H=4.31; calcd C=71.85, H=4.22. Reference K. Krohn, A. Michel, U. Fl6rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins CI-C16 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

75

Common/Systematic Name Palmarumycin CP4 (8R *,8 aS*)-4, 8-Dihydroxy-6, 7,8,8a-tetrahydro spiro [naphthalene- 1( 5H),2'-naphtho [ 1,8de][ 1,3 ]dioxin]-5-one Molecular Formula/Molecular Weight C20H1605, M W = 336.09977

OH

0

11III

General Characteristics Colorless prisms from methylene chloride; mp., 186~ (dec.); mp., 193~ (diethyl ether); [Ct]D2~= + 495 ~ (C=0.2, in CH2C12). Isolation/Purification The palmarumycins were extracted 3 times with ethyl acetate. The combined extracts were dried (MgSO4), filtered, and concentrated under reduced pressure. The crude extract was subjected to preparative liquid chromatography four successive times using ethyl acetatemethylene chloride (1:1, v/v); diethyl ether-methylene chloride (5:15, v/v); diethyl ethermethylene chloride (1:1, v/v); and diethyl ether-methylene chloride (2:98, v/v). The polar fractions of the chromatography were further separated by PLC (Chromatotron, 2mm, CH2C12-CH3OH; 99:1, v/v) to afford pure palmarumycin CP4. Fungal Source Coniothyrium palmarum (internal strain number 2014), an endophytic fungus isolated from Lamium purpureum. Biological Activity Palmarumycin CP3 showed biological activity against a variety of bacteria and fungi. Spectral Data UV:

~, Cm~~176176 283(1og e=3.62), 295(4.03), 301(4.07), 304(4.07), 314(4.10), and 328nm (4.08).

76

6.

Palmarumycins

IP-~

(KBr) 3551(OH), 3063, 2932, 2905, 1638, 1611, 1580, 1412, 1375, 1323, 1252, 1252, 1213, 1103, 1082, 1065, 1036, 959, 930, 919, 826, and 762cm"1. 1H N1VIR:

(CDC13) 15.5(1H, s, OH); 7.55(1H, dd, ds,6~8.2Hz, Jy,7~-O.6Hz,H-5'); 7.54(1H, d, J3,4~8.2Hz, H-4'); 7.48(1H, t(dd), dz,3~7.4Hz, d3,4~8.3Hz, H-Y); 7.4[ 1H, pseudo-t (dd), Js,6,~-8.2Hz,J6,7,=7.7Hz, H-6']; 7.04(1H, dd, Jz,3~-8.2Hz, Jz,4,~O.6Hz, H-2']; 6.91(1H, d, J6,,7~7.5Hz, H-7'); 6.44(1H, d, Jz3=10.3Hz, H-2); 6.13(1H, d, dz3=10.3Hz, H-3); 4.86[1H, pseudo-t (dd), dT~,8=2.3Hz,ds,oH=2.3Hz, H- 8]; 3.77(2H, d, Js,~,sa=2.3Hz, H-8a); 3.4(1H, s, OH); 2.98(1H, ddd, J6~, 6eq=19.1Hz, J6~,TAX=12.4Hz,d6,~,6~q=6.8Hz,H-6ax); 2.41 (1H, dd, J6~, 6~q=l9.1Hz, J~q,7~=5.5Hz, H-6eq); 2.25(1H, m, H-7eq); and 1.82ppm (1H, m, H-7ax).

13CNMR:

(CDCI3) C-4, 181.3(s); C-5, 188.4(s); C-8', 146.5(s); C-I', 145.9(s); C-2, 137.3(d); C4a', 134.2(s); C-3, 130.6(d); C-3', 127.7(d); C-6', 127.4(d); C-5', 121.3(d); C-4', 121.7(d); C-8a', 113.6(s); C-2', 110.1(d); C-7', 110.1(d); C-l, 99.3(s); C-8, 63.4(d); C8a, 46.0(d); C-6, 27.6(d); and C-7, 27.1ppm (t). Mass Data: 336(M § 100%), 318(94, M+ - n20), 289(15, 318 - CHO), 279(76, 318 - C3H3), 160(24), 159(76, 1,8-dihydroxynaphthalene-H), 144(19), 131(50), and 115re~e; HREI]~S: C20H1605,obsd 336.0997re~e; calcd, 336.0998; anal, C, 71.26, H, 4.61; calcd, C, 71.42, H, 4.79. Reference K. Krohn, A. Michel, U. Fl6rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1-C16 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

77

Common/Systematic Name Palmarumycin C1 3-Chloro-5-hydroxyspiro[naphthalene- 1(4H),2'-naphtho [ 1,8-de] [ 1,3 ] dioxin]-4-one Molecular Formula/Molecular Weight C20HllCIO4; ~

Cl

0

OH

4

~

1111

,

~ 6'

6

7

8

8,(. r

= 350.03459

' 4'

General Characteristics Slightly yellow needles; mp., 280~ (dec.). Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The first fraction was further separated by thin-layer chromatography to afford palmarumycin C1 and palmarumycin C2. Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Spectral Data UV: Chloroform ~, max

285(log 6=3.52), 296(3.96), 301(4.05), 314(3.81), and 329nm (3.71).

IR:

(KBr) 3438(OH), 3202, 3067, 1734, 1718, 1580, 1704, 1653(C=O), 1628, 1611, 1458, 1412, 1381, 1271(Ar-O), 1231, 1167, 1070, 939, 903, 845, 826, 802, and 756cm -1.

78

6. Palmarumycins

~H NMR: (CDCI3) I 1.84(IH, s, OH); 7.67[IH, pseudo-t (dd), J6,z=8.4I-Iz, dT,s=7.9Hz, H- 7]; 7.59(2H, d, J3,,4,a,,69=8.3Hz, H-4', H-5'); 7.47121-1,pseudo-t (dd), d2;3,(a;79=7.61-1z, Js,,4,(5,,6.)=8.3Hz,H-3', H-6']; 7.45(IH, dd, JT,~7.9Hz, J6,8=l.01-Iz, H-8); 7.16(IH, (dd), J6,7=8.41-Iz, d6,s=l.OHz, H-6); 7.16(2H, s, H-2); and 6.98ppm (2H, d, d2,3,(6,7,)=7.61-Iz, H-2', H-7'). 13CNMP-~:

(CDC13) C-4, 181.7(s); C-5, 161.9(s), C-8', 146.5(s); C-I', 146.5(s); C-8a, 137.8(s); C-7, 137.1(d); C-2, 135.5(d); C-4a', 113.0(s); C-3, 133.2(d); C-Y, 127.5(d); C-6', 127.5(d); C-4a, 113.0(s); C-8a', 112.4(s), C-2', 109.9(d); C-7', 109.9(d); and C- 1, 93.3ppm (s). Mass Spectrum: LREIMS: 352(M+, 35%), 350(100), 315(32, M+- CI), 287(20), 259(13), 116(29), 96(42), 71(29), and 56role (53); HREIMS: C2oHl1C104,obsd 350.0345m/e, calcd 350.0346. Reference K. Krohn, A.Michel, U. FlSrke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1C~6 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

79

Common/Systematic Name Palmarumycin C 2 (2S*, 3R *)- 5-Hydroxyspiro [2,3-epoxynaphthalene- 1(2H),2'-naphtho [ 1,8-de] [ 1,3 ]dioxin]4(3H)-one Molecular Formula/Molecular Weight C20H1205; M'W = 332.0647

0

OH

General Characteristics Crystallized in long yellow needles; mp., 228~ (dec.); [ ~ ] D with 2,4-dinitrophenylhydrazine and bromocresol green.

20 - - -

341 ~ Positive reaction

Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fembach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The first fraction was further separated by thin-layer chromatography to afford palmarumycin C1 and palmarumycin Cz. Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Spectral Data UV:

c~o~oro,. 295(log 6-=4.11), 300(4.13), 314(4.08), and 328nm (4.10).

80

6. Palmarumycins

IR:

(KBr) 3432(OH), 3052, 1651(C=O), 1611, 1355, 1410, 1381, 1333, 1269(Ar-O), 1240, 1179, 1115, 1105, 1065, 1016, 970, 918, 874, 818, 506, 758, 752, 739, and 506cml. :H NMR:

(CDCla) 11.26(1H, s, OH); 7.6411H, pseudo-t (dd), J6,z=8.2Hz, Jzs-=7.9Hz, H- 7]; 7.59(1H, dd, J3,4~8.3Hz, Jz,4~0.7Hz, H-4'); 7.56(1H, d, Jy, D~8.3Hz, H-5'); 7.52[1H, pseudo-t (dd), J2,,3~7.SHz, Jy,4~-8.3Hz, H-3']; 7.4511H, pseudo-t (dd), Jy,6~8.3Hz, JD,,7~7.4Hz, n-6']; 7.45(1H, dd, Jzs=7.9Hz, JD,8=l.OHz,H-8); 7.16(1H, (dd), JD,7=8.4Hz,J6,,r=l.OHz,n-6); 7.44(1H, dd, J7,,s~7.9Hz, J6,,s~l.OHz, H- 8); 7.18(1H, dd, Jz,3~7.4Hz, Jz,4~0.7Hz,H-2'); 7.13(1H, dd, JD,7=8.4Hz,J6,s=l.0Hz, H-6); 6.92(1H, dd, J6,,7~7.4Hz, Jy,7~0.6Hz, H-7'); 4.08(1H, d, Jz3=4.0Hz, H-3); and 3.68ppm (2H, d, Jz3=4.0Hz, n-2).

13C NMR:

(CDC13) C-4, 196.5(s), C-5, 161.9(s); C-8', 146.9(s); C-I', 146.7(s); C-8a, 136.9(s); C-7, 137.7(d); C-2, C-3, 53.2(d); C-4a', 134.2(s); C-3', 127.8(d); C-6', 127.6(d); C-4a, 112.2(s); C-8a', 112.8(s); C-2', 109.3(d); C-7', 109.3(d); C-l, 96.0(s); C-4', 121.4(d); C-5', 121.3(d); C-6, 120.1(d); and C-8, 119.1ppm (d). Mass Data: LREIMS: 332(NV, 100%), 287(20), 246(19), 232(50), 204(30), 176(20), 173(33), 159(22), 154(20), 145(20), 130(20), and 43m/e (55); HREIMS: C20H1205,obsd 332.0660m/e; calcd 332.0685; calcd, C=72.29, H=3.64; found C=72.16, H=3.59. Reference K. Krohn, A. Michel, U. F16rke, H.-J. Aust, S. Draeger, and B. Schulz, Palmarumycins C1-C16 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

81

Common/Systematic Name Palmarumycin C3 (2S*,3R*)-5,8-Dihydroxyspiro [2,3-epoxynaphthalene- 1(2/~,2'-naphtho [ 1,8de][1,3 ] dioxin]-4(3H)-one Molecular Formula/Molecular Weight C2oH1206, MW

0

-- 3 4 8 . 0 6 3 3 9

OH

I

General Characteristics Crystallized as greenish needles; mp., 220~

[0~]D 20 --

-300 ~

Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins Ca and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The first fraction was further separated by thin-layer chromatography to afford palmarumycin C1 and palmarumycin C2. Column chromatography was continued with a gradient of methylene chloride-methanol (0-10%). The second fraction was further purified by column chromatography with petroleum ethermethylene chloride (1:1, v/v) as eluant to afford additional palmarumycin C2 and palmarumycin C3. Funsal Source v An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Spectral Data UV:

X c~o~ofo~m296(1og e=4.14), 299(4.14), 312(4.01), 327(3.91), and 380nm (3.91).

82

6. Palmarumycins IR:

(KBr) 3499(OH), 3060, 1651(C=O), 1613, 1595, 1476, 1410, 1375, 1335, 1264(ArO), 1237, 1223, 1175, 1098, 1067, 1026, 954, 893, 876, 818, 797, 764, and 754cm1 1H NMR: (CDC13) 11.48(1H, s, OH); 7.67(1H, d, J3,4~8.4Hz, H-4'); 7.61(1H, d, Jy,o~8.2Hz, H5'); 7.54[ 1H, pseudo-t (dd), Jz.3~7.7Hz, J3,4~8.3Hz, H-3']; 7.49[ 1H, pseudo-t (dd), Jy,o~-8.2Hz, J6, 7~-7.7Hz, H-6']; 7.35(1H, s, OH); 7.29(1H, d, Jo,7=9.2Hz, H-7); 7.25(1H, d, Jz,3~7.5Hz, H-2'); 7.11(1H, d, Jo.7=9.3Hz,H-6); 7.03(1H, d, Jo,7~-7.6Hz, H-7'); 4.03(1H, d, Jz3=4.0Hz, H-3); and 3.63ppm (1H, d, Jz3=4.0Hz, H-Z). 13CNMR: (CDC13) C-4, 195.5(s); C-5, 157.0(s), C-8, 149.1(s); C-8', 146.3(s); C-l', 144.9(s); C4a', 134.0(s); C-7, 129.6(d); C-6', 127.8(d); C-3', 127.5(d); C-4', 122.7(d); C-6, 122.3,(d); C-5', 121.6(d); C-8a, 115.9(s); C-8a', 115.9(s); C-4a, 111.0(s); C-2', 111.0(d); C-7', 110.2(d); C-l, 98.8(s); C-2, 53.0 (d); and C-3, 52.5ppm (d). Mass Data: LREIMS: 348(M§ 100%), 303(11), 189(9), 171(9), 125(11), 111(17), 97(22), 85(20), 83(22), 71(27), and 57m/e (35); calcd, C=68.97, H=3.47, found C=68.75, H=3.43. Reference K. Krohn, A. Michel, U. F16rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1-C16from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6.

Palmarumycins

83

Common/Systematic Name Palmarumycin C4 (4aRS, 8aRS)3-Chlorospiro [4a, 8a-epoxynaphthalene- 1(4H),2'-naphtho [ 1,8de][1,3 ]dioxin]-4,5,8-trione Molecular Formula/Molecular Weight C20H9C106; M~W = 380.00877

O

O

CI

(,Z) ~ General Characteristics An orange amorphous material; [a]D2~-285 ~ (C=I.0, in CH2C12). Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 331 g of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins Ca and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The first fraction was further separated by thin-layer chromatography to afford palmarumycin C1 and palmarumycin C2. Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Spectral Data UV~

c~o~oro,, 285(log c=3.52), 296(3.96), 301(4.05), 314(3.81), and 329nm (3.71).

84

6. Palmarumycins

IR~

(KBr) 3438(OH), 3202, 3067, 1734, 1718, 1580, 1704, 1653(C=O), 1628, 1611, 1458, 1412, 1381, 1271(Ar-O), 1231, 1167, 1070, 939, 903, 845, 826, 802, and 756cm1. 1H NMR:

(CDC13) 7.62(1H, d, J3,4~8.3Hz, H-4'); 7.60(1H, d, Jy,D~8.2Hz, H-5'), 7.52[ 1H, pseudo-t (dd), J2,3~7.6Hz, J3,4~8.2Hz, H-3']; 7.45[ 1H, pseudo-t (dd), Jy, D~8.2Hz, J6,z~7.6Hz, H-6']; 7.12(1H, dd, J2,3~7.SHz, J2,4~-O.5Hz,H-2'), 6.91(1H, d, J6,z~7.6Hz, H-7'); 6.75(s, H-2); 6.72(1H, d, J6,7=10.8Hz, H-7); and 6.71ppm (1H, d, J6,7=10.8Hz, H-6).

13C NMR:

(CDCI3) C-4, 178.6(s); C-5, 183.3(s); C-8, 184.4(s); C-8', 184.5(s); C-I', 145.0(s); C4a', 134.5(s); C-7, C-6, 136.8(d); C-6', 127.8(d); C-3', 128.0(d); C-4', 122.2 (d); C-5', 122.2 (d); C-8a, 55.3(s); C-8a', 112.6(s); C-4a, 62.1(s); C-2', 110.1(d); C-7', lO9.8(d); C-I, 95.2(s); C-2, 133.6(d); and C-3, 132.2ppm (s). Mass Spectrum: LREIMS: 382(36), 380(M§ 100), 352(M§ CO, 5), 317(4), 289(10), 230(12), 195(42), and 114m/e (28); HREIMS: C2oH9CIO6,found 380.O087m/e; calcd, 380.0088. Reference K. Krohn, A. Michel, U. Fl6rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins CI-C16 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

85

Common/Systematic Name Palmarumycin C5 (4R *)-3,4-Dihydro-4-methoxyspiro [2,3-epoxynaphthalene- 1,(2H)2'-naphtho [ l, 8de][1,3 ]dioxin]-5,8-dione Molecular Formula/Molecular Weight C21H1605; ~

- 348.09977

OMe 0

I ,

11III

General Characteristics The compound slowly decomposed on exposure to air. Isolotion/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The first fraction was further separated by thin-layer chromatography to afford palmarumycin C1 and palmarumycin C2. Column chromatography was continued with a gradient of methylene chloride-methanol (0-10%). The second fraction was further purified by column chromatography with petroleum ethermethylene chloride (1:1, v/v) as eluant to afford additional palmarumycin C2 and palmarumycin C3. The third fraction was subjected to low-pressure column chromatography with methylene chloride as eluant to afford additional palmarumycin C3 and palmarumycin C5. Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo.

86

6. Palmarumycins

Spectral Data UV;

l, Chl m~176176286(1og e=3.35), 299(4.02), 313(4.20), 317(4.16), and 329nm (4.04). IR;

(KBr) 3050, 2930, 1665(C=O), 1608, 1583, 1414, 1381, 1354, 1333, 1271(Ar-O), 1237, 1183, 1119, 1084, 1047, 1034, 959, 926, 851,818, and 750cmq 1H ~ :

(CDCI3) 7.50(1H, dd, Js,4~-8.5Hz, J2,4~0.8Hz, H-4'); 7.49(1H, dd, Jy,6~8.3Hz, Jy,7~0.8Hz, H-5'); 7.42[ 1H, pseudo-t (dd), Jz,3~7.4Hz, Js,4~8.3Hz, H-3']; 7.41[ 1H, pseudo-t (dd), Jy,6~8.4Hz, J6,7~7.4Hz, H-6']; 6.79(1H, d, J6,7=10.2Hz, H- 7); 6.89(1H, dd, J2,s~7.3Hz, J2,4~0.9Hz, H-2'); 6.86(1H, d, J6,7=10.3Hz, H-6); 6.87(1H, dd, J6,7~-7.4Hz, Jy,7~0.8Hz, H-7'); 4.44[ 1H, pseudo-t (dd), J3eq,,,=3.0Hz, J3,~,t=3.0I-Iz, H-4]; 3.47(3H, s, OCH3, H-9); 2.22(2H, m, H-2ax, H-2eq); 2.04(1H, dq, J3eq,s== 14.6Hz, Jseq,4eq=3.0Hz, H-3eq]; and 1.87ppm (1H, dddd, JS~q,.~==14.6Hz, J~=,3=--11.3Hz, J~q,3=,~5 .9Hz, J3,=,4=3 .0Hz, H-3ax). 13C NMR:

(CDCI3) C-4, 68.4(d); C-5, C-8, 186.5(s); C-8', 147.6(s); C-l', 146.3(s); C-4a', 134.2(s), C-7, 137.8(d), C-6', 127.2(d), C-3', 127.4(d); C-4', 120.7(d), C-6, 135.3(d); C-5', 120.4(d); C-Sa, 142.3(s); C-8a', 112.9(s); C-4a, 138.6(s); C-2', 109.2(d); C-7', 109.18(d); C-I, 98.7(s); C-2, 22.4(0; C-9, 58.3(q); and C-3, 26.6ppm (t). Mass Spectrum: LREIMS: 350(10%), 349(14), 348(M+, 56), 318(18), 317(24), 316(M§ CH3OH, 100), 299(10), 287(19), 271(10), 159(11), 131(14), 115(16), 114(14), 71(15), 57(13), and 43m/e (16); HREIMS: C21H1605,found 348.0997re~e, calcd 348.0998. Reference K. Krohn, A. Michel, U. Fl6rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins CI-C16 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

87

Common/Systematic Name Palmarumycin C6 4,7-Dihydroxyspiro [ 1H-inden- 1, 2'-naphtho [ 1,8-de] [ 1,3 ] dioxin]-3 (2/O-one Molecular Formula/Molecular Weight C19H12Os; MW = 320.06847

0

OH

General Characteristics Obtained as a microcrystalline powder that slowly turned dark upon contact with air; mp., 191-192~ (dec.). Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of soluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ethermethylene chloride (1:1, v/v) as eluant. The fourth fraction from the column chromatography contained the majority of the secondary metabolites. Column chromatography was repeated with this fraction using a gradient of methylene chloridemethanol (0-10%) to afford six new fractions. From the first fraction an additional 20 mg of palmarumycin C3 was isolated. From the second fraction 22mg of palmarumycin C6 was crystallized (petroleum ether). From fraction four and five liquid chromatography (Chromatotron, 99.5% methylene chloride-0.5% methanol) an additional 15mg of palmarumycin C6 and 30mg of the mixture of palmarumycin C7 and Cs could be isolated. Repeated liquid chromatography of fraction five (Chromatotron, 99.5% methylene chloride-0.5% methanol) afforded an additional 920mg of a mixture of diastereomeric diepoxides (palmarumycin C9), 66mg of palmarumycin C10, 30mg of palmarumycin Cll, and 198mg of palmarumycin Cn. An additional 246mg of palmarumycin C12 was isolated by repeated fractional crystallization from fraction six.

88

6.

Palmarumycins

Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Spectral Data UV: ~Cm~~176176286(1og e=3.47), 296(3.76), 300(3.74), 314(3.55), 346(3.52), and 329nm (3.57). IR: (KBr) 3468(OH), 3376(OH), 3064, 2926, 1638(C=O), 1609, 1584, 1561, 1509, 1476, 1412, 1377, 1264(Ar-O), 1235, 1181, 1092, 1021, 938, 909, 874, 820, 793, 762, and 681 cm~. 1H NMR: (DMSO-d6) 9.83(1H, OH); 9.56(1H, OH); 7.61(2H, d, J3,,4yr,6~=8.1Hz, H-4', H- 5'); 7.5012H, pseudo-t (dd), J2;3y6,,7,~=7.6Hz, J3,,4ys:6~=8.2Hz, H-3', H-6']; 7.14(1H, d, Js,6=8.8Hz, H-6); 7.00(2H, d, ff2,3y6,,79=7.5Hz,J2,4~O.9Hz, H-2', H-7'); 6.96(1H, d, Js,6~8.8Hz, H-5); and 2.76ppm (2H, s, H-2). 13CNMR: (DMSO-d6) C-4, 148.1(s); C-5, 120.4(d); C-8', C-I', 148.26(s); C-4a', 134.0(s); C- 7, 147.5(s); C-6', C-3', 127.6(d); C-4', C-5', 120.8(d); C-6, 125.7(d); C-3, 196.9(s), C-8a', 113.5(s); C-3a, 123.1(s); C-2', C-7', 109.6(d); C-l, 103.5(s); C-2, 49.1(0; and C-7a, 131.8ppm (s).

Mass Spectrum: LREIMS: 320(M § 100%), 303(M+ - OH, 55), 160(10), and 115m/e (11); LRCIMS: (NH3, positive ion) 338(M ++ NH4, 55%), 321(M + + H, 100), 320(M+, 22), 236(21), and 161m/e (30); HREIMS: C19H1205,found 320.0684m/e, calcd 320.0685. Reference K. Krohn, A. Michel, U. F16rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1- C~6 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

89

Common/Systematic Name Palmarumycin C7 (4aRS, 8aRS)-3-Chloro-5-hydroxyspiro [4a, 8a-epoxynaphthalene- 1(4H),2'-naphtho [ 1,8de][ 1,3]-dioxin]-4,8(5H)-dione Molecular Formula/Molecular Weight C2oH1]C106, MW = 382.02442 0 Cl

OH :

General Characteristics Obtained as the major component of a mixture with palmarumycin C8. Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 331 g of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether/methylene chloride (1:1, v/v) as eluant.The fourth fraction from the column chromatography contained the majority of the secondary metabolites. Column chromatography was repeated with this fraction using a gradient of methylene chloridemethanol (0-10%) to afford six new fractions. From the first fraction an additional 20mg of palmarumycin C3 was isolated. From the second fraction 22mg of palmarumycin C6 crystallized (petroleum ether). From fractions four and five liquid chromatography (Chromatotron, 99.5% methylene chloride- 0.5% methanol) an additional 15mg of palmarumycin C6 and 30mg of the mixture of palmarumycin C7 and C8 could be isolated. Repeated liquid chromatography of fraction five (Chromatotron, 99.5% methylene chloride- 0.5% methanol) afforded an additional 920mg of a mixture of diastereomeric diepoxides (palmarumycin C9), 66mg of palmarumycin C~0, 30mg of palmarumycin CI~, and 198mg ofpalmarumycin C12. An additional 246mg ofpalmarumycin C12 was isolated by repeated fractional crystallization from fraction six.

90

6. Palmarumycins

Fungal Source An unidentified Coniothyrium sp.(intemal strain number 812) isolated from soil from a forest on West Borneo. Biological Activity Exhibited biological activity against a variety of bacteria and fungi, as well as biological activity against growth of garden cress. Spectral Data IH NMR: (CDC13) 7.58(2H, m, H-4', H-5'); 7.50[ 1H, pseudo-t (dd), J2,3~7.5Hz, J3,4=8.2Hz, H3']; 7.45[1H, pseudo-t (dd), J6,7=7.5Hz, Jy.6~8.2Hz, H-6']; 7.11(1H, d, J2,3=7.5Hz, H2'), 6.95(1H, d, J6,,7=7.5t-Iz, H-7'), 6.85(1H, s, H-2), 6.78(1H, d, J6,z=10.6Hz, J5,6 = 5.1Hz, H-6); 6.16(1H, d, J6,7 = 10.6Hz, J5,7= 0.9Hz, H-7); and 5.32ppm (1H, m, H-5). 13C NMR: (CDC13) C-8, 184.8(s); C-4, 184.3(s); C-8', 145.1(s); C-I', 144.4(s); C-6, 140.8(d); C2, 135.9(d); C-4a', 134.1(s); C-3, 131.3(s); C-3', 127.9(d); C-7, 127.6(d); C-6', 127.5(d); C-4', 121.7(d); C-5', 121.6(d); C-4a', 112.3(s); C-2', 110.4(d); C- 7', 109.5(d); C-l, 95.0 (s); C-4a, 63.2(s); C-5, 61. l(d), and C-Sa, 60.4ppm (s). Reference K. Krohn, A. Michel, U. FlOrke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1-C16 from Coniothyrium sp." Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

91

Common/Systematic Name Palmarumycin C8 (4aRS, 8aRS)-3-Chloro-6,7-dihydro-5-hydroxyspiro [4a,8a-epoxynaphthalene- 1(4H),2'nap htho [ 1,8-de ][ 1,3]-dioxin ]-4, 8(5H)-dione Molecular Formula/Molecular Weight C20H13C106; MW = 384.04007 0

OH

C

General Characteristics Obtained as the minor component of a mixture with palmarumycin C7. Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The fourth fraction from the column chromatography contained the majority of the secondary metabolites. Column chromatography was repeated with this fraction using a gradient of methylene chloridemethanol (0-10%) to afford six new fractions. From the first fraction an additional 20mg of palmarumycin C3 was isolated. From liquid chromatography of the second fraction 22mg of palmarumycin C6 crystallized (petroleum ether). From liquid chromatography of fractions four and five (Chromatotron, 99.5% methylene chloride-0.5% methanol) an additional 15mg of palmarumycin C6 and 30mg of the mixture of palmarumycin C7 and C8 were isolated. Repeated liquid chromatography of fraction five (Chromatotron, 99.5% methylene chloride-0.5% methanol) afforded an additional 920mg of a mixture of diastereomeric diepoxides (palmarumycin C9), 66mg of palmarumycin C10, 30mg of palmarumycin Cll, and 198mg of palmarumycin C12. An additional 246mg of palmarumycin C12 was isolated by repeated fractional crystallization from fraction six.

92

6. Palmarumycins

Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Biological Activity Exhibited biological activity against a variety of bacteria and fungi, as well as biological activit3 against growth of garden cress. Spectral Data 1H NMR: (CDC13) 7.58 0(2H, m, H-4', H-5'); 7.50 [1H, pseudo-t(dd), J2,,3,=7.5Hz, ,]3,4,- 8.2Hz, H-3']; 7.43 [1H, pseudo-t(dd), J6,,7,-7.5Hz, Js,,6,-8.3Hz, H-6']; 7.11(1H, d, J2',s' =7.5Hz, n-2'); 6.93(1H, d, JD,7,-7.6Hz, H-7'); 6.78(1H, s, H-E); 5.12(1H, s, H-5); 2.81(1H, ddd, J7ax,7eq=18.6Hz,J6~,7ax=l 1.4Hz, J6eq,7ax=3.6Hz, H-7ax); 2.5 I(1H, td, J7ax,7eq-18.6Hz, J6ax,7eq(6eq,7eq)=3.6Hz,H-7eq); and 2.09ppm (2H, m, H-6ax, H-6eq). 13C NMR: (CDC13) C-8, 194.4(s); C-4, 185.3(s); C-8', 145.1(s); C-I', 144.5(s); C-6, 23.3(0; C-2, 135.4(d); C-4a', 134.1(s); C-3, 131.4(s); C-3', 127.6(d); C-7, 32.7(0; C-6', 127.4(d); C4', 121.6(d); C-5', 121.55(d); C-8a', 112.4(s); C-2', 110.4(d); C- 7', 109.5(d); C-I, 95.5(s); C-4a, 62.9(s); C-5, 61.4(d); and C-Sa, 61.3ppm (s). Reference K. Krohn, A. Michel, U. Fl6rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1-C~6 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

93

Common/Systematic Name Palmarumycin C10

(2S*,3S*,4R*,4aR *,8aS)-3 ,4-Dihydro-4-hydroxyspiro[2,3 :4a,8a-diepoxynaphthalene1,(2H) 2'-naphtho[ 1,8-de][ 1,3 ]-dioxin]-5,8-dione

Molecular Formula/Molecular Weight C20H1207; MW = 364.05830 OH

0

O

CL General Characteristics A yellow solid; mp., 236~ (dec.); [a]D 2~=-48.2 ~ Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The fourth fraction from the column chromatography contained the majority of the secondary metabolites. Column chromatography was repeated with this fraction using a gradient of methylene chloridemethanol (0-10%) to afford six new fractions. From the first fraction an additional 20mg of palmarumycin Cs was isolated; from the second fraction 22mg of palmarumycin C6 crystallized (petroleum ether).Liquid chromatography (Chromatotron, 99.5% methylene chloride-0.5% methanol) of fractions four and five produced an additional 15mg of palmarumycin C6 and 30mg of the mixture of palmarumycin C7 and C8. Repeated liquid chromatography of fraction five (Chromatotron, 99.5% methylene chloride-0.5% methanol) afforded an additional 920mg of a mixture of diastereomeric diepoxides (palmarumycin C9), 66mg ofpalmarumycin C10, 30mg ofpalmarumycin Cll, and 198mg of palmarumycin C12. An additional 246mg of palmarumycin C12 was isolated by repeated fractional crystallization from fraction six.

94

6.

Palmarumycins

Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Biological Activity Exhibited biological activity against a variety of bacteria and fungi, as well as biological activity against growth of garden cress. Spectral Data UV~

EmtOaxH/10%MethyleneChl~293(log e =4.19), 299(4.31), 307(4.22), 313(4.24), and 327nm (4.16). IR: (KBr) 3480(OH), 1692(C=O), 1609, 1414, 1381, 1275(Ar-O), 1111, 1042, 951,818, and 754cm1. 1H NMR: (DMSO--d6) 7.66(1H, dd, Jy4~-8.3nz, H-4'); 7.65(1H, d, Js:6,=8.3Hz, H-5'); 7.53(2H, m, Jy,6,=8.3Hz, H-3', H-4'); 7.13(1H, d, J2,,.~,=7.5Hz, H-2'); 7.03(1H, d, J6, 7,= 7.5Hz, H-7'); [1H, pseudo-t(dd), Js,,6,=8.2Hz,J6.,7,=7.6Hz, H-6']; 6.77(1H, d, J6,7= 10.7Hz, H6); 6.66(1H, d, J6.7=lO.7Hz, H-7); 5.02(1H, d, J3.4=3.0nz, H-4); 3.46(11-1, d, J2,3 =4.1Hz, H-2); and 3.40ppm (1H, d,[pseudo-t(dd), J2.3~3,.0=3.6Hz, H-3). 13C NMR:

(DMSO-d6) C-8, 190.1(s); C-5, 187.3(s); C-8', 145.2(s); C-I', 144.9(s); C-7, 136.4(d); C-6, 135.7, (d); C-4a', 133.8(s); C-3', 127.9(d); C-6', 127.8(d); C-4', C-5', 121.0(d); C8a', 111.50(s); C-2', 109.2(d); C-7', 109.0(d); C-l, 94.5(s); C-4a, 65.4(s); C- 8a, 63.5(s); C-4, 59.4(d); C-2, 53.2(d); and C-3, 55.2ppm (d). Mass Data: LREIMS: 366(M++ 2H, 22%), 365(M*+ H, 100), 364(M§ 86), 211(6), 171(8), 159(8), 115(14), and 114m/e (18); HREIMS: C20H1207calcd. 364.058303; found 364.058305; calcd. C=65.92, H=3.32, found C=65.85, H=3.40. Reference K. Krohn, A. Michel, U. FlOrke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1-C16 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6.

Palmarumycins

95

Common/Systematic Name Palmarumycin all (2S*,3R*,4S*)-3,4-Dihydrospiro [2,3-epoxynaphthalene- 1,(2H) 2'-naphtho [ 1,8-de] [ 1,3 ] dioxin]-4,5-diol Molecular Formula/Molecular Weight C20H1405; ~ = 334.08412 OH

OH

General Characteristics Crystallized as colorless needles; mp., 207-208~

[Q~]D20"- -153 ~

Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fembach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 331 g of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The fourth fraction from the column chromatography contained the majority of the secondary metabolites. Column chromatography was repeated with this fraction using a gradient of methylene chloridemethanol (0-10%) to afford six new fractions. From the first fraction an additional 20mg of palmarumycin C3 was isolated. From the second fraction 22mg of palmarumycin C6 crystallized (petroleum ether). Liquid chromatography (Chromatotron, 99.5% methylene chloride-0.5% methanol) of fractions four and five provided an additional 15mg of palmarumycin C6 and 30mg of the mixture of palmarumycin C7 and Cs. Repeated liquid chromatography of fraction five (Chromatotron, 99.5% methylene chloride-0.5% methanol) afforded an additional 920 mg of a mixture of diastereomeric diepoxides (palmarumycin C9), 66mg of palmarumycin C10, 30mg of palmarumycin Cll, and 198mg of palmarumycin C12. An additional 246mg of palmarumycin C12 was isolated by repeated fractional crystallization from fraction six.

96

6. Palmarumycins

Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Biological Activity Exhibited biological activity against a variety of bacteria and fungi, as well as biological activity against growth of garden cress. Spectral Data UV: ~,~e,,c,.o,d~ 293(log e=4.05), 296(4.09), 300(4.10), 314(3.83), and 327nm (3.62). IR: (KBr) 3540(OH), 3070(OH), 3000, 2900, 1619, 1590, 1467, 1412, 1381, 1327, 1232, 1180, 1130, 1111, 1072, 1028, 968, 872, 822, and 798cm"1. 1H N1V[R: (CDC13) 8.28(1H, s, OH), 7.57(1H, dd, J3,.4~8.1Hz, J2,,4~-0.9Hz, H-4'), 7.55(1H, d, Jy6 ,= 8.1Hz, H-5'); 7.51[1H, pseudo-t(dd), Jz3,=7.4Hz, Jy4,=8.3Hz, H-3']; 7.44[1H, pseudo-t(dd), Jy,6,=8.2nz, J6,,7,-_7.6Hz,H-6']; 7.37(1H, d, [pseudo-t(dd), J7.8,=7.9Hz, Ja,7=7.5Hz, H-7); 7.14(1H, dd, J2,3,=7.3Hz, J2,.4,=O.9Hz, H-2'); 7.04(1H, dd, J6,7=7.0Hz, J6,s=2.4Hz, H-6); 6.92(1H, d, J6, 7,=7.4Hz, H-7'); 3.74(1H, dd, Jz3=4.4Hz, J~,4=2.7Hz, H-3), 3.ppm(1H, d, Jz3=4.Hz, H-2); 7.41(1H, dd, J7,s=8.0Hz, J6,s=2.3Hz); 5.44(1H, dd, J4.on=l 1.3Hz, J3,4=2.7Hz, H-4); and 3.15ppm (1H, d, J4,OH= 11.3Hz, OH). 13C NMR: (CDCl3) C-4, 66.7(d), C-5, 156.5(s); C-8, 119.3(d); C-8', 147.3(s); C-l', 147.2(s); C-4a', 134.1(s), C-7, 130.6(d), C-6', 127.4(d), C-3', 127.7(d), C-4', 121.3(d), C-6, 118.9(d), C-5', 120.96(d); C-8a, 132.0(s); C-4a, 118.5(s); C-2', 109.9(d); C-7', 109.0(d); C-l, 96.7(s); C-2, 54. l(d); and C-3, 52.8ppm (d).

Mass Data: LREIMS: 335(M ++ H, 27%), 334(M § 8), 285(17), 209(70), 169(100), and 152m/e (72); HREIMS: C2oH~405calcd. 334.0841 found 334.084 lm/e; calcd. C= 71.85, H, 4.22; found C, 71.57, H, 4.07. Reference K. Krohn, A. Michel, U. Fl6rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1-C16 from Coniothyrium sp. Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

97

Common/Systematic Name Palmarumycin C~2 (2S*,3S*,4S*)-3,4-Dihydrospiro[2,3: 4a,8a-epoxynaphthalene- 1,(2/-/) 2'-naphtho[ 1,8de][1,3 ]~-diordn]-4,5 , 8-triol Molecular Formula/Molecular Weight C20H1406; ~

= 350.07904

OH

OH

I

General Characteristics Colorless prisms with poor solubility in methylene chloride; mp., 207-208~ (dec.); [a]o 2~ = _179.6 o. Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fembach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The fourth fraction from the column chromatography contained the majority of the secondary metabolites. Column chromatography was repeated with this fraction using a gradient of methylene chloridemethanol (0-10%) to afford six new fractions. From the first fraction an additional 20mg of palmarumycin C3 was isolated. Palmarumycin C6 crystallized (petroleum ether) from the second fraction. Liquid chromatography (Chromatotron, 99.5% methylene chloride-0.5% methanol) of fractions four and five provided an additional 15mg of palmarumycin C6 and 30mg of the mixture of palmarumycin C7 and C8. Repeated liquid chromatography of fraction five (Chromatotron, 99.5% methylene chloride-0.5% methanol) afforded an additional 920mg of a mixture of diastereomeric diepoxides (palmarumycin C9), 66mg of palmarumycin C~0, 30mg ofpalmarumycin Cll, and 198mg ofpalmarumycin C12. An additional 246mg of palmarumycin C12 was isolated by repeated fractional crystallization from fraction six.

98

6. Palmarumycins

Fungal Source An unidemified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Biological Activity Exhibited biological activity against a variety of bacteria and fungi, as well as biological activity against growth of garden cress. Spectral Data UV: ~,

1096Methylene Chloride max

288(log e=3.64), 292(3.97), 299(4.10), 302(4.10), 313(3.99), and

327nm (3.46). IR:

(KBr) 3476(OH), 1609, 1586, 1474, 1445, 1377, 1304, 1265(Ar-O), 1190, 1183, 1069, 1061, 1049, 1030, 947, 909, 870, 835, 820, 793,760, and 739cm1. 1H NMR: (CDCI3/CD3OD) 7.54(1H, d, J3,4~8.4Hz, H-4'), 7.49(1H, d, Jy,o,=8.4Hz, H-5'), 7.43[ 1H, pseudo-t(dd), J2,~,=7.5Hz, J3,r H-3']; 7.39[ 1H, pseudo-t(dd), Jy,6,=8.4Hz, d6,,v'=7.5Hz, H-6']; 7.08(1H, d, d2,~,=7.5Hz, H-2'); 6.92(1H, d, ,I6,7, =7.5Hz, H-7'), 6.87(1H, d, Jo,7=8.9Hz, H-7); 6.82(1H, d, J6,7=8.9Hz, H-6); 5.29 (1H, d, J3,4=2.3Hz, H-4); 3.67(1H, d, J2,3=4.4Hz, H-2); and 3.57ppm (1H, dd, J2,r~4.3Hz, ,]3,4=2.7Hz, H-3). ~3CNMR: (CDCla/CDaOD) C-8, 149.6(s); C-5, 149.3(s); C-8', 147.0(s); C-I', 145.7(s); C-4a', 134.0(s), C-3', 127.5(d); C-6', 127.4(d); C-4', 122.4 (d); C5', 121.2(d); C-4a, 120.0(s); C-7, 119.9(d), C-6, 118.9(d); C-8a, 115.4(s), C-8a', 113.0(s); C-2', 110.6(d), C-7', 109.8(d); C-l, 99.4(s); C-4, 65.5(d), C-2, 51.5(d); and C-3, 54.0ppm (d). Mass Data: LREIMS: 351(M++ H, 59%), 350(M+, 90), 332(NC- H20, 100), 316(21), 304(332CO, 34), 303(332 - CHO, 34), 287(20), 275(16), 159(41), 131(23), 115(36), and 114m/e (33), HREIMS: C20H1406calcd. 350.0790, found 350.0790m/e. Reference K. Krohn, A. Michel, U. Fl6rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C~-C16from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

99

Common/Systematic Name Palmarumycin C13; Diepoxide rl (2S*,3 S*,4R*,4aR *,5R*,8aS*)-3,4-Dihydro-4, 5-dihydroxyspiro [2,3:4a, 8adiepoxynaphthalene- 1(2H),2'-naphtho [ 1,8-de] [ 1,3 ] -dioxin]-8(5H)-one Molecular Formula/Molecular Weight C20H1607; MW -- 366.07395

OH

OH

General Characteristics Isolated as a 1:1 mixture with palmarumycin C14. Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fembach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 331 g of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The fifth fraction from the column chromatography was first subjected to column chromatography and then twice to liquid chromatography (Chromatotron, 2mm silica gel, 93% methylene chloride-7% methanol) to afford palmarumycins C13 and C14 as a 1:1 mixture, along with palmarumycins C15, and C16. Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Biological Activity Exhibited biological activity against a variety of bacteria, fungi and biological activity against growth of garden cress.

100

6. Palmarumycins

Spectral Data ~HNMR: (CD3OD) 7.57(2H, m, J~,4,o,6,)=8.4Hz, H-4', H-5'); 7.50[ 1H, pseudo-t(dd), J2,3,=7.4Hz, J3,4,=8.4Hz, H-3']; 7.46[ 1H, pseudo-t(dd), Jy,o,=8.4Hz, Jo,7,=7.5Hz, H6']; 7.04(1H, dd, Jz,3,=7.4Hz, Jz,4,= 1.0Hz,H-2'); 6.94(1H, dd, Je,7~-7.5Hz, Jy,7,=0.6Hz, H-7'); 5.91(1H, dd, Jo,7=lO.6Hz, Js.7=O.9Hz, H-7); 6.74(1H, dd, Jo,7=lO.6Hz, Js,6=4.9Hz, H-6); 5.11(1H, m, H-4); 4.82(1H, dd, Js,o=4.9Hz, J5,7= 0.9Hz, H-5); and 3.45ppm (2H, m, H-2, H-3). 13C NMR:

(CD3OD) C-8, 190.1(s); C-5, 62.8(d); C-8', 147.5(s); C-I', 145.2(s); C-4a', 136.0(s); C-3', 129.0(d); C-6', 128.8(d); C-4', 122.2(d); C-5', 122.1(d); C-4a, 72.3(s); C- 7, 127.4(d); C-6, 145.1(d); C-8a, 65.4(s); C-8a', 113.7(s); C-2', 110.5(d); C-7', 110.0(d); C-l, 96.8(s); C-4, 62.5(d); C-2, 54.9(d); and C-3, 57.0ppm (d). Reference K. Krohn, A. Michel, U. Fl6rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1C16 from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumyeins

101

Common/Systematic Name Palmarumycin C~4; Diepoxine

(2S*,3S*,4R*,4aR *,5R *,8aS*)-3 ,4,6,7-Tetrahydro-4,5-dihydroxyspiro[2,3 :4a, 8a_

diepoxynaphthalene- 1(2/-/), 2'-naphtho[ 1,8-de] [ 1,3 ]-dioxin]-8(5H)-one Molecular Formula/Molecular Weight C20H1607, ~

OH

= 368.08960

!

OH

General Characteristics Isolated as a 1:1 mixture with palmarumycin C13. Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fembach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins Ca and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The fifth fraction from the column chromatography was first subjected to column chromatography and then twice to liquid chromatography (Chromatotron, 2 mm silica gel, 93% methylene chloride-7% methanol) to afford palmarumycins C~3 and C14 as a 1:1 mixture, along with palmarumycins C]5, and C16.

Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Biological Activity Exhibited biological activity against a variety of bacteria and fungi, as well as biological activity against growth of garden cress.

102

6. Palmarumycins

Spectral Data ~HNMR: (CD3OD) 7.56(2H, m, Jy,4,(y69=8.4Hz, H-4', H-5'); 7.49[ 1H, pseudo-t(dd), J2,,3~7.5Hz, Jy,4,=8.4Hz, H-3']; 7.45 [ 1H, pseudo-t(dd), Js,,6,=8.4I-Iz, J6,,7,=7.5I-'Iz, H6']; 7.03(1H, dd, J2,,3,=7.5Hz, J2,,4,=l.OHz, H-2'); 6.95(1H, dd, J6,,7~7.5Hz, Jy,7,=0.6Hz, H-7'); 4.87(1H, dd, J3,4=2.5Hz, H-4); 4.71(1H, t, Js,6ax(5,6eq)=3.0nz,H-5), 3.45(2H, m, H-2, H-3); 2.57(1H, ddd, J7~x,7eq=18.1Hz,J6~,Tax--12.4Hz, J7eq,7~x=6.5Hz, H-7ax); 2.32(1H, ddd, J7a~,7eq=l8.1Hz, J6ax,7eq=5.6I--Iz,J6eq,7eq=2.4Hz, H-7eq); 2.04(1H, dddd, J6ax,6eq = 14.2Hz, J6~,7~-12.4Hz, J6ax,Teq=5.6nz, Js,6ax=3.0nz, H-6ax); and 1.83ppm (1H, dddd, J6ax,6eq= l 4.2Hz, J6eq,7ax=6. 5Hz, Js,6eq --3 .0 n z , J6eq,Teq=2.4Hz, H-6eq). 13C NMR: (CD3OD) C-8, 200.2(s); C-8', 147.5(s); C-I', 147.2(s); C-4a', 136.0(s); C-3', 129.0(d), C-6', 128.8(d), C-4', 122.2,(d); C-5', 122.1(d), C-4a, 71.3(s), C-7, 33.9(0; C-6, 25.7(0; C-8a, 64.1(s); C-8a', 113.7(s); C-2', 1lO.5(d); C-7', 1lO.O(d); C-l, 96.8(s); C-4, 63.2(d); C-2, 55.0(d); C-5, 64. l(d); and C-3, 56.9ppm (d).

Reference K. Krohn, A. Michel, U. F16rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C~-C16from Coniothyrium sp.: Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

103

Common/Systematic Name Palmarumycin C15 (2S*,3S* 4R* 4aR*,5R* 8RS, 8aS*)-3,4,5,8-Tetrahydrospiro[2,3:4a,8adiepoxynaphthalene- 1(2H),2'-naphtho [ 1,8-de ] [ 1,3 ]-dioxin]-4, 5,8-tri ol Molecular Formula/Molecular Weight C20H1607; M~V = 367.08178

OH

OH

General Characteristics Isolated as a white powder; mp., 150~ (dec.); [a]D2~ -18.1 ~ Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The fifth fraction from the column chromatography was first subjected to column chromatography and then twice to liquid chromatography (Chromatotron, 2mm silica gel, 93% methylene chloride-7% methanol) to afford palmarumycins Cls and C14 as a 1:1 mixture, along with palmarumycins C15, and C16.

Fungal Source An unidentified Coniothyrium sp. (internal strain number 812) isolated from soil from a forest on West Borneo. Biological Activity Exhibited biological activity against a variety of bacteria and fungi, as well as biological activit3 against growth of garden cress.

104

6. Palmarumycins

Spectral Data UV: ~, ~.,.Ch,o,d. 287(1og e = 3.79), 297(4.07), 312(3.71), and 327nm (3.44). IR: (KBr) 3426(OH), 2361, 1611, 1414, 1381, 1273(Ar-O), 1136, 1088, 1047, 1032, 941,822, and 758cm1. 1H NNtR: (CDC13/CD3OD) 7.51(1H, d, J3,~8.2Hz, H-4'); 7.50(1H, d, Js,,6~8.3Hz, H-5'); 7.42(2H, m, H-3', H-6'); 7.11(1H, d, Jz,3,=7.3Hz, H-2'); 6.96(1H, d, J6.,7.=7.4Hz, H7'); 5.75(1H, ddd, J6,7=lO.SHz, J s,6=4.SHz, ff6,s=l.7Hz, H-6); 5.60(1H, dd, ,]6,7= 10.5Hz, JT,s-2.60Hz, H-7); 5.18(1H, s, H-8); 4.93(1H, d, J3,~2.8Hz, H-4); 4.61(1H, d, J4,5=4.5Hz, H-5); 3.48(1H, d, Jz3=4.3Hz, H-2); and 3.42ppm(1H, dd, Jz3=4.3Hz, J3,r=2.SHz, H-3). 13CNMR: (CDC13/CD3OD) C-8, 63.0(d); C-5, 61.0(d); C-8', 145.8(s); C-l', 145.1(s), C-4a', 134.0(s), C-3', 127.7(d), C-6', 127.3(d), C-4', 121.5(d); C-5', 121.3(d), C-4a, 68.3(s); C-7, 126.9(d); C-6, 125.6(d); C-8a, 66.2(s); C-8a', 112.7(s); C-2', 110.3(d); C-7', 109.4(d); C-l, 96.6(s); C-4, 61.4(d); C-2, 53.2(d), and C-3, 55.8ppm (d).

Mass Data: LREIMS: 368(M § 100%), 160(21), 125(26), 111(38), 97(51), 82(48), 71(55), and 57role (68); HREIMS C2oH1607calcd. 368.0896; found 368.089m/e; calcd. C,65.22, H, 4.42; found C, 65.14, H, 4.42. Reference K. Krohn, A. Michel, U. Flfrke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1C16 from Coniothyrium sp." Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

6. Palmarumycins

105

Common/Systematic Name Palmarumycin C 16 (2S*, 3S*,4R*,4aR *, 5R *, 8RS, 8 aS*)-3,4,5,6, 7, 8-Hexahydro spiro [2,3: 4a, 8adiepoxynaphthalene- 1(2H),2'-naphtho [ 1,8-de ][ 1,3] -dio~n]-4, 5,8-triol Molecular Formula/Molecular Weight C20H1807; ~

OH

= 369.09743

OH

General Characteristics Crystals; mp., 187-188~ (dec.);

[I~]D 20 -'-

-43.3 ~

Isolation/Purification Liquid fungal cultures were shaken for 14-22 days in 2L Fernbach flasks at 25~ and 125 rpm. The 50L of culture material was filtered and extracted three times with ethyl acetate. The combined extracts were dried (Na2SO4), filtered, and concentrated at reduced pressure to afford 33 lg of crude extract. The residue was treated with methanol and filtered. The precipitate was extracted with ethyl acetate to afford 2.0g of dissoluble material that was investigated later (palmarumycins C4 and C9). The filtrate from the methanol treatment was further separated by column chromatography using petroleum ether-methylene chloride (1:1, v/v) as eluant. The fifth fraction from the column chromatography was first subjected to column chromatography and then twice to liquid chromatography (Chromatotron, 2mm silica gel, 93% methylene chloride-7% methanol) to afford palmarumycins C13 and C14 as a 1:1 mixture, along with palmarumycins C15 and C16.

Fungal Source An unidentified Coniothyriumsp. (internal strain number 812) isolated from soil from a forest on West Borneo. Biological Activity Exhibited biological activity against a variety of bacteria and fungi, as well as biological activit2 against growth of garden cress.

106

6. Palmarumycins

Spectral Data UV: L ~.o.c,,o,d, 287 (log e=3.60), 298 (3.81), 307 (3.66), 313 (3.68), and 327nm (3.60). IR:

(KBr) 3426(OH), 2938, 2361, 1611, 1412, 1381, 1273(Ar-O), 1090, 1047, 1034, 1005, 965, 939, 820, and 758cm1. :H ~ :

(CD3OD) 7.59(1H, dd, J~,,4~8.SHz, J~,,4~l.lHz, H-4'); 7.58(1H, d, Js,,6~8.SHz, H-5'); 7.52 [1H, pseudo-t(dd), J2,,3,=7.4Hz, J2,,4,=8.SHz, H-3']; 7.49[1H, pseudo-t(dd), Js,a,=8.5Hz, J6,,7.=7.5Hz, H-6']; 7.14(1H, dd, J2,~,=7.3Hz, J2,.4,=l.OHz, H-2'); 7.04(1H, dd, J6,z,--7.5Hz, ds,,7,=0.7Hz, H-7'); 4.82(1H, d, d3.4=2.8Hz, H-4); 4.68(1H, m, H-8); 4.47(1H, m, H-5); 3.45(1H, d, Jz3=4.3Hz, H-2); 3.41(1H, dd, Jz3=4.3Hz, J3,4=2.8Hz, H-3); 1.94(2H, m, J=l 1.4Hz, H-6eq, H-7eq); and 1.48ppm (2H, m, J= 11.4Hz, H-6ax, H-7ax).

13CNMR: (CDCI3/CD3OD) C-8, 63.4(d), C-5, 64.3(d); C-8', 147.9(s); C-I', 147.6(s); C-4a', 135.9(s); C-3', 129.1(d); C-6', 128.9(d); C-4', C-5', 122.4(d), C-4a, 69.3(s); C-7, 26.0(0; C-6, 24.0(0; C-8a, 68.0(s); C-8a', 114.3(s), C-2', 111.4(d); C-7', 110.5(d); C-I, 98.4(s); C-4, 63.2(d); C-2, 54.0(d), and C-3, 57.2ppm (d). Mass Data: LREIMS 370(M§ 100%), 352(11), 213(16), 172(20), 160(72), 159(20), 139(31), 121(27), 116(26), 99(38), 86(60), and 56m/e (37); HR IMS: C20H1807calcd. 370.1052; found 370.1052re~e; calcd. C, 64.86, H, 4.90; found C, 64.31, H, 4.76. Reference K. Krohn, A. Michel, U. Fl6rke, H.-J. Aust, S. Draeger, and B. Schulz; Palmarumycins C1-C16 from Coniothyrium sp. Isolation, Structure Elucidation, and Biological Activity; Liebigs Ann. Chem., pp. 1099-1108(1994).

Phomosines Phomosine A Phomosine B Phomosine C

107


7. Phomosines

109

Common/Systematic Name Phomosine A Molecular Formula/Molecular Weight C18H1807; ~

= 346.10525

H

OH

0

Me 5//.,. i ~ 0 HO

OH

~'~J "Me "~, OMe

Me

General Characteristics Long, pale yellow needles from dichloromethane-petroleum ether; mp., 203-204~ Isolation/Purification Culture medium from Phomopsis sp. was homogenized with a Waring blender, diluted with water and then extracted four times with ethyl acetate. The organic extracts were dried (Na2SO4), filtered, and evaporated m vacuo. The main compounds were detected in the residue on TLC at Rf values: phomosine A: Rf=0.71; phomosine B: Rf=0.53; phomosine C: Rf=0.48, 5% methanol-methylene chloride detected by UV irradiation at 254nm. Column chromatography on silica gel (methylene chloride-20% ethyl ether) gave four fractions. The second fraction gave material that was further purified by crystallization from dichloromethane-petroleum ether to afford phomosine A. The third fraction was further separated by Chromatotron chromatography (methylene chloridepetroleum ether; 1:1, v/v) into four fractions. The second fraction was purified by thinlayer chromatography (5% methanol-methylene chloride) and crystallization from dichloromethane-petroleum ether to afford additional phomosine A. From the fourth fraction, phomosine B and phomosine C were isolated by the same procedure. Fungal Source Phomopsis sp. Biological Activity All three metabolites showed moderate fungicidal and antibacterial activity against four fungal and two bacterial (one Gram-positive and one Gram-negative) test organisms. Sp,,ectral Data UV:

~mM~y~=~176176232(1og C = 5.23) and 269nm (4.52).

110

7.

Phomosines

IR~

(KBr) 3320(OH), 2955(CH3), 1653(CO), 1637(CO), 1607, 1582, 1437, 1331, 1302, 1211, 1200, 1096, and 808cm~ 1H NMR: (CDCI3) 2.10(3H, s, H-3a); 2.16(3H, s, H-5'a); 2.28(3H, s, H-2a); 3.92(3 H, s, H-lb); 5.80(1H, s, H-6'); 6.36(1H, s, H-4'), 8.92(1H, OH); 10.43(1H, s, H-2'a); 11.87(1H, s, OH), and 11.92ppm (1H, s, OH). 13C NMR: (CDC13) 8.4, C-Sa, (q); 15.3, C-2a (q); 22.5, C-5'a (q); 52.3, C-lb (q); 104.3, C-6' (q); 104.9, C-1 (s); 109.2, C-2' (s); 111.2, C-5 (s); 111.6, C-4' (d); 131.6, 133.0, C-2, C-3 (2s); 150.9, C-5' (s); 153.8, C-4 (s); 161.0, C-3' (s); 161.1, C-6 (s); 164.1, C-I' (s); 172.9, C-la (s); and 194.3ppm C-2a (s). Mass Data: 346(M+, 100%), 314(98), 286(54), 258(26), 165(32), and 151m/e (53); HREIMS: (ClsHIsO7): 346.1050; calcd. 346.1052; found C=62.29, H=5.44; calcd. C=62.41, H=5.24. Reference K. Krohn, A. Michel, E. R6mer, U. F16rke, H.-J. Aust, S. Draeger, B. Schulz, and V. Wray, Biologically Active Metabolites from Fungi 6, Phomosines A-C, Three New Biaryl Ethers from Phomopsis sp.; Natural Product Letters, Vol. 6, pp. 309-314(1995).

7.

Phomosines

111

Common/Systematic Name Phomosine B Molecular FormulafMolecular Weight C19H2207; 1VIW = 362.13655

OH

HO"

"~ 0-"

CH2OMe

"Me "OMe

, Me

General Characteristics Colorless powder from dichloromethane-petroleum ether; mp., 204-206~ Isolation/Purification Culture medium from Phomopsis sp. was homogenized with a Waring blender, diluted with water and then extracted four times with ethyl acetate. The organic extracts were dried (Na2SO4), filtered, and evaporated m vacuo. The main compounds were detected in the residue on TLC at Rf values: phomosine A: Rf=0.71; phomosine B: Rf=0.53; phomosine C: Rf=0.48, 5% methanol-methylene chloride detected by UV irradiation at 254nm. Column chromatography on silica gel (methylene chloride-20% ethyl ether) gave four fractions. The second fraction gave material that was further purified by crystallization from dichloromethane-petroleum ether to afford phomosine A. The third fraction was further separated by Chromatotron chromatography (methylene chloridepetroleum ether; 1:1, v/v) into four fractions. The second fraction was purified by thinlayer chromatography (5% methanol-methylene chloride) and crystallization from dichloromethane-petroleum ether to afford additional phomosine A. From the fourth fraction, phomosine B and phomosine C were isolated by the same procedure. Fungal Source Phomopsis sp. Biological Activity All three metabolites showed moderate fungicidal and antibacterial activity against four fungal and two bacterial (one Gram-positive and one Gram-negative) test organisms. Phomosines B and C also inhibited the algal organism, Chlorellafusca.

112

7.

Phomosines

Spectral Data UV:

~,mM~y~'~176

232(1og e = 4.51), 267(4.25), and 312nm (3.74).

IR: (KBr) 3413(OH), 2953(CH3), 1647, 1622, 1599, 1456, 1422, 1337, 1300, 1285, 1223, 1088, 1047,and 934cm 1. 1H N-MR:

(CDC13) 2.08(3H, s, H-3a), 2.11(3H, s, 5'a-H); 2.44(3H, s, H-6a), 3.47(3H, s, H2'b); 3.97(3H, s, H-lb); 4.78(2H, s, H-2'a); 5.94(1H, s, H-6'a); 6.46(1H, s, H-4'); 8.69(1H, s, OH), 9.10(1H, s, OH); and 11.94ppm (1H, s, OH). lSc NMR:

(CDCI3) 8.3, C-3a (q); 15.8, C-6a (q); 21.5, C-5'a (q), 52.4, C-lb (q); 58.5, C-2'b (q); 64.6, C-2'a (t), 104.3, C-1 (s); 107.1, C-6' (d); 111.1, C-3 (s), 111.5, C-2' (s); 111.5, C-4' (d); 132.3, 135.5, C-2, C 5 (2s); 140.9, C-5' (s), 154.7, C-4 (s), 157.2, C-3' (s), 158.5, C-I' (s); 161.1, C-2 (s); and 173.4ppm, C-la (s). Mass Spectrum: 362(M +, 20%), 330(62), 298(100), 283(26), and 270m/e (18); HREIMS: (C19H2207) 362.136Ore~e; calcd, 362.1365. Reference K. Krohn, A. Michel, E. R0mer, U. FlOrke, H.-J. Aust, S. Draeger, B. Schulz, and V. Wray; Biologically Active Metabolites from Fungi 6; Phomosines A-C, Three New Biaryl Ethers from Phomopsis sp.; Natural Product Letters, Vol. 6, pp. 309-314(1995).

7.

Phomosines

113

Common/Systematic Name Phomosine C Molecular Formula/Molecular Weight C17H1607; M W -- 3 3 2 . 0 8 9 6 0

o.

.yo

HO-~~ I~Me 1 ~L~s,I 0~ ~OMe Me General Characteristics White powder from dichloromethane-petroleum ether; mp., 192-193~ Isolation/Purification Culture medium from Phomopsis sp. was homogenized with a Waring blender, diluted with water and then extracted four times with ethyl acetate. The organic extracts were dried (Na2SO4), filtered, and evaporated in vacuo. The main compounds were detected in the residue on TLC at Re values: phomosine A, Rf=0.71; phomosine B, Rf=0.53; phomosine C, Rf=0.48, 5% methanol-methylene chloride detected by UV irradiation at 254nm. Column chromatography on silica gel (methylene chloride-methylene chloride20% ethyl ether) gave four fractions. The second fraction gave material that was further purified by crystallization from dichloromethane-petroleum ether to afford phomosine A. The third fraction was further separated by Chromatotron chromatography (methylene chloride-petroleum ether; 1:1, v/v) into four fractions. The second fraction was purified by thin-layer chromatography (5% methanol-methylene chloride) and crystallization from dichloromethane-petroleum ether to afford additional phomosine A. From the fourth fraction, phomosine B and phomosine C were isolated by the same procedure. Fungal Source Phomopsis sp. Biological Activity All three metabolites showed moderate fungicidal and antibacterial activity against four fungal and two bacterial (one Gram-positive and one Gram-negative) test organisms. Phomosines B and C also inhibited the algal organism, Chlorellafusca.

114

7.

Phomosines

Spectral Data UV:

~,~yl~oC,o,~ 232(log e = 4.43), 263(4.00), and 273nm (3.93). IR:

(KBr) 1420(OH), 3195, 2955, 1647, 1623, 1431, 1329, 1285, 1254, 1198, and 1074cm1. 1H NMR:

(CDC13) 2.21(3H, s, H-5'a), 2.38(3H, s, H-2a); 3.96(3 H, s, H-lb), 5.93(1H, s, H-6'); 6.42(1H, s, H-4'); 6.46(1H, s, H-5), 9.15(1H, OH); 10.50(1H, s, H-Z'a); 11.43(1H, s, OH); and 11.95ppm (1H, s, OH). 13C NMR:

(CDC13) 15.4, C-2a (q); 22.5, C-5'a (q); 52.5, C-lb (q); 103.1, C-5 (d); 105.0, C-1 (s); 105.0, C-6' (d), 109.6, C-2' (s), 111.7, C-4' (d), 130.7, 135.8, C-2, C-3 (2s), 151.4, C-5' (s), 156.4, C-4 (s), 161.8, C-3' (s); 162.8, C-6 (s); 164.3, C-l' (s); 172.5, C-la (s), and 194.Sppm, C-2a (s). Mass spectrum: 332(M +, 100%), 300(71), and 272m/e (36); HREIMS: (C]7H]607): 332.0890; calcd, 332.0896. Reference K. Krohn, A. Michel, E. ROmer, U. F1Orke, H.-J. Aust, S. Draeger, B. Schulz, and V. Wray; Biologically Active Metabolites from Fungi 6; Phomosines A-C, Three New Biaryl Ethers from Phomopsis sp. Natural Product Letters, Vol. 6, pp. 309-314(1995).

Fumiquinazolines Fumiquinazoline A Fumiquinazoline B Fumiquinazoline C Fumiquinazoline D Fumiquinazoline E Fumiquinazoline F Fumiquinazoline G

115


8. Fumiquinazolines

117

Common/Systematic Name Fumiquinazoline A Molecular Formula/Molecular Weight C24H23NsO4; MW' = 445.17500 16

d."'~.c,I~NH 7

-

H_

H,"';4N~, "0

II

o . ,,,,

.,Me

H "ItOH

H

179

ll,,, 20

N

\

N

~

//

\xj

General Characteristics Fumiquinazoline A was obtained as a pale yellow powder; mp., 178-182~ (c=0.47, in CHCI3).

[(I]D-214.5 ~

Isolation/Purification The fungal strain was grown in a liquid medium containing 2% glucose, 1% peptone and 0.5% yeast extract in artificial seawater adjusted to pH 7.5 for two weeks at 27~ The culture was filtered under suction and the mycelium collected was extracted three times with methanol. The combined extracts were evaporated under reduced pressure. The resulting extract was partitioned between methylene chloride and water and removal of solvents gave the ethylene chloride fraction. This fraction was passed through Sephadex LH2o using methanol as the eluant. The third fraction was ehromatographed on a silica gel column with a methylene chloride-methanol gradient as the eluant and 3 fractions were collected. The methylene chloride eluant yielded fumiquinazoline C alter purification by HPLC (methanol-water; 7:3, v/v). The methanol-methylene chloride (1:99, v/v) eluate was purified by HPLC (methanol-water; 7:3, v/v) to afford fumiquinazoline A and fumiquinazoline D. The methanol-methylene chloride (2:98, v/v) eluant gave fumiquinazolines G, F, B, and E after purification by HPLC (methanol-water; 7:3, v/v). Fungal Source A strain of Aspergillusfumigatus originally isolated from the marine fish Pseudolabrus

japonicus.

Biological Activity Exhibited moderate cytotoxicity against cultured P388 cells.

118

8.

Fumiquinazolines

Spectral Data UV"

~,M~n 208(1og e = 4.58), 226(4.29), 233(4.44), 256(4.17), 265(4.13), 277(4.01), 305(3.54), and 327nm (3.44). CD: (c=1.47 x 10-5 mol/dm3 in ethanol) 230(Ae -11.82), 245(0), 251(+2.11), 261(0), 278(-3.53), 284(-2.99), 290(-2.65), 297(-2.31), 305(-2.45), 312(-1.90), 316(- 0.20), and 334nm (0). IR:

3349(OH, NH), 1680(CON), and 1608em1 (Ar-C-C). 1H NMR: (CDCI3) 66.1(1H, dd, J-0.9, 0.3Hz, NH-2); 4.88(1H, qd, J=7.1, 0.3Hz, H-3); 7.67(1H, dd, J=8.2, 1.0Hz, H-7); 7.75(1H, ddd, J-8.2, 7.0, 1.0Hz, H-8); 7.49(1H, ddd, J=7.9, 7.0, 1.0Hz, H-9); 8.23(1H, dd, J=7.9, 1.0Hz, H-10); 5.97(1H, ddd, ,/=10.9, 6.0, 0.9Hz, H-14); 2.28(1H, dd, J-13.7, 6.0Hz, H-15A); 2.51(1H, dd, J=13.7, 10.9Hz, H-15B); 1.79(1H, d, J=7.1Hz, H-16); 5.49(1H, s, H=18); 2.79(1H, br s, NH19); 4.22(1H, q, J=6.THz, H=20); 7.52(1H, dd, J=7.5, 1.0Hz, H-24); 7.31(1H, td, J=7.5, 1.0Hz, H-25); 7.16(1H, ddd, J=7.5, 6.8, 1.0Hz, H-26); 7.61(1H, dd, J=6.8, 1.0Hz, H-27); 1.35(3H, d, J=6.7Hz, H-29); and 4.89ppm (1H, s, OH). 13C NMR:

(CDCI3) 172.36(C-1, q); 49.15(C-3, t); 150.78(C-4, q); 146.87(C-6, q); 127.57(C-7, t); 134.80(C-8, t); 127.45(C-9, t); 126.77(C-10, t); 120.18(C-11, q); 160.48(C-12, q); 52.98(C-14, t); 36.72(C-15, s); 16.75(C-16, p); 80.20(C-17, q); 86.28(C-18, t); 59.01(C-20, t); 170.53(C-21, q); 136.17(C-23, q); 115.01(C- 24, t); 129.76(C-25, t); 125.58(C-26, t); 124.85(C-27, t); 138.59(C-28, q); and 18.63ppm(C-29, p). Mass Spectrum: EIMS: 445(M+, 5%), 428(M+- OH, 36), 229(13), 228(4), 217(100), 199(20), and 146m/e (22); HREIMS: 445.1769re~e;C24Hz3NsO4,requires 445.1750. Reference C. Takahashi, T. Matsushita, M. Doi, K. Minoura, T. Shingu, Y. Kumeda and A. Numata; Fumiquinazolines A-G, Novel Metabolites of a Fungus Separated from a Pseudolabrus Marine Fish; J. Chem. Soc. Perkin Trans. 1, pp. 2345-2353(1995).

8. Fumiquinazolines

119

Common/Systematic Name Fumiquinazoline B Molecular Formula/Molecular Weight C24H23NsO4; M W = 445.17500

0 General Characteristics Fumiquinazoline B was obtained as a pale yellow powder; mp., 174-176~ (C=0.38, in CHCIa).

[a]D-196.6 ~

Isolation/Purification The fungal strain was grown in a liquid medium containing 2% glucose, 1% peptone and 0.5% yeast extract in artificial seawater adjusted to pH 7.5 for two weeks at 27~ The culture was filtered under suction and the mycelium collected was extracted three times with methanol. The combined extracts were evaporated under reduced pressure. The resulting extract was partitioned between methylene chloride and water and removal of solvents gave the ethylene chloride fraction. This fraction was passed through Sephadex LH20 using methanol as the eluant. The third fraction was chromatographed on a silica gel column with a methylene chloride-methanol gradient as the eluant and 3 fractions were collected. The methylene chloride eluant yielded fumiquinazoline C after purification by HPLC (methanol-water; 7:3, v/v). The methanol-methylene chloride (1:99, v/v) eluate was purified by HPLC (methanol-water; 7:3, v/v) to afford fumiquinazoline A and fumiquinazoline D. The methanol-methylene chloride (2:98, v/v) eluant gave fumiquinazolines G, F, B, and E after purification by I-IPLC (methanol-water; 7:3, v/v). Fungal Source A strain of Aspergillus fumigatus originally isolated from the marine fishPseudolabrus

japonicus.

Biological ,Activity Exhibited moderate cytotoxicity against cultured P388 cells.

120

8. Fumiquinazolines

Spectral Data UV:

~M~, 206(log 6 = 4.77), 225(4.69), 232(4.65), 256(4.38), 266(4.32), 277(4.24), 305(3.79), and 317rim (3.68). CD: (c=2.22 x 10.5 mol/dm-3 in ethanol) 227(Ae -11.04), 246(0), 252(+2.32), 259(0), 280(-7.36), 287(-5.31), 297(-4.36), 305(-4.77), 318(-3.13), and 329nm (0). IR:

3350(OH, NH), 1673(CON), and 1604cm1 (Ar-C-C). 1H M R : (CDCI3) 7.34(1H, dd, J=4.9, 0.9Hz, NH-2); 4.72(1H, qd, J=7.2, 4.9Hz, H-3), 7.56(1H, dd, J=8.0, 1.0Hz, n-7); 7.73(1H, ddd, J=8.0, 7.0, 1.0Hz, H-8); 7.45(1H, ddd, J=7.8, 7.0, 1.0Hz, H-9), 8.19(1H, dd, J=7.8, 1.0Hz, H-10), 5.79(1H, ddd, J=l 1.2, 4.8, 0.9Hz, H-14); 2.48(1H, dd, J=13.3, 4.8Hz, H-15A); 2.61(1H, dd, J=13.3, ll.2Hz, H-15B); 1.83(1H, d, J=7.2Hz, H-16); 5.42(1H, br s, H-18); 2.75(1H, br s, NH-19); 4.14(1H, q, J=6.7Hz, n-20); 7.51(1H, dd, J=7.5, 1.0Hz, H-24); 7.30(1H, td, J=7.5, 1.0Hz, H-25); 7.17(1H, td, J=7.5, 1.0Hz, H-26); 7.61(1H, dd, J=7.5, 1.0Hz, H-27); 1.29(3H, d, J=6.7Hz, H-29); and 5.47ppm (1H, s, OH). 13C NMR:

(CDC13) 170.69(C-1, q), 52.73(C-3, t), 150.72(C-4, q), 147.00(C-6, q), 126.88(C-7, t), 134.97(C-8, t), 127.27(C-9, t); 126.88(C-10, t), 120.01(C-11, q), 160.30(C-12, q); 52.01(C-14, t); 38.97(C-15, s), 24.88(C-16, p), 80.20(C-17, q), 80.43(C-18, t), 59.07(C-20, t), 170.56(C-21, q), 136.56(C-23, q), 114.86(C-24, t), 129.73(C-25, t), 125.50(C-26, t), 125.02(C-27, t), 138.62(C-28, q), and 18.14ppm (C-29, p). Mass Spectrum: EIMS: 445(M+, 5%), 229(10), 228(2), 217(100), 199(6), and 146m/e (15); HREIMS: 445.174 lm/e; C2aH23NsO4,requires 445.1750. Reference C. Takahashi, T. Matsushita, M. Doi, K. Minoura, T. Shingu, Y. Kumeda and A. Numata; Fumiquinazolines A-G, Novel Metabolites of a Fungus Separated from a Pseudolabrus Marine Fish, J. Chem. Soc. Perkin Trans. 1, pp. 2345-2353(1995).

8. Fumiquinazolines

121

Common/Systematic Name Fumiquinazoline C Molecular Formula/Molecular Weight C24H21NsO4; M W = 443.15935

0 General Characteristics Fumiquinazoline C was obtained as colorless prisms, 244-246~ in CHC13).

[a]D -193.7 ~ (C=0.31,

Isolation/Purification The fungal strain was grown in a liquid medium containing 2% glucose, 1% peptone and 0.5% yeast extract in artificial seawater adjusted to pH 7.5 for two weeks at 27~ The culture was filtered under suction and the mycelium collected was extracted three times with methanol. The combined extracts were evaporated under reduced pressure. The resulting extract was partitioned between methylene chloride and water and removal of solvents gave the ethylene chloride fraction. This fraction was passed through Sephadex LH20 using methanol as the eluant. The third fraction was chromatographed on a silica gel column with a methylene chloride-methanol gradient as the eluant and 3 fractions were collected. The methylene chloride eluant yielded fumiquinazoline C after purification by HPLC (methanol-water; 7:3, v/v). The methanol-methylene chloride (1:99, v/v) eluate was purified by HPLC (methanol-water; 7:3, v/v) to afford fumiquinazoline A and fumiquinazoline D. The methanol-methylene chloride (2:98, v/v) eluant gave fumiquinazolines G, F, B, and E after purification by HPLC (methanol-water; 7:3, v/v). Fungal Source A strain of Aspergillus fumigatus originally isolated from the marine fish Pseudolabrus

japonicus.

Biological Acti~ty Exhibited moderate cytotoxicity against cultured P388 cells.

122

8.

Fumiquinazolines

Spectral Data UV:

~M~, 207(1og e = 4.56), 225(4.48), 260(4.06), 271(4.02), 282(3.98), 304(3.61), and 317nm (3.50). CD: (c = 4.02 x 10-5 mol/dm3 in ethanol) 226 (Ae -15.84), 238(0), 243(+5.28), 247(+ 3.77), 253(+ 4.90), 269(0), 303(-13.58), 310(-10.18), 314(-10.56), and 328nm (0). IR;

3343, 3257(NH), 1715(CON), and 1611cm-1 (Ar-C-C). 1H NMR:

(CDCI3) 8.04(1H, br s, NH-2); 7.78(1H, dd, J=7.4, 1.7Hz, H-7); 7.81(1H, ddd, J-7.4, 6.3, 1.THz, H-8); 7.60(1H, ddd, J=7.4, 6.3, 1.THz, H-9); 8.35(1H, dd, J=7.4, 1.7Hz, H-10); 5.72(1H, dd, J=10.9, 6.0Hz, H-14); 2.14(1H, dd, J-13.7, 6.0Hz, H15A); 2.98(1H, dd, J=13.7, 10.9Hz, H=15B); 2.06(1H, s, H-16); 5.34(1H, d, J=6.9Hz, H=18), 1.04(1H, dd, J=6.9, 6.THz, NH-19); 3.71(1H, qd, J--6.9, 6.THz, H- 20); 7.45(1H, dd, J-7.4, 1.0Hz, H=24); 7.32(1H, td, ,/=7.4, 1.0Hz, H=25); 7.19(1H, td, J=7.4, 1.0Hz, H-26); 7.37(1H, dd, J-7.4, 1.0Hz, H-27); and 1.06ppm (3H, d, J=6.9Hz, H-29). 13C NMR.:

(CDCI3) 171.02(C-I, q), 84.16(C-3, q); 150.39(C=4, q), 146.32(C-6, q), 128.45(C-7, t), 134.91(C-8, t), 128.56(C-9, t), 126.9S(C-10, t), 121.34(C-11, q); 159.53(C-12, q), 51.39(C-14, t), 31.36(C-15, s), 24.43(C-16, p), 87.07(C-17, q), 87.07(C-18, t), 58.61(C-20, t), 170.90(C-21, q), 135.73(C-23, q), 115.46(C-24, t), 130.23(C-25, t), 126.17(C-26, t), 124.88(C-27, t), 138.41(C-28, q), and 18.71ppm (C-29, p). Mass Spectrum: HREIMS: 443.159 lm/e; C24H21NsO4,requires 443.1594. Reference C. Takahashi, T. Matsushita, M. Doi, K. Minoura, T. Shingu, Y. Kumeda and A. Numata; Fumiquinazolines A-G, Novel Metabolites of a Fungus Separated from a Pseudolabrus Marine Fish, J. Chem. Soc. Perkin Trans. 1, pp. 2345-2353(1995).

8. Fumiquinazolines

123

Common/Systematic Name Fumiquinazoline D Molecular Formula/Molecular Weight C24H21N504, M ~ = 443.1593 5

O General Characteristics Fumiquinazoline D was obtained as colorless prisms from acetone, mp., 214-216~ + 68.9 ~ (C=0.27, in CHC13).

[a]D

Isolation/Purification The fungal strain was grown in a liquid medium containing 2% glucose, 1% peptone and 0.5% yeast extract in artificial seawater adjusted to pH 7.5 for two weeks at 27~ The culture was filtered under suction and the mycelium collected was extracted three times with methanol. The combined extracts were evaporated under reduced pressure. The resulting extract was partitioned between methylene chloride and water and removal of solvents gave the ethylene chloride fraction. This fraction was passed through Sephadex LH20 using methanol as the eluant. The third fraction was chromatographed on a silica gel column with a methylene chloride-methanol gradient as the eluant and 3 fractions were collected. The methylene chloride eluant yielded fumiquinazoline C after purification by HPLC (methanol-water; 7:3, v/v). The methanol-methylene chloride (1:99, v/v) eluate was purified by HPLC (methanol-water; 7:3, v/v) to afford fumiquinazoline A and fumiquinazoline D. The methanol-methylene chloride (2:98, v/v) eluant gave fumiquinazolines G, F, B, and E after purification by HPLC (methanol-water; 7:3, v/v). Fungal Source A strain of Aspergillus fumigatus originally isolated from the marine fish Pseudolabrus

japonicus.


124

8. Fumiquinazolines

Spectral Data UV~

ZM~" 205(1og e = 4.30), 225(4.26), 232(4.24), 254(3.91), 265(3.84), 276(3.77), 304(3.43), and 316nm (3.54). CD: (c = 4.33 x 10.5 mol/dm3 in ethanol) 232(Ae -1.75), 235(0), 247(+6.12), 258(0), 268(- 4.90), 275(- 5.25), 294(- 2.45), 303(-2.80), 310(-2.10), 315(-2.10), and 326nm

(o). IR~

3418(OH, NH), 1703(CON), and 1611em1 (At-C-C). ~H NMR: (CDCI3) 9.16(1H, br s, NH-2); 7.66(1H, dd, J=8.3, 1.2Hz, H-7); 7.75(1H, ddd, J=8.3, 6.8, 1.2Hz, H-8); 7.50(1H, ddd, J=7.9, 6.8, 1.2Hz, H-9); 8.19(1H, dd, J=7.9, 1.2Hz, n-10), 5.65(1H, d, J=10.3Hz, n-14); 2.27(1H, d, J=15.1Hz, H-15A); 3.38(1H, dd, ,/=15.1, 10.3Hz, H-15B); 2.02(1H, s, n-16); 5.52(1H, d, J=l.3Hz, U-18); 3.96(1H, qd, J=6.5, 1.3Hz, n-20); 7.41(1H, dd, J=7.4, 1.0Hz, H-24); 7.23(1H, td, J=7.4, 1.0Hz, H-25); 7.05(1H, td, ,/=7.4, 1.0Hz, H-26); 7.44(1H, dd, J=7.4, 1.0Hz, H-27); 1.08(3H, d, J=6.5Hz, H-29); and 5.27ppm (1H, br s, OH). 13C NMR:

(CDCI3) 172.58(C-1, q), 70.84(C-3, q), 152.14(C-4, q), 146.34(C-6, q), 127.79(C-7, t); 134.83(C-8, t); 127.68(C-9, t), 126.85(C-10, t), 120.35(C-11, q), 160.86(C-12, q); 52.76(C-14, t); 43.44(C-15, s), 18.77(C-16, p); 84.13(C-17, q); 85.59(C-18, t), 59.11(C-20, t), 171.41(C-21, q), 137.60(C-23, q), 115.43(C-24, t), 130.07(C-25, t), 125.77(C-26, t), 124.31(C-27, t); 137.38(C-28, q); and 17.41ppm (C-29, p). Mass Spectrum: HR IMS: 443.1588re~e; C24I"I21N504,requires 443.1594. Reference C. Takahashi, T. Matsushita, M. Doi, K. Minoura, T. Shingu, Y. Kumeda and A. Numata; Fumiquinazolines A-G, Novel Metabolites of a Fungus Separated from a Pseudolabrus Marine Fish; J. Chem. Soc. Perkin Trans. 1, pp. 2345-2353(1995).

8. Fumiquinazolines

125

Common/Systematic Name Fumiquinazoline E Molecular Formula/Molecular Weight CzaH25NsOs; MW = 475.18557

o ,,

HN--V

O General Characteristics Fumiquinazoline E was obtained as a pale yellow powder, mp., 168-172~ (C=0.18, in CHC13).

[tt]D-143.3 ~

Isolation/Purification / The fungal strain was grown in a liquid medium containing 2% glucose, 1% peptone and 0.5% yeast extract in artificial seawater adjusted to pH 7.5 for two weeks at 27~ The culture was filtered under suction and the mycelium collected was extracted three times with methanol. The combined extracts were evaporated under reduced pressure. The resulting extract was partitioned between methylene chloride and water and removal of solvents gave the ethylene chloride fraction. This fraction was passed through Sephadex LH20 using methanol as the eluant. The third fraction was chromatographed on a silica gel column with a methylene chloride-methanol gradient as the eluant and 3 fractions were collected. The methylene chloride eluant yielded fumiquinazoline C after purification by HPLC (methanol-water; 7:3, v/v). The methanol-methylene chloride (1:99, v/v) eluate was purified by HPLC (methanol-water;7:3,v/v) to afford fumiquinazoline A and fumiquinazoline D. The methanol-methylene chloride (2:98, v/v) eluant gave fumiquinazolines G, F, B, and E after purification by HPLC (methanol-water; 7:3, v/v).

Fungal Source A strain of Aspergillus fumigatus originally isolated from the marine fish Pseudolabrus

japonicus.

126

8.

Fumiquinazolines

Biological Activity Exhibited moderate cytotoxicity against cultured P388 cells. Spectral Data UV:

ZM~" 210 (log e = 4.54), 226 (4.48), 233 (4.44), 256 (4.16), 278 (4.06), 304 (3.62), and 317nm (3.52). CD: (c = 4.33 x 10-5 mol/dm3 in ethanol) 219(Ae -2.69), 221(-2.34), 233(-7.73), 245(0), 251(+ 1.70), 259(0), 278(- 3.98), 290(- 2.87), 296(-2.34), 312(-2.58), 310(-2.05), 314(-1.99), and 329nm (0). IR:

3427(OH, NH), 1685(CON), and 1608cma (Ar-C-C). 1H NMR:

(CDC13) 7.57(1H, br d, NH-2); 7.74(1H, dd, J=7.7, 1.5Hz, H-7); 7.79(1H, ddd, J=8.2, 7.7, 1.5Hz, H-8); 7.53(1H, ddd, J=8.2, 6.8, 1.5Hz, H-9), 8.25(1H, dd, J=6.8, 1.5Hz, H-10); 5.93(1H, ddd, J=8.9, 5.2, 1.0Hz, H-14), 2.34(1H, dd, J=14.4, 5.2Hz, H-15A), 2.80(1H, dd, J=14.4, 8.9Hz, H-15B); 1.97(1H, s, H-16); 5.45(1H, s, H-18), 4.16(1H, q, J=6.6Hz, H-20), 7.56(1H, dd, J=7.2, 0.8Hz, H-24); 7.33(1H, ddd, J=8.0, 7.2, 1.0Hz, H-25); 7.16(1H, ddd, J=8.0, 7.2, 0.8Hz, H-26); 7.57(1H, dd, J=7.2, 1.0Hz, H-27); 1.33(3H, d, J=6.6I-Iz, H-29); 3.33(3H, s, OMe); and 4.55ppm (1H, s, OH-17). 13C NMR:

(CDC13) 172.70(C-1, q); 84.83(C-3, q), 148.17(C-4, q), 146.18(C-6, q), 127.96(C-7, t), 134.82(C-8, t); 127.96(C-9, t); 126.95(C-10, t); 120.60(C-11, q); 161.01(C-12, q); 53.38(C-14, t); 38.91(C-15, s); 20.88(C-16, p), 80.11(C-17, q), 86.29(C-18, t); 59.20(C-20, t); 171.28(C-21, q); 136.78(C-23, q); 115.09(C-24, t); 129.74(C-25, t), 125.16(C-26, t); 124.69(C-27, t); 138.61(C-28, q); 17.85(C-29 p); and 50.80ppm (COMe, p). Mass Spectrum: FAB: 476(MH +, 11%), 442(8), 228(18), 217(38), 199(34), 154(100), and 136m/e

(90). Reference C. Takahashi, T. Matsushita, M. Doi, K. Minoura, T. Shingu, Y. Kumeda and A. Numata; Fumiquinazolines A-G, Novel Metabolites of a Fungus Separated from a Pseudolabrus Marine Fish; J. Chem. Soc. Perkin Trans. 1, pp. 2345-2353(1995).

8. Fumiquinazolines

127

Common/Systematic Name Fumiquinazoline F Molecular Formula/Molecular Weight C21HlaN402, MW = 358.14298

N

General Characteristics Fumiquinazoline F was obtained as a pale yellow powder, mp., 88-90~ (c= 1.36, in CHCI3).

[a]D -411.2 ~

Isolation/Purification The fungal strain was grown in a liquid medium containing 2% glucose, 1% peptone and 0.5% yeast extract in artificial seawater adjusted to pH 7.5 for two weeks at 27~ The culture was filtered under suction and the mycelium collected was extracted three times with methanol. The combined extracts were evaporated under reduced pressure. The resulting extract was partitioned between methylene chloride and water and removal of solvents gave the ethylene ehioride fraction. This fraction was passed through Sephadex LH20 using methanol as the eluant. The third fraction was chromatographed on a silica gel column with a methylene chloride-methanol gradient as the eluant and 3 fractions were collected. The methylene chloride eluant yielded fumiquinazoline C after purification by HPLC (methanol-water; 7:3, v/v). The methanol-methylene chloride (1:99, v/v) eluate was purified by I-IPLC (methanol-water; 7:3, v/v) to afford fumiquinazoline A and fumiquinazoline D. The methanol-methylene chloride (2:98, v/v) eluant gave fumiquinazolines G, F, B, and E after purification by HPLC (methanol-water; 7:3, v/v). Fungal Source A strain of Aspergillus fumigatus originally isolated from the marine fish Pseudolabrus

japonicus.


128

8.

Fumiquinazolines

Spectral Data UV;

~,Mm ~H ~x 207(1og e = 4.71), 219(4.73), 270(4.13), 277(4.13), 289(3.99), 306(3.78), and 320nm (3.66). IR;

3264(NH), 1679(CON), and 1610cmq (At-C-C). IH NMR: (CDCI3) 6.60(1H, br s, NH-2); 3.14(1H, q, J=6.8, 0.3Hz, H-3); 7.60(1H, dd, J=7.8, 1.0Hz, H-7); 7.77(1H, td, ,/=7.8, 1.8Hz, H-8); 7.53(1H, td, ,/=7.8, 1.0Hz, H-9); 8.37(1H, dd, J=7.8, 1.8Hz, H-10); 5.68(1H, dd, J=5.2, 3.6Hz, H-14); 3.64(1H, dd, 3'=15.0, 5.2Hz, H-15A); 3.71(1H, dd, 3'=15.0, 3.6Hz, H-15B); 1.37(1H, d, J=6.8Hz, H16); 6.71(1H, d, J=2.5Hz, H-18); 8.26(1H, br s, NH-19); 7.30(1H, dd, 3'=8.0, 0.8Hz, H-21); 7.13(1H, td, J=8.0, 0.8Hz, H-22); 6.92(1H, td, 3'=8.0, 0.8Hz, H-23); and 7.40ppm (1H, dd, J=8.0, 0.8Hz, H-25). X3CNMR: (CDCI3) 169.33(C-1, q); 49.18(C-3, t); 151.68(C-4, q); 147.08(C-6, q); 127.30(C-7, t); 134.70(C-8, t); 127.12(C-9, t); 126.85(C-10, t); 120.24(C-11, q); 160.82(C-12, q); 57.53(C-14, t); 27.04(C-15, s); 19.08(C-16, p); 109.39(C-17, q); 123.55(C-18, t); 135.98(C-20, q); 111.22(C-21, t); 122.57(C-22, t); 120.01(C-23, t); 118.48(C-24, t); and 127.30ppm (C-25, q). Mass Spectrum: EIMS: 358(M +, 3%), 229(4), 228(1), and 130m/e (100); HRElMS: C21HlsN402, requires 358.1430.

358.1436re~e;

Reference C. Takahashi, T. Matsushita, M. Doi, K. Minoura, T. Shingu, Y. Kumeda and A. Numata, Fumiquinazolines A-G, Novel Metabolites of a Fungus Separated from a Pseudolabrus Marine Fish; J. Chem. Soc. Perkin Trans. 1, pp. 2345-2353(1995).

8. Fumiquinazolines

129

Common/Systematic Name Fumiquinazoline G Molecular Formula/Molecular Weight C21HIsN402; MW = 358.14298

N

General Characteristics Fumiquinazoline G was obtained as a pale yellow powder; mp., 119-121~ - 462.8 ~ (C=0.61, in CHCI3).

[a]D =

Isolation/Purification The fungal strain was grown in a liquid medium containing 2% glucose, 1% peptone and 0.5% yeast extract in artificial seawater adjusted to pH 7.5 for two weeks at 27~ The culture was filtered under suction and the mycelium collected was extracted three times with methanol. The combined extracts were evaporated under reduced pressure. The resulting extract was partitioned between methylene chloride and water and removal of solvents gave the ethylene chloride fraction. This fraction was passed through Sephadex LH20 using methanol as the eluant. The third fraction was ehromatographed on a silica gel column with a methylene chloride-methanol gradient as the eluant and 3 fractions were collected. The methylene chloride eluant yielded fumiquinazoline C after purification by HPLC (methanol-water; 7:3, v/v). The methanol-methylene chloride (1:99, v/v) eluate was purified by HPLC (methanol-water; 7:3, v/v) to afford fumiquinazoline A and fumiquinazoline D. The methanol-methylene chloride (2:98, v/v) eluant gave fumiquinazolines G, F, B, and E after purification by HPLC (methanol-water; 7:3, v/v). Fungal Source A strain of Aspergillus fumigatus originally isolated from the marine fish Pseudolabrus

japonicus.

Biological Activity Exhibited moderate cytotoxicity against cultured P388 cells. w

Ganomastenols

g

Ganomastenol A Ganomastenol B Ganomastenol C Ganomastenol D

131


9.

Ganomastenols

133

Common/Systematic Name Ganomastenol A

rel-3a,8,9a-Trihydroxycadina-4,10(15)-diene

Molecular Formula/Molecular Weight C15H2403; M~cV = 2 5 2 . 1 7 2 5 4 15

H

,

|,

~

R

-

13"i~14

i

General Characteristics Isolated as a white powder. Isolation/Purification The fungus was cultured on soytone medium. The mycelia were cultured statically at 25~ in the dark for 51 days. After 52 days culture, the mycelia were harvested with nylon cloth and homogenized with methanol and then allowed to stand for 10 days at room temperature. The culture broth, concentrated under reduced pressure and adjusted to pH 2 with 2 M HC1, was extracted with ethyl acetate. The ethyl acetate layer was dried over Na2SO4 and then evaporated to dryness to afford the crude extract. After 10 days the methanol-homogenized mycelium was filtered and the residue re-extracted with methanol and the organic solvent removed under reduced pressure. The residue was extracted with CHCI3 and the CHCI3 fraction was dried and evaporated to dryness. The combined CHC13 extracts of the mycelia and ethyl acetate extract of the culture broth were subjected to silica gel column (Wako-gel C-200) eluted with an ethyl acetate-n-hexane solvent system and separated into 6 fractions. Fraction 1 eluted with n-hexane and ethyl acetate-n-hexane (1:4, v/v); fraction 2 eluted with ethyl acetate-n-hexane (1:4, v/v; 200ml); fraction 3 with ethyl acetate-n-hexane (1:1, v/v; 500ml); fraction 4 with ethyl acetate-n-hexane (1:1, v/v, 100ml); fraction 5 with ethyl acetate-n-hexane (1:1, v/v, 940ml); and fraction 6 with ethyl acetate-n-hexane (1:1, v/v; 200ml), ethyl acetate-n-hexane, (3:2, v/v; 800ml), ethyl acetate (300ml), and methanol (500ml). Repeated purification of fraction 3 by HPLC eluted with 40% acetonitrile in 2% acetic acid yielded purified ganomastenols A, B, C, and D. Fungal Source

Ganoderma mastoporum.

Spectral Data IR:

(KBr) 3425ernq (OH).

134

9.

Ganomastenols

1HN:VIR: (CDC13) 1.89(1H, ddd, J=12.0, 11.0, 2.5Hz, H-I), 1.48(1H, ddd, J=12.0, 12.0, 10.0Hz, H-2~); 2.24(1H, ddd, J=12.0, 6.0, 2.5Hz, H-2a); 4.16(1H, dd, J=10.0, 6.0Hz, H-313); 5.61(1H, dd, J=l.0, 1.0Hz, H-5); 1.62(1H, ddd, J=l 1.0, 11.0, 1.0Hz, H-6); 1.24(1H, ddd, J=l 1.0, 11.0, 1.0Hz, H-713); 3.12(1H, dd, J=l 1.0, 10.0Hz, H-8a); 3.80(1H, ddd, J=10.0, 1.5, 1.5Hz, H-913); 1.76(1H, br s, H-11); 2.27(1H, qqd, J=7.0, 7.0, 1.0Hz, H-12); 1.00(3H, d, J=7.0Hz); 1.09(3H, d, J=7.0Hz, H-14); 4.76 91H, dd, J=3.0, 1.SHz, H-15a); and 5.09ppm (1I-I, dd, J=3.0, 1.SHz, H-15b). 13CNMR: (CDCI3) 43.1, C-l; 37.2, C-2; 71.6, C-3; 139.4, C-4; 127.3, C-5; 44.9, C-6, 52.7, C7; 79.0, C-8; 79.1, C-9; 152.2, C-10; 20.1, C-11; 28.0, C-12; 19.9, C-13; 22.1, C-14 and 103.2ppm, C- 15. Mass Spectrum: FAB-MS: 275m/e (M + Na)+; HREIMS: 234.1625m/e (M - H20) +, C15H2403,requires 252.1725; EIMS: 234(97%), 191(49), and 173m/e (100). Reference M. Hirotani, C. Ino, A. Hatano, H. Takayanagi, and T. Furuya; Ganomastenols A, B, C, and D, Cadinene sesquiterpenes from Ganoderma mastoporum; Phytochemistry, Vol. 40, pp. 161-165(1995).

9.

Ganomastenols

135

Common/Systematic Name Ganomastenol B rel- 313,8[3,9a-Trihydroxycadina-4,10( 15)-diene Molecular Formula/Molecular Weight C15H2403; MW" = 252.17254

HO

.,

~,,-

: v FI _

H

0

~DH

General Characteristics Isolated as needles; mp., 139-140~ Isolation/Purification The fungus was cultured on soytone medium. The mycelia were cultured statically at 25~ in the dark for 51 days. After 52 days culture, the mycelia were harvested with nylon cloth and homogenized with methanol and then allowed to stand for 10 days at room temperature. The culture broth, concentrated under reduced pressure and adjusted to pH 2 with 2M HC1, was extracted with ethyl acetate. The ethyl acetate layer was dried over Na2SO4 and then evaporated to dryness to afford the crude extract. After 10 days the methanol-homogenized mycelium was filtered and the residue re-extracted with methanol and the organic solvent removed under reduced pressure. The residue was extracted with CHC13 and the CHC13 fraction was dried and evaporated to dryness. The combined CHCI3 extracts of the mycelia and ethyl acetate extract of the culture broth were subjected to silica gel column (Wako-gel C-200) eluted with an ethyl acetate-n-hexane solvent system and separated into 6 fractions. Fraction 1 eluted with n-hexane and ethyl acetate-n-hexane (1:4, v/v); fraction 2 eluted with ethyl acetate-n-hexane (1:4, v/v; 200ml); fraction 3 with ethyl acetate-n-hexane (1:1, v/v; 500ml); fraction 4 with ethyl acetate-n-hexane (1:1, v/v; 100ml); fraction 5 with ethyl acetate-n-hexane (1:1, v/v; 940ml); and fraction 6 with ethyl acetate-n-hexane (1:1, v/v; 200ml), ethyl acetate-n-hexane (3:2, v/v; 800ml), ethyl acetate (300ml), and methanol (500ml). Repeated purification of fraction 3 by HPLC eluted with 40% acetonitrile in 2% acetic acid yielded purified ganomastenols A, B, C, and D. Fungal Source Ganoderma mastoporum. Spectral Data IR:

(KBr) 3320(OH) and 2880cm ~.

136

9.

Ganomastenols

1H NMR:

(CDC13) 2.05(1H, ddd, J-11.0, 11.0, 2.0Hz, H-I); 1.63(1H, ddd, J-13.0, 11.0, 4.0Hz, H-213);2.00(1H, ddd, ./=13.0, 2.0, 2.0Hz, H-2a); 3.99(1H, dd, ./- 4.0, 2.0Hz, H-3a); 5.72(1H, dd, ./--1.0, 1.0I-Iz, H-3[~); 1.48(1H, ddd, ./=11.0, 11.0, 1.0I-lz, H-6); 1.3 I(1H, ddd, ./--11.0, 11.0, 1.0Hz, H-713); 3.14(1H, dd, J--11.0, 10.0Hz, H-8a); 3.85(1H, ddd,./-- 10.0, 1.5, 1.5Hz, H-9[~); 1.81(1H, br s, H-11); 2.31(1H, qqd, J=7.0, 7.0, 1.0Hz, H-12); 1.02(3H, d, `/-7.0Hz, H-13); 1.09(3H, d, J-7.0Hz, H- 14); 4.73 (1H, rid,`/=3.0, 1.5Hz, H-15a); and 5.08ppm (1H, rid,`/-3.0, 1.5Hz, H-15b). 13CNMR: (CDC13) 37.7, C-l; 36.2, C-2; 68.7, C-3; 137.1, C-4; 128.6, C-5; 44.9, C-6; 55.2, C7; 79.2, C-8; 79.2, C-9; 152.8, C-10; 21.8, C-11; 27.7, C-12; 19.9, C-13; 22.0, C-14 and 103.1ppm C-15. Mass Spectrum: FAB-MS: 275m/e (M + Na)+; HREIMS: 234.1614m/e (M - H20) +, C15H2403requires 252.1725; EIMS: 234(10%), 216(25), 173(70), and 145m/e (100). Reference M. Hirotani, C. Ino, A. Hatano, H. Takayanagi, and T. Furuya; Ganomastenols A, B, C, and D, Cadinene sesquiterpenes from Ganoderma mastoporum; Phytochemistry, Vol. 40, pp. 161-165(1995).

9.

Ganomastenols

137

Common/Systematic Name Ganomastenol C

rel-313,813,9tt-Trihydroxycadina-10(15)-ene

Molecular Formul .a/Molecular Weight C15H2603; M W -- 2 5 4 . 1 8 8 1 9

HO

H v - v R :

"OH

General Characteristics Isolated as needles; mp., 155.5-157~ Isolation/Purification The fungus was cultured on soytone medium. The mycelia were cultured statically at 25~ in the dark for 51 days. After 52 days culture, the mycelia were harvested with nylon cloth and homogenized with methanol and then allowed to stand for 10 days at room temperature. The culture broth, concentrated under reduced pressure and adjusted to pH 2 with 2 M HC1, was extracted with ethyl acetate. The ethyl acetate layer was dried over Na/SO4 and then evaporated to dryness to afford the crude extract. After 10 days the methanol-homogenized mycelium was filtered and the residue re-extracted with methanol and the organic solvent removed under reduced pressure. The residue was extracted with CHC13 and the CHC13 fraction was dried and evaporated to dryness. The combined CHC13 extracts of the mycelia and ethyl acetate extract of the culture broth were subjected to silica gel column (Wako-gel C-200) eluted with an ethyl acetate-n-hexane solvent system and separated into 6 fractions. Fraction 1 eluted with n-hexane and ethyl acetate-n-hexane (1:4, v/v); fraction 2 eluted with ethyl acetate-n-hexane (1:4, v/v; 200ml); fraction 3 with ethyl acetate-n-hexane (1:1, v/v; 500ml); fraction 4 with ethyl acetate-n-hexane (1:1, v/v; 100ml); fraction 5 with ethyl acetate-n-hexane (1:1, v/v; 940ml); and fraction 6 with ethyl acetate-n-hexane (1:1, v/v; 200ml), ethyl acetate-n-hexane (3:2, v/v; 800ml), ethyl acetate (300ml), and methanol (500ml). Repeated purification of fraction 3 by HPLC eluted with 40% acetonitrile in 2% acetic acid yielded purified ganomastenols A, B, C, and D. Fungal Source


Spectral Data IR:

(KBr) 3360(OH) and 2890cm1

138

9.

Ganomastenols

1HNIV[R:

(CDCI3) 2.03(1H, ddd, J=13.0, 11.0, 2.0Hz, H-l); 1.54(1H, ddd, J=13.0, 11.0, 2.5Hz, H-213); 1.95(1H, ddd, J=13.0, 4.0, 2.0Hz, H-2a); 3.85(1H, dd, J= 4.0, 2.5Hz, n-3a); 1.47(1H, m, H-4); 1.13(1H, ddd, J=13.0, 12.0, 12Hz, H-5); 1.67(1H, ddd, J=13.0, 3.5, 3.0Hz, H-5); 0.93(1H, dddd, J=13.0, 12.0, 11.0, 3.0Hz, H-6); 1.26(1H, ddd, J=12.0, 11.0, 1.5Hz, H-713); 3.07(1H, dd, J-11.0, 9.0Hz, H-Sa); 3.79(1H, ddd, J= 9.0, 1.5, 1.5Hz, H-913); 0.96(1H, d, Jr--7.0Hz, H-11); 2.24(1H, qqd, J-7.0, 7.0, 1.0Hz, H-12); 0.98(3H, d, J=7.0Hz); 1.04(3H, d, J=7.0Hz, H-14); 4.70(1H, dd, J=2.5, 1.5Hz, H-15a); and 5.06ppm (1H, dd, J=2.5, 1.5Hz, H-15b). 13CNMR: (CDC13) 38.7, C-l; 37.8, C-2; 71.1, C-3; 37.6, C-4; 35.4, C-5; 45.2, C-6; 53.8, C-7; 78.9, C-8; 79.2, C-9; 153.3, C-10; 19.3, C-11; 27.7, C-12; 20.3, C-13; 21.6, C-14 and 103.0ppm C- 15. Mass Spectrum: FAB-MS: 277m/e (M + Na)+; HREIMS: 236.1778m/e (M- H20)§ C~sH2603requires 254.1881; EIMS: 236(38%), 218(40), 175(100), and 91m/e (28). Reference M. Hirotani, C. Ino, A. qatano, H. Takayanagi, and T. Furuya; Ganomastenols A, B, C, and D, Cadinene sesquiterpenes from Ganoderma mastoporum; Phytochemistry, Vol. 40, pp. 161-165(1995).

9.

Ganomastenols

139

Common/Systematic Name Ganomastenol D

rel-8[~,9a-Dihydroxy-4-hydroxymethylcadina-4,10(15)-diene

Molecular Formula/Molecular Weight C15H2403; M W = 2 5 2 . 1 7 2 5 4

.,

H

HOH2C" "~" ~ ~..~ ~)H

General Characteristics Isolated as a white powder; mp., 155.5-157~ Isolation/Purification The fungus was cultured on soytone medium. The mycelia were cultured statically at 25~ in the dark for 51 days. A_fter 52 days culture, the mycelia were harvested with nylon cloth and homogenized with methanol and then allowed to stand for 10 days at room temperature. The culture broth, concentrated under reduced pressure and adjusted to pH 2 with 2 M HC1, was extracted with ethyl acetate. The ethyl acetate layer was dried over Na2SO4 and then evaporated to dryness to afford the crude extract. After 10 days the methanol-homogenized mycelium was filtered and the residue re-extracted with methanol and the organic solvent removed under reduced pressure. The residue was extracted with CHCI3 and the CHC13 fraction was dried and evaporated to dryness. The combined CHC13 extracts of the mycelia and ethyl acetate extract of the culture broth were subjected to silica gel column (Wako-gel C-200) eluted with an ethyl acetate-n-hexane solvent system and separated into 6 fractions. Fraction 1 eluted with n-hexane and ethyl acetate-n-hexane (1:4, v/v); fraction 2 eluted with ethyl acetate-n-hexane (1:4, v/v; 200ml); fraction 3 with ethyl acetate-n-hexane, 1: lv/v (500ml); fraction 4 with ethyl acetate-n-hexane (1:1, v/v; 100ml); fraction 5 with ethyl acetate-n-hexane (1:1, v/v; 940ml); and fraction 6 with ethyl acetate-n- hexane (1:1, v/v; 200ml), ethyl acetate-n-hexane (3:2, v/v; 800ml), ethyl acetate (300ml), and methanol (500ml). Repeated purification of fraction 3 by HPLC eluted with 40% acetonitrile in 2% acetic acid yielded purified ganomastenols A, B, C, and D. Fungal Source


.Spectral Data IR:

(KBr) 3360(OH) and 2890cm ~.

140

9.

Ganomastenols

1H NMR: (CDC13) 2.03(1H, ddd, J=13.0, 11.0, 2.0Hz, H-I); 1.54(1H, ddd, J=13.0, 11.0, 2.5Hz, H-213); 1.95(1H, ddd, J=13.0, 4.0, 2.0Hz, H-2a); 3.85(1H, dd, J = 4.0, 2.5Hz, H-3a); 1.47(1H, m, H-4); 1.13(1H, ddd, J=13.0, 12.0, 12Hz, H-5); 1.67(1H, ddd, J=13.0, 3.5, 3.0Hz, H-5); 0.93(1H, dddd, J=13.0, 12.0, 11.0, 3.0Hz, H-6); 1.26(1H, ddd, J=12.0, 11.0, 1.5Hz, H-713); 3.07(1H, dd, J=l 1.0, 9.0Hz, H-8a); 3.79(1H, ddd, J=9.0, 1.5, 1.5Hz, H-913); 0.96(1H, d, J=7.0Hz, H-11); 2.24(1H, qqd, J=7.0, 7.0, 1.0Hz, H-12); 0.98(3H, d, J=7.0Hz); 1.04(3H, d, J=7.0Hz, H-14); 4.70(1H, dd, J=2.5, 1.5Hz, H-15a); and 5.06ppm (1H, dd, J=2.5, 1.5Hz, H-15b). 13CNMR: (CDC13) 38.7, C-l; 37.8, C-2; 71.1, C-3; 37.6, C-4; 35.4, C-5; 45.2, C-6; 53.8, C- 7; 78.9, C-8; 79.2, C-9; 153.3, C-10; 19.3, C-11; 27.7, C-12; 20.3, C-13; 21.6, C-14 and 103.0ppm, C- 15. Mass Spectrum: FAB-MS: 277m/e (M + Na)§ HREIMS: 236.1778m/e (M- H20) § C15H2403requires 252.1725; ELMS: 236(38%), 218(40), 175(100), and 91m/e (28). Reference M. Hirotani, C. Ino, A. Hatano, H. Takayanagi, and T. Furuya; Ganomastenols A, B, C, and D, Cadinene sesquiterpenes from Ganoderma mastoporum; Phytochemistry, Vol. 40, pp. 161-165(1995).

Memnobotrins Memnobotrin A Memnobotrin B Memnoconone Memnoconol

141


10.

Memnobotrins

143

Common/Systematic Name Memnobotrin A Molecular Formula/Molecular Weight C25H33NOs; MW = 427.23587 O NH

~~

""II I I 12

3

AcO

\n 14

13

General Characteristics Crystalline solid; m.p., 240-250~ (dec.);

[ a ] D 20 --

+ 9.6 ~ (C=I. 14, in MeOH).

Fungal Source Memnoniella echinata (JS6308). Isolation/Purification A culture ofM. echinata grown for 5 weeks on rice was air dried, ground, and extracted with methanol-chloroform, followed by partition between water and chloroform. Chromatography of the chloroform-soluble fraction over polyethyleneimine (methylene chloride with increasing methanol) gave 7 fractions. Fraction 4 was applied to a 2mm Chromatotron plate which was eluted with increasing proportions of methanol in methylene chloride to give memnobotrin A (22.8mg) and memnobotrin B (25.0mg). An earlier fraction from the plate required one further passage through a 2mm Chromatotron plate (gradient elution with 25-50% ethyl acetate-hexane) to yield pure memnoconone (3.7mg). Fraction 5 was dissolved in the minimum methylene chloride-methanol (1:1, v/v), diluted with a small volume of hexane, and the emulsion applied to a silica column. Elution with ethyl acetate-hexane gave a total of 90.2mg of memnoconol. Spectral Data UV:

~.~"

217.0 (e= 26,200), 257.5 (5,600), and 300nm (2,300).

IR:

(CHC13) 3596, 3455, 3316,2931,2854, 1716, 1692, 1612, 1458, 1368, 1136, and 1071cm 1

144

10.

Memnobotrins

1H NNtIR:

[(CD3)3CO)] 0.71(3H, s, H-15); 0.84(3FL s, n-13); 0.89(3H, s, n-14); 1.13(1H, m, H-5), 1.19(3H, s, H-12); 1.20(1H, m, H-1B); 1.55(1H, m, H-6B); 157 (1n, m, H-9), 1.61(2H, m, H-2); 1.70(2H, m, H-6A, H-7B); 1.89(1H, dt, J=3.4, 13.2Hz, H-1A), 1.98(3H, s, Ac), 2.16(1H, br, d, J= 9.60Hz, H-7A), 2.71(1H, ABX, J=8.2, 18.8Hz, H-11B); 2.87(1H, AB, J=18.SHz, H-1A); 4.21(2H, br AB, d=16.9Hz, H-8'); 4.46(1H, br dd, J=6.3, 9.3Hz, H-3); and 6.80ppm (1H, s, H-3'). 13C NMR:

[(CD3)3CO] 38.4, C-1, 24.1, C-2; 80.9, C-3; 38.3, C-4; 54.8, C-5; 18.5, C-6, 40.8, C-7, 76.3, C-8, 49.0, C-9; 38.3, C-10; 18.9, C-11, 27.2, C-12; 17.2, C-13, 28.7, C-14; 14.6, C-15; 114.7, C-I', 156.3, C-2', 100.6, C-3', 132.3, C-4'; 123.6, C-5'; 151.3, C-6'; 171.8, C-7'; 43.0 C-8'; 21.0, Ac-CH3; and 170.8ppm Ac-CO. Reference S. F. Hinkley, J. C. Fettinger, K. Dudley, and B. B. Jarvis; Memnobotrins and Memnoconols: Novel Metabolites from Memnoniella echinata; J. Antibiotics, Vol. 52, pp. 988-997 (1999).

10.

Memnobotrins

145

Comm0n/Systematic Name Memnobotrin B Molecular Formula/Molecular Weight C27H37NO6; M W -- 4 7 1 . 2 6 2 0 9

O NCH2CH2OH 15

Aco

\ 14

13

General Characteristics Cream colored powder; m.p., 186-192~

[0~]D20"- + 1 1 . 7

~ (c=1.25, in MeOH).

Fungal Source Memnoniella echinata (JS6308).

Isolation/Purification A culture ofM. echinata grown for 5 weeks on rice was air dried, ground, and extracted with methanol-chloroform, followed by partition between water and chloroform. Chromatography of the chloroform-soluble fraction over polyethyleneimine (methylene chloride with increasing methanol) gave 7 fractions. Fraction 4 was applied to a 2mm Chromatotron plate which was eluted with increasing proportions of methanol in methylene chloride to give memnobotrin A (22.8mg) and memnobotrin B (25.0mg). An earlier fraction from the plate required one further passage through a 2mm Chromatotron plate (gradient elution with 25-50% ethyl acetate-hexane) to yield pure memnoconone (3.7mg). Fraction 5 was dissolved in the minimum methylene chloride-methanol (1:1, v/v), diluted with a small volume of hexane, and the emulsion applied to a silica column. Elution with ethyl acetate-hexane gave a total of 90.2mg of memnoconol. Biological Activity Cytotoxic. Spectral Data UV:

MeOH max

218.5 (c = 37,200), 259.0 (10,600), and 302nm (3,300).

146

10.

Memnobotrins

IR:

(CHC13) 3598, 3323, 2933,2855, 1717, 1669, 1616, 1458, 1368, 1317, 1135, and 1071cm1. ~HNMR: [(CD3)3CO] 0.69(3H, s, H-15); 0.82(3H, s, H-13); 0.87(3H, s, H-14); 1.13(1H, br d, J=9.8Hz, H-5); 1.17(3H, s, H-12); 1.20(1H, m, H-1B); 1.57(1H, m, H-9); 1.61(2H, m, H-2); 1.63(2H, m, H-6); 1.70(1H, m, 7B); 1.90(1H, dt, J=3.4, 13.3Hz, H-1A); 1.96(3H, s, Ac); 2.16(1H, br, dd, J=2.0, 10.4Hz, H-TA); 2.71(1H, ABX, J=8.1, 18.9Hz, H-11B); 2.87(1H, AB, J=18.9Hz, H-1A); 3.65(2H, t, J=5.4Hz, H-9'); 3.77(2H, t, J=5.4Hz, H-10'); 4.36(2H, br AB, J=l 7.0Hz, H-8'); 4.46(1H, br dd, J=6.7, 9.8Hz, H-3); and 6.81ppm (1H, s, H-3'). 13CNMR: [(CD3)3CO] 38.4, C-l; 24.1, C-2; 80.9, C-3; 38.3, C-4; 54.7, C-5; 18.5, C-6; 40.8, C-7; 76.4, C-8; 49.0, C-9; 38.7, C-10; 18.8, C-11; 27.2, C-12; 17.1, C-13; 28.6, C-14; 14.6, C-15; 114.4, C-I'; 156.2, C-2'; 100.6, C-3'; 132.5, C-4'; 121.6, C-5'; 151.0, C-6'; 169.6, C-7', 49.2 C-8'; 46.0, C-9', 61.2, C-10'; 21.0, Ac-CH3; and 170.8ppm At-CO. Reference S. F. Hinkley, J. C. Fettinger, K. Dudley, and B. B. Jarvis; Memnobotrins and Memnoconols: Novel Metabolites from Memnoniella echinata.; J. Antibiotics, Vol. 52, pp. 988-997 (1999).

10.

Memnobotrins

147

Common/Systematic Name Memnoconone Molecular Formula/Molecular Weight C23H3005; M W -- 386.20932

Ii~ "I

13'

0

7

, I

-

HO.o,i,~~ x,~O

14'

I

15'

O

oH

General Characteristics Light oil. Fungal Source Memnoniella echinata (JS6308).

Isolation/Purification A culture ofM. echinata grown for 5 weeks on rice was air dried, ground, and extracted with methanol-chloroform, followed by partition between water and chloroform. Chromatography of the chloroform-soluble fraction over polyethyleneimine (methylene chloride with increasing methanol) gave 7 fractions. Fraction 4 was applied to a 2mm Chromatotron plate which was eluted with increasing proportions of methanol in methylene chloride to give memnobotrin A (22.8mg) and memnobotrin B (25.0mg). An earlier fraction from the plate required one further passage through a 2mm Chromatotron plate (gradient elution with 25-50% ethyl acetate-hexane) to yield pure memnoconone (3.7mg). Fraction 5 was dissolved in the minimum methylene chloride-methanol (1:1, v/v), diluted with a small volume of hexane, and the emulsion applied to a silica column. Elution with ethyl acetate-hexane gave a total of 90.2mg of memnoconol. Biological Activity Cytotoxic. Spectral Data UV: , ~ MeOH max

219.0(c = 38,000), 261.0(14,600), and 289nm (2,900).

IR:

(CHCI3) 3587, 3435, 3934, 2972, 1761, 1639, 1612, 1462, 1353, 1384, 1355, 1316, and 1124cm"1.

148

10.

Memnobotrins

1H NMR:

[(CD3)3CO] 1.1 l(6H, s, H-12', H-13'); 1.26(1H, dddd, J=4.4, 10.0, 10.1, and 13.6Hz, H-9'A); 1.55(3H, s, H-15'); 1.58(1H, dddd, J=l.6, 6.8, 9.9, and 13.6Hz, H-9'B); 1.76(3H, s, H-14'); 1.95(2H, br t, J=7.SHz, H-4'); 2.02(2H, m, H-5'); 2.10(2H, br t, J=7.5Hz, H-8'A); 2.49 (2H, m, H-9'); 2.60(1H, hept, J=7.0Hz, H-11'); 3.36 (2H, br d, J= 7.1Hz, H-I'); 5.07(1H, dt, J=l.1, 6.SHz, H-6'); 5.22(2H, s, H-3); 5.25(1H, dt, J=l. 1, 6.8Hz, H-2'); and 6.62ppm (1H, s, H-7). 13CNMR:

[(CD3)3CO] 173.0, C-l; 70.7, C-3; 103.7, 3a; 163.8, C-4; 114.2, C-5; 155.7, C-6; 101.5, C-7; 147.5, C-Ta; 22.1, C-I'; 123.0, C-2'; 135.4, C-3'; 40.3, C-4'; 27.1, C-5'; 125.0, C-6'; 134.9, C-7'; 34.1, C-8'; 39.5, C-9'; 213.7, C-10'; 41.0, C-11'; 18.4, C-12'; 18.4, C-13'; 16.2, C-14'; and 16.1ppm, C-15'. Reference S. F. Hinkley, J. C. Fettinger, K. Dudley, and B. B. Jarvis; Memnobotrins and Memnoconols: Novel Metabolites from Memnoniella echinata.; J. Antibiotics, Vol. 52, pp. 988-997 (1999).

10.

Memnobotrins

149

Common/Systematic Name Memnoconol Molecular Formula/Molecular Weight C23H3206; M W = 404.21989

O

OH OHI_~H 13'

-

I

14'

,

HO~6.,~ "1/ . "]~ \0

I

lo.

1~'

General Characteristics Light brown powder; m.p., 60-63~

[tZ]D2~= -14.0 ~ (C=1.08, in MeOH).

Fungal Source Memnoniella echinata (JS6308). Isolation/Purification A culture ofM. echinata grown for 5 weeks on rice was air dried, ground, and extracted with methanol-chloroform, followed by partition between water and chloroform. Chromatography of the chloroform-soluble fraction over polyethyleneimine (methylene chloride with increasing methanol) gave 7 fractions. Fraction 4 was applied to a 2mm Chromatotron plate which was eluted with increasing proportions of methanol in methylene chloride to give memnobotrin A (22.8mg) and memnobotrin B (25.0mg). An earlier fraction from the plate required one further passage through a 2mm Chromatotron plate (gradient elution with 25-50% ethyl acetate-hexane) to yield pure memnoconone (3.7mg). Fraction 5 was dissolved in the minimum methylene chloride-methanol (1:1, v/v), diluted with a small volume of hexane, and the emulsion applied to a silica column. Elution with ethyl acetate-hexane gave a total of 90.2mg of memnoconol. Biological Activity Cytotoxic. Spectral Data UV:

)tmM~" 219.0(e = 28,700), 261.5(11,100), and 290nm (3,000). IR: (CHCI3) 3587, 3440, 3005,2978,2932, 1728, 1637, 1612, 1451, 1353, 1316, 1196, and 1161 cm1.

150

10.

Memnobotrins

1H~ : [(CD3)3CO] 1.1 l(6H, s, H-12', H-13'); 1.26(1H, dddd, J=4.4, 10.0, 10.1, and 13.6Hz, H-9'B); 1.55(3H, s, H-15'); 1.58(1H, dddd, J--1.6, 6.8, 9.9, and 13.6Hz, H-9'A); 1.76(3H, s, H-14'); 1.87(1H, m, H-8'B); 1.91(2H, br t, J=7.4Hz, H-4'); 2.02(2H, m, H-5'); 2.15(1H, br ddd, ,/=4.4, 9.9, and 13.7Hz, H-8'A); 3.18(1H, dd, J=l.6, 10.1Hz, H-10'); 3.30(2H, d, J=6.9Hz, H-I'); 5.07(1H, br t, J=6.SHz, H-6'); 5.15(2H, s, H-3); 5.20(1H, br t, J=6.9Hz, H-2'); and 6.52ppm (1H, s, H-7). 13C NMR: [(CD3)3CO] 172.9, C-l, 70.6, C-3; 103.6, 3a; 163.8, C-4; 115.6, C-5; 155.7, C-6; 101.5, C-7; 147.4, C-7a; 22.1, C-I'; 123.0, C-2'; 135.4, C-3'; 40.3, C-4'; 27.0, C-5'; 124.6, C-6'; 135.7, C-7'; 37.4, C-8'; 30.6, C-9'; 78.4, C-10'; 72.9, C-11'; 25.7, C-12'; 24.9, C-13'; 16.2, C-14'; and 16.1ppm, C-15'. Reference S. F. Hinkley, J. C. Fettinger, K. Dudley, and B. B. Jarvis, Memnobotrins and Memnoconols: Novel Metabolites from Memnoniella echinata.; J. Antibiotics, Vol. 52, pp. 988-997(1999).

Tsugicolines Tsugicoline A Tsugicoline B Tsugicoline C Tsugicoline D

151


11. Tsugicolines

153

Common/Systematic Name Tsugicoline A Molecular Formula/Molecular Weight C 15H2204; M W -- 266.15181

H,.

OH

H

H O H 2 C ~

15

O '''~ %58

",14

H OH Fungal Source

Laurilia tsugicola = Echinodontium tsugicola (CBS 248.51, Centraal Bureau voor

Schimmel Cultures, Baarn). General Characteristics White crystals from methylene chloride-hexane; mp., 168-170~ [a]o - 156 ~ (c=0.15, in MeOH) ; triacetate, an oil; [a]D - 43.2 ~ (C=0.1, in CHC13); acetonide, white crystals from chloroform; mp., 146-148~ Isolation/Purification The culture filtrate which was separated from the mycelium was extracted twice with ethyl acetate. Then the extracts were dried (Na2SO4) and evaporated to yield a mixture of sesquiterpenes. The mixture was treated with ethyl ether; the residue was composed of pure tsugicoline A; the mother liquid was chromatographed by flash silica gel column chromatography eluted with hexane-ethyl acetate (2:1, v/v) to give a mixture of tsugicoline B and D that was further purified by preparative TLC using hexane-ethyl acetate (1:2, v/v) and methylene chloride-methanol (15:1, v/v) to yield pure tsugicoline B and D; Re, 0.10, 0.30 for tsugicoline A, respectively. Spectral Data

UV; ;~

260nm (c=8,500).

IR; (KBr) 3370(OH) and 1730crn1 (earbonyl).

154

11. Tsugicolines

1H NMR: (acetone-d6) 4.46(1H, J~,lb=16Hz, J~a=l.4Hz, H-la); 4.42(1H, Jlb,a=l.5Hz, H-lb); 4.23(1H, Ja,la=8.7Hz, H-a); 4.36(1H, n-6); 1.00(3H, n-8); 2.54(1H, Jg,lO~=10.4Hz, Ja,lo~=7.9Hz, J9,~3=11.5Hz, H-9); 1.5 I(1H, Jwa,~ol3=12.4Hz,Jw~,ls=0.8Hz, H-10a); 1.55(1H, Jlo13,1213=l.8Hz,H-10I]); 1.30(1H, JiE~A2fl=12.2Hz,J1Ea,]a=10.4Hz, J12~,ls=0.8Hz, H-12a); 1.86(1H, J121~,13=7.3Hz,H-1213); 2.32(1H, H-13); 1.13(3H, H14); 1.01(3H, n-15); 3.82(1H, OH-I); 4.57(1H, OH-3); and 5.16ppm (1H, OH-6); (CDC13) 4.57(1H, H-la); 4.57(1H, n-lb); 4.24(1H, n-3); 4.39(1H, H-6); 1.02(3H, n-8); 2.51(1H, n-9); 1.48(1H, U-10a); 1.62(1H, n-1013); 1.23(1H, H-lEa); 1.88(1H, H-1213); 2.33(1H, n-13); 1.15(3H, U-14); 1.00(3H, H-15); 1.55"(1H, OH-l); 2.15"(1H, OH-3); and 2.95ppm* (1H, OH-6). * Assignments may be interchanged. 13CNMR: 61.45, t, C-l; 153.02, s, C-2; 74.93, d, C-3; 143.98, s, C-4; 200.15, s, C-5; 90.33, d, C-6; 42.70, s, C-7; 14.66, q, C-8; 47.02, d, C-9; 42.50, t, C-10; 40.89, s, C-11; 47.51, t, C-12; 50.90, d, C-13; 29.79, q, C-14; and 27.23ppm, q, C-15. Mass Data: LREIMS: 266role (M+); CIMS: (isobutane) 267(MH+), 249(MH+ - H20, base peak), 23 I(MH+ - 2H20), and 203role (231 - 28), found: C, 67.5, H, 8.3. C15H2204requires C, 67.64; H, 8.33%. Reference A. Amone, U. Brambilla, G. Nasini, and O. Vajna de Pava; Isolation and Structure Elucidation of Tsugicolines A-D, Novel ProtoiUudane Sesquiterpenes from Laurilia tsugicola; Tetrahedron; Vol. 51, pp. 13357-13364(1995).

11. Tsugicolines

155

Common/Systematic Name Tsugicoline B Molecular Formula/Molecular Weight C15H2103; MW = 250.15689 OH

HOH

H �9. ,,,%

O ~

\H

Fungal Source Laurilia tsugicola = Echinodontium tsugicola (CBS 248.51; Centraal Bureau voor

Schimmel Cultures, Baam). General Characteristics Obtained as an oil; [a]D - 71.5 ~(C=4.5, in CHC13). Isolation/Purification The culture filtrate which was separated from the mycelium was extracted twice with ethyl acetate. Then the extractswere dried (Na2SO4) and evaporated to yield a mixture of sesquiterpenes. The mixture was treated with ethyl ether; the residue was composed of pure tsugicoline A; the mother liquid was chromatographed by flash silica gel column chromatography eluted with hexane-ethyl acetate (2:1, v/v) to give a mixture of tsugicoline B and D that was further purified by preparative TLC using hexane-ethyl acetate (1:2, v/v) and methylene chloride-methanol (15:1, v/v) to yield pure tsugicoline B and D; Rf, 0.50; 0.30 for tsugicoline B, respectively. Spectral Data UV:

Xm~x 254nm (e=5,100). IR:

(chloroform) 1730cm"1 (conjugated carbonyl). 1H NMR:

(CDCI3) 4.54(1H, H-la); 4.50(1H, Jlb,3-1.9Hz, H-lb); 4.24(1H, J3,~3-9.1Hz, H-3); 2.82(1H, H-6); 1.17(3H, H-8); 2.48(1H, J9,~o~-10.2Hz, Jo,lop-8.3I-Iz, J9,13=11.9Hz, H9); 1.46(1H, J~o~,~op=12.6I-~J~o~,lS=0.8H~ H-10a); 1.56(1H, J~op,~2p=l.8Hz, H-1013); 1.22(1H, J12a,12p-12.3Hz, J12~,13=11.3Hz, Jl2~,lS=0.8Hz, H-12a); 1.85(1H, J12p,~3=7.2Hz, H-12~); 2.44(1H, H-13); 1.14(3H, H-14); 0.99(3H, H-15); 3.00"(3H, OH-l); and 3.80ppm* (3H, OH-3). * Assignments may be interchanged.

156

11. Tsugicolines

13C NM~: 61.17, t, C-l; 149.99, s, C-2; 74.51, d, C-3; 148.67, s, C-4; 197.77, s, C-5; 60.31, t, C6; 36.35, s, C-7; 20.41, q, C-8; 46.40, d, C-9; 41.27, t, C-10; 40.81, s, C-11; 46.56, t, C-12; 52.45, d, C-13; 29.46, q, C-14; and 26.82ppm, q, C-15. Mass Data: ELMS: (isobutane) 251(MI-F, 100%), 233(MIT - H20, 95), 215(38), 205(30), 203(22), 187(28), and 173role (25); found: C, 71.6, H, 8.7; C~5H2203 requires C, 71.97; H, 8.86%. Reference A. Arnone, U. Brambilla, G. Nasini, and O. Vajna de Pava; Isolation and Structure Elucidation of T sugicolines A-D, Novel Protoilludane Sesquiterpenes from Laurilia tsugicola; Tetrahedron, Vol. 51, pp. 13357-13364(1995).

11. Tsugicolines

157

Common/Systematic Name Tsugicoline C Molecular Formula/Molecular Weight C15H2404; M W "-" 2 6 8 . 1 6 7 4 6

OH HOH2

H

HO"~,~. - 171 H I L : H OH

Fungal Source Laurilia tsugicola = Echinodontium tsugicola (CBS 248.51 Centraal Bureau voor

Schimmel Cultures, Baarn). General Characteristics Obtained as white crystals from methylene ehloride-hexane; mp., 74-760C; [a]D - 39.6* (c=3.00, in CHCI3). Isolation/Purification The culture filtrate which was separated from the mycelium was extracted twice with ethyl acetate. Then the extracts were dried (Na2SO4) and evaporated to yield a mixture of sesquiterpenes. The mixture was treated with ethyl ether. The residue was composed of pure tsugicoline A. The mother liquid was chromatographed by flash silica gel column chromatography eluted with hexane-ethyl acetate (2:1, v/v) to give a mixture of tsugicoline B and D that was further purified by preparative TLC using hexane-ethyl acetate (1:2, v/v) and methylene chloride-methanol (15:1, v/v) to yield pure tsugicoline B, C, and D; Re, 0.50; 0.30 for tsugicoline C. Spectral Data UV: ~.mx 208nm (e=5,600). IR:

(chloroform) 3400cm1 (OH). 1H N]~IR: (CDCla) 4.28(1H, Jla,lb=13.4Hz, Jl~a=l.0Hz, H-la); 4.26(1H, Jlb,a=l.2Hz, H-lb); 4.15(1H, d3,13=8.5Hz, H-a); 3.70(1H, n-6); 1.04(3H, n-8); 2.30(1H, Jg,lo~=10.2Hz, Jg,lO~=8.2Hz, Jg,la=12.0Hz, H-9); 1.35(1H, Jloa,lO~=12.6Hz, Jloa,15=0.8Hz, H-10a);

158

11. Tsugicolines

1.39(1H, Jlop,~2p=l.6Hz, H-10~); 1.17(1H, d~2~,~2p=12.6Hz, J12=,13=10.2Hz, J~2~,~5=0.8Hz,n-12a); 1.78(1I-I,J~2p,~a=7.2Hz,H-1213); 2.25(1H, H-13); 1.08(3H, H- 14); 0.97(3H, H- 15); 3.97"(1H, OH- 1); 4.50"(1H, 3-OH); and 3.95ppm* (1H, 6OH). * Assignments may be interchanged. 13CNMR: 59.437, t, C-l; 139.59, s, C-2; 74.16, d, C-3; 141.03, s, C-4; 70.90, d, C-5; 76.92, d, C-6; 51.03, s, C-7; 15.481, q, (~-8; 46.59, d, C-9; 41.82, t, C-10; 40.50, s, C-11; 47.70, t, C-12; 51.58, d, C-13; 29.87, q, C-14; and 27.35ppm, q, C-15. Mass Data: CIMS: (isobutane) 269(MH+, 100%) and 233m/e (MIT - 2H20, base peak). Reference A. Amone, U. Brambilla, G. Nasini, and O. Vajna de Pava, Isolation and Structure Elucidation of Tsugicolines A-D, Novel Protoilludane Sesquiterpenes from Laurilia tsugicola; Tetrahedron, Vol. 51, pp. 13357-13364(1995).

11. Tsugicolines

159

Common/Systematic Name Tsugieoline D Molecular Formul.dMolecular Weight C15H2003; M W = 248.14124 H

H ~

'".,,

H OH Fungal Source

l_xmrilia tsugicola = Echinodontium tsugicola (CBS 248.51; Centraal Bureau voor

Schimmel Cultures, Baarn). General Characteristics Obtained as a white solid; mp., 107-110~

[0~]D +

161.8~(c=0.1, in CHCh).

Isolation/Purification The culture filtrate which was separated from the mycelium was extracted twice with ethyl acetate. Thenthe extracts were dried (Na2SO4) and evaporated to yield a mixture of sesquiterpenes. The mixture was treated with ethyl ether; the residue was composed of pure tsugicoline A; the mother liquid was chromatographed by flash silica gel column chromatography eluted with hexane-ethyl acetate (2:1, v/v) to give a mixture of tsugicoline B and D that was further purified by preparative TLC using hexane-ethyl acetate (1:2, v/v) and methylene chloride-methanol (15:1, v/v) to yield pure tsugicoline B and D; Re, 0.70, 0.30 for tsugicoline D, respectively. Soe~ral Data UV" ~,~ 215(e=6,0,50) and 240nm (e=4,800). IR:

(KBr) 3400(OH), 1780, and 1680em1 (earbonyl). 1H ~ . (CDCI3) 9.47(1H, H-la), 6.77(1H, Ja,13=2.0Hz, H-3); 4.71(1H, H-6); 1.13(3H, H-8); 2.64(1H, J9,1oa=12.7Hz, J9,1Ol~=6.9Hz,J9,1a=7.4Hz, H-9); 1.07(1H, J1oa,1o13=12.6Hz, Jloa,15=~ 0Hz, n-10a), 1.59(1H, J1o1~,12~=~ 0Hz, n-1013); 1.72(1H, J12,~12~=la.5Hz, J12a,la=2.3Hz, J12~,lS=-q3Hz,n-12a); 2.10(1H, J121~,13=9.2Hz,n-12~); 3.16(1H, n-13); 1.08(3H, H-14); 1.01(3H, H-15); and 5.14ppm (1H, OH-6).

160

11. Tsugicolines

13CNMR: 193.97, t, C-l; 134.54, s, C-2; 154.21, d, C-3; 54.55, d, C-4; 203.02, s, C-5; 85.83, d, C-6; 38.38, s, C-7; 18.82, q, C-8; 43.02, d, C-9; 37.61, t, C-10; 44.40, s, C-11; 37.61, t, C-12; 47.26, d, C-13; 32.00, q, C-14; and 31.61pprn, q, C-15. Mass Data: CIMS: (isobutane) 249(MH+, 80%), 231(MH+ - H20, 100), 220(20), and 203m/e (25); found: C, 72.2, H, 8.0; C15H2003requires C, 72.55; H, 8.12%. Reference A. Arnone, U. Brambilla, G. Nasini, and O. Vajna de Pava; Isolation and Structure Elucidation of Tsugicolines A-D, Novel Protoilludane Sesquiterpenes from Laurilia tsugicola; Tetrahedron, Vol. 51, pp. 13357-13364(1995).

Radicinins Radicinin Radicinin Diastereomer (2S*, 3R*) Radicinol Diasteromer (2S*,3S*,4S*)

2S,3 S, 4S-epi-Radicino l

Radicinol

161


12.

Radicinins

163

Common/Systematic Name Radicinin; Stemphylone Molecular Formula/Molecular Weight C12H1205; MW = 236.06847

SO,~~~dOH O

11

0

8

O

12

General Characteristics Needles from methanol; mp., 238-240~ (dec,),[t~]D2~-236 ~ (c--0.1, in CHCI3). Isolation/Purification The fungus was grown without shaking at 24~ for 21 days in the dark in liquid medium made up of glucose (3.0g/L), peptone (3.0g/L) and the extract from 50g~ of malt and water. The culture filtrate was acidified to pH 2.0 with HC1, after which the metabolites in the culture filtrate were extracted with ethyl acetate (3x). The ethyl acetate extracts were dried over Na2SO4 and concentrated. The residue was applied to a silica gel column and the column washed with 1500ml of 10% acetone in n-hexane, then developed successively with 750ml each 20, 30, and 40% acetone in n-hexane. Each 150ml eluate constituted one fraction. Fractions 10 and 11 were combined and evaporated. Recrystallization of the residue from methanol afforded radicinin as needles. The mother liquor was purified by Sephadex LH20 column chromatography. The column was washed with 500ml of methanol and fractions consisting of 7ml eluates were collected. Fractions 33-48 were combined and evaporated to dryness. Recrystallization of the residue from methanol afforded radicinin as needles. The diastereoisomer of radicinin (2S*,3R*) (Rf=0.38) in the mother liquor was separated from the mixture of the diastereoisomer of radicinol (2S*,3S*,4S*) and epiradicinol (2S*,3S*,4R*) bypreparative TLC [acetone-benzene (2:8, v/v) Rf=0.26]. The crude radicinin diastereoisomer was further purified with preparative TLC (ethyl acetaten-hexane; 7:3, v/v) which gave the radicinin diastereoisomer as needles [ethyl acetate-nhexane (7:3, v/v) Rf=0.63]. The diastereoisomer of radieinol (2S*,3S*,4S*) and epiradicinol (2S*,3S*,4R*) and radicinol epoxide were purified after preparative TLC (ethyl acetate-n-hexane; 7:3, v/v) of the mixture. Fungal Source

Bipolaris coicis, a phytopathogen which causes a serious leaf blight on Job's tears (Coix lachryma-jobi L), Alternaria radicina, A. chrysanthemL A. helianthL Cochliobolus lunata, and Sternphylium radicinum.

Biological Activity In a leaf spot assay of Job's tears, radicinin caused necrotic lesions at 0.31zg/leaf but not active at 0. ll,tg/leaf.

164

12.

Radicinins

Spectral Data UV:

X~ff2 203(1oge = 4.00), 221(4.14), 270(3.66), 280(3.61), and 341nm (4.15). IR:

(KBr) 3462, 3098, 3034, 2990, 2924, 2918, 1760, 1659, 1605, 1522, 1456, 1435, 1379, 1325, 1228, 1172, and 1056cm~. ~HN M R : (CDCIa) 4.36(1H, dq, J=12.4, 6.3Hz, H-2); 3.98(1H, d, J=12.4Hz, H=3); 5.84(1H, s, H-8); 6.03(1H, dq, J=15.5, 1.8Hz, H-9); 6.95(1H, dq, ,/=15.5, 7.0Hz, H-10); 1.95(3H, dq, J=7.0, 1.8Hz, H-11); and 1.64ppm (3H, d, J=6.3Hz, H-12). 13CNMR: (CDCI3) C-2, 80.0; C-3, 72.0; C-4, 188.6; C4a, 97.9; C-5, 156.7; C-7, 164.3; C-8, 98.0; C-Sa, 176.3; C-9, 122.6; C-10, 141.0; C-11, 18.8; and C-12, 18.1ppm. Mass Spectrum: CIMS (isobutane): 236m/e (M)'; 236.0682m/e exact mass calcd for C~2H1205, 236.0684. Reference H. Nakajima, T. Ishida, Y. Otsuka, T. Hamasaki, and M. Ichinoe; Phytotoxins and Related Metabolites Produced by Bipolaris coicis, The Pathogen of Job's Tears; Phytochemistry, Vol. 45, pp. 41-45(1997).

12.

Radicinins

165

Common/Systematic Name Radicinin Diastereomer (2S*, 3R*) Molecular Formula/Molecular Weight C12H1205, ~

-- 236.06847

0

O ~

0

,,,OH

General Characteristics Colorless needles from methanol; mp., 202-220~ EtOH).

(dee,); [et]D2~ = -105 ~ (C=0.25, in

Isolation/Purification See radicinin for isolation/purification. Fungal Source

Bipolaris coicis, a phytopathogen which causes a serious leaf blight on Job's tears (Coix lachryma-jobi L).

Biological Activity In a leaf spot assay of Job's tears, radicinin diastereomer caused necrotic lesions at 1.0~g/leaf but was not active at 0.3~g/leaf. Spectral Data UV: Z Emt~ 203(1og e = 4.00), 220(4.14), 269(3.67), 280(3.64), and 338nm (4.14). IR: (KBr) 3420, 2928, 2858, 1742, 1655, 1605, 1531, 1458, 1438, 1381 1263, 1170, 1118, and 1044cm1. 1H

NMR:

(CDCI3) 5.07(1H, dq, J-- 6.0Hz, 6.5Hz, H-2), 4.52(1H, d, J=6.0Hz, H-3); 5.83(1H, s, H-8), 6.03(1H, dq, J=15.3Hz, 1.3Hz, H-9), 6.96(1H, dq, J=15.3Hz, 7.0Hz, H-10), 1.96(3H, dd, J=7.0Hz, 1.3Hz, H-11), and 1.35ppm (3H, d, J=6.5Hz, H-12). 13C N M R :

(CDCI3) C-2, 78.9; C-3, 70.1; C-4, 188.0; C-4a, 97.6; C-5, 156.7; C-7, 164.5; C-8, 98.4; C-8a, 174.9; C-9, 122.6; C-10, 141.0; C-11, 18.8; and C-12, 12.1ppm. Mass Spectrum: CIMS (isobutane): 236role(M); 236.0682role exact mass calcd for C~2H1205, 236.0684

166

12.

Radicinins

Reference H. Nakajima, T. Ishida, Y. Otsuka, T. Hamasaki, and M. Ichinoe; Phytotoxins and Related Metabolites Produced by Bipolaris coicis, The Pathogen of Job's Tears; Phytochemistry, Vol. 45, pp. 41-45(1997).

12.

Radicinins

167

Common/Systematic Name Radicinol diasteromer (2S*,3S*,4S*) Molecular Formula/Molecular Weight C12H1405; M~W -- 238.08412

0

O~

OH

,,OH

General Characteristics An oil; [a]D2~-19 ~ (C----0.85,in EtOH). Isolation/Purification See radicinin for isolation/purification. Fungal Source

Bipolaris coicis, a phytopathogen which causes a serious leaf blight on Job's tears (Coix lachryma-jobi L).

Biological Activity In a leaf spot assay of Job's tears, radicinol diasteromer caused no necrotic lesions up to 10~g/leaf. Spectral Data UV:

%Em ~~

224 (log e = 4.45), 260 (3.51), 271 (3.56), and 315nm (3.95).

IR:

(KBr) 3408, 2988, 2942, 1690, 1618, 15742, 1437, 1383, 1315, 1278, 1214, 1160, 1141, 1071, and 1025cm1. 1H NMR:

(CDC13) 4.37(1H, dq, J=l.1, 6.3Hz, H-2); 3.88(1H, br, H-3); 4.60(1H, br, H-4OH); 5.79(1H, s, H-8); 5.97(1H, dq, J=15.7, 1.5Hz, H-9); 6.72(1H, dq, J=15.7, 6.8I-Iz, H10); 1.91(3H, dd, J=6.8, 1.5Hz, H-11); and 1.50ppm (3H, d, J=6.3Hz, H-12). 13C NMR:

(CDC13) C-2, 72.4; C-3, 69,1; C-4, 63.2; C4a, 99.5; C-5, 165.4; C-7, 158.5; C-8, 99.3; C-8a, 166.6; C-9, 122.6; C-10, 135.2; C-11, 18.4; and C-12, 15.8ppm. Mass Spectrum: EIMS: 238(M+, 18%), 181(100), 152(7), 137(8), and lllm/e (26); 238.0845m/e exact mass calcd for C12H1405,238.0841.

168

12.

Radicinins

Reference H. Nakajima, T. Ishida, Y. Otsuka, T. Hamasaki, and M. Ichinoe; Phytotoxins and Related Metabolites Produced by Bipolaris coicis, The Pathogen of Job's Tears; Phytochemistry, Vol. 45, pp. 41-45(1997).

12.

Radicinins

169


2S,3S, 4S-epi-Radicinol

Molecular Formula/Molecular Weight C12H1406, M W = 254.07904

0

O ~ A

O--

Me"/>

/

"~

L

OH

"0"

,,OH "'Me

General Characteristics An oil; [0~]D20 -92 ~ (c= 1.0, in EtOH). Isolation/Purification See radicinin for isolation/purification. Fungal Source

Bipolaris coicis, a phytopathogen which causes a serious leaf blight on Job's tears (Coix lachryma-jo bi L).

Biological Activity In a leaf spot assay of Job's tears, epi-radicinol caused no necrotic lesions up to 10~tg/leaf. Spectral Data UV: Era'~ 215 (log e = 4.34) and 288nm (4.31). IR: (Thin film) 3376, 3000, 2930, 1698, 1653, 1580, 1444, 1383, 1212, 1139, 1071 and 1033cm1. 1H N M R :

(CDC13) 4.37(1H, brq, J=6.7Hz, H-2), 3.84(1H, br, H-3); 4.56(1H, d, J=2.4Hz, H-4); 6.05(1H, s, H-8), 3.33(1H, d, J=l.9Hz, H-9), 3.21(1H, dq, J=l.9Hz, 5.4Hz, H-10), 1.42(3H, d, J=5.40Hz, H-11); and 1.49ppm (3H, d, J=6.7Hz, H-12). 13C N M R :

(CDC13) C-2, 72.6; C-3, 69,1; C-4, 63.3; C-4a, 100.4; C-5, 164.8; C-7, 160.4; C-8, 99.7; C-8a, 165.9; C-9, 55.1; C-10, 57.3; C-11, 17.3; and C-12, 15.7ppm. Mass Spectrum: ELMS: 254(M+, 28%), 197(100), 182(10), 153(79), and 139m/e (13);254.0795m/e exact mass calcd for C~2H1406,254.0790.

170

12.

Radicinins

Reference H. Nakajima, T. Ishida, Y. Otsuka, T. Hamasaki, and M. Ichinoe, Phytotoxins and Related Metabolites Produced by Bipolaris coicis, The Pathogen of Job's Tears, Phytochemistry, Vol. 45, pp. 41-45(1997).

12.

Radicinins

171

Common/Systematic Name Radicinol Molecular Formula/Molecular Weight C12H1405; MW" = 2 3 8 . 0 8 4 1 2 III ii1,.

HO

OH

0

General Characteristics Colorless viscous oil; [a]D 31 -175 ~ (c--1.02, in CHCI3). Isolation/Purification A mixture of radicinol and radicinin was obtained from the ethyl acetate extract of the fungal culture aider column chromatography using 60% ethyl acetate in n-hexane. The mixture was purified using Sephadex LH20 with acetone as eluanl. Fungal Source Cochliobolus lunata (IFO 6288). Spectral Data UV~ ,~ EtOH max

226(~ =39,300), 262(3,100), 271 (3,200), and 318nm (10,800).

IR~

(CHC13) 3425(OH) and 1680cmq (a-pyrone). 1H N M R :

(CDC13) 6.72(1H, dq, J=7.0, 16.0Hz, H-10); 3.76(1H, dd, ./=6.5, 8.0Hz, H-3); 4.75(2H, bs, 2 OH's); 5.85(1H, s, H-8); 6.02(1H, dq, ./=16.0, 2.0Hz, H-9); 1.92(3H, dd, J=7.0, 2.0Hz, H-11); 4.70(1H, d, J=6.5Hz, H-4); 4.23(1H, dq, ./=8.0, 6.6Hz, H-2); and 1.52ppm (3H, d, J=6.5Hz, H-12). 13C NMR: (CDCla) C-2, 76.8; C-3, 72.5; C-4, 68.0; C-4a, 100.6; C-5, 165.4; C-7, 158.9; C-8, 99.1; C-8a, 164.5; C-9, 122.7; C-10, 135.7; C-11, 18.4; and C-12, 17.0ppm. Mass Spectrum: HREIMS: 238.082re~e; exact mass calcd for C12H1405, 238.084.

172

12.

Radicinins

Reference M. Nukina and S. Marumo, Radicinol, A New Metabolite of Cochliobolus lunata, and Absolute Stereochemistry of Radicinin, Tetrahedron Letters, pp. 3271-3271 (1977).

Stachybotrylactone and Related Metabolites Stachybotrylactone K-76 Stachybotrylactone Acetate 2a-Hydroxystaehybotrylactone 2~t-Acetoxystachybotrylactone Acetate Stachybotrylactam Stachybotrylactam Acetate 2~t-Acetoxystachybotrylactone Acetate Stachybotramide Stachybotrydial Stachybotrin A Stachybotrin B

173


13. Stachybotrylactone and Related Metabolites

175

Common/Systematic Name Stachybotrylactone Molecular Formula/Molecular Weight C23H3005; M W -- 3 8 6 . 2 0 9 3 2

O HO I

"

r

11

,,,.J 12

HO' .....

General Characteristics White or pale yellow crystalline solid; mp., 280~ (dec.); [0~]D -6.4 ~ (c=0.09, in acetone). Isolation/Purification Rice cultures of Stachybotrys chartarum (Egypt 1) were air dried, ground, and extracted with methanol followed by extraction with chloroform. The extracts were combined and evaporated to dryness. The crude extract was dissolved in methanol and filtered through a pad of ClS silica gel followed by elution with methanol to give a black gum alter removal of solvent. The gum was dissolved in methylene chloride and chromatographed on a PEI silica gel column eluted with methylene chloride-hexane (4:1, v/v), methylene chloride, and 10% methanol in methylene chloride. A combination of preparative HPLC and TLC gave purified satratoxin H, S-isosatratoxin H, stachybotrylactone, stachybotrylactone acetate, 2a-acetoxystachybotrylactone acetate and stachybotrylactam. Fungal Source

Stachybotrys chartarum and S. cylindrospora. Note: Ayer and Miao (1993) believed stachybotrylactone to be an artifact formed from stachybotrydial.

Biological Activity Cytotoxic and immunosuppressive. Spectral Data UV: ~ Aoetonitrile max

218(c=28,000), 268(5,3000), and 309nm (2,600).

176


IR~

(CHsC1) 3600-3100, 2939, 1742, 1623, 1469, 1389, 1356, 1332, 954, 943,772, and 737cmq. 1H NMR:

(CDCls) 1.90(1H, H-la); 1.06 (1H, H-113); 1.60 (1H, H-2~); 1.44(1H, H-213); 3.32(1H, br s, H-313); 2.17(1H, dd, J=l 1.6, 2.7Hz, H-5~); 1.46(1H, H-6a); 1.55(1H, H-613); 1.60 (1H, H-7a); 1.45(1H, H-713); 1.86(1H, H-813); 3.26(1H, d, J=17.3Hz, HI la, proton on the side of C-10); 2.90(1H, d, J=17.3Hz, H-1 lb, proton on the side of C-8); 0.72(3H, d, J=6.5Hz, H-12a); 0.97(3H, s, H-141~); 0.86(3H, s, H-1413); 1.06(3H, s, H-1513); 6.76(1H, s, H-3'); 5.23(1H, d, J=14.7Hz, H-8'a); and 5.17ppm (1H, d, J=14.7Hz, H-8"o). ~SCNMR: (acetone-d6) 24.91, t, C-I; 21.60, t, C-2; 75.21, d, C-3; 38.29, s, C-4; 40.65, d, C-5; 25.91, t, C-6; 31.81, t, C-7; 37.78, d, C-8; 100.00, s, C-9; 43.09, s, C-10; 32.53, t, C11; 15.86, q, C-12; 28.94, q, C-13; 22.80, q, C-14; 16.36, q, C-15; 120.72, s, C-I'; 156.65, s, C-2'; 103.00, d, C-3'; 119.2, s, C-4'; 128.07, s, C-5'; 155.47, s, C-6'; 171.29, s, C-7'; and 67.84ppm, t, C-8'. Mass Spectrum: HREIMS: 386.2095(C23H3005,53%), 368.1977(C23H2sO4, 11), 353.1753(C22H2504, 9), 244.0730(C14H1204, 9), 230.0579(ClsH1004, 100), 217.049(C~2H904, 18), 207.1747(C14H230, 38), and 189.1642m/e (C~,I-I2~,35). References W. A. Ayer and S. Miao; Secondary Metabolites of the Aspen Fungus Stachybotrys cylindrospora; Canad. J. Chem., Vol. 71, pp. 487-493(1993). B. B. Jarvis, J. Salemme, and A. Morais; Stachybotrys Toxins 1. Natural toxins; Vol. 3, pp. 10-16 (1995).

13.

Stachybotrylactone and Related Metabolites

177

Common/Systematic Name K-76 Molecular Formula/Molecular Weight C23H3oO6; MW = 402.20424

HO 1 . ~ - , , , . / . C HO

H ~

r'lU,

....

' .... '

.... , 1 2

General Characteristics Crystals; mp., 176 o; [tt]D20 _ 48 ~ (in MeOH). Fungal Source Stachybotrys complementi (ATCC 20511). Biological Activity Strongly inhibited the complement C5 step at ca. 10pg/ml in vitro and preliminary experiments indicated that it improved the symptoms of experimental glomerulonephritis. Spectral Data UV:

~

EtOH max

246(e=16,500), 307(6,700) and 359nm (5,400).

IR:

(KBr) 1600, 1585, 1385, and 880cm~. 1H NNIR:

(C6HsN) 0.82(3H, d, J=5.0Hz, H-12); 0.93, 1.04, and 1.27(3H each, s); 3.72(1H, d, J=2.0Hz, H-3); 4.30(1H, J=20.4Hz, H-2); 7.41(1H, s, H-3'); and 10.75, 11.02ppm (1H each, s, CHO). X-Ray: Structure confirmed by X-ray analysis of the p-bromobenzenesulfonate. References E. J. Corey and J. Das; Total Synthesis of the Complement Inhibitor K-76 in Racemic Form. Structural Assignment to "1(-76 monocarboxylic Acid"; J. Am. Chem. Sot., Vol. 104, pp. 5551-5553(1982).

178

13.


H. Kaise, M. Shinohara, W. Smiyazaki, T. Izawa, Y. Nakano, M. Sugawara, K. Sugiura, and K. Sasaki; Structure of K-76, A Complement Inhibitor Produced by Stachybotrys complementi nov. sp. K-76; J. C. S. Chem. Comm,pp. 726-727(1979).

13.


179

Common/Systematic Name Stachybotrylactone acetate Molecular Formula/Molecular Weight C25H3206; M W -- 428.21989

O

�9 /__

Aoo

.......

14~

7 ; ........' 12 13

General Characteristics Crystals; mp., 105-106~

[~]D -12 ~ (c=0.5, in MeOH).

Isolation/Purification Rice cultures of Stachybotrys chartarum (Egypt 1) were air dried, ground, and extracted with methanol followed by extraction with chloroform. The extracts were combined and evaporated to dryness. The crude extract was dissolved in methanol and filtered through a pad of C~s silica gel followed by elution with methanol to give a black gum after removal of solvent. The gum was dissolved in methylene chloride and chromatographed on a PEI silica gel column eluted with methylene chloride-hexane (4:1, v/v), methylene chloride, and 10% methanol in methylene chloride. A combination of preparative HPLC and TLC gave purified satratoxin H, S-isosatratoxin H, stachybotrylactone, stachybotrylactone acetate, 2~-acetoxystachybotrylactone acetate and stachybotrylactam. F ungal Source

Stachybotrys chartarum.

Biological Activity Cytotoxic and immunosuppressive. Spectral Data UV:

maxA~~ 218, 265, and 306nm. IR:

(CHaC1) 3280(OH), 1754(C=0), and 1724crn1 (C = 0).

180


1H NMR: (CDCI3) 0.74(3H, d, J=6.4Hz, H-12); 0.90(3H, s, H-13); 0.91(3H, s, H-14); 0.98(3H, s, H-15); 1.4-1.9(9H, m, H-I, 2, 6, 7, 8); 2.02(3H, s, acetate); 2.05(1H, m, H-5); 2.90 and 3.30(1H each, AB, J=17.4, H-11); 4.60(1H, bs, H-3); 5.13 and 5.27(1H each, AB, J= 14.5, H-8'); and 6.90ppm (1H, s, H-3'). 13C NMR: 25.0, C-I; 20.7, C-2; 77.8, C-3; 37.0, C-4; 41.0, C-5; 22.3, C-6; 30.9, C-7; 36.8, C-8; 99.6, C-9; 42.2, C-10; 31.6, C-11; 15.5, C-12, 27.8, C-13; 21.3, C-14; 16.0, C-15; 118.9, C-I'; 153.9, C-2'; 103.3, C-3'; 120.0, C-4'; 127.5, C-5'; 155.7, C-6'; 170.4, C-7'; 67.6, C-8'; 21.9, and 170.5ppm (acetates). Mass Spectrum: CIMS (CH4): 429(7%), 369(12), 299(12), 281(19), and 69m/e (100); HREIMS: 428.2195m/e for C25H3206;calcd, 428.2199. Reference B. B. Jarvis, J. Salemme and A. Morals; Stachybotrys Toxins. 1; Natural Toxins, Vol. 3, pp. 10-16(1995).

13.


181

Common/Systematic Name 2tt-Hydroxystachybotrylactone Molecular Formula/Molecular Weight C23H3006; ~

= 402.20424

O 2'

0

Te

--

HO.......(3 ;kl/~,,,,,() 14@" : ~ \

8'8'

/'//

13

General Characteristics Amorphous solidi [tt]D - 40 ~ (C=I.0, in MeOH). Fungal Source Stachybotrys chartarum. Biological Activity Cytotoxic and immunosuppressive. Spectral Data UV:

Ao~om~em,x 218, 265, and 306nm. IR:

(CH3C1) 3296(OH) and 1750cm~ (C = O). ~H NMR: (CDC13) 0.67(3H, d, J=6.2Hz, H-10); 0.83(3H, s, H-14); 0.97(3H, s, H-13); 1.01(3H, s, H-15); 1.3-1.8(7H, m, H-I, 6, 7, 8); 2.05(1H, m, H-5); 2.85 and 3.2(1H each, AB, J=17.3Hz, H-11); 3.40(1H, bs, H-3); 4.02(1H, bd, J=l 1Hz, H-2); 5.10 and 5.24(1H each, AB, J=14.9Hz, H-8'); and 6.78ppm (1H, s, H-3'). 13C NMR: 30.9, C-l; 66.7, C-2; 78.7, C-3; 39.2, C-4; 38.3, C-5; 20.6, C-6; 30.9, C-7; 36.8, C-8; 98.9, C-9; 43.4, C-10; 33.0, C-11; 15.4, C-12; 28.4, C-13; 21.9, C-14; 16.9, C-15; 119.0, C-I'; 153.9, C-2'; 103.3, C-3'; 120.1, C-4'; 127.1, C-5'; 155.4, C-6'; 172.0, C-7'; and 68.1 pprn, C-8'.

182


Mass Spectrum: CIMS (El-h): 403(15%), 299(8), 257(2), 173(100), and 145m/e (43); HREIMS: 402.2047m/e for C23H3oO6;calcd, 402.2042. Reference B. B. Jarvis, J. Salemme and A. Morals; Stachybotrys Toxins. 1 Natural Toxins, Vol. 3, pp. 10-16(1995).


183

Common/Systematic Name 2tt-Acetoxystachybotrylactone acetate Molecular Formula/Molecular Weight C27H3408; M W = 4 8 6 . 2 2 5 3 7

O

AcQ \.

z

,s % / ~ . , ) dl T~

0

8'8'

A o O .......

14 ~

7//~........I 12 13

General Characteristics Amorphous solid; [tt]D -15 ~ (c=l.0, in MeOH). Fungal Source Stachybotrys chartarum. Isolation~urification The black gummy crude extract derived from a rice culture of S. chartarum S-17 was triturated with hexane washes until no further color was extracted. The remaining black gum was partitioned between water and chloroform. The chloroform soluble portion was dried (Na2SO4) and concentrated to dryness on the rotary evaporator to give a black gum. The gum was subjected to flash chromatography, over silica gel with a gradient of hexaneCH2C12-MeOH, and collected in 124 fractions. Fractions 83-84 (3% MeOH-CH2C12) were combined to give A; fractions 85-87 (3% MeOH/CH2C12) were combined to give B; fractions 88-89 (3% MeOH/CH2C12) and 90-99 (4% MeOH/CH2C12) were combined to give C; fractions 100-103 (5% MeOH/CH2C12) and fractions 104-110 (6% MeOH/CH2C12) were combined to give D; fractions 111-112 (6% MeOH-CH2C12) were combined to give E; fractions 113-115 (10% MeOH-CH2C12) were combined to give F; and fractions 116-119 were combined to give G. Fraction A was subjected to high speed countercurrent distribution [Vo = 850ml, MeOH (3)-1-120 (2)-CC14 (3.5)-CH2C12 (0.5)hexane (1), organic mobile phase at 3.2ml/min] to give 10 fractions (F1-F10). Fraction F5 was subjected to preparative TLC (Chromatotron, 2mm plate, 20% hexane in EtOAc) to give pure 2a-acetoxystachybotrylactone acetate. Biological Activity Cytotoxic and immunosuppressive.

184

13.


Spectral Data UV:

~,Am~~

218, 265, and 306nm.

IR:

(CH3C1) 3280(OH)and 1744cm1 (C = O). 1H NMR:

(CDC13) 0.73(3 H, d, J=6.4, H-12); 0.93(3H, s, H-14); 1.00(3H, s, H-13); 1.10( 3H, s, H-15); 1.3-1.8(7H, m, H-I, 6, 7, 8); 2.05(1H, m, H-5); 1.90 and 2.05(3H each, s, acetates); 2.95 and 3.35(1H each, AB, J=17.4Hz, H-11); 3.50(1H, bs, H-3); 4.95(1H, bd, J=10Hz, H-2); 5.18 and 5.37(1H each, AB, J=14.5Hz, H-8'); and 6.92ppm (1H, s, H-3'). 13CNMR: (CDC13) 30.7, C-l; 68.3, C-2; 77.0, C-3; 38.2, C-4; 40.6, C-5; 20.4, C-6; 30.6, C-7; 36.8, C-8; 99.0, C-9; 43.6, C-10; 32.0, C-11; 15.5, C-12; 27.7, C-13; 21.6, C-14; 16.9, C-15; 118.8, C-I'; 153.9, C-2', 103.6, C-3'; 119.9, C-4'; 127.6, C-5'; 155.4, C-6'; 171.6, C-7'; 67.8, C-8'; 21.1, 20.9, 170.4, and 170.5 ppm, acetates. Mass Spectrum: ELMS: 486(M+, 18%), 384(100), 351(3), 279(7), and 230m/e (21); HREIMS: 486.2250m/e for CzvH34Os;calcd, 486.2254. Reference B. B. Jarvis, J. Salemme and A. Morais; Stachybotrys Toxins. 1" Natural Toxins, Vol. 3, pp. 10-16(1995).

13.


185

Common/Systematic Name Stachybotrylactam Molecular Formula/Molecular Weight C23H31NO4; ~ "- 385.22531 O

HOrN H .o

....

~

14,"

-

~\

_, /2 " '

12

13

General Characteristics Amorphous solid. Fungal Source Stachybotrys chartarum. Isolation/Purification The black gummy crude extract derived from a rice culture of S. chartarum S-17 was triturated with hexane washes until no further color was extracted. The remaining black gum was partitioned between water and chloroform. The chloroform soluble portion was dried (Na2SO4) and concentrated to dryness on the rotary evaporator to give a black gum. The gum was subjected to flash chromatography, over silica gel with a gradient of hexaneCH2CI2-MeOH, and collected in 124 fractions. Fractions 83-84 (3% MeOH-CH:C12) were combined to give A; fractions 85-87 (3% MeOH-CH2C12) were combined to give B; fractions 88-89 (3% MeOH-CH:C1:) and 90-99 (4% MeOH/CH2C1:) were combined to give C; fractions 100-103 (5% MeOH-CH2C12) and fractions 104-110 (6% MeOH/CH2C12) were combined to give D; fractions 111-112 (6% MeOH/CHzC12) were combined to give E; fractions 113-115 (10% MeOH-CH:C12) were combined to give F; and fractions 116-119 were combined to give G. Fraction D was subjected to reversedphase MPLC (Rainin, 18g, 81~, C-18, 4ml/min) with increasing MeOH, starting with 70% MeOH in H20, to give several fractions one of which upon reversed-phase HPLC (semipreparative Cls, 70% MeOH in 1-120) gave pure stachybotrylactam. Biological Activity Cytotoxic and immunosuppressive. Spectral Data UV~

XA~~

216, 257, and 298nm.

186

13.


(CH3CI) 3450(NH), 3290(OH), and 1690cm1 (C=O). 1H NMR: (CDCls) 0.73(3H, d, J=6.0Hz, H-12), 0.87(3H, s, H-14), 0.98(3H, s, H-13), 1.03(3H, s, H-15), 1.5-1.9(9H, m, H-l, -2, -6, -7, -8), 2.20(1H, m, H-5), 2.83 and 3.18(1H each, AB, J=17.2Hz, H-11); 3.36(1H, bs, H-3), 4.25 and 4.35(1H each, AB, J=16.5Hz, H-8'), 6.80(1H, s, H-3'), and 8.0ppm (1H, bs, NH). 13CNMR: (CDC13) 24.9, C-l; 21.7, C-2; 75.5, C-3; 38.3, C-4; 40.6, C-5; 25.9, C-6; 31.9, C-7; 37.7, C-8; 99.0, C-9; 43.0, C-10; 32.6, C-11; 16.0, C-12; 28.6, C-13; 22.8, C-14; 16.4, C-15; 118.0, C-I'; 153.9, C-2'; 102.3, C-3'; 116.0, C-4'; 135.0, C-5'; 154.7, C-6'; 172.1, C-7'; and 43.3ppm, C-8'.

Mass Spectrum: EIMS: 386(8%), 385(Nf, 23), 279(10), 230(18), 179(15), and 149m/e (100), HREIMS: 385.2215m/e for C23H31NO4,calcd, 385.2253. Reference B. B. Jarvis, J. Salemme and A. Morais; Stachybotrys Toxins. 1; Natural Toxins, Vol. 3, pp. 10-16(1995).


187

Common/Systematic Name Stachybotrylactam acetate Molecular Formula/Molecular Weight C25H33NOs, ~ = 427.23587 O

HOz~/NH AcO

.......~

5 ~

''''0

14 13 General Characteristics Amorphous solid. Fungal Source


Isolation/Purification A shake culture of Stachybotrys complementi (ATCC 20511) yielded a crude extract from which were isolated stachybotrylactam, stachybotrylactam acetate, and 2t~-hydroxystachybotrylactam acetate by a series of chromatographic steps similar to those described for stachybotrylactam. Biological Activity Cytotoxie and immunosuppressive. Spectral Data UV~

Aeetomtrilemax216, 257, and 298nm.

IR~ (CH3C1) 3450(NH), 3248(OH), 1730(C=O), and 1696cm~ (lactam). 1H NMR: (CDC13) 0.70(3H, d, J=6.0Hz, H-12); 0.89(3H, s, H-14); 0.97(3H, s, H-13); 1.23(3H, s, H-15); 1.4-1.9(9H, m, H-I, -2, -6, -7, -8); 1.99(3H, s, acetate); 2.00(1H, m, H-5); 2.84 and 3.19(1H each, AB, d=17.0Hz, H-11); 4.22 and 4.38(1H each AB, J=l 5.0Hz, H-8'); 4.58(1H, s, H-6); 6.78(1H, bs, NH); and 6.90ppm (1H, s, H-3').

188

13.


13C NMR: (CDC13) 24.9, C-I; 20.8, C-2; 77.9, C-3; 36.8, C-4; 40.9, C-5; 22.3, C-6; 30.8, C-7; 37.0, C-8; 98.6, C-9; 42.0, C-10; 31.8, C-11; 15.5, C-12; 27.8, C-13; 21.2, C-14; 16.0, C-15; 117.8, C-I'; 153.3, C-2'; 102.3, C-3'; 115.3, C-4'; 135.0, C-5'; 156.4, C-6'; 172.4, C-7'; 43.0, C-8'; 21.7, acetate; and 170.4ppm, acetate. Mass Spectrum: HREIMS: 427.2350m/e for C25Ha3NOs; calcd 427.2359. Reference B. B. Jarvis, J. Salemme and A. Morais, Stachybotrys Toxins. 1" Natural Toxins, Vol. 3, pp. 10-16(1995).


189

Common/Systematic Name 2a-Acetoxystachybotrylactam acetate Molecular Formula/Molecular Weight C27H35NO7;M ~ = 485.24135 O AcO -~-

~5 ~ N H ~

AcO....... (3[, F ' ~oP. 1,~'""06' - ~1 ,

14 ~

~-8'

_

7 / ........, 12 13

General Characteristics Amorphous solidi [0~]D -29 ~ (c=0.1, in MeOH). Fungal Source

Stachybotrys chartarum (MRC 1422).

Isolation/Purification A rice culture of S. chartarum yielded 3.2g of crude extract. Verrucarin J, roridin E, and satratoxins F, G, and H were isolated from this extract by a series of chromatographic procedures (see isolation of stachylbotrylactone acetate). Also isolated was 13mg of stachybotrylactone and 25mg of 2t~-acetoxystachybotrylactam acetate. Biological Activity Cytotoxic and immunosuppressive. Spectral Data UV; ~,Am~r~~ 216, 257, and 298nm. IR~ (CHaC1) 3450(NH), 3280(OH), 1744(C=O, acetate), and 1696cm"1 (C=O, lactam). 1H NMR: (CDC13) 0.71(3H, d, J-6.4Hz, H-12); 0.93(3H, s, H-14); 1.02(3H, s, H-13); 1.10(3H s, H-15); 1.4-1.8(7H, m, H-I, -6, -7, -8); 2.05(1H, m H-5); 1.90 and 2.07(3H each, s, acetates); 2.90 and 3.24(1H each, AB, J=17.0Hz, H-11); 4.25 and 4.42(1H each AB, J=16.5Hz, H-8'); 4.95(1H, bs, H-3); 5.21(1H, bd, J=l 1Hz, H-2); 7.01(1H, s, H-3'); and 8.0ppm (1H, bs, NH).

190

13.


13C NMR: (CDCI3) 30.5, C-l; 68.2, C-2; 76.7, C-3; 38.1, C-4; 40.5, C-5; 20.4, C-6; 30.7, C-7; 36.7, C-8; 98.1, C-9; 43.5, C-10; 32.0, C-11; 15.4, C-12; 27.7, C-13; 20.9, C-14; 16.9, C-15; 117.6, C-I'; 153.4, C-2'; 102.5, C-3'; 115.3, C-4'; 133.0, C-5'; 156.1, C-6'; 172.0, C-7'; 43.5, C-8'; 21.0, 21.6; and 170.3, 170.6ppm, acetates.

Mass Spectrum: EIMS: 487(7%), 486(20), 485(60), 383(62), 279(14), and 229m/e (24); HREIMS: 485.2404m/e for C27H35NO7;calcd, 485.2414). Reference B. B. Jarvis, J. Salemme and A. Morais; Stachybotrys Toxins. 1" Natural Toxins, Vol. 3, pp. 10-16(1995).

13.


191

Common/Systematic Name Stachybotramide Molecular Formula/Molecular Weight C25H35NOs; M W = 429.25152 0

is ~NCH2CH2OH HO....~ , , , O

il

14- ~ 1 4 \

,/

[~

8'

....... , 1 2

13


[0~]D -16 ~ (c= 0.1, in MeOH).

Fungal Source

Stachybotrys chartarum and S. cylindrospora.

Isolation/Purification The black gummy crude extract derived from a rice culture of S. chartarum S-17 was triturated with hexane washes until no further color was extracted. The remaining black gum was partitioned between water and chloroform. The chloroform soluble portion was dried (Na2SO4) and concentrated to dryness on the rotary evaporator to give a black gum. The gum was subjected to flash chromatography, over silica gel with a gradient of hexaneCH2C12-MeOH, and collected in 124 fractions. Fractions 83-84 (3% MeOH-CH2C12) were combined to give A; fractions 85-87 (3% MeOH-CH2C12) were combined to give B; fractions 88-89 (3% MeOH-CH2C12) and 90-99 (4% MeOH-CH2C12) were combined to give C; fractions 100-103 (5% MeOH-CH2C12) and fractions 104-110 (6% MeOHCH2C12) were combined to give D; fractions 111-112 (6% MeOH-CH2C12) were combined to give E; fractions 113-115 (10% MeOH-CH2C12) were combined to give F; and fractions 116-119 were combined to give G. Fraction F was filtered through a short PEI silica gel column which was eluted with increasing concentrations of MeOH in CH2C12 to give two fractions. The first fraction was crystallized from EtOAc-hexane to give stachybotramide. Acetylation of staehybotramide gave the triacetate derivative, which was identical to the compound reported by Ayer and Miao [ 1993]. Biological Activity Cytotoxic and immunosuppressive.

192

13.


Spectral Data UV~

~,A~on~o 216, 257, and 300nm. max IR;

(CH3C1) 3270(OH), and 1695cm1 (C=O, lactam). 1H M R :

(pyridin-ds) 0.84(3H, d, J-5.5Hz, H-12); 0.91(3H, s, H-14); 0.98(3H, s, H-13); 1.22(3H s, H-15); 1.20-1.90(9H, m, H-I, -2, -6, -7, -8); 2.05(1H, m, H-5); 3.10 and 3.52(1H each, d, J=l 7.0Hz, H-11); 3.60(1H, br s, H-2); 3.65(2H, m, H-9'); 3.90 (2H, m, H-10'); 4.07 and 4.35 (1Heach, AB, J=16.8Hz, H-8'); and 7.35ppm (1H, s, H-3'). 13C NMR:

(pyridin-ds) 25.7, C-I; 24.5, C-2; 74.6, C-3; 38.0, C-4; 40.2, C-5; 21.2, C-6; 31.4, C7; 37.2, C-8; 98.5, C-9; 42.5, C-10; 32.6, C-11; 15.7, C-12; 28.9, C-13; 22.5, C-14; 16.0, C-15; 117.4, C-I'; 155.2, C-2'; 101.6, C-3'; 113.0, C-4'; 135.0, C-5'; 156.7, C-6'; 168.8, C-7'; 48.3, C-8'; 45.7, C-9'; and 60.3ppm, C-10'. Mass Spectrum: HREIMS: 4292534m/e for C27H35NO7,calcd, 429.2515. References W. A. Ayer and S. Miao; Secondary Metabolites of the Aspen Fungus Stachybotrys cylindrospora; Can. J. Chem., Vol. 71, pp. 487-493(1993). B. B. Jarvis, J. Salemme and A. Morals; Stachybotrys Toxins. 1 Natural Toxins, Vol. 3, pp. 10-16(1995).


193

Common/Systematic Name Stachybotrydial Molecular Formula/Molecular Weight Cz3H30Os, ~

= 386.20932

HO,..v/~y,,...GHO

General Characteristics Crystals; mp., 266~ (dec.). Fungal Source

Stachybotrys chartarum and S. cylindrospora.

Isolation/Purification The black gummy crude extract derived from a rice culture of S. chartarum S-17 was triturated with hexane washes until no further color was extracted. The remaining black gum was partitioned between water and chloroform. The chloroform soluble portion was dried (Na2SO4) and concentrated to dryness on the rotary evaporator to give a black gum. The gum was subjected to flash chromatography, over silica gel with a gradient of hexaneCH2C12-MeOH, and collected in 124 fractions. Fractions 83-84 (3% MeOH-CH2C12) were combined to give A; fractions 85-87 (3% MeOH-CH2C12) were combined to give B; fractions 88-89 (3% MeOH-CH2C12) and 90-99 (4% MeOH-CH2C12) were combined to give C; fractions 100-103 (5% MeOH-CH2CI2) and fractions 104-110 (6% MeOHCH2C12) were combined to give D; fractions 111-112 (6% MeOH-CH2C12) were combined to give E; fractions 113-115 (10% MeOH/CH2C12) were combined to give F; and fractions 116-119 were combined to give G. Fraction B was subjected to CCC (Vc = 850ml, MeOH (2)-H20(2)-EtOAc(2)-hexane(3), organic mobile phase at 3.2ml/min) to give a major fraction which was subjected to preparative TLC (Chromatotron, 4mm plate, increasing EtOAc in hexane) to yield a semisolid material. This material was subjected to silica gel HPLC (semipreparative column, 2.5% MeOH in CH2C12 [40%]-hexane [60%], 4ml/min, 0.2g/injection to give stachybotrylactone and stachybotrydial). _ _ Biological Activity Cytotoxic and immunosuppressive. Spectral Data UV:

~on~e

232, 254, 248, and 360nm.

194

13.


IR~

(CHaC1) 3450(OH)and 1690cm1 (C=O). 1H NMR:

(CDC13) 0.72(3H, d, J=6.4Hz, H-12), 0.85(3H, s, H-14), 0.98(3H, s, H-13), 0.98(3H, s, H-15); 1.5-2.0(9H, m, H-l, -2, -6, -7, -8); 2.05(1H, dd, J-13 and 3Hz); 2.79 and 3.15(1H each, AB, J=17.4Hz, H-11); 3.39(1H, bs, H-3), 6.87(1H, s, H-3'), 10.34(1H, s, H-7'); and 10.57ppm ( 1H, s, H-8'). 13C NMR: (CDC13) 24.6, C-I, 24.0, C-2, 75.7, C-3; 37.5, C-4; 40.0, C-5; 20.8, C-6; 30.3, C-7; 36.9, C-8; 100.6, C-9; 42.1, C-10; 30.9, C-11; 15.4, C-12; 28.2, C-13; 22.2, C-14; 16.0, C-15; 111.0, C-I'; 158.0, C-2'; 109.3, C-3'; 137.9, C-4'; 119.3, C-5'; 167.5, C-6'; 188.7, C-7'; and 193.4ppm, C-8'. Mass Spectrum: HREIMS: 386.4878m/efor C25H3005; calcd, 386.4882. References W. A. Ayer and S. Miao; Secondary Metabolites of the Aspen Fungus Stachybotrys cylindrospora; Can. J. Chem., Vol. 71, pp. 487-493(1993). B. B. Jarvis, J. Salemme and A. Morais; Stachybotrys Toxins. 1; Natural Toxins, Vol. 3, pp. 10-16(1995).

13.


195

Common/Systematic Name Stachybotrin A Molecular Formula/Molecular Weight C23H31NOs; M"W = 401.22022

25 OH 22.

i

23

C H20H 24

7

0,,,~

OH

12 13

H/N--~O General Characteristics Isolated as a yellow oil; [tt]D + 8.8 ~ (C=0.612, in MeOH). Fungal Source

Stachybotrys sp. (ATCC 90017) collected from brackish water in Florida.

Isolation/Purification The culture filtrate was extracted with ethyl acetate and the organic phase was dried (MgSO4) and concentrated to afford a red oil. The extract was dissolved in acetone and divided into four equal portions. Each portion was subjected to preparative TLC on silica gel GF plates developed twice with 9:1, v/v, chloroform-ethanol. Major bands observed under illumination (254nm) were collected and extracted with methanol. Bands 2 (Rf 0.18) and 4 (Rf 0.36) were further purified with the same TLC conditions to afford stachybotrin A (a yellow oil) and stachybotrin B (as a white solid). Biological Activity Stachybotrins A and B each showed activity in disk assays against Bacillus subtilis (ATCC 6051) at 10~g/disk (8- and 10-mm zones, respectively). They also inhibited the radial growth of the filamentous fungi Ascobolusfurfuraceus (NRRL 6460) and Sordaria fimicola (NRRL 6459) by 50% in centerpoint inoculation assays at 20 and 10ktg/disk, respectively. Disk assays for activity against a strain of Candida albicans (ATCC 14053) were negative at 1001.tg/disk. Stachybotrin A was also evaluated for cytotoxicity against three human solid tumor cell lines (nonsmall carcinoma A-549, breast adenocarcinoma MCF-7, and colon adenocarcinoma HT-29), but afforded only mild activity (EDs0 values 20-30~g/ml). Spectral Data UV:

~,mM~" 220(e=17,000), 254(C=6,400) and 302nm (c=2,900).

196


IR:

(neat) 3262,2918, 1684, 1611, 1462, 1358, 1049, 1025 and 1006cmq. 1H NMR:

(CDC13) 6.74(1H, s, H-4); 2.65(1H, dd, J=17.6, 7.2Hz, H-7); 2.97(1H, dd, J=17.6, 5.5Hz, H-7); 3.88(1H, dd, J=7.2, 5.SHz, H-8); 4.24(2H, br s, H-13); 1.69(2H, m, H14); 2.27 (2H, m, H-15); 5.30(1H, dd, J=7.4, 7.4Hz, H-16); 2.08(2H, m, H-18); 2.07(2H, m, H-19); 5.07(1H, dd, J-1.4, 1.4Hz, H-20); 1.63(3H, s, H-22); 1.55(3H, s, H-23); 4.08(1H, d, J=12.2Hz, H-24); 4.05(1H, d, J=12.2Hz, H-24); and 1.26ppm (3H, s, H-25). 13CNMR: (CDC13) 174.3, C-2; 132.5, C-3; 101.1, C-4; 158.4, C-5; 113.6, C-6; 27.7, C-7; 68.4, C-8; 80.1, C-9; 150.1, C-11; 123.8, C-12, 44.2, C-13; 38.9, C-14; 22.1, C-15; 128.8, C-16; 139.7, C-17; 36.0, C-18; 27.9, C19; 125.4, C-20; 132.2, C-21; 25.5, C-22; 17.7, C-23; 59.9, C-24; and 18.7ppm, C-25. Mass Spectrum: EIMS: 401(M+, 24%), 383(10), 332(7), 314(4), 248(12), 232(26), 216(18), 190(23), 178(57), 147(19), 121(21), 93(24), and 69m/e (base peak); HREIMS: obsd for C23H31NOs, 401.2189, calcd, 401.2202. HPLC Data Beckman C18 Ultrasphere column; 250 x 4.6 mm; 90:10, v/v, MeOH-water, 1.0ml/min;tR 2.95 min. Reference X. Xu, F. S. de Guzman, and J. B. Gloer; Stachybotrins A and B: Novel Bioactive Metabolites from a Brackish Water Isolate of the Fungus Stachybotrys sp.; J. Org. Chem., Vol. 57, pp. 6700-6703(1992).

13.


197

Common/Systematic Name Stachybotrin B Molecular Formula/Molecular Weight C23H31NO4; MW" = 385.22531 r5 OH

o H/N General Characteristics Isolated as a white solid; mp., 178-180~

o. O [~]D q- 39.1 ~ (c=0.11, in MeOH).

Fungal Source

Stachybotrys sp. (ATCC 90017) collected from brackish water in Florida.

Isolation/Purification The culture filtrate was extracted with ethyl acetate and the organic phase was dried (MgSO4) and concentrated to afford a red oil. The extract was dissolved in acetone and divided into four equal portions. Each portion was subjected to preparative TLC on silica gel GF plates developed twice with 9:1, v/v, chloroform-ethanol. Major bands observed under illumination (254nm) were collected and extracted with methanol. Bands 2 (Re 0.18) and 4 (Rf 0.36) were further purified with the same TLC conditions to afford stachybotrin A (a yellow oil) and stachybotrin B (as a white solid). Biological Activity Stachybotrins A and B each showed activity in disk assays against Bacillus subtilis (ATCC 6051) at 10~g/disk (8- and 10-mm zones, respectively). They also inhibited the radial growth of the filamentous fungi Ascobolusfurfuraceus (NRRL 6460) and Sordaria fimicola (NRRL 6459) by 50% in centerpoint inoculation assays at 20 and 101,tg/disk, respectively. Disk assays for activity against a strain of Candida albicans (ATCC 14053) were negative at 100~g/disk. Stachybotrin A was also evaluated for cytotoxicity against three human solid tumor cell lines (nonsmall carcinoma A-549, breast adenocarcinoma MCF-7, and colon adenocarcinoma HT-29), but afforded only mild activity (EDs0 values 20-30~g/ml).

198

13.


Spectral Data IR~

(neat) 3293, 2919, 1653, 1608, 1457, 1350, and 1080cm1. 1H NMR.:

(CDCls) 6.74(1H, s, H-4); 2.64(1H, dd, J=18.0, 6.0Hz, H-7); 2.98(1H, dd, J=18.0, 6.0Hz, H-7), 3.87(1H, dd, J=6.0, 6.0Hz, H-8), 4.23(1, d, J=18.0Hz, H-13); 4.20(1H, d, d=18.0Hz, H-13); 1.67(2H, m, H-14); 2.18(2H, m, H-15); 5.14(1H, m, H-16); 1.95(2H, m, H-18); 2.02(2H, m, H-19); 5.05(1H, m, H-20); 1.63(3H, s, H-22); 1.55(3H, s, H-23), 1.57(3H, s, H-24); and 1.27ppm (3H, s, H-25). 13C NMR: (CDCI3) 174.2, C-2; 132.5, C-3; 100.8, C-4; 158.0, C-5; 113.5, C-6; 27.8, C-7; 68.4, C-8; 80.2, C-9; 150.2, C-11; 124.1, C-12, 44.2, C-13; 38.5, C-14; 22.6, C-15; 125.5, C-16; 136.2, C-17; 40.8, C-18; 27.8, C19; 125.3, C-20; 132.2, C-21; 25.8, C-22; 17.7, C-23; 15.9, C-24; and 18.8ppm, C-25. Mass Spectrum: EIMS: 385(M+, 36%), 311(22), 232(13), 216(16), 190(23), 178(38), 177(30), 147(9), 123(13), 109(15), 95(16), 81(25), and 69m/e (base peak); HREIMS: obsd for C23H31NO4, 385.2270m/e; calcd, 385.2254. HPLC Data Beckman Cls Ultrasphere column; 250 x 4.6 mm; 90:10, v/v, MeOH-water; 1.0ml/min; tR 4.05 min. Reference X. Xu, F. S. de Guzman, and J. B. Gloer; Stachybotrins A and B: Novel Bioactive Metabolites from a Brackish Water Isolate of the Fungus Stachybotrys sp.; J. Org. Chem., Vol. 57, pp. 6700-6703(1992).

Trichothecenes and Related Metabolites Culmorin Trichothecene 813-Hydroxytrichothecene Trichodermin Isotrichodermin 7-Hydroxyisotrichodermin 8a-Hydroxyisotrichodermin 7,8-Dihydroxyisotrichodermin NT- 1 Toxin 8-Ketoisotrichodermin Calonectrin 8-Ketocalonectrin 15-Deacetylcalonectrin 3-Deacetylcalonectrin 7a -Hydroxycalonectrin 8a -Hydroxycalonectrin 7a,8a -Dihydroxycalonectrin 4-Acetoxyscirpenediol 15-Acetoxyscirpenetriol 4,15-Diacetoxyscirpenol Scirpenetriol 7a-Hydroxydiacetoxyscirpenol 7a,8a-Dihydroxydiacetoxyscirpenol 3,15-Diacetoxyscirpenetriol 3,4,15-Triacetoxyscirpenetriol T-2 Tetraol T-2 Toxin HT-2 Toxin Acetyl T-2 Toxin 4-Propanyl HT-2 Toxin 3'-Hydroxy T-2 Toxin 3'-Hydroxy HT-2 Toxin 3'-Hydroxy T-2 Triol Neosolaniol Isoneosolaniol (Acuminatum) 8-Acetoxyneosolaniol 8-Propanylneosolaniol 8-Butyrylneosolaniol 8-Isobutyrylneosolaniol 15-Deacetylneosolaniol (NT-2 Toxin) 4-Deacetylneosolaniol Sporotrichiol 413,8a,15-Triacetoxy-3a,7a-dihydroxy- 12,13-epoxytrichothec-9-ene

199

200

14. Trichothecenes and Related Metabolites

3-Acetyl-4-deoxynivalenol Trichodermol 7a-Hydroxytrichodermol Verrucarol 4,15-Deacetylverrucarol Crotocin (Antibiotic T) Crotocol Trichothecolone Trichothecin Trichothecinol A Trichothecinol B Trichothecinol C Trichodermone Nivalenol 4-Deoxynivalenol (Rd Toxin, Vomitoxin) 3-Acetyl-4-deoxynivalenol 413,15-Diacetoxynivalenol 413,15-Diacetyl-7-deoxynivalenol

15-Acetyl-4-deoxynivalenol

3,15-Diaeetyl-4-deoxynivalenol Fusarenon-X (Nivalenol Monoacetate)


201

Common/Systematic Name Culmorin Molecular Formula/Molecular Weight C15H2602; MW

-- 2 3 8 . 1 9 3 2 8 4

3

1

HO/

14

7

OH

General Characteristics Colorless needles from tetrahydrofuran-cyclohexane; mp., 178-179~ (c=0.60, in CHC13).

[ ~ ] D 24

-

15~

Fungal Source Fusarium roseum (ATCC 28114), F. culmorum, F. graminearum, and F. crookwellense.

Isolation/Purification Fusarium roseum cultures were harvested, and the broth was extracted as described by Greenhalgh et al. (1984). Some of the oil from the methanol phase after it had been

partitioned and concentrated was dissolved in methylene chloride and passed through a Florisil column eluted with 750ml of (A) methylene chloride, (B) 0.5% methanol in methylene chloride, and (C) 5% methanol in methylene chloride. Fraction A afforded an oil from which 3-acetyldeoxynivalenol was obtained. Fractions B and C also afforded yellow oils. The oil which remained in fraction A alter removal of 3-acetyldeoxynivaienol was chromatographed on silica gel (Kieselgel 60). The column was eluted first with 10% toluene in methylene chloride and then with 0.5% methanol in methylene chloride, 1.5% methanol in methylene chloride, 3% methanol in methylene chloride, and finally 13% methanol in methylene chloride. Fractions were collected and monitored by TLC; fractions 6-10, 11-12, 13-17, and 18-20 were combined to give mixtures A1, A2, A3, and A4, respectively. Further purification of mixtures A2 and A3 was accomplished with a Chromatotron (Model No. 7924, Harrison Research, Inc.) with a 2mm silica gel plate (Kieselgel 60). Culmorin was isolated from mixture A3 by elution with 50% ethyl acetate in hexane followed by recrystallization from tetrahydrofuran-cyclohexane.

202


Spectral Data 1H ~ : (CDC13) 0.70(3H, H-13), 0.86(3H, H-12), 0.90(3H, H-14), 1.02(3H, H-15), 1.32.1(6H, H-3, H-4, H-5, m), 1.64(2H, J1o,l~=6.8Hz, H-10), 1.74(1H, J7,s=5.0Hz, H-7), 1.90(1H, Y1,11=4.5Hz, H-l), 3.86(1H, J2,s=8.0Hz, H-8), and 4.35ppm (1H, dlo,~1=6.8, Yll,l=4.5Hz, H-11). Mass Spectrum: HR IMS: 238.1922m/e (M+); calcd for C15H2602,238.1922. References R. Greenhalgh, R. M. Meier, B. A. Blackwell, J. D. Miller, A. Taylor, and J. W. Apsimon; Minor Metabolites ofFusarium roseum (ATCC 28114); J. Agric. Food Chem., Vol. 32, pp. 1261-1264(1984). D. R. Lauren, A. Ashley, B. A. BlaekweU, R. Greenhalgh, J. D. Miller, and G. A. Neish, Tilchothecenes Produced by Fusarium crookwellense DAOM 193611, J. Agile. Food Chem., Vol. 35, pp. 884-889(1987).

14. Trichothecenes

and Related Metabolites

203

Common/Systematic Name Trichothecene 12,13-Epoxytrichothec-9-ene Molecular Formula/Molecular Weight C15H2202; M W = 234.16198 ~o H

~ 6 ~ 0 . ~

~

I-

"1

H

13

2 3 I .~

~,~"u

i~le ~ ~ 15

4

14

General Characteristics Crystals from aqueous methanol; mp., 54-57~ Fungal Source Trichothecium roseum.

TLC Data Preparative TLC using silica gel developed with benzene-ethyl acetate (10:2, v/v). References Y. Fujimoto, S. Yokuna, T. Nakamura, T. Morikawa, and T. Tatsuno; Total Synthesis of 12,13-Epoxytrichothec-9-ene; Tetrahedron Letters, pp. 2523-2526(1974). N. Masouka and T. Kawikawa; A Synthesis of 12,13-Epoxytrichothec-9-ene; Tetrahedron Letters, pp. 1691-1694(1976).

204


Common/Systematic Name 8[3-Hydroxytrichothecene 813-Hydroxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C15H2203; MW = 250.15689

~o H

H

[-8 ';l

H O f ~

~2 14

Fungal Source

Fusarium sporotrichioides (MC-72083).

Spectral Data 1H NMR:

(CDCI3) 3.65(1H, d, J=3.9Hz, H-2); 1.91(1H, m, H-3); 1.57(1H, dd, J=13.0, 7.0Hz, H-3); 2.78(1H, dd, J=13.0, 7.0Hz, H-4); 1.68(1H, dd, J=13.6, 7.0Hz, H-4); 2.03(1H, dd, J=12.8, 5.5Hz, H-7); 1.15(1H, dd, J=12.5, 9.3Hz, H-7); 4.12(1H, m, H-8); 5.27(1H, m, H-10); 4.66(1H, m, H-11); 3.37(1H, d, J=4.1Hz, H-13); 3.06(1H, d, J=4.1Hz, H-13); 0.96(3H, s, CH3-14); 0.96(3H, s, CH3-15); and 1.77ppm (3H, s, CH316). 13CNMR: (CDC13) 81.6, C-2; 30.2, C-3; 36.8, C-4; 46.8, C-5; 44.8, C-6; 39.9, C-7; 69.2, C-8; 136.2, C-9; 128.9, C-10; 71.6, C-11; 69.1, C-12; 49.3, C-13; 20.4, C-14; 13.5, C-15; and 18.2ppm, C- 16. Mass Spectrum: HREIMS: 250.150m/e (M+); calcd for C15H2203, 250.156. References D. G. Corley, G. E. Rottinghaus, and M. S. Tempesta; Novel Trichothecenes from Fusarium sporotrichioides; Tetrahedron Letters, Vol. 27, pp. 427-430(1986). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).


205

Common/Systematic Name Trichodermin 413-Acetoxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2404; ~

-- 2 9 2 . 1 6 7 4 6

lo H

H

I~le J s 14

'~A

C

General Characteristics Synthetic trichodermin; mp., 58-60~ natural trichodermin crystallized from pentane; mp., 45-46~ [a]D2~ - 11.0~ (C=I.0, in CHC13). Insoluble in water; soluble in all common organic solvents. Fungal Source

Trichoderma lignorum, T. viride, and Stachybotrys cylindrospora, a fungal pathogen of aspen (Populus tremuloides) that has been found to be strongly antagonistic to the bluestain fungus, Ceratocystiopsis crassivaginata.

Biological Activity A potent inhibitor of protein synthesis in mammalian cells. Trichodermin inhibited the elongation and/or termination steps in protein synthesis. In vitro, it was a potent inhibitor of the peptidyl transferase activity required for elongation and/or termination. LDs0 in mice dosed SC was 500-1000mg/kg. Trichodermin is also a potent antifungal antibiotic. Spectral Data UV:

205nm (e=2,400). I_R:

(KBr) 1730, 1682, 1245, 1225, and 1085cm1; (CHC13) 2970, 1735, 1374, 1244, 1220, 1081, 1031,996, and 970cm1. 1H NMR: (CDC13) 3.80, H-2; 1.9-2.5, H-3; 5.60, H-4; 1.9-2.5, H-7; 1.9-2.5, H-8; 5.4, H-10; 3.7, H-11; 2.80, ttH-13; 3.10, 13H-13; 0.95, H-14; 0.75, H-15; 1.70, H-16; and 2.05ppm, CH3COO-.

206


13C NMR: 79.1 d, C-2; 36.7 t, C-3; 75.0 d, C-4; 48.9 s, C-5; 40.4 s, C-6; 24.5 t, C-7; 28.0 t, C-8; 140.1 s, C-9; 118.6 d, C-10; 70.5 d, C-11; 65.5 s, C-12; 47.8 t, C-13; 5.8 q, C-14; 16.0 q, C-15; 23.3 q, C-16; 21.1 q, CH3COO-; and 170.8ppm, s, CH3COO. Mass Spectrum: HREIMS: 292.1676(C17H2404, Amm~=0.2, 100), 277.1441(C16H2104, 78), and 217.1230m/e (C14H1702, 10%). TLC Data A. Adsorbent, silica gel G; solvent, chloroform-methanol, 98:2, v/v; Re, 0.67; detection: H2SO4 spraying and heating at 110~ for 5 minutes. B. Adsorbent, silica gel G; solvent: benzene-tetrahydrofuran, 85:15, v/v; Rf, 0.51; detection, H2SO4 spraying and heating at 110~ for 5 minutes. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 162 (1981). W.O. Godtfredsen and S. Vangedal; Trichodermin, A N e w Antibiotic, Related to

Trichothecin; Proc. Chem. Sot., Vol. 19, pp. 188-189(1964). W. A. Ayer and S. Miao; Secondary Metabolites of the Aspen Fungus Stachybotrys cylindrospora; Can. J. Chem., Vol. 71, pp. 487-493(1993).


207

Common/Systematic Name Isotrichodermin 3 a-Acetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2404; MW

= 292.16746

lo H: c~ H�9

16

-.-

~"

13

~el~ ~ 15

2

H I

3

"'OAc

iI

4

14

General Characteristics Colorless crystals; mp, 97-97.5~

[a]D 24 d- 9.5 ~ (c=0.09,

in CHC13).

Fungal Source Fusarium culmorum, F. roseum, F. graminearum, and F. crookwellense.

Isolation/Purification Fusarium roseum culture broth was extracted with methylene chloride alter being treated

with sodium chloride and adjusted to pH 9.3 with sodium hydroxide. The methylene chloride extract was evaporated to dryness and the residue partitioned between aqueous methanol and petroleum ether. Some of the oil from the methanol phase after it had been partitioned and concentrated was dissolved in methylene chloride and passed through a Florisil column eluted with 750ml of (A) methylene chloride, (B) 0.5% methanol in methylene chloride, and (C) 5% methanol in methylene chloride. Fraction A afforded an oil from which 3-acetyldeoxynivalenol was obtained. Fractions B and C also afforded yellow oils. The oil which remained in fraction A alter removal of 3-acetyldeoxynivalenol was chromatographed on silica gel (Kieselgel 60). The column was eluted first with 10% toluene in methylene chloride and then with 0.5% methanol in methylene chloride, 1.5% methanol in methylene chloride, 3% methanol in methylene chloride, and finally 13% methanol in methylene chloride. Fractions were collected and monitored by TLC; fractions 6-10, 11-12, 13-17, and 18-20 were combined to give mixtures A1, A2, A3, and A4, respectively. Further purification of mixtures A2 and A3 was accomplished with a Chromatotron (Model No. 7924, Harrison Research, Inc.) with a 2mm silica gel plate (Kieselge160). Isotrichodermin was isolated from mixture A2 by elution with 20% ethyl acetate in hexane. Spectral Data 1H N]VIR:

(CDCI3) 0.74(3H, s, H-15); 0.80(3H, s, H-14); 1.0-2.0(6H, m, H-7, H-4, H-8);

2.08


1.71(3H, br s, H-16); 2.1 l(3H, s, Ac-CH3); 2.84, 3.07(2H, Jab=4.0Hz, H-13); 3.73(1H, d, JE,a=4.6Hz, H-2); 3.96(1H, d, JlO,ll=5.5Hz, H-11); 5.15(1H, dt, J24=10.0, JE,a=4.6Hz, H-3); and 5.44ppm (1H, dq, J1o,ll=5.5Hz, Jlo,16=l.5Hz, H-10). 13C NMR: (CDC13) 78.2, C-2; 71.7, C-3; 38.6, C-4; 45.3, C-5; 40.2, C-6; 24.4, C-7; 28.2, C-8; 139.5, C-9; 119.4, C-10; 71.5, C-11; 65.2, C-12; 48.4, C-13; 11.0, C-14; 15.9, C-15; 23.1, C-16; 20.9, CH3COO-; and 170.6ppm, C=O. Mass Spectrum: HREIMS: 293.174m/e (M+ + 1); calcd for C17H2504,293.168. References R. Greenhalgh, R. M. Meier, B. A. Blackwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Minor Metabolites ofFusarium roseum (ATCC 28114); J. Agric. Food Chem., Vol. 32, pp. 1261-1264(1984). D. R. Lauren, A. Ashley, B. A. Blackwell, R. Greenhalgh, J. D. Miller, and G. A. Neish; Trichothecenes Produced by Fusarium crookwellense DAOM 193611; J. Agric. Food Chem., Vol. 35, pp. 884-889(1987).


209

Common/Systematic Name 7-Hydroxyisotrichodermin 7a-Hydroxy-3a-acetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2405; M'W -- 308.16237

1 6 ~ ~ 0 ~lo2 H

3,

, "4 -

" '

15

OH

14

General Characteristics Crystalline solid from ether-petroleum ethers; mp., 154-155.5oC; in CHC13).

[ a ] D 22 -

10.6 ~ (c=0.095,

Fungal Source Fusarium culmorum, F. roseum (ATCC 28114), and F. crookwellense.

Isolation/Purification Fusarium roseum culture broth was extracted with methylene chloride after being treated

with sodium chloride and adjusted to pH 9.3 with sodium hydroxide. The methylene chloride extract was evaporated to dryness and the residue partitioned between aqueous methanol and petroleum ether. Some of the oil from the methanol phase after it had been partitioned and concentrated was dissolved in methylene chloride and passed through a Florisil column eluted with 750ml of (A) methylene chloride, (B) 0.5% methanol in methylene chloride, and (C) 5% methanol in methylene chloride. Fraction A afforded an oil which was subjected to medium pressure liquid chromatography on a LiChroprep Si 60 column.The column was eluted with the following solvents: 10% ethyl acetate-hexane; 20% ethyl acetate-hexane; 30% ethyl acetate-hexane; and ethyl acetate. A metabolite eluting in the 10% ethyl acetate fractions was further purified by elution initially with chloroform and then 2% methanol-chloroform. The metabolite was recrystallized from ether-petroleum ether to give a crystalline solid of 7-hydroxyisotrichodermin. Spectral Data IH ~ : (CDC13) 0.82(3H, s, CH3-15); 1.04(3H, s, CH3-14); 1.09(1H, 7-OH, J=6.3Hz); 1.70(3 H, br s, CH3-16); 2.10(3H, s, Ac-CH3); 1.9, 2.2(2H, dd, J=17.2, 10.0; 17.2, 6.0Hz, H-8); 2.07, 2.10(2H, m, 2H-4); 3.08, 3.13(2H, d, J=4.4, 4.4Hz, H-13); 3.76(1H, d, J2,3=4.5Hz, H-2); 4.10(1H, d, J11,1o=5.4Hz, H-11); 4.46(1H, JT,s=5.9, 10.1, J7,1o=6.3Hz); 5.16(1H, ddd, J=9.8, 5.2, 4.5Hz, H-3); and 5.40ppm (1H, br d, J=5.4Hz, H-10).

210


13C NMR: (CDC13) 79.0, C-2; 74.1, C-3; 39.5, C-4; 44.6, C-5; 46.2, C-6; 74.7, C-7; 41.4, C-8; 138.0, C-9; 119.6, C-10; 69.0, C-11; 64.9, C-12; 47.7, C-13; 10.2, C-14; 15.0, C-15; 22.4, C-16; 20.9, CH3COO-; and 170.6ppm, C=O. Mass Spectrum: HREIMS: 308.163m/e (M~); calcd for C17I-I2405, 308.162. References R. Greenhalgh, R. M. Meier, B. A. Blackwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Minor Metabolites ofFusarium roseum (ATCC 28114); J. Agric. Food Chem., Vol. 34, pp. 115-118(1986). D. R. Lauren, A. Ashley, B. A. Blackwell, R. Greenhalgh, J. D. Miller, and G. A. Neish; Trichothecenes Produced by Fusarium crookwellense DAOM 193611; J. Agric. Food Chem., Vol. 35, pp. 884-889(1987).


211

Common/Systematic Name 8tt-Hydroxyisotrichodermin 8t~-Hydroxy-3t~-acetoxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2405,

16

~

= 308.16237

10 H U

2

HO'"[~~~~I~" ' 191e~ ~

3

o

....

15

14

General Characteristics Crystals from ethyl acetate-hexane; mp., 153-155.5~

[tg]D 21 -

18.2~ (c=0.11, in CHC13).

Fungal Source Fusarium culmorum, F. roseum (ATCC 28114), and F. crookwellense. Isolation/Purification Fusarium roseum culture broth was extracted with methylene chloride after being treated with sodium chloride and adjusted to pH 9.3 with sodium hydroxide. The methylene chloride extract was evaporated to dryness and the residue partitioned between aqueous methanol and petroleum ether. Some of the oil from the methanol phase after it had been partitioned and concentrated was dissolved in methylene chloride and passed through a Florisil column eluted with 750ml of (A) methylene chloride, (B) 0.5% methanol in methylene chloride, and (C) 5% methanol in methylene chloride. Fraction A afforded an oil which was subjected to medium pressure liquid chromatography on a LiChroprep Si 60 column. The column was eluted with the following solvents: 10% ethyl acetate-hexane; 20% ethyl acetate-hexane; 30% ethyl acetate-hexane; and ethyl acetate. An oil eluting in the 10% ethyl acetate fractions was further purified with a Chromatotron eluted with chloroform followed by 5% methanol in chloroform. 8-Hydroxyisotrichodermin and 8hydroxy-3tt-acetoxy-12,13-epoxytrichothec-9-ene were purified by preparative TLC with 7% methanol in chloroform. Spectral Data 1H NMR: (CDC13) 3.72(1H, d,J=4.6Hz, H-2); 5.16(1H, ddd, J=9.8, 5.5, 4.6Hz, H-3); 2.09(2H, m, J=9.8, 5.5Hz, H-4); 1.70(1H, dd, J=14.3, 1.4Hz, H-7); 2.21(1H, dd, J=14.3, 5.7Hz, H-7); 4.10(1H, br d, J=5.7Hz, H-8); 5.59(1H, d, J=5.7Hz, H-10); 4.04(1H, d, J=5.7Hz, H-11); 2.87(1H, d, J=4.1Hz, H-13); 3.06(1H, d, J=4.1Hz, H-13), 0.77(3H, s, CHs-14); 0.98(3H, s, CH3-15); 1.86(3H, br s, CH3-16); and 2.1 lppm (3H, s, CH3COO-).

212


13C NMR: (CDC13) 78.1, C-2; 71.5, C-3; 38.1, C-4; 45.4, C-5; 39.6, C-6; 33.6, C-7; 68.0, C-8; 138.8, C-9; 122.1, C-10; 71.3, C-11; 65.2, C-12; 48.5, C-13; 11.0, C-14; 19.0, C-15; 20.4, C-16; 20.9, CH3COO-; and 170.6ppm, C=O. Mass Spectrum: HREIMS: 308.166m/e (Mr); ealcd for C17H2405,308.162. References R. Greenhalgh, R.M. Meier, B. A. Blaekwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Minor Metabolites ofFusarium roseum (ATCC 28114); J. Agrie. Food Chem., Vol. 34, pp. 115-118(1986). D. R. Lauren, A. Ashley, B. A. Blackwell, R. Greenhalgh, J. D. Miller, and G. A. Neish; Trichotheeenes Produced by Fusarium crookwellense DAOM 193611; J. Agrie. Food Chem., Vol. 35, pp. 884-889(1987).


213

Common/Systematic Name 7,8-Dihydroxyisotrichodermin 7,8-Dihydroxy-3 t~-acetoxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2406; MW : 324.15729 16

10 8

H 6

OH

13~

,,~

I

....OAc

14

General Characteristics White solidi mp., 80-84 ~ Fungal Source Fusarium crookwellense.

Isolation/Purification The fungal culture filtrate was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and evaporated to dryness to yield a yellow gum. The yellow gum was chromatographed on a silica gel column eluted initially with 10% ethyl acetate-chloroform and increasing the percentage of ethyl acetate. Ethyl acetate eluted an oil that was further purified by preparative HPLC using a cyano-bonded phase column with 3% 2-propanolhexane as mobile phase to give pure 7,8-dihydroxyisotrichodermin. Spectral Data 1H NIV[R:

(CDC13) 3.76(1H, d, J=4.SHz, H-2); 5.17(1H, m, J=8.8, 6.9, 4.5Hz, H-3); 2.05(2H, m, Jt~=6.9, 7.8Hz, J][3=8.8, 7.8Hz, H-4); 4.41(1H, dd, J=9.7, 5.5Hz, H-7); 3.98(1H, bt, J=6.0Hz, H-8); 5.59(1H, dq, J=5.9, 1.6Hz, H-10); 4.16(1H, d, J=5.9Hz, H-11); 3.10(1H, d, J=4.3Hz, H-13); 3.17(1H, d, J=4.3Hz, H-13); 1.06(3H, s, CH3-14); 0.89(3H, s, CH3-15); 1.87(3H, br s, CH3-16); and 2.10ppm (3H, s, CH3COO-). 13C NMR: 78.9 C-2; 71.5, C-3; 40.6, C-4; 46.4, C-5; 44.2, C-6; 73.8, C-7; 71.1, C-8; 138.8, C-9; 122.6, C-10; 69.8, C-11; 65.0, C-12; 47.8, C-13; 13.2, C-14; 14.7, C-15; 20.3, C-16; 20.9, CH3COO-; and 170.6ppm, C-O. Mass Spectrum: LREIMS: 324(NV, 27%), 165(24), 148(47), 123(49), 135(61), 95(51), 107(75), and 100m/e (100).

214


Reference D. R. Lauren, A. Ashley, B. A. Blackwell, R. Greenhalgh, J. D. Miller, and G. A. Neish; Trichothecenes Produced by Fusarium crookwellense DAOM 193611; J. Agile. Food Chem., Vol. 35, pp. 884-889(1987).

14. Trichothecenes and

Related Metabolites

215

Common/Systematic Name NT- 1 Toxin 413,8a-Diacetoxy-3 a, 15-dihydroxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C19H2608; MW = 382.16277 lo

..

H

H

1~)~O-,~

H

2 3 i .... 'OH

H

'2"0 -

l

- 14 CH2OH 15 General Characteristics

'I ,,H OAc

Crystals from benzene-n-hexane; mp., 172.5-173.5~ Fungal Source

Fusarium sporotrichioides (strain M- 1-1), F. tricinctum, F. equiseti, and F. acuminatum.

Isolation/Purification Extracted by charcoal absorption and elution with methanol. Extraction of methanolchloroform soluble materials produced a pale yellow powder. The powder was chromatographed on a silica gel column eluted with n-hexane, n-hexane-acetone, 12:5, 2:1, 1:1, 1:2 (v/v), acetone and methanol followed by a second silica gel column eluted with benzene-acetone (3:2) and n-hexane-acetone (1:1, v/v). NT-1 was crystallized with benzene-n-hexane. Biological Activity Inhibited uptake of [14C]leucine in protein synthesis with rabbit reticulocytes; IDs0 at concentrations of 0.23 and 0.251.tg/ml by NT-1 and NT-2, respectively. These activities were similar to that of neosolaniol (0.25~g/ml) in the same assay. LDs0in rats dosed IP was 1.2mg/Kg. Spectral Data Im: (KBr) 3450, 2940, 1720, and 1230cm"1. 1H NMP< (CDCl3) 3.66(H-2); 4.24(H-3), 5.40(H-4); m.96(H-7cx);2.34(H-7~); 5.36(H-8); 5.82(H-10); 4.27(H-11); 2.80(H-13a); 3.04(H-13~); 0.84(H-]4); 3.60(H-15a); 3.91(H-1513); 1.75(H-16); and 2.09, 2.14ppm (3H each, CHACO-).

216


Mass Data: LREIMS: 382m/e (M+); found: C, 59.69; H, 6.82; O, 33.49%; calcd for C19H2608 ; C, 59.65; H, 6.85; O, 33.50%. TLC Data Adsorbent: silica gel; solvent: ethyl acetate-petrol (3:1, v/v); Re, 0.4; detection: sky-blue fluorescence in UV after spraying withp-anisaldehyde and heating. References K. Ishii and Y. Ueno; Isolation and Characterization of Two New Trichothecenes from Fusarium sporotrichioides Strain M-1-1; Applied and Environmental Microbiology, Vol. 42, pp. 541-543(1981). A. Visconti, C. J. Mirocha, A. Logrieco, A. Bottalico, and M. Solfrizzo; Mycotoxins Produced by Fusarium acuminatum: A New Trichothecene; J. Agric. Food Chem., Vol. 37, pp. 1348-1351(1989).


217

Common/Systematic Name 8-Ketoisotrichodermin 8-Keto-3 tt-acetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2205; l~trW = 306.14672

~o H

~6~o.,J

H

2

3.,,"OAc

,,,o I

J: 15 14

General Characteristics White solidi mp., 122-124~ Fungal Source

Fusarium crookwellense.

Isolation/Purification The fungal culture filtrate was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and evaporated to dryness to yield a yellow gum. The yellow gum was chromatographed by a silica gel column eluted initially with 10% ethyl acetate-chloroform and increasing the percentage of ethyl acetate. Ethyl acetate eluted an oil that was further purified by preparative HPLC using a cyano-bonded phase column with 3% 2-propanolhexane as mobile phase. Spectral Data 1H ~ :

(CDCI3) 3.83(1H, d, J=4.6Hz, H-2); 5.22(1H, m, ,/--4.6, 10.9, 4.6Hz, H-3); 2.03(2H, dd, Jtt=14.8, 10.9Hz, JI3=14.8, 4.6Hz, H-4); 2.26(1H, dd, J=15.3, 1.1Hz, H-7); 2.87(1H, d, J=15.3Hz, H-7); 6.51(1H, dd, J=5.7, 1.5Hz, H-10); 4.32(1H, d, J=5.7Hz, H-11); 2.86(1H, d, J=3.9Hz, n-13); 3.10(1H, d, J=3.9Hz, n-13); 0.75(3H, s, CH3-14); 0.92(3H, d, d=l. 1Hz,CH3-15); 1.81(3H, d, J=l.5Hz, CH3-16); and 2.14ppm (3H, s, cn3coo-). 13CNMR: (CDC13) 78.5, C-2; 71.4, C-3; 42.1, C-4; 45.4, C-5; 43.4, C-6; 37.8, C-7; 189.0, C-8; 137.7, C-9; 137.6, C-10; 71.6, C-11; 57.5, C-12; 48.1, C-13; 10.6, C-14; 18.3, C-15; 15.3, C-16; 20.8, CH3COO-; and 172.5ppm, C=O. Mass Spectrum: LREIMS: 306(M+, 3%), 216(27), 253(35), 138(92), 123(100), 107(42), and 79m/e (49).

218


Reference D. R. Lauren, A. Ashley, B. A. Blackwell, R. Greenhalgh, J. D. Miller, and G. A. Neish; Trichothecenes Produced by Fusarium crookwellense DAOM 193611" J. Agric. Food Chem., Vol. 35, pp. 884-889(1987).

14.

T r i c h o t h e c e n e s and Related M e t a b o l i t e s

219

Common/Systematic Name Calonectrin 3 t~, 15-Diacetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C 19H2606; MW -- 350.17294 16

1o H

H

s

r. 14

H

~i'''H H

CH2OAc 15

General Characteristics Prisms from ethyl ether-petroleum ether; mp., 83-85~

[ ~ ] D 27 -

14.6 ~ (c=l.0, in CHCI3).

Fungal Source Fusarium culmorum, F. roseum (ATCC 28114), and Calonectria nivalis.

Isolation/Purification. Fusarium roseum culture broth was extracted with methylene chloride atter being treated

with sodium chloride and adjusted to pH 9.3 with sodium hydroxide. The methylene chloride extract was evaporated to dryness and the residue partitioned between aqueous methanol and petroleum ether. Some of the oil from the methanol phase atter it had been partitioned and concentrated was dissolved in methylene chloride and passed through a Florisil column eluted with (A) methylene chloride, (B) 0.5% methanol in methylene chloride, and (C) 5% methanol in methylene chloride. Fraction A afforded an oil which was subjected to medium pressure liquid chromatography on a LiChroprep Si 60 column. The column was eluted with the following solvents: 10% ethyl acetate-hexane; 20% ethyl acetate-hexane; 30% ethyl acetate-hexane; and ethyl acetate. The fractions collected (651) were reduced to 52 samples by combining appropriate fractions. Calonectrin was isolated from an oil eluted in fraction 13 and purified by column chromatography on silica gel eluted with 5% methanol in chloroform. Spectral Data l-R:

(KBr) 1745, 1725, 1240, and 965cm "1. 1H N:V[R: (CDCI3) 3.82(2H, d, J=12.2Hz, ttCH2-15); 4.06(2H, d, J=12.2Hz, 13CH2-15); 0.81(3 H, s, CH3-14); 1.78-2.19(2H, m, H-7); 1.70(3 H, br s, CH3-16); 2.01(3H, s, Ac-CH3);

220


1.7-2.19(2H, m, H-8); 2.1-2.19(2H, m, H-4); 2.84, 3.08(2H each, d, J=4.0, 4.0Hz, 2H13t~ and 2H-1313, respectively); 3.74(1H, d, J=4.6Hz, H-2); 4.00(1H, d, J=5.6Hz, HI 1; 5.15(1H, ddd, J=9.6, 7.4, 4.6Hz, H-3); 2.01(3H, s, CH3COO); 2.08(3H, s, CH3COO); and 5.45ppm (1 H, dq, J=5.4, 1.3Hz, H-10). 13CNMR: (CDC13) 78.0, C-2; 71.2, C-3; 39.35, C-4; 45.3, C-5; 42.9; C-6; 20.9, C-7; 28.1, C-8; 140.3, C-9; 118.9, C-10; 68.0, C-11; 64.9, C-12; 48.4, C-13; 12.0, C-14; 63.5, C-15; 23.1, C-16; 20.9, 21.0, CH3COO-; and 170.6, 170.7ppm, C=O. References R. Greenhalgh, D. Lavandier, W. Adams, J. D. Miller, B. A. Blackwell, A. J. McAlees, and A. Taylor; Production and Characterization of Deoxynivalenol and Other Secondary Metabolites of Fusarium culmorum (CMI 14764, HLX 1503); J. Agric. Food Chem., Vol. 34, pp. 98-102(1986). R. Greenhalgh, R.M. Meier, B. A. Blackwell, J. D. Miller, A. Taylor, and J.W. ApSimon; Minor Metabolites ofFusarium roseum (ATCC 28114), Part 2; J. Agile. Food Chem., Vol. 34, pp. 115-118(1986). D. R. Lauren, A. Ashley, B. A. BlackweU, R. Greenhalgh, J. D. Miller, and G. A. Neish; Triehothecenes Produced by Fusarium crookwellense DAOM 193611; J. Agile. Food Chem., Vol. 35, pp. 884-889(1987). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 192 (1981).


221

.Common/Systematic Name 8-Ketocalonectrin 3tz, 15-Diacetoxy-12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C19H2407; M W -- 3 6 4 . 1 5 2 2 0

16

lo

H

_

~'i''O .... 'OAc -

C)O~2

:

14

i

4

~H2OAc 15

General Characteristics Isolated as an oil. Fungal Source

Fusarium culmorum.

Isolation/Purification Mycelia were extracted with methylene chloride, reduced to dryness, redissolved in methanol-water and partitioned against petroleum ether. The methanol raffinate was evaporated to dryness, redissolved in hot absolute alcohol and hexane added slowly. On cooling the solution deposited crystals of 3-acetyldeoxynivalenol which were removed for further purification. The brown viscous oil from the mother liquor was dissolved and chromatographed on a silica gel column eluted with the following: 25% ethyl acetate in methylene chloride; 2% methanol in methylene chloride; 5% methanol in methylene chloride; and methanol. Fractions containing 8-ketocalonectrin were combined and obtained pure by preparative TLC. Spectral Data ~H NMR: (CDC13) 0.81(3H, s, CH3-14), 1.82(3H, d, J16,1o=l.6Hz, CH3-16), 1.96(3H, s, CH315Ac); 2.14(3H, s, CHa-3Ac); 1.95-2.23(2H, m, H-4); 2.51, 2.85(2H, dd, JAB=16.0Hz, J7~,ls=l.5Hz, n-7); 2.88, 3.12(2H, d, JAB=3.8Hz, n-13); 3.84(2H, d, JE,a=4.6Hz, n-2); 4.07(2H, m, H-15); 4.44(1H, d, Jl1,~o=5.1Hz, H-11); 5.23(1H, ddd, da,E=4.6Hz, d3,4-5.5, 10.4Hz, H-3); and 6.55ppm (1H, dd, JLO,11=5.1Hz,Jll,16=l.6Hz, H-10).

13CNMR: (CDC13) 78.1, C-2, 71.2, C-3; 38.5, C-4; 45.0, C-5; 47.5, C-6; 38.4, C-7; 198.7, C-8, 138.3, C-9; 137.5, C-10; 68.2, C-11; 65.3, C-12; 48.3, C-13; 11.3, C-14; 64.2, C-15; 15.4, C-16, 20.9, CHaCOO-, and 170.5ppm, C=O.

222


Mass Spectrum: LREIMS: 364(11%, M§ 262 (18), 203, (13), 109 (19); 121, and 43m/e (100). Reference R. Greenhalgh, D. Lavandier, W. Adams, J. D. Miller, B. A. Blackwell, A. J. McAlees, and A. Taylor; Production andCharacterization of Deoxynivalenol and Other Secondary Metabolites of Fusarium culmorum (CMI 14764, HLX 1503); J. Agric. Food Chem., Vol. 34, pp. 98-102(1986).

14.

Trichothecenes and Related Metabolites

223

Common/Systematic Name 15-Deacetylcalonectrin 3 tt-Acetoxy- 15-hydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2405; M W = 3 0 8 . 1 6 2 3 7

~6

lo H

H

H

- O...]2 [.,3,,,OAc :

l~

l'~,'''H

-

14

H

~H2OH

15

General Characteristics Colorless prisms from ethyl acetate-hexane, mp., 181-183~ [~]D 26 q- 10.2 ~ (c=0.8, in CHC13); prisms from ethyl ether-light petroleum; mp., 184-186~ Fungal Source Fusarium culmorum, F. roseum, F. graminearum, and Calonectria nivalis.

Isolation/Purification Fusariurn roseum was cultured, harvested, and the broth extracted as described by Greenhalgh et al. (1984). Some of the oil from the methanol phase alter it had been

partitioned and concentrated was dissolved in methylene chloride and passed through a Florisil column eluted with 750mL of(A) methylene chloride, (B) 0.5% methanol in methylene chloride, and (C) 5% methanol in methylene chloride. Fraction A afforded an oil from which 3-acetyldeoxynivalenol was obtained. Fractions B and C also afforded yellow oils. The oil which remained in fraction A alter removal of 3-acetyldeoxynivalenol was chromatographed on silica gel (Kieselgel 60). The column was eluted first with 10% toluene in methylene chloride and then with 0.5% methanol in methylene chloride, 1.5% methanol in methylene chloride, 3% methanol in methylene chloride, and finally 13% methanol in methylene chloride. Fractions were collected and monitored by TLC; fractions 6-10, 11-12, 13-17, and 18-20 were combined to give mixtures A1, A2, A3, and A4, respectively. Further purification of mixtures A2 and A3 was accomplished with a Chromatotron (Model No. 7924, Harrison Research, Inc.) with a 2mm silica gel plate (Kieselgel 60). Isotrichodermin was isolated from mixture A2 by elution with 20% ethyl acetate in hexane. Changing the solvent to 20% ethyl acetate in hexane gave pure 15deacetylcalonectrin.

224


Spectral Data 1H NMR: (CDCI3) 3.72(1H, d, J2,3=4.68Hz, H-2); 5.13(1H, ddd, J=10, 5.0, 4.6Hz, H-3); 1.732.30(2H, m, H-4); 1.73-2.30(2H, m, H-7); 1.73-2.30(2H, m, H-8); 5.46(1H, dd, J=5.3, 1.5Hz, H-10); 3.96(1H, d, J=5.3Hz, H-11); 2.84, 3.07(1H each, d, J=4.0Hz, H-13); 0.90(3H, s, CH3-14); 3.47(1H, d, J=l 1.6Hz, H-15); 3.66(1H, ddd, J=l 1.6, 4.5, 3.2Hz, H-15); 1.71(3H, d, J=l.5Hz, H-16); and 2.09ppm (3H, s, CH3COO-). 13CNMR: (CDC13) 78.2, C-2; 71.5, C-3; 39.4, C-4; 45.3, C-5; 44.2, C-6; 20.8, C-7; 28.5, C-8; 140.5, C-9; 119.5, C-10; 68.3, C-11; 65.2, C-12; 48.5, C-13; 12.3, C-14; 62.8, C-15; 23.1, C-16; 20.8, CH3COO-; and 170.6ppm, C=O. Mass Spectrum: HREIMS: 309.167m/e

( M ++

1); calcd for C17H2505, 309.162.

References R. Greenhalgh, D. Levandier, W. Adams, J. D. Miller, B. A. Blackwell, A. J. McAlees, and A. Taylor; Production and Characterization of Deoxynivalenol and Other Secondary Metabolites o f F u s a r i u m culmorum (CMI 14764, HLX 1503); J. Agric. Food Chem., Vol. 34, pp. 98-102(1986). R. Greenhalgh, R. M. Meier, B. A. Blackwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Minor Metabolites o f F u s a r i u m roseum (ATCC 28114); J. Agric. Food Chem., Vol. 32, pp. 1261-1264(1984).


225

Common/Systematic Name 3-Deacetylcalonectrin 3 a-Hydroxy- 15-acetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2405; M ' W -- 3 0 8 . 1 6 2 3 7

16

lo

H

,OH

,, i'" = 14

(~H2OAc 15 Fungal Source Fusarium graminearum and F. culmorum. Isolation/Purification Mycelia were extracted with methylene chloride, reduced to dryness, redissolved in methanol-water and partitioned against petroleum ether. The methanol raffinate was evaporated to dryness, redissolved in hot absolute alcohol and hexane added slowly. On cooling the solution deposited crystals of 3-acetyldeoxynivalenol which were removed for further purification. The brown viscous oil from the mother liquor was chromatographed on a silica gel column eluted with the following: 25% ethyl acetate in methylene chloride; 2% methanol in methylene chloride; 5% methanol in methylene chloride; and methanol. Fractions containing 3-deacetylcalonectrin were combined and further purified by preparative TLC using 10% acetone in chloroform to yield pure 3-deacetylcalonectrin (Re, 0.62). Biological Activity The trichothecenes as a group show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukocytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-free systems. Some trichothecenes inhibit the initiation step of protein synthesis on polyribosomes and others inhibit the elongation and/or termination step in protein synthesis. The trichothecenes have been strongly implicated in natural intoxications of

226


animals and man. Spectral Data 1H NMR: (CDC13) 0.79(3H, s, CH3-14); 1.70(3H, s, CH3-16); 2.02(3H, s, CH3-15Ac); 1.92.3(6H, unresolved m, H-4, -7, -8); 2.82, 3.06(2H, d, JA,B=4.0Hz, H-13); 3.48(1H, d, J2,3=4.6Hz, H-2); 3.82, 4.06(2H, d, JA,B=12.2Hz, H-15); 4.13(1H, d, Jll,10=5.4Hz, H11); 4.43(1H, ddd, J3,2=4.6, J3,4=ca 6, 9.5Hz, H-3); and 5.48ppm (1H, dd, Jlo,ll=5.4Hz, J10,16=l.4Hz, H-10). 13CNMR: (CDCI3) 68.4, C-2; 69.1, C-3; 42.4, C-4; 45.9, C-5; 45.9; C-6; 21.2, C-7; 28.4, C-8; 140.1, C-9; 119.3, C-10; 80.0, C-11; 65.6, C-12; 48.4, C-13; 12.3, C-14; 62.8, C-15; 23.2, C-16; 21.0, CH3COO-; and 170.6ppm, C=O. Mass Spectrum: LREIMS: 308(M+), 106(70%), 248(80), 123(82); 220(82), and 91m/e (100). Reference R. Greenhalgh, D. Lavandier, W. Adams, J. D. Miller, B. A. Blackwell, A. J. McAlees, and A. Taylor; Production and Characterization of Deoxynivalenol and Other Secondary Metabolites of Fusarium culmorum (CIVIl 14764, HLX 1503); J. Agric. Food Chem., Vol. 34, pp. 98-102(1986).


227

Common/Systematic Name 7a-Hydroxycalonectrin 3a, 15-Diacetoxy-7a-hydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C19H2607; M W -- 3 6 6 . 1 6 7 8 5

lo

H

1 6 " ~ O ~

2

13 :

HO

3..,"OAc

I ,,O

~5

4

- 14 ~H2OAc

15

General Characteristics Crystals from ethyl acetate-hexane; mp., 174-175~

[a]D 23

- 14.3 o (c=0" 14, in CHC13).

Fungal Source Fusarium roseum.

Isolation/Purification Fusarium roseum Greenhalgh et al.

was cultured, harvested, and the broth was extracted as described by (1984). Some of the oil from the methanol phase atter it had been partitioned and concentrated was dissolved in methylene chloride and passed through a Florisil column eluted with 750mL of (A) methylene chloride, (B) 0.5 methanol in methylene chloride, and (C) 5% methanol in methylene chloride. Fraction A afforded an oil from which 3-acetyldeoxynivalenol was obtained. Fractions B and C also afforded yellow oils. The oil which remained in fraction A aider removal of 3-acetyldeoxynivalenol was chromatographed on silica gel (Kieselgel 60). The column was eluted first with 10% toluene in methylene chloride and then with 0.5% methanol in methylene chloride, 1.5% methanol in methylene chloride, 3% methanol in methylene chloride, and finally 13% methanol in methylene chloride. Fractions were collected and monitored by TLC (75% ethyl acetate in chloroform; Re, 0.50); fractions 6-10, 11-12, 13-17, and 18-20 were combined to give mixtures A1, A2, A3, and A4, respectively. Further purification of mixtures A2 was accomplished with a Chromatotron (Model No. 7924, Harrison Research, Inc.) with a 2mm silica gel plate (Kieselgel 60) eluted with chloroform followed by 5% methanol in chloroform. Final purification of 7-hydroxycalonectrin was by preparative TLC using ethyl acetate as developing solvent.

Biological Activity The trichothecenes as a group show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range

228


of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukocytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-free systems. Some trichothecenes inhibit the initiation step of protein synthesis on polyribosomes and others inhibit the elongation and/or termination step in protein synthesis. The trichothecenes have been strongly implicated in natural intoxications of animals and man. Spectral Data 1H NMR: (CDC13) 3.78(1H, d, J2,3=4.4Hz, H-2); 5.17(1H, ddd, J=10, 8.0, 4.5Hz, H-3); 1.972.18(2H, m, H-4); 4.55(1 H, dd, J=5.9, 11.2Hz, H-7); 1.4 I(1H, d, J= 11.2Hz, OH-7); 1.90-2.0(2H, m, H-8), 2.41(dd, J=17.6, 5.9Hz H-8); 5.38(1H, dd, J=5.4, 1.3Hz, H10); 4.11(1H, d, J=5.4Hz, H-11); 3.09, 3.23(1H each, d, J=4.4Hz, H-13); 1.14(3H, s, CH3.14); 4.05(1H, d, J=12.2Hz, H-15); 4.3 I(1H, m, H-15); 1.72(3H, br s, CHs-16); and 2.08, 2.09ppm (each 3H, s, CH3COO-). 13C NMR: (CDC13) 78.9, C-2; 71.1, C-3; 39.6, C-4; 46.1, C-5; 46.4, C-6; 71.0, C-7; 41.7, C-8; 139.4, C-9; 119.0, C-10; 70.0, C-11; 64.3, C-12; 47.8, C-13; 15.9, C-14; 64.4, C-15; 22.4, C-16; 21.0 and 20.8, CH3COO-; and 170.0 and 170.1ppm, C=O. Mass Spectrum: HREIMS: 306.148m/e (M+ - AcOH); calcd for C17H2205, 306.147. TLC Data Silica gel 60 F-254 plates developed with 75% ethyl acetate in chloroform; Rf, 0.50. References R. Greenhalgh, D. Levandier, W. Adams, J. D. Miller, B. A. Blackwell, A. J. McAlees and A. Taylor; Production and Characterization of Deoxynivalenol and Other Secondary Metabolites ofFusarium culmorum (CMI 14764, HLX 1503); J. Agric. Food Chem., Vol. 34, pp. 98-102(1986). R. Greenhalgh, R. M. Meier, B. A. Blackwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Minor Metabolites ofFusarium roseum (ATCC 28114); J. Agric. Food Chem., Vol. 32, pp. 1261-1264(1984).


229

Common/Systematic Name 8 t~-Hydroxycalonectrin 3et, 15-Diacetoxy-8t~-hydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C19H2607; M W

~6

~o H -

= 366.16785

0

2

HO""~~2 __.

3r

,OAc

4

14

(~H2OAc 15


[0~]D 21 -

6.67 ~ (c=0.15, in CHC13).

Fungal Source Fusarium roseum, F. culmorum, and F. graminearum.

Isolation/Purification Mycelia were extracted with methylene chloride, reduced to dryness, redissolved in methanol-water and partitioned against petroleum ether. The methanol raffinate was evaporated to dryness, redissolved in hot absolute alcohol and hexane added slowly. On cooling the solution deposited crystals of 3-acetyldeoxynivalenol which were removed for further purification. The brown viscous oil from the mother liquor was chromatographed on a silica gel column eluted with the following: 25% ethyl acetate in methylene chloride; 2%methanol in methylene chloride; 5% methanol in methylene chloride; and methanol. Fractions containing 8-hydroxycalonectrin were combined and the mixture was separated by preparative TLC (developed with 1% ethanol in ethyl ether; Rf, 0.27) to yield pure 8hydroxycalonectrin. Biological Activity The trichothecenes as a group show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukocytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-free systems. Some trichothecenes inhibit the initiation step of protein synthesis on

230


polyribosomes and others inhibit the elongation and/or termination step in protein synthesis. The triehothecenes have been strongly implicated in natural intoxications of animals and man. Spectral Data 1H NMR: (CDC13) 3.73(1H, d, J2,3=4.6Hz, H-2); 5.15(1H, ddd, J=10, 5.1, 5.0Hz, H-3); 2.232.28(2H, m, H-4); 1.95(1H, d, J=14.6Hz, H-7), 2.19(1H, dd, J=14.6, 10.8Hz, H-7); 4.08-4.18(2H, m, H-8), 5.58(dd, J=5.7, 1.9Hz H-10); 4.08-4.18(1H, m, H-11); 2.86, 3.08(1H each, d, J=4.0Hz, H-13); 0.83(3H, s, CH3-14); 4.08-4.18(2H, m, H-15); 1.85(3H, d,J=O.6Hz, CH3-16); and 2.03, 2.1 lppm (each 3H, s, CH3COO-). 13CNMR: (CDC13) 77.8, C-2; 71.1, C-3; 39.1, C-4; 45.4, C-5; 42.3, C-6; 30.0, C-7; 67.8, C-8; 139.5, C-9; 121.5, C-10; 67.0, C-11; 64.9, C-12; 48.6, C-13; 11.9, C-14; 65.0, C-15; 20.5, C-16; 20.9 and 21.8, CH3COO-; and 170.3 and 170.4ppm, C=O. Mass Spectrum: HREIMS: 306.145m/e (M§ AcOH); calcd for C17H2205,306.147. TLC Data Silica gel 60 F254plates developed with 75% ethyl acetate in chloroform; Rf, 0.37. References R. Greenhalgh, D. Levandier, W. Adams, J. D. Miller, B. A. Blackwell, A. J. McAlees, and A. Taylor; Production and Characterization of Deoxynivalenol and Other Secondary Metabolites ofFusarium culmorum (CMI 14764, HLX 1503); J. Agile. Food Chem., Vol. 34, pp. 98-102(1986). R. Greenhalgh, R. M. Meier, B. A. Blackwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Minor Metabolites ofFusarium roseum (ATCC 28114); J. Agile. Food Chem., Vol. 32, pp. 1261-1264(1984).

14.


231

Common/Systematic Name 7 tt, 8 t~-Dihydroxycalonectrin 3 tt, 15-Diacetoxy-7 tt, 8 tt-dihydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C19H2608; ~ 16

m,~,., IVl~-

= 382.16277

10

n

A

2

13

H

,UAC A

~

A

3 .,~

i II

e (2H2OAc 15

General Characteristics Needles from ethyl ether; mp., 190-192~

[t~]D26 + 7.2 ~ (C=0.07, in CHC13).

Fungal Source Fusarium roseum (ATCC 28114), F. culmorum, and F. graminearum.

Isolation/Purification Fusarium roseum was cultured, harvested, and the broth was extracted as described by Greenhalgh et al. (1984). Some of the oil from the methanol phase aider it had been

partitioned and concentrated was dissolved in methylene chloride and passed through a Florisil column eluted with 750mL of (A) methylene chloride, (B) 0.5 methanol in methylene chloride, and (C) 5% methanol in methylene chloride. Fraction A afforded an oil from which 3-acetyldeoxynivalenol was obtained. Fractions B and C also afforded yellow oils. The oil which remained in fraction A aider removal of 3-acetyldeoxynivalenol was chromatographed on silica gel (Kieselgel 60). The column was eluted first with 10% toluene in methylene chloride and then with 0.5% methanol in methylene chloride, 1.5% methanol in methylene chloride, 3% methanol in methylene chloride, and finally 13% methanol in methylene chloride. Fractions were collected and monitored by TLC (75% ethyl acetate in chloroform; Re, 0.31); fractions 6-10, 11-12, 13-17, and 18-20 were combined to give mixtures A1, A2, A3, and A4, respectively. Further purification of mixtures A2 and A3 was accomplished with a Chromatotron (Model No. 7924, Harrison Research, Inc.) with a 2mm silica gel plate (Kieselgel 60). 3a,15-diacetoxy-7a,813dihydroxy-12,13-epoxytrichothec-9-ene was isolated from mixture A3 by elution with 50% ethyl acetate in hexane followed by recrystallization from ethyl ether.

232


Spectral Data 1HNMR:

(CDCI3) 1.12(3H,H-14); 1.86(3H, H-16, Jxo,16=l.0Hz); 2.02(3H, AC-CH3); 2.10(3H, AC-CH3); 2.13, 2.34(2H, H-4, ,/4,3=11.1, 4.6Hz); 2.71(1H, 8-OH, Js,ou=8.0Hz); 3.0(1H, 7-OH, JT,on=9.7Hz); 3.09, 3.20(2H, H-13, Jab=4.3Hz); 3.77(1H, H-Z, J2,3=4.4Hz); 3.99(1H, H-8, JT,s=5.8, Js,on=7.5Hz); 4.14, 4.44(2H, H-15, Jab=lZ.3Hz); 4.32(1H, H-11, J11,lO=5.8Hz); 4.50(1H, H-7, JT,on=9.6Hz,JT,s=5.4Hz); 5.15(1H, H-3, J3,2=4.4Hz, J3,4=4.5, 11.1Hz); and 5.58ppm (1H, H-10, Jlo,ll=5.8Hz, Jlo,a6=0.9Hz). 13CNMR: (CDC13) 78.5, C-2; 70.4, C-3; 41.3, C-4; 46.0, C-5; 46.2, C-6; 70.2, C-7; 71.0, C-8; 139.3, C-9; 122.1, C-10; 69.7, C-11; 64.6, C-12; 47.8, C-13; 15.0, C-14; 63.3, C-15; 21.1, C-16; 20.9, 20.3, CH3COO-; and 170.4, 170.0ppm, C=O. Mass Spectrum: HREIMS: 383.179role

( M ++

1); calcd for C19H27Os,383.171.

References R. Greenhalgh, R. M. Meier, B. A. Blackwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Part 2. Minor Metabolites o f F u s a r i u m r o s e u m (ATCC 28114); J. Agric. Food Chem., Vol. 32, pp. 115-118(1984). R. Greenhalgh, R. M. Meier, B. A. Blaekwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Minor Metabolites o f F u s a r i u m r o s e u m (ATCC 28114); J. Agrie. Food Chem., Vol. 32, pp. 1261-1264(1984). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from F u s a r i u m Fungi; In Myeotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).


233

Common/Systematic Name 4-Acetoxyscirpenediol 413-Acetyl-3 tt, 15-dihydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2406; M W = 324.15729

1o _H "T"7 11"~ 8

~

6 13

H I

~ ~ ' 2 ~s _- Me 14

H

I..

~ '"OH

,,O 4

....,H OAc

CH2OH 15

General Characteristics Amorphous solid from benzene-hexane; mp., 100-110 oC. Fungal Source Fusarium roseum (NRRL 1181), F. sulphureum, and F. sambucinum (NRRL 13495). Isolation/Purification The mycelium was extracted with ethyl acetate, followed by silica gel column chromatography eluted with benzene-acetone (2:1, v/v), acetone, and methanol. 4-Acetoxyscirpenediol was purified by preparative TLC using silica gel G plates developed with hexane-acetone (1:1, v/v). Biological Activity 4-Acetoxyscirpenediol caused dermal necrosis in white rats. The trichothecenes as a group show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukocytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-free systems. Some trichothecenes inhibit the initiation step of protein synthesis on polyribosomes and others inhibit the elongation and/or termination step in protein synthesis. The trichothecenes have been strongly implicated in natural intoxications of animals and man.

234


Spectral Data IR~ 3450, 2950, 1720, 1435, 1375, 1240, 1165, 1110, 1080, 1050, and 960cm1. 1H NMR: (CDC13) 3.68(1H, d,J=4.8Hz, H-2); 4.26(1H, dd, J-3.1, 4.1Hz, H-3); 5.53(1H, d, J-3.1Hz, H-3); 1.7-2.2(4H, m, H-7 and H-8); 5.58(1H, br d, J=5.0Hz, H-10); 4.20(1H, br d, J-5.0Hz, H-11); 2.78(1H, d, J=4.0Hz, H-13); 3.06(1H, d, J-4.0Hz, H13); 0.86(3H, s, CH3-14); 3.64, 3.69(2H, d, J=12.0Hz, CH2-15); 1.74(3H,br s, CH316); and 2.170ppm (3H, s, CH3-Ae).

Mass Data: LREIMS: 306(M+ - H20), 278, 234, 219, and 43re~e; TMS ether: 468(M+); anal. calcd for C17H2406: C, 62.95; H, 7.46%; found: C, 60.93; H, 6.65%. References K. Ishii, S. V. Pathre, and C. J. Mirocha; Two New Trichothecenes Produced by Fusarium roseum; J. Agile. Food Chem., Vol. 26, pp. 649-653(1978). K. E. Richardson, G. E. Toney, C. A. Haney, and P. B. Hamilton; Occurrence of Seirpentriol and Its Seven Acetylated Derivatives in Culture Extracts of Fusarium sambucinum NRRL 13495; J. Food Protect., Vol. 52, pp. 871-876(1989). D. R. Sanson, D. G. Corley, C. L. Barnes, S. Searles, E. O. Schlemper, and M. S. Tempesta; New Mycotoxins from Fusarium sambucinum; J. Org. Chem., Vol. 54, pp. 4313-4318(1989). M. E. Savard and B. A. Blaekwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Myeotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

14.


235

Common/Systematic Name 15-Acetoxyscirpenetriol 15-Acetoxy-3 ~,4[3-dihydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2406; MW = 324.15729

',,o ',',',,OH

~o H

H

-

15

=

Me

-

14

H

'H

4 OH

_

CH2OAc 15

General Characteristics Crystals from isooctane-ethyl acetate; mp., 172-173~ Fungal Source F u s a r i u m r o s e u m and F. sambucinum.

Isolation/Purification The dried cultures were moistened with water to a moisture content of 30% and extracted with ethyl acetate. The extract was concentrated to a gum and redissolved in acetonitrile and partitioned against petroleum ether; the petroleum ether layer was discarded. The acetonitrile layer was concentrated and then chromatographed on a column of silica gel. The components were eluted off the column with ethyl acetate-nhexane (1:3 to 1:7, v/v), ethyl acetate-methanol (5:1, v/v), and methanol. Each 100ml fraction was tested for toxicity by topical application to the shaved skin of the white rat. Fractions 4 through 12 were toxic and were pooled and rechromatographed on a column of silica gel using chloroform-methanol (98:2, v/v) as the eluting solvent. The fractions from this column were tested for toxicity and the one found toxic was purified by preparative TLC on silica gel G, developed in chloroform-methanol (98: ~, v/v) and made visible by charring with concentrated H2SO4. The monoacetoxyscirpenol was crystallized from methylene chloride-petroleum ether. Two recrystallizations from isooctane-ethyl acetate gave an analytical sample. Biological Activity Clinical signs in birds ingesting the toxin were bilateral inflammation of the beak area and gastrointestinal hemorrhaging. Application to shaved skin of rat resulted in hyperkeratosis and petechial hemorrhaging. The LDs0 in 20-day-old white female weanling rats dosed SC was 0.752mg/kg. A single 20kg female pig injected IV with 20mg monoacetoxyscirpenol (lmg/kg) exhibited emesis within 0.5 hr, lethargy, a staggered gait, and death within 13 hr.

236


Spectral Data IR:

(KBr) 3400,1715, and 1250 cm "1. 1H NMR: (CDCI3) 3.60(1H, d, J=4.6Hz, H-2); 4.21(1H, dd, J--2.7, 4.6Hz, H-3); 4.26(1H, br d, J=2.7Hz, H-4); 1.76(1H, dd, J=l 1.3, 4.8Hz, H-7); 2.01(1H, m, H-7); 1.98-2.00(2H, m, H-8); 5.46(1H, d, J=5.4Hz, H-10); 3.92(1H, br d, J=5 4Hz, H-11); 2.73(1H, d, J=3.9Hz, n-13); 3.01(1H, d, J=3.9Hz, n-13); 0.79(3H, s, CH3-14); 3.87(1H, d, J=12.3Hz, H-15); 4.18(1H, d, J=12.3Hz, H-15); 1.70(3H, br s, CH3-16); and 2.04ppm (s, CHaCOO-). 13C NMR: (CDCI3) 78.9, C-2; 80.0, C-3; 81.9, C-4; 49.2, C-5; 43.5, C-6; 21.1, C-7; 28.0, C-8; 140.7, C-9; 118.4, C-10; 68.0, C-11; 64.7, C-12; 46.9, C-13; 6.9, C-14; 63.6, C-15; 23.2, C-16; 21.3, CH3COO-; and 170.8ppm, CH3COO-. Mass Data: 264(36), 205(100), 191(9), 189(11), 187(30), and 177role (24); anal. calcd for C17H2406: C, 62.96; H, 7.41%; found: C, 63.42; H, 7.61%. TLC Data Adsorbent: silica gel G; Solvent: chloroform-methanol (90:10, v/v); Re, 0.37. Detection: H2SO4 or p-anisaldehyde spraying and heating at 110~for 10 min. Purple to brown color with H2SO4; purple color with p-anisaldehyde. GLC Data Liquid phase: 3% OV-1 on Gas Chrom Q (100-120 mesh); TMS, 9-10 min.; retention time: trifluoroaeetate, 5-6 min. (Derivative for GLC: trimethylsilyl ether or trifluoroacetate). References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 168(1981). S. V. Pathre, C. J. Mirocha, C. M. Christensen, and J. Behrens; Monoacetoxyscirpenol. A New Mycotoxin Produced by Fusarium roseum Gibbosum; J. Agile. Food Chem., Vol. 24, pp. 97-103(1976). K. E. Richardson, G. E. Toney, C. A. Haney, and P. B. Hamilton; Occurrence of Scirpentriol and Its Seven Acetylated Derivatives in Culture Extracts of Fusarium sambucinum N R R L 13495; J. Food Protection, Vol. 52, pp.871-876(1989). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257(1994).


237

Common/Systematic Name 4,15-Diacetoxyscirpenol; Anguidine 4~, 15-Diacetyl-3 t~-hydroxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C19H2607; M W -- 3 6 6 . 1 6 7 8 5

16

lo

H

H

~O.,,J2 13-

~ ~ ' 2 --

I

H

31. "OH

,,0 !" . ,,H

14

OAc

CH2OAc 15 General Characteristics Crystals from ether; mp., 162-164~ Benzoate: prisms from ethanol; mp., 178~ [t~]D24 - 27 ~ (C=1.28). Fungal Source

Fusarium roseum (NRRL 1181), F. sambucinum (NRRL 13495) = Gibberella pulicaris, F. tricinctum, F. equiseti, F. lateritium, F. graminearum, F. semitectum, F. sulphureum, F. diversisporum, F. scirpL and Gibberella intricans.

Isolation/Purification The mycelium was extracted with ethyl acetate, followed by silica gel column chromatography eluted with benzene-acetone (2:1, v/v), acetone, and methanol. 4,15Diacetoxyscirpenol was purified by repetitive preparative TLC using silica gel G plates developed with hexane-acetone (1:1, v/v). Biological Activity The LDs0 in mice dosed IP was 23.0mg/kg; LDs0 in chicken embryo test was 0.091,tg per egg. IDs0 of protein synthesis in rabbit reticulocytes (whole cell) was 0.03~g/ml; cell-free was 5.01.tg/ml; tumor cells were 0.30Ftg/ml; rat liver (cell-free) was >501.tg/ml. Dermal toxicity to rabbit, mouse, and guinea pig. Diacetoxyscirpenol was also highly phytotoxic. Spectral Data 1H NMR: (CDCI3) 3.65(1H, d, J=4.9Hz, H-2); 4.10(1H, dd, ,/--2.9, 4.9Hz, H-3); 5.1 l(1H, d, J=2.9Hz, H-4); 2.00(1H, d, J=l 1Hz, H-7); 1.71(1H, d, J=l 1.0Hz, U-7); 1.93(2H, m, H-8); 5.50(1H, d, J=5.3Hz, H-10); 4.02(1H, d, J=5.3Hz, H-11); 3.00(1H, d, J=4.0nz, n-13); 2.74(1H, d, J= 4.0Hz, n-13); 0.79(3H, s, CH3-14); 4.10, 3.90(2H, d, J=12.3Hz, CH2-15); 1.68(3H, s, CH3-16); and 2.00, 2.09ppm (3H, s, CHa-Ac).

238


13C NMR: 79.0, d, C-2; 76.3, d, C-3; 83.0, d, C-4; 49.1, s, C-5; 44.2, s, C-6; 21.1, t, C-7; 27.9, t, C-8; 139.4, s, C-9; 119.3, d, C-10; 67.4, d, C-11; 64.8, s, C-12; 46.6, t, C-13; 6.8, q, C-14; 63.4, t, C-15; 23.2, q, C-16; 21.1, q, and 21.1, q, CH3C = O; 170.3, s, and 170.3, s, CH3C__= O. Mass Data: LREIMS (TMS derivative): 439(M § + 1), 438(M+), 324, 379(base peak), 319, 301, 289, and 229m/e. TLC Data Adsorbent: Kieselgel G; solvent: chloroform-methanol, 97:3 (v/v); Rf, 0.55; detection: H2SO4 spraying and heating at 110~ for 5 min. Adsorbent: alumina; solvent: chloroformmethanol, 98:2 (v/v); Re, 0.86; detection: H2SO4 spraying and heating at 110~ for 5 min. GLC Data Support: Shimalite W; liquid phase: OV-17 (1.5%); retention time: 5.95; relative retention time: (relative to nivalenol) 1.67. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 171-172(1981). K. E. Richardson, G. E. Toney, C. A. Haney and P. B. Hamilton; Occurrence of Scirpentriol and Its Seven Acetylated Derivatives in Culture Extracts of Fusarium sambucinum NRRL 13495; J. Food Protect., Vol. 52, pp. 871-876(1989). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp.59-257 (1994).


239

Common/Systematic Name Scirpenetriol 3 tt,4 fl, 15-Trihydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C15H2205; MW" = 282.14672 ~o

H

H

- ~ -

14

H

'~

,,H OH

CH2OH 15

General Characteristics Prisms from ethyl acetate; mp., 193~

[tg]D 23 0 ~

(c=1.27, in acetone).

Fungal Source Fusarium roseum and F. equiseti.

Isolation/Purification The dried cultures were rehydrated to a moisture content of 30% and extracted with ethyl acetate. The extract was concentrated to a gum and redissolved in acetonitrile and partitioned against petroleum ether; the petroleum ether layer was discarded. The acetonitrile layer was concentrated and then chromatographed on a column of silica gel. The components were eluted off the column with ethyl acetate-n-hexane (1:3 to 1:7, v/v), ethyl acetate-methanol (5:1, v/v), and methanol. Each 100ml fraction was tested for toxicity by topical application to the shaved skin of the white rat. Fractions 4 through 12 were toxic and were pooled and rechromatographed on a column of silica gel using chloroform-methanol (98:2, v/v) as the eluting solvent. Scirpenetriol was purified by preparative TLC on silica gel G, developed in chloroform-methanol (98:5, v/v) and made visible by charring with concentrated H2SO4. Biological Activity LDs0 in rats dosed IP was 0.8 lmg/kg (single dose). Spectral Data UV~

End absorption.

(Nujol) 3480, 3455, 3405, 1676, and 830cm 1.

240


1H NMR: 3.78, H-2; 4.08, H-3; 4.12, H-4; NR, H-7; NR, H-8; NR, H-10; NR, H-11; 2.70, H13a; 3.05, H-1313; 0.90, H-14; 3.59, H-15; and 1.72ppm, H-16. References P. W. Brian, A. W. Dawkins, J. F. Grove, H. G. Hemming, D. Lowe, and G. L. F. Norris; Phytotoxic Compounds Produced by Fusarium equiseti; J. Exp. Botany, Vol. 12, pp. 116123(1966). R. J. Cole and R. H. Cox, Handbook of Toxic Fungal Metabolites; Academic Press, New

York, p. 160 (1981). S. V. Pathre, C. J. Mirocha, C. M. Christensen, and J. Behrens; Monoacetoxyscirpenol. A New Mycotoxin Produced by Fusarium roseum Gibbosum; J. Agric. Food Chem., Vol. 24, p. 97(1976)


241

Common/Systematic Name 7tt-Hydroxydiacetoxyscirpenol 3a,7 tt-Dihydroxy-4~3,15t~-diacetoxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C19H2608; MW

=

382.16277

~o H

H

~ > ~ , , , ~111I_.:- H131, ost. I in, "'I''OHAc (~H2OAc 15

General Characteristics Needles from chloroform-hexane; mp., 201-203~ tetraacetoxy derivative, needles from benzene-n-hexane; mp., 147-149~ Fungal Source

Fusarium lateritium and Fusarium

spp. (K-5036).

Isolation/Purification The culture filtrate was mixed with activated charcoal, the charcoal washed with water and the materials adsorbed to the charcoal eluted with methanol. The methanol extract was concentrated and chloroform added which resulted in a precipitate. The chloroformmethanol was filtered and the filtrate evaporated to dryness. The residue was extracted with hot acetone, the acetone soluble materials were chromatographed on a silica gel column eluted with benzene-acetone 5:2, 2:1, 11 (v/v), acetone and methanol. The fractions were monitored by TLC (Kieselgel G) and the fractions containing 7ahydroxydiacetoxyscirpenol were combined and chromatographed on a silica gel column eluted with n-hexane-acetone, 1:1 (v/v); the metabolite at Rf0.5 was crystallized from chloroform-n hexane to give needles of 7a-hydroxydiacetoxyscirpenol. Biological Activity The LDs0 to mice dosed IP was about 3.5mg/kg; IDs0 of protein synthesis in rabbit reticulocytes was 0.31.tg/ml. Spectral Data UV:

End absorption. IR:

(KBr) 3500, 2950, 1720, 1380, 1280, and 1060cm.1.

242


1H NMR: (CDC13) 3.70(H-2); 4.25(H-3); 5.20(H-4); 4.62(H-7); 2.00(H-8a); 2.43(H-813); 5.50(H-10); 4.25(H-11); 3.07(H-13a); 3.17(H-1313); 1.13(H-14); 4.24(H-15a); 4.42(H-1513); 1.75(H-16); and 2.07, 2.15ppm (C_H_3H CO-). Mass Data: LREIMS: 364(M + - 18), 322(M + - 60), 304, and 263m/e; found: C, 59.25; H, 6.78%; C19H2608 requires: C, 59.65; H, 6.85%. TLC Data Adsorbent: silica gel; solvent: acetone-hexane (1:1, v/v); Rf, 0.5; detection: spraying with 20% H2SO4 and heating at 110~ References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 190(1981). K. Ishii; Two New Trichothecenes Produced by Fusarium sp.; Phytochemistry, Vol. 14, pp. 2469-2471(1975).


243

Common/Systematic Name 7a,8a-Dihydroxydiacetoxyscirpenol 3 a,7a,8 a-Trihydroxy-4[3,15 ~-diacetoxy- 12,13-epoxytrichothec-9-ene M.Qlecular Formula/Molecular Weight C19H2609, ~

-- 3 9 8 . 1 5 7 6 8

~o H

16

-

HO"

v

O

H

q

2

... -,~

H~) i ~

3

,.., o .

I

~J 0Ac

~-,H2OAc 15

General Characteristics Needles from ethanol-ether-n-hexane; mp., 167-169~ Fungal Source Fusarium lateritium, and F. spp. (K-5036). Isolation/Purification The culture filtrate was mixed with activated charcoal, the charcoal washed with water and the materials adsorbed to the charcoal eluted with methanol. The methanol extract was concentrated; chloroform was added which resulted in a precipitate. The chloroformmethanol was filtered and the filtrate evaporated to dryness. The residue was extracted with hot acetone, the acetone soluble materials were chromatographed on a silica gel column eluted with benzene-acetone 5:2, 2:1, 1:1 (v/v), acetone and methanol. The fractions were monitored by TLC (Kieselgel G) and the fractions containing 7t~hydroxydiacetoxyscirpenol were combined and chromatographed on a silica gel column eluted with n-hexane-acetone (1:1, v/v); the metabolite at Rf 0.38 was crystallized from EtOH-ether-n hexane to give needles of 7a,8ec-dihydroxydiacetoxyscirpenol. Biological Activity The LDs0 to mice dosed IP was about 6.0mg/kg; IDs0 of protein synthesis in rabbit reticulocytes was 0.61.tg/ml. Spectral Data UV:

End absorption. IR:

(KBr) 3500, 2950, 1750, 1390, 1260, and 1050cml.

244


1H NMR: (CDC13) 3.70(H-2); 4.20(H-3), 5.45(H-4), 4.01(H-7), 4.50(H-8); 5.65(H-10); 4.50(H-11), 3.05(H-13a); 3.15(H-1313); 1.10(H-14); 4.29(H-15a); 4.50(H-1513); 1.85(H-16); and 2.02, 2.13ppm (CH_H_3_CO-). TLC Data Adsorbent, silica gel; solvent, acetone-n-hexane (1:1, v/v); Rf, 0.38; detection, spraying with 20% H2SO4and heating at 110~ References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press' New York, p. 191 (1981). K. Ishii; Two New Trichothecenes Produced by Fusarium sp.; Phytochemistry, Vol. 14, pp. 2469-2471(1975).


245

Common/Systematic Name 3,15-Diacetoxyscirpenetriol 3t~, 15-Diacetyl-413-hydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C19H2607; MW = 366.16785

16

~o H

H

"-0

2 i

o

_ :

is

4

.... ,o

c

~OH

14

~_,H2OAc 15

General Characteristics Colorless glass. Fungal Source Fusarium sambucinum.

Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit over night, filtered,and the solid residue was re-extracted with acetone. The acetone extract was combined with the chloroform-acetone extract, concentrated under vacuum, and the nonpolar materials were removed by hexane drip. The solvent was decanted, concentrated and chromatographed by flash chromatography with toluene, toluene-acetone (4:1, 2:1, 1:1, v/v), acetone, and acetone-methanol (1:1, v/v). The toluene-acetone (4:1, v/v) fraction was further purified by flash chromatography eluting with toluene-acetone (4:1, v/v) This resulted in purified 3,15-diacetoxyscirpenol Biological Activity The trichothecenes as a group show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukocytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-free systems. Some trichothecenes inhibit the initiation step of protein synthesis on polyribosomes and others inhibit the elongation and/or termination step in protein

246


synthesis. The trichothecenes have been strongly implicated in natural intoxications of animals and man. Spectral Data UV: XAm~ 199nm(e=8,200, ~ to r~* transition). IR~ (film) 3403 and 1731cm~ (C=O). 1H N]VIR: (CDC13) 3.81(1H, d, J=4.9Hz, H-2); 4.85(1H, dd, J=2.9, 4.9Hz, H-3); 4.31(1H, d, J=2.9Hz, H-4); 1.7-2.1; 5.47(1H, b d, J=5.5Hz, H-10); 3.75(1H, b d, J=5.5Hz, H-11); 2.79(1H, H-13); 3.05(1H, d, J=4.0Hz, H-13); 0.87(3H, s, CH3-14); 3.90(1H, CH215); 4.13(1H, d, J=12.3Hz, CH2-15); 1.73(3H, b s, CH3-16); and 2.05, 2.20ppm (3H each, s, CH3-Ac).

13C NMR: 77.6, C-2; 83.5, C-3; 79.1, C-4; 48.9, C-5; 43.6, C-6; 20.9, C-7; 28.0, C-8; 141.0, C9; 118.2, C-10; 68.1, C-11; 64.0, C-12; 46.8, C-13; 6.7,C-14; 63.5, C-15; 23.2, C-16; 21.0, and 21.2, CH3C = O; not observed due to small sample size, CH3C = O. Mass Data: HR IMS: 307.154m/e; calcd for C17H2305 (M + - OAc); 307.174; LREIMS: 307(M + OAc, 5%), 205(68), 124(base peak), 105(97), and 81m/e (55). Reference D. R. Sanson, D. G. Corley, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta, and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucinum; J. Org. Chem., Vol. 54, pp. 4313-4318(1989).


247

Common/Systematic Name 3,4,15-Triacetoxyscirpenetriol 3~,413,15-Triacetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C21H2808; MW = 408.17842

- ~

10 H

16

O'~2l,,O3[....,OAc H

.- 5 ?

~'2 4- -=OAc

(~H2OAc 15 --

14

H

FI

General Characteristics Colorless crystals from benzene-n-hexane; mp., 124-126~ Fungal Source Fusarium sulphureum (MRC-514). Isolation/Purification Fungal cultures were extracted with chloroform-methanol (1:1, v/v), concentrated, and partitioned between 90% methanol-n-hexane. The aqueous methanol solution was concentrated and partitioned between chloroform and water. The chloroform solution was dried, filtered, evaporated to dryness, and chromatographed on a silica gel H column eluted with chloroform-methanol (19:1, v/v). The first fraction was filtered through a short alumina column (activity II-III) using chloroform; the yellow oil obtained was recrystallized from benzene-n-hexane to give colorless crystals of triacetoxyscirpenol. Spectral Data IH NMR: (CDC13) 0.76(3H, s, C-14 Me); 1.71(3H, s, C-16 Me); 2.04(3H, s, C-15 OAc); 2.09(3H, s, C-30Ac); 2.12(3H, s, C-40Ac); 2.77; 3.05(1H,each d, J~3,~4=4Hz C-13 H); 3.84(1H, d, ,/2,3=5 Hz, C-2 H); 3.99(d, 1H, d, Jlo, l~=5Hz, C-11 H); 4.03 and 4.25(11H, each d, Jls,ls=12Hz, C-15 H); 5.17(1H, dd, J3,a=3.5Hz, J2,3=5Hz, C-3 H); 5.46(1H, br d, Jlo, lt=5Hz, C-10 H); and 5.74ppm (1H, d, J3,4-3.5Hz, C-4 H). Mass Data: Found: C, 61.89; H, 6.68%; calcd for C21H2808"C, 61.75- H, 6.91%.

248


References R. J. Cole and R. H. Cox, Handbook of Toxic Fungal Metabolites, Academic Press, New York, p. 201(1981). P. S. Steyn, R. Vleggaar, C. J. Rabie, N. P. J. Kriek, and J. S. Harington, Trichothecene Mycotoxins from Fusarium sulphureum, Phytochemistry, Vol. 17, pp. 949-951(1978).

14.

Trichothecenes


249

Common/Systematic Name T-2 Tetraol 3a,413,8a, 15-Tetrahydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C15H2206; M W = 298.14164 ~o H I-

1~ OH

H I n o4 ~%1 .... , v

"1 13 ~1

Is - 1, CH2OH

i

.... , O H

4~ H

15

Fungal Source

Fusarium poae and F. sporotrichioides.

Spectral Data UV:

End absorption. IR:

(KBr) 3400, 2930, 1625, 1450, and 1380cm1. 1H NMR: (CDC13) 3.60(1H, d, J=4.8Hz, H-2); 4.07(1H, dd, J=4.8, 3.3Hz, H-3); 4.12(1H, d, J=3.3Hz, H-4); 1.97(1H, dr, J=14.4, 1.6Hz, ttH-7); 2.16(1H, dd, J=14.4, 5.3Hz, I]H7); 5.52(1H, dq, J=5.7, 1.5Hz, H-10); 3.80(1H, d, J=5.7Hz, H-11); 2.89(1H, d, J=4.1Hz, H-13); 2.76(1H, d, J=4.1Hz, H-13); 0.84(3H, s, CH3-14); 3.72(1H, d, J=2.4Hz, H-15); 3.40(1H, d, J=12.4Hz, H-15); and 1.82ppm (3H, s, CH3-16). 13C NMR: 78.5, C-2; 79.1, C-3; 80.0, C-4; 48.0, C-5; 45.1, C-6; 28.5, C-7; 64.8, C-8; 138.1, C-9; 121.6, C-10; 67.6, C-11; 64.3, C-12; 45.1, C-13; 6.3, C-14; 61.0, C-15; and 19.9ppm, C-16. (CDC13) 80.6, C-2; 80.8, C-3; 82.3, C-4; 50.4, C-5; 45.9, C-6; 29.9, C-7; 67.0, C-8; 141, C-9; 122.6, C-10; 70.0, C-11; 65.8, C-12; 47.2, C-13; 7.3, C-14; 63.1, C-15 and 20.7ppm, C- 16. TLC Data Adsorbent: silica gel G or Hi solvent: ethanol-ethyl acetate-acetone, 1:4:4, v/v/v; Re, 0.52; detection: H2SO4 spraying and heating at 150~ for 5 min.

250


References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 162 (1981). R. A. Ellison and F. N. Kotsonis; Carbon-13 Nuclear Magnetic Resonance Assignments in the Trichothecene Mycotoxins; J. Org. Chem., Vol. 41, p. 576(1976). E. B. Smalley and F. M. Strong; Toxic Trichothecenes. In Mycotoxins; I. F. H. Purchase, ed.; pp. 199-228, Elsevier Scientific Pub. Co., New York (1974). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

14.


251

Common/Systematic Name T-2 Toxin 3 et-Hydroxy-413,15-diacetoxy-8tt- [3-methylbutyryloxy]- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C24H3409; M W = 4 6 6 . 2 2 0 2 8

16~ Me

18

O_

~CHCH2~O

Me/ 21

H

H

042

"

~~

:

,4

(~H2OAc

,iO lH ,~

OAc

15

General Characteristics White needles from benzene-Skellysolve B; mp., 151-152~ [~]D 26 "at- 15~ (c=2.58, in EtOH). Acetate derivative: amorphous solid from ether-pentane; [a]D3~ +27 ~ (C=0.9, in 95% EtOH). Fungal Source Fusarium tricinctum, F. culmorum (F. roseum), F. solanL F. poae, F. sporotrichioides, and Trichoderma lignorum. Biological Activity Acute LDs0 in rats and swine dosed orally was 4mg/kg; LDs0 in mice dosed IP was 3.04mg/kg; LDs0 in chicks dosed orally was 1.84mg/kg; IDso of rabbit reticulocytes (whole cell) was 0.031,tg/ml; IDs0 of rabbit reticulocytes (cell-free) was 0.151.tg/ml. T-2 toxin inhibited the initiation step of protein synthesis on polyribosomes. It had emetic properties when administered orally, IP or IV. Minimum SC dosage to induce vomiting in ducklings was 0. l mg/kg; in cat, 0.1-0.2mg/kg. Major gross clinical signs in cats from T-2 toxin were emesis, vomiting, diarrhea, anorexia, ataxia of the hind legs, discharge from the eyes, and ejection of hemorrhagic fluid. Consecutive administration at sublethal dosages caused a marked decrease in white cells. Necropsy showed extensive cellular damage in the bone marrow, intestine, spleen, and lymph nodes. Also evident were meningeal hemorrhage of the brain, bleeding in the lungs, and vacuolic degeneration of the renal tubes. T-2 caused dermal necrosis when applied to the surface of the skin. It caused primary oral lesions in animals ingesting it. Spectral Data UV~

End absorption.

252


1H NMR: (CDC13) 3.68(1H, d, J=4.9Hz, H-2); 4.13(1H, ddd, d=4.9, 2.8, 2.8Hz, H-3); 3.18(1H, d, J=2.8Hz, 3-OH); 5.24(1H, d, J=2.8Hz, H-4); 2.38(1H, dd, d=15.1, 5.8Hz, all-7); 1.87(1H, dd, J=15.3, 1.5Hz, [3H-7); 5.27(1H, d, J=5.8Hz, H-8); 5.72(1H, dt, J=5.8, 1.2Hz, H-10); 4.33(1H, d, J=5.8Hz, H-11); 3.04(1H, d, J=3.9Hz, H-13); 2.78(1H, d, J=3.9Hz, H-13); 0.79(3H, s, CH3-14); 4.04(1H, d, J=12.6Hz, all-15); 4.27(1H, d, J=12.6Hz, 13H-15); 1.72(3H, br s, CH3-16); 2.10(2H, m, H-18); 2.01(1H, m, H-19); 0.94, 0.93(3H each, d, J=6.5Hz, 20 and 21 CH3); and 2.01, 2.12ppm (3H each, s, CH3COO-). 13C NMR: 78.7, C-2; 78.2, C-3; 84.3, C-4; 48.3, C-5; 42.9, C-6; 27.7, C-7, 68.0, C-8; 136.3, C-9; 123.8, C-10; 67.3, C-11; 64.2, C-12; 47.0, C-13; 6.6, C-14; 64.5, C-15; 20.1, C-16; 172.7, C-17; 43.4, C-18; 25.6, C-19; 22.2, C-20 and C-21; 20.8(3H, CH3COO-); and 170.2, 170.5ppm, (3H each, CH3COO-).

TLC Data A. Adsorbent: silica gel G; solvent: chloroform-methanol, 98:2, v/v; Rf, 0.40; detection: H2SO4 spraying and heating at 110~ for 5 minutes. B. Adsorbent: alumina; solvent: chloroform-methanol, 98:2, v/v; Rf, 0.88; detection: H2SO4 spraying and heating at 110~ for 5 minutes. GLC Data (TMS derivatives) Support: Shimalite W; liquid phase: OV-17 (1.5 %); retention time 8.55; relative retention time: (relative to nivalenol) 2.40. References J. R. Bamberg and F. M. Strong; Mycotoxins of the Trichothecene Family Produced by Fusarium tricinctum and Trichoderma lignorum; Phytochemistry, Vol. 8, pp. 24052410(1969). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 185-186(1981). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).


253

Common/Systematic Name HT-2 Toxin 3a,413-Dihydroxy-15-acetoxy-8a- [3-methylbutyryloxy]-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C22H32Og; MW

= 424.20972

H

H .... , O H

Me. 20

18

0 ~

U

Me/CHCH2CO 21

~

_

~5

- ,, ~_,H2OAcO

4~)H

15

General Characteristics Pale yellow oil. Fungal Source

Fusarium sporotrichioides, F. poae, and F. culmorum.

Biological Activity LDs0 dosed IP to mice was 9.0mg/kg; LDs0 in chicken embryo assay was 0.5~g per egg. IDs0 of protein synthesis in rabbit reticulocytes (whole cell) was 0.03~g/ml. It inhibited the initiation step in protein synthesis. It also caused dermal necrosis similar to that of T-2. Spectral Data IR:

3400, 2950, 1720, 1635, and 1240cm1. 1H NMR: (CDC13) 3.60(1H, d, J=4.9Hz, H-2); 4.22(1H, m, H-3); 4.13(1H, br d, J=3.0Hz, H4); 2.00(1H, dr, J=15.0, 1.5Hz, all-7); 2.32(1H, dd, J=lS.0, 5.7Hz, 13H-7); 5.25(1H, br d, J=5.1Hz, H-8); 5.73(1H, br d, J=6.0Hz, H-10); 4.36(1H, br d, J=6.0Hz, H-11); 2.74(1H, d, J=4.0Hz, H-13); 3.00(1H, d, J=4.0Hz, H-13), 0.75(3H, s, CH3-14); 3.97(1H, d, J=12.5Hz, all-15); 4.29(1H, d, J=12.5Hz, ~H-15); 1.71(3H, s, CH3-16); 2.05-2.10(1H, m, H-19); 0.93, 0.92(3H each, d, J=6.4Hz, 20- and 21-CH3); and 2.01ppm (3H, s, CH3COO-). lSC NMR: 78.7, C-2; 80.6, C-3; 81.6, C-4; 48.9, C-5; 42.5, C-6; 27.6, C-7; 68.1, C-8; 136.4, C-9; 123.7, C-10; 67.3, C-11; 64.7, C-12; 46.9, C-13; 6.9, C-14; 64.5, C-15; 20.3, C-16; 172.7, C-17; 43.6, C-18; 25.7, C-19; 22.4, C-20 and C-21; 21.1(3H, C__H3COO-), and

254


170.4ppm, (3H, CH3COO-). Mass Spectrum: LREIMS: 424(M+), 322, 249, 203, 121,105, 85, and 57m/e (base peak). TLC Data A. Adsorbent: silica gel G; solvent: chloroform-methanol, 98:2, v/v; Rf, 0.09; detection: H2SO4 spraying and heating at 110~ for 5 min. B. Adsorbent: alumina; solvent: chloroform-methanol, 98:2, v/v; Rf, 0.32; detection: H2SO4 spraying and heating at 110~ for 5 min. References J. R. Bamberg and F. M. Strong; Mycotoxins of the Trichothecene Family Produced by Fusarium tricinctum and Trichoderma lignorum; Phytochemistry, Vol. 8, pp. 2405-2410 (1969). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 181-182(1981). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

14. T r i c h o t h e c e n e s and Related M e t a b o l i t e s

255

Common/Systematic Name Acetyl T-2 Toxin 3 a,4~, 15-Triacetoxy-8a-[3-methylbutyloxy]- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C26H36010; M W --- 5 0 8 . 2 3 0 8 5

lo

~

H -

H O..J~ ,

[~ 20 Me

0

II\CHCH2CO Me/ ,8

-

21

~1 ~

" ~ 1 .... ,v ~ ,

l

--~4 CH2OAc

OAc

15

General Characteristics An oil. Fungal Source

Fusarium poae (NRRL 3287).

Biological Activity Acetyl T-2 toxin was much less toxic than T-2 toxin in pigeons. It caused emesis at higher dosages (18.2mg/kg oral). No deaths occurred at tiffs dosage level. Spectral Dat.a UV:

End absorption. 1H NMR: (CDC13) 3.78, H-2; 5.07, H-3; 5.85, H-4; 1.85-2.1, H-7; 5.20, H-8; 5.65, H-10; 4.14, H-11; 2.75, all-13; 2.95, ~H-13; 0.65, H-14; 4.00, all-15; 4.27, 13H-15; 1.66, H16;1.93, H-18; 2.0, H-19; 0.96, H-20; 0.96, H-21; and 2.03ppm, (3H, CH3-COO-). 13CNMR: 76.2, C-2; 78.7, C-3; 78.2, C-4; 47.7, C-5; 42.0, C-6; 26.7, C-7; 67.2, C-8; 135.0, C-9; 124.0, C-10; 66.3, C-11; 63.6, C-12; 41.4, C-13; 6.1, C-14; 63.6, C-15; 19.4, C-16; 172.2, C-17; 42.4, C-18; 25.3, C-19; 21.7, C-20; 21.7, C-21; 170.6, CH3CO; and 20.3ppm, CH3CO. TLC Data Adsorbent: BrinkmanF254 silica gel; solvent: ethyl acetate-ethanol, 6:1, v/v; Rf, 0.74; detection: a quenching spot under UV light.

256


References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 185-186(1981). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).


257

Common/Systematic Name 4-Propanyl HT-2 413-Propanyl-3a-hydroxy- 15-acetoxy-8a-[3-methylbutyryloxy]-12,13-epoxytrichothec-9ene Molecular Formula/Molecular Weight C25H3609; M W = 480.23 593

H .-

4

Me.

a" d'

,"

I-

.[

111

6 1

H v"J i

13

"~i~,, I

"~CHCH2COO'....- , , , G / i ~ / 1 2

Me/ 5"

~ ~e

-=

.... O H

,-,

ii u

1'

4

2'

3'

--~'OCOCH2Me

14

~;H2OAc 15

General Characteristics Isolated as white needles; mp., 141-142~ Fungal Source

Fusarium sporotrichioides (MC-72083) and F. sambucmum.

Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), followed by Florisil column chromatography eluted with benzene-hexane (2:1, v/v), methylene chloride, and chloroform-methanol (95:5, v/v) to give an oil highly enriched with trichothecenes. The oil was further purified by normal phase preparative HPLC using benzene-acetone followed by preparative RPTLC to yield several trichothecenes including 4-propanyl HT-2 toxin. Spectral Data IR:

(film) 3430(OH), 1741, and 1730cm] (ester). 1H NMR:

(CDCla) 6.24(1H, t, J=-l.5Hz, H-2); 3.10(1H, d, ,/--18.2, H-4), 2.03(1H, d, J=18.2Hz, H-4); 1.95(1H, dd, ,/=15.0, 4.6Hz, n-7); 1.79(1H, d, J=15.0Hz, H-7); 1.45(2H, m, J=4.6Hz, H-8); 5.05(1H, br s, H-10); 4.11(1H, br s, H-11); 4.55(1H, d, d=17.3Hz, H 13); 4.34(1H, d, J=17.3Hz, n-13); 1.24(3H, s, CH3-14); 0.84(3H, s, CHa-15); and 1.61ppm (3H, br s, CH3-16). 13CNMR: (CDCI3) 129.5, C-2; 208.4, C-3; 50.4, C-4; 53.1, C-5; 41.9, C-6; 29.9, C-7; 27.4, C8, 135.7, C-9; 125.0, C-10; 71.3, C-11; 188.0, C-12; 61.0, C-13; 22.4, C-14; 13.1, C-

258


15; and 22.5ppm C-16. Mass Spectrum: CIMS: 470.236m/e (h/V); calcd for C25H3609,470.235. References D. G. Corley, G. E. Rottinghaus, J. K. Tracy, and M. S. Tempesta; New Trichothecene Mycotoxins of Fusarium sporotrichioides (MC- 72083); Tetrahedron Letters, pp. 41334136(1986). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).


259

Common/Systematic Name 3'-Hydroxy T-2 toxin 3~-Hydroxy-413,15-diacetoxy-8a-[3-hydroxy-3-methylbutyryloxy]- 12,13-epoxytrichothec9-ene Molecular Formula/Molecular Weight C24I-I3401o;,M W -- 4 8 2 . 2 1 5 2 0 10

H

16~0.,j~--2 o _g "

o, ,,,' ,'

HO

5'

I A

-

H

I

3...., O H

14

(~�89 15

General Characteristics An oil. Fungal Source Fusarium sporotrichioides (MC-72083 and DAOM 165006). Isolation/Purification Fungal material was extracted with ethyl ether, chromatographed in two equal portions on a column containing activated LiChroprep Si-60 eluted with methylene chloride, methylene chloride-methanol (79:1, v/v), methylene chloride-methanol (59:11, v/v), methylene chloride-methanol (24:1, v/v), methylene chloride-methanol (4:1, v/v), and methanol. Appropriate fractions were combined to give seven fractions. Fraction five was further purified, first by activated LiChroprep Si-60 eluted with methylene chloride containing increasing amounts of ethyl acetate followed by preparative HPLC using 9% isopropanol-hexane to yield 3'-hydroxy T-2 toxin as an oil. Spectral Data UV:

End absorption. 1H NMR: (CDC13) 3.67(1H, d, J=4.9Hz, H-2), 4.14(1H, dd, J=4.9, 2.9Hz, H-3), 5.27(1H, d, J=2.9Hz, H-4), 2.40(1H, dd, J=lh.0, 5.2Hz, ~H-7); 1.90(1H, br d, J=lh.0Hz, ~H-7); 5.26(1H, d, J=5.3Hz, H-8); 5.79(1H, br d, J=5.9Hz, H-10); 3.04(1H, d, J=3.9Hz, H13); 2.78(1H, d, J=3.9Hz, H-13); 0.78(3H, s, CH3-t4); 4.04(1H, d, J=2.6Hz, all-15);

260


4.26(1H, d, J=12.6Hz, [3H-15); 1.73(3H, br s, CH3-16); 2.42(2H, s, 2'-CH2); 1.27(3H, s, 4' and 5'-CH3); and 2.02, 2.12ppm (3H each, s, CH3COO-). 13C NMR: 78.8, C-2; 78.4, C-3; 84.5, C-4; 48.4, C-5; 42.9, C-6; 27.7, C-7; 68.5, C-8; 135.8, C-9; 124.3, C-10; 67.2, C-11; 64.2, C-12; 47.1, C-13; 6.7, C-14; 64.6, C-15; 20.2, C-16; 172.8, C-I'; 46.6, C-2'; 69.1, C-3'; 29.0, 29.5, C-4', C-5'; 20.9, 21.0(3H each,_ CH3COO-); and 170.4, 172.6ppm, (3H each, CH3COO-).

Mass Spectrum: CIMS: (methane, positive ion) (TFA derivative) 601(M+ + 1), 561,541,501,451, 403,401(base peak), 341,311,281,215, 207, 151, and ll5m/e. EIMS: 185(48%), 180(45), 105(57), 121(100), and 59m/e (92). TLC Data Silica gel. Solvent system A: Benzene-acetone (12:7, v/v), Rf0.48; solvent system B: toluene-ethyl acetate-formic acid (6:3:1, v/v/v); Re, 0.19. Solvent system C: chloroformmethanol (9:1, v/v); Re, 0.59. References R. Greenhaugh, B. A. Blackwell, M. Savard, J. D. Miller, and A. Taylor; Secondary Metabolites Produced by Fusarium sporotrichioides DAOM 165006 in Liquid Culture; J. Agrie. Food Chem., Vol. 36, pp.216-219(1988). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Myeotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994). A. Visconti, C. J. Mirocha, A. Bottalico, and J. Chelkowski; Trichothecene Mycotoxins Produced by Fusarium sporotrichioides Strain P-11; Mycotoxin Research, Vol. 1, pp. 310(1985). T. Yoshizawa, T. Sakamoto, Y. Ayano, and C. J. Miroeha; 3'-Hydroxy T-2 and 3' Hydroxy HT-2 Toxins: New Metabolites ofT-2 Toxin, A Trichothecene Myeotoxin, In Animals; Agrie. Biol. Chem., Vol. 46, pp. 2613-2615(1982).


261

Common/Systematic Name 3'-Hydroxy HT-2 toxin 3 t~,413-Dihydroxy-15-acetoxy-8 t~-[3-hydroxy-3-methylbutyryloxy]- 12,13-epoxytrichothee9-ene Molecular Formula/Molecular Weight C22H3209; M W -- 440.20463

,~

,0 _H

H ....... o .

,'_~

.... 1'

"

"

1 ~

I

I

';1

CN2OAc 15

Fungal Source Fusarium acuminatum misidentified as F. heterosporum. The F. acuminatum was isolated from Claviceps paspali honey dew on Paspalum distichum. Isolation/Purification Fungal cultures were extracted with chloroform; the crude chloroform extract was chromatographed on a column containing silica gel eluted with toluene, ethyl ether, ethyl acetate, acetone, and methanol. The acetone fraction was further purified on a silica gel column eluted with a linear gradient from ethyl acetate to acetone. The 3'-hydroxy HT-2 toxin-containing fractions were combined, reduced in volume and the toxin was precipitated from solution. Biological Activity The LDs0 value for day-old chicks dosed orally was 8.5mg/kg; the toxin was inhibitory in the wheat coleoptile assay down to 10-5 M; EDs0 values for dermal toxicity on back skin of rabbits was >1.28~g compared to < 0.16~g for T-2 toxin in the same assay. Spectral Data UV~

End absorption. 1H NMR: (CDCI3) 3.28(1H, J=4.6Hz, H-2); 4.02(1H, H-3); 4.02(1H, H-4); 2.09(2H, H-7); 5.18(1H, n-8); 5.65(1H, J=5.EHz, n-10); 4.10(1n, n-11); 2.66(1H, J=4.0Hz, n-13); 2.88(1H, J=4.0Hz, n-13); 0.65(3H, s, CH3-14); 3.84(1H, J=12.0Hz, H-15); 4.10(1H, J=12.0Hz, H-15); 1.67(3H, s, CH3-16); 2.31(2H, s, 2'-CH2); 1.19(3H each, s, 4', 5'CH3); and 1.99ppm (3H, s, CHaCOO-). 13C N M R [

262


78.43, d, C-2; 78.87, d, C-3; 79.45, d, C-4; 47.95, s, C-5, 41.90, s, C-6; 26.40, t, C-7; 67.58, d, C-8; 134.43, s, C-9; 124.19, d, C-10; 66.22, d, C-11; 64.28, s, C-12; 45.44, t, C-13; 6.93, q, C-14; 63.85, t, C-15; 19.81, q, C-16; 169.89, s, C-I'; 47.86, t, C-2'; 68.05, s, C-3'; 29.17, 29.37, C-4', C-5'; 20.83, s, CH3COO-, and 169.89ppm, s, CH3COO-. Mass Spectrum: CIMS: 441(M § + 1), 423,405, 381,363,263,203, and 121m/e. EIMS: 381,203, and 121m/e. Reference R. J. Cole, J. W. Dorner, R. H. Cox, B. M. Cunfer, H. G. Cutler, and B. P. Stuart; The Isolation and Identification of Several Trichothecene Mycotoxins from Fusarium heterosporum; J. Nat. Prod., Vol. 44, pp. 321-330(1981).


263

Common/Systematic Name 3'-Hydroxy T-2 triol 3 tt,4~, 15-Trihydroxy-8tt-[3-hydroxy-3-methylbutyryloxy]- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C2oH3oO8; M W -- 3 9 8 . 1 9 4 0 7

,~

,0 _H

O

Is

'

....

H

sl ~ 1

!

.,l~J I

I

(~H2OH 15

Fungal Source Fusarium acuminatum misidentified as F. heterosporum. The F. acuminatum was isolated from Clavicepspaspali honey dew on Paspalum distichum. Isolation/Purification Fungal cultures were extracted with chloroform; the crude chloroform extract was chromatographed on a column containing silica gel eluted with toluene, ethyl ether, ethyl acetate, acetone, and methanol. The acetone fraction was further purified on a silica gel column eluted with a linear gradient from ethyl acetate to acetone. The 3'-hydroxy T-2 triol-containing fractions were combined, reduced in volume and final purification was on a C~s reversed-phase column eluted with a linear gradient from 10-50% acetonitrile-water. Biological Activity The toxin was inhibitory in the wheat coleoptile assay down to 10-5 M; EDs0 values for dermal toxicity on back skin of rabbits was >l.281,tg compared to < 0.16~g for T-2 toxin in the same assay: Spectral Data UV:

End absorption.

IH N]VIR: (CDCI3) 3.21(IH, d, J=4.8Hz, H-2); 3.84(II--I,J=4.8, 2.9Hz, H-3), 4.42(IH, J=2.9Hz, H-4); 2.05(2H, H-7); 5.15(IH, J=5.81--Iz,H-8); 5.63(IH, J=5.4Hz, H-10); 4.05(IH, J=5.4Hz, H-11), 2.83(1H, J=4.2Hz, H-13), 2.61, 2.83(2H, J=4.2Hz, H-13), 0.68(3H, s, CH3-14); 4.24(1H, J=12.0Hz, H-15); 3.64(1H, J=12.0Hz, H-15); 1.66(3H, s, CH316); 2.40(2H, s, 2'-CH2); and 1.20ppm (3H, s, 4', 5'-CH3).

13C N M R :

264


78.49, d, C-2; 79.18, d, C-3; 79.18, d, C-4; 48.06, s, C-5; 43.37, s, C-6; 26.66, t, C-7; 68.12, d, C-8; 133.93, s, C-9; 124.76, d, C-IO; 66.45, d, C-11; 64.75, s, C-12; 45.58, t, C-13; 6.92, q, C-14; 61.37, t, C-15; 19.78, q, C-16; 170.11, s, C-I'; 47.81, t, C-2'; 67.91, s, C-3'; 29.08, q, C-4', and 29.64ppm, q, C-5' Reference R. J. Cole, J. W. Domer, R. H. Cox. B. M. Cunfer, H. G. Cutler, and B. P. Stuart; The Isolation and Identification of Several Trichothecene Mycotoxins from Fusarium heterosporum; J. Nat. Prod., Vol. 44, pp. 321-330(1981).


265

Common/Systematic Name Neosolaniol; Solaniol 413,15tt-Diacetoxy-3 t~,8a-dihydroxy- 12,13-epoxytrich9thee-9-ene Molecular Formula/Molecular Weight C19H2608;

MW

~o

I I~

o.

-" 3 8 2 . 1 6 2 7 7

H

H

~I 13 Iic~ ~I ~ I ...." "

i l

"

_--

14

..... ,OH

Ac

CH2OAc 15

General Characteristics Crystals from ethyl acetate-n-hexane; mp., 170-172~ F_ungal Source

Fusarium culmorum, F. solanL F. poae, F. sporotrichioides, F. lateritium, F. equiseti, and F. averaceum.

Biological Activity LDs0 to mice dosed IP was 14.5mg/kg; LDs0 in chicken embryo test was 5.01,tg per egg. IDs0 of protein synthesis in rabbit reticulocytes (whole cell) was 0.25~g/ml; rat liver (cellflee) was 20~g/ml. Clinical signs in animals receiving a fatal dose were marked cellular degeneration and karyorrhexis in actively dividing cells of thymus, lymph nodes, spleen, bone marrow, intestine, and testes reflecting the so-called "radiomimetic" property. Minimum dose for skin-irritant toxicity to rabbits was 1.0~g. Spectral Data 1H NMR: (CDC13) 3.66(1H, d, d-4.9Hz, H=2); 4.14(1H, dd, J-4.9, 2.9Hz, H-3); 5.25(1H, d, J=2.9Hz, H-4); 1.89(1H, dd, J=14.5, 1.4Hz, H-7); 2.33(1H, dd, J-14.5, 5.7Hz, H-7); 4.10(1H, d, d=5.7Hz, H-8); 5.65(1H, d, d-5.9Hz, H-10); 4.25(1H, d, J=5.9Hz, H-11); 2.78(1H, d, J=4.0Hz, H-13); 3.04(1H, d, J=4.0Hz, H-13); 0.83(3H, s, CH3-14); 4.29(1H, d, J=12.6Hz, H-15); 4.21(1H, d, J-12.6Hz, H-15); 1.82(3H, s, CH3-16); and 2.02, 2.13ppm (3H each, s, CH3COO-). 13C NMR: 78.7, C-2; 78.4, C-3; 84.7, C-4; 48.7, C-5; 43.4, C-6; 30.4, C-7; 66.7, C-8; 139.6, C-9; 121.1, C-10; 67.7, C-11; 64.4, C-12; 47.2, C-13; 6.7, C-14; 64.8, C-15; 19.9, C-16; 21.4 and 21.0 (3H each, CH3COO-); 169.8 and 172.0ppm (3H each, CH3COO-).

266


TLC Data Adsorbent: Kieselgel G; solvent: ethyl acetate-n-hexane, 3:1 (v/v); Rf, 0.15; detection: H2SO4 spraying and heating at 110~ for 5 min. GLC Data Solid Support: Shimalite W; liquid phase: OV-17 (1.5%); retention time: 7.50 (relative to nivalenol). References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 177(1981). M. Mesilaakso, M. Moilanen, and E. Rahkamaa; 1H and 13CNMR Analysis of Some Trichothecenes; Arch. Environ. Contam. Toxicol., Vol. 18, pp. 365-373(1989). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).


267

Common/Systematic Name Isoneosolaniol; Acuminatum; 8a,15-Diacetoxy-T-2 tetraol 8a, 15-Diacetoxy-3 a,413-dihydroxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C19H2608, ~

"- 3 8 2 . 1 6 2 7 7

H

~ O . , J

H

18 1:1

6Ac

i A5

2

3 ,

,,o l'''~ '~H

CH2OAc 15

Fungal Source Fusarium acuminatum, F. tricinctum, F. compactum, and F. equiseti. Isolation/Purification Extraction was performed with MeOH- 1% aqueous NaC1 (55:45, v/v); after filtration, the residue was extracted with the MeOH-NaCI solution. The filtrate was concentrated, defatted with n-hexane and then extracted with methylene chloride. The methylene chloride extract was dried over anhydrous Na2SO4 and concentrated to dryness. The residue was reconstituted with MeOH and chromatographed on a silica gel 60 column eluted sequentially with CH2C12, CH2C12-MeOH (95:5, v/v), CH2C12-MeOH (90:10, v/v), CH2C12-MeOH (80:20, v/v), and MeOH. Eighteen fractions were collected and tested by TLC for trichothecenes. The compounds of major interest that were positive for trichotheeenes accumulated in fractions F6 and F7. Fraction F6 contained acuminatin which was further purified by preparative HPLC using an RP18 column with MeOH-H20 (40:60, v/v) as mobile phase. Biological Activity Highly toxic to brine shrimp larvae (Artemia salina) and inhibited the growth of tomato seedlings. Spectral Data 1H NMR: (CDC13) 3.64(1H, d, J=4.9Hz, H-2); 4.17(1H, dd, J=4.9, 3.0Hz, H-3); 4.94(1H, d, J=3.0Hz, H-4); 2.06(1H, dr, J=14.3, 1.6Hz, all-7); 2.19(1H, dd, J=14.3, 5.2Hz, 13H7); 4.12(1H, m, H-8); 5.65(1H, dq, J=5.8, 1.5Hz, H-10); 3.88(1H, d,J=5.8Hz, H-11); 2.79(1H, d, J=4.0Hz, H-13); 3.02(1H, d, J=4.0Hz, H-13); 0.89(3H, s, CH3-14); 3.48(1H, d, J-12.6Hz, H-15); 3.76(1H, d,J-12.6Hz, H-15); 1.85(3H, br s, CH3-16); and 2.13 ppm (3H, s, CH3COO-).

268


13CNMR: 78.7, C-2; 78.3, C-3; 84.9, C-4; 49.1, C-5; 44.9, C-6; 27.9, C-7; 66.8, C-8; 139.0, C-9; 122.0, C-10; 68.5, C-11; 64.4, C-12; 47.0, C-13; 7.0, C-14; 62.2, C-15; 20.59, C-16; 21.0, CH3COO-; and 172.8ppm CH3COO-. References K. Ishii and Y. Ueno; Isolation and Characterization of Two New Trichothecenes from Fusarium sporotrichioides Strain M-1-1; Applied and Environmental Microbiology; Vol. 42, pp. 541-543(1981 ). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994). A. Visconti, C. J. Mirocha, A. Logrieco, A. Bottalico, and M. Solfrizzo; Mycotoxins Produced by Fusarium acuminatum: A New Triehothecene; J. Agrie. Food Chem., Vol. 37, pp. 1348-1351(1989).

14.


269

Common/Systematic Name 8-Acetoxyneosolaniol 413,8a, 15-Triacetoxy-3 tz-hydroxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C21H2sO9; M W = 4 2 4 . 1 7 3 3 3

~o H H ~ 6 ~ 0 . . J 2

I-

"1 -

:

13

I

i

1.

3 ,r~

.... ,OH

AC

CH2OAc 15

Fungal Source

Fusarium acuminatum, F. tricinctum, F. compactum, and F. roseum.

Isolation/Purification Extraction was performed with MeOH- 1% aqueous NaC1 (55:45); after filtration, the residue was extracted with the MeOH-NaC1 solution. The filtrate was concentrated, defatted with n-hexane, and extracted with methylene chloride. The methylene chloride extract was dried over anhydrous NazSO4 and concentrated to dryness. The residue was reconstituted with MeOH and chromatographed on a silica gel 60 column eluted sequentially with CHuC12, CH2CIz-MeOH (95:5, v/v), CH/Clz-MeOH (90:10, v/v), CH2C12-MeOH (80:20, v/v), and MeOH. Eighteen fractions were collected and tested by TLC for trichothecenes. The compounds of major interest that were positive for trichothecenes accumulated in fractions F6 and F7. Fraction F6 contained 8acetoxyneosolaniol which was further purified by preparative HPLC using an RPI8 column with MeOH-H20 (40:60, v/v) as mobile phase. Biological Activity Highly toxic to brine shrimp larvae (Artemia salina) and inhibited the growth of tomato seedlings. LDs0 in day-old cockerels dosed via crop intubation was 0.789mg/kg; LD50 of T-2 toxin (dosed simultaneously) was 1.84mg/kg. Also, showed inhibition in wheat coleoptile assay down to 106 M. It showed a linear, rather than a curvilinear, response. Spectral Data 1H NMR: (CDC13) 3.67(1H, d, J=4.9Hz, H-2); 4.12(1H, dd, J=4.9, 2.9Hz, H-3); 5.21(1H, d, J=2.9Hz, H-4); 1.97(1H, d, J=15.1Hz, tzH-7); 2.35(1H, dd, J=15.1, 5.7Hz, [3H-7); 5.24(1H, d, J=5.7Hz, H-8); 5.79(1H, d, J=5.9Hz, H-10); 4.28(1H, d, J=5.9Hz, H-11); 2.77(1H, d, J=4.0Hz, H-13); 3.03(1H, d, J=4.0Hz, H-13); 0.80(3H, s, CH3-14);

270


4.05(1H, d, J=12.5Hz, H-15); 4.26(1H, d, J=12.5Hz, H-15); 1.73(3H, s, CH3-16); and 2.00, 2.01, 2.12ppm (3H, s, CH3COO-). ~3CNMR: 78.7, C-2; 78.4, C-3; 84.6, C-4; 48.6, C-5; 43.0, C-6; 27.4, C-7; 68.5, C-8; 136.2, C-9; 123.8, C-10; 67.4, C-11; 64.3, C-12; 47.2, C-13; 7.0, C-14; 64.4, C-15; 20.3, C-16; 21.0, 21.1, 21.1(3H each, CH3COO-); and 172.6, 170.5, 170.0ppm (3H each, CH3CO0-). Mass Data: Positive CIMS (TFA derivative), 521(8%), 461 (10), 401 (100), and 341m/e (10). TLC Data A. Benzene-acetone, 12:7 (v/v); Re, 0.6. B: Chloroform-methanol, 9:1 (v/v); Rf, 0.85. C: Toluene-ethyl acetate-formic acid, 6:3:1 (v/v/v); Re, 0.28. GLC Data (TFA derivative) DB5 capillary column, with covalently bonded liquid phase; injector temperature, 275~ detector temperature, 300~ and temperature programming from 150-280~ at 10~ followed by 5 min. at 280~ retention time, 10.51 min. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites, Academic Press, New York, p. 178(1981). K. Ishii and Y. Ueno; Isolation and Characterization of Two New Trichothecenes from

Fusarium sporotrichioides Strain M-1-1; Applied and Environmental Microbiology, Vol.

42, pp. 541-543(1981). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994). A. Visconti, C. J. Mirocha, A. Logrieco, A. Bottalico and M. Solfrizzo; Mycotoxins Produced by Fusarium acuminatum: A New Trichothecene; J. Agile. Food Chem., Vol. 37, pp. 1348-1351(1989).

14.

Trichothecenes


271

Common/Systematic Name 8-Propionylneosolaniol 8 t~-Propanyl-3 et-hydroxy-413,15-diacetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C22H3009; M W -- 4 3 8 . 1 8 8 9 8

~0 H H ",,,,,,,,~",,,,~/.-- O,,,~2

16

H 18

I-. 1ol

~ O II -

MeCH2CO

'

~

:

~5

_ 14

3......OH

,,o / 4

c

r189 15

General Characteristics Crystals; mp., 182-183~ Fungal Source

Fusarium sporotrichioides (MC-72083 and DAOM 165006).

Isolation/Purification Fungal material was extracted with ethyl ether, chromatographed in two equal portions on a column containing activated LiChroprep Si-60 eluted with methylene chloride, methylene chloride-methanol (79:1, v/v), methylene chloride-methanol (59:11, v/v), methylene chloride-methanol (24:1, v/v), methylene chloride-methanol (4:1, v/v), and methanol. Appropriate fractions were combined to give seven fractions. Further purification was achieved by preparative HPLC using various isocratic solvent systems of 2-propanol-hexane. Fraction three was further purified by preparative HPLC using 5% isopropanol-hexane followed by 3% isopropanol-hexane to yield crystalline 8-propionylneosolaniol in pure form. Biological Activity The trichothecenes as a group show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukoeytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-free systems. Some trichothecenes inhibit the initiation step of protein synthesis on

272


polyribosomes and others inhibit the elongation and/or termination step in protein synthesis. The trichothecenes have been strongly implicated in natural intoxications of animals and humans. Spectral Data 1H NMR: (CDC13) 3.68(J2,3=4.9Hz, H-2); 4.12(J3,2=4.9, J3,4=2.8Hz, H-3); 5.27(J4,3=2.8Hz, H4); 1.93(JAB=15.1Hz, H-70; 2.38(JT,s=5.8Hz, H-7); 5.27(Js,7=5.SHz, H-8); 5.80(J10,11-6.0nz, J10,16-l.2Hz, H-10); 4.33(J11,10=6.0Hz, H-11); 2.78(JAB=3.9Hz, H-13); 3.04(H13); 0.79(H-14); 4.03(JAB=12.6Hz, H-15); 4.27(H-15); 1.72(J16,10=l.2Hz, H-16); 2.28(2H, q, J2,,3.=7.5Hz, H-2'); 1.13(1H, t, Jy,2,=7.5Hz, H-3'); 2.01; and 2.13ppm (CH3Ac). 13C NMR: 78.7, C-2; 78.2, C-3; 84.3, C-4; 48.3, C-5; 42.9, C-6; 27.7, C-7; 68.0, C-8; 136.3, C-9; 123.8, C-10; 67.3, C-11; 64.2, C-12; 47.0, C-13; 6.6, C-14; 64.5, C-15; 20.1, C-16; 174.1, C-17; 27.6, C-18; 8.9, C-19; 20.8, 20.1(3H, CH3COO-); and 170.2, 170.5ppm (3H each, CH3COO-).

Mass Spectrum: EIMS: (lacked a significant M +) 185(35%), 180(61), 121(89), 105(48), and 57role (100). GLC Data DB-5 fused capillary column (20m x 0.32mm i.d., 0.25l.tm film); temperature programmed from 140-260~ at 15~ using helium as carrier gas. References R. Greenhaugh, B. A. Blackwell, M. Savard, J. D. Miller, and A. Taylor; Secondary Metabolites Produced by Fusarium sporotrichioides DAOM 165006 in Liquid Culture; J. Agric. Food Chem., Vol. 36, pp. 216-219(1988). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; Plant Research Centre, Agriculture Canada, Ottawa, Ontario, K1A 0C6.

14. Trichothecenesand Related Metabolites

273

Common/Systematic Name 8-Butyrylneosolaniol 8 t~-Butyryloxy-3 t~-hydroxy-413,15-diacetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C23H3209, ~

= 452.20463

~o H

H

16~O-,~2

] ....,OH

o 11=113" I,,O H-~;O......k~'v>~'~:l'2 M CH C i i '\OAc -

14

(3�89 15



Isolation/Purification Fungal material was extracted with ethyl ether and chromatographed in two equal portions on a column containing activated LiChroprep Si-60 eluted with methylene chloride, methylene chloride-methanol (79:1, v/v), methylene chloride-methanol (59:11, v/v), methylene chloride-methanol (24:1, v/v), methylene chloride-methanol (4:1, v/v), and methanol. Appropriate fractions were combined to give seven fractions. Further purification was achieved by preparative HPLC using various isocratic solvent systems of 2-propanol-hexane. Fraction three was further purified by preparative HPLC using 5% isopropanol-hexane followed by 3% isopropanol-hexane. Pure 8-butyrylneosolaniol was obtained as an oil. Biological Activity The trichothecenes as a group show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukocytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-free

274


systems. Some trichothecenes inhibit the initiation step of protein synthesis on polyribosomes and others inhibit the elongation and/or termination step in protein synthesis. The trichothecenes have been strongly implicated in natural intoxications of animals and humans. Spectral Data 1H NMR: (CDC13) 3.68(,J2,3=4.9Hz,H-2); 4.13(J3,2=4.9Hz, J3,4=2.9Hz, H-3); 5.27(.A,3=2.9Hz, H-4); 1.91(J~=15.9Hz, H-7); 2.39(JT,S=5.3Hz, H-7); 5.27(,Js,7=5.5Hz, H-8); 5.79(Jlo,1~= 4.9Hz, H-10); 4.30(,J~1,lo=4.9Hz, H-11); 2.78(J,~=3.9Hz, H-13); 3.04(H13); 0.79(H-14); 4.03(,L~=12.5Hz, H-15); 4.27(H-15); 1.72(J~6,1o=l.2Hz, H-16); 2.33(2H, q, Jz,r=7.4Hz, H-2'); 1.64(Jz,3,=,J3,,a,=7.4Hz, H-3'); 0.93(J4,,3~-7.4Hz, H-4'); 2.01; and 2.13ppm (CH3-Ac). Mass Spectrum: EIMS: (lacked a significant M+) 185(26%), 180(45), 121(100), 105(48), and 71m/e

(80).

GLC Data DB-5 fused capillary column (20m x 0.32mm i.d., 0.251.tm film); temperature programmed from 140-260~ at 15~ using helium as cartier gas. References R. Greenhaugh, B. A. Blackwell, M. Savard, J. D. Miller, and A. Taylor; Secondary Metabolites Produced by Fusarium sporotrichioides DAOM 165006 in Liquid Culture; J. Agric. Food Chem., Vol. 36, pp. 216-219(1988). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).


275

Common/Systematic Name 8-Isobutyrylneosolaniol 3 t~-Hydroxy-413, 15-diacetoxy-8 tt-[(2-methylpropionyl)oxy]- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C23H3209; MW

16

Me\

-- 4 5 2 . 2 0 4 6 3

~o H -~?',~2 _[8

CHCOO~'"'~

Me /

H

3.... ,OH

61 '" ~1., ;''0 4L~ 12

i ~5 7. = 14

OAc

6�89 15

Fungal Source


Isolation/Purification Fungal material was extracted with ethyl ether, chromatographed in two equal portions on a column containing activated LiChroprep Si-60 eluted with methylene chloride, methylene chloride-methanol (79:1, v/v), methylene chloride-methanol (59:11, v/v), methylene chloride-methanol (24:1, v/v), methylene chloride-methanol (4:1, v/v), and methanol. Appropriate fractions were combined to give seven fractions. Further purification was achieved by preparative HPLC using various isocratic solvent systems of 2-propanol-hexane. Fraction three was further purified by preparative HPLC using 5% isopropanol-hexane followed by 3% isopropanol-hexane. Pure 8-isobutyrylneosolaniol was obtained atter double development preparative TLC using hexane-acetone-formic acid (65:33:2, v/v/v; Rf 0.42). Biological Activity The trichothecenes as a group show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukoeytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-free systems. Some trichothecenes inhibit the initiation step of protein synthesis on polyribosomes and others inhibit the elongation and/or termination step in protein synthesis. The trichothecenes have been strongly implicated in natural intoxications of animals and humans.

276


_Spectral Data 1H NMR: (CDCI3) 3.69(J2,3=4.9Hz, H-2); 4.14(J3,2=4.9Hz, J3,4=2.9Hz, H-3); 5.35(J4,3=2.9Hz, H-4); 1.83(JAB=15.0Hz, H-7); 2.40(H-7); 5.26(Js,7=5.6Hz, H-8); 5.80(Jx0,~=5.4Hz, H10); 4.38(Jlx,10=5.4Hz, H-11); 2.78(JAB=3.9Hz, H-13); 3.05(H-13); 0.79(H-14); 4.02(JAB=lZ.6Hz, H-15); 4.3 l(H-15); 1.73(J16,~0=l.2Hz, H-16); 2.45(2H, q, J2,,3~J2,,4~=7.4Hz, H-2'); 1.16(J2,,3~7.0Hz, H-3'); 1.16(J2,,4'=7.0Hz, H-4'); 2.01; and 2.13ppm (CH3-Ac). Mass Spectrum: EIMS: (lacked a significant M +) 195(38%), 191(62), 180(60), 121(100), 105(57), and 71m/e (98). GLC Data DB-5 fused capillary column (20m x 0.32mm i.d., 0.2511m film); temperature programmed from 140-260~ at 15~ using helium as carrier gas. References R. Greenhaugh, B. A. Blackwell, M. Savard, J. D. Miller, and A. Taylor; Secondary Metabolites Produced by Fusarium sporotrichioides DAOM 165006 in Liquid Culture; J. Agrie. Food Chem., Vol. 36, pp. 216-219(1988). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).


277

Common/Systematic Name 15-Deacetylneosolaniol; NT-2 Toxin; 4-Acetyl-T-2 tetraol 4~-Acetoxy-3a,8a, 15-trihydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2407; MW

=

~o H I-

H

"1 13

-

340.15220

~ .c~ l .... ,OH

14

AC

(~H2OH 15

General Characteristics Needles from ethyl acetate-n-hexane; mp., 172-173~ FunRal Source Fusarium sporotrichioides (strain M-1-1), F. tricinctum, F. compactum, and F. averaceum. Isolation/Purification Extracted by charcoal absorption and elution with methanol. Extraction of methanolchloroform soluble materials produced a pale yellow powder. The powder was chromatographed on a silica gel column eluted with n-hexane, n-hexane-acetone (12: 5, 2:1, 1:1, 1:2, v/v), acetone and methanol followed by a second silica gel column eluted with benzene-acetone (3:2, v/v), and n-hexane-acetone (1:1, v/v). NT-2 was crystallized with ethyl acetate-n-hexane. Biological Activity Inhibited uptake of [14C]leucine in protein synthesis with rabbit reticulocytes; IDs0 at concentrations of 0.23 and 0.251,tg/ml by NT-1 and NT-2, respectively. These activities were similar to that of neosolaniol (0.25tzg/ml) in the same assay. Spectral Data IR:

(KBr) 3380, 2960, 1720, 1370, 1240, and 1025cm"1. ]H NMR: (CDCI3) 3.64(1H, d, J=4.9Hz, H-2); 4.17(1H, dd, J=4.9, 3.0Hz, H-3); 4.94(1H, d, J=3.0Hz, H-4); 2.06(1H, dt, J=14.3, 1.6Hz, all-7); 2.19(1H, dd, J=14.3, 5.2Hz, [~H7); 4.12(1H, m, H-8); 5.65(1H, dq, J=5.8, 1.5Hz, H-10); 3.88(1H, d, J=5.8Hz, H-11);

278


2.79(1H, d, J=4.0Hz, H-13); 3.02(1H, d, J=4.0Hz, H-13); 0.89(3H,s, CH3-14); 3.48(1H, d,J=12.6Hz, H-15); 3.76(1H, d, J=12.6Hz, H-15); 1.85(3H, br s, CH3-16); and 2.13 ppm (3H, s, CH3COO-). 13C NMR: 78.7, C-2; 78.3, C-3; 84.9, C-4; 49.1, C-5; 44.9, C-6; 27.9, C-7; 66.8, C-8; 139.0, C-9; 122.0, C-10; 68.5, C-11; 64.4, C-12; 47.0, C-13; 7.0, C-14; 62.2, C-15; 20.59, C-16; 21.0, CH3COO-; and 172.8ppm, 3H, CH3COO-. Mass Data: LREIMS: 340m/e (M+); found: C, 60.04; H, 7.10; O, 32.86%; calcd for C17H2407;C, 59.97; H, 7.11; O, 32.92%. References K. Ishii and Y. Ueno; Isolation and Characterization of Two New Trichothecenes from Fusarium sporotrichioides Strain M-1-1; Applied and Environmental Microbiology, Vol. 42, pp. 541-543(1981). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994). A. Visconti, C. J. Mirocha, A. Logrieco, A. Bottalico and M. Solfrizzo; Mycotoxins Produced by Fusarium acuminatum: A New Trichothecene; J. Agric. Food Chem., Vol. 37, pp. 1348-1351(1989).


279

Common/Systematic Name 4-Deacetylneosolaniol 3a,4~,8a-Trihydroxy- 15-acetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C17H2407; M W = 3 4 0 . 1 5 2 2 0

H H oJ2 ~r,~,,O

"

,o

] "OH ......

-" ~' OH (~H2OAc 15

Fungal Source Fusarium acuminatum misidentified as F. heterosporum. The F. acuminatum was isolated from Claviceps paspali honey dew on Paspalum distichum. Isolation/Purification Fungal cultures were extracted with chloroform; the crude chloroform extract was chromatographed on a column containing silica gel eluted with toluene, ethyl ether, ethyl acetate, acetone, and methanol. The acetone fraction was further purified on a silica gel column eluted with a linear gradient from ethyl acetate to acetone. The 15-acetoxy T-2 triol-containing fractions were combined, reduced in volume and final purification was on a Cls reversed-phase column eluted with a linear gradient from 50% acetonitrile to water. Biological Activity The toxin was inhibitory in the wheat coleoptile assay down to 10"3 M; EDs0 values for dermal toxicity on back skin of rabbits was > 1.28~g compared to < 0.161,tg for T-2 toxin in the same assay. The LDs0 value for day-old chicks dosed orally was > 10mg/kg. Spectral Data UV:

End absorption. :H NMR: (CDCI3) 3.24(1H, J=4.9Hz, n-2); 3.89(1H, H-3); 4.06(1H, H-4); 2.06(2H, n-7); 5.19(1H, H-8); 5.40(1H, H-10), 4.03(1H, H-I 1); 2.84(1H, J=4.1Hz, H-13); 2.61(1H, J=4.1Hz, H-13); 0.67(3H, s, CHa-14); 3.89, 4.03(2H, H-15); 1.72(3H, s, CH3-16); and 1.97ppm (3H, s, CHaCOO).

~3C NMR:

280


(CDC13) 78.48, d, C-2; 79.01, d, C-3; 79.49, d, C-4; 47.86, s, C-5; 42.57, s, C-6; 30.09, t, C-7; 64.82, d, C-8; 138.93, s, C-9; 120.38, d, C-10; 66.65, d, C-11; 64.61, s, C-12; 45.58, t, C-13; 6.78, q, C-14; 64.61, t, C-15; 20.34, q, C-16; 20.82, q, CH3COO) and 169,60ppm, s, CH3COO. Mass Spectrum: EIMS: 340(M+), 322, 203, 175, and 121m/e (base peak). Reference R. J. Cole, J. W. Domer, R. H. Cox, B. M. Cunfer, H. G. Cutler, and B. P. Stuart; The Isolation and Identification of Several Trichothecene Mycotoxins from Fusarium heterosporum; J. Nat. Prod., Vol. 44, pp. 321-330(1981).


281

Common/Systematic Name Sporotrichiol Molecular Formula/Molecular Weight C20H3006;MW = 366.20424

~o H 16

-

O

2

.,,,,," ~ ~ ' 2

Me2CHCH2COO

!

-_.

~5

,,OH 'O

14

(~H2OH 15

General Characteristics Isolated as an oil. Fungal Source Fusarium sporotrichioides (MC-72083). Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), followed by Florisil column chromatography eluted with benzene-hexane (2:1, v/v), methylene chloride, and chloroform-methanol (95:5, v/v) to give an oil highly enriched with trichothecenes. The oil was further purified by normal phase preparative HPLC using benzene-acetone followed by preparative RPTLC to yield several trichothecenes including sporol and sporotrichiol. Spectral Data IR: (thin film) 3422(OH) and 1726cm1 (ester). 1HN:V[R: (CDCI3) 3.49(1H, d, H-2), 4.49(1H, m, H-3); 2.05-2.25(2H, m, H-4); 1.96(1H, d, n-7), 2.30(1H, dd, n-7), 5.50(1H, d, n-8), 5.79(1H, d, n-10), 4.17(1H~ d, n-11), 2.57(1H, d, J=4.1Hz, H-13); 3.06(1H, d, d=4.1Hz, H-13), 0.87(3H, s, CH3-14); 3.53(1H, d, J=12.7Hz, n-15), 3.70(1H, d, J=12.7Hz, H-15), 1.75(3H, s, CH3-16); 2.21(2H, m, H-18); 2.05-2.30(1H, m, H-19); and 0.95ppm (3H each, d, J=7.0Hz, CH3-20 and -21).

13CNMR: (CDCI3) 79.6 C-2; 69.1, C-3, 42.1, C-4, 45.8, C-5, 43.3, C-6; 26.8, C-7, 68.4, C-8, 135.7, C-9; 125.4, C-10, 68.1, C-11, 65.1, C-12; 48.5, C-13; 12.44, C-14; 63.1, C-15; 20.4, C-16; 171.9, C-17; 43.7, C-18, 25.7, C-19; and 22.3ppm, C-20 and C-21.

282


Mass Spectrum: HREIMS: 366.204m/e; calcd for C20H3006,366.206. References D. G. Corley, G. E. Rottinghaus, and M. S. Tempesta; Novel Trichothecenes from Fusarium sporotrichioides; Tetrahedron Letters, Vol. 27, pp. 427-430(1986). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).


283

Common/Systematic Name 413,8a, 15-Triacetoxy-3 t~,7a-dihydroxy-12,13-epoxytriehothec-9-ene Molecular Formula/Molecular Weight C21H28Olo;

~6.

~

M 3 v V -- 4 4 0 . 1 6 8 2 5

~o H

o

.

H

J

-

2

5

Ac0 ~)H 141 C;H2OAc

3

OAc

15

General Characteristics Crystals; mp., 185-188~ Fungal Source Fusarium equiseti. Biological Activity LDs0 in rats dosed IP was 1.2mg/kg. Reference R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 200(1981).

284


Common/Systematic Name 3 a-Acetoxy- 15-hydroxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C17H2206, M W = 322.14164

,,,OAc

.... .

;

o I" 4

14

~H20H 15

General Characteristics Colorless needles; mp., 202-203~

[a]D 26 q- 41.7 ~ (c=0.40,

in CHCI3).

Fungal Source Fusarium roseum

(ATCC 28114).

Isolation/Purification Fusarium roseum Greenhalgh et al.

cultures were harvested and the broth was extracted as described by (1984). Some of the oil from the methanol phase after it had been partitioned and concentrated was dissolved in methylene chloride and passed through a Florisil column eluted with 750ml of (A) methylene chloride, (B) 0.5% methanol in methylene chloride, and (C) 5% methanol in methylene chloride. Fraction A afforded an oil from which 3-acetyldeoxynivalenol was obtained. Fractions B and C also afforded yellow oils. The oil which remained in fraction A after removal of 3-acetyldeoxynivalenol was chromatographed on silica gel (Kieselgel 60). The column was eluted first with 10% toluene in methylene chloride and then with 0.5% methanol in methylene chloride, 1.5% methanol in methylene chloride, 3% methanol in methylene chloride, and finally 13% methanol in methylene chloride. Fractions were collected and monitored by TLC; fractions 6-10, 11-12, 13-17, and 18-20 were combined to give mixtures A1, A2, A3, and A4, respectively. Further purification of mixtures A2 and A3 was accomplished with a Chromatotron (Model No. 7924, Harrison Research, Inc.) with a 2mm silica gel plate (Kieselgel 60). 3 a-Acetoxy- 15-hydroxy- 12,13-epoxytrichothec-9-en-8-one was isolated from mixture A2 by elution with 20% ethyl acetate in hexane followed by rechromatography using the same solvent.

Spectral Data 1H NMR: (CDC13) 0.83(3H, H-14); 1.80(3H, H-16,J16,~o=l.4Hz); 2.12(3H, AC-CH3); 2.40, 2.17(2H, H-4, Jab=12.8Hz, J4,3=4.4Hz); 2.86, 2.42(2H, H-7, J~=16.8Hz); 2.86,


285

3.09(2H, H-13, Jab=3.9Hz); 3.64(2H, H-15); 3.81(1H, H-2, Jz,3=4.6Hz); 4.52(1H, H11, J11,10=5.7Hz), 5.18(1H, H-3, J3,z=4.6Hz, J3,4=lO.2, 4.4Hz); and 6.55 ppm (1H, HI0, J10,11=5.7Hz, J10,a6=l.4Hz). 13C NMR: (CDCI3) 78.1, C-2; 71.2, C-3; 38.5, C-4; 45.0, C-5; 47.5, C-6; 38.3, C-7; 198.7, C-8; 138.3, C-9; 137.7, C-10; 68.2, C-11; 65.3, C-12; 48.3, C-13; 11.2, C-14; 64.1, C-15; 15.3, C- 16; 20.9, CH3COO-; and 170.5ppm, C=O. Mass Spectrum: HREIMS: 323.149m/e (M ++ 1); calcd for C17H2306,323.150. References R. Greenhalgh, R.M. Meier, B. A. Blaekwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Minor Metabolites ofFusarium roseum (ATCC 28114); J. Agile. Food Chem., Vol. 32, pp. 1261-1264(1984). D. R. Lauren, A. Ashley, B. A. Blackwell, R. Greenhalgh, J. D. Miller, and G. A. Neish; Triehothecenes Produced by Fusarium crookwellense DAOM 193611; J. Agile. Food Chem., Vol. 35, pp. 884-889(1987).

286


Common/Systematic Name 3-Acetyl-4-deoxynivalenol 3 a-Acetoxy-Ta, 15-dihydroxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C17H2207; ~

= 338.13655

1 6 ~ 02q 1H 0

HO

H

"" " c 3.,",uP,

: 14 ~H2OH

15

General Characteristics Colorless needles from ethyl ether-n-pentane; mp.,185.5-1860C; [a]D 20 + 43.0* (c=1.3, in EtOH); [a]D25 + 40.5~ tetracetate; mp., 168-1700C. Isolation/Purification Fungal mycelium was extracted with methylene chloride, concentrated and the residue dissolved in methanol. The methanol phase was extracted with petroleum ether and evaporated to dryness. The residue was chromatographed on a silica gel column eluted with chloroform-ethanol (50:1, v/v) and then chloroform-ethanol (400:1, v/v). 3-Acetyl-4deoxynivalenol was recrystallized from hot ethyl ether. Fungal Source Fusarium culmorum (HLX 1503 ATCC 28114), F. graminearum (HLX 1506), and F. roseum. Biological Activity LDs0 in male ddS strain of mice dosed IP was 76.7mg/kg; females, 49.9mg/kg; ducklings, 37mg/kg. Minimum SC dosage to induce vomiting in ducklings was 10.0 mg/kg; dogs, S.C., 0.2mg/kg. Inhibited Tetrahymena pyriformis, 29.0~g/ml. Spectral Data UV:

~, E~. max

219nm (6=5,900).

IR:

(KBr) 3480, 3400, 1720, and 1680cml.


287

Spectral Data UV:

~,.=~~ 220nm (c=7,100). IR:

(Nujol) (needles) 3500, 3420, 1740, 1683, and 1668cm1; (needles or plates) 3480 br, 1750, 1690, 1672, and 1660cm"1. 1H NMR: (CDC13) 6.10(1H, d, J=4.5Hz, H-2); 4.72(1H, m, H-3); ca. 7.75(2H, m, H-4); 5.12(1H, d, J=l.5Hz, H-7); 3.36(1H, dd, J=5.5, 1.5Hz, H-10); 5.29(1H, d, J=5.5Hz, H-11); 6.84(2H, s, H-13); 8.81(1H, s, H-14); 6.14(2H, d, J=l.5Hz, H-15); 8.12(3H, s, H-16); 7.90(3H, OAt); and 6.15, 8.0ppm (1H each, OH). Mass Data: HREIMS: 338.1360, C17H2207requires 338.1365; found: C, 60.4; H, 7.0%; C17H2207 requires C, 60.35; H, 6.55%. Reference M. M. Blight and J. F. Grove; New Metabolic Products of Fusarium culmorum: Toxic Trichothec-9-en-8-ones and 2-Acetylquinazolin-4(3H)-one; J. C. S. Perkin I, pp. 16911693(1974).

288


Common/Systematic Name Trichodermol 4 I]-Hydroxy-12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C15H2203; MW = 250.15689 ~o H

~~

~.;-

0 ~H

~

,~,,'-" 15

14

H

General Characteristics Needle crystals from light petroleum; mp., 116-119~ crystals from ether-n-hexane; mp., 117.5~176 [a] D2~ - 33.5~ in CHC13). Fungal Source Myrothecium roridum, Trichoderma polysporum, T. sporulosum, and Hypocrea austrograndis. Spectral Data UV~

End absorption. 1H NMR: (CDC13) 3.51, H-2; 1.7-2.5, H-3; 4.3, H-4; 1.7-2.5, H-7; 1.7-2.5, H-8; 5.41, H-10; 3.81, H-11; 2.95, H-12; 0.80, H-14; 0.85, H-15; and 1.70ppm, H-16. 13C NMR: (CDCh) 78.8, C-2; 40.2, C-3; 74.0, C-4; 49.2, C-5; 39.8, C-6; 24.5, C-7; 28.0, C-8; 140.1, C-9; 118.8, C-10; 70.4, C-11; 65.8, C-12; 47.6, C-13; 6.2, C-14; 15.8, C-15; and 23.2ppm, C- 16. Mass Data: HREIMS: 250.1569(C~5H2203),235.1335(C14H~903), 207.1384(C~3H~902), and 188.1200m/e (C13H160). TLC Data A. Adsorbent: silica gel G. Solvent: chloroform-methanol, 98:2, v/v; Re, 0.36; detection: H2SO4 spraying and heating at 110~ for 5 minutes. B. Adsorbent: alumina. Solvent: chloroform-methanol, 98:2, v/v; Rf, 0.86; detection: H2SO4 spraying and heating for 5 minutes.


289

References W. A. Ayer and S. Miao; Secondary Metabolites of the Aspen Fungus Stachybotrys cylindrospora; Can. J. Chem., Vol. 71, pp. 487-493(1993). J. R. Hanson, T. Marten, and M. Sivems; Studies in Terpenoid Biosynthesis. Part XII. Carbon-13 Nuclear Magnetic Resonance Spectra of the Trichothecenes and the Biosynthesis of Trichothecolone from [2-13C]Mevalonic Acid; J. Chem. Soc. Perkin I, pp. 1033-1036(1974).

290


Common/Systematic Name 7 t~-Hydroxytrichodermol 413,7a-Dihydroxy- 12,13-epoxytrichothec-9-ene Molecular formulamolecular Weight C15H2204; MW = 266.15181 lo

H

H

~I~

~

H

.......H

~1 '~ ~-;~.,,,,v ~

-IA ~e ~

H~)

~

"H

'H

15

General Characteristics Amorphous colorless solidi [a]D = +2.4* (C=0.76, in MeOH). Plant Source Myrothecium roridum.

Isolation/Purification An aqueous shake culture ofMyrothecium roridum (M4582) as filtered through Whatman # 1 filter paper, and the filtrate was extracted three times with ethyl acetate. The mycelia remaining from filtration were soaked in methanol overnight and filtered. The methanol filtrate was concentrated in vacuo until only an aqueous phase remained which was extracted three times with ethyl acetate. The ethyl acetate extracts were pooled and concentrated m vacuo to yield 4g of gum. The gum was subjected to filtration chromatography (50g of silica gel) with increasing amounts of ethyl acetate in hexane to yield five fractions: 1 (800mg, eluted with hexane), 2 (200mg, eluted with 10% ethyl acetate in hexane), 3 (400mg, eluted with 30% ethyl acetate in hexane), 4 (500mg, eluted with 50 to 70% ethyl acetate in hexane), and 5 (1.5g, eluted with ethyl acetate). Fractions 3 and 4 were subjected to purification on a Chromatotron (2-mm silica gel plates, ethyl acetate-hexane) to yield, after recrystallization (ethyl acetate-hexane), 25mg of trichodermol and 50 mg of 7a-hydroxytrichodermol. 7a-Hydroxytrichodermol also was isolated from rice culture: ca. 200mg/kg of flee. Spectral Data ~H NMR: (CDC13) 0.86(3H, s, H-14); 1.09(3H, s, H-15); 1.69(3H, s, H-16); 1.8 to 2.1(2H, m, H-3~ and H-8~); 2.24(1H, dd, J= 12.6 and 6.0Hz, H-8a); 3.06 and 3.1 0(1H each, AB, J=4.3 Hz, H-13); 3.61(1H, d, J-5.5Hz, H-11); 3.82(1H, d, J-5.0Hz, H-2); 4.26(1H, dd, J=7.6 and 4.0Hz, H-4); 4.46(1H, dd, J=12.0 and 6.0Hz, H-7); and 5.36ppm (1H, br d, J=5Hz, H- 10).


Mass Spectrum:

HREIMS: 266.1526m/e found for C15H2204;calcd 266.1512. Reference B. B. Jarvis, Y.-W. Lee, C. S. Yatawara, D. B. Mazzocchi, J. L. Flippen-Anderson, R. Gilardi, and C. George; 7a-Hydroxytrichodermol, A New Trichothecene from Myrothecium roridum; Appl. Environ. Microbiol., Vol. 50, pp. 225-228(1985).

291

292


Common/Systematic Name Verrucarol 413,15-Dihydroxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C15H2204; MW - - 266.15181 10

[8

H -

H

61,3.,~

....

iu

. 14

OH20H 15

General Characteristics Crystals from ether-methylene chloride; mp., 158-159~ fine needles from acetone-ether; mp., 155-156~ [a]D22 -39 ~ (C= 1.069, in CHC13). Di-O-acetylverrucarol, colorless needles from acetone-ether-petroleum ether; mp., 148-150~ [a]D25 -17 ~ (C=1.22, in CHC13). Di-O-benzoylverrucarol, crystals from ether-petroleum ether; mp., 151-152~ [a]D24 - 64 ~ (c=l. 130, in acetone). Dihydroverrucarol, crystals from acetone-etherpetroleum ether; mp., 149-151~ [a]D25 - 6 ~ (C=I.101, in CHCI3). Fungal Source Formed as one of the products of alkaline hydrolysis of verrucarin A which is produced by Myrothecium vemwaria. Spectral Data UV~ EtOH

~. max 195nm (e=7,900). IR;

(CH2C12) 3610, 3570, 1675, 1380, 1335, 1079, 1045, 965, and 820cm"1. 1H NMR: 3.70, H-2; 1.7-2.5, H-3; 4.7, H-4; 1.7-2.5, H-7; 1.7-2.5, H-8; 5.45, H-10; 3.80, H-11; 2.95, H-12; 0.92, H-14; 3.70, H-15; and 1.72ppm, H-16. 13C NMR: 78.5, C-2; 39.5, C-3; 74.0, C-4; 48.7, C-5; 43.7, C-6; 20.9, C-7; 28.0, C-8; 140.4, C-9; 118.7, C-10; 66.4, C-11; 65.6, C-12; 47.4, C-13; 6.8, C-14; 62.1, C-15; and 22.9ppm, C-16.


293

TLC Data A. Adsorbent: silica gel G. Solvent: chloroform-methanol (98:2, v/v); Rf, 0.06; detection: H2SO4 spraying and heating at 110~ for 5 minutes. B. Adsorbent: alumina. Solvent: chloroform-methanol (98:2, v/v); Rf, 0.34; detection: H2SO4 spraying and heating at 110~ for 5 minutes. References J. Gutzwiller and C. Tamm; Uber die Verrucarine und Roridine. Struktur von Verrucarol; Helv. Chim. Acta, Vol. 46, pp. 1786-1790(1963). J. Gutzwiller and C. Tamm; Uber die Struktur von Verrucarin A, Helv. Claim. Acta, Vol. 48, pp. 157-176(1965).

294


Common/Systematic Name 4,15-Diacetylverrucarol 4[3,15-Diacetoxy- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C19H2606; M W -- 350.17294 H 61

-

H

,~.,,,

,

14

CH2OAc

AC

15

General Characteristics White prisms from benzene-methanol; mp., 147-148~ [0~]D 25 - 14~ (c=l.01, in MeOH). Readily soluble in most organic solvents such as methanol, acetone, chloroform, ether, and benzene; sparingly soluble in hexane and petroleum ether. Fungal Source

Myrothecium spp.

Biological Activity Antifungal activity against Trichophytonasteroides (EDso, O.1ktg/ml) and T. interdigitale (EDs0, 10~g/ml). Spectral Data 1H NMR: (CDCI3) 3.80(H-2); NR(H-3); 3.24(H-4); NR(H-7); NR(H-8); 5.39(H-10); 3.75(H-11); 2.79(H-13e0; 3.09(H-1313); 0.79(H-14); 4.05(H-15a); 4.15(H-1513); and 1.70ppm (H- 16). 13C NMR: (CDC13) 79.0, C-2; 36.7, C-3; 75.4, C-4; 48.9, C-5; 43.4, C-6; 21.4, C-7; 28.1, C-8; 138.7, C-9; 119.7, C-10; 66.8, C-11; 65.2, C-12; 47.3, C-13; 6.8, C-14; 63.7, C-15; 23.0, C-16; 20.2; 2 each CH3CO; and 169.6, 169ppm CH3CO. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 189 (1981). M. Okuchi, M. Itoh, Y. Kaneko, and S. Doi; A New Antifungal Substance Produced by

Myrothecium; J. Agric. Biol. Chem. Jpn., Vol. 32, pp. 394(1968).


295

Common/Systematic Name Crotocin; Antibiotic T Molecular Formula/Molecular Weight C19H2405; M W "- 3 3 2 . 1 6 2 3 7

~ 0

~,D ;s

CCH=:CHMe

General Characteristics Colorless prisms from methanol; mp., 126-128~ 118~ [a]D25 + 7.17 ~ (C=3.98, in CHC13).

needles from ether-benzene; mp., 116-

Fungal Source

Cephalosporium crotocmigenum and Trichothecium roseum.

Biological Activity LDs0 of crotocin in mice dosed IV was 700mg/kg; fungistatic; negligible antitumor activity. Spectral Data UV:

Xmx 211(e=21,800) and 209nm. IR:

1710 and 1640cm~. 13C NMR: (CDCI3) 74.3, C-2; 36.2, C-3; 78.5, C-4; 47.7, C-5; 41.3, C-6; 58.1, C-7; 50.4, C-8; 137.2, C-9; 122.9, C-10; 69.4, C-11; 65.9, C-12; 46.8, C-13; 6.2, C-14; 15.9, C-15; 21.0, C-16; 166.4, C-17; 120.2, C-18; 146.5, C-19; and 14.8ppm, C-20. TLC Data Adsorbent, silica gel; solvent, ethanol-ethyl acetate-acetone, 1:4:4, v/v/v; Re, 0.67; detection: 50% H2SO4 spraying and heating at 110~ for 5 minutes. References B. A. Achilladelis and J. R. Hanson; Minor Terpenoids of Trichothecium roseum; Phytochemistry, Vol. 8, p. 765 (1969).

296


R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 195 (1981).

J. Gyimesi and A. Melera, On the Structure of Crotocin, an Antifungal Antibiotic, Tetrahedron Letters, p. 1665 (1967).


297

_Common/Systematic Name Crotocol 4 [3-Hydroxy-7, 8,12,13-diepoxytrichothec-9-ene Molecular Formula/Molecular Weight C 1 5 H 2 0 0 4 ; M W -- 264.13616

~ "

o

--

I s

General Characteristics Crystals; mp., 154~

4

[ ~ ] D 20 -

6.4 ~ (c=2.01, in CHC13); acetate derivative; mp., 128~

Fungal Source Formed upon mild alkaline hydrolysis of crotocin. Spectral Data UV~

Zm~, 210nm (e=4,830). TLC Data Adsorbent: silica gel; solvent, ethanol-ethyl acetate-acetone, 1:4:4, v/v/v; Rf, 0.50; detection: 50% H2SO4 spraying and heating at 110~ for 5 minutes. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 196 (1981). J. Gyimesi and A. Melera; On the Structure of Crotocin an Antifungal Antibotic; Tetrahedron Letters, p. 1665 (1967).

298


Common/Systematic Name Trichothecolone 4[3-Hydroxy-12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C15H2004; ~ = 264.13616 10

H

-

15

H

~5

'""~

14

4"%OH

General Characteristics Colorless needles from benzene-light petroleum; mp., 183-184~ [a]D 19"5 + 22.5 ~(c=l.0, in CHC13); [et]Ds + 17.0~ (C=I.0, in EtOH). Acetyl derivative, prisms from methanol; mp., 148-149~ 2,4-dinitrophenylhydrazone, deep orange rods from chloroform-ethanol; mp., 261-262~ Fungal Source F u s a r i u m r o s e u m and Trichothecium roseum.

Also, hydrolysis product of trichothecin.

Spectral Data UV:

)t, mLxMeOH226nm (e=8,000); acetate derivative: ~ maxM~O" 227nm (e=8,000). IR~

3560 and 1680cml. 1H NMR:

(CDC13) 3.90(1H, H-2); 1.93(1H, H-3); 2.43(1H, H-3); 4.35(1H, H-4); 6.49(1H, H10); 3.94(1H, H-11); 2.81, 3.12(2H, H-13); 1.00(3H, CH3-15); and 1.80ppm (3H, s, CH3-16). 13C NMR: 70.2, C-2; 40.2, C-3; 79.6, C-4; 49.5, C-5; 43.6, C-6; 42.3, C-7; 199.4, C-8; 138.5, C9; 137.7, C-10; 73.1, C-11; 66.0, C-12; 47.3, C-13; 6.2, C-14; 15.6, CH3-15; and 18.5ppm, CH3-16. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 222 (1981).


299

M. E. Savard and B. A. Blackwell; A. Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

300


Common/Systematic Name Trichothecin 4 I]-Crotonoyloxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C19H2405; ~ ~6

lo

IJ~

= 332.16237 H

H

~1 61 is

13

I

,n

"~1 ....,v

14

General Characteristics Crystals; mp., 118~

i'OAd --

II 0

- - C 2'H = C \

Me 4'

[a]D 10 Jr 44 ~ (c=l.0, in CHCI3).

Fungal Source Fusarium roseum.

Biological Activity LDs0 in mice dosed IV was-~300mg/kg; IDs0 of protein synthesis in rabbit reticulocytes (whole cell) was 0.151xg/ml. Inhibited the elongation or termination steps in protein synthesis on polyribosomes. Spectral Data UV: /~, maxMeOH 215nm (c = 19,000).

1H ~ : 3.95(1H, H-2); 2.0-2.5(2H, H-3); 5.70(1H, H-4); 2.0-2.5(2H, H-7); 6.50(1H, H-10); 3.95(1H, n-11); 2.80(1H, n-laa), 3.10(1H, n-13b); 1.05(3H, n-14); 0.80(3H, n=15); 1.80(3H, n-16); 5.80(1H, n-2'); 6.30(1H, n-3'); and 2.10ppm (an, n-4'). 13C NMR: (CDCI3) 73.3, d, C-2; 36.9, t, C-3, 79.5, d, C-4; 49.0, s, C-5; 43.7, s, C-6, 42.1, t, C7; 198.5, s, C-8, 138.1, s, C-9, 137.0, d, C-10, 1,70.1, d, C-11, 65.4, s, C-12; 47.4, t, C-13, 5.7, q, C-14; 15.4, q, C-15, 18.5, q, C-16; 166.1, s, C-1', 120.3, d, C-2'; 145.9, d, C-3'; and 15.4ppm, q, C-4'.


301

TLC Data A. Adsorbent, silica gel G; solvent, chloroform-methanol, 98:2, v/v; Rf, 0.66; detection: H2SO4 spraying and heating at 110~ for 5 minutes. B. Adsorbent, silica gel G; solvent, benzene-tetrahydrofuran, 85:15, v/v; Rf, 0.53; detection, H2SO4 spraying and heating at 110~ for 5 minutes. GLC Data Support, Shimalite W; liquid phase, OV-17 (1.5%); retention time: 8.55min.; relative retention time (relative to nivalenol): 2.40 minutes. Reference R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 217(1981).

302

14.


Common/Systematic Name Trichothecinol A Molecular Formula/Molecular Weight C19H2406, ~ 16

"- 3 4 8 . 1 5 7 2 9

lo H

H TM

0r I~lOl 5

is ~4

'

i

.... O H

'1

~'

2'

3',,H

O--C--CH=C,, II Me 0 4'

General Characteristics [a]D 18 "4-81.5 ~ (c=0.70, in MeOH). Fungal Source Trichothecium roseum (TMI-32358 Supplied from the Tottori Mycological Institute; Tottori, Japan). Biological Activity Trichothecinol A was quite a potent antitumor promoter on Epstein-Barr virus early antigen activation induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate in Raji cells in comparison with t-carotene. Spectral Data UV: MeOH max

218nm (E=1.4 x 104).

IR: (CHC13) 3450, 1720, and 1680 c m "l. 1H NMR: (CDCI3) 3.78(d, d=5.0Hz, H-2); 4.29(ddd, d=2.6, 3.0, 5.0Hz, H-30); 4.99(d, J=3.0Hz, H-4); 2.3 l(dd, J=l.6, 15.2Hz, H-7a); 2.95(dd, ,/=1.2, 15.2Hz, H-713); 6.595(dq, `/=1.4, 5.8Hz, H-10); 4.41(dd, J=0.8, 5.8Hz, H-11); 2.81(d, `/=3.9Hz, H-13 pro-R); 3.08(d, ,/=3.9Hz, H-13 pro-S); 0.77(s, 5-Me); 1.05(d, ,/=l.2Hz, 6-Me); 1.84(dd, ,/=0.8, 1.4Hz, 9-Me); 3.50(d, ,/=2.6Hz, 3-OH); 5.88(dq, ,/=1.8, 11.5Hz, H-2'); 6.45(dq,,/=7.3, 11.5Hz, H-3'); and 2.17ppm (dd,,/=l.8, 7.3Hz, 3'-Me). 13C N M R :

(CDCI3) 79.30(d, C-2), 78.77(d, C-3), 83.17(d, C-4), 48.87(s, C-5), 44.39(s, C-6); 42.03(t, C-7); 198.46(s, C-8), 137.77(s, C-9), 137.24(d, C-10); 70.97(d, C-11); 64.50(s, C-12), 46.63(t, C-13), 5.90(q, C-14), 18.40(q, C-15); 15.33(q, C-16),


303

167.81(s, C-I'), 119.89(d, C-2'); 147.05(d, C-3'); and 15.57ppm (q, C-4'). Mass Spectrum: HR-CIMS: 349.1656re~e; MH+; calcd mass, 349.1651. Reference A. Iida, K. Konishi, H. Kubo, K. Tomioka, H. Tokuda, and H. Nishino; Trichothecinols A, B, C, Potent anti-Tumor Promoting Sesquiterpenoids from the Fungus Trichothecium roseum; Tetrahedron Letters, Vol. 37, pp-9219-9220.

304


Common/Systematic Name Trichothecinol B Molecular Formula/Molecular Weight C19H2605; ~

= 334.17802

IH6~%31 o H

I~lel14 15

H

s

4~0

'

2'

3'/H

C--CH--C,, II

O

Me 4'

General Characteristics [tt]D19 - 14.1 o (C=0.10, in MeOH). Fungal Source

Trichothecium roseum (TMI-32358 Supplied from the Tottori Mycological Institute;

Tottori, Japan). Biological Activity Trichothecinol B was quite a potent antitumor promoter on Epstein-Barr virus early antigen activation induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate in Raji cells in comparison with [}-carotene. Spectral Data UV: ~M~. 210nm (e-l.5 x 104). I-R:

(neat) 3450 and 1720cm "1. 1H NMR:

(CDC13) 2.58 (dd, J=7.8, 15.5Hz, H-3a); 2.04 (ddd, J=3.6, 5.2, 15.5Hz, H-313); 4.15 (d, J=5.8Hz, H-313). Compare spectrum with trichothecinol A. 13C NMR:

(CDCI3) 36.91 (t, C-3) and 67.83ppm (d, C-8). Compare spectrum with trichothecinol A. Mass Spectrum: HR-CIMS: 335.1859re~e; MI-F, calcd mass, 335.1858.


305

Reference A. Iida, K. Konishi, H. Kubo, K. Tomioka, H. Tokuda, and H. Nishino; Trichothecinols A, B, C, Potent Anti-Tumor Promoting Sesquiterpenoids from the Fungus Trichothecium roseum; Tetrahedron Letters, Vol. 37, pp-9219-9220, 1996.

306

14.

Trichothecenes

and R e l a t e d M e t a b o l i t e s

Common/Systematic Name Trichothecinol C Molecular Formula/Molecular Weight C 19H2606; ~

-" 3 5 0 . 1 7 2 9 4

~o H

H

HO,,'~k~"O ! ~el~ '6 ~ 0 . . . . . ]

1~

2

1,

3.,,,OH

'

--

d

II 0

2'

--CH=C\

3' /

H Me 4'

General Characteristics [a]DTM +28.8 ~ (c=0.2, in MeOH). Fungal Source Trichothecium roseum

(TMI-32358 Supplied from the Tottori Mycological Institute;

Tottori, Japan). Biological Activity Trichothecinol C was quite a potent anti-tumor promoter on Epstein-Barr virus early antigen activation induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate in Raji cells in comparison with l-carotene. Spectral Data UV: ~M~.max 211nm(e = 1.2x 104). IR:

(CHC13) 3420 and 1705cm"1. 1H NMR: (CDC13) 4.22(ddd, ./-2.7, 3.0, 4.9I-Iz, H-3[~); and 4.13ppm (d, J-4.9Hz, H-813). Compare spectrum with trichothecinol A. 13C N M R :

(CDC13) 78.81(d, C-3) and 67.73ppm (d, C-8). Compare spectrum with trichothecinol A. Mass Spectrum: HR-CIMS: 351.1805m/e; MH +, calcd mass, 351.1807.


307

Reference A. Iida, K. Konishi, H. Kubo, K. Tomioka, H. Tokuda, and H. Nishino; Trichothecinols A, B, C, Potent anti-Tumor Promoting Sesquiterpenoids from the Fungus Trichothecium roseum; Tetrahedron Letters, Vol. 37, pp-9219-9220 (1996).

308


Common/Systematic Name Trichodermone 4-Keto- 12,13-epoxytrichothec-9-ene Molecular Formula/Molecular Weight C15H2oO3, M W -- 248.14124 ~o H

H

,% 15

14

General Characteristics Crystals from ether-hexane; mp., 154-155~

[ a ] D z0 -

42.3 ~ (c=l.0, in CHC13).

Fungal Source Obtained from the oxidation of trichodermin. Spectral Data UV:

maxE~O" 205nm (e=2,800). IR.:

(KBr) 1735cm"1. ~H NMR: (CDC13) 4.22(1H, H-2); 2.80(2H, H-3); 2.01(2H, H-7); 2.01(2H, H-8); 5.41(1H, H10); 4.38(1H, H-11); 3.15(2H, H-13); 0.84(3H, CH3-14); 0.39(3H, CH3-15); and 1.70ppm (3H, s, CH3-16). 13CNMR: 76.2, C-2; 42.0, C-3; 214.9, C-4; 55.3, C-5; 42.7, C-6; 23.9, C-7; 27.9, C-8; 140.9, C9; 118.7, C-10; 71.4, C-11; 65.3, C-12; 50.1, C-13; 5.4, C-14; 15.4, C-15; and 23.4ppm, C- 16. Reference R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites, Academic Press, New York, p. 224(1981).


309

Common/Systematic Name Nivalenol 3tz,413,7tt,15-Tetrahydroxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula]Molecular Weight C15H2007; M W -- 3 1 2 . 1 2 0 9 0

~o H

.1o

0

"12

I

,,o.

~ ......~=. ,~I

~ - :~ H(3

H

-

~,

OH

CH2OH 15

General Characteristics Crystals from methanol; mp., 80-90~ dried in presence of P205 in reduced pressure; mp., 222-223~ (dec.); [tZ]D24 + 21.54 ~ (C=1.3, in EtOH); tetraacetate; mp., 168-170~ Funsal Source

Fusarium nivale (Fn-2B) = F. sporotrichioides, F. graminearum, and F. crookwellense.

Biolozical Activity LDs0 in mice dosed IP was 501~g/g (injured proliferating cells) and in chick embryo, 4.0ktg/egg Minimum SC dosage to induce vomiting in ducklings was 1.0 mg/kg. 110t,tg/ml inhibited uptake of [14C]leucine and [14C]thymidine into protein and DNA in Ehrlich ascites tumor cells. However, uptake of [~4C]uracil into RNA was not affected. Inhibition of protein synthesis occurred before inhibition of DNA synthesis. It inhibited the initiation step of protein synthesis on polyribosomes. IDs0 in rabbit reticulocytes was 3.0~g/ml (whole cell); 0.5~g/ml (cell free); rat liver was 8.0ktg/ml (whole cell); and tumor cell was 6.0~tg/ml (whole cell). Dermally toxic in rabbit and guinea pig. Emetic to laboratory animals; in ducklings emetic at lmg/kg (S.C.). Caused cell degeneration of bone marrow, lymph nodes, intestines, testes and thymus. Soectral Data ~

UV:

~, m~M"~ 218nm (E=7,500); tetraacetate, 227nm (e=7,900).

~H NMR: (CDC13) 3.61(1H, d, d=4.8Hz, H-2); 4.14(1H, dd, J=4.8, 3.4Hz, H-3); 4.43(2H, d, J=3.4Hz, H-4); 4.81(1H, s, H-7); 6.57(1H, dd, J=6.0, 1.SHz, H-10); 4.72(1H, d, d=6.0Hz, H-11); 3.01(1H, d, J=4.4Hz, H-13); 2.97(1H, d, J=4.4Hz, H-13); 1.08(3H, s, CH3-14); 3.74, 3.79(2H, d, J=12.1Hz, CH2OH-15); and 1.84ppm (3H, d, d=!.5I-Iz, CH3-16).

310


13C NMR: 81.6, C-2; 81.3, C-3; 80.8, C-4; 50.6, C-5; 54.5, C-6; 75.1, C-7; 201.5, C-8; 137.1, C9; 139.2, C-10; 71.0, C-11; 66.0, C-12; 46.3, C-13; 8.3, C-14; 61.5, C-15; and 15.2ppm, CH3-16. TLC Data A: Adsorbent, Kieselgel G; solvent, chloroform-methanol, 5:1 v/v; Rf, 0.45; detection, brown color after H2SO4 spraying and heating at 110~ for 5 minutes. B: Adsorbent, silica gel G; solvent, ethyl acetate-toluene, 3:1 v/v; Rf, 0.09; detection; brown color after H~SO4 spraying and heating at 110~ for 5 minutes. GLC Data Support, Shimalite W; liquid phase, OV-17 (1.5%); retention time, 3.55 minutes. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 206-207( 1981). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994). R. F. Vesonder and P. Golinski; Metabolites of Fusarium; In Fusarium Mycotoxins, Taxonomy and Pathogenicity; J. Chelkowski, ed.; Elsevier Pub. Co., New York, pp. 1323(1989).


311

Common/Systematic Name 4-Deoxynivalenol; Rd Toxin; Vomitoxin 3 c~,7tt,15-Trihydroxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C]5H2006; M W = 2 9 6 . 1 2 5 9 9

10

H

H

16~O~1

J8 HO

2 3.,,,OH

6J13~1 ,,0 -

14

(~H2OH 15

General Characteristics Needles from ethyl acetate-petroleum ether; mp., 151-153~ and 176-178~ (bound water); semicarbazone derivative; mp., 199-201 ~ triacetate, colorless needles from ethyl acetate-petroleum ether; mp., 155-157~ [t~]Dz5 + 6.35 ~ (C=0.07, in EtOH). Fungal Source Fusarium graminearum, F. culmorum, and F. roseum. Isolation/Purification Contaminated corn was extracted with aqueous methanol, concentrated and allowed to sit for 4 hours at 4~ The supernatant liquid was recovered,and basic and acidic components were removed from the neutral components. The neutral material was chromatographed on a Florisil column eluted with chloroform-methanol, 50:50 and 25:75, v/v. The deoxynivalenol containing fractions were further fractionated by flash chromatography, preparative TLC (silica gel F254developed with chloroform-methanol-water, 80:20:0.1, v/v/v) and Sephadex LH-20 column chromatography. Biological Activity LDso in male mice (ddY strain) dosed IP was 70mg/kg; females, 49.4mg/kg; ducklings (10 day old) 27mg/kg. Also, inhibited the multiplication of culture cells of Tetrahymena pyriformis at same level as fusarenon-X (4.6ktg/ml); IDs0 in rabbit reticulocytes was 2ktg/ml(whole cells); caused emesis in swine (7mg/601b via intubation), duckling (10 day old, 10mg/kg), dogs (0. lmg/kg, s.c.) and food refusal in swine at 40mg/kg. Spectral Data UV:

~, ~m~

218nm(e=4,500); semicarbazone derivative, 268nm (6=18,200).

312


IR~

(KBr) 3470, 3430, 3350, and 1680cm-1. 1H N1V[R:

(CDC13) 3.62(1H, d, J-4.5Hz, H-2); 4.53(1H, dt, J=10.7, 4.5Hz, H-3); 2.21(1H, dd, Jt~=14.8, 4.5Hz, H-4); 2.07(1H, dd, J[]=14.8, 10.7Hz, H-4); 4.83(1H, d, J=2.0Hz, H7); 6.61(1H, dq, J=5.9, 1.5Hz, H-10); 4.80(1H, d, J=5.9Hz, H-11); 3.07(1H, d, J=4.3Hz, H-13); 3.15(1H, d, J=4.3Hz, H-13); 1.13(3H, s, CH3-14); 3.73, 3.89(2H, d, J=l 1.7Hz, CH2OH-15); and 1.86ppm (3H, br s, CH3-16). 13CNMR: 80.6, C-2; 68.5, C-3; 43.0, C-4; 46.0, C-5; 52.1, C-6; 74.3, C-7; 200.1, C-8; 135.7, C9; 138.3, C-10; 70.2, C-11; 65.7, C-12; 47.3, C-13; 14.1, C-14; 61.4, C-15; and 15.01ppm, CH3-16. TLC Data A: Adsorbent, silica gel G; solvent, chloroform-methanol, 97:3, v/v; Rf, 0.09; detection, H2SO4 spraying and heating at 110~ for 5 minutes. B: Adsorbent; silica gel G; solvent, ethyl acetate-toluene, 3:1, v/v; Rf, 0.21; detection, H2SO4 spraying and heating at 110~ for 5 minutes. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 202-203(1981 ). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; Plant Research Centre, Agriculture Canada, Ottawa, Ontario, K1A 0C6. R. F. Vesonder, A. Ciegler, and A. H. Jensen; Isolation of the Emetic Principle from Fusarium-Infected Corn; Applied Microbiology, Vol. 26, pp. 1008-1010(1973).

R. F. Vesonder and P. Golinski; Metabolites of Fusarium; In Fusafi'um Mycotoxins, Taxonomy and Pathogenicity; J. Chelkowski, ed.; Elsevier Pub. Co., New York, pp. 1323(1989).


313

Common/Systematic Name 3-Acetyl-4-deoxynivalenol 3a-Acetoxy-7a, 15-dihydroxy-12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C17H2207, M W -- 3 3 8 . 1 3 6 5 5

16

~ O ' ~10l J8 HO

H

H

2

3

,,,OAc

6|13~ .... ,0 -

14

(~H2OH

15

General Characteristics Colorless needles from ethyl ether-n-pentane; mp.,185.5-186~ EtOH); [t~]D25 + 40.5~ tetraacetate; mp., 168-170~

[0g]D20 q-

43.0 ~(c=1.3, in

Isolation/Purification Fungal mycelium was extracted with methylene chloride, concentrated and the residue dissolved in methanol. The methanol phase was extracted with petroleum ether and evaporated to dryness. The residue was chromatographed on a silica gel column eluted with chloroform-ethanol (50:1, v/v) and then chloroform-ethanol (400:1, v/v). 3-Acetyl-4deoxynivalenol was recrystallized from hot ethyl ether. Fungal Sour.ce Fusarium culmorum (HLX 1503; ATCC 28114), F. graminearum (HLX 1506), and F. roseum. Biological Activity LDs0 in male ddS strain of mice dosed IP was 76.7mg/kg; females, 49.9mg/kg; ducklings, 37mg/kg. Minimum SC dosage to induce vomiting in ducklings was 10.0 mg/kg; dogs, S.C., 0.2mg/kg. Inhibited Tetrahymena pyriformis, 29.0~g/ml. Spectral Data UV:

~m~

219nm (e=5,900).

IR:

(KBr) 3480, 3400, 1720, and 1680cm].

314


1H NIVIR: (CDC13) 3.87(1H, d, Jr= 4.5Hz, H-2); 5.18(1H, dt, J=l 1.2, 4.5Hz, H-3); 2.35(1H, dd, Ja=15.1, 4.5Hz, H-4); 2.12(1H, dd, 313=15.1, 11.2Hz, H-4); 4.79(1H, d, J=2.1Hz, H7); 6.56(1H, dq, J=5.9, 1.5Hz, H-10); 4.66(1H, d, J=5.9Hz, H-11); 3.08(1H, d, Ja=4.3Hz, H-13); 3.14(1H, d, J=4.3Hz, H-13); 1.12(3H, s, CH3-14); 3.75(a), 3.79(13) (2H, d, J=l 1.7Hz, CH2OH-15); 1.85(3H, d, J=l.5Hz, CH3-16) ;and 2.10ppm (3H, s, CH3COO-). 13C NMR: 79.1, C-2; 71.3, C-3; 40.5, C-4; 45.9, C-5; 52.0, C-6; 74.5, C-7; 199.8, C-8; 135.8, C9; 138.4, C-10; 70.2, C-11; 65.1, C-12; 47.5, C-13; 14.1, C-14; 62.2, C-15; 15.2, CH316; 20.9, CH3-Ac; and 170.3ppm (CH3C=O). Mass Data: LREIMS: 338(M+), 278, 290, 248, 241,231,223(100), 203, 189, 181, and 175re~e; anal. calcd for C17H2207: C, 60.3; H, 6.6; O, 33.1%; found C, 60.1; H, 6.9; O, 32.9%. TLC Data A: Adsorbent, silica gel G; chloroform-methanol, 93:7 v/v; Rf, 0.40; detection, H2SO4 spraying and heating at 110~ for 5 minutes. B: Adsorbent, silica gel G; solvent, ethyl acetate-toluene, 3:1 v/v; Re, 0.43; detection, H2SO4 spraying and heating at 110~ for 5 minutes. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 209(1981). R. Greenhalgh, A. W. Hanson, J. D. Miller, and A. Taylor; Production and X-ray Crystal Structure of 3a-Acetoxy-7a, 15-dihydroxy-12,13-epoxytrichothec-9-en-8-one; J. Agric. Food Chem., Vol. 32, pp. 948-952(1984). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994). R. F. Vesonder and P. Golinski; Metabolites of Fusarium; In Fusarium Mycotoxins, Taxonomy and Pathogenicity; J. Chelkowski, ed.; Elsevier Pub. Co., New York, pp. 1323(1989).

14.


315

Common/Systematic Name 413, 15-Diacetylnivalenol 4[3,15-Diacetoxy-3 a,7a-dihydroxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C19H2409; MW

--- 3 9 6 . 1 4 2 0 3

16

o4

lo H

J: :l

H

I

HO ~ A ''OAo ~H2OAc 15

General Characteristics Crystals from acetone-n-hexane; mp., 135-136~ [a]D25 + 72 ~ (in acetone).

[a]D 26 -I- 64.3 ~ (c--1.0,

in EtOH);

Isolation/Purification The fungal culture filtrate was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and evaporated to dryness to yield a yellow gum. The yellow gum was chromatographed by a silica gel column eluted initially with 10% ethyl acetate-chloroform and increasing the percentage of ethyl acetate. Ethyl acetate eluted an oil that was further purified by preparative HPLC using a cyano-bonded phase column with 3% 2-propanolhexane as mobile phase. Fungal Source

Fusarium sporotrichioides, F. crookwellense, F. scirpi (CMI 45490), and Gibberella mtricans = F. equiseti.

Biological Activity Caused skin necrosis and emesis in laboratory animals (emetic in ducklings); S. C., 0.4mg/kg; LDs0 in male mice (ddY strain) dosed IP was 3.5mg/kg; chick embryo, 0.9mg/egg; IDs0 in rabbit reticulocytes, 0.10~g/ml (whole cell). Spectral Data UV: ~, M~oa 220nm (e=6,200). max IR:

(KBr) 3420-3450, 2980-2920, 1735, 1680, 1235, and 1040cm1.

316


1H NMR: (CDCI3) 3.82(1H, d, J=4.7Hz, H-2); 4.22(1H, ddd, J=4.8, 3.1, 2.7Hz, H-3); 3.25(1H, d, J=2.7Hz, OH-3); 5.16(1H, d, J=3.1Hz, H-4); 4.86(1H, d, J=2.0Hz, H-7); 3.80(1H, d, J=2.0Hz, OH-7); 6.63(1H, dd, J=5.9, 1.6Hz, H-10); 4.72(1H, d, J=5.9Hz, H-11); 3.07(1H, d, .]=4.3Hz, H-13); 3.08(1H, d, J=4.3Hz, n-13); 1.09(3H, s, CH3-14); 4.19, 4.32(2H, d, J=12.3Hz, CH2OAc-15); 1.88(3H, d, J=l.6Hz, CH3-16); 1.90(3H, s, CHaAc- 15); and 2.14ppm (3 H, s, CHaCOO-). 13C NMR: 79.7, C-2; 78.4, C-3; 83.9, C-4; 49.4, C-5; 52.2, C-6; 73.2, C-7; 198.9, C-8; 136.0, C9; 138.3, C-10; 69.3, C-11; 64.3, C-12; 46.1, C-13; 7.6, C-14; 61.8, C-15; 15.3, CH316; 20.6, CH3-Ac-15; 21.0, CH3-Ac-4; and 170.0, 172.7ppm (CH3C=O). Mass Spectrum: [,REIMS: 396(M+), 366, 336, 294, 277, 247, 189, 179(100), 151,137, and 123m/e. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 221(1981). D. R. Lauren, A. Ashley, B. A. Blackwell, R. Greenhalgh, J. D. Miller, and G. A. Neish; Trichothecenes Produced by Fusarium crookwellense DAOM 193611; J. Agric. Food Chem., Vol. 35, pp. 884-889(1987). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994). R. F. Vesonder and P. Golinski; Metabolites of Fusarium; In Fusarium Mycotoxins, Taxonomy and Pathogenicity; J. Chelkowski, ed.; Elsevier Pub. Co., New York, pp. 1323(1989).

14. T r i c h o t h e c e n e s and Related Metabolites

317

Common/Systematic Name 413, 15-Diacetyl-7-deoxynivalenol 413,15-Diacetoxy-3 t~-hydroxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C19H2408; M W " - 3 80.14712 16

~o H

: .--

0

H "~

"

2

i ~5

:_ 14

,OH

L,

3,,"

' OAc

~H2OAc 15

Fungal Source Fusarium sporotrichioides and F. crookwellense. Isolation/Purification The fungal culture filtrate was extracted with ethyl acetate, dried over anhydrous sodium sulfate, and evaporated to dryness to yield a yellow gum. The yellow gum was chromatographed by a silica gel column eluted initially with 10% ethyl acetate-chloroform and increasing the percentage of ethyl acetate. Ethyl acetate eluted an oil that was further purified by preparative HPLC using a cyano-bonded phase column with 3% 2-propanolhexane as mobile phase. Biological Activity The trichothecenes, as a group, show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukocytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-flee systems. Some trichothecenes inhibit the initiation step of protein synthesis on polyribosomes and others inhibit the elongation and/or termination step in protein synthesis. The trichothecenes have been strongly implicated in natural intoxications of animals and humans.

318


Spectral Data 1H NMR: (CDCI3) 3.77(1H, d, J=4.9I-Iz, H-2); 4.21(1H, ddd, J=4.9, 2.9, 2.6Hz, H-3); 3.21(1H, d, J=2.6Hz, OH-3); 5.06(1H, d, J=2.9Hz, H-4); 2.46(1H, dd, J=15.9, 1.6Hz, H-7); 2.92(1H, dd, J=15.9, 1.6Hz, H-7); 6.59(1H, dd, J=5.9, 1.5Hz, H-10); 4.51(1H, d, J=5.9Hz, H-11); 2.79(1H, d, J=3.9Hz, H-13); 3.07(1H, d, J=3.9Hz, H-13); 0.80(3H, s, CH3-14); 4.10, 4.17(2H, d, J=12.4Hz, CHaOAc=15); 1.82(3H, d, J=l.5Hz, CH3-16); 1.97(3H, s, CH3Ac-15); and 2.14ppm (3H, s, CH3COO-). 13CNMR: 79.0, C-2; 78.5, C-3; 83.8, C-4; 47.5, C-5; 48.8, C-6; 38.2, C-7; 196.7, C-8; 138.9, C9; 136.9, C-10; 68.4, C-11; 64.4, C-12; 46.7, C-13; 6.1, C-14; 64.5, C-15; 15.3, CH316; 20.8, CH3-Ac-15; 20.5, C__H3-Ac-4;and 170.2, 172.7ppm (CH3C=O). Mass Spectrum: LREIMS: 380(M+),338, 320, 292, 278, 247, 189, 173, 121,109, and 43m/e (100). References D. R. Lauren, A. Ashley, B. A. Blackwell, R. Greenhalgh, J. D. Miller, and G. A. Neish; Trichothecenes Produced by Fusarium crookwellense DAOM 193611; J. Agric. Food Chem., Vol. 35, pp. 884-889(1987). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).


319

Common/Systematic Name 15-Acetyl-4-deoxynivalenol 15-Acetoxy-3 tt,7tt-dihydroxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C17Hz207, ~

= 338.13655

10 H

16~O~1

H

2

3.,-"OH

.... ,o

HO

: 14 ~H2OAc

15


[a]D 19 +

79 ~ (in CHC13).

Fungal Source Fusarium grammearum (DAOM 17; 8148). Biological Activity The trichothecenes as a group show a wide range of biological activity such as antibacterial, antiviral, antifungal, and cytostatic activity; some are phytotoxic and all show some degree of animal toxicity, including insecticidal activity. There exists a broad range of toxicity in vertebrate animals and the acute toxicities vary considerably. General gross clinical signs reported for trichothecenes are vomiting, diarrhea, anorexia, ataxia, hematuria, leukocytosis soon followed by severe leukopenia, inflammation of the gastrointestinal tract, degeneration of nerve cells in the central nervous system, degeneration and hemorrhaging of cardiac muscle, and lesions of lymph nodes, testes, and thymus. Topical application or contact with skin causes dermal necrosis. Biochemically, they are potent inhibitors of protein and DNA synthesis in whole-cell and cell-free systems. Some trichothecenes inhibit the initiation step of protein synthesis on polyribosome and others inhibit the elongation and/or termination step in protein synthesis. The trichothecenes have been strongly implicated in natural intoxications of animals and humans. Spectral Data 1H N]VIR:

(CDCI3) 3.60(IH, d, J=4.4Hz, H-2); 4.50(IH, ddd, J=I0.6, 4.4, 4.51--Iz,H-3); 2.20(IH, dd, Ja=14.8, 4.SHz, H-4), 2.07(IH, dd, J[3=14.8, 10.6Hz, H-4); 4.81(IH, d, J=l.9Hz, H-7); 6.58(IH, dq, J=5.8, 1.31--Iz,H-10), 4.87(IH, d, J=5.81--Iz,H-I I); 3.11(1H, d, Jtt=4.2Hz, H-13); 3.06(1H, d, J=4.2Hz, H-13); 1.04(3H, s, CH3-14); 4.21(2H, s, CH2OAc-15); 1.86(3H, br s, CH3-16) and 1.85ppm (3H, s, CH3COO-).

320


13C NMR: 80.7, C-2; 68.9, C-3; 43.3, C-4; 46.4, C-5; 51.4, C-6; 73.5, C-7; 199.4, C-8; 135.5, C9; 138.6, C-10; 70.1, C-11; 65.4, C-12; 47.3, C-13; 13.8, C-14; 62.2, C-15; 15.3, CH316; 20.6, CH3-Ac; and 170.1ppm (CH3C=O). References M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994). R. F. Vesonder and P. Golinski; Metabolites of Fusarium; In Fusarium Mycotoxins, Taxonomy and Pathogenicity; J. Chelkowski, ed., Elsevier Pub. Co., New York, pp. 1323(1989).


321

Common/Systematic Name 3,15-Diacetyl-4-deoxynivalenol 3a, 15a-Diacetoxy-7tt-hydroxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C19H2408; M W -- 380.14712

10 H

1 6 ~ O " ~

H

2

3.,,,OAc

'~

CH2OAc 15

General Characteristics Crystals from ethyl acetate-hexane; mp., 119-120oC. Fungal Source Fusarium roseum (ATCC 28114) and F. grammearum.

Spectral Data 1H NIVIR:

(CDC13) 3.90(1H, d, J=4.5Hz, H-2); 5.22(2H, ddd, J=l 1.2, 4.5, 4.4Hz, H-3); 2.17(1H, dd, J=l 1.2, 15.1Hz, H-4); 2.30(1H, dd, ,/--15.1, 4.4Hz, H-4); 3.81(1H, d, J=l.6Hz, H-7), 3.76(1H, d, J=l.6Hz, OH-7); 6.57(1H, dd, J=5.8, 1.6Hz, H-10); 4.70(1H, d, J=5.8Hz, H-11); 3.10(1H, d, J=4.3Hz, H-13); 3.15(1H, d, J=4.3Hz, H13); 1.09(3H, s, CH3-14); 4.22, 4.28(2H, d, J=12.0Hz, CH2OAc-15); 1.89(3H, d, J=0.8Hz, CH3-16); 1.88(3H, s, CH3Ac-15); and 2.13ppm (3H, s, CH3COO-). 13CNMR: 78.9, C-2; 71.1, C-3; 40.4, C-4; 45.8, C-5; 51.5, C-6; 73.4, C-7; 199.2, C-8; 135.6, C9; 138.4, C-10; 70.1, C-11; 64.9, C-12; 47.4, C-13; 13.6, C-14; 62.1, C-15; 15.3, CH3-16; 20.9, CH3-Ac-15; 20.6, CH3-Ac-4; and 170.2, 170.7ppm (CH3C=O). Mass Spectrum: LREIMS: 380(M+), 320, 291, 231,203, 181, and 163m/e. References R. Greenhalgh, R. M. Meier, B. A. Blackwell, J. D. Miller, A. Taylor, and J. W. ApSimon; Part 2. Minor Metabolites o f Fusarium roseum (ATCC 28114); J. Agric. Food Chem., Vol. 32, pp. 115-118(1986).

322


M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).


323

Common/Systematic Name Fusarenon-X; Nivalenol monoacetate; Fusarenon 413-Acetoxy-3 a,7 a, 15a-trihydroxy- 12,13-epoxytrichothec-9-en-8-one Molecular Formula/Molecular Weight C17H2208; ~

-- 3 5 4 . 1 2 1 4 5

~o H O

~

~

i

H ~

....,o I

- 12

HO i 1{5 CH2OH 15

4\OAc

General Characteristics Hexagonal bipyramid crystals from dichloromethane-n-pentane; mp., 91-92~ crystals from dichloroethane; mp., 181-182~ [a]o 25 + 58 ~ (c=l.0, in MeOH.) Fungal Source

Fusarium nivale, Fn-2B = sporotrichioides, F. crookwellense, F. graminearum, and F. equiseti.

Isolation/Purification The toxin in the fungal broth was absorbed on active carbon, followed by elution with methanol. After evaporation of the solvent, 5 volumes of chloroform were added to the methanol extract. The methanol-chloroform soluble fraction, herein referred to as crude toxin, was chromatographed on Kieselgel. Development with chloroform-methanol (97:3 to 5:1, v/v) yielded a highly toxic fraction that eluted before the nivalenol fraction. Rechromatography on Kieselgel with chloroform-acetone (5:1, v/v) gave a white powder. This material, when crystallized from dichloromethane-n-pentane, gave hexagonal bipyramid crystals. Biological Activity Application of 0.2-1.0~g to dehaired back skin of rabbit, guinea pig, and mouse caused cytotoxic effects on epidermis, dermis, and hair follicles; no change in the subcutaneous tissues or muscle fibers. LDs0 in male mice (ddY strain) dosed IP (0.9% saline) was-3.3 mg/kg; nonlethal doses produced mucoidal secretion around the eye one week after treatment. LDs0 in chick embryos, 2.6mg/egg; IDs0 in rabbit reticulocytes, 0.25~g/ml (whole cell); 0.51~g/ml (cell flee); tumor cells, 6.01~g/ml (whole cell); and rat liver, 8.0~tg/ml (cell flee). Emetic, minimum S.C. dose to induce vomiting in ducklings was 0.40.5 mg/kg; in cats, 0.3-0.5 mg/kg; and in pigeons, 0.5-1.0 mg/kg (IV and PO).

324


Spectral Data UV: X m~,M~~ 220nm (e=6,500). 1H NMR: (CDC13) 3.79(1H, d, J=4.7Hz, H-2); 4.32(2H, m, H-3); 5.53(1H, d, J=3.4Hz, H-4); 4.83(1H, d, J=2.0Hz, H-7); 3.71(1H, d, J=2.0Hz, OH-7); 6.66(1H, d, J=6.0Hz, H-10); 4.81(1H, d, J=6.0Hz, H-11); 3.04(1H, d, J=4.2Hz, H-13); 3.09(1H, d, J=4.2Hz, H-13); 1.08(3H, s, CH3-14); 3.59(1H, dd, J=12.5, 4.7Hz, H-15); 4.01(1H, br d, J=12.5Hz, H-15); 1.88(3H, br s, CH3-16); and 2.14ppm (3H, s, CH3COO-). 13CNMR: 77.5, C-2; 80.0, C-3; 83.5, C-4; 49.4, C-5; 52.9, C-6; 75.8, C-7; 199.6, C-8; 135.9, C-9; 138.3, C-10; 69.1, C-11; 64.7, C-12; 46.3, C-13; 7.8, C-14; 61.6, C-15; 15.2, CH3-16; 21.0, CH3-Ac-15; and 172.9ppm (CH3C=O).

Mass Data: Found: C, 57.62; H, 6.22; O, 36.16%; calcd for C, 57.62; H, 6.22; O, 36.16%. TLC Data A. Adsorbent: silica gel G; Solvent: chloroform-methanol, 97:3, v/v; Rf, 0.19; detection: purple to yellow-green with H2SO4 spraying and heating at 110~ for 5 minutes. B. Adsorbent: silica gel G; solvent: toluene-ethyl acetate, 1:3, v/v; Rf, 0.36; detection: purple to yellow-green with H2SO4 spraying and heating at 110~ for 5 minutes. GLC Data Support: Shimalite W; liquid phase: OV-17 (1.5%); retention time: 3.95; relative retention time (relative to nivalenol): 1.11. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 213 (1981). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from F u s a r i u m Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994). Y. Sugiura, Y. watanabe, T. Tanaka, S. Yamamoto, and Y. Ueno; Occurrence of Strains That Produce Both Nivalenol and Deoxynivalenol; Appl. Environ. Microbiol.; Vol. 56, pp. 3047-3051 (1990). Gibberella zoeae

Y. Ueno, I. Ueno, T. Tatsuno, K. Okubo, and H. Tsunoda; Fusarenon-X, A Toxic Principle o f F u s a r i u m n i v a l e - Culture Filtrate, Experientia, Vol. 25, pp. 1062 (1969).

Modified Trichothecenes 2-Deoxy- 11-epi- 12-acetyl-3~t-hydroxysambucoin 2-Deoxy- 11-epi-3ct-hydroxysambucoin Trichodiol A Trichodiene FS-1 FS-2 FS-3 FS-4 3-Ketoapotrichothecene 3ct-Hydroxyapotrichothecene 313-Hydroxyapotrichothecene

325


15.

ModifiedTrichothecenes

327

Common/Systematic Name 2-Deoxy- 11-epi- 12-acetyl-3 a-hydroxysambucoin Molecular Formula/Molecular Weight C17H2604; M W = 294.18311 Me

_H

OAc

M~Mel4~//2 14

OH General Characteristics Isolated as an oil. Fungal Source

Fusarium sporotrichioides isolated from Ethiopian wheat.


F. sporotrichioides was cultured on ground corn grits and the fermented corn grits were blended with chloroform-acetone (85:15, v/v) and the mixture allowed to stand overnight. After filtration the remaining solid residue was blended with acetone; the mixture was filtered and the acetone extract and the chloroform-acetone extract were combined and concentrated under reduced pressure, which yielded a dark green oil. The oil was dissolved in 2 liters benzene-n-hexane (2:1, v/v) and aliquots were applied to Florisil columns. Columns were eluted successively with benzene-n-hexane (2:1, v/v), methylene chloride, chloroform-acetone (9:1, v/v), and acetone. The residue from the chloroformacetone fraction was recrystaUized from acetone-n-hexane to yield a mixture of trichothecenes. Reversed-phase flash column chromatography of the solid trichothecene mixture using MeOH-H20-acetic acid (35:20:1, v/v/v) yielded neosolaniol, NT-1, 8acetylneosolaniol, 8-propionyl-neosolaniol, 8-isobutyrylneosolaniol, 8-nbutyrylneosolaniol, T-2 toxin, 8-n-pentanoyl-neosolaniol, and 8-n-hexanoylneosolaniol. The acetone fraction was dissolved in benzene-acetone (2:1, v/v) and subjected to Florisil column chromatography. The column was eluted successively with benzene-acetone (2:1, v/v), chloroform-acetone (4:1, v/v), and acetone. The residue from the acetone fraction was chromatographed using flash column chromatography, eluting with solvent mixtures of increasing polarity: benzene-acetone (65:35, v/v), benzene-acetone (57:63, v/v), acetone, acetone-methanol (1:1, v/v), and MeOH. Fractions were collected and similar fractions were combined. The polar fractions of the silica gel column chromatography were combined, concentrated at reduced pressure, and subjected to reversed-phase column chromatography eluting with methanol-water-acetic acid (35:20:1, v/v/v). Similar fractions were combined. Acuminatin, FS-1, scirpenol, T-2 tetraol, T-2 triol, 15acetylscirpenol, DON (deoxynivalenol), 8a-hydroxytrichothecolone, verrucarol, 2-deoxy11-epi-3 a-hydroxysambucoin and 2-deoxy- 11-epi- 12-acetyl-3 a-hydroxysambucoin were

328

15.


isolated in pure form. Biological Activity 2-Deoxy- 11-epi-3a-hydroxysambucoin and 2-deoxy- 11-epi- 12-acetyl-3 ~-hydroxysambucoin were screened for relative cytotoxicity in cultured baby hamster kidney (BHK' 21) cells and found to be non-toxic (LC~00=1000 ng/ml). Spectral Data IR:

(NaCI) 1736(acetyl group) and 3408cm"1 (OH group). 1H NMR:

(CDC13) 1.40(1H, dd, J=14.9, 2.0Hz, H-2); 2.32(1H, dd, J=14.9, 8.4Hz, H-2); 4.4(1H, m, H-3); 1.91(1H, m, H-4); 2.12(1H, m, H-4); 1.68(1H, dd, J=5.5, 2.0Hz, H7); 1.95(1H, dd, J=9.0, 2.0Hz, H-7); 1.95-2.0(2H, m, 2H-8); 5.45(1H, dd, J=6.0, 1.0Hz, H-10); 3.6(1H, bs, H-11); 3.58(1H, d, d=15.1Hz, H-13); 3.75(1H, d, d=15.1Hz, H-13); 0.96(3H, s, H-14); 0.73(3H, s, H-15), 1.68(3H, s, H-16); and 2.05ppm (3H, s, COCH3). 13C NMR: (CDC13) 45.9, t, C-2; 67.5, d, C-3; 44.8, t, C-4; obscured by solvent, C-5; 35.2, s, C6; 24.5, t, C-7; 29.2, t, C-8; 140.4, s, C-9; 120.8, d, C-10; 75.9, d, C-11; 86.9, s, C-12; 74.5, t, C-13; 16.8, q, C-14; 16.3, q, C-15; 23.0, q, C-16; and 22.4ppm, q, COCH3. (COCH3, not observed). Mass Spectrum: CIMS (TFA derivative): 391(M + + 1, 10%), 331(17), 301(38), 277(22), 217(55), and 111role (100). Reference D. M. Fort, C. L. Barnes, M. S. Tempestra, H. H. Casper, E. Bekele, A. A. Rottinghaus, and G. E.Rottinghaus; Two New Modified Trichothecenes from Fusarium sporotrichioides; J. Natural Products, Vol. 56, pp. 1890-1897(1993).

15. ModifiedTrichothecenes

329

Common/Systematic Name 2-Deoxy- 11-epi-3 a-hydroxysambucoin Molecular Formula/M01ecular Weight C15H2403; MW

Me 16

-- 2 5 2 . 1 7 2 5 4

H .= O .

9

is M6 E..~. 14

OH

General Characteristics Colorless crystals from methanol-H20, mp., 177-178 oC. Funsal Source v

Fusarium sporotrichioides isolated from Ethiopian wheat.


F. sporotrichioides was cultured on ground corn grits at 10~ for 28 days and the fermented corn grits were blended with chloroform-acetone (85:15, v/v) and the mixture allowed to stand overnight. After filtration, the remaining solid residue was blended with acetone. The mixture was filtered and the solid autoclaved and discarded. The acetone and the chloroform-acetone extract were combined and concentrated under reduced pressure, which yielded a dark green oil. The oil was dissolved in 2 liters benzene-n-hexane (2:1, v/v), and aliquots were applied to Florisil columns. Columns were eluted successively with benzene-n-hexane (2:1, v/v), methylene chloride, chloroform-acetone (9:1, v/v), and acetone. The residue from the chloroform-acetone fraction was recrystallized from acetone-n-hexane to yield a mixture of trichothecenes. Reversed-phase flash column chromatography of the solid trichothecene mixture using methanol-water-acetic acid (35:20:1, v/v/v) yielded neosolaniol, NT- 1, 8-acetylneosolaniol, 8-propionyl-neosolaniol, 8-isobutyrylneosolaniol, 8-n-butyrylneosolaniol, T-2 toxin, 8-n-pentanoylneosolaniol, and 8-n-hexanoylneosolaniol. The acetone fraction was dissolved in benzene-acetone (2:1, v/v) and subjected to Florisil column chromatography. The column was eluted successively with benzene-acetone (2:1, v/v), chloroform-acetone (4:1, v/v), and acetone. The residue from the acetone fraction was chromatographed using flash column chromatography, eluting with solvent mixtures of increasing polarity: benzene-acetone (65:35, v/v), benzene-acetone (57:63, v/v), acetone, acetone-methanol (1:1, v/v), and MeOH. Fractions were collected and similar fractions were combined. The polar fractions of the silica gel column chromatography were combined, concentrated at reduced pressure, and subjected to reversed-phase column chromatography eluting with methanol-water-acetic acid (35:20:1, v/v/v). Similar fractions were combined. Acuminatin, FS-1, scirpenol, T - 2 tetraol, T-2 triol, 15-acetylscirpenol, DON (deoxynivalenol), 8a-hydroxytrichothecolone,

330

15.


verrucarol, 2-deoxy- 11-epi-3a-hydroxysambucoin and 2-deoxy- 11-epi- 12-acetyl-3tthydroxysambucoin were isolated in pure form. Biological Activity 2-Deoxy-11-epi-3a-hydroxysambucoin and 2-deoxy-11-epi-12-acetyl-3a-hydroxysambucoin were screened for relative cytotoxicity in cultured baby hamster kidney (BHK21) cells and found to be non-toxic (LC100= 1000ng/ml). Spectral Data IR:

(NaC1) 3412cm1 (OH group). 1H ~ :

(CDC13) 1.46(1H, dd, J-15.0, 2.4Hz, H-2); 2.28(1H, dd, .]=15.0, 9.4Hz, H-2); 4.44(1H, ddd, J=15.6,7.8, 2.4Hz, H-3); 1.85(1H, m, H-4); 2.02(1H, m, H-4); 1.53 (1H, dd, J-13.3, 2.0Hz, H-7); 1.91(1H, dd, J=8.0, 2.0Hz, H-7); 1.95-2.0(2H, m, H8); 5.52(1H, dd, J=5.6, 0.8Hz, H-10); 3.69(1H, bs, H-11); 3.66(1H, d, J=12.6Hz, H13); 3.95(1H, d, J=12.6Hz, H-13); 0.97(3H, s, 3H-14); 0.71(3H, s, H-15); and 1.70ppm (3H, s, H-16). 13C NMR: (CDC13) 44.1, t, C-2; 67.0, d, C-3; 44.2, t, C-4; 48.6, s, C-5; 35.0, s, C-6; 24.3, t, C-7; 28.2, t, C-8; 139.5, s, C-9; 119.5, d, C-10; 75.1, d, C-11; 77.6, s, C-12; 74.0, t, C-13; 16.6, q, C-14; 16.1, q, C-15; and 23.0ppm, q, C-16. Mass Spectrum: LREIMS (TMSi derivative): 396(NY, 1%), 378(4), 258(100), 108(63), and 73m/e (60); ElMS (TMSi derivative): 397(M++ 1, 18%), 381 (15), 307(22), 291 (100), 263(42), 217(53), 199(32), 73role (21); and CIMS (TFA derivative): 445(M § + 1, 45%), 445(45), 331(100), 217(13), 115(67), and 93role (80). Reference D. M. Fort, C. L. Barnes, M. S. Tempestra, H. H. Casper, E. Bekele, A. A. Rottinghaus, and G. E. Rottinghaus; Two New Modified Trichothecenes from Fusarium sporotrichioides; J. Natural Products, Vol. 56, pp. 1890-1897(1993).


331

Common/Systematic Name Trichodiol A Molecular Formula/Molecular Weight C15H2403,1VIW' = 252.17254

Me

Me~/.O

H

General Characteristics Colorless crystals from ether-hexane; mp., 81-830C;

[tg]D =

+52 ~ (CHC13).

Isolation/Purification The ethyl acetate extract of the fermentation broth from Trichothecium r o s e u m was saponified with 10% ethanolic KOH at room temperature. The mixture was concentrated to ca. 1/3 volume below 40~ under reduced pressure. This material was extracted with ether and the extract washed with water, dried and the solvent evaporated to dryness. Repeated column chromatography of the nonsaponifiable material using Wakogel C-200 (60% ether-benzene) gave trichodiol A which crystallized from ether-hexane to give a pure sample. Fungal Source T r i c h o t h e c i u m roseum.

Spectral Data UV:

E~H

End absorption, 210nm (e = 360).

IR:

(CHCI3) 3460cm1 (OH). 1H NMR: (CDC13) The NMR spectrum revealed the presence of three tertiary methyl groups (singlets at 0.86, 0.96, and 1.15ppm, 3H each), a CHzOH group (3.48, 4.06, 2H, Abq, J = 12.0Hz) attached to a non-hydrogen atom, and a-CH=CHgroup (5.46, 5.73ppm, 2H, Abq, J = 10Hz). Both carbon atoms are attached to quaternary carbon atoms and a methine proton (3.17ppm, 1H, bs). The signals at 3.48 and 4.06ppm shifted to 4.33 and 4.54ppm upon acetylation. Mass Data: HREIMS: 234.162(C15H2202, M § - 1-120, calcd. 234.162), 127.074(C7Hl102, calcd 127.076), 125.099(CsHx30, ealcd 125.097), 108.095(CsH12, calcd 108.094), 107.088(C8H11, calcd 107.086), 109.065(C7H90, calcd 109.065), 9 6 . 0 5 6 m / e (C6HsO, calcd 96.058). Found: O, 18.84; C15H2403 requires O, 19.02%.

332

15.


Reference S. Nozoe and Y. Machida; The Structures of Trichodiol and Trichodiene; Tetrahedrort, Vol. 28, pp. 5105-5111(1972).

15.


333

Common/Systematic Name Trichodiene Molecular Formula/Molecular Weight C15H24; M W --" 204.18780

CH2

General Characteristics Colorless oily material; [a]D--" q- 21 ~ (CHCI3). Isolation/Purification The myeelium from the fermentation of T. r o s e u m was suspended in warm acetone for 3 hours. After filtration, the mycelium was resuspended in acetone and allowed to stand overnight at room temperature and then filtered. The combined acetone extracts were concentrated to ca. ~/3 volume, to which water was added. Extraction with ethyl acetate followed by successive washing with water, drying, and removal of solvent afforded a brown residue, which was saponified with 10% KOH at room temperature to obtain the nonsaponifiable material. Column chromatographic separation was first afforded using ether-benzene. The first fraction (100% benzene) was further chromatographed using a AgNO3 impregnated silica gel column (hexane elution) to obtain pure trichodiene. Fungal Source Trichothecium roseum.

Spectral Data IR:

(CHC13) 3065, 1645, and 890cm"]. ]H NMR: (CDCI3) The NMR spectrum revealed the presence of ringlets at 0.85 and 1.04 ppm due to two tertiary methyl groups, a broad ringlet at 1.63ppm due to an olefinic methyl group, a multiplet at 5.23ppm due to an olefinic proton, and two broad singlets at 4.71 and 4.92ppm due to olefinic protons. Mass Spectrum: EIMS: 204(M +, C15H24); intense peaks at 189, 161,133, 121, 119, 109 (base peak CsHI3), 95 (Cyril0, 93, and 67m/e.

334

15.

Mo d i fi ed T r i ch o th ecen es

Reference S. Nozoe and Y. Machida; The Structures of Trichodiol and Trichodiene; Tetrahedron, Vol. 28, pp. 5105-5111(1972).


335

Common/Systematic Name FS-1 Molecular Formula/Molecular Weight C15H2203; MW = 250.15689 16

~,'

,,OH

13CH20H

0 General Characteristics Isolated as an oil. Fungal Source

Fusarium sporotrichioides (MC-72083) and F. sambucinum.

Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), followed by Florisil column chromatography eluted with benzene-hexane, 2:1 (v/v), methylene chloride, and chloroform-methanol (95:5, v/v) to give an oil highly enriched with trichothecenes. The oil was further purified by normal phase preparative HPLC using benzene-acetone followed by preparative reversed-phase TLC to yield several trichothecenes including FS-1. Spectral Data IR~

(thin film) 1684(enone) and 3352cm"1 (OH). 1H NMR:

(CDC13) 6.24(1H, t, J-1.5Hz, H-2); 3.10(1H, d, J-18.2Hz, H-4), 2.03(1H, d, J=18.2Hz, H-4); 1.95(1H, dd, ,/=15.0, 4.6Hz, H-7); 1.79(1H, d, J=15.0Hz, H-7); 1.45(2H, m, J-4.6Hz, H-8); 5.05(1H, br s, H-10); 4.11(1H, br s, H-11); 4.55(1H, d, J= 17.3I-Iz, H-13); 4.34(1H, d, J-17.3Hz, H-13); 1.24(3H, s, CH3-14); 0.84(3H, s, CH3-15); and 1.61ppm (3H, br s, CH3-16). 13C NMR: (CDC13) 129.5, C-2; 208.4, C-3; 50.4, C-4; 53.1, C-5; 41.9, C-6; 29.9, C-7; 27.4, C-8; 135.7, C-9; 125.0, C-10; 71.3, C-11; 188.0, C-12; 61.0, C-13; 22.4, C-14; 13.1, C-15; and 22.5ppm; C-16.

336

15.


Mass Spectrum: HREIMS: 250.150m/e (M+); calcd for C15H2zO3,250.156; LREIMS: 250, 232, 219, 204, 167(base peak), 126, 107, 84, and 55m/e. References D. G. Corley, G. E. Rottinghaus, J. K. Tracy, and M. S. Tempesta; New Trichothecene Mycotoxins of Fusarium sporotrichioides (MC- 72083); Tet. Lett., pp. 4133-4136 (1986). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

15.


337

Common/Systematic Name FS-2 Molecular Formula/Molecular Weight C15H2403, M W = 2 5 2 . 1 7 2 5 4 16

9 All

HO,,,"~ "~

13CH20H

OH Fungal Source Fusarium sporotrichioides (MC-72083). Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), followed by acetone. The combined extracts were concentrated to yield a dark red oil which was treated with n-hexane to remove non-polar materials. The resulting orange-red oil was further purified by Florisil column chromatography eluted with benzene-hexane, 2:1 (v/v), methylene chloride, and chloroform-methanol (95:5, v/v) followed by acetone. The latter two were combined to give an oil highly enriched with trichothecenes. Flash chromatography followed by normal phase preparative HPLC using toluene-acetone and preparative reversed-phase TLC yielded several purified triehotheeenes including FS-2. Spectral Data UV: max

194.5nm (e=12,000; two ene 7: to re* transitions).

:H NMR: (CDCI3) 5.75(1H, dd, J=l.5, 3.3Hz, H-2); 4.79(1H, m, H-3), 2.10(1H, m, H-4); 1.75(1H, m, H-7); 1.31(1H, m, H-7); 1.98, 1.73(2H, m, H-8); 5.58(1H, d, J=10.EHz, n-10); 5.67(1H, dd, J=l.7, 10.2Hz, n-11); 4.35(1H, br d, J=14.5Hz, n-13); 4.23(1H, br d, J=14.5Hz, H-13); 1.09(3H, s, CH3-14); 0.95(3H, s, CH3-15); and 1.27ppm (31-1, s, CH3-16). 13C N M R :

(CDCI3) 133.0, C-2, 73.4, C-3; 47.4, C-4, 54.6, C-5, 40.0, C-6; 27.8, C-7, 35.0, C-8; 65.7, C-9, 135.6, C-10, 133.2, C-11,154.0, C-12; 60.7, C-13; 21.3, C-14, 22.0, C-15; and 30.9ppm (C- 16).

338

15.


Mass Spectrum: HREIMS: 252.173m/e (M+); calcd for C15H2403,252.173. CIMS: 253(M § + 1, 2%), 235(M + - OH, 50), 127(cleavage ofC-6/C-6 bond, 85), 125(90), 109(127 - I410, 100), and 107m/e (125 - 1-/20, 50), References D. G. Corley, G. E. Rottinghaus, J. K. Tracy, and M. S. Tempesta; New Trichothecene Mycotoxins ofFusarium sporotrichioides (MC- 72083); Tet. Lett., pp. 4133-4136 (1986). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

15.

Modified Trichothecenes

339

Common/Systematic Name. FS-3 Molecular Formula/Molecular Weight C 1 5 H 2 0 0 3 ; M W -- 248.14124 16

0

13CH20H

General Characteristics Isolated as a colorless glass. Fungal Source

Fusarium sambucmum.

Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit overnight, filtered and the solid residue re-extracted with acetone. The acetone extract was combined with the chloroform-acetone extract, concentrated under vacuum, and the nonpolar material~were removed by hexane drip. The solvent was decanted, concentrated and chromatographed by flash chromatography with toluene, toluene-acetone 4:1, 2:1, 1"1 (v/v), acetone, and acetone-methanol, 11 (v/v). The toluene-acetone (4:1, v/v) fraction was further purified by flash chromatography eluting with toluene-acetone (4:1v/v). This resulted in purified diacetoxyscirpenol, 3,15-diacetoxyscirpenol, 3,4,15triacetoxyscirpenol, neosolaniol, sambucoin, 4-monoacetoxyscirpenol, 15monoacetoxyscirpenol, and FS-3. Spectral Data UV:

max^~176 229nm (e = 13,000). IR:

(film) 3470(OH)and 1675crn1 (C=O). 'H NMR:

(CDCI3) 6.29(IH, t,J=1.5I-Iz,H-2); 2.32(11-1,d, J=18.8, H-4), 2.60(IH, d, J=I8.8Hz, H-4); 1.73(IH, dd, J=I2.0, 2.5Hz, H-7); 1.87(IH, dd, J=I2.0, 2.51-Iz,H-7); 2.20(IH, dd, J=19.0, 2.51-1z,H-8); 1.63(IH, dd, J=19.0, 2.SHz, H-8); 5.75(IH, br s, H-10), 4.49(IH, dd, d=I7.0, 1.5Hz, H-13), 4.56(IH, dd, J=17.0, 1.5Hz, H-13), 1.50(3H, s, CH3-14); 1.17(3H, s, CH3-15); and 1.93ppm (3H, s, CH3-16).

340

15.


13C NMR: (CDC13) 129.6, C-2; 207.0, C-3; 50.6, C-4; 53.3, C-5; 47.1, C-6; 30.9, C-7; 28.3, C8; 160.1, C-9; 126.4, C-10; 202.5, C-11; 186.3, C-12; 61.6, C-13; 23.8, C-14; 18.2, C15; and 29.0ppm; C- 16. Mass Spectrum: HREIMS: 248.141m/e (M+); calcd for C15H2003,248.154; LREIMS: 248(M+, 1%), 181(14), 131(31), 124(26), and 69m/e (100). References D. R. Sanson, D. G. Corley, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta, and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucinum; J. Org. Chem., Vol. 54, pp. 4313-4318(1989). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

15.

Modified Trichothecenes

341

Common/Systematic Name FS-4 Molecular Formula/Molecular Weight C15H2203; M W -- 2 5 0 . 1 5 6 8 9

16

HO"~

13CH2H 0 0

General Characteristics Isolated as a colorless glass. Fungal Source

Fusarium sambucinum.

Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit overnight, filtered and the solid residue re-extracted with acetone. The acetone extract was combined with the chloroform-acetone extract, concentrated under vacuum and the nonpolar materials removed by hexane drip. The solvent was decanted, concentrated and chromatographed by flash chromatography with toluene, toluene-acetone 4:1, 2:1, 1:1 (v/v), acetone, and acetone-methanol, 1:1 (v/v). The toluene-acetone (1:1, v/v) fraction was further purified by preparative reversed-phased HPLC with water-acetonitrile (5:1, v/v) as mobile phase. This resulted in purified FS-4. Spectral Data UV: ,~ maxACa~ 198nm (e = 10,400 rr - ~:* transition). IR:

(film) 3450(OH) and 1690cm1 (C--O). 1H N~IR: (CDCla) 6.27(1H, t, J=l.5Hz, H-2); 2.15(1H, d, ,/--19.0, H-4), 2.73(1H, d, J=19.0Hz, H-4); 1.40(1H, m, n-7); 1.70(1H, m, n-7); 1.70(1H, m, n-8); 1.85(1H, m, n-8); 5.25(1H, d, J=10.0Hz, H-10); 5.61(1H, dd, J=10.0, 1.5Hz, H-11); 4.69(1H, d, J--17.0, n-13); 4.35(1H, d, J=17.0, n-13); 1.29(3H, s, CH3-14); 0.98(3H, s, CHa-15); and 1.25ppm (3H, s, CH3-16).

342

15.


13C NMR: (CDC13) 128.1, C-2; 205.0, C-3; 49.0, C-4; 28.2, C-7; 32.5, C-8; 133.6, C-10; 130.9, C-11; 183.0, C-12; 62.0, C-13; 19.2, C-14; 18.0, C-15; and 29.0ppm (C-16). Mass Spectrum: EIMS: 235(4%, M + - Me), 125(86), 108(100), and 81m/e (83); exact mass for C8H130 ( M + - C7H902) calcd. 125.097; found 125.096m/e; C7H902 ( M + - C s n l 3 0 ) calcd 125.060, found 125.059; CsH11 (CsH130 - 1-120)calcd. 107.086, found 107.086. References D. R. Sanson, D. G. Corley, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta, and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucinum; J. Org. Chem., Vol. 54, pp. 4313-4318(1989). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

15.


343

Common/Systematic Name 3-Ketoapotrichothecene Molecular Formula/Molecular Weight C15H2203; M W -" 2 5 0 . 1 5 6 8 9 13

H

CH2OH

15

14

General Characteristics Colorless glass. Fungal Source Fusarium sambucmum.

Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit over night, filtered and the solid residue re-extracted with acetone. The acetone extract was combined with the chloroform-acetone extract, concentrated under vacuum and the nonpolar materials removed by hexane drip. The solvent was decanted, concentrated and chromatographed by flash chromatography with toluene, toluene-acetone 4:1, 2:1, 1:1 (v/v), acetone, and acetone-methanol, 1:1 (v/v). The toluene-acetone (2:1, v/v) fraction was further purified by preparative normal-phase TLC which resulted in purified 3ketoapotrichothecene. Spectral Data UV: ~. maxA'~~176 197nm(e=8,600 rr to n* transition). Im: (film) 3420(OH) and 1737cm1 (C=O). 1H NMR: (CDC13) 2.60(1H, dd, J=20.0, 2.2Hz, H-2); 2.79(1H, dd, J=20.0, 1.4Hz, H-2); 2.14(1H, dd, J=19.2, 1.4Hz, H-4), 2.68(1H, dd, J=19.2, 2.2Hz, H-4); 1.48(1H, m, H7); 1.63(1H, m, H-7); 2.05, 2.15(2H, m, H-8); 5.55(1H, m, H-10); 4.23(1H, m, H-11); 3.53(1H, d, J=l 1.3Hz, H-13); 3.83(1H, d, J=l 1.3Hz, H-13); 1.16(3H, s, CH3-14); 0.76(3H, s, CH3-15); and 1.68ppm (3H, s, CH3-16).

344

15.


13CNMR: (CDC13) 49.6, C-2; 215.5, C-3; 49.6, C-4; 51.3, C-5; 46.2, C-6; 28.8, C-7; 26.8, C-8; 135.7, C-9; 210.4, C-10; 80.1, C-11; 90.8, C-12; 65.0,.C-13; 19.2, C-14, 15.3, C-15; and 22.6ppm (C- 16).

Mass Spectrum: HR IMS: 250.158m/e (M+); calcd for C15H2203,250.156. LREIMS: 250(M+,15%), 235(38), 124(45), 107(50), and 43m/e (100). References D. R. Sanson, D. G. Codey, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta, and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucinum; J. Org. Chem., Vol. 54, pp. 4313-4318(1989). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi; In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

15.


345

Commort/Systematic Name 3 a-Hydroxyapotrichothecene 3a, 13-Dihydroxy- 11-epiapotrichothec-9-ene Molecular Formula/Molecular Weight C15H2403; MW = 252.17254 13

H

CH2OH

15

14

General Characteristics Crystals from 2-propanol-hexane; mp., 167-169~

[a]D - 33.2 ~ (in ~EtOH).

Fungal Source Fusarium sambucmum, F. graminearum (ATCC 28114), F. crookwellense, F. culmorum, and F. sporotrichioides. Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit over night, filtered and the solid residue re-extracted with acetone. The acetone extract was combined with the chloroform-acetone extract, concentrated under vacuum and the nonpolar materials removed by hexane drip. The solvent was decanted, concentrated and chromatographed by flash chromatography with toluene, toluene-acetone 4:1, 2:1, 1:1 (v/v), acetone, and acetone-methanol, 1:1 (v/v). The toluene-acetone (1:1, v/v) fraction was further purified by reversed-phase TLC using methanol-water, 7:3 (v/v), which resulted in purified 3a-hydroxyapotrichothecene. Alternatively, the crude extract from F. graminearum was chromatographed on Florisil; after removal of 3-aeetyldeoxynivalenol, an oil remained which was chromatographed by medium-pressure liquid chromatography using LiChroprep silica gel column eluted with the following: 10% ethyl acetate-hexane; 20% ethyl acetate-hexane; 30% ethyl acetate-hexane; and ethyl acetate. The later fractions eluting with ethyl acetate contained the 3a and 3 [3-isomers; these were separated by preparative HPLC using a CSC-S nitrile column with a 6% 2-propanol-hexane mobile phase and a flow rate of 4 ml/minute. Spectral Data UV~ ~ Aoetonilrilr max

196nm (e=7,200 ~ to rr* transition).

346


IR:

(film) 3400cm1 (OH). 1H NMR:

(CDC13) 1.74(1H, m, H-2); 2.62(1H, ddd, J-1.7, 6.1, 12.3Hz, H-2); 4.30(1H, b m, H-3), 1.64(1H, m, H-4); 2.15(1H, dd, J=10.3, 13.0Hz, H-4); 1.40(1H, dddd, J=2.0, 4.0, 5.8, 13.0Hz, H-7); 1.58(1H, m, H-7); 2.02(1H, m, H-8); 1.62(1H, b m, H-8); 5.55(1H, b sptet, H-10); 4.18(1H, b sptet, H-11); 3.18(1H, b t, J=l 1.1Hz, H-13); 3.77(1H, dd, J=8.0, ll.lHz, H-13); 1.02(3H, s, CH3-14); 0.95(3H, s, CH3-15); and 1.50ppm (3H, q,J=l.4Hz, CH3-16). 13C NMR: (CDC13) 44.4, C-2; 72.8, C-3; 43.5, C-4; 52.5, C-5; 44.7, C-6; 27.8, C-7; 29.3, C-8; 135.5, C-9; 21.4, C-10; 81.3, C-11; 92.5, C-12; 63.3, C-13; 19.2, C-14; 17.9, C-15; and 22.6ppm (C-16). Mass Spectrum: HREIMS: 252.173m/e (M+); calcd for C15H2403, 252.186. LREIMS: 252(M § , 28%), 237(17), 140(100), and 124role (51). References R. Greenhalgh, D. A. Fielder, L. A. Morrison, J-P Charland, B. A. Blaekwell, M. E. Savard, and J. W. Apsimon; Secondary Metabolites of Fusarium species: Apotriehothecene Derivatives; J. Agile. food Chem., Vol. 37, pp. 699-705(1989). D. R. Sanson, D. G. Corley, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta, and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucinum; J. Org. Chem., Vol. 54, pp. 4313-4318(1989). M. E. Savard and B. A. Blackwell; A Compilation of Spectral Characteristics of Secondary Metabolites from Fusarium Fungi, In Mycotoxins in Grain; Compounds Other Than Aflatoxin; J. D. Miller and H. L. Trenholm, eds., Eagan Press, St. Paul, pp. 59-257 (1994).

15.


347

Common/Systematic Nam_e 313-Hydroxyapotrichothecene Molecular Formula/Molecular Weight C15H2403; M W = 252.17254

H

15

13

,H2OH

14

General Characteristics Colorless glass. Fungal Source


Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit over night, filtered and the solid residue re-extracted with acetone. The acetone extract was combined with the chloroform-acetone extract, concentrated under vacuum and the nonpolar materials removed by hexane drip. The solvent was decanted, concentrated and chromatographed by flash chromatography with toluene, toluene-acetone 4:1, 2:1, 1:1 (v/v), acetone, and acetone-methanol, 1:1 (v/v). The toluene-acetone (1:1, v/v) fraction was further purified by reversed-phase TLC using methanol-water, 7:3 (v/v), which resulted in purified 3[3-hydroxyapotrichothecene. Spectral Data UV:

~. maxA~176176196nm(e=7,800 z~to n* transition). IR:

(film) 3420 and 1048cm1 (-C-O-C-). 1H NMR: (CDC13) 1.38(1H, m, H-2); 2.45(1H, ddd, 3"=1.3, 5.8, 6.4Hz, H-2); 4.51(1H, b m, H3), 2.10(1H, H-4); 2.23(1H, H-4); 1.36(1H, m, H-7); 1.57(1H, m, H-7); 1.99(2H, m, H-8); 5.50(1H, H-10); 4.11(1H, H-11); 3.56(1H, d, J=l 1.4Hz, H-13); 3.78(1H, d, J=l 1.4Hz, H-13); 1.07(3H, s, CH3-14); 0.52(3H, s, CH3-15); and 1.63ppm (3H, b s, CH3-16).

348

15.


13C NMR: (CDC13) 47.0, C-2; 74.2, C-3; 45.7, C-4; not observed, C-5; not observed, C-6; 27.5, C-7; 29.3, C-8; not observed, C-9; 122.5, C-10, 81.3, C-11; not observed, C-12; 65.5, C-13; 20.0, C-14; 16.5, C-15; and 22.5ppm (C-16). Mass Spectrum: HREIMS: 252.181m/e (M+); calcd for C15H2403,252.186. LREIMS: 252(M~, 15%), 237(15), 124(38), 107(92), and 83m/e (100). Reference D. R. Sanson, D. G. Corley, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta, and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucinum; J. Org. Chem., Vol. 54, pp. 4313-4318(1989).

Macrocyclic Trichothecenes and Related Metabolites Trichodermadiene Trichodermadienediol A Trichodermadienediol B

12,13-Deoxytrichodermadiene

Verrol Tfichoverrol A (C6'R,C7'R)-Isotrichoverrol A (2'E, C6'R, C7'R)-Isotrichoverrol A Trichoverrol B (C6'R,C7'S)-Isotrichoverrol B (2'E, CCR,C7'S)-Isotrichoverrol B Trichoverrin A (2'E,4'Z)-Isotrichoverrin A (C6'R,C7'R)-Isotrichoverrin A 913,1013-Epoxyisotrichoverrin A 8~-Hydroxyisotrichoverrin A Trichoverrin B (C6'R,C7'R)-Isotrichoverrin B (2'E,4'Z)-Isotrichoverrin B 913,10j3-Epoxyisotrichoverrin B (2'E)- 12,13-Deoxyisotrichoverrin B (9'E)-Roridin L-2 Roridin L-2 Verrucarin A Verrucarin A 13-D-glucoside 2'-Dehydroverrucarin A Verrucarin B Verrucarin J Verrucarin K Verrucarin L Verrucarin L Acetate Roridin A Roridin A 1313-D-glucoside 8a-Butoyloxyroridin A 8a-Crotonyloxyroridin A Isororidin A Rofidin D Roridin D 13-D-glucoside Roridin E Roridin E 13-D-glucoside Isororidin E Epiroridin E Epiisororidin E 713,813-Epoxyisororidin E 349

350

16. MacrocyclicTrichothecenes and Related Metabolites

Roridin K Acetate Roridin H (Verruearin H) Roridin J 7J3,8J3-Epoxyroridin H 7j3,Sj3,2',3'-Diepoxyisororidin H Satratoxin F Satratoxin G Isosatratoxin F Isosatratoxin G Satratoxin H S-Isosatratoxin H 12'-Hydroxy-2'-isoverruearin J (PD 113,325) M-Isosatratoxin H (PD 113,326) Vertisporin

16.

Macrocyclic Trichothecenes and Related Metabolites

351

Common/Systematic Name Trichodermadiene Molecular Formula/Molecular Weight C23H3005; ~

"- 3 8 6 . 2 0 9 3 2

~o H

16

~s

H

j

14

[;,~,,0

g

H 0

Me

' 'bCCH=CHCH=CH---Ca'C:. II 2' 0

3'

4'

s'

6'

r

General Characteristics Crystallization from ether gave needles; mp., 145-146~ [~]D 27 -I-17.7~

in CHC13).

Fungal Source

Myrothecium verrucaria (ATCC 24571).

Isolation/Purification Trichodermadiene was isolated by a methanol extraction of the mycelium of a fermentation ofMyrothecium verrucaria. Chromatography on silica gel gave a series of the less polar trichothecenes (roridin H and verrucarins B and J) and a previously uncharacterized trichothecene which had an Rf value higher than any heretofore reported macrocyclic trichothecene. Crystallization from ether gave needles. Biological Activity Trichodermadiene showed a T/C = 143 at 26mg/kg in vivo against p388 mouse leukemia (PS). Spectral Data UV:

~, ~f~

264nm (log e = 4.41).

IR:

(CHC13) 1700, 1640, and 1600cm"1. 1H NMR: (CDC13) 3.83(1H, d, J=5.0Hz, H-2); 2.1(1H, m, H-313); 2.66(1H, dd, J=7.5, 15.0Hz, H-3a); 5.7(1H.dd, J=4.0, 7.5Hz, H-4); 5.43(1H, d, J=5.5Hz, H-10); 3.64(1H, d, J=5.5Hz, H-11); 2.97(2H, JAB=4.0Hz, H-13); 0.74(3H, s, H-14); 1.0(3H, s, H-15); 1.72(3H, s, H-16); 5.7(1H, d, J=l 1.0Hz, H-2'); 6.57(1H, t, J=l 1.0Hz, H-3'); 7.83(1H, dd, J=ll, 15.5Hz, H-4'); 5.7(1H, dd, J=8, 15.5Hz, H-5'); 3.21(1H, dd, J=2.0, 8.0Hz,

352


H-6'); 2.97(1H. dq, J= 2.0, 5Hz, H-7'); and 1.36ppm (1H, d, Jr- 5.0Hz, H-7'). 13CNMR: (CDC13) 79.2 d, C-2; 36.9 t, C-3; 75.0 d, C-4; 49.2 s, C-5; 40.5 s, C-6; 24.6 t, C-7; 28.1 t, C-8; 140.0 s, C-9; 118.9 d, C-10; 70.6 d, C-11; 65.5 s, C-12; 47.8 t, C-13; 6.0 q, C-14; 16.1 q, C-15; 23.2 q, C-16; 165.8 s, C-1'; 118.4 d, C-2'; 143.1 d, C-3'; 130.3 d, C-4'; 140.4 d, C-5'; 58.8 d, C-6'; 56.8 d, C-7'; and 17.5ppm q, C-8'. Mass Data: EIMS: 386m/e (M+); anal. calcd for C23H3005: C, 71.48; H, 7.82%; found: C, 71.38, H, 8.07%. Reference B. B. Jarvis, J.O. Midiwo, G. P. Stahly, G. Pavanasasivam, and E.P. Mazzola; Trichodermadiene: A New Trichothecene; Tetrahedron Letters, Vol. 21, pp. 787788(1980).

16.


353

Common/Systematic Name Trichodermadienediol A Molecular Formula/Molecular Weight C23H3206; M W -" 4 0 4 . 2 1 9 8 9

16

10 H

Me

H

I

0

I~Me

i~i Me

General Characteristics Crystallization from dichloromethane-hexane; mp., 184-185~ [a]D28 +26.3 ~ (C=0.39, in CHC13). Fungal Source Myrothecium verrucaria (ATCC 24571). Isolation/Purification The fungal culture broth was passed through XAD-2 resin; the resin was eluted with methanol yielding an aqueous methanol fraction that was concentrated to yield an aqueous concentrate. A portion of the aqueous concentrate was extracted with ethyl acetate. The organic extract was washed with water and saturated salt solution and dried. Removal of the solvent by rotary evaporation yielded a dark brown gum. HPLC analysis indicated that the principal trichothecenes present were verrucarin J, verrucarin B, verrucarin A, isororidin E, roridin E, roridin A, trichodermadienediols A and B, trichoverrols A and B, and trichoverrins A and B in the order of increasing retention time on a silica column (ethyl acetate-hexane eluent). The gum was subjected to flash chromatography (silica gel) with increasing amounts of methanol in dichloromethane to yield four fractions: I (eluted with 0-1% methanol in methylene chloride), II (eluted with 1-5% methanol in methylene chloride), III (eluted with 5-7.5% methanol in methylene chloride), and IV (eluted with methanol). Fraction II was subjected to partition chromatography. The sample was applied to a column of Celite impregnated with 83% aqueous methanol and eluted with 1-5% dichloromethane in petroleum ether to give a fraction rich in the trichothecenes. This was chromatographed to yield an oil, Re 0.15-0.20 (silica gel, 3% methanol in methylene chloride). This material was subjected to HPLC (1-4% methanol-methylene chloride gradient) followed by HPLC with 70% ethyl acetate in hexane to yield triehodermadienediol A and triehodermadienediol B.

354


Spectral Data UV;

maxMeOH260nm (log e=4.53). 1HN]VIR: (CDCI3) 0.76(3H, s, 14-H); 0.98(3H, s, 15-H); 1.13(3H, d, J=6I-Iz, 8'-H); 1.74(3H, s, 16-H); 2.60(1H, dd, J=8 and 15Hz, 3a-H); 3.03(2H, AB, d=4Hz, 13-H), 3.66(1H, d, J=5 Hz, 1l-H); 3.87(1H, d, J=5Hz, 2-H); 4.06(1H, m, 6'-H); 5.44(1H, d, J=5Hz, 10H), 5.73(1H, d, J=l 1Hz, 2'-H), 6.09(1H, dd, J=6, 16Hz, 5'-H); 6.64(1H, dd, J=l 1Hz, 3'-H); and 7.65ppm (1H, dd, J =11.0, 16Hz, 4'-H.

13CN M ~ :

(CDCI3) 79.1 d, C-2; 37.0 t, C-3; 75.0 d, C-4; 49.3 s, C-5; 40.5 s, C-6; 24.5 t, C-7; 28.1 t, C-8; 140.2 s, C-9; 118.7 d, C-I0; 70.6 d, C-11; 66.1 s, C-12; 48.1 t, C-13; 6.2 q, C-14; 16.1 q, C-15; 23.2 q, C-16; 166.0 s, C-I'; 118.7 d, C-2'; 143.4 d, C-3'; 127.9 d, C-4'; 141.9 d, C-5'; 76.6 d, C-6'; 70.6 d, C-7'; and 18.9ppm q, C-8'.

Mass Spectrum: HRCIMS (methane gas reagent): 4 0 5 . 2 2 6 3 m / e 4 8 9 . 2 4 5 5 m / e (M+ + H, calcd 489.2488).

(M ++

H, calcd 405.2277); diacetate,

Reference B. B. Jarvis, G. P. Stably, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E.P. Mazzola and R. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verruearins, J. Org. Chem.,Vol. 47, pp. 1117-1124(1982).

16. Macrocyclic Trichothecenes and Related Metabolites

355

Common/Systematic Name Trichodermadienediol B Molecular Formula/Molecular Weight C23H3206; M W -- 4 0 4 . 2 1 9 8 9 16

Me~.---

I-

1o H

�9 13

'~./'~--7

H

OJ2

I

3

.... ,o

o

~

HO

I1~'

.

Me

General Characteristics Isolated as an oil; [a]D 28 - 15.8 ~ (C=0.80, in CHC13).

Fungal Source


Isolation/Purification The fungal culture broth was passed through XAD-2 resin; the resin was eluted with methanol yielding an aqueous methanol fraction that was concentrated to yield an aqueous concentrate. A portion of the aqueous concentrate was extracted with ethyl acetate. The organic extract was washed with water and saturated salt solution and dried. Removal of the solvent by rotary evaporation yielded a dark brown gum. HPLC analysis indicated that the principal trichothecenes present were verrucarin J, verrucarin B, verrucarin A, isororidin E, roridin E, roridin A, trichodermadienediols A and B, trichoverrols A and B, and trichoverrins A and B in the order of increasing retention time on a silica column (ethyl acetate-hexane eluent). The gum was subjected to flash chromatography (silica gel) with increasing amounts of methanol in dichloromethane to yield four fractions: I (eluted with 0-1% methanol-methylene chloride), II (eluted with 1-5% methanol-methylene chloride), III (eluted with 5-7.5% methanol-methylene chloride), and IV (eluted with methanol). Fraction II was subjected to partition chromatography. The sample was applied to a column of Celite impregnated with 83% aqueous methanol and eluted with 1-5% dichloromethane in petroleum ether to give a fraction rich in the trichothecenes. This was chromatographed to yield an oil, Re, 0.15-0.20 (silica gel, 3% methanol-methylene chloride). This material was subjected to HPLC (1-4% methanol-methylene chloride gradient) followed by HPLC with 70% ethyl acetate in hexane to yield trichodermadienediol A and trichodermadienediol B.

356

16.. Macrocyclic Trichothecenes and Related Metabolites

Spectral Data UV~

~, maxM~~ 260rim(log C=4.55). 1H NMR: (CDCI3) 0.74(3H, s, 14-H); 0.98(3H, s, 15-H); 1.16(3H, d, J=6Hz, 8'-H); 1.73(3H, s, 16-H); 2.62(1H, dd, J=8,15Hz, 3a-H); 3.04(2H, AB, J=4Hz, 13-U); 3.66(1H, d, J=5Hz, 1l-H); 3.88(1H, d, J=5Hz, 2-H), 4.30(1H, m, 6'-H), 5.44(1H, d, J=5Hz, 10-H); 5.72(1H, d, d=l 1Hz, 2'-H); 6.16(1H, dd, J=6, 16Hz, 5'-H); 6.66(1H, dd, J's=l 1Hz, 3'-H); and 7.64ppm (1H, dd, J=l 1, 16Hz, 4'-H); trichodermadienediol B diacetate, 0.74(3H, s, 14-H); 0.98(3H, s, 15-H); 1.23(3H, d, J=6Hz, 8'-H); 1.74(3H, s, 16-H); 2.06 and 2.12(3H each, s, acetates); 2.60(1H, dd, J=8, 15Hz, 3a-H); 3.00(2H, AB, J=4Hz, 13-H); 3.64(1H, d, J=5Hz, 1l-H); 3.86(1H, d,J=5 Hz, 2-H); 5.16(1H, dq, J=4, 6Hz, 7'-H); 6.60(1H, dd, 3's=l 1Hz, 3'-H); and 7.68ppm (1H, dd, J=l 1, 16Hz, 4'H). 13C NMR: (CDC13) 79.1 d, C-2; 36.9 t, C-3; 74.9 d, C-4; 49.2 s, C-5; 40.5 s, C-6; 24.5 t, C-7; 28.0 t, C-8; 140.3 s, C-9; 118.6 d, C-10; 70.6 d, C-11; 66.1 s, C-12; 48.1 t, C-13; 6.1 q, C-14; 16.0 q, C-15; 23.2 q, C-16; 166.0 s, C-I'; 118.2 d, C-2'; 143.7 d, C-3'; 128.0 d, C-4'; 141.3 d, C-5'; 75.6 d, C-6', 70.3 d, C-7'; and 17.7ppm q, C-8'. Mass Spectrum: HRCIMS (methane gas reagent): 405.2252m/e (M§ + H, calcd 405.2277); diacetate, 489.2436role (M+ + H, caled 489.2488). Reference B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E. P. Mazzola, and R. F. Creoghegan; Isolation and Characterization of the Tdchoverroids and New Roridins and Verrucarins; J. Org. Chem,Vol. 47, pp. 1117-1124(1982).

16.


357

Common/Systematic Name 12,13-Deoxytrichodermadiene Molecular Formula/Molecular Weight C23H3004; MW = 370.21441

16

10H

3

H 13

O

,,,Z'.~ 0 8' General Characteristics Isolated as an oil;

[ a ] D 25 -

H

5.6 ~ (c=0.95, in CHC13).

Fungal Source Myrothecium verrucaria (ATCC 24571). Isolation/Purification The fungal culture broth was passed through XAD-2 resin; the resin was eluted with methanol yielding an aqueous methanol fraction that was concentrated to yield an aqueous concentrate. A portion of the aqueous concentrate was extracted with ethyl acetate. The organic extract was washed with water and saturated salt solution and dried. Removal of the solvent by rotary evaporation yielded a dark brown gum. The gum was subjected to flash chromatography (silica gel) with increasing amounts of methanol in dichloromethane to yield four fractions: I (eluted with 0-1% methanol-methylene chloride), II (eluted with 1-5% methanol-methylene chloride), III (eluted with 5-7.5% methanol-methylene chloride), and IV (eluted with methanol). Fraction I was passed through a silica gel flash column and eluted with dichloromethane to yield several fractions. The first fraction was subjected to preparative TLC (20% hexane in methylene chloride) on 2-mm silica gel plates to yield an oil. Further purification was done on a Magnum-9 column (silica gel) under gradient conditions (90-100% methylene chloride in hexane) to g ive 12,13deoxytrichodermadiene as an oil.

358

16.

MacrocyclicTrichothecenes and Related Metabolites

Spectral Data UV:

k maxMeOH265nm (log r 1H NMR: (CDC13) 0.98(3H, s, 14-H); 0.98(3H, s, 15-H); 1.40(3H, d, J=5Hz, 8'-H); 1.72(3H, br s, 16-H); 2.00(1H, m, H-3[3); 2.62(1H, dd, J=7 and 15Hz, 3a-H); 4.75, 5.16(1H each, s, 13-H); 3.68(1H, d, J=5 Hz, 1I-H); 4.47(1H, d, J=5Hz, 2-H); 3.21(1H, dd, J=2.0, 8.0Hz, 6'-H); 5.40(1H, d, J=5Hz, 10-H); 5.80(1H, d, J=l 1Hz, 2'-H); 5.70(1H, dd, J=8, 15Hz, 5'-H); 6.60(1H, t, J-11Hz, 3'-H); 7.75(1H, dd, J=l 1.0, 15Hz, 4'-H); 5.70(1H, dd, J=8, 15Hz, 5'-H); 3.21(1H, dd, J=2, 8Hz, 6'-H), and 3.01ppm (1H, dq, J=2, 5Hz, 7'-H). 13CNMR: (CDC13) 75.7, C-2; 38.0, C-3; 75.1, C-4; 52.1, C-5; 40.4, C-6; 23.6, C-7; 28.1, C-8, 139.8, C-9; 119.0, C-10; 70.6, C-11; 152.7, C-12; 104.9, C-13; 10.2, C-14; 16.2, C15; 23.3, C-16; 169.4, C-I'; 118.3, C-2'; 143.2, C-3'; 129.9, C-4'; 140.3, C-5'; 58.8, C6'; 57.0, C-7'; and 17.5ppm, C-8'. Mass Spectrum: HRCIMS (methane gas reagent), 371.2216m/e (M+ + H, calcd 371.2222). Reference B. B. Jarvis, V. M. Vrudhula, J. O. Mikiwo, and E. P. Mazzola; New Trichoverroids from Myrothecium verrucaria: Verrol and 12,13-Deoxytrichodermadiene; J. Org. Chem., Vol. 48, pp. 2576-2578 (1983).

16.


359

Common/Systematic Name Verrol Molecular Formula/Molecular Weight C21H3006; M W = 3 7 8 . 2 0 4 2 4

~ 0

~o H

H

O,J~

I

n

~ 6'

CH2CH2OH 5'



Isolation/Purification The fungal culture broth was passed through XAD-2 resin; the resin was eluted with methanol yielding an aqueous methanol fraction that was concentrated to yield an aqueous concentrate. A portion of the aqueous concentrate was extracted with ethyl acetate. The organic extract was washed with water and saturated salt solution and dried. Removal of the solvent by rotary evaporation yielded a dark brown gum. HPLC analysis indicated that the principal trichothecenes present were verrucarin J, verrucarin B, verrucarin A, isororidin E, roridin E, roridin A, trichodermadienediols A and B, trichoverrols A and B, and trichoverrins A and B in the order of increasing retention time on a silica column (ethyl acetate-hexane eluent). The gum was subjected to flash chromatography (silica gel) with increasing amounts of methanol in dichloromethane to yield four fractions: I (eluted with 0-1% methanol-methylene chloride), II (eluted with 1-5% methanol-methylene chloride), III (eluted with 5-7.5% methanol-methylene chloride), and IV (eluted with methanol). Fraction III of the crude extract was subjected to flash chromatography (silica gel, 0-8% methanol in methylene chloride) to yield three major fractions rich in roridin A, verrol, and trichoverrins, respectively. The verrol containing fraction was subjected to flash chromatography again under the conditions described above to yield a fraction which was mostly verrol and trichoverrols. This was further purified by column chromatography on silica (10-25% 2-propanol in hexane) to yield verrol as an oil.

360


Spectral Data 1HNMR: (CDCI3) 0.87(3H, s, 14-H), 1.70(3H, br s, 16-H); 2.60(1H, dd, J=7.5, 15.7Hz, 3a-H); 2.00(1H, m, 31~-H);2.96(2H, AB, J=3.9Hz, 13-H); 5.42(1H, d, J=5.4Hz, 10-H); 5.73(1H, q, J=l.2Hz, 2'-H); 2.42(1H, t, J=6.1, 4'-1-1);3.82(1H, d, J=5.1Hz, 2-H); 1.9(2H, m, 7-H); 2.0(2H, m, 8-n); 3.64(1H, d, J=5.4Hz, 1l-H); 2.96(2H, AB, J=3.9Hz, 13-H); 4.05(2H, AB, J=12.4Hz, 15-H); 3.80(2H, t, J=6.1Hz, 5'-H); and 2.21ppm (3H, d, J=l.2Hz, 6'-H). 13CNMR: (CDCI3) 78.8, C-2; 39.9, C-3; 74.5, C-4; 49.2, C-5; 42.8, C-6; 21.4, C-7; 28.1, C-8; 140.7, C-9; 118.6, C-10; 66.7, C-11, 65.6, C-12; 47.6, C-13; 7.1, C-14; 62.9, C-15; 23.1, C-16; 166.0, C-I'; 117.2, C-2'; 157.3, C-3'; 43.9, C-4'; 60.3, C-5'; and 18.8ppm, C-6'. Mass Spectrum: HRCIMS (methane gas reagent): 379.2112m/e (M++ H, calcd 379.2120). Reference B. B. Jarvis, V. M. Vrudhula, J. O. Mikiwo, and E. P. Mazzola; New Trichoverroids from Myrothecium verrucaria: Verrol and 12,13-Deoxytrichodermadiene; J. Org. Chem., Vol. 48, pp. 2576-2578(1983).


361

Common/Systematic Name Trichoverrol A Molecular Formula/Molecular Weight C23H3207; M W = 4 2 0 . 2 1 4 8 0

H H M e ~ . , , ~. ~ O ~- l _. _ II....iO ~"~I/~'/'~'HOH2(~ ~Me

0

"~0,~

I Me 7-~---. OH General Characteristics Crystals from ethyl acetate-petroleum ether; mp., 177-179~ [a]D2s +37.7 ~ (C=0.45, in CHCI3). Fungal Source Myrothecium verrucaria (ATCC 24571). Isolation/Purification The fungal culture broth was passed through XAD-2 resin; the resin was eluted with methanol yielding an aqueous methanol fraction that was concentrated to yield an aqueous concentrate. A portion of the aqueous concentrate was extracted with ethyl acetate. The organic extract was washed with water and saturated salt solution and dried. Removal of the solvent by rotary evaporation yielded a dark brown gum. HPLC analysis indicated that the principal trichothecenes present were verrucarin J, verrucarin B, verrucarin A, isororidin E, roridin E, roridin A, trichodermadienediols A and B, trichoverrols A and B, and trichoverrins A and B in the order of increasing retention time on a silica column (ethyl acetate-hexane eluent). The gum was subjected to flash chromatography (silica gel) with increasing amounts of methanol in dichloromethane to yield four fractions: I (eluted with 0-1% methanol-methylene chloride), II (eluted with 1-5% methanol-methylene chloride), III (eluted with 5-7.5% methanol-methylene chloride), and IV (eluted with methanol). Fraction III of the crude extract was subjected to partition chromatography. The sample was applied to a column of Celite impregnated with 83% aqueous methanol and eluted with 15% dichloromethane in petroleum ether to give a gum. Flash chromatography (90% ethyl acetate-petroleum ether) yielded two major fractions, III-A (Re, 0.35 in 90% ethyl acetate-petroleum ether) and III-B (Re, 0.30 in 90% ethyl acetatepetroleum ether). After filtration through TLC grade silica gel, each of these fractions was purified by HPLC (1-6% methanol-methylene chloride, gradient, 30 min) to give trichoverrol A and trichoverrol B from fraction III-A and trichoverrin A and trichoverrin B from fraction III-B.

362

16.


Spectral Data

UV~ ~" maxMeOH260nm (log e=4.56). 1H NMR: (CDC13) 0.84(3H, s, 14-H); 1.19(3H, d, J=6I-Iz, 8'-H); 1.72(3H, s, 16-H); 2.46(1H, dd, ,/=8, 15Hz, 3a-H); 3.00(2H, AB, J=4H_z, 13=H); 5.49(1H, d, J=5Hz, 10-H); 5.73(1H, d, J--11Hz, 2'-H); 6.66(1H, dd, d's=l 1Hz, 3'-H); 7.62(1H, dd, J-11, 16Hz, 4'-H). 13CNMR: (CDCI3) 79.0d, C-2; 36.3 t, C-3; 75.5 d, C-4; 49.1 s, C-5; 44.4 s, C-6; 21.3 t, C-7; 28.2 t, C-8; 140.6 s, C-9; 118.8 d, C-10; 66.9 d, C-11; 66.1 s, C-12; 48.3 t, C-13; 6.8 q, C-14; 62.7 t, C-15, 23.3 q, C-16; 166.9 s, C-I'; 118.2 d, C-2'; 144.2 d, C-3'; 127.8 d, C-4'; 142.5 d, C-5'; 76.5 d, C-6'; 70.7 d, C-7'; and 19.0ppm q, C-8'. Mass Spectrum: HRCIMS (methane gas reagent): 421.2226m/e

(M + +

H, calcd 421.2226).

Reference B. B. Jarvis, G. P. Stably, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E. P. Mazzola and g. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 1117-1124(1982).

16.


363

Common/Systematic Name (C6'R, C7'R)-Isotrichoverrol A Molecular Formula/Molecular Weight C23H3207; M W -- 4 2 0 . 2 1 4 8 0

~o H I

~I

H ~3

i14

I

.~

~,~,"

I

O

~.~ 2

HO' ~ : ~8' H General Characteristics Isotrichoverrol A was recrystallized from methylene chloride-hexane to give crystals; mp., 180-183~ [a]D2~+54.0 ~ (C=1.65, in CHC13). Fungal Source Myrothecium verrucaria (ATCC 20540). Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Rf fractions from silica gel chromatography (methanol in methylene chloride), given in the order of increasing polarity. To sample 1, which contained some solid material, was added cold ethyl acetate. The solid was collected and recrystallized from methylene chloride-hexane to give (C6'R, C7'R)-isotrichoverrol A. Spectral Data 1H NMR: (CDC13) 0.81(3, s, H-14); 1.19(3H, d, J=6.3 Hz, H-8'); 1.54(1H, brd, J=7.6Hz, H-7A); 1.70(3H, s, H-16); 1.98(3H, m, H-7B, H-8); 2.08(1H, ddd, J=4.0, 5.1, 15.4I~, H-313), 2.49(1H, dd, J=8.0, 15.4Hz, H-3a); 2.83(1H, d, J=4.0Hz, H-13A); 3.14(1H, d, J=4.0Hz, H-13B); 3.63(1H, d, J-12.3Hz, H-15A); 3.67(1H, dq, J=6.3, 6.3Hz, H-7'); 3.80(1H, d, J=12.3Hz, H-15B); 3.83(1H, d, J=5.1Hz, H-2'); 3.88(1H, br d, J=5.0Hz, H-11); 4.03(18, m, H-6'); 5.47(1H, br d, J=5.0Hz, H-10); 5.70(1H, d, J=l 1.5Hz, H-2'); 6.06(1H, dd, J=5.7, 15.5Hz, H-5'); 6.08(1H, m, H-4); 6.61(1H, dd, J=11.3, 11.5Hz, H-3'); and 7.59ppm (1H, dd, J= 11.3, 15.5Hz, H-4').

364

16.


13C NMR: (CDC13) 79.0d, C-2; 36.3 t, C-3; 75.5 d, C-4; 49.1 s, C-5; 44.4 s, C-6; 21.3 t, C-7; 28.2 t, C-8; 140.6 s, C-9; 118.8 d, C-10; 66.9 d, C-11; 66.1 s, C-12; 48.3 t, C-13; 6.8 q, C-14; 62.7 t, C-15; 23.3 q, C-16; 166.9 s, C-I'; 118.2 d, C-2'; 144.2 d, C-3'; 127.8 d, C-4'; 142.5 d, C-5'; 76.5 d, C-6'; 70.7 d, C-7'; and 19.0ppm q, C-8'. Mass Spectrum: HRCIMS: calcd for C23H3307 ([M + + H] +) 421.2226; found 421.221 lm/e. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E.P. Mazzola and R. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins, J. Org. Chem.,Vol. 47, pp. 1117-1124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261 (1996).


365

Common/Systematic Name (2'E, C6'R, C7'R)-Isotrichoverrol A Molecular Formula/Molecular Weight C23H3207, MW = 420.21480 16

10

H -

~l

H

2

3

NO

.17'

8'

_

General Characteristics Isolated as an oil. Fungal Source Myrothecium verrucaria (ATCC 20540). Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Rf fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. To sample 1, which contained some solid material, was added cold ethyl acetate. The solid was collected and recrystallized from methylene chloride-hexane to give isotrichoverrol A. The remaining portion of S 1 was subjected to semipreparative CCC (V~=355ml) with a solvent system of methanol-water-chloroform-hexane (3:2:3 1.2, v/v/v/v), flow rate was 2.4ml/min (ca. 400mg/injection, total 10 injections) to give isotrichoverrol A and isotrichoverrol B. Another fraction from this CCC was subjected to CCC (semipreparative column) with a solvent system of methylene chloride-carbon tetrachloride-methanol-water (2:3:3:2, v/v/v/v) (ca. 400mg/injection). The major fraction of this sample was eluted with little retention from the column, and there were no detectable trichothecenes in this fraction, according to TLC analysis. Seven additional fractions were collected: S1Fla, S1Flb, S1Flc, S1Fld, S1Fle, S1Flf, and S1Flg. Fraction S1F2 contained mainly trichoverrols according to TLC analysis. The sample was subjected to preparative CCC with a solvent system of chloroform-hexane-methanol-water (3:1:3:2, v/v/v/v) and a flow rate of 3.2ml/min to give six fractions: S 1F2a, S 1F2b, S 1F2c, S1F2d, S 1F2e, and S 1F2f. S1F2e was further chromatographed on CCC with a solvent system of of chloroformhexane-methanol-water (3:1:3:2, v/v/v) and a flow rate of 2.0ml/min. The chromatogram

366

16.


appears as a single peak; however, the eluent was collected into two parts, A and B. Fraction A was subjected to CCC (analytical column) with a solvent system of methylene chloride-carbon tetrachloride-methanol-water (3:5:2:6:4, v/v/v/v/v) and a flow rate of 1 ml/min (ca. 40mg/injection) to give (2'E, C6'R,C7'R)-isotrichoverrol A and isotrichoverrol B as oils. Spectral Data UV~

~, M~o, 262nm (log e=4.36). max

IR~

(CHCI3) 3600, 3470(OH), 1703(C=O), 1644(C=C), and 1620cm~ (C=C). :H NMR: (CDCI3) 0.80(3H, s, H-14), 1.19(3H, d, J=6.3Hz, H-8'); 1.70(3H, s, H-16); 2.47(1H, dd, J=8.1, 15.3Hz, H-3a),(1H, d, J=4.0Hz, H-13A); 3.12(1H, d, J=4.0Hz, H-13B); 3.64(1H, d, J=12.5Hz, H-15A); 3.67(1H, dd, J-6.3Hz, H-7'); 3.80(1H, d, ,/=12.5Hz, H-15B); 3.83(1H, d, J=5.0Hz, H-2); 3.92(1H, d, J=5.2Hz, H-11); 3.97(1H, dd, J=6.1, 6.3Hz, H-6'); 5.48(1H, bd, J=5.2Hz, H-10); 5.94(1H, d, J=15.4Hz, H-2'), 6.10(1H, dd, J=6.1, 15.4Hz, H-5'); 6.12(1H, dd, J=3.7, 8.2Hz, H-4); 6.45(1H, dd, J=l 1.3, 15.4Hz, H-4'); and 7.29ppm(1H, dd, J=l 1.3, 15.4Hz, H-3'). ~3CNMR: (CDCI3) 6.4, C-14; 19.1, C-8'; 21.2, C-7; 23.3, C-16; 28.0, C-8; 35.9, C-3; 44.3, C-6, 48.2, C-13; 48.9, C-5; 62.9, C-15; 65.6, C-12; 66.8, C-11; 70.6, C-7'; 75.6, C-4; 76.5, C-6'; 79.0, C-2; 118.7, C-10; 121.6, C-2'; 129.6, C-4'; 140.4, C-9; 141.5, C5'; 144.4, C-3'; and 167.7ppm, C-I' Mass Spectrum: HREIMS calcd for C23H3207(M§ 420.2148; found, 420.2164m/e. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261(1996).

16.


367

Common/Systematic Name Trichoverrol B Molecular Formula/Molecular Weight

Meoj

C23H3207; M W = 4 2 0 . 2 1 4 8 0

H

H

.....iO

O

Me ~0~

HOH26 I

General Characteristics Isolated as an oil; [a]D 25 -3.3 ~ (c=0.39, in CHC13). Fungal Source Myrothecium verrucaria (ATCC 24571). Isolation/Purification The fungal culture broth was passed through XAD-2 resin; the resin was eluted with methanol yielding an aqueous methanol fraction that was concentrated to yield an aqueous concentrate. A portion of the aqueous concentrate was extracted with ethyl acetate. The organic extract was washed with water and saturated salt solution and dried. Removal of the solvent by rotary evaporation yielded a dark brown gum. HPLC analysis indicated that the principal trichothecenes present were verrucarin J, verrucarin B, verrucarin A, isororidin E, roridin E, roridin A, trichodermadienediols A and B, trichoverrols A and B, and trichoverrins A and B in the order of increasing retention time on a silica column (ethyl acetate/hexane eluent). The gum was subjected to flash chromatography (silica gel) with increasing amounts of methanol in dichloromethane to yield four fractions: I (eluted with 0-1% methanol-methylene chloride), II (eluted with 1-5% methanol-methylene chloride), III (eluted with 5-7.5% methanol-methylene chloride), and IV (eluted with methanol). Fraction III of the crude extract was subjected to partition chromatography. The sample was applied to a column of Celite impregnated with 83% aqueous methanol and eluted with 15% dichloromethane in petroleum ether to give a gum. Flash chromatography (90% ethyl acetate-petroleum ether) yielded two major fractions, III-A (Rf, 0.35 in 90% ethyl acetate-petroleum ether) and III-B (Re, 0.30 in 90% ethyl acetatepetroleum ether). After filtration through TLC grade silica gel, each of these fractions was purified by HPLC (1-6%methanol-methylene chloride, gradient, 30 min) to give trichoverrol A and trichoverrol B from fraction III-A and trichoverrin A and trichoverrin B from fraction III-B.

368


Spectral Data

UW: ~bmaxMeOH260nm (log e=4.53). 1H NMR: (CDC13) 0.84(3H, s, 14-H); 1.16(3H, d, J=6Hz, 8'-H); 1.74(3H, 8, 16-H); 2.47(1H, dd, J=8, 15Hz, 3a-H); 3.07(2H, AB, J=4Hz, 13-H); 4.18 (1H, m, 6'-H); 5.50(1H, d, J=5Hz, 10-H); 5.74(1H, d, J=l 1Hz, 2'-H); 6.67(1H, dd, J=l 1, 11Hz, 3'-H); 7.61(1H, dd, J=l 1, 16Hz, 4'-H). 13C NMR: (CDC13) 79.0 d, C-2; 36.2 t, C-3; 75.5 d, C-4; 49.1 s, C-5; 44.4 s, C-6; 21.2 t, C-7; 28.12 t, C-8; 140.6 s, C-9; 118.8 d, C-10; 66.9 d, C-11; 66.0 s, C-12; 48.3 t, C-13; 6.8 q, C-14; 62.6 t, C-15; 23.2 q, C-16; 167.0 s, C-I'; 117.9 d, C-2'; 144.3 d, C-Y; 128.0 d, C-4'; 141.6 d, C-5'; 75.5 d, C-6'; 70.3 d, C-7'; and 17.8ppm q, C-8'. Mass Spectrum: HRCIMS (methane gas reagent): 421.2223m/e

(M ++

H, calcd 421.2226).

Reference B. B. Jarvis, G. P. Stably, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E. P. Mazzola and R. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem.,Vol. 47, pp. 1117-1124(1982).

16.


369

Common/Systematic Name (C6'R, C7'S)-Isotrichoverrol B Molecular Formula/Molecular Weight C23H3207; M W -- 4 2 0 . 2 1 4 8 0

10 H

~

I-

111 13

H

I ,iO

~

0

HOH2~ ,,

HI0 H1

3'

General Characteristics Isotrichoverrol B was isolated as an oil; [a]D2~- 4.0 ~ (C=1.50, in CHel3). Fungal Source Myrothecium verrucaria (ATCC 20540). Isolation/Purification The fermemation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Re fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. To sample 1, which contained some solid material, was added cold ethyl acetate. The solid was collected and recrystallized from methylene chloride-hexane to give isotrichoverrol A. The remaining portion of S 1 was subjected to semipreparative high speed countercurrent chromatography (Vc=355ml) with a solvent system of methanol-water-chloroform-hexane (3:2:3:1.2, v/v/v/v); flow rate was 2.4ml/min (ca. 400mg/injection, total 10 injections) to give isotrichoverrol A and isotrichoverrol B. Spectral Data 1H NMR: (CDCI3) 0.80(3H, s, H-14); 1.12(3H, d, J-6.41-Iz, H-8'); 1.57(IH, m, H-7A); 1.70(3H, s, H=16); 1.98(31-1, m, H-7B, H-8); 2.08(IH, ddd, d=4.0, 5.2, ]5.41-Iz, H-313); 2.49(1H, dd, J=7.8, 15.4Hz, H-3tt); 2.82(1H, d, J=3.9Hz, H-13A); 3.12(1H, d, J=3.9Hz, H-13B); 3.61(1H, d, J=12.3Hz, H-15A); 3.81(1H, d, J=12.3Hz, H-15B); 3.83(1H, d, J=5.2Hz, H-2); 3.86(1H, br d, J=5.1Hz, H-11); 3.90(1H, dq, J=3.5, 6.5Hz, n-7'); 4.25(1H, m, n-6'); 5.45(1H, br d, J=5.1Hz, H-10); 5.70(1H, d,

370


J=l 1.4Hz, H-2'); 6.09(1H, m, H-4); 6.11(1H, dd, ,/-6.0, 15.4Hz, H-5'); 6.63(1H, dd, ,/--11.3, 11.4Hz, H-3'); and 7.55ppm (1H, dd, J=l 1.3, 15.4Hz, H-4'). 13C NMR: The 13C NMR data are identical to those reported earlier for (C6'S,C7'R)-trichoverrol B.

Mass Spectrum: HRCIMS: calcd for C23H3307; [M+ + H] +, 421.2226; found, 421.2206m/e. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E.P. Mazzola and R. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins, J. Org. Chem.,Vol. 47, pp. 1117-1124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261 (1996).

16.


371

Common/Systematic Name (2'E, C6'R,C7'S)-Isotrichoverrol B Molecular Formula/Molecular Weight C23H3207; M W -- 4 2 0 . 2 1 4 8 0 ~o

H

H

~ ~

''"

HOH2l,, "

5

15

0

4

II

IL HO ....i 7'

8'

H General Characteristics Isolated as an oil. Fungal Source


Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Rf fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. To sample 1, which contained some solid material, was added cold ethyl acetate. The solid was collected and recrystallized from methylene chloride-hexane to give isotrichoverrol A. The remaining portion of S1 was subjected to semipreparative CCC (Vc=355ml) with a solvent system of methanol-water-chloroform-hexane (3:2:3:1.2, v/v/v/v); flow rate was 2.4ml/min (ca. 400mg/injection, total 10 injections) to give isotrichoverrol A and isotrichoverrol B. Another fraction from this CCC was subjected to CCC (semipreparative column) with a solvent system of methylene chloride-carbon tetrachloride-methanol-water (2:3:3:2, v/v/v/v; ca. 400mg/injection). The major fraction of this sample was eluted with little retention from the column, and there were no detectable trichothecenes in this fraction, according to TLC analysis. Seven additional fractions were collected: S1Fla, S1Flb, S1Flc, S1Fld, S1Fle, S1Flf, and S1Flg. Fraction S1F2 contained mainly trichoverrols according to TLC analysis. The sample was subjected to preparative CCC with a solvent system of chloroform-hexane-methanol-water (3:1:3:2, v/v/v/v) and a flow rate of 3.2ml/min to give six fractions: S 1F2a, S 1F2b, S 1F2c, S 1F2d, S 1F2e, and S 1F2f. S1F2fwas subjected to CCC (analytical column) with a solvent system of methylene chloride-carbon tetrachloride-methano!-wat~r (2:3:3:2, v/v/v/v) and a flow rate of 1

372


ml/min to give a mixture of (2'E)-isotrichoverrol B and another unknown trichoverroid. This mixture was chromatographed on CCC (analytical column) with a solvent system of methylene chloride-carbon tetrachloride-hexane-methanol-water (3:5:2:6:4, v/v/v/v/v) and a flow rate of lml/min to give pure (2'E)-isotrichoverrol B, as an oil. Spectral Data UV~ ~, M~o. 262nm (log e = 4.44). max

IR~ (CHCI3) 3600, 3470(OH), 1705(C=O), 1645(C=C), and 1621cm1 (C=C). 1H NMR: (CDC13) 0.79(3H, s, H-14); 1.12(3H, d, J=6.5 Hz, H-8'); 1.69(3H, s, H-16); 2.47(1H, dd, J=8.0, 15.3Hz, H-3a); 2.80(1H, d, J=4.0Hz, H-13A); 3.11(1H, d, J=4.0Hz, H13B); 3.63(1H, d, J=12.ZHz, H-15A); 3.80(1H, d, J=lZ.ZHz, H-15B); 3.82(1H, 3, J=5.1Hz, H-2); 3.88-3.93(2H, m, H11 and H-7'); 4.21(1H, m, H-6'); 5.47(1H, bd, J=4.5Hz, H-10); 5.93(1H, d, J=15.3Hz, H-2'); 6.10(1H, dd, J=3.6, 8.2Hz, H-4); 6.13(1H, dd, J=5.9, 15.9Hz, H-5'); 6.42(1H, dd, J=10.9, 15.9Hz, H-4'); and 7.29ppm (1H, dd, J=10.9, 15.3Hz, H-3').

13C NMR: (CDC13) 6.4, C-14; 17.6, C-8'; 21.2, C-7; 23.2, C-16; 28.0, C-8; 35.9, C-3; 44.3, C-6; 48.1, C-13; 48.9, C-5; 62.8, C-15; 65.6, C-12; 66.8, C-11; 70.1, C-7'; 75.3, C-6'; 75.6, C-4; 79.0, C-2; 118.8, C-10; 121.5, C-2'; 129.6, C4'; 140.5, C-5'; 140.5, C-9; 144.5, C3'; and 167.7ppm, C-I'. Mass Spectrum: HR IMS: calcd for C23H3207 (M+), 420.2148; found, 420.2188m/e. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261 (1996).


373

Common/Systematic Name Trichoverrin A Molecular Formula/Molecular Weight C28I--I3809; M W -- 5 1 8 . 2 5 1 5 8

H

LA.y

{)l

H

.. ,o i ,

Me

'

.CH2 HO--~8' (~H20H Me. !;',OH _

General Characteristics Crystals from ethyl acetate-petroleum ether; mp., 78-79~ [t~]D2s -21.5 ~ (C=0.39, in CHC13). Fungal Source


Isolation/Purification The fungal culture broth was passed through XAD-2 resin; the resin was eluted with methanol yielding an aqueous methanol fraction that was concentrated to yield an aqueous concentrate. A portion of the aqueous concentrate was extracted with ethyl acetate. The organic extract was washed with water and saturated salt solution and dried. Removal of the solvent by rotary evaporation yielded a dark brown gum. HPLC analysis indicated that the principal trichothecenes present were verrucarin J, verrucarin B, verrucarin A, isororidin E, roridin E, roridin A, trichodermadienediols A and B, trichoverrols A and B, and trichoverrins A and B in the order of increasing retention time on a silica column (ethyl acetate-hexane eluent). The gum was subjected to flash chromatography (silica gel) with increasing amounts of methanol in dichloromethane to yield four fractions: I (eluted with 0-1% methanol-methylene chloride), II (eluted with 1-5% methanol-methylene chloride), III (eluted with 5-7.5% methanol-methylene chloride), and IV (eluted with methanol). Fraction III of the crude extract was subjected to partition chromatography. The sample was applied to a column of Celite impregnated with 83% aqueous methanol and eluted with 15% dichloromethane in petroleum ether to give a gum. Flash chromatography (90% ethyl acetate-petroleum ether) yielded two major fractions, III-A (Rf, 0.35 in 90% ethyl acetate-petroleum ether) and III-B (Re, 0.30 in 90% ethyl acetatepetroleum ether). Atter filtration through TLC grade silica gel, each of these fractions was purified by HPLC (1-6% methanol-methylene chloride, gradient, 30 min) to give trichoverrol A and trichoverrol B from fraction III-A and trichoverrin A and trichoverrin

374


B from fraction III-B. Spectral Data

Lv~ ,~ maxMeOH 260nm (log e=4.60). ~H NMR:

(CDCI3) 0.82(3H, s, 14-H); 1.21(3H, d, J=6I-Iz,8'-H); 1.74(3;H, s, 16-H); 2.53(I;H, dd, ,/=8, 15Hz, 3(x-H);3.04(2H, AB, J=4Hz, 13-H); 4.18(2H, s, 15-H); 5.52(IH, d, J=5Hz, 10-H); 5.75(IH, d, J=l IHz, 2'-H); 5.90(IH, s, 2'-H);6.67(IH, dd, Js=l IHz, 3'-H); and 7.63ppm (IH, dd.,J=l I, 16Hz, 4'-H). 13C NMR: (CDC13) 79.1 d, C-2; 36.9 t, C-3; 75.1 td, C-4; 48.6 s, C-5; 42.9 s, C-6; 21.9 t, C-7; 27.9 t, C-8; 140.5 s, C-9; 118.5 d, C-10; 66.7 d, C-11; 65.8 s, C-12; 48.2 t, C-13; 6.7 q, C-14; 63.5 t, C-15; 23.2 q, C-16; 166.0 s, C-I'; 118.1 d, C-2'; 144.1 d, C-3'; 127.5 d, C-4'; 142.4 d, C-5'; 76.5 d, C-6'; 70.5 d, C-7'; 18.9 q, C-8'; 166.0 s, C-l"; 116.9 d, C2"; 157.3 s, C-3"; 43.6 t, C-4"; 59.7 t, C-5"; and 19.1ppm q, C-6". Mass Spectrum: HRCIMS (methane gas reagent): 533.2754m/e (M § + H, calcd 533.2754). Reference B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E. P. Mazzola and R. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 1117-1124(1982).

16.


375

Common/Systematic Name (2'E,4'Z)-Isotrichoverrin A Molecular Formula/Molecular Weight C29H4009;

"

Me

MW

,0

-- 5 3 2 . 2 6 7 2 3

U

H

~;;

I

I-

O.3

0

,,,0

H2~~Me ~ 0 14

"0~

I

07~~~'H Me'~CH2

1'

,

HO i, HO3,J:L-.7 Me _

~H2OH 5" General Characteristics Isolated as an amorphous solid. Fungal Source Myrothecium verrucaria (ATCC 20540). Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Rf fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. To sample 1, which contained some solid material, was added cold ethyl acetate. The solid was collected and recrystallized from methylene chloride-hexane to give isotrichoverrol A. The remaining portion of S 1 was subjected to semipreparative CCC (Vc=355ml) with a solvent system of methanol-water-chloroform-hexane (3:2:3:1.2, v/v/v/v); flow rate was 2.4ml/min (ca. 400mg/injeetion, total 10 injections) to give isotrichoverrol A and isotrichoverrol B. Another fraction from this CCC was subjected to CCC (semipreparative column) with a solvent system of methylene chloride-carbon tetrachloride-methanol-water (2:3:3:2, v/v/v/v; ca. 400mg/injection). The major fraction of this sample was eluted with. little retention from the column, and there were no detectable trichothecenes in this fraction, according to TLC analysis. Seven additional fractions were collected: S IF 1a, S1Flb, S1Flc, S1Fld, S1Fle, S1Flf, and S1Flg. S1Fle was subjected to C18 reversedphase TLC (70% methanol in 0.5 M aqueous NaC1) to give (2'E,4'Z)-isotrichoverrin A.

376


S.pectral Data UV:

), mx,M~~ 261nm(log e=4.49). (CHC13) 3600(OH), 1710(C=O), and 1652cm-1 (C=C). 1H NMR: (CDC13) 0.78(3H, s, H-14); 1.12(3H, d, J=6.3Hz, 8'-H); 1.69(3H, s, 16-H); 2.17(3H, d, J-1.0Hz, 6"-H); 2.40(2H, m, H-4"); 2.55(1H, dd, J=7.9, 16.0Hz, H-3~); 2.81(1H, d, J-4Hz, H-13A); 3.13(1H, d, J=4.0Hz, H-13B); 3.66(1H, dq, J values-~ 6.6Hz, H-7'); 3.83(1H, d, J=5.3Hz, H-2); 3.94(1H, bd, J=4.8Hz, H-11); 4.10(2H, s, H-15); 4.37(1H, dd, J=6.7, 8.1Hz, H-6'); 5.46(1H, bd, J-=4.8Hz, H-10); 5.79(1 H, dd, J=8.3, 10.7Hz, H-5'); 5.81(1H, d, J=l.0Hz, H-2"); 5.95(1H, d, J=15.2Hz, H-2'); 6.13(1H, dd, J=3.4, 7.9Hz, H-4); 6.24(1H, dd, J=10.7, 11.9Hz, H-4'); and 7.64ppm(1H, dd, J= 11.9, 15.2Hz, H-3'). 13CNMR: (CDC13) 6.6, C-14; 18.6, C-8'; 19.0, C-6"; 21.9, C-7; 23.2, C-16; 27.8, C-8; 36.7, C3; 43.0, C-6; 43.7, C-4"; 48.1, C-13; 48.6, C-5; 59.7, C-5"; 63.5, C-15; 65.5, C-12; 66.8, C-11; 70.7, C-7'; 72.4, C-6'; 75.7, C-4; 79.1, C-2; 117.1, C-2"; 118.5, C-10; 123.1, C-2'; 129.2, C4'; 138.6, C-5'; 139.8, C-3'; 140.5, C-9; 157.0, C-3"; 165.9, C-I"; and 166.9ppm, C- 1' Mass Spectrum: HRCIMS: caled for C29H4109

( M + d-

1), 533.2751; found, 533.2772m/e.

References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; J. Org. Chem., Vol. 47, pp. 1117-1124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261(1996).


377

Common/Systematic Name (C6'R, C77~)-Isotrichoverrin A Molecular Formula/Molecular Weight C29H4009; M W --- 5 3 2 . 2 6 7 2 3

H

H

,,,,0 |

0

i

5

General Characteristics Isolated as an amorphous solid; [~]D2~ + 5.6 ~ (C=2.10, in CHel3). Fungal Source


Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Re fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. Sample 2 ($2) contained mainly trichoverrins according to TLC analysis. This sample was chromatographed on CCC (semipreparative column, Vc=355ml) with a solvent system of chloroform-hexane-methanol-water (31:3:2, v/v/v/v) and a flow rate of 3.2ml/min (ca. 400mg/injection). Like fractions were combined to give seven fractions: S2F 1, $2F2, $2F3, $2F4, $2F5, $2F6, and $2F7. A portion of $2F2 was subjected to CCC (semipreparative column) with a solvent system of methylene chloride-carbon tetrachloride-methanol-water (2:3:3:2, v/v/v/v) and a flow rate of 2.8ml/min. The components of the mobile phase (organic layer, methylene chloride-carbon tetrachloride) were varied from 2:3 to 5:2 (v/v), starting at t = 120 min and going to t = 160 min. Like portions were combined to give seven fractions: I, (C6'R, C7'R)-isotrichoverdn A; II, (C6'R, C7'S)-isotrichoverrin B; III, a mixture of isotrichoverrin B and trichoverdn C); IV, V, VI, 2~E-isotrichoverdn A; VII, 2'E-isotrichoverrin B.

378

16.


Spectral Data 1H NMR: (CDC13) 0.79(3H, s, H-14); 1.19(3H, d, J=6.3Hz, H-14'); 1.70(3H, s, H-16); 2.17(3H, d, J= 1.0Hz, H-6"); 2.40(2H, m, H-4"); 2.56(1H, dd, J=7.6, 15.5Hz, H-3a); 2.82(1H, d, J=4.0Hz, H-13A); 3.14(1H, d, J=4.0Hz, H-13B); 3.66(1H, dq, ,/=6.3, 6.3Hz, H-7'); 3.80(2H, m, H-5"); 3.84(1H, d, J=5.4Hz, H-2); 3.98(1H, d, J=4.7Hz, H11); 4.03(1H, m, H-6'); 4.07(1H, d, J=12.5Hz, H-15A); 4.14(1H, d, J=12.5Hz, H15B); 5.46(11-1, d, J=4.7Hz, H-10); 5.67(1H, d, J=l 1.3Hz, H-2'); 5.85(1H, d, J=l.0Hz, H-2"); 6.07(1H, dd, J=4.7, 15.5Hz, H-5'); 6.20(1H, dd, ,/=7.6, 15.5Hz, H-4), 6.59(1H, dd, J= 11.3, 11.3Hz, H-3'); and 7.54ppm(1H, dd, J= 11.3, 15.5Hz, H-4'). 13CNMR: (CDC13) 6.7, C-14; 18.9, C-8'; 19.1, C-6"; 21.9, C-7; 23.2, C-16; 27.9, C-8; 36.9, C3; 42.9, C-6; 43.6, C-4"; 48.2, C-13; 48.6, C-5; 59.7, C-5"; 63.4, C-15; 65.8, C-12; 66.6, C-11; 70.6, C-7'; 75.0, C-4; 76.1, C-6'; 79.1, C-2; 117.0, C-2"; 118.2, C-2'; 118.5, C-10; 127.1, C-4'; 140.4, C-9; 142.1, C-5'; 143.7, C-3'; 157.0, C-3"; 165.9, C1"; and 166.0ppm, C- 1'. Mass Spectrum: HRCIMS: calcd for C29H4109 (M q- H) +, 533.2751; found, 533.2759role. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261 (1996).


379

Common/Systematic Name 913,1013-Epoxyisotrichoverrin A Molecular Formula/Molecular Weight C29H40010; MW = 548.26215

o. Yo3

.,,,,o

I

?

Me

0/

"i~2'

',/

y)l,

'e/\cH2CH225

General Characteristics Isolated as an oil; [~]D2~ - 16~ (C=0.12, in CHC13). Fungal Source Myrothecium verrucaria (ATCC 20540). Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Rf fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. Sample 3 ($3) fromM, verrucaria isolate ATCC 20540, which was more polar than those that contained trichoverrols (S 1) and trichoverrins ($2), was partitioned between chloroform and methanol-water mixture (50%). The organic fraction was subjected to preparative TLC (Chromatotron, 2 mm, silica gel) with methanol-methylene chloride (310%). The most polar fraction was subjected to high speed countercurrent distribution (CCC) with a solvent system of chloroform-hexane-methanol-water (12:8:15:5, v/v/v/v) at a flow rate of 1.8ml/min to give 913,1013-epoxyisotrichoverrin A and 913,1013epoxyisotrichoverrin B.

380


Spectral Data 1H NMR: (CDC13) 0.72(3H, s, H-14); 1.20(3H, d, J=6.5Hz, H-8'); 1.34(3H, s, H-16); 1.602.00(5H, m, H-7, H-8, S-3~); 2.20(3H, d, J=0.9Hz, S-6"); 2.41(2H, t, J - 6.0Hz, H-4"); 2.53(1H, dd, J=7.9, 15.5Hz, H-3a); 2.78(1H, d, J=4.0Hz, H-13A); 3.11(1H, d, J=5.5Hz, H-10); 3.19(1H, d, J=4.0Hz, H-13B); 3.67(1H, dq J=6.5, 6.5Hz, H-7'); 3.81(2H, m, H-5"); 3.89(1H, br d, J=5.5Hz, H-11);3.93(1H, d, J-5.1Hz, H-2); 4.03(1H, m, H-6'); 4.04(1H, d, J=12.5Hz, H-15A); 4.15(1H, d, J=12.5Hz, H-15B); 5.68(1H, d, J-11.3Hz, H-2'; 5.82(1H, d, J=0.9Hz, H-2"); 6.03(1H, dd, J=3.5, 7.5Hz, H-4); 6.08(1H, dd, ,]=5.8, 15.4Hz, H-5'); 6.60(1H, dd, J=l 1.3, 11.3Hz, H-3'); and 7.55ppm (1H, dd, J=l 1.3, 15.4Hz, H-4'). 13CNMR: (CDC13) 6.8, C-14; 18.9, C-8'; 19.2, C-6"; 19.4, C-7; 22.4, C-16; 26.5, C-8; 26.5, C8; 36.6, C-3; 42.6, C-6; 43.6, C-4"; 48.0, C-13; 48.5, C-5; 57.3, C-10; 57.5, C-9; 59.7, C-5"; 63.1, C-15; 65.2, C-12; 66.9, C-11; 70.6, C-7'; 74.8, C-4; 76.2, C-6'; 78.7, C-2; 116.5, C-2"; 118.1, C-2'; 127.5, C-4'; 142.2, C-5'; 143.9, C-3'; 158.1, C-3"; 165.8, C1'; and 166.0ppm, C- 1" Mass Spectrum: HRCIMS: calcd for C29H41Olo(M + H)+, 549.2700; found, 549.2732m/e. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261(1996).


381

Common/Systematic Name 8tt-Hydroxyisotrichoverdn A Molecular Formula/Molecular Weight C29H400]0; MW = 548.26215

Me~

HO'

H

H ....,0

H ?IoMe

V,Jl:

J

M~> General Characteristics Isolated as an oil;

0

HO,,, ~,

,

HO-4.

~

"CH2CH20H H ,, 5

[ ~ ] D 20 -

22.0 ~ (c=0.37, in CHC13).

Fungal Source Myrothecium verrucaria (ATCC 20540). Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carded out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Re fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. Sample 4 ($4) was triturated with methanol. The soluble portion was concentrated and dissolved in 50% aqueous methanol solution, and the solution was washed with carbon tetrachloride-hexane, chloroform-hexane (7:3, v/v), and chloroform. The aqueous phase was concentrated by rotary evaporation and extracted with chloroform. The chloroform extract was subjected to high-speed countercurrent distribution (CCC) with a solvent system of chloroform-hexane-methanol-water (7:3:5:5, v/v/v/v) to give 16hydroxyisotrichodermadienediols A and B and 8a-hydroxyisotrichoverrin A.

382


Spectral Data 1H NMR: (CDC13) 0.81(3H, s, H-14); 1.19(3H, d,J=6.3Hz, H-14'); 1.69(1H, br d,J=14.4Hz, H-7~); 1.84(3H, s, H-16); 2.02(1H, m, H-313); 2.17(3H, s, H-6"); 2.32(1H, dd, J=6.5, 14.4Hz, H-7t~); 2.39(2H, m, H-4"); 2.58(1H, dd, J=7.8, 15.5Hz, H-3t~); 2.85(1H, d, J=4.0Hz, H-13A); 3.14(1H, d, J=4.0Hz, H-13B); 3.66(1H, dq, ,/=6.3, 6.3Hz, H-7'); 3.78(2H, m, H-5"); 3.83(1H, d, J=5.3Hz, H-2); 4.03(1H, br d, J=5.7Hz, H-11); 4.09(1H, m, H-6'); 4.11 (1H, br d, J=6.5Hz, H-8); 4.24(1H, d, Jr=13.0Hz, H- 15A); 4.39(1H, d, J=13.0Hz, H-15B); 5.58(1H, br d, J=5.7Hz, H-10); 5.68(1H, d, J=l 1.3Hz, H-2'); 5.85(1H, s, H-2"); 6.07(1H, dd, J=5.1, 15.5Hz, H-5'); 6.30(1H, dd, J=3.1, 7.8Hz, H-4); 6.60(1H, dd, J= 11.3, 11.3Hz, H-3'); and 7.53ppm (1H, dd, J= 11.3, 15.5Hz, H-4'). 13C NMR: (CDC13) 6.6, C-14; 18.9, C-8'; 19.2, C-6"; 20.4, C-16; 31.3, C-7; 36.8, C-3; 42.9, C6; 43.5, C-4"; 48.3, C-13; 48.5, C-5; 59.6, C-5"; 64.6, C-15; 65.8, C-12; 66.5, C-8; 66.8, C-11; 70.5, C-7'; 74.8, C-4; 76.4, C-6'; 79.0, C-2; 116.9, C-2"; 118.1, C-2'; 120.9, C-10; 127.3, C-4', 139.8, C-9; 142.2, C-5'; 144.0, C-3'; 157.2, C-3"; 165.2, C1'; and 166.0ppm, C- 1" Mass Spectrum: HRCIMS: calcd for C29H41010(M + H)+, 549.2700; found, 549.2710m/e. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod.,Vol. 59, pp. 254-261 (1996).

16.


383

Common/Systematic Name Trichoverrin B Molecular Formula/Molecular Weight C29I--I4009; M W = 5 3 2 . 2 6 7 2 3

H

H

M e,,,,,,,.,,,~,,~/..~ O.,,,J " I

0

iO

I Me

U '1 M e ' 7 " " C H2 (L H20H ~" General Characteristics Isolated as an oil;

HO HO,--+~. Me 8' H

[a]D 28 - 3 2 . 2 ~

(c=0.57, in CHC13).

Fungal Source Myrothecium verrucaria (ATCC 24571). Isolation/Purification The fungal culture broth was passed through XAD-2 resin; the resin was eluted with methanol yielding an aqueous methanol fraction that was concentrated to yield an aqueous concentrate. A portion of the aqueous concentrate was extracted with ethyl acetate. The organic extract was washed with water and saturated salt solution and dried. Removal of the solvent by rotary evaporation yielded a dark brown gum. HPLC analysis indicated that the principal trichothecenes present were verrucarin J, verrucarin B, verrucarin A, isororidin E, roridin E, roridin A, trichodermadienediols A and B, trichoverrols A and B, and trichoverrins A and B in the order of increasing retention time on a silica column (ethyl acetate-hexane eluent). The gum was subjected to flash chromatography (silica gel) with increasing amounts of methanol in dichloromethane to yield four fractions: I (eluted with 0-1% methanol-methylene chloride), II (eluted with 1-5% methanol-methylene chloride), III (eluted with 5-7.5% methanol-methylene chloride), and IV (eluted with methanol). Fraction III of the crude extract was subjected to partition chromatography. The sample was applied to a column of Celite impregnated with 83% aqueous methanol and eluted with 15% dichloromethane in petroleum ether to give a gum. Flash chromatography (90% ethyl acetate-petroleum ether) yielded two major fractions, III-A (Re, 0.35 in 90% ethyl acetate-petroleum ether) and III-B (Rf, 0.30 in 90% ethyl acetatepetroleum ether). After filtration through TLC grade silica gel, each of these fractions was purified by HPLC (1-6% methanol-methylene chloride, gradient, 30 min) to give trichoverrol A and trichoverrol B from fraction III-A and trichoverrin A and trichoverrin

384


B from fraction III-B. Spectral Data UV;

maxM~O" 260nm (log e=4.53). 1H NMR: (CDCI3) 0.82(3H, s, 14-H), 1.15(3H, d, J=6Hz, 8'-H); 1.70(3H s, 16-H); 2.48(1H, dd, J=8, 15Hz, 3a-H); 3.04(2H, AB, J=4Hz, 13-H); 4.18(2H, s, 15-H); 5.51(1H, d, J=5Hz, 10-H), 5.73(1H, d, J=l 1Hz, 2'-H); 5.93(1H, 8, H-2'); 6.68(1H, dd, d's=l 1Hz, H-3'); and 7.58ppm (1H, dd, J= 11, 16Hz, 4'-H).

13C NMR: (CDC13) 79.2 d, C-2, 36.9 t, C-3, 75.1 d, C-4; 48.6 s, C-5; 42.9 s, C-6, 21.9 t, C-7; 27.9 t, C-8; 140.5 s, C-9; 118.6 d, C-10; 66.7 d, C-11; 65.8 s, C-12; 48.3 t, C-13; 6.7 q, C-14; 63.5 t, C-15; 23.2 q, C-16; 166.1 s, C-I'; 117.9 d, C-2'; 144.1 d, C-3'; 127.6 d, C-4'; 141.4 d, C-5'; 75.4 d, C-6'; 70.3 d, C-7'; 17.9 q, C-8'; 166.0 s, C-I"; 116.9 d, C2"; 157.2 s, C-3"; 43.6 t, C-4"; 59.7 t, C- 5"; and 19.2ppm q, C-6". Mass Spectrum: HRCIMS (methane gas reagent):

5 3 3 . 2 7 2 2 m / e ( M ++

H, ealed 533.2754).

Reference B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E. P. Mazzola and R. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 1117-1124(1982).

16.


385

_Common/Systematic Name (C6'R,C7'S)-Isotrichoverrin B Molecular Formula/Molecular Weight C29H4009; MW

16

= 532.26723

10 H

H

H2Cl 15 14 :

I'

5

..

CH2CH2OH

~

"

~J

3'

Ho..IJ

HO" 1. "8' H

General Characteristics Isolated as an oil; [tZ]D2~ - 25.0 ~ (C=2.20, in CHC13). Fungal Source


Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carded out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Rf fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. Sample 2 ($2) contained mainly trichoverrins according to TLC analysis. This sample was chromatographed on CCC (semipreparative column, V~=355ml) with a solvent system of chloroform-hexane-methanol-water (3:1:3:2, v/v/v/v) and a flow rate of 3.2ml/min (ca. 400rag/injection). Like fractions were combined to give seven fractions: S2F 1, $2F2, $2F3, $2F4, $2F5, $2F6, and $2F7. A portion of $2F2 was subjected to CCC (semipreparative column) with a solvent system of of methylene chloride-carbon tetrachloride-methanol-water (2:3:3:2, v/v/v/v) and a flow rate of 2.8ml/min. The components of the mobile phase (organic layer, CH2C12/CCIa) were varied from 2:3 to 5:2 (v/v), starting at t = 120 min and going to t = 160 min. Like portions were combined to give seven fractions: I, (C6'R, CT'R)-isotriehoverrin A, II, (C6'R, C7'S)-isotrichoverrin B, III, a mixture ofisotrichoverrin B and trichoverrin C), IV, V, VI, 2'E-isotrichoverrin A, VII, 2'E-isotrichoverrin B.

386

16. MacrocyclicTrichothecenes and Related Metabolites.

Spectral Data

1HN/VIR: (CDC13) 0.78(3H, s, H-14); 1.12(3H, d, J=6.5Hz, H-14'); 1.69(3H, s, H-16); 2.16(3H, d, J=l. 1Hz, H-6"); 2.39(2H, t, J=6.0Hz, H-4"); 2.55(1H, dd, ,/--7.7, 15.5Hz, H-3a); 2.82(1H, d, J= 4.0Hz, H-13A); 3.14(1H, d, J=4.0Hz, H-13B); 3.75(1H, dt, J=6.0; 11.5Hz, H-5"A); 3.83(1H, dt, J=6.0, 11.5Hz, H-5"B); 3.84(1H, d, J=5.1Hz, H2); 3.89(1H, dq, J=3.6, 6.5Hz, H-7'); 3.97(1H, d, J=5.4Hz, H-11); 4.10(2H, s, H-15); 4.23(1H, rn, H-6'); 5.46(1H, d, J=5.4Hz, H-10); 5.66(1H, d, J=l 1.3Hz, H-2'); 5.83(1H, d,J=l.OHz, H-6"); 6.11(1H, dd, J=5.2, 15.5Hz, H-5'); 6.19(1H, dd, J=-3.3, 7.7Hz, H-4); 6.60(1H, dd, J= 11.3, 11.3Hz, H-3'); and 7.52ppm(1H, dd, J=l 1.3, 15.5Hz, H-4'). 13CNMR: (CDCI3) 6.7, C-14; 17.9, C-8'; 19.2, C-6"; 21.9, C-7; 23.2, C-16; 27.9, C-8; 36.9, C3 42.9, C-6; 43.6, C-4"; 48.2, C-13" 48.6, C-5; 59.7, C-5"" 63.4, C-15 65.8, C-12; 66.6, C-11" 70.2, C-7'; 75.0, C-4; 75.4, C-6'; 79.1, C-2; 116.9, C-2"; 118.0, C-2'; 118.5, C-10; 127.6, C-4'; 140.4, C9; 141.1, C-5'; 143.9, C-3'; 157.0, C-3"; 165.9, C-I"; and 166.0ppm, C- 1'. Mass Spectrum: HRCIMS: calcd for C29H4109(M + H) +, 533.2751; found, 533.2786role. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261 (1996).

16.

Macrocyclic Trichothecenes


387

Common/Systematic Name (2'E, 4'Z)-Isotrichoverrin B Molecular Formula/Molecular Weight C29H4009; M W = 532.26723

16

~

~o H

H

-

0 . 1 2

" 13

1"

~;'~

O

I 15

0

3

14

6"

3'

H

HOWl',

HO" ,I, "8' CH2CH2OH

I"1

5"

General Characteristics Isolated as an amorphous solid. Fung.al Source


Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Rf fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. Sample 2 ($2) contained mainly trichoverrins according to TLC analysis. This sample was chromatographed on semipreparative countercurrent chromatography (CCC; Vc=355ml) with a solvent system of chloroform-hexane-methanol-water (3:1:3:2, v/v/v/v) and a flow rate of 3.2ml/min (ca. 400mg/injection). Like fractions were combined to give seven fractions: S2F1, $2F2, $2F3, $2F4, $2F5, $2F6, and $2F7. A portion of $2F2 was subjected to semi-preparative CCC with a solvent system of methylene chloride-carbon tetrachloride-methanol-water (2:3:3:2, v/v/v/v) and a flow rate of 2.8ml/min. The components of the mobile phase (organic layer, methylene chloride-carbon tetrachloride) were varied from 2:3 to 5:2 (v/v), starting at t = 120 min and going to t = 160 min. Like portions were combined to give seven fractions: I, (C6'R, C7'R)-isotrichoverrin A, .II, (C6'R,C7'S)-isotrichoverrin B, III, a mixture of isotrichoverrin B and trichoverrin C, IV, V, VI, (2'E)-isotrichoverrin A), VII, (2'E)-isotrichoverrin B). Fractions IV and V were subjected to CCC (semi-preparative column) separately with the solvent system of methylene chloride-carbon tetrachloride-methanol-water (2:3:3:2, v/v/v/v) and a flow rate of 1.8ml/min to yield fractions rich in (2T, 4'Z)-isotrichoverrin B. These fractions were

388


combined and purified on TLC (silica gel, 5% methanol in methylene chloride, developed three times) to give pure (2'E, 4'Z) isotrichoverfin B. Spectral Data UV~

~, maxM~~ 262nm (log 6=4.38). IR;

(CHCI3) 3600(OH), 3470(OH), 1710(C=O), and 1646cm~ (C=C). ~H NMR: (CDC13) 0.77(3H, s, H-14); 1.1 l(3H, d, J=6.4Hz, H-8'); 1.69(3H, s, H-16); 2.17(3H, s, H-6"); 2.40(2H, t, J=5.7Hz, H-4"); 2.55(1H, dd, J=7.8, 15.5Hz, H-3a), 2.81(1H, d, J=4.0Hz, H-13A); 3.13(1H, d, J=4.0Hz, H-13B); 3.75-3.92(3H, m, H-7', H-5"); 3.83(1H, d, J=5.1Hz, H-2); 3.95(1H, bd, J=4.8Hz, H-11); 4.1 l(2H, s, H-15); 4.60(1H, dd, J=3.3, 8.6Hz, H-6'); 5.46(1H, bd, J=4.8Hz, H-10); 5.83(1H, s, H-2"); 5.88(1H, dd, J=8.6,11.0Hz, H-5'); 5.94(1H, d, J=15.1Hz, H-2'); 6.16(1H, dd, J=3.5, 7.8Hz, H-4); 6.26(1H, dd, J=l 1.0, 11.8Hz, H-4'); and 7.59ppm(1H, dd, J=l 1.8,

15.1Hz, H-3').

13C NMR: (CDCla) 6.6, C-14; 17.3, C-8'; 19.0, C-6"; 21.9, C-7; 23.2, C-16; 27.9, C-8; 36.7, C3; 42.9, C-6; 43.7, C-4"; 48.1, C-13; 48.7, C-5; 59.7, C-5"; 63.5, C-15; 65.5, C-12; 66.8, C-11; 70.4, C-7'; 71.6, C-6'; 75.6, C-4; 79.1, C-2; 117.2, C-2"; 118.5, C-10; 122.7, C-2'; 128.9, C-4; 138.1, C-5'; 139.9, C-3'; 140.5, C-9; 156.9, C-3"; 165.9, C-I"; and 167.0ppm, C-I'. Mass Spectrum: HRCIMS: calcd for C29H4109,(M + H) +, 533.2751; found, 533.2768role. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod.,Vol. 59, pp. 254-261 (1996).


389

Common/Systematic Name 913,1013-Epoxyisotrichoverrin B Molecular Formula/Molecular Weight C29H40010; M W = 548.26723

01o

H

H

.....

I

....

H CI :

15

5

o

4 %

14

CH2CH2OH

HO'"lx ' H

5"

General Characteristics Isolated as an oil;

[ ( g ] D 20

-

21.0 ~ (c= 0.13, in CHC13).

Fungal Source Myrothecium verrucaria (ATCC 20540). Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Rf fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. Sample 3 ($3) fromM, verrucaria isolate ATCC 20540, which was more polar than those that contained trichoverrols (S 1) and trichoverrins ($2), was partitioned between chloroform and methanol-water mixture (50%). The organic fraction was subjected to preparative TLC (Chromatotron, 2mm, silica gel) with methanol-methylene chloride (310%). The most polar fraction was subjected to high speed countercurrent distribution (CCC) with a solvent system of chloroform-hexane-methanol-water (12: 8:15:5, v/v/v/v) at a flow rate of 1.8ml/min to give 913,1013-epoxyisotrichoverrin A and 9[},1013epoxyisotrichoverrin B. Spectral Data 1H NMR: (CDC13) 0.74(3H, s, H-14); 1.13(3H, d, J-6.4Hz, H-8'); 1.34(3H, s, H-16); 1.702.03(5H, m, H-3~, H-7, H-8); 2.20(3H, d, d=0.9Hz, H-6"); 2.41(2H, t, J=6.1Hz, H-

390


4"); 2.53(1H, dd, J=7.9, 15.5Hz, H-3a); 2.78(1H, d, J=4.0Hz, H-13A); 3.10(1H, d, J=5.6Hz, H-10); 3.19(1H, d, J=4.0Hz, H-13B); 3.81(2 H, m, H-5"); 3.90(2H, m, H11, H-7'); 3.93(1H, d, J=5.2Hz, H-2); 4.04(1H, d, J=12.6Hz, H-15A); 4.15(1H, d, J=12.6Hz, H-15B); 5.67(1H, d, J=l 1.3Hz, H-2'); 5.81(1H, d, J=0.9Hz, H-2"); 6.03(1H, dd, J=3.4, 7.9Hz, H-4); 6.12(1H, dd, J=5.3, 15.5Hz, H-5'); 6.62(1H, dd, J=l 1.3, 11.3Hz, H-3'); and 7.53ppm (1H, dd, J=l 1.3, 15.5Hz, H-4'). 13CNMR: (CDC13) 6.7, C-14; 17.9, C-8'; 19.2, C-6"; 19.4, C-7; 22.3, C-16; 26.4, C-8; 36.6, C3; 42.6, C-6; 43.6, C4"; 48.0, C-13; 48.5, C-5; 57.3, C-10; 57.5, C-9; 59.7, C-5"; 63.1, C-15; 65.2, C-12; 66.7, C-11; 70.2, C-7'; 74.7, C-4; 75.4, C-6'; 78.7, C-2; 116.5, C-2"; 118.0, C-2'; 127.7, C-4'; 142.1, C-5'; 143.9, C-3'; 158.4, C-3"; 165.8, CI'; and 165.9ppm, C- 1" Mass Spectrum: HRCIMS: calcd for C29H41010 (M d- H)§ 549.2700; found, 549.2754m/e. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261 (1996).


391

Common/Systematic Name (2'E)- 12,13-Deoxyisotrichoverrin B Molecular Formula/Molecular Weight C29H40Os; MW = 516.27230

MemO.,,]

H

H

LL Lc I H2~ Mel 0 I O=~1"~ H

o "~

'

M~'~CH2 I CH2OH ~,,

OH H

GeneralCharacteristics Isolatedasan oil.

Fungal Source Myrothecium verrucaria (ATCC 20540). Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Rf fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. To sample 1, which contained some solid material, was added cold ethyl acetate. The solid was collected and recrystallized from methylene chloride-hexane to give isotrichoverrol A. The remaining portion of S 1 was subjected to semipreparative CCC (Vc=355ml) with a solvent system of methanol-water-chloroform-hexane (3:2:3:1.2, v/v/v/); flow rate was 2.4ml/min (ca. 400mg/injection, total 10 injections) to give isotrichoverrol A and isotrichoverrol B. Another fraction from this CCC was subjected to CCC (semipreparative column) with a solvent system of methylene chloride-carbon tetrachloride-methanol-water (2:3:3:2, v/v/v/v; ca. 400mg/injection). The major fraction of this sample was eluted with little retention from the column, and there were no detectable trichothecenes in this fraction, according to TLC analysis. Seven additional fractions were collected: S IF 1a, S 1F 1b, S 1F 1c, S 1F 1d, S 1F 1e, S 1F 1f, and S 1F 1g. S 1F 1f was subjected to reversed-phase TLC on Cs (55% methanol in water) to give (2'E)12,13-deoxyisotrichoverrin B as an oil.

392


Spectral Data UV~

maxM~~ 260rim(log e=4.44). IR~

(CHC13) 1707(C=O) and 1652cm~ (C=C). 1H NMR: (CDCI3) 1.02(3H, s, H-14); 1.12(3H, d, J-6.5Hz, H-8'); 1.66(3H, s, H-16); 2.18(3H, d, J=l.0Hz, H-6"); 2.39(2H, t, J=5.6Hz, H-4"); 2.57(1H, dd, J=7.7, 15.5Hz, H-3~); 3.76-3.90(3H, m, H-7', H-5"); 3.96(1H, bd, J=5.7Hz, H-11); 4.1 l(2H, s, H-15); 4.18(1H, m, H-6'); 4.42(1H, d, J=5.1Hz, H-2); 4.71(1H, s, H-13A); 5.13(1H, s, H13B); 5.41(1H, bd, J=5.7Hz, H-10); 5.83(1H, d, J=15.4Hz, H-2'); 5.84(1H, d, J=l.lHz, H-2"); 6.06(1H, dd, J=3.0, 7.7Hz, H-4); 6.11(1H, dd, J=5.9, 15.6Hz, H-5'); 6.39(1H, dd, J=10.9, 15.6Hz, H-4'); and 7.21ppm(1H, dd, J=10.9, 15.4 Hz, H-3'). 13C NMR: (CDC13) 11.1, C-14; 17.6, C-8'; 19.0, C-6"; 20.9, C-7; 23.2, C-16; 28.0, C-8; 37.9, C3; 42.8, C-6; 43.8, C-4"; 51.6, C-5; 59.8, C-5"; 63.7, C-15; 66.6, C-11; 70.2, C-7'; 75.4, C4; 75.6, C-6'; 78.8, C-2; 105.4, C-13; 117.3, C-2"; 118.8, C-10; 121.8, C-2'; 129.8, C-4'; 140.2, C-5'; 140.1, C-9; 143.9, C-3'; 152.3, C-12; 156.7, C-3"; 166.2, C1"; and 166.5ppm, C- 1'. Mass Spectrum: HRCIMS: calcd for C29I-'I4109, (M+d " 1), 517.2801; found, 517.2849role. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod., Vol. 59, pp. 254-261 (1996).


393

Common/Systematic Name (9'E)-Roridin L-2 Molecular Formula/Molecular Weight C29H3809, ~

= 530.25158

~~ 0 ~ ~o H

13

H 3 I

HOH2(~ i~, 15

General Characteristics Isolated as an oil. Fungal Source Myrothecium verrucaria (ATCC 20540). Isolation/Purification The fermentation procedures with Myrothecium verrucaria (ATCC 20540) were carried out in a manner similar to those described previously (Jarvis et al., 1982). Samples 1-4 (S 1, $2, $3, and $4) from this isolate were the lower Re fractions from silica gel chromatography (methanol-methylene chloride), given in the order of increasing polarity. To sample 1, which contained some solid material, was added cold ethyl acetate. The solid was collected and recrystallized from methylene chloride-hexane to give isotrichoverrol A. The remaining portion of S 1 was subjected to semipreparative high-speed countercurrent chromatography (CCC, V~=355ml) with a solvent system of methanol-waterchloroform-hexane (3:2:3:1.2, v/v/v/v); flow rate was 2.4ml/min (ca. 400 mg/injection, total 10 injections) to give isotrichoverrol A and isotrichoverrol B. Another fraction from this CCC was subjected to CCC (semipreparative column) with a solvent system of methylene chloride-carbon tetrachloride-methanol-water (2:3:3:2, v/v/v/v) (ca. 400mg/injection). The major fraction of this sample was eluted with little retention from the column, and there were no detectable trichothecenes in this fraction, according to TLC analysis. Seven additional fractions were collected: S1Fla, S1Flb, S1Flc, S1Fld, S1Fle, S1Flf, and S1Flg. S1Fld was subjected to reversed-phase TLC on Cs plates (60% methanol in water) to give isotrichoverrin A and (9'E)-roridin L-2.

394


Spectral Data 1H N1VIR: (CDC13) 0.81(3H, s, H-14); 1.1 l(3H, d, J=6.3Hz, H-14'); 1.71(3H, s, H-16); 2.49(1H, dd, J=7.9, 15.5Hz, H-3a); 2.70(2H, t, J=6.0Hz, H-4'); 2.81(1H, d, J=4.0Hz, H-13A), 3.13(1H, d, J=4.0Hz, H-13B); 3.83(1H, d, J=5.1Hz, H-2); 3.92(1H, bd, J=5.7Hz, H-11); 4.77(2H, d, J=l.5Hz, H-12'); 5.48(1H, bd, J=5.7Hz, H-10); 5.90(1H, d, J=l.5Hz, H-2'); 5.90(1H, dd, J=8.2, 15.3I-Iz, H-7'); 5.97(1H, d, J=15.4Hz, H-10'); 6.12(1H, dd, J=3.6, 7.9Hz, H-4); 6.36(1H, dd, J=l 1.0, 15.3Hz, H-8'); and 7.27ppm (1H, dd, J=l 1.0, 15.4Hz, H-9'). 13CNMR: (CDCI3) 6.6, C-14; 18.4, C-14'; 19.0, C-12'; 23.2, C-16; 28.0, C-8; 29.3, C-4'; 31.3, C-7; 35.9, C-3; 44.2, C-6; 48.2, C-13; 49.0, C-5; 63.0, C-15; 65.6, C-12; 66.2, C-5'; 66.8, C-11; 71.4, C-13'; 75.8, C-4; 79.0, C-2; 85.9, C-6'; 116.7, C-2'; 118.8, C-10; 122.6, C-10'; 132.4, C-8'; 138.6, C-7'; 140.5, C-9; 143.4, C9'; 166.8, C-11'; 167.4, C3'; and 173.2ppm, C- 1'. Mass Spectrum: HREIMS: calcd for C29H3sO9(1W), 530.2516; found, 530.2516m/e. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. DeSilva, C. E. Holmlund, E. P. Mazzola, and R. F. Geoghegan, Jr.; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 11171124(1982). B. B. Jarvis, S. Wang, and H. L. Ammon; Trichoverroid Stereoisomers; J. Nat. Prod.,Vol. 59, pp. 254-261 (1996).


395

Common/Systematic Name Roridin L-2 Molecular Formula/Molecular Weight C29H3809; ~ 16

lo

= 530.25158 H

~ O . , , J

H

2

HOH2~s 11s, O

3

~O'~

0

IJg'

HO ~; General Characteristics Isolated as a white, homogeneous solidi rap., 93-97~

[a]D + 83.6~ (c= 1.0, in CHC13).

Fungal Source Myrothecium roridum. Isolation~urification Fermented beer was extracted with ethyl acetate, concentrated, and chromatographed by silica gel high performance liquid chromatography eluted with methylene chloride-ethyl acetate, 50:50 (v/v) and by C]8-reversed phase chromatography eluted with methanolwater, 50:50 (v/v). Spectral Data UV: )i, M~H

~x

259nm (e=24,650).

IR:

(CC14) 3500, 3500, 1785, 1750, 1710, 1640, and 1600cm"1. ]H NMR: (CDC13) 0.84(3H, s, H-14); 1.14(3H, d, J=6.0Hz, H-14'); 1.72(3H, s, H-16); 2.50(1H, dd, J=8.0, 15.5Hz, H-3a); 2.0(1H, m, H-313); 2.70(2H, m, H-4'); 2.81(1H, d, J=4.0Hz, H-13A); 3.13(1H, d, J=4.0Hz, H-13B); 3.70(1H, m, H-2); 3.70(1H, m, H-11); 4.78(1H, dd, J=l.5, 17.5Hz, H-12'A); 4.84(1H, dd, J=l.5Hz, 17.5Hz, H-12'B); 5.48(1H, d, J=5.0 Hz, H-10); 5.92(1H, br, J=l.3Hz, H-2'); 5.86(1H, dd, J--7.0, 15.5Hz, H-7'); 5.78(1H, d, J=l 1.5Hz, n-10'); 6.10(1H, dd, J=3.5, 8.0Hz, n-4);

396


7.61(1H, dd, J=l 1.5, 15.SHz, H-8'); and 6.61ppm (1H, t, J=l 1.SHz, H-9'). 13CNMR: (CDCI3) 6.7, C-14; 18.5, C-14'; 73.6, C-12'; 23.3, C-16; 28.1, C-8; 29.3, C-4'; 21.2, C-7; 36.3, C-3; 44.3, C-6; 6.7, C-13; 48.9, C-5; 62.7, C-15; 65.7, C-12; 66.4, C-5'; 66.9, C-11; 69.8, C-13'; 75.5, C-4; 79.0, C-2; 85.4, C-6'; 116.7, C-2'; 118.9, C-10; 118.8, C-10'; 130.6, C-8'; 139.1, C-7'; 140.6, C-9; 143.5, C9'; 166.5, C-11'; 167.8, C3'; and 174.2ppm (C- 1'). Mass Spectrum: EIMS: 530m/e (M+). References R. J. Bloem, T. A. Smitka, R. H. Bunge, J. C. French, and E. P. Mazzola; Roridin L-2, A New Trichothecene; Tet. Lett., Vol. 24, pp. 249-252(1983). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991).

16.


397

Common/Systematic Name Verrucarin A Molecular Formula/Molecular Weight C27H3409; M W = 502.22028

~o H

H2r o

H

! 15

4 "~O"'~

, & "

HO

II O

O

12'

General Characteristics Crystals; mp., >360 ~ (dec.);

[a]D 22 + 2 0 6 ~

(c=1.012, in dioxane); +260 ~ (in CHC13).

Fungal Source

Myrothecium verrucaria and M. roridum.

Biological Activity Rats given verrucarin A developed creatinuria; m vitro it inhibited ATP-creatine phosphotransferase. It caused severe edema in body cavities. Major clinical signs in several different experimental animals were diarrhea, hematuria, vomiting, anorexia, loss of weight, ataxia, and thirst. At relatively low dosages, it caused leukocytosis followed by severe leukopenia. The LDs0 values in mice were 1.5mg/kg (IV) and 0.5mg/kg (IP). It caused dermal toxicity: necrosis of epidermis with suppuration and extensive necrosis of dermal tissue with damage extending into subcuticulis. IDs0 of protein synthesis in rabbit reticulocytes was 10-15ktg/ml. Spectral Data UV:

~.~a 260nm (6=17,700).

max

~H NMR: (CDCI3) 5.83(H-4); 5.46(H-10); 2.97(H-13a); 0.87(H-14); 1.79(H-16); 4.20(H-2'), 6.06(H-7'); 8.08(H-8'); 6.70(H-9'); 6.17(H-10'); and 0.89ppm (H-12').

398


13CNMR: (CDC13) 78.6, C-2; 34.6, C-3; 75.3, C-4; 49.2, C-5; 43.9, C-6; 19.7, C-7; 27.2, C-8; 140.7, C-9; 117.7, C-10; 66.6, C-11; 64.9, C-12; 47.4, C-13; 7.0, C-14; 63.1, C-15; 22.6, C-16; 174.3, C-I'; 73.8, C-2'; 32.9, C-3'; 31.9, C-4'; 60.8, C-5'; 165.8, C-6'; 127.2, C-7'; 138.6, C-8'; 138.6, C-9'; 125.5, C-10'; 165.1, C-11'; and 9.8ppm, C-12'. TLC Data A. Adsorbent: silica gel G; solvent system, chloroform-methanol, 98:2 v/v; Rf, 0.70; detection, H2SO4spraying and heating at 110~ for 5 minutes. B. Adsorbent: alumina; solvent system, chloroforrrl-methanol, 98:2 v/v; Re, 0.28; detection, HzSO4 spraying and heating at 110~ for 5 minutes. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 247-248(1981 ). J. Gutzwiller and Ch. Tamm; Uber die Struktur von Verrucarin A; Helvetica Chimica Acta, Vol. 48, pp. 157-176(1965). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991).

16.


399

Common/Systematic Name Verrucarin A [~-D-glucoside Molecular Formula/Molecular Weight C33H44014; MW

"-

664.27311

H

H 13 ~ ; ,,it) I

o

H2(~ i s

O

~0/'~

I ,,

II

1

~-glucose

0

General Characteristics Amorphous solidi [~]D + 98~ (c=0.63, in CHC13). Fungal Source Baccharis coridifolia. Isolation/Purification The crude extract (10% methanol-chloroform) of female B. coridifolia plant was partitioned (hexane-10% water in methanol) and the aqueous methanol, atter solvent removal,was chromatographed (silica gel) with ethyl ether-hexane, ethyl ether-hexane, and methanol-methylene chloride. The 20% methanol-methylene chloride was subjected to further flash chromatography, preparative TLC (Chromatotron), and finally preparative C ls reversed phase HPLC to give verrucarin A ~-D-glucoside. Biological Activity Cytotoxic: IC60 ca. 100 ng/ml. Spectral Data IR:

(CH2C12) 3440, 1720, 1660, 1640, 1595, 1270, 1195, and 1080cm"1. 1H N]h/[R: (CDCls) 7.99(1H, dd, J=12.0, 15.5Hz, H-8'); 6.65(1H, dd, ,/--12.0, 15.5Hz, H- 8'); 6.65(1H, dd, J=l 1.1, 12.0Hz, H-9'); 6.14(1H, d, J=l 1.1Hz, H-10'); 6.02(1H, d, J=15.5Hz, H-7'), 5.77(1H, dd, J=3.9, 8.1Hz, H-4); 5.42(1I-I, br d, J=5.0Hz, H-10); 4.75 and 4.20(1H each, AB, J=12.1Hz, H-15); 4.46(1H, ddd, J=H-5~B); 4.20(1H, d,

400


J=2.0Hz, H-2'); 4.12(1H, d, J=7.6Hz, H-I"); 3.97(1H, ddd, J-11.7, 11.7, 3.5Hz, H5'A); 3.90(1H, dd, J=3.6, 11.8Hz, H-6"B); 3.84(1H, d, J=5.1Hz, H-2); 3.79(1H, d, J=5.1, 11.SHz, H-6"A); 3.54-3.57(3H, m, H-11, H-3", H-4"); 3.40(1H, dd, ,]-7.6, 9.2Hz, H-2"); 3.36(1H, ddd, J=3.6, 5.1, 7.SHz, H-5"); 3.11 and 2.78(1H each, AB, J=3.9Hz, H-13); 2.46(1H, dd, J-8.1, 15.4Hz, H-3Gt); 2.44(1H, m, H-3'); 2.20(1H, ddd, J=3.9, 5.1, 15.4Hz, H-313); 1.99(1H, m, H- 8B); 1.80-1.90(4H, m, H-4', H-7B, H-8A); 1.75(3H, s, H-16); 1.69(1H, m, H-7A); 0.94(3H, d, J=5.9Hz, H-12'); and 0.82ppm (an, s, n-14). 13CNMR: (CDC13) 78.9(C-2), 34.9(C-3), 75.6(C-4), 49.5(C-5), 44.1(C-6), 20.1(C-7), 27.5(C8), 141.0(C-9), 118.0(C-10), 66.8(C-11), 65.2(C-12), 47.8(C-13), 7.4(C-14), 63.6(C15), 23.4(C-16), 172.4(C-1'), 80.6(C-2'), 32.4(C-3'), 32.1(C-4'), 61.0(C-5'), 166.1(C6'), 127.3(C-7'), 138.8(C-8'), 138.7(C-9'), 125.9(C-10'), 165.4(C-11'), 11.2(C-12'), 102.6(C-1"), 73.6(C-2"), 76.5(C-3"), 70.0(C-4"), 75.7(C-5"), and 62.3ppm (C-6"). TLC Data Adsorbent: Silica gel, solvent system: 10% methanol-methylene chloride; Re, 0.38. Reference B. B. Jarvis, S. Wang, C. Cox, M. M. Rao, V. Philip, M. S. Varaschin, and C. S. Barros; Brazilian Baccharis toxins: Livestock Poisoning and the Isolation of Macrocyclic Trichothecene Glucosides; Natural Toxins, Vol. 4, pp. 58-71(1996).


401

Common/Systematic Name 2'-Dehydroverrucarin A Molecular Formula/Molecular Weight C27H3209; M~V = 500.20463

H

I.

H

~l'~',~i ....,u I

_~v"T~, ~

H27,~!,5

o~,

I

o

II~,'

''0%

/, ,

Id

o

General Characteristics Needles from acetone-ethyl ether; mp., 233-240~

[~]D 25 +

118~ (in CHCI3).

Fungal Source Myrothecium roridum. Biological Activity Antibiotic activity similar to that of verrucarin A. Spectral Data UV:

X maxEtO" 262nm (e=23,400). IR:

(KBr) 1710, 1725, 1590, and 1630cm1. Reference B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991).

402

16.

Macrocyclic T r i c h o t h e c e n e s and Related Metabolites

Common/Systematic Name Verrucarin B Molecular Formula/Molecular Weight C27H3209; MW = 500.20463 ~

O I

l~

1o

H 2

H

~1 13

I

d

61

~

3 r

....i " l

I

0

Fungal Source Myrothecium verrucaria and M. roridum.

General Characteristics Crystals; mp., >330~

[a]D + 94 ~ (in CHCI3), + 101 ~ (in dioxane); + 147~ (in benzene).

Biological Activity Typical trichothecene type activity: dermal toxicity, antifungal, cytostatic, and acute toxicity to vertebrate animals. The LDs0 in mice dosed IV was 7.0mg/kg. The EDs0 for cytostatic activity in vitro against Ehrlich ascites tumor cells was 0. 003 ~tg/ml. Spectral Data UV: maxE~O" 258.5nm (e=23,400). 1H NIV[R: (CDCI3) 5.90(H-4); 5.47(H-10), 3.00(H-13a); 0.88(H-14); 1.74(H-16), 3.41(H-2'); 6.10(H-7'); 7.98(H-8'), 6.69(H-9'); 6.19(H-10'); and 1.56ppm (H-12'). ~3CNMR: (CDCI3) 78.7,C-2; 34.8, C-3; 75.4, C-4; 49.0, C-5; 43.6, C-6; 19.8, C-7; 27.4, C-8; 140.4, C-9; 118.0, C-10; 66.9, C-I I; 64.9, C-12; 47.5, C-13; 7.3, C-14; 63.6,C-15; 22.8, C-16; 167.4, C-I'; 58.0, C-2'; 61.1, C-3'; 36.9, C-4'; 60.4, C-5'; 165.8, C-6'; 127.2, C-7'; 138.0, C=8'; 138.0, C-9'; 125.6, C=I0'; 164.8; CI I', and 15.8ppm, C-12'.

16.


403

TLC Data Absorbent: Kieselgel G; solvent, ethyl ether (two consecutive runs); Re, 0.37; detection, iodine vapors. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 253-254(1981). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991).

404


Common/Systematie Name Verrucarin J Molecular Formula/Molecular Weight C27H3208; M W - 4 8 4 . 2 0 9 7 2 ,0 H

HC

H

2l,sa4

0

'

15

,~0

'~

,~'

.

Jl

j~r

0 Fungal Source Myrothecium verrucaria. General Characteristics Colored needles from chloroform-ether; acetone-ether; mp., >315~ (dec.); (c=l.011, in CHCI3); [a]D23 + 41 ~ (C=0.784, in benzene).

[a]D 22 +22 ~

Biological Activity Antibiotic activity similar to that of verrucarin A. Spectral Data UV:

~, ~m~ ~

196(e = 15,500), 219(19,900), and 262nm (14,500).

IR:

(CH2C12) 2810, 1710, 1650, 1630, 1588, 1352, 1221, 1180, 1147, 1070-1088, 1042, 995, 968, 877, and 820cm "~. ~H NMR: (CDCI3) 5.90(H-4); 5.47(H-10); 2.98(H-13a); 0.83(H-14); 4.30(H-lSa); 1.72(H-16); 3.50(H-2'); 2.50(H-4'a); 3.82(H-5'); 6.05(H-7'); 8.12(H-8'); 6.60(H-9'); 6.10(H-10'); and 2.28ppm (H- 12'). TLC Data Adsorbent: Kieselgel G; solvent was ethyl ether (two consecutive runs); Rf, 0.42; detection by iodine vapors.

16.


405

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 259 (1981). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991).

406

16.


Common/Systematic Name Verrucarin K 12,13-Deoxyverrucarin A Molecular Formula/Molecular Weight C27H3408; M'W -- 486.2253 7 ,0

H

H

'

'

0

I

,

H

Fungal Source

Myrothecium verrucaria.

General Characteristics Crystals from dichloromethane-ether; mp., >320~ (dec.); [a]D23 +218 ~ (C=0.58, in CHCI3). Monoacetyl derivative, colorless needles from acetone-ether-petroleum ether; mp., 199-202~ [a]D23 + 143~ (C=0.83, in CHC13). Spectral Data UV:

~, maxS~n 259nm (6=15,500). IR:

171O, 3550, 1630, and 1585cm~. 1H NMR:

(CDCI ) 5.810-1-4),5.a9(H-10), 4.71(H-13a); 5.18(H-13b); 1.09(H-14); 3.66(H-15); 1.70(H-16); 6.05(H-7'); 8.05(H-8'); 6.67(H-9'); 6.08(H-10'); and 0.89ppm (H-12'). 13C NMR:

(CDCIa) 78.5, C-2; 35.9, C-3; 75.5, C-4; 52.0, C-5; 44.2, C-6; 18.7, C-7; 27.4, C-8; 140.5, C-9; 118.2, C-10; 66.5, C-11; 151.6, C-12; 106.3, C-13; 12.1, C-14; 63.5, C15; 23.2, C-16; 174.5, C-I'; 74.0, C-2'; 61.1, C-3'; 32.1, C-4'; 61.1, C-5'; 165.9, C-6'; 127.4, C-7'; 138.7, C-8'; 138.7, C-9'; 125.6, C-10'; 165.3, C-11'; and 10.0ppm, C-12'.


407

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 260(1981). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991).

408


Common/Systematic Name Verrucarin L Molecular Formula/Molecular Weight C27H3209; MW

16

= 500.20463

1o H

I

H

~I

~

I ,,I I,r~ I v

1

o,

o9 0 -/

i

g

'C----C(CN2)2OC"

H

~

General Characteristics Crystals from dichloromethane-hexane, mp., 230-23 5~ [tt]D 27 + 15.0 ~ (c = 0.92 in chloroform). Acetate derivative, crystals from dichloromethane-ether, m.p. 132-135~ [a]D27 + 29.7 ~ (C=0.52, in CHC13). Isolation/Purification Spores ofMyrothecium verrucaria grown on N-Z amine agar were added to Czapek-Dox media and allowed to grow at 28~ in a shake culture. After 2 days, the solution was divided into three equal portions and added to three separate Fernback flasks containing 1L of production media. After 3 days of growth, the mycelium in each flask was separately centrifuged, washed, and resuspended in 1L of sterile water. Trichoverrin A and trichoverrin B were added separately to flasks one and two; flask three was used as the control. After 7 days, the mycelium was removed and extracted with ethyl acetate. The mycelium extract was subjected to partition chromatography on 500g Celite impregnated with 250ml of 18% water in methanol. The column was eluted with petroleum ether followed by increasing amounts of dichloromethane in petroleum ether up to 40% dichloromethane in petroleum ether. Fractions were combined on the basis of TLC analysis to give a total of eight fractions: A, B, C, D, E, F, G, and H (methanol wash). Flash chromatography of fraction G (3% MeOH in CH2C12) gave a fraction rich in roridin A, which upon crystallization from dichloromethane-hexane yielded roridin A. The mother liquor was subjected to HPLC (2% methanol-methylene chloride) to give verrucarin L which elutes just before roridin A. Fungal Source


16.


409

Spectral Data UV:

~E,o. max

262nm (log e = 4.42).

1H NIV[R: (CDCI3) 0.86(3H, s, 14-H); 1.87(3H, s, 16-H); 2.26(3H, d, J = l H z , 12'-H); 2.99(2H, AB, J=4Hz, 13-H); 3.80(1H, d, J=5Hz, 1 I-H), 4.45(2H AB, J=12 Hz, 15-H); 5.2(1H, m, 8-H); 5.58 (1H, d, J=5Hz 10-H); 6.00 (1H, d, J=16Hz, 7'-H); 6.12 (1H, d, J=l 1Hz, 10'-H); 6.65(1H, dd, d's=l 1Hz, 9'-H); and 8.10ppm (1 H, dd, J = l l , 16Hz, 8'H). Verrucarin L acetate, 0.86(3H, s, 14-H); 1.80(3H, s, 16-H); 1.94(acetate); 2.27(3H, d, J=lHz, 12'-H); 2.97(2H, AB, d=4Hz, 13-H); 3.75(1H, d, J=5Hz, 1 I-H); 4.40(2H, AB, J=12 Hz, 15-H), 5.70(1H, d , J = 5.0Hz, 10-H); 5.96(1H, d, J=16Hz, 7'H); 6.07(1H, d, J=l 1Hz, 10'-H); 6.61(1H, dd, 3's=l 1Hz, 9'-H); and 8.01ppm (1H, dd, J=l 1, 16Hz, 8'-H). 13C NMP~: (CDCI3) 79.0 d, C-2; 35.3 t, C-3; 75.1 d, C-4; 48.8 s, C-5; 42.5 s, C-6; 30.1 t, C-7; 66.8 d, C-8; 139.7 s, C-9; 120.9 d, C-10; 67.2 d, C-11; 65.5 s, C-12; 48.1 t, C-13; 6.9 q, C-14; 65.0 t, C-15; 20.6 q, C-16; 165.7 s, C-I'; 118.2 d, C-2'; 156.6 C-3'; 40.3 t, C4'; 60.5 t, C-5'; 165.5 s, C-6'; 127.2 d, C-7'; 139.3 d, C-8', 139.3 d, C-9'; 125.6 d, C10', 165.7 s, C-11'; and 17.3ppm q, C-12'. Mass Spectrum: HRCIMS (methane gas reagent), 501.2112m/e (M § + H, calcd 501.2124). Verrucarin L acetate (chemical ionization, methane gas reagent), 543.2217m/e (M ++ H; calcd for 543.2230). Reference B. B. Jarvis, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E.P. Mazzola and R. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 1117-1124(1982).

410


Common/Systematic Name Verrucarin L Acetate Molecular Formula/Molecular Weight C29I-I34010;MW = 542.21520 16

10 H

ACO

;I

H

5

4%~

2 "C--C(CH2)20~ , 2 /

0 ~"

H

O

General Characteristics Crystals from ether-hexane, mp., 132-135~

[a]D27+ 29.7 ~ (c= 0.52 in CHCI3).

Isolation/Purification Spores ofMyrothecium verrucaria grown on N-Z amine agar were added to Czapek-Dox media and allowed to grow at 28~ in a shake culture. After 2 days, the solution was divided into three equal portions and added to three separate Fernback flasks containing 1L of production media. After 3 days of growth, the mycelium in each flask was separately centrifuged, washed, and resuspended in 1L of sterile water. Trichoverrin A and trichoverrin B were added separately to flasks one and two; flask three was used as the control. After 7 days, the mycelium was removed and extracted with ethyl acetate. The mycelium extract was subjected to partition chromatography on 500g Celite impregnated with 250ml of 18% water in methanol. The column was eluted with petroleum ether followed by increasing amounts of dichloromethane in petroleum ether up to 40% dichloromethane in petroleum ether. Fractions were combined on the basis of TLC analysis to give a total of eight fractions: A, B, C, D, E, F, G, and H (methanol wash). Fraction E was subjected to MPLC using 30-40% ethyl acetate in hexane to give verrucarin B, verrucarin L acetate, and a mixture of verrucarin A and roridin J. Fungal Source


Spectral Data

UV: maxE~n

261 nm (log e = 4 .28 ).


411

1H NMR: (CDC13) 0.86(3H, s, 14-H); 1.80(3H, s, 16-H); 194 (acetate); 2.27(3H, d, J=lHz, 12'-H), 2.97(2H, AB, J=4 Hz, 13-H); 3.75(1H, d, J=SHz 1 l-H); 4.40(2H, AB, J=12Hz, 15-H); 5.70(1H, d, J=5.0Hz, 10'-H); 5.96(1H, d, J=16Hz, 7'-H); 6.07(1H, d, J=l 1Hz, 10'-H); 6.61(1H, dd, J's=l 1Hz, 9'-H); and 8.01ppm (1H, dd, J=l 1, 16Hz, 8'-

H).

~3C NMR: (CDCls) 79.0 d, C-2; 34.9 t, C-3; 74.1 d, C-4; 49.0 s, C-5; 42.2 s, C-6; 26.5 t, C-7; 68.8 d, C-8; 136.5 s, C-9; 123.9 d, C-10; 67.0 d, C-11; 65.3 s, C-12; 47.9 t, C-13; 7.0 q, C-14; 64.5 t, C-15; 21.0 q, C-16; 165.5 s, C-I'; 117.8 d, C-2'; 156.9 C-3'; 40.2 t, C4'; 60.4 t, C-5'; 165.4 s, C-6'; 127.8 d, C-7'; 138.8 d, C-8'; 139.9 d, C-9'; 125.2 d, C10'; 165.8 s, C-11'; and 17.1 q, C-12'; 170.9, s, CH3C=O; and 20.4ppm q, CH3C=O. Mass Spectrum: HRCIMS (methane gas reagent), 543.2217m/e (M + + H, calcd 543.2230). Reference B. B. Jarvis, G. P. Stably, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E.P. Mazzola and R. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 1117-1124(1982).

412


Common/Systematic Name Roridin A Molecular Formula/Molecular Weight C29H4009; M W ~- 532.26723

1 6 ~H

2 3 ,;,,0 ]

H

0

I " 0 .....

h

14'

General Characteristics Crystals from ether; mp., 198-204~ [aiD 22 +130 ~ (c=1.36, in CHCI3); [~]D 22 + 192~ (c=1.38 in dioxane); di-O-acetylroridin A, needles from ether-petroleum ether; mp., 165166~ [tt]D22 +168.5 ~ (C= 1.108, in CHCI3). Fungal Source Myrothecium verrucaria and M. roridum. Biological Activity The LDs0 value of roridin A in mice dosed IV was 1mg/kg. In mouse tumor cells, it caused 50% inhibition of cell growth at 1ng/ml. Spectral Data UV~

m,~E~~ 263nm (e=18,600). IR: (Kbr) 3546, 3472, 1742, 1709, 1704, 1701, 1637, 1631, and 1597cm "1. 1H NMR:

(CDCI3) 5.85(IH, H-4); 5.44(IH, H-10); 2.96(IH, H-13a); 0.80(3H, H-14); 4.44(IH, H-15a); 1.74(3H, H-16); 4.09(IH, H-2');6.00(IH, H-7');7.68(IH, H-8');6.66(IH, H9'0;5.78(IH, H-10'); 1.08(3H, H-12'); and 1.17ppm (3H, H-14').

16.


413

13C NMR: (CDC13) 78.8, C-2; 34.6, C-3; 74.2, C-4; 49.1, C-5; 43.6, C-6; 20.0, C-7; 27.5, C-8; 140.4, C-9; 118.2, C-10; 66.9, C-11; 64.9, C-12; 47.4, C-13; 7.2, C-14; 64.2, C-15; 22.9, C-16; 174.5, C-I'; 75.3, C-2'; 36.7, C-3'; 33.0, C-4'; 69.5, C-5'; 83.7, C-6'; 139.0, C-7'; 126.0, C-8'; 143.6, C-9'; 117.2, C-10'; 166.3, C-11', 14.4, C-IT; 70.4, C-13', and 18.0ppm C- 14'. TLC Data A. Adsorbent: silica gel G; solvent: chloroform-methanol, 98:2 v/v; Re, 0.70; detection: H2SO4spraying and heating at 110~ for 5 minutes; B. Adsorbent: alumina; solvent: chloroform-methanol, 98:2 v/v, Rf, 0.18, detection: H2SO4 spraying and heating at 110~ for 5 minutes. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 230-231 (1981 ).

414


Common/Systematic Name Roridin A 13' [3-D-glucoside Molecular Formula/Molecular Weight C35H50014; MW = 694.32006 H

H

3

o

!.

Ho'" ~

-o '

0

I H

General Characteristics Amorphous solid; [a]D + 90 ~ (c=1.6, in CHCI3). Fungal Source Female Baccharis coridifolia. Isolation/Purification The crude extract (10% methanol-chloroform) of female B. coridifolia plant was partitioned (hexane-10% water in methanol) and the aqueous methanol, after solvent removal was chromatographed (silica gel) with ethyl ether-hexane, ethyl ether-hexane, and methanol-methylene chloride. The first methanol fraction from the chromatography of the crude extract of the plant (see verrucain A glucoside isolation) was slurried with 70% methanol-water and filtered through a pad of C-18 silica (ca. 10 g, 40pm), washed with 70% methanol-water. The filtrate was concentrated by rotary evaporation to give a gum. The gum was subjected to CCC (semi-preparative column, Vc = 3 55ml) with a solvent system of methanol-water-chloroform-hexane (6:4: 7:3, v/v/v/v) and the lower organic phase as the mobile phase to give five fractions A, B, C, D, E. Fraction E from a reversed phase filtration chromatography was subjected to C18 reversed phase semipreparative HPLC (250 x 10 mm, 63% methanol-water, 4.0mL/min) to yield roridin A 13-glucoside. Biological Activity Cytotoxic: IC90ca. lng/ml.


415

Spectral Data IR:

(CH3CI) 3406, 1732, 1718, 1637, 1600, 1181, and 1081cmq. 1H NMR:

(pyridine-ds) 7.92(1H, dd, J-- 11.5, 15.7Hz, H-8'); 6.58(1I-I, dd, J=-11.1, 11.5Hz, H-9'); 6.41(1H, dd, J=2.5, 15.7Hz, H-7'); 6.03(1H, dd, J=4.0, 8.1Hz, H-4); 5.81(1H, d, d=l 1.1Hz, H-10'); 5.49(1H, br d, J=4.3Hz, H-10); 4.96(1H, d, J=7.7Hz, H-I"); 4.66 and 4.60(1H each, AB, J=12.2Hz, H-15); 4.57(1H, rn, H-6"B); 4.55(1H, m, H-6'); 4.51(11-1, d, J=3.5Hz, H-2'); 4.40(1H, dd, J=5.4, 11.8Hz, H-6"A); 4.37(1H, dq, J=6.2, 6.2Hz, H-lY); 4.27(1H, dd, J=8.7, 8.9Hz, H-4"); 4.25(1H, dd, J=8.5, 8.9Hz, H-3"); 4.03(11-1, dd, J=7.7, 8.5Hz, H-2"); 3.97(1H, m, H-5"); 3.93(1H, d, d=4.9Hz, H-2); 3.70(1H, d, J=4.9Hz, H-11); 3.55(1H, m, H-5'B); 3.48(1H, m, H-5'A); 3.10 and 2.86(1H each, AB, d=4.1Hz, H-13); 2.51(1H, m, H-3'); 2.43(1H, dd, ,/--8.1, 15.2Hz, H-3a); 2.26(11-1, ddd, d=4.0, 4.9, 15.2Hz, H-313); 2.00(1H, m, H-4'); 1.90(2H, m, H-4', H-7B); 1.83(1H, m, H-8B); 1.77(2H, m, H-7A, H-8A); 1.53(3H, s, H-16); 1.42(3H, d, J=6.6Hz, H-12'); 1.192(3H, d, d=6.2Hz, H-14'); and 1.08ppm(3H, s, H-14). 13C NMR:

(pyfidine-ds) 79.1, C-2; 35.3, C-3; 75.3, C-4; 49.8, C-5; 44.2, C-6; 20.5, C-7; 27.8, C-8; 139.6, C-9; 119.7, C-10; 67.3, C-11; 65.8, C-12; 47.7, C-13; 7.6, C-14; 63.6, C15; 23.1, C-16; 175.0, C-I'; 76.2, C-2'; 36.8, C-3'; 34.7, C-4'; 69.5, C-5'; 80.9, C-6'; 141.6, C-7'; 126.5, C-8'; 144.3, C-9'; 117.0, C-10'; 166.9, C-11'; 14.7, C-12'; 76.6, C13'; 14.8, C-14' 104.1, C-I"; 75.2, C-2"; 78.6, C-3"; 71.6, C-4"; 78.6, C-5"; and 62.8ppm, C-6". TLC Data Adsorbent: Silica gel; solvent system: 10% methanol-methylene chloride; Re, 0.20. Reference B. B. Jarvis, S. Wang, C. Cox, M. M. Rao, V. Philip, M. S. Varaschin, and C. S. Barros; Brazilian Baccharis toxins: Livestock Poisoning and the Isolation of Macrocyclic Trichothecene Glucosides; Natural Toxins, Vol. 4, pp. 58-71(1996).

416


Common/Systematic Name 8a-Butoyloxyroridin A; YM-47525 Molecular Formula/Molecular Weight C33H46Oll; M W --" 618.30401 16

~ o . j10~H

o

H

"ol .~.~I ..o I

Io

u , . u , . ,"'. , , ,. , 'r,.~ i 5 4" n2bn2Hv MeC ,

:l~

o

/ ..,,,I,Lll ., 4'~,--0"'0

.I/ 12 General Characteristics Crystalline solid;

[a]D

--

HO" ~I ~ 4 '

+ 152.4 ~ (C=0.84, in MeOH).

Fungal Source Unidentified fungal species (likely a deuteromycete in the Agonomycetes) isolated from a deadwood sample ofAcer sp. Isolation/Purification The filtrate from a submerged culture was extracted at pH 3 (ethyl acetate) and the extract subjected to silica gel chromatography (methanol in chloroform). The active fraction was crystallized from hexane and the solid subjected to reversed phase HPLC (wateracetonitrile) to give 6.5mg of 8a-crotonyloxyroridin A and 10.6mg of 8a-butoyloxyroridin A. Biological Activity Inhibited growth of Candida albicans at 6.25mg/mL. Spectral Data UV:

~M~O.m~x 262nm (e =18,100). IR:

(CHCI3) 3450, 2970, 1720, 1640, 1600, and 1440cml.

16.


417

1HNMR: (CDC13) 0.80(3H, s, H-14); 0.93(3H, t, J=7.6Hz, H-4"); 1.10(3H, d, J=6.8Hz, H12'); 1.19(3H, d, J=6.1Hz, H-14'); 1.60(1H, tq, J=7.6 and 7.6Hz, H-3"); 1.74(2H, m, H-4'); 1.74(3H, br s, H-16); 1.99 br dq, ,/=2.7 and 6.8Hz, H-3'); 2.11(1H, br d, J=14.0Hz, H-7B); 2.14(1H, t, J=7.6Hz, H-2"); 2.21(1H, dd, ,/--5.5 and 14.0Hz, H7A); 2.22(1H, ddd, J=4.8, 8.2", and 15.5Hz, H-313); 2.47(1H, dd, J=8.2 and 15.5Hz, H-3tt); 2.81 and 3.09(1H each, AB, J=3.6Hz, H-13); 3.54(2H, t, J=5.8Hz, H-5'); 3.60(1H, br q, J=6.1Hz, H-13'); 3.64(1H, br d, J=3.4Hz, H-6'); 3.69(1H, d, J=5.5Hz, H-11); 3.84(1H, d, J=4.8Hz, H-2); 4.02(1H, dd, ,/--2.7 and 7.6Hz, H-2'); 4.36 and 4.58(1H each, AB, J=12.2Hz, H-15); 5.28(1H, br d, J=5.5Hz, H-8); 5.67(1H, br d, J=5.5Hz, H-10); 5.77(1H, d, J=l 1.6Hz, H-10'); 5.78(1H, dd, ,/--8.2 and 8.2Hz*, H-4); 5.98(1H, dd, ,/=3.4, 15.9Hz, H-7'); 6.64(1H, dd, J=l 1.6 and 11.6Hz, H-9'); and 7.63(1H, dd, J=l 1.6, 15.9Hz, H-8'). * As reported, but value should be closer to 5Hz.

13CNMR: (CDC13) 78.8, C-2; 34.8, C-3; 73.7, C-4; 49.3, C-5; 42.3, C-6; 26.2, C-7; 67.8, C-8; 136.7, C-9; 123.5, C-10; 66.6, C-11; 65.1, C-12; 47.6, C-13; 7.4, C-14; 65.5, C-15; 20.4, C-16; 174.3, C-I'; 75.5, C-2'; 37.4, C-3'; 33.5, C-4'; 70.2, C-5'; 84.0, C-6', 139.4, C-7'; 126.0, C-8'; 144.1, C-9'; 117.3, C-10'; 166.3, C-11'; 14.5, C-12'; 70.8, C-13'; 18.2, C-14'; 173.5, C-I"; 36.0, C-2"; 18.5, C-3"; and 13.6ppm, C-4". Reference T. Sugawara, A. Tanaka, K. Nagai, K. Suzuki, and G. Okada; New Members of the Trichothecene Family; J. Antibiotics, Vol. 50, pp. 778-780(1997).

418

16.

Macrocyclic

Trichothecenes

and

Related

Metabolites

Common/Systematic Name 8a-Crotonyloxyroridin A YM-47524 Molecular Formula/Molecular Weight C33H46Oll; M W -- 618.30401 lo H

H 2

3

o

. .

"C /

H.-.(~ Is

1'

Me 4

"~,",""~

,

H

~2'

"

H

i

0,,, HO ~

-

H

~

,

~'

General Characteristics Crystalline solid; [a]D -k-100.5~ (c=0.58, in MeOH). Fungal Source Unidentified fungal species (likely a deuteromycete in the Agonomycetes) isolated from a deadwood sample ofAcer sp. Isolation/Purification The filtrate from a submerged culture was extracted at pH 3 (ethyl acetate) and the extract subjected to silica gel chromatography (methanol in chloroform). The active fraction was crystallized from hexane and the solid subjected to reversed phase HPLC (wateracetonitrile) to give 6.5mg of 8t~-crotonyloxyroridin A and 10.6mg of 8ttbutoyloxyroridin A. Biological Activity Inhibited growth of Candida albicans at 6.25mg/mL. Spectral Data UV; ~b MeOH max

207(e = 18,100) and 262nm (17,300).

IR:"

(CHCI3) 3500, 2980, 1710, 1640, 1600, and 1440cm"l.


419

1HNMR: (CDCI3) 0.79(3H, s, H-14); 1.08(3H, d, J=7.3Hz, H-12'), 1.19(3H, d, J=6.1Hz, H14'); 1.7(2H, m, H-4'); 1.76(3H, br s, H-16), 1.88(3H, dd, J=6.7 and 1.3Hz, H-4"), 2.19(2H, br s, H-7), 2.23(1H, ddd, J=4.9, 4.9, and 15.SHz, H-3fl); 2.47(1H, dd, J= 7.9 and 15.SHz, H-3a); 2.82 and 3.09(1H each, AB, J=3.9Hz, H-13), 3.5(2H, m, H- 5'); 3.59(1H, br q, J=6.1Hz, H-13'); 3.63(1H, br d, J=3.0Hz, H-6'), 3.71(1H, d, J - 5.5Hz, H-11); 3.84(1H, d, J=4.9Hz, H-2); 3.91(1H, dd, J=2.4 and 7.3Hz, H-2'), 4.40 and 4.57(1H each, AB, J=12.2Hz, H-15); 5.25(1H, br s, H-8); 5.67(1H, dd, J = 1.3 and 15.3Hz, H-2"); 5.70(1H, br d, d=5.5Hz, H-10), 5.77(1H, d, J=l 1.3Hz, H - 10'), 5.78(1H, dd, J=4.9, 7.9Hz, H-4); 5.98(1H, dd, J=3.0, 15.2Hz, H-7'), 6.64(1H, dd, J=l 1.3 and 11.6Hz, H-9'), 6.94(1H, dq, J=6.7 and 15.3Hz, H-3"); and 7.62ppm (1H, dd, J-11.6, 15.2Hz, H-8'). 13CNMR: (CDC13) 78.8, C-2; 34.8, C-3; 73.7, C-4; 49.3, C-5; 42.4, C-6; 26.2, C-7; 68.0, C-8; 136.8, C-9; 123.5, C-10; 66.6, C-11; 65.0, C-12; 47.6, C-13; 7.5, C-14; 65.6, C-15; 20.5, C-16; 174.6, C-I'; 75.7, C-2'; 37.6, C-3'; 33.2, C-4'; 70.3, C-5'; 84.0, C-6'; 139.3, C-7'; 125.9, C-8'; 144.0, C-9'; 117.4, C-10'; 166.3, C-11'; 14.6, C-IT; 70.8, C-13'; 18.2, C-14'; 166.1, C-I"; 121.8, C-2"; 146.7, C-3"; and 18.1ppm, C-4". Reference T. Sugawara, A. Tanaka, K. Nagai, K. Suzuki, and G. Okada; New Members of the Trichothecene Family; J. Antibiotics, Vol. 50, pp. 778-780(1997).

420

16.


Common/Systematic Name Isororidin A Molecular Formula/Molecular Weight C29H4oO9; M W = 532.26723

10 H '~

-

H o.,j~

.l: . o ......

5

HO/~.~3'

General Characteristics Clear spherical crystals from methylene chloride-hexane; mp., 183-185~ [a]D25 +6.7 ~ (C=3.3, in CHC13). Fungal Source Myrothecium verrucaria (ATCC 24571). Isolation/Purification Partition chromatography of the mycelial extract gave a fraction rich in roridin A. This fraction was subjected to flash chromatography on silica gel eluting with 3% methanol in methylene chloride to give three principal fractions: A, B, and C, in the order of elution. Fraction A was composed of roridin D and roridin K acetate. Fraction C was composed mostly of trichothecenes of lower Rf than that of roridin A. Fraction B was crystallized from methylene chloride-hexane to give roridin A. The mother liquor was subjected to preparative HPLC (2% methanol-methylene chloride) to give verrucarin L, roridin A, and isororidin A in their order of elution. Spectral Data ~H NMR: (CDCI3) 0.83(3H, s, H-14); 1.09(3H, d, J=7Hz, H-IT); 1.16(3H, d, J=7Hz, H-14'); 1.75(3H, s, H-16); 2.48(1H, dd, J=8 and 15Hz, H-3a); 2.96(2H, AB, J=4Hz, H-13); 3.60(1H, d, J=5Hz, H-11); 3.86(1H, d, J=5Hz, H-2); 4.11(1H, dd, J=3 and 6Hz, H-2'); 4.44(2H, s br, H-15); 5.41(1H, d, J=SHz, H-10); 5.80(1H, m, H-4); 5.80(1H, d, J=l 1Hz, H-10'); 6.00(1H, dd, J=2 and 15.5Hz, H-7'); 6.66(1H, dd, J's=l 1Hz, H-9'); and 7.64ppm (1H, dd, J= 11 and 15.SHz, H-8').


421

13C NMR: (CDCI3) 7.5, q, C-14; 14.4, q, C-12'; 17.9, q, C-14'; 20.3, t, C-7; 23.3, q, C-16; 27.7, t, C-8; 33.3, t, C-4'; 34.9, t, C-3; 37.0, d, C-3', 43.8, s, C-6; 47.8, t, C-13; 49.4, s, C-5; 64.4, t, C-15; 65.3, s, C-12; 67.2, d, C-11; 69.4, t, C-5'; 70.0, d, C-13'; 74.4, d, C-4; 75.5, d, C-2'; 79.1, d, C-2, 82.6, d, C-6'; 117.3, d, C-10'; 118.3, d, C-10; 126.4, d, C8', 139.0, d, C-7'; 140.9, s, C-9; 143.9, d, C-9'; 166.6, s, C-11', and 174.9ppm, s, C-I' References B. B. Jarvis, Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. W. Jarvis, J. O. Midiwo, J. L. Flippen-Anderson, and E. P. Mazzola; Stereochemistry of the Roridins; J. Nat. Prod., Vol. 45, pp. 440-448(1982).

422


Common/Systematic Nam.e Roridin D Molecular Formula/Molecular Weight C29H3809, ~

l ~ ~ o ,~o ,j

= 530.25158

H

H2(~ 15

I ,, o..~.o

H

~ 4"~O~

.7

II

H 117

-"

........

o1 lz

1r / | ~OH H

General Characteristics Needles from acetone-ether; mp., 232-235~ [a]D 23 +29~ = 2.71, in CHC13). Fungal Source

Myrothecium verrucaria and M. roridum.

Biological Activit_y Antibiotic activity. Spectral Data UV~

Z EmtO~ 260(~=21,400). IR~

(KBr) 3540-3440,2970, 1747, 1711, 1641, 1601, 1420, 1190, 1105, 1083, 968, 818, 753, and 660cm1. 13C NMR:

(CDC13) 78.8, C-2; 34.9, C-3; 74.3, C-4; 49.0, C-5; 43.1, C-6; 20.4, C-7; 27.4, C-8; 140.1, C-9; 118.4, C-10; 66.9, C-11; 65.1, C-12; 47.4, C-13; 6.8, C-14; 64.3, C-15; 22.9, C-16; 167.8, C-I'; 57.9, C-2'; 62.9, C-3'; 39.4, C-4'; 67.3, C-5'; 85.3, C-6'; 138.1, C-7'; 126.2, C-8'; 142.9, C-9'; 117.8, C-10'; 166.1, C-11', 17.2, C-12'; 70.5, C-13'; and 17.9ppm, C- 14'.

16.


423

TLC Data Absorbent: Kieselgel G; solvent system, ethyl ether (two consecutive runs); Rf, 0.18; Detection, iodine vapors. References B, Bohner, E. Fetz, E. Harri, H. P. Sigg, and C. Tamm; Helv. Claim. Acta., Vol. 48, pp.

1079(1965).

R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 237-238(1981). B. B. Jarvis, Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991).

424

16.

MacrocyclicTrichothecenes


Common/Systematic Name Roridin D 13-D-glucoside Molecular Formula/Molecular Weight C35H48014; M W -- 692

~6

~o H

---

0

H

2

o ,o 2

I.' /

~.'0 H

l]'glucose

General Characteristics Amorphous solid; [0~]D q- 27* (c=0.80, in CHC13). Fungal Source Female Baccharis coridifolia.. Isolation~urification The first methanol fraction from the chromatography of the crude extract of the plant (see verrucain A glucoside isolation) was slurried with 70% methanol-water and filtered through a pa~ of C-18 silica (ca. 10 g, 40 ~tm), washed with 70% methanol-water. The filtrate was concentratec by rotary evaporation to give a gum. The gum was subjected to CCC (semi-preparative column, Vc = 355 ml) with a solvent system of methanol-water-chloroform-hexane (6:4:7:3 v/v/v/v) and the lower organic phase as the mobile phase to give five fractions A, B, C, D, E. Fraction C was subjected to PTLC (Chromatotron, silica gel, 2 mm) with 3-10% methanol-methylene chloride tt give three portions CI, CII and CIII. Roridin D glucoside was isolated by semipreparative HPLC (silica gel, 250 xl 0 mm, 6% methanol-methylene chloride, 4.0 mL/min) from all three of these fractions. Biological Activity Cytotoxic: ICs5 ca. 1 ng/ml.

Spectral Data IR: (CH2C12) 3418, 1750, 1718, 1650, 1600, 1181, and 1081cm"l.

16.


425

1H NMR: (pyridine-d5) 7.63 (1H, dd, d= 11.9, 15.4Hz, H-8'); 6.50 (1H, dd, J= 11.2, 11.9Hz, H-9'); 6.38 (1H, dd, J= 2.3, 15.4Hz, H-7'); 6.00 (1H, dd, J= 3.9, 7.9Hz, H-4); 5.79 (1H, d , J = 11.2Hz, H-10'); 5.49 (1H, br d, J= 4.1Hz, H-10); 4.99 (1H, d , J = 7.THz, H-I"); 4.71 and 4.40 (1H each, AB, J = 12.4Hz, H-15); 4.58 (1H, m, H-6"B); 4.56 (1H, m, H-6'); 4.37 (1H, m, H-6"A); 4.35 (1H, dq, J= 6.2, 6.2Hz, H-13'); 4.26 (1H, dd, J= 8.0, 7.7Hz, H-4"); 4.24 (1H, dd, J= 8.6, 7.7Hz, H-3"); 4.04 (1H, dd, J= 7.7, 8.6Hz, H-2"); 4.00 (1H, m, H-5"); 3.95 (1H, d, J= 4.9Hz, H-2); 3.73 (1H, d , J = 4.THz, H-11); 3.82 (1H, dd, d= 10.0, 10.3Hz H-5'B); 3.73 (1H, d, J= 4.7Hz, H-11); 3.62 (1H, s, H-2'); 3.26 (1H, m, H-5'A); 3.12 and 2.89 (1H each, AB, J= 4.0Hz, H13); 2.47 (1H, dd, J= 8.3, 15.3Hz, H-3ot); 2.42 (1H, dd, J= 10.0, 15.0Hz, H-4'B); 2.26 (1H, ddd, J = 3.9, 4.9, 15.3Hz, H-313); 1.88-1.95 (4H, m, H-7, H-8); 1.90 (3H, s, H-12'); 1.57 (3H, s, H-16); 1.37 (1H, dd, J= 3.9, 15.0Hz, H4'A); 1.20 (3H, d, d= 6.2Hz, H-14'); and 1.07ppm (3H, s, H-14). 13C NMR: (CDC13) 79.1, C-2; 35.3, C-3; 75.3, C-4; 49.8, C-5; 43.7, C-6; 20.6, C-7; 27.7, C-8; 139.5, C-9; 119.8, C-10; 67.2, C-11; 65.8, C-12; 47.7, C-13; 7.4, C-14; 64.7, C-15; 23.1, C-16; 168.7, C-I'; 58.6, C-2'; 63.8, C-3'; 40.6, C-4'; 68.4, C-5'; 82.6, C-6'; 141.4, C-7'; 125.9, C-8'; 143.9, C-9'; 117.2, C-10'; 169.9, C-11'; 17.6, C-12'; 77.4, C-13'; 17.9ppm, C- 14'. TLC Data Adsorbent: Silica gel; solvent system: 10% methanol-methylene chloride; Rf, 0.26; detection with iodine vapors. Reference B. B. Jarvis, S. Wang, C. Cox, M. M. Rao, V. Philip, M. S. Varaschin, and C. S. Barros; Brazilian Baccharis toxins: Livestock Poisoning and the Isolation of Macrocyclic Trichothecene Glucosides; Natural Toxins, Vol. 4, pp. 58-71 (1996).

426

16.

MaerocyclicTrichothecenes and Related Metabolites

Common/Systematic Name Roridin E Molecular Formula/Molecular Weight C29H3808, M ~ r = 5 1 4 . 2 5 6 6 7

10 H

H

o

'

12'

1; /

1 "OH H

General Characteristics Colored needles from ether-pentane; mp., 177-1780C or 211-212~ needles from ether, 220-221~ [tt]D23 -27 ~ (C=1.3, in CHCI3); [tt]D25 -21" (C=1.852, in CHCI3). Fungal Source Myrothecium verrucaria and M. roridum.

Biological Activity Antibiotic activity. Spectral Data UV:

~, ~~

195(e=15,800), 223(25,100), and 263nm (19,900).

IR:

(CH2C12) 3570, 3050, 2975, 1712, 1647, 1603, 1365, 1220, 1180, 1148, 1142, 1096, 1090, 1080, 966, and 814cm4. 1H NMR: (CDCI3) 3.82, (1H, d , J = 5.0Hz, H-2); 2.04, (1H, ddd, J= 4.2, 5.0, 15.5Hz, H-313; 2.53, (1H, dd, J= 8.2, 15.0Hz, H-3a); 6.20, (1H, dd, J= 4.0, 8.2Hz, H-4); 5.47 (1H, br d, Jr= 5.0Hz, H-10); 3.89 (1H, d, J=- 5.0Hz, H-11); 2.81 and 3.12(1H each, AB, J= 4.1Hz, H-13); 0.79 (3H, s, H-14); 3.93 and 4.32 (1H each, AB, J= 12.5Hz, H-15); 1.71 (3H, br s, H-16); 5.95(1H, q, d=- 1.5Hz, H-2'), 3.70 (1H, m, H-6'); 5.89 (1H, dd, or-- 2.0, 16.0Hz, H-7'); 7.51(1H, dd, Jr= 11.2, 16.0Hz, H-8'); 6.56 (1H, dd, J= 11.0,

16.


427

11.2Hz, H-9'); 5.73 (1H, d, J= 11.0Hz, H-10'); 2.25 (3H, d, Jr= 1.5Hz, H-12'); 3.70 (1H, m, H-13'; and 1.19ppm (3H, d, Jr= 6.0Hz, H-14'). 13CNMR: (CDCla) 79.3, C-2; 35.8, C-3; 74.2, C-4; 48.4, C-5; 42.8, C-6; 21.6, C-7; 27.7, C-8; 140.0, C-9; 117.8, C-10; 67.2, C-11; 65.6, C-12; 48.1, C-13; 6.7, C-14; 63.7, C-15; 23.2, C-16; 165.8, C-I'; 119.0, C-2'; 159.0, C-3'; 41.3, C-4'; 69.8, C-5'; 83.8, C-6'; 138.1, C-7'; 126.6, C-8'; 143.7, C-9'; 117.2, C-10'; 166.4, C-11'; 20.2, C-12'; 70.5, C13'; 18.3, C-14'. TLC Data Adsorbent: Kieselgel G; solvent system: ethyl ether (two consecutive runs); Re, 0.24; detection with iodine vapors. References B. Bohner, E. Fetz, E. Harri, H. P. Sigg, and C. Tamm; Helv. Chim. Acta.; Vol. 48, pp. 1079(1965). R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 237-238(1981). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). M. Matsumoto; Structures oflsororidin E, Epoxyisororidin E, Epoxyroridin H, and Diepoxyroridin H, New Metabolites Isolated from a Species of Cylindrocarpon; J. Sci. Hinoshima; University serial A; Vol. 43, pp. 107-118 (1979).

428

16.


Common/Systematic Name Roridin E [3-D-glucoside Molecular Formula/Molecular Weight C35I-I48013;MW = 676.30949 H

18

H

01 13~.;,,,io

I

o O ~ ~ ~ 9'

'3

~'

O

[7'

glucose General Characteristics Amorphous solid; [a]D+ 37 ~ (C=2.0, in CHC13). Fungal Source Female Baccharis coridifolia. Isolation/Purification The first methanol fraction from the chromatography of the crude extract of the plant (see verrucain A glucoside isolation) was slurried with 70% methanol-water and filtered through a pad Of Cls silica (ca. 10 g, 40 lam), washed with 70% methanol-water. The filtrate was concentrated by rotary evaporation to give a gum. The gum was subjected to CCC (semi-preparative column, Vc = 355 ml) with a solvent system of methanol-waterchloroform-hexane (6:4:7:3 v/v/v/v) and the lower organic phase as the mobile phase to give five fractions A, B, C, D, E. Fraction B was subjected to Cls reversed phase HPLC (250 x 10 mm, 68% methanol-water, 4.0 mL/min) to yield roridin E 13-glucoside. Biological Activity Cytotoxic: ICs5 ca. 1 ng/ml. Spectral Data IR:

(CH2CI2) 3412, 1706, 1643, 1600, 1181, and 1081cm~.

16.


429

1H NMR: (CDCI3) 7.39(1H, dd, ,/--12.0, 15.0Hz, H-8'); 6.59(1H, dd, J=l 1.2, 12.0Hz, H-9'); 6.08(1H, dd, J=3.9, 7.6Hz, H-4); 5.98(1H, dd, J=2.7, 15.0Hz, H-7'); 5.86(1H, s, H-2'); 5.69(1H, d, J=l 1.2 Hz, H-10'); 5.44(1H, d, J=4.4Hz, H-10); 4.38(1H, d, J=7.4Hz, HI"); 4.31 and 3.90(1H each, AB, J=12.4Hz, H-15); 4.10(1H, m, H-6'); 3.98(1H, dq, d =5.5, 6.0Hz, H-13'); 3.80-3.83(3H, m, n-6", n-11); 3.80(1H, d, J=5.2Hz, H-2); 3.70(1H, m, H-5'B); 3.56(1H, dd, ,/--8.6, 9.4Hz, H-4"); 3.51(1H, dd,J=8.0, 9.4Hz, n3"); 3.48(1H, m, H-5'A); 3.33(1H, dd, ,/--7.4, 8.0Hz, n-2"); 3.28(1H, bd, J=8.6Hz, n5"); 3.09 and 2.78(1H each, AB, J=3.7Hz, H-13); 2.48(1H, dd, ,/--7.6, 15.0Hz, H-3'); 2.43(2H, m, n-4'); 2.21(3H, s, U-12'); 2.04(1H, ddd, ,/--4.0, 5.0, 15.0Hz, H-3B); 1.98(4H, m, n-7, n-8); 1.68(3H, s, n-16); 1.08(3H, d, J=6.0Hz, n-14'); and 0.76ppm(3H, s, H-14). (pyridine-d5) 7.71(1H, dd, J=l 1.8, 15.5Hz, H-8'); 6.49(1H, dd, J=l 1.1, 11.8Hz, H-9'); 6.34(1H, dd, ,/=2.8, 15.5Hz, H-7');6.23(1H, dd, ,1=4.0, 8.0Hz, H-4); 6.02(1H, s, U-2'); 5.74(1H, d, J=l 1.1Hz, H-10'); 5.5 I(1H, br d, `/=5.0Hz, H-10); 4.96(1H, d, J=7.8Hz, H-I"); 4.75 and 4.09(1H each, AB, J=12.5Hz, H-15); 4.57(1H, dd, J=2.2, 11.8Hz, H-6"B); 4.40(1H, dq, J=6.3, 6.3Hz, H-13'); 4.37(1H, m, H-6"A); 4.26(2H, m, H-3", H-4"); 4.03(1H, dd, ,/--7.8, 8.0Hz, H-2"); 3.99(1H, m, H5"); 3.92(1H, d, J=5.0Hz, H-2); 3.83(1H, br d, J=5.0Hz, H-11); 3.48(1H, m, H-5'B); 3.38(1H, m, H-5'A); 3.10 and 2.85(1H each, AB, J=4.0Hz, H-13); 2.47(1H, dd, J=8.0, 15.0Hz, H-3a); 2.41(3H, s, H-12'); 2.31(2H, m, H-4'); 2.19(1H, ddd, J=4.0, 5.0, 15.0Hz, H-313); 1.92-1.99(3H, m, H-7B, H-8); 1.79(1H, br d, J=7.5Hz, H-7A); 1.56(3H, s, H-16); 1.12(3H, d,J=6.3Hz, H-14'); and 1.05ppm (3h, s, H-14). 13CNMR: (CDC13) 79.2, C-2; 35.6, C-3; 74.3, C-4; 48.5, C-5; 42.8, C-6; 21.3, C-7; 27.6, C-8; 140.2, C-9; 117.2, C-10; 67.2, C-11; 65.6, C-12; 48.1, C-13; 6.8, C-14; 63.6, C-15; 23.3, C-16; 166.5, C-I'; 118.8, C-2'; 159.0, C-3'; 41.4, C-4'; 69.0, C-5'; 80.4, C-6'; 138.6, C-7'; 126.9, C-8'; 143.9, C-9'; 117.2, C-10'; 166.2, C-11'; 19.5, C-12'; 76.7, C13'; 15.0, C-14' 101.8, C-I"; 73.2, C-2"; 76.2, C-3'; 69.7, C-4"; 75.5, C-5"; and 61.7ppm, C-6". TLC Data Adsorbent: Silica gel; solvent system: 10% methanol-methylene chloride; Rf, 0.26; detection with iodine vapors. Reference B. B. Jarvis, S. Wang, C. Cox, M. M. Rao, V. Philip, M. S. Varaschin, and C. S. Barros; Brazilian Baccharis toxins: Livestock Poisoning and the Isolation of Macrocyclic Trichothecene Glucosides; Natural Toxins, Vol. 4, pp. 58-71(1996).

430


Common/Systematic Name Isororidin E Molecular Formula/Molecular Weight C29H3808; M W = 5 1 4 . 2 5 6 6 7

10 H

H

HO"

1 ~ 14'

H

General Characteristics Colorless prisms from ethyl acetate; mp., 200-202~

[tt]D - 65.1 o (in CHC13).

Fungal Source

Cylindrocarpon spp.

Spectral Data UV~

~

EtOH max

223(e=24,000) and 262nm (16,000).

IR~

(CHaCI) 3757, 1713, 1644, and 1598cm"l. ~H NMR: 3.84(H-2); 2.03(H-3a); 2.58(H-3b); 6.35(H-4); 5.50(H-10); 4.09(H-11); 2.83(H-13a); 3.15(H-13b); 0.80(H-14); 4.06(H-15a); 4.16(H-15b); 1.71(H-16); 5.83(H-2'); 3.74(H6'); 5.71(H-7'); 7.55(H-8'); 6.60(H-9'); 5.82(H-10'); 2.22(H-12'); 3.70(H-13'); and 1.17ppm (H- 14'). 13CNMR: (CD3C1) 79.2, C-2; 36.6, C-3; 75.3, C-4; 48.5, C-5; 42.6, C-6; 22.7, C-7; 27.7, C-8; 140.1, C-9; 118.9, C-10; 66.7, C-11; 65.7, C-12; 47.7, C-13; 6.4, C-14; 64.5, C-15; 23.2, C-16; 166.3, C-I'; 119.5, C-2'; 158.0, C-3'; 40.0, C-4'; 67.0, C-5'; 83.2, C-6'; 135.3, C-7'; 131.0, C-8'; 142.0, C-9'; 117.1, C-10'; 166.3, C-11'; 19.8, C-12'; 67.6, C-13'; and 18.5ppm, C-14'.


431

References R. J. Cole and R. H. Cox, Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 242 (1981). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds., CRC Press, Boca Raton, Florida, pp. 361-421 (1991). M. Matsumoto, H. Minato, K. Tori, and M.Ueyama, Structures oflsororidin E, Epoxyisororidin E, and Epoxy- and Diepoxyroridin H, New Metabolites Isolated from Cylindrocarpon Species Determined by Carbon-13 and Hydrogen-1 NMR Spectroscopy. Revision of C-2':C-3' Double Bond Configuration of the Roridin Group; Tet. Lett.; pp. 4093-4096(1977).

432

16.


Common/Systematic Name Epiroridin E Molecular Formula/Molecular Weight C29H3808; M W = 5 1 4 . 2 5 6 6 7

10 H

H

~,-r162 "OH H General Characteristics Amorphous solid; [~]D 30-~- q- 0.70 ~ (c--1.8, in chloroform). Fungal Source Myrothecium verrucaria (ATCC 24571 ). Isolation/Purification A chromatography fraction from an earlier fermentation ofM. verrucaria ATCC 24571 [Jarvis et al.; J. Org. Chem.; Vol. 47,pp. 1117-1124 (1982)] was subjected to CCC (Vc = 850ml) with a solvent system of carbon tetrachloride-methanol-water (5:3:2, v/v/v), the lower organic phase was the mobile phase and the flow rate was 3.3ml/min, to give verrucarin A (23mg), roridin D (1 lmg), a fraction rich in roridin E (400mg), and roridin A (67mg). The roridin E fraction was subjected to CCC (Vc 355mL) with a solvent system of carbon tetrachloride-hexane-methanol-water (4:1:3:2, v/v/v/v), the lower organic phase was the mobile phase, and the flow rate was 1.8ml/min, to give 310mg of a mixture of roridin E and isororidin E and a later fraction of pure epiroridin E (50mg). Spectral Data IR:

(CHCI3) 2491,1713, 1652, and 1601cm"1. 1H NMR: (CDCI3) 0.76(3H, s, H-14); 1.13(3H, d, J=6.0Hz, H-14'); 1.67(3H, s, H-16); 1.92-2.20(5H, m, H-7, H-8, H-313); 2.24(3; H, s, H-12'); 2.42-2.54(3H, m, H-4', H3tt); 2.83(1H, d, J=4.0Hz, H-13B); 3.15(1H, d, J=4.0Hz, H-13A); 3.50(1H, dt,

16.


433

J=l 5.0, 6.0Hz, H-5B); 3.69(1H, dt, J=15.0, 6.0Hz, H-5A); 3.87(1H, d, J=5.0Hz, H-2); 3.79-3.90(2H, m, H-6', H-13'), 3.89(1H, d, J=12.0Hz, H-15B); 3.91(1H, d, J=5.0Hz, H-11); 4.31(1H, d, J=12.0Hz, H-15A); 5.44(1H, d, J=5.0Hz, H-10); 5.70(1H, d, J=l 1.0Hz, H-10'); 5.86(1H, dd, J=3.0, 15.0Hz, H-7'); 5.98(1H, s, H-2'); 6.15(1H, dd, J=4.0, 8.0Hz, H-4); 6.54(1H, dd, J=l 1.0Hz, H-9'); and 7.45ppm (1H, dd, J=l 1.0, 15.0Hz, H-8'). 13CNMR: (CDC13) 6.7, C-14; 17.8, C-14'; 19.8, C-12'; 21.5, C-7; 23.2, C-16; 27.2, C-8; 35.7, C-3; 41.4, C-4'; 42.7, C-6; 48.1, C-13; 48.4, C-5; 63.6, C-15; 65.6, C-12; 67.2, C-11; 69.1, C-13'; 69.5, C-5'; 74.2, C-4; 79.2, C-2; 82.5, C-6'; 117.2, C-10'; 117.6, C-10; 118.9, C-2'; 126.8, C-8'; 137.8, C-7'; 140.8, C-9; 143.6, C-9'; 159.0, C-3'; 165.9, C-I'; and 166.4ppm, C- 11' Reference B. B. Jarvis and S. Wang; Stereochemistry of the Roridins. Diastereomers of Roridin E; J. Nat. Prod., Vol. 62, pp. 1284-1289(1999).

434

16.


Common/Systematic Name Epiisororidin E Molecular Formula/Molecular Weight C29H3808; ~

= 514.25667

lo H

16

H

2

3

, , ' / 1 "OH H General Characteristics Amorphous solid;

[ a ] D 30 --

-29.2 ~ (c=1.5, in CHCI3).

Fungal Source Myrothecium verrucaria (ATCC 24571).

Isolation/Purification Extraction of a submerged culture ofM. verrucaria [see Jarvis, B. B., Armstrong, C. A., Zeng, M. J.; Antibiotics; Vol. 43, pp. 1502-1504 (1990)] with organic solvent gave 7.5g of crude extract atter solvent removal. A series of chromatographies (silica gel and CCC) gave 353mg of a mixture of roridin E and isororidin E, 17mg of mixture of roridin E, isororidin E and epiisororidin E and 20mg of pure epiisororidin E. The roridin E diastereomers can be separated by RP-HPLC on a phenyl column (Phenomenex, 4.6 x 250mm) with 40% acetonitrile in water at a flow rate of 1.2ml/min. The observed retention times under these conditions were: roridin E (27.7 min), isororidin E (23.3 min), epiroridin E (26.1 min), and epiisororidin E (22.1 min). Spectral Data IR:

(CHC13) 3486,1713, 1647, and 1599cm q. 1H NM:R: (CDC13) 0.77(3H, s, H-14); 1.12(3H, d, J-6.5Hz, H-14'); 1.68(3H, s, H-16); 1.962.13(5H, m, H-313, H-7, H-8); 2.21(3H, d, J=l.2Hz, H-12'); 2.29(1H, m, H-4'B); 2.502.56(2H, m, H-3~, H-4'A); 2.81(1H, d, J=4.0Hz, H-13B); 3.13(1H, d, J-4.0Hz, H-

16.


435

13A); 3.56(1H, ddd, J=5.0, 7.4, 10.1 Hz, H-5'B); 3.73(1H, ddd, J=7.4, 7.5, 10.1Hz, H-5'A); 3.83(1H, d, J=5.0Hz, H-2); 3.89(1H, m, H-6'); 3.98(1H, dq, J=3.0, 6.5Hz, H13'); 4.01(1n, d, J=12.5Hz, H-15B); 4.03(1H, d, J=5.5Hz, n-11); 4.17(1H, d, J=12.5Hz, H-15A); 5.47(1H, d,J = 5.5Hz, H-10); 5.80(1H, d,J=l 1.0Hz, H-10'); 5.82(1H, d, J=l.2Hz, H-2'); 5.87(1H, dd, J=6.1, 15.6Hz, H-7'); 6.29(1H, dd, J-4.1, 8.0Hz, H-4); 6.60(1H, dd, J=l 1.0, 11.0Hz, H-9'); and 7.54ppm (1H, dd, J = 11.0, 15.6Hz, H-8'). 13CNMR: 6.5, C-14; 17.9, C-14'; 19.2, C-12'; 22.3, C-7; 23.2, C-16; 27.7, C-8; 36.4, C-3; 40.3, C-4'; 42.6, C-6; 47.8, C-5; 48.4, C-13; 64.3, C-15; 66.3, C-5'; 66.6, C-12; 67.0, C-11; 68.5, C-13'; 75.0, C-4; 79.2, C-2; 81.9, C-6'; 117.2, C-10'; 117.6, C-10; 119.0, C-2'; 131.0, C-8'; 134.7, C-7'; 140.2, C-9; 142.3, C-9'; 157.9, C-3'; 166.3, C-I'; and 166.5ppm, C-11'. Reference B. B. Jarvis and S. Wang; Stereochemistry of the Roridins. Diastereomers of Roridin E; J. Nat. Prod., Vol. 62, pp. 1284-1289(1999).

436


Common/Systematic Name 713,813-Epoxyisororidin E Molecular Formula/Molecular Weight C29H3609; MW

-- 5 2 8 . 2 3 5 9 3

~oH

H

~~

0 H271~!~ -" .,0,~11i 0=:~--~' , / 01

3

H General Characteristics Colorless prisms from acetone; mp., 216-219~

[ a ] D 24

-

69.9 ~ (c= 0.438 in CHC13).

Fungal Source

Cylindrocarpon spp.

Spectral Data UV: ~, E~

221 (C=26,300) and 262nm (16,700).

IR: (CH3C1) 3567, 1712, 1644, and 1598cm1. 1H NMR: (CDCI3) 3.90(1H, d , J - 4.7Hz, H-2), 2.06(1H, ddd, J-- 4,3, 4.7, 15.0Hz, H-3~), 2.55(1H, dd, J = 8.1, 15.0Hz, H-3tx), 6.30(1H, dd, J= 4.3, 8.1Hz, H-4); 3.34(1H, dd, Jr-- 3.2, 4.0Hz, H-7); 3.17(1H, dd, J-2.1, 4.0Hz, H-8); 5.75(1H, ddd, J = 1.5,2.1, 7.0Hz, H-10); 4.21(1H, dd, J= 3.2, 7.0Hz, H-11); 2.99 and 3.21(1H each, AB, J 3.4Hz, H-13); 1.02(3H, s, H-14), 3.79 and 4.07(1H each, AB, J = 12.5Hz, H-15), 2.00(3H, d,J = 1.5Hz, H-16); 5.80(1H, q , J - 1.0Hz, H-2'), 3.75(1H, m, H-6'); 5.77(1H, dd, J - 5.0, 15.9Hz, H-7'), 7.54(1H, dd, J = 11.0, 15.9Hz, H-8'); 6.62(1H, dd, J= 11.0, 11.1Hz, H-9'); 5.83(1H, d, Jr-- 11.1Hz, H-10'); 2.24(3H, d, Jr- 1.0Hz, H-12'); 3.70(1H, m, H-13'); and 1.16ppm (3H, d, J= 6.0Hz, H-14').


437

13C NMR: (CDCI3) 79.2, C-2; 36.6, C-3; 74.6, C-4; 47.0, C-5; 44.0, C-6; 50.8, C-7; 56.1, C-8; 138.2, C-9; 123.0, C-10; 66.7, C-11; 65.7, C-12; 48.3, C-13; 7.2, C-14; 62.9, C-15; 21.9, C-16; 165.9, C-I'; 119.1, C-2'; 159.0, C-3'; 40.1, C-4'; 67.0, C-5'; 83.3, C-6'; 135.5, C-7'; 130.5, C-8'; 142.4, C-9'; 116.7, C-10'; 166.2, C-11'; 20.0, C-12'; 69.7, C-13', and 18.5ppm, C-14'. References R. J. Cole and R. H. Cox; Handbook of Toxic Fun_galMetabolites; Academic Press, New York, pp. 243(1981). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). M. Matsumoto, H. Minato, K. Toil,and M. Ueyama; Structures oflsororidin E, Epoxyisororidin E, and Epoxy- and Diepoxyroridin H, New Metabolites Isolated from Cylindrocarpon Species Determined by Carbon-13 and Hydrogen-1 NMR Spectroscopy. Revision of C-2': C-3' Double Bond Configuration of the Roridin Group, Tet. Lett.; pp. 4093-4096(1977).

438


Common/Systematic Name Roridin K acetate Molecular Formula/Molecular Weight

C31H40010;M W ~o H

-- 5 7 2 . 2 6 2 1 5

H

,

, -

,.

13' 14'~07'H H General Characteristics Crystals from dichloromethane-hexane, rap., 255-257~

[a]D2~ + 2.1 ~ (C= 5.6 in CHel3).

Isolation/Purification Spores ofMyrothecium verrucaria grown on N-Z amine agar were added to Czapek-Dox media and allowed to grow at 28~ in a shake culture. After 2 days, the solution was divided into three equal portions and added to three separate Fernback flasks containing 1L of production media. After 3 days of growth, the mycelium in each flask was separately centrifuged, washed, and resuspended in 1L of sterile water. Trichoverrin A and trichoverrin B were added separately to flasks one and two; flask three was used as the control. After 7 days, the mycelium was removed and extracted with ethyl acetate. The mycelium extract was subjected to partition chromatography on 500g Celite impregnated with 250ml of 18% water in methanol. The column was eluted with petroleum ether followed by increasing amounts of dichloromethane in petroleum ether up to 40% dichloromethane in petroleum ether. Fractions were combined on the basis of TLC analysis to give a total of eight fractions: A, B, C, D, E, F, G, and H (methanol wash). Fraction F was subjected to MPLC (30-60% ethyl acetate in hexane) to yield a fraction composed principally of a mixture of roridin E and isororidin E in a ratio of ca. 1:4, v/v. The following fraction was subjected to HPLC (20% ethyl acetate in hexane) to give roridin D and roridin K acetate. Fungal Source


Spectral Data UV:

max

~. E~.

263nm (log e= 4.21).

16.


439

~H N M R : (CDCI3) 0.78(3H, s, 14-H); 1.19(3H, d , J = 6.0Hz, 14'-H); 1.76(3H, s, 16-H); 1.19(acetate); 2.30(3H, d, J=l.2Hz, 12'-H); 2.52(1H, dd, J=7, 15Hz, 3tt-H); 2.97(2H, d=4Hz, 13-H); 3.84(11-1, d, J=5Hz, 2-H); 3.90(1H, d, J=5Hz, 1 l-H), 4.31(2H AB, d=12Hz, 15-H); 5.75(1H, d, J=l 1Hz, 10'-H), 5.78(1H, d, d=16Hz, 7'-H); 6.10(1H, dd, J=4, 8Hz, 4-H); 6.57(1H, dd, Js=llHz, 9'-H); and 7.47ppm (1H, dd, J=ll, 16Hz, 8'H). 13C NMR: (CDCIa) 79.1 d, C-2; 35.5 t, C-3; 73.8 d, C-4; 48.6 s, C-5; 42.2 s, C-6; 27.4 t, C-7; 68.8 d, C-8; 136.4 s, C-9; 124.0 d, C-10; 67.0 d, C-11; 65.4 s; C-12; 48.0 t, C-13; 6.8 q, C-14; 64.7 t, C-15; 21.1 q, C-16; 165.9 s, C-I'; 117.6 d, C-2'; 159.7 s, C-3'; 41.2 t, C-4'; 70.3 t, C-5'; 84.1 d, C-6'; 138.5 d, C-7', 126.5 d, C-8'; 143.0 d, C-9'; 116.7 d, C10'; 166.2 s, C-11'; 20.2 q, C-12'; 70.7 d, C-13'; 18.3 q, C-14'; 170.8 s, COCH3; and 20.7ppm q, COCH3. Mass Spectrum: HRCIMS (methane gas reagent): 573.2692m/e(M + + H, calcd 573.2700). Reference B. B. Jal~S, G. P. Stahly, G. Pavanasasivam, J. O. Midiwo, T. Desilva, C. E. Holmlund, E.P. Mazzola and R. F. Geoghegan; Isolation and Characterization of the Trichoverroids and New Roridins and Verrucarins; J. Org. Chem., Vol. 47, pp. 1117-1124(1982).

440


Common/Systematic Name Roridin H; Verrucarin H Molecular Formula/Molecular Weight C29H3608, M W -- 5 1 2 . 2 4 1 0 2 ~o H

H

,,,o

,

General Characteristics Crystals from acetone-ether, mp >325~ (dec.), [~]D 23 +31 ~ (c=1.16, in CHC13), [{~]D23 + 40.3 ~ (c=1.065, in benzene); [~]Dz3 +31.5 ~ (C=0.855, in dioxane). Fungal Source

Myrothecium verrucaria.

Biological Activity Antibiotic activity. Spectral Data UV:

195(e=15,800), 224 (24,500), and 260nm (18,200). IR;

(CH2CH2) 2970,2910, 1710, 1645, 1600, 1380, 1355, 1220, 1175, 1115, 1101, 1087, 1071, 1033, 1005, 992, 971, and 832cm 1. 1H NMR: (CDC13) 3.80(1H, d, H-2); 5.90(H-4); 5.42(1H, d, J=4.0Hz, H-10); 3.64(H-11); 2.96(2H, AB, J=4.0Hz, H-13); 0.85(H-14); 4.15(2H, AB, J=12.0Hz, H-15); 1.69(H16); 5.67(H-2'); 2.74(1H, m, H-4'); 5.58(1H, J= 3.5, 8.0Hz, H-5'); 4.03(1H, m, H-6'); 5.90(1H, m, H-7'); 7.68(1H, dd, J=l 1.0, 15.5Hz, H-8'); 6.55(1H, t,J=l 1.0Hz, H-9'); 5.79(1H, d, J=l 1.0Hz, H-10'); 2.27(3H, d, J=l.5Hz, H-12'); 3.65(1H, m, H-13'); and 1.32ppm(3H, d, J-6.0Hz, H-14').


441

13CNMR: (CDC13) 79.0,C-2; 34.8,C-3; 74.0, C-4; 48.9,C-5, 43.2, C-6; 20.5, C-7; 27.6, C-8; 139.9, C-9; 118.6, C-10; 67.6, C-11; 65.3, C-12; 47.3, C-13; 7.0, C-14; 63.0, C-15; 22.9, C-16; 166.0, C-I'; 119.0, C-2'; 154.4, C-3'; 47.7, C-4'; 100.8, C-5'; 81.9, C-6'; 134.6, C-7'; 126.2, C-8'; 142.5, C-9'; 118.9, C-10'; 166.0, C-11'; 18.2, C-12'; 76.8, C13'; and 16.3ppm, C-14'. TLC Data Absorbent: Kieselgel G; solvent, ethyl ether (two consecutive runs); Rf, 0.51; detection: iodine vapors. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 239-240(1981). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). M. Matsumoto; Structures of Isororidin E, Epoxyisororidin E, Epoxyroridin H, and Diepoxyroridin H, New Metabolites Isolated from a Species of Cylindrocarpon; J. Sci. Hinoshima, University serial A, Vol. 43, pp. 107-118 (1979).

442

16.


Common/Systematic Name Roridin J Molecular Formula/Molecular Weight C29H3609; M W -- 5 2 8 . 2 3 5 9 3

~o H

H

Ii

i,o H2C I s

O ~ OH

1

o

L

U,,,

4~ 0 ~ ~ .

J

12

General Characteristics Crystals from dichloromethane-hexane; mp., 281-285~ [a]D2s + 21.8 ~ (in CHCla). Acetate derivative, crystals from diehloromethane-ether; mp., 230-235~ [a]D2s -40.6 ~ (in CHC13). Fungal Source


Isolation/Purification The metabolite was isolated by a combination of column chromatographies (adsorption and partition) and recrystallizations from an ethyl acetate extract of the fermentation broth. Roridin J, is closely related to roridin H, but can be separated by careful chromatography using alumina with hexane-methylene chloride as eluant. B.i..ological Activity Exhibited substantial in vivo activity against P388 mouse leukemia (PS). It was toxic at 10mg/kg and exhibited the following T/C activities (dose level) in PS: 158 (Smg/kg), 149 (2.5mg/kg), 140 (1.25mg/kg), and 125 (0.62mg/kg). Spectral Data UV"

~.~H

max

26 lnm (log 6=4.28).

16.

MacrocyelicTrichothecenes and Related Metabolites

443

IR:

(KBr) 3535(OH), 1715(C=O), 1645, and 1595cm1 (diene); acetate, 1745 and 1715(C=O's), 1655, 1605cm"1 (diene). 1H ~ : (CDCI3) 3.85(1H, d, J=5.0Hz, H-2); 2.1(1H, m, H-313); 2.48(1H, dd, J=8.0, 15Hz, H3ct); 6.0(1H, dd, J=4.0, 8.0Hz, H-4); 2.0(2H, m, n-7); 2.0(2H, m, n-8); 5.44(1H, d, J=5.0Hz, H-10); 3.63(1H, d, J=5.0Hz, n-11); 2.97(2H, AB, J=4.0Hz, H-13); 0.87(3H, H-14); 4.21(2H, AB, J=2.0Hz, n-15); 1.74(3H, H-16); 5.84(1H, d, J=l, 2Hz, n-2'); 3.85(1H, d, J=7.0Hz, H-4'); 5.24(1H, d, J=7.0Hz, n-5'); 3.87(1H, n-6'); 5.8(1H, d, J=15, 5.0Hz, H-7'); 7.70(1H, dd, J=l 1.5, 15.5Hz, H-8'); 6.54(1H, t, J=l 1.5Hz, H-9'); 5.9(1H, d, J=l.5Hz, n-10'); 2.28(3H, d, J=l, 2.0Hz, H-12'); 3.70(1H, q, J=6.0Hz, H13'); and 1.36ppm (3H, d, J=-6.0Hz, H-14'). 13CNMR: (CDC13) 79.2, d, C-2; 34.7, t, C-3; 73.9, d, C-4; 49.2, C-5; 43.3, C-6; 20.4, t, C-7; 27.6, t, C-8; 140.4, C-9; 118.6, d, C-10; 67.9, d, C-11; 65.6, C-12; 47.9, t, C-13; 7.4, q, C-14; 63.4, t, C-15; 23.3, q, C-16; 165.9, C-I'; 119.8, d, C-2'; 155.4, C-3'; 79.8, d, C-4'; 103.4, d, C-5'; 82.3, d, C-6'; 134.5, d, C-7'; 126.1, d, C-8'; 143.1, d, C-9'; 118.9, d, C-10'; 166.2, C-11'; 13.1, q, C-12'; 76.5, d, C-13' and 16.0ppm, q, C-14'. Mass Spectrum: EIMS: 528m/e (M~). Reference B. B. Jarvis, G. P. Stahly, and G. Pavanasasivam, E. P. Mazzola; Structure ofRoridin J. A New Macrocyclic Trichothecene from Myrothecium verrucaria; Joumal of Antibiotics; Vol. 33, pp. 256-258(1980).

444

16.


Common/Systematic Name 7[3,8[3-Epoxyroridin H Molecular Formula/Molecular Weight C29H3409; MW - 526.22028

10 H

16

H

141 '

(D 011~*

a. ~ General Characteristics Colorless, amorphous powder. Fungal Source

Cylindrocarpon spp.

Spectral Data IR:

(CHCI3) 1717, 1646, and 1601cmq.

IH N~/[R: (CDCIs) 5.91 (IH, d, J= 4.5Hz, H-2), 2.15, (IH, ddd, ~-- 4.0, 4.5, 14.0Hz, H-3~; 2.40 (IH, dd, ~= 8.0, 14.0I-Iz,H-3c0, 5.87 (IH, dd, J= 4.0, 8.0Hz, H-4), 3.75 (IH, dd, J= 3.0, 4.0Hz, H-7); 3.17 (IH, rid,J= 2.1, 4.0I-Iz,H-8); 5.69 (IH, m, H-10); 4.08 (II-I,m, H-11); 2.98 and 3.18(IH each, AB, J= 3.6I-Iz,H-13), 1.12 (3H, s, H-14), 3.41 and 4.43 (IH each, AB, J= 12.3Hz, H-15), 1.98 (3H, d , J = 1.4Hz, H-16), 5.69(1H, q,J= 1.0Hz, H-2'); 2.32 and 2.41 (1H each, m, H-4'), 5.54 (1H, dd, J= 3.4, 8.4Hz, H-5'), 4.06 (1H, ddd, J = 2.0,2.3, 8.0Hz, H-6'); 5.95 (1H, dd, J = 2.3, 15.4Hz, H-7'), 7.76(1H, ddd, J = 2.0, 11.4, 15.4Hz, H-8'), 6.58 (1H, dd, J = 11.1, 11.4Hz, H-9'), 5.79 (1H, d,J-- 11.1Hz, H-10'), 2.28 (3H, d, J= 1.0Hz, H-12'), and 1.34ppm (3H, d, J - 5.9Hz, H-14').

16.


445

13C NMR: (CDC13) 79.3, C-2; 35.0, C-3; 73.6, C-4; 48.2, C-5; 44.2, C-6; 50.9, C-7; 57.2, C-8; 138.0, C-9, 123.0, C-10; 67.9, C-11; 65.5, C-12; 47.9, C-13; 8.2, C-14; 65.8, C-15; 22.0, C-16; 165.9, C-I'; 118.7, C-2'; 155.6, C-3'; 47.7, C-4'; 101.0, C-5'; 82.0, C-6'; 135.4, C-7'; 126.2, C-8'; 143.4, C-9'; 118.4, C-10'; 165.9, C-11'; 18.4, C-12'; 77.1, C13'; and 16.5ppm, C-14'.

References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 244(1981). B. B. Jarvis, Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). M. Matsumoto; Structures of Isororidin E, Epoxyisororidin E, Epoxyroridin H, and Diepoxyroridin H, New Metabolites Isolated from a Species of Cylindrocarpon;J. Sci. Hinoshima, University serial A, Vol. 43, pp. 107-118 (1979).

446

16.


Common/Systematic Name 7 p,813,2',3'-Diepoxyisororidin H Molecular Formula/Molecular Weight C29H3401o; M W = 5 4 2 . 2 1 5 2 0

lo H

H

';I o

,,o I

o

I10,

12

General Characteristics Colorless prisms from ethyl acetate, mp 291-293~ (dec.). Fungal Source

Cylindrocarpon spp.

Spectral Data IR:

(CHCI3) 1755, 1710, 1645, and 1601cm ~. ]H NMR: (CDCI3) 3.90 (1H, d, J= 4.5Hz, H-2), 2.26 (1H, ddd, J= 4.5, 5.0, 15.0Hz, H-313; 2.45 (1H, dd, J = 8.0, 15.0Hz, H-3ot); 5.89 (1H, dd, J = 5.0, 8.0Hz, H-4), 3.62 (1H, dd, J = 3.0, 3.9Hz, H-7), 3.18 (1H, dd, J= 1.9, 3.9Hz, H-8), 5.67 (1H, ddd, J= 1.5, 1.9, 6.3Hz, H-10); 3.75 (1H, dd, J = 3.0, 6.3Hz, H-11); 2.97 and 3.17(1H each, AB, J = 3.5Hz, H13); 1.12 (3H, s, H-14), 4.44 (2H, s, H-15), 2.01 (3H, d , J = 1.5Hz, H-16), 3.29(1H, s, H-2'), 1.57 (1H, dd, J = 9.2, 14.0Hz, H-4'A), 2.32 (1H, dd, J = 2.7, 14.0Hz, H-4'B); 5.37 (1H, dd, J = 2.7, 9.2Hz, H-5'); 4.17 (1H, ddd, J = 1.8, 2.2, 8.0Hz, H-6'), 5.98 (1H, ddd, J = 2.2, 3.0, 15.5Hz, H-7'); 7.60(1H, ddd, J = 1.8, 11.3, 15.SHz, H-8'); 6.62 (1H, dd, J= 11.3, 11.7Hz, H-9'), 5.88 (1H, d, J - 11.7Hz, H-10'), 1.60 (3H, s, H-12'), 3.70 (1H, m, H-13'), and 1.33ppm (3H, d, J= 6.0Hz, H-14').


447

13C NMR: (CDC13) 79.2, C-2; 34.8, C-3; 73.9, C-4; 48.5, C-5; 44.7, C-6; 50.9, C-7; 56.3, C-8; 138.6, C-9; 122.4, C-10; 67.3, C-11; 65.3, C-12; 48.0, C-13; 8.5, C-14; 65.6, C-15; 22.0, C-16; 167.2, C-I'; 59.2, C-2'; 60.8, C-3'; 44.0, C-4'; 101.1, C-5'; 82.7, C-6'; 134.6, C-7'; 126.8, C-8'; 142.7, C-9'; 119.0, C-10'; 166.9, C-11'; 17.1, C-12', 76.3, C13'; and 15.8ppm, C-14'.

References R. J. Cole and R. H. Cox, Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 245 (1981). B. B. Jarvis; Macrocyclic Trichothecenes, In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). M. Matsumoto; Structures of Isororidin E, Epoxyisororidin E, Epoxyroridin H, and Diepoxyroridin H, New Metabolites Isolated from a Species of Cylindrocarpon;J. Sci. Hinoshima, University serial A, Vol. 43, pp.107-118 (1979).

448


Common/Systematic Name Satratoxin F Molecular Formula]Molecular Weight C29H3401o; M W -- 5 4 2 . 2 1 5 2 0

O32

3

ok, o

o

,o. o

General Characteristics Crystals; m.p., 140-143~ Fungal Source Stachybotrys chartarum (S. atra) ATTC 26303. Isolation/Purification A fraction from the isolation of satratoxin H (see R. M. Eppley et al.; J. Org. Chem, Vol. 42, p. 240, 1977) was crystallized from CHCl3-hexane. Biological Activity Cytotoxic. Spectral Data IR~

(CHaC1) 3460(OH), 1748(C=O), 1715, and 1183cm1. 1H NMR: (CDC13) 0.83(3H, s, H-14); 1.73(3H, s, H-16); 2.0(5H, m, H-313, H-7 and H-8); 2.5(1H, m, H-3a); 2.98(2H, center of AB system, J=4Hz, H- 13); 3.38(1H, s, H-2'); 3.59(1H, d, J=5Hz, H-11); 4.24(1H, s, H-12'); 3.85(1H, d, J=5Hz, H-2); 4.15(2H, m, H-5'); 3.88(2H, center of AB system, H- 15); 5.43(1H, d, J=5Hz, H-10); 5.60(1H, d, J=15.5Hz, H-7'); 5.9(1H, m, H- 4); 5.92(1H, d, J=10.5Hz, H-10'); 6.57(1H, dd, J=6


449

and 10.5Hz, H-9'); and 6.81ppm (1H, dd, 3--6 and 15.5Hz, H-8'). 13C NMR: 79.2, C-2; 34.6, C-3; 74.3, C-4; 49.5, C-5; 43.2, C-6; 20.1, C-7; 27.5, C-8; 140.4, C-9; 118.6, C-10; 67.9, C-11; 65.3, C-12; 48.0, C-13; 8.0, C-14; 65.1, C-15; 23.3, C-16; 166.1, C-I'; 58.9, C-2'; 63.9, C-3'; 22.7, C4'; 61.2, C-5'; 87.1, C-6; 130.2, C-7'; 130.5, C-8'; 143.2, C-9'; 121.2, C-10'; 166.9, C-11'; 73.7, C-12'; 217.8, C-13'; and 29.7ppm, C-14'. Reference R. M. Eppley, E. P. Mazzola, M. E. Stack, and P. A. Dreifuss; Structures of Satratoxin F and Satratoxin G, Metabolites of Stachybotrys atra: Application of Proton and Carbon-13 Nuclear Magnetic Resonance Spectroscopy; J. Org. Chem., Vol. 45, pp. 2522-523(1980).

450

16.


Common/Systematic Name Satratoxin G Molecular Formula/Molecular Weight C29H36010; M W --- 5 4 4 . 2 3 0 9

~o H

H

~ ~ , ~ , , O

H2~. 14

0.

~

0"-~

14'

General Characteristics Crystals; m.p., 132-136~ Fungal Source Stachybotrys chartarum (S. atra) ATTC 26303. Isolation/Purification A fraction from the isolation of satratoxin H (see R. M. Eppley et al.; J. Org. Chem, Vol. 42, p. 240, 1977) was crystallized from CHCls-hexane. Biological Activity Cytotoxic. Spectral Data IR:

(CHCIs) 3450(OH), 1747(C=O), 1710, and 1185cm1. ~HNMR: (CDCIs) 0.87(1H, s, H=14); 1.12(3H, d, J=7Hz, H=14'); 1.74(3H, s, H-16); 2.0(5H, m, H-3[}, H-7 and H-8); 2.5(1 H, m, H-3a); 2.5(2H, m, H-4'); 2.98(2H, center of AB system, J=4Hz, H-13); 3.61(1H, d, J=5Hz, H- 11); 3.43(1H, s, H-2'); 3.90(1H, d, J=5Hz, H-2); 3.9(2H, m, H-5'); 4.02(2H, center of AB system, J=12Hz, H-15); 4.35(1H, s, H-12'); 4.45(1H, q, J=7Hz, H-13'); 5.46(1H, d, J=5Hz, H-10); 5.90(1H, d,


451

J-16.5 Hz, H-7'); 5.93(1H, d, J=10.5Hz, H-10'); 6.0(1H, m, H-4); 6.68(1H, dd, J=7.5 and 10.5Hz, H-9'); and 7.00ppm (1H, dd, J=7.5 and 16.5Hz, H-8'). 13C NMR: (CDC13) 79.3, C-2; 34.4, C-3; 73.7, C-4; 49.3, C-5; 43.3, C-6; 20.2, C-7; 27.5, C-8; 140.3, C-9; 118.8, C-10; 67.1, C-11; 65.4, C-12; 48.1, C-13; 8.0, C-14; 64.9, C-15; 23.3, C-16; 166.9, C-I'; 61.0, C-2'; 65.4, C-3'; 22.7, C-4'; 60.3, C- 5'; 81.5, C-6'; 132.0, C-7'; 131.5, C-8'; 144.2, C-9' 120.0, C-10'; 166.9, C-11'; 72.6, C-12'; 70.1, C13'; and 16.1ppm, C-14'. Reference R. M. Eppley, E. P. Mazzola, M. E. Stack, and P. A. Dreifuss; Structures of Satratoxin F and Satratoxin G, Metabolites of Stachybotrys atra: Application of Proton and Carbon-13 Nuclear Magnetic Resonance Spectroscopy; J. Org. Chem., Vol. 45, pp. 2522-523(1980).

452


Common/Systematic Name Isosatratoxin F Molecular Formula/Molecular Weight C29H34010; M V ~ -- 542.21518

~o H I

H

" I 13

I

r~

~ ~ ' ~ " "

0

") 0

~'

~'

,~r

14'

General Characteristics Crystals; m.p., 153-155~

[a]D = + 46.4 ~ (C---0.40, in acetone).

Fungal Source Stachybotrys atra (S. chartarum) ATTC 26303. Isolation/Purification Methanol extraction of a culture of S. atra (JS5106) grown at room temperature for 30 days gave 7.5g of crude extract. This material was subjected to MPLC over silica gel. Elution with dichloromethane gave 520mg in the first fraction which was subjected to high speed countercurrent chromatography (methanol-water-carbon tetrachloride-hexanemethylene chloride, 6:4:8:1:1, v/v/v/v/v) to give five fractions. Fraction 4 was recrystallized from methylene chloride-hexane to give 20mg of isosatratoxin F. Spectral Data IR:

(CHCl3) 3478(OH), 1747(C-O), 1713, and l188cm "1. 1H NMR: (CDC13) 0.80(3H, s, H-14); 1.70(3H, s, H-16); 1.80-2.00(4H, m, H-7 and H- 8); 2.20(ddd, 1H, J=4.7, 5.1, and 15.5Hz, H-3~), 2.50(1H, dd, J-8.0, 15.5Hz, H-3a); 2.80 and 3.12(1H each, AB, J= 4.0 Hz, H-13); 3.38(1H, s, H-2'); 3.54(1H, d, J=5.0Hz, H-11); 3.62(1H, s, H-12'); 3.83(1H, d, J=5.1Hz, H-2); 4.14(2H, m, H-5'); 4.20(2H, s, H-15); 5.39(1H, d, J=5.0Hz, H-10); 5.55(1H, dd, J=l.6 and 16.4Hz, H-7');


453

5.84(1H, dd, d=4.7 and 8.0Hz, H-4); 5.90(1H, dd, ,]=1.6 and 11.6Hz, H-10'); 6.54(1H, ddd, J=l.6, 5.7, and 11.6Hz, H-9'); and 6.72ppm (1H, ddd, ,]--1.6, 5.7, and 16.4Hz, H8'). 13CNMR: (CDCI3) 79.1, C-2~ 34.4, C-3, 74.1, C-4~ 49.4, C-5~ 43.0, C-6, 20.0, C-7~ 27.4, C-8; 140.5, C-9~ 118.5, C-10, 67.7, C-11, 65.3, C-12, 47.9, C-13~ 7.9, C-14~ 65.0, C-15~ 23.3, C-16~ 166.0, C-I', 58.8, C-2', 63.8, C-3', 22.5, C4'~ 61.1, C- 5', 87.0, C-6, 129.8, C-7'~ 130.5, C-8', 143.2, C-9', 121.1, C-10', 166.9, C-11', 73.5, C-12', 208.6, C-13'~ and 27.4ppm, C-14'. Reference B. B. Jarvis, W. G. Sorenson, E.-L. Hintikka, M. Nikulin, Y. Zhou, J. Jiang, S. Wang, S. Hinkley, R. A. Etzel, and D. Dearborn; Studies of Toxin Production by Isolates of Stachybotrys chartarum and Memnoniella echinata Isolated During a Study of Pulmonary Hemosiderosis in Infants; Appl. Environ. Microbiol., Vol. 64, pp. 3620-3625(1998).

454

16.


Common/Systematic Name Isosatratoxin G Molecular Formula/Molecular Weight C29H36010; M~W = 5 4 4 . 2 3 0 8 5

10 H 16~O ~

~

H

o,

2 3 .~,,O

H2(7-'~A O'~~U2'~

~O~7,

O OH~OH 14'



Isolation/Purification The black gummy crude extract derived from a rice culture of S. chartarum S-17 was triturated with hexane washes until no further color was extracted. The remaining black gum was partitioned between water and chloroform. The chloroform soluble portion was dried (Na2SO4) concentrated to dryness on the rotary evaporator to give a black gum. The gum was subjected to flash chromatography, over silica gel with a gradient of hexanemethanol-methylene chloride, and collected in 124 fractions. Fractions 83/84 (3% methanol-methylene chloride) were combined to give A; fractions 85-87 (3% methanolmethylene chloride) were combined to give B, fractions 88-89 (3% methanol-methylene chloride) and 90-99 (4% methanol-methylene chloride) were combined to give C, fractions 100-103 (5% methanol-methylene chloride) and fractions 104-110 (6% methanolmethylene chloride) were combined to give D; fractions 111-112 (6% methanol-methylene chloride) were combined to give E; fractions 113-115 (10% methanol-methylene chloride) were combined to give F; and fractions 116-119 were combined to give G. Fraction A was subjected to CCC (Vc = 850ml, methanol -water-carbon tetrachloride-methylene chloridehexane(3: 2: 3.5:0.5:1 v/v/v/v/v) organic mobile phase at 3.2ml/min) to give 10 fractions (F1-F10). Fraction F5 was subjected to TLC (Chromatotron, 2 mm plate, 20% hexane in ethyl acetate) to give 2tt-acetoxystachybotrylactone acetate. The stationary phase of this CCC was concentrated to dryness to give a gum which upon preparative TLC (Chromatotron, 2 mm plate, -~ 20% hexane in ethyl acetate) yielded fractions from which

16.


455

were isolated satratoxin G and isosatratoxin G. Biological Activity Cytotoxic. Spectral Data UV: ~, maxMeOH255nm. IR~ (CH3C1) 3450(OH), 1745(C=O), and 1712cm1 (C=O). 1H NMR: (CDC13) 0.80(1H, s, H-14); 1.23(3H, d, J-6.5 Hz, H-14'); 1.70(3H, s, H-16); 1.84(2H, m, H-7); 2.03(2H, m, H-8); 2.17(1H, dt, J=5.0 and 15.2 Hz, H-313; 2.44(1H, dd, J-8.5 and 15.2Hz, H-3~); 2.08(1H, dd, J=5.2 and 14.1Hz, H-4'A); 2.35(1 H, bd, J=14.1Hz, H-4'B); 2.81 and 3.12 (1H each, AB, J-4.0Hz, H=13); 3.55(1H, d, J=5.0 Hz, H-11); 3.74(1H, s, H-2'); 3.82(1I-I, d, J=5.0 Hz, H-2); 3.83(1H, m, H-5'B); 4.08(1H, dt, J=2.0 and 11.4Hz, H-5'B); 3.87(1H, q, J=6.5Hz, H=13'); 4.16 and 4.28(1H each, AB, J=12.5Hz, H-IS); 4.30(1H, s, H-IT); 5.40(1H, d, J-5.0Hz, H-10); 5.80(1H, d, J-16.SHz, H-7'); 5.90(1H, d, J-11.4Hz, H-10'); 5.90(1H, dd, ,/-5.0 and 8.5Hz, H-4); 6.65(1H, dd, J-6.8 and 11.4Hz, H-9'); and 6.88ppm (1H, dd, J-6.8 and 16.8Hz, H-8'). 13C NMR: (CDC13) 79.2, C-2; 34.5, C-3; 73.5, C-4; 49.5, C-5; 43.2, C-6; 20.0, C-7; 27.4, C-8; 140.5, C-9; 118.5, C-10; 67.8, C-11; 65.3, C-12; 48.0, C-13; 8.1, C-14; 64.7, C-15; 23.4, C-16; 166.5, C-I'; 54.2, C-2'; 65.0, C-3'; 27.4, C-4'; 60.9, C-5'; 81.5, C-6'; 130.9, C-7'; 130.1, C-8'; 144.0, C-9'; 120.3, C-10'; 168.5, C-11'; 68.7, C-12'; 73.0, C-13'; and 17.3ppm, C-14'.

Mass Spectrum: HREIMS: 544.2301m/e for C29H36010;calcd., 544.2308. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). B. B. Jarvis, J. Salemme and A. Morais; Stachybotrys Toxins 1; Natural Toxins, Vol. 3, pp. 10-16(1995).

456

16.


Common/Systematic Name Satratoxin H Molecular Formula/Molecular Weight C29H3609, M~IV = 528.23 593

2'. ?o. 14'


Stachybotrys atra.

Biological Activity Toxic to brine shrimp and positive in rabbit skin tests. Details not reported. Spectral Data UV:

Z ~n

225(c=14,700 ) and 255nm (10,400).

~HNMR: (CDCI3) 3.90(1H, m, H-2); 2.20(1H, ddd, J=4.0, 5.0, 15Hz, H-313);2.45(1H, dd, J=7.5, 15Hz, n-3a); 5.90(1H, m, n-4); 1.90(2H, m, n-7); 2.10(2H, m, n-8); 5.46(1H, d, J=5.0Hz, H-10); 3.62(1H, d, J=5.0Hz, H-11); 2.81 and 3.12(1H each, AB, J=4.0Hz, H-13); 3.12(1H, d, J=4.0Hz, H-13b); 0.83(3H, s, H-14); 2.88 and 4.56(1H each, AB, J=12.0Hz, H-15); 1.74(3H, s, n-16); 5.85(1H, d, J=2.0Hz, n-2'), 2.60(1H, m, U-4'a); 3.74(1H, dt, J=3.0, 10.0Hz, H-4'b); 3.90(2H, m, H-5'); 6.09(1H, d, J=17.5Hz, H-7'); 7.36(1H, dd, J--10.5, 17.5Hz, H-8'); 6.63(1H, t, J=-10.5Hz, H-9'); 5.91(1H, d, J=10.5Hz, H-10'); 3.97 1n, s, H-12') 4.38(1H, q, J=7.0Hz, H-13'); and 1.16ppm (3H, d, J=7.0Hz, H- 14').


457

13C NMR: (CDC13) 79.1, C-2; 34.4, C-3; 74.2, C-4; 49.0, C-5; 43.4, C-6; 20.4, C-7; 27.6, C-8, 140.2, C-9; 119.0, C-10; 68.2, C-11; 65.4, C-12; 48.0, C-13; 7.6, C-14; 64.2, C-15; 23.3, C-16; 166.2, C-I'; 119.0, C-2'; 155.1, C-3'; 25.3, C-4'; 60.4, C-5'; 81.4, C-6'; 134.2, C-7'; 132.2, C-8'; 143.0, C-9'; 120.4, C-10'; 167.0, C-11'; 73.7, C-12'; 69.7, C-13'; and 15.7pprn, C-14'. TLC Data Adsorbent: silica gel; solvent: isopropanol-chloroform, 2:98, v/v; Re, not reported; detection method: not reported. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, pp. 227-229(1981). R. M. Eppley, E. P. Mazzola, R. J. Highet, and W. J. Bailey; Structure of Satratoxin H, a Metabolite ofStachybotrys atra. Application of Proton and Carbon-13 Nuclear Magnetic Resonance; J. Org. Chem., Vol. 42, pp. 240-243(1977). B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991).

458

16.


Common/Systematic Name S-Isosatratoxin H Molecular Formula/Molecular Weight C29H3609; M ' W -- 5 2 8 . 2 3 5 9 3

lo H H ~O.,,12

~

~~I,0 . H2~

!~

0

General Characteristics Crystals, m.p., 180-183~ Fungal Source


Isolation/Purification In a manner similar to that described for extraction of S. chartarum, a rice culture of S. chartarum Egypt I gave a crude extract that was taken up in 10ml of methanol and filtered through a pad Of Cls silica gel in a Biichner funnel. Elution with methanol gave, aRer solvent removal, a black gum. This material was dissolved in a small amount of methylene chloride and loaded onto a PEI silica gel column and eluted with methylene chloridehexane(4:1, v/v), methylene chloride and 10% methanol in methylene chloride. A combination of preparative HPLC and TLC gave pure satratoxin H, S-isosatratoxin H, stachybotrylactone, and stachybotrylactam. Biological Activity Cytotoxic. Spectral Data UV:

)t maxM~O" 228 and 255nm.

16.


459

IR:

(CH3C1) 3440(OH) and 1712cm1 (C=O). 1H N]VIR:

(CDCI3) 0.80(1H, s, H-14); 1.35(3H, d, J=6.5 Hz, H-14'), 1.70(3H, s, H-16); 1.92(2H, m, n-7); 2.01(2H, m, n-8); 2.18(1H, dt, d=5.0 and 15.2Hz, H-313);2.45(1H, dd, J=8.0 and 15.2Hz, H-3a); 2.30(1H, m, H-4'A), 4.15(1H, bd, d=14.1Hz, H-4'B); 2.80 and 3.12(1H each, AB, d=4.0Hz, H-13); 3.57(1H, d, J=5.0Hz, H-11); 3.80(1H, d, J=5.0Hz, H-2); 3.83(2H, m, H-5'); 3.95(1H, q, J=6.SHz, H-13'); 3.85 and 4.55(1H each, AB, d=12.5 Hz, H-15); 4.65(1H, s, H-12'); 5.41(1H, d, d=5.0Hz, H-10); 5.70(1H, d, d=17.0Hz, H-7'); 5.90(1H, d, J=l 1.4Hz, H-10'); 5.87(1H, dd, J=4.5 and 8.0Hz, H-4); 6.10(1H, s, H-2'), 6.52(1H, t, J=l 1.4Hz, H-9'); and 7.45ppm (1H, dd, J= 11.4 and 17.0Hz, H-8'). 13CNMR: (CDCI3) 79.2, C-2; 34.5, C-3; 74.1, C-4; 49.0, C-5; 43.4, C-6; 20.3, C-7; 27.7, C-8; 140.3, C-9; 119.0, C-10; 68.2, C-11; 65.5, C-12; 48.0, C-13; 7.5, C-14; 63.9, C-15; 23.3, C-16, 166.8, C-I'; 112.9, C-2'; 158.4, C-3'; 29.3, C-4', 60.5, C-5'; 80.6, C-6'; 133.8, C-7'; 134.5, C-8'; 142.5, C-9'; 120.6, C-10'; 166.8, C-11'; 73.8, C-12'; 71.6, C13'; and 17.3ppm, C-14'. Mass Spectrum: HREIMS: 528.2374m/efor C29H3609,calcd 528.2359. References B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421(1991). B. B. Jarvis, J. Salemme and A. Morais; Stachybotrys Toxins 1; Natural Toxins, Vol. 3, pp. 10-16(1995).

460

16.


Common/Systematic Name 12'-Hydroxy-2'-isoverrucarin J PD 113,325 Molecular Formula/Molecular Weight C27H3209; M W = 500.20463

H

I~

H

~1 '~-~i~ u I

, L

H2C,s l '

~

o

U,,,

4" ~ 0 ~ ' ~ ,

~

i, I'

General Characteristics Crystals; mp., >250~ Plant Source

Myrothecium roridum (ATCC 20605).

Isolation/Purification A large scale submerged fermentation was extracted (ethyl acetate) and concentration of the organic layer gave 650g of gum. Large scale preparative HPLC of 118g of this gum over silica gel (Prep-500) gave 18 fractions. A portion (7.2g) from the 25% ethyl acetatemethylene chloride fraction (12g) was further chromatographed (MPLC, isopropanolhexane) to give a fraction (1.37g) from which 568mg of 12'-hydroxy-2'-isoverrucarin J was crystallized (methylene chloride-cyclohexane). Biological Activity In vim P388 mouse anti-leukemic activity: T/C x 100 at 5mg/kg = 129. Cytotoxic and skin irritant. Spectral Data UV:

~, M~,

217(e = 23,500) and 262nm (18,900).

IR:

(CHCI3) 3640, 2980, 2920, 1745, 1660, 1270, 1210, and l l90cm "l.


461

1H NMR: (CDCI3) 0.64(1H, s, H-14); 1.72(3H, s, H-16); 1.95(2H, m, H-7);-~2.05(2H, m H- 8); 2.19(1H, dt, J=6 and 17Hz, H-3~3);2.45(1H, dd, J=8 and 17Hz, H-3a); 2.3(1H, m, H4'A); 4.14(1H, m, H-4'B); 2.83 and 3.14(1H each, AB, J=4Hz, H-13); 3.7(1H, d, J=5 Hz, H-11); 3.86(1H, d, J=6Hz, H-2); 4.20 and 4.27(1H each, AB, J=16Hz, H-12'); -~4.47(2H, m, H-5'); 3.7 and 5.07(1H each, AB, J=12Hz, H-15); 5.44(1H, dd, J=5 and 2Hz, H-10); 5.9(1H, d, J=16Hz, H-7'); 5.98(1H, dd, J=4 and 8Hz, H-4); 6.10(1H, d, J=l 1Hz, H-10'); 6.1(1H, d, J=2 Hz, H-2'); 6.65(1H, t, J=l 1Hz, H- 9'); and 8.2ppm (1H, dd, Jr=11 and 16Hz, H-8'). 13CNMR: 79.2, C-2; 34.9, C-3; 75.2, C-4; 49.4, C-5; 43.7, C-6; 20.7, C-7; 26.9, C-8; 143.3, C-9; 118.3, C-10; 67.3, C-11; 65.2, C-12; 48.1, C-13; 7.0, C-14; 62.7, C-15; 23.2, C-16; 165.3, C-I'; 116.7, C-2'; 157.4, C-3'; 28.1, C-4'; 61.8, C-5'; 165.9, C-6'; 127.3, C-7'; 139.4, C-8'; 140.0, C-9'; 125.0, C-10'; 166.1, C-11'; and 64.4ppm, C-12'. Reference T. A. Smitka, R. H. Bunge, R. J. Bloem, and J. C. French; Two New Trichothecenes, PD 113,325 and PD 113,326; J. Antibiotics, Vol. 37, pp. 823-828(1984).

462


Common/Systematic Name M-Isosatratoxin H; PD 113,326 Molecular Formula/Molecular Weight C29H3609; ~

= 528.23593

,o H

H O.,,]

~

r

6

[

0

II

TM

6H

~t~-OH

General Characteristics Crystals mp., 168-171 ~ Fungal source Myrothecium roridum (ATCC 20605). Isolation/Purification A large scale submerged fermentation of M. roridum was extracted with ethyl acetate. Concentration of the organic layer gave 650g of gum. Large scale preparative HPLC of 118g of this gum over silica gel (Prep-500, Waters Assoc.) gave 18 fractions. A portion (7.2g) from the 25% ethyl acetate-methylene chloride fraction was further chromatographed (MPLC, isopropanol-hexane) to give a fraction rich in M-isosatratoxin H. This fraction was further purified by reversed phase HPLC (C~8, water-acetonitrile) to give, after crystallization from methylene chloride-cyclohexane, 139mg of M-isosatratoxin H. Biological Activity In vivo P388 mouse anti-leukemic activity: T/C x 100 at 5mg/kg = 198. Cytotoxic and skin irritant. S,.pectral Data UV:

~, ra~. 227(e = 19,500) and 255nm (12,500).

16.


463

IR~

(CHC13) 3560, 3440,2975,2930, 1730, 1660, 1595, 1195, 1165, 1145, 1080, and 970cm-a. 1H NMR:

(CDCI3) 0.83(1H, s, H-14); 1.35(3H, d, J=7Hz, H-14'); 1.72(3H, s, H-16); 1.92(2H, m, H-7); -~2(2H, m H-8); 2.18(1H, dt, J=5 and 15Hz, H-3~); 2.45(1H, dd, J=8 and 15I-Iz, H-3tt); 2.7(1H, m, H-4'A); 3.78(1H, m, H-4'B); 2.83 and 3.14(1H each, AB, J=4 z, H-13); 3.59(1H, d, J=5Hz, H-11); 3.9(1H, m, H-2); 3.9(2H, m, H-5'); 4.0(1H, q, J=6.5Hz, H-13'); 4.22(1H, s, H-12'); 4.54 and 5.10(1H each, AB, J=13Hz, H-15); 5.39(1H, d, J-17Hz, H-7'); 5.43(1H, d,J--5 Hz, H-10); 5.93(1H, d, J=12Hz, H-10'); 5.9(1H, m, H-4); 5.80(1H, d, J=2Hz, H-2'); 6.52(1H, dd, J=10 and 12Hz, H-9'); and 7.26ppm (1H, dd, J= 10 and 17Hz, H-8'). 13CNMR~ 79.1, C-2; 34.5, C-3; 74.2, C-4; 49.0, C-5, 43.4, C-6; 20.3, C-7, 27.6, C-8; 140.4, C-9, 118.9, C-10, 68.2, C-11; 65.5, C-12, 48.1, C-13; 7.5, C-14; 64.0, C-15; 23.3, C-16, 165.4, C-I'; 118.2, C-2'; 154.9, C-3'; 25.5, C-4'; 61.1, C-5'; 78.7, C-6'; 133.6, C-7'; 134.2, C-8'; 142.4, C-9'; 121.0, C-10'; 166.7, C-11', 75.4, C-12', 74.7, C-13'; and 15.8ppm, C-14'. Reference T. A. Smitka, R. H. Bunge, R. J. Bloem, and J. C. French; Two New Trichothecenes, PD 113,325 and PD 113,326; J. Antibiotics, Vol. 37, pp. 823-828(1984).

464

16.


Common/Systematic Name Vertisporin Molecular Formula/Molecular Weight C29H36010; M W -- 544.23085

10 H

H

~.~o.,J

1-8 "61

~

/

~ ~ ' ~ H2r

Is

I

9

4 k Al1'

d~oH .

_

OH General Characteristics A colorless amorphous powder; mp., 176-183~ [a]D26 + 62.5~ diacetate; mp., 145155~ Fungal Source Verticimonosporium diffractum. Biological Activity Cytotoxic and antibiotic activity: EDs0 to HeLa cells was 0.00 l mg/ml; limited antifungal activity against Trichophyton asteroides. Spectral Data UV:

~, maxE~"216rim (e=19,500). IR:

(CHCI3) 1723 and 1711cm"l. 1H NMR: (diacetate derivative) (C6Dr) 3.82(H-2); 5.80(H-4); 5.40(H-10); 3.57(H-11); 2.77(H-13a); 3.09(H-13b); 0.80(H-14); 3.97(H-15a); 4.24(H-15b); 1.70(H-16); 5.81(H-2'); 6.41(H-9'); 5.82(H10'); 4.19(H-12'); 5.10(H-13'); and 6.61ppm (H-14').


465

13C NMR: (CDC13) 79.0, C-2; 35.0, C-3; 74.0, C-4; 49.7, C-5; 43.2, C-6; 20.7, C-7; 27.7, C-8; 138.4, C-9; 119.9, C-10; 67.6, C-11; 65.3, C-12; 47.2, C-13; 8.0, C-14; 64.7, C-15; 23.0, C-16; 165.7, C-I'; 118.5, C-2'; 152.6, C-3'; 22.8, C-4'; 64.9, C-5'; 86.7, C-6'; 23.6, C-7'; 26.4, C-8', 149.4, C-9'; 121.1, C-10'; 166.0, C-11'; 86.0, C-12'; 75.7, C-13'; and 97.5ppm, C- 14'. References R. J. Cole and R. H. Cox; Handbook of Toxic Fungal Metabolites; Academic Press, New York, p. 241 (1981). 0 B. B. Jarvis; Macrocyclic Trichothecenes; In Mycotoxins and Phytoalexins; R. P. Sharma and D. K. Salunkhe, eds.; CRC Press, Boca Raton, Florida, pp. 361-421 (1991). H. Minato, T. Katayama, and K. Toni: Vertisporin, A New Antibiotic from Verticimonosporim diffractum; Tetrahedron Lett., Vol. 16, pp. 2579-2582 (1975)

Miotoxins Miotoxin A Miotoxin A 13' 13-D-glucoside Miotoxin B Miotoxin C Miotoxin D Isomiotoxin D Miotoxin E Miotoxin F Miotoxin G

467


17.

Miotoxins

469

Common/Systematic Name Miotoxin A Molecular Formula/Molecular Weight C29H3809; M W -- 5 3 0 . 2 5 1 5 8

~o H

,,

H

,.1.o

.j

~.'/_-" "OH H

General Characteristics Crystals mp., 159-161~ [(~]D = +100.0 ~ (c=1.40, in CH2C12). Rf=0.52 (CH3-MeOH, 100:4) and 0.66 (ethyl acetate). Plant Source Baccharis coridifolia. Isolation/Purification Inflorescences and seeds ofB. coridifolia were extracted with CHC13 and the extract flash chromatographed over silica gel. Repeated flash chromatography followed by PTLC led to the isolation of miotoxins A-D and iso-rrflOtOXJnD. Spectral Data ~,Em~x~H220 and 263nm. IR: (KBr) 3410, 2960, 1710, 1635, 1220, and 1185cm"1. 1H N]VIR: (CDCI3) 0.78(3H, s, H-14); 1.20(3H, d, J=6.0Hz, H-14'), 1.71(3H, s, H-16); 2.01(2H, m, H-7), 2.03(2H, m, H-8), 2.10(1H, ddd, no Jvalues given, H-313), 2.30(3H, d, J=l.5Hz, H-12'); 2.51(1H, d, J=8Hz, H-3a); 2.81, 3.13(1H each, AB pattern, J=4Hz, U-13), 3.72(1H, d, J=6.7Hz, n-5'), 3.74(1H, t, J=7Hz, n-6'), 3.84(1H, m, H-11), 4.32(1H, br s, H-4'); 3.84(1H, d, no Jvalue given, H-2), 3.93, 4.38(1H each,

470

17.

Miotoxins

AB pattern, J=12nz, H-15); 3.74(1H, t, J=7Hz, H-6'); 5.47(1H, d, J=5Hz, H-10); 5.75(1H, d, J=l 1Hz, H-10'); 5.88(1H, dd, J=3 and 16Hz, H-7'); 6.07(1H, d, J=l.5Hz H-2'); 6.11(1H, dd, J=4 and 8Hz, H-4); 6.55(1H, dd, Js.,9~J9,,~o~=l1Hz, H- 9'); and 7.43 ppm (1H, dd, Jr--11 and 16Hz, H-8'). 13CNMR: (CDC13) 79.1, C-2; 35.6, C-3; 74.4, C-4; 48.6, C-5; 42.8, C-6; 21.3, C-7; 27.7, C-8; 140.1, C-9; 118.9, C-10; 67.2, C-11; 65.5, C-12; 48.0, C-13; 6.7, C-14; 63.7, C-15; 23.2, C-16; 165.9, C-I'; 115.2, C-2'; 160.0, C-3'; 74.4, C-4'; 73.7, C-5'; 83.9, C-6'; 137.5, C-7'; 126.8, C-8'; 143.3, C-9'; 118.0, C-10'; 166.4, C-11'; 15.8, C-12'; 70.8, C13'; and 18.6ppm C-14'. Reference G. G. Habermehl, L. Busam, and J. Stegemann; Miotoxin-A: A Novel Macrocyclic Trichothecene from the Brazilian Plant Baccharis coridifolia; Z. Naturforsch., Vol. 39c, pp.212-216(1983).

17.

Miotoxins

471

Common/Systematic Name Miotoxin A 13' 13-D-glucoside _Molecular Formula/Molecular Weight C35H48014; M W -- 6 9 2 . 3 0 4 4 1

~o H

H

0~

H2~s 15

o@:

4 ~0 ~

o

fi-glucose 0

= H

14'

General Characteristics An oil; [~]D + 50 (C--1.13, in CHC13). Plant Source Female Baccharis coridifofia. Isolation/Purification Fraction D from a reversed phase filtration chromatography (see isolation of roridin E glucoside) was subjected to CCC (Vc = 355mL) with a solvent system of methanol-waterchloroform-carbon tetrachloride-hexane (6:4:6:1:3, v/v/v/v/v). The lower organic phase was the mobile phase and the flow rate was 1.8ml/min to yield roridin A glucoside and a fraction containing miotoxin A glucoside, which was further purified on C18 reversed phase HPLC (250 x 10mm, 60% methanol-water, 4.0mL/min) to give miotoxin A 13-D-glucoside Biological Activity Cytotoxic: ICs5 ca. 1 ng/ml. Spectral Data IR:

(KBr) 3431, 1712, 1650, 1600, 1181, and 1081cm1. 1H NMR: (pyridine-d5) 7.73(1H, dd, J=12.0, 15.6Hz, H-8'); 6.51(1H, dd, J-11.1, 12.0Hz, H-9'); 6.35(1H, dd, J-2.9, 15.6Hz, H-7'); 6.18(1H, s, H-2'); 6.10(1H, dd, J = 4.2, 8.1Hz, H-4);

472

17.

Miotoxins

5.76(1H, d, J=l 1.1Hz, H-10'); 5.48(1H, d, J=4.5Hz, H-10); 4.90 and 4.06(1H each, AB, J=12.5Hz, H-15); 4.89(1H, d, J=7.7Hz, H-I"); 4.62(1H, m, H-6'), 4.52(1H, rn, H6"B); 4.51(1H, rn, H-4'); 4.39(1H, dq, J=6.3, 6.3Hz, H-13'), 4.37(1H, m, H-6"A); 4.26(1H, dd, J=8.5, 9.6Hz, H-4"); 4.23(1H, dd, J=8.5, 9.6Hz, H-3"); 4.01(11-1, dd, J=7.7, 8.5Hz, H-2"); 3.95(2H, m, H-5'B, H-5"); 3.91(1H, d, J=5.1Hz, H-2); 3.76(1H, dd, J=4.3, 8.1Hz, H-5'A); 3.72(1H, d, J=5.0Hz, H-11); 3.08 and 2.84(1H each, AB, J=4.1Hz, H-13); 2.72(3H, s, H-12'), 2.42(1H, dd, J=8.1, 15.1Hz, H-3); 2.22(1H, ddd, J=4.2, 5.1, 15.1Hz, H-3), 1.86-1.93(4H, m, H-7, H-8); 1.56(3H, s, H-16); 1.11(3H, d, J=6.3Hz, H-14'); and 1.09ppm (3H, s, H-14). 13CNMR: (pyridine-d5) 79.2, C-2; 35.2, C-3; 74.9, C-4; 49.4, C-5; 43.6, C-6; 20.7, C-7; 27.7, C-8; 139.0, C-9; 120.1, C-10; 67.7, C-11; 66.0, C-12; 47.8, C-13; 7.5, C-14; 63.7, C15; 23.1, C-16; 166.7, C-I'; 116.9, C-2'; 161.7, C-3'; 75.5, C-4', 72.9, C-5'; 81.3, C-6'; 141.3, C-7'; 126.7, C-8'; 144.7, C-9'; 116.9, C-10'; 166.7, C-11'; 14.2, C-12'; 75.7, C13'; 14.6, C-14'; 104.1, C-l"; 75.2, C-2"; 78.5, C-3"; 71.5, C-4"; 78.5, C-5"; and 62.7ppm, C-6". Reference B. B. Jarvis, S. Wang, C. Cox, M. M. Rao, V. Philip, M. S. Varaschin, and C. S. Barros; Brazilian Baccharis toxins: Livestock Poisoning and the Isolation of Macrocyclic Trichothecene Glucosides; Natural Toxins, Vol. 4, pp. 58-71(1996).

17. Miotoxins

473

Common/Systematic Name Miotoxin B Molecular Formula/Molecular Weight C29H3809; M W - 530.25158

~o H

~~: ~ O . . J

13

H

2

3

I"o

L

0

~,11'

0

12'

HO" I " 1; H

General Characteristics Amorphous solidi Rf=0.45 (chloroform-methanol, 100:6) and 0.38 (ethyl acetate-hexane; 3:1, v/v). Plant Source Baccharis coridifolia. Isolation/Purification Inflorescences and seeds ofB. coridifolia were extracted with CHCI3and the extract flash chromatographed over silica gel. Repeated flash chromatography followed by PTLC led to the isolation of miotoxins A-D and iso-miotoxJn D. Spectral Data UV: ~,max~~" 252nm (log e=3.9). IR:

(KBr) 3460, 2960, 1725, 1680, 1630, 1590cm]. 1H Nlh4R: (CDCI3) 0.77(3H, s, H-14); 1.17(3H, d,J=6Hz, H-14'); 1.19(3H, d,J=7I-Iz, H-12'); 1.75(3H, s, H-16); 1.84(2H, m, H-7); 1.95(2H, m, H-8); 2.09(1H, dt, J=5 and 15Hz, H-3I}); 2.3 I(1H, dd, J=5 and 17Hz, H-3a); 2.60, 2.98(1H each, d ofAB, J=8 and 15I-Iz, H-2'); 2.83, 3.14(1H each, AB pattern, J=4Hz, H-13); 3.38(1H, m, H- 3'); 4.26(2H, AB, J=l 8Hz, H-5'); 3.79(2H, m, H-6' and H-13'); 3.70(1H, d, J=6Hz, H-11);

474

17.

Miotoxins

3.51(1H, d, J=5Hz, H-2); 3.73, 4.78(1H each, AB pattern, J=12Hz, H-15); 3.79(1H, m, H-6'); 5.44(1H, d, J=6Hz, H-10); 5.58(1H, dd, J=4.5 and 8Hz, H-4), 5.80(1H, d, J=l 1Hz, H-10'), 5.87(1H, dd, J=6 and 15Hz, H-7'); 6.61(1H, dd, Jg.,9,=J9,,lo,=l1Hz, H9'); and 7.52ppm (1H, m, H-8'). 13C NMR: (CDC13) 79.0, C-2; 36.5, C-3; 76.1, C-4; 48.9, C-5; 43.9, C-6; 20.6, C-7; 28.0, C-8; 141.0, C-9; 119.7, C-10; 67.3, C-11; 65.3, C-12; 48.0, C-13; 7.5, C-14; 63.0, C-15; 23.2, C-16; 172.1, C-I'; 37.1, C-2'; 38.1, C-3'; 211.1, C-4'; 73.2, C-5'; 85.1, C-6'; 136.2, C-7'; 129.8, C-8'; 141.5, C-9'; 118.3, C-10'; 166.5, C-11'; 18.7, C-12'; 70.2, C13'; and 16.8ppm C-14'. References G. G. Habermehl, L. Busam, P. Heydel, D. Mebs, C. H. Tokarnia, J. Dobereiner, and M. Spraul; Macrocyclic Trichothecenes: Cause of Livestock Poisoning by the Brazilian Plant Baccharis coridifolia; Toxicon, Vol. 23, pp. 731-745(1985). G. G. Habermehl and L. Busam; Miotoxins B and C, Two New Macrocyclic Trichothecenes from Baccharis coridifolia DC; Liebigs Ann. Chem., pp. 17461754(1984).

17.

Miotoxins

475

Common/Systematic Name Miotoxin C Molecular Formula/Molecular Weight C31H42011; MW --" 590.27271

~

H

H

~ O ~ J 2

0

~

3

I~ ,,0

, L~ 0~1'' 0

0

12

r

HO"

I

H

~ 14'

General Characteristics Amorphous solid; Rf=0.45 (chloroform-methanol, 100:6) and 0.30 (ethyl acetate-hexane; 3:1, v/v). Plant Source

Baccharis coridif o lia.

Isolation/Purification Inflorescences and seeds ofB. coridifolia were extracted with CHC13 and the extract flash chromatographed over silica gel. Repeated flash chromatography followed by PTLC led to the isolation of miotoxins A-D and iso-nfiotoxdn D. Spectral Data UV:

~,maxEt~ 271nm (log e=4.2). IR:

(KBr) 3400, 2960, 1735, 1712, 1630, 1590cm-1. 1H NMR: (CDC13) 0.79(3H, s, H-14); 1.27(3H, d, J-6Hz, H-14'); 1.20(3H, s, H-12'); 1.76(3H, s, H-16); 1.85-2.12(4H, m, H-7 and H-8); 2.09(3H, s, acetate); 2.22(1H, dt, J=5 and 15Hz, H-3~); 2.49(1H, dd, J=8 and 17Hz, H-3tt); 2.49, 2.67(1H each, AB, J=5Hz, H2'); 2.86, 3.18(1H each, AB pattern, J=4Hz, H-13); 3.60(2H, m, H-5'); 4.05(1H, m, H6'); 3.83(1H, br s, H-2); 3.90(1H, d, J=5Hz, H-11); 4.08, 4.55(1H each, AB pattern,

476

17.

Miotoxins

J=12Hz, H-15); 5.47(1H, d, J=5Hz, H-10); 5.88(1H, dd, J=4.5 and 8Hz, H-4), 5.86(1H, d, J=l 1Hz, H-10'); 5.91(1H, dd, J=3 and 16Hz, H-7'); 6.74(1H, dd, Js, 9,=J9,~o,=11Hz,H-9'); and 7.65ppm (1H, m, H-8'). 13CNMR: (CDCI3) 79.0, C-2; 34.9, C-3; 74.4, C-4; 49.1, C-5; 43.4, C-6; 21.1, C-7; 27.7, C-8; 140.7, C-9; 118.4, C-10; 67.2, C-11; 65.2, C-12; 47.9, C-13; 7.1, C-14; 64.0, C-15; 23.2, C-16; 171.6, C-I'; 43.4, C-2'; 72.2, C-3'; 74.2, C-4'; 72.1, C-5'; 80.8, C-6'; 137.8, C-7'; 126.9, C-8'; 143.7, C-9'; 117.5, C-10'; 166.6, C-11'; 23.2, C-12'; 71.2, C-13'; 15.4, C-14'; 170.3(acetate C=O), and 20.5ppm (acetate Me). References G. G. Habermehl, L. Busam, P. Heydel, D. Mebs, C. H. Tokarnia, J. Dobereiner, and M. Spraul; Macrocyclic Trichothecenes: Cause of Livestock Poisoning by the Brazilian Plant Baccharis coridifolia; Toxicon, Vol. 23, pp. 731-745(1985). G. G. Habermehl and L. Busam; Miotoxins B and C, Two New Macrocyclic Trichothecenes from Baccharis coridifolia DC; Liebigs Ann. Chem., pp. 17461754(1984).

17.

Miotoxins

477

Common/Systematic Name Miotoxin D Molecular Formula/Molecular Weight C29H4009; MW

I-

I:

: 532.26723

~o H

H

"113

I

,~

o

_

H2C I s 0

~,

11'

4~0"~

M

H General Characteristics Amorphous solid. Rf=0.56 (chloroform-methanol, 100:4) and 0.71 (ethyl acetate). Plant Source

Baccharis coridifolia.

Isolation/Purification Inflorescences and seeds ofB. coridifolia were extracted with CHC13 and the extract flash chromatographed over silica gel. Repeated flash chromatography followed by PTLC led to the isolation of miotoxins A-D and iso-rrfioto~dn D. Spectral Data IR:

(KBr) 3420, 1730, 1710, 1640, and 1600cm~. IH NMR: (CDC13) 0.77(3H, s, H-14); 0.98(3H, d, d=7Hz, H-12'); 1.20(3H, d, 3=6Hz, H- 14'); 1.71(3H, s, H-16); 1.82-2.04(4H, m, H-7 and H-8); 2.16(1H, dt, J=5 and 15Hz, H-313); 2.23-2.39(3H, m, H-2' and H-3'); 2.44(1H, dd, d=8 and 15Hz, H-3a); 2.81, 3.13(1H each, AB pattern, J-4Hz, H-13); 3.4-3.6(2H, m, H-5'); 3.70(1H, m, H-6'); 3.6(1H, m, H-11); 3.90, 3.84(1H, m, H-2); 3.90(1H, m, H-4'); 4.62(1H each, AB pattern, J=12Hz, H-15); 5.43(1H, dd, 3=1.1 and 5Hz, H-10); 5.84(1H, dd, 3=5.0, 8.0I-Iz, H-4); 5.77(1H, d, 3=11Hz, H-10'); 6.0(1H, dd, J=3 and 16Hz, H-7'); 6.65(1H, dd, ds. 9~J~,10~11Hz, H-9'); and 7.75ppm (1H, m, 8'-H).

478

17.

Miotoxins

13C NMR: (CDC13) 79.2, C-2; 35.1, C-3; 74.5, C-4; 49.2, C-5; 43.5, C-6; 20.6, C-7; 27.9, C-8; 140.7, C-9; 118.6, C-10; 67.5, C-11; 65.4, C-12; 48.0, C-13; 7.2, C-14; 63.8, C-15; 23.3, C-16; 172.7, C-I'; 37.5, C-2'; 32.9, C-3'; 73.5, C-4'; 71.7, C-5'; 84.8, C-6'; 138.8, C-7'; 126.9, C-8'; 144.0, C-9'; 117.7, C-10'; 166.6, C-11'; 16.1, C-12'; 70.9, C-13'; and 18.5ppm C- 14'. References G. G. Habermehl, L. Busam, P. Heydel, D. Mebs, C. H. Tokarnia, J. Dobereiner, and M. Spraul; Macrocyclic Trichothecenes: Cause of Livestock Poisoning by the Brazilian Plant Baccharis coridifolia; Toxicon, Vol. 23, pp. 731-745(1985). G. G. Habermehl, L. Busam, and M. Spraul; Miotoxin D and iso-MiotoxJn D, Two New Macrocyclic Trichothecene from Baccharis coridifolia DC; Liebigs Ann. Chem., pp. 633639(1985).

17.

Miotoxins

479

Common/Systematic Name Isomiotoxin D Molecular Formula/Molecular Weight C29H4009; M W -- 532.26723

~o H

16"~~~~'~

OJ

I~1 13 I~~J~~;,'"

o

H

2

I

3 r~

!' ~,

0

o-],

H

9

O, 12'

HO"

: " 1; H

General Characteristics Amorphous solid. Rf=0.56 (chloroform-methanol, 100:4) and 0.65 (ethyl acetate). Plant Source Baccharis coridifolia. Isolation/Purification Inflorescences and seeds ofB. coridifolia were extracted with CHC13 and the extract flash chromatographed over silica gel. Repeated flash chromatography followed by PTLC led to the isolation of miotoxins A-D and isomiotoxin D. Spectral Data IR:

(KBr) 3460, 1725, 1710, 1640, and 1600cm~. 1H N M R : (CDCI3) 0.77(3H, s, H-14); 0.98(3H, d, J=7Hz, H-12'); 1.20(3H, d, J=6Hz, H- 14'); 1.71(3H, s, H-16); 1.84-2.04(4H, m, H-7 and H-8); 2.14-2.22(1H, m, H- 3~3);2.382.49(4H, m, H-3a, H-2', and H-3'); 2.81, 3.12(1H each, AB pattern, J=4Hz, H-13); 3.57-3.70(2H, m, H-5'); 3.57-3.70(1H, m, H-11); 3.90, 3.84(1H, m, H-2); 3.743.79(2H, m, H-4' and 6'); 4.62(1H each, AB pattern, J=12Hz, H-15); 5.42(1H, dd, d=l.2 and 5Hz, H-10); 5.80-5.82(1H, rn, H-4); 5.79(1H, d, J=llHz, H-10'); 6.04(1H, dd, J=3 and 16Hz, H-7'); 6.66(1H, dd, Js,9.=J9.lO.=11Hz, H- 9'); and 7.81ppm (1H, m, H-8').

480

17.

Miotoxins

13C NMR: (CDC13) 79.2, C-2; 35.0, C-3; 74.7, C-4; 49.3, C-5; 43.5, C-6; 20.6, C-7; 27.9, C-8; 140.8, C-9; 118.6, C-10; 67.5, C-11; 65.4, C-12; 47.9, C-13; 7.0, C-14; 63.6, C-15; 23.3, C-16; 172.7, C-I'; 38.3, C-2'; 32.4, C-3'; 73.0, C-4'; 74.7, C-5'; 86.0, C-6'; 139.5, C-7'; 126.7, C-8'; 143.8, C-9'; 117.9, C-10', 166.8, C-11'; 14.7, C-12'; 71.0, C-13'; and 18.6ppm C-14'. References G. G. Habermehl, L. Busarn, P. Heydel, D. Mebs, C. H. Tokarnia, J. Dobereiner, and M. Spraul; Macrocyclic Trichothecenes: Cause of Livestock Poisoning by the Brazilian Plant Baccharis coridifolia; Toxicon, Vol. 23, pp. 731-745(1985). G. G. Habermehl, L. Busam, and M. Spraul; Miotoxin D and iso-Miotordn D, Two New Macrocyclic Trichothecene from Baccharis coridifolia DC; Liebigs Ann. Chem., pp. 633639(1985).

17.

Miotoxins

481

Common/Systematic Name Miotoxin E Molecular Formula/Molecular Weight C29H3sO10; MW = 546.24650

~.

~0 H

H

,,iO

O

~2

,

II,

o

0 12

HO" ~1 ~ 1,'

General Characteristics An oil; [0~]D-25 ~ (c=0.40, in CHCI3). Plant Source Female Baccharis coridifolia. Isolation/Purification The 70-100% ethyl acetate in hexane and 10% methanol-methylene chloride fractions from the initial column chromatography of the crude extract (see description under verrucarin A 13-glucoside) were subjected to flash chromatography (silica, 80g, methanolmethylene chloride) to yield various trichothecene-containing fractions which were combined, based on TLC analysis, and subjected to further chromatographic procedures including preparative TLC, filtration reversed phase chromatography, preparative HPLC, and countercurrent chromatography (CCC). From these operations were isolated miotoxins E, F, and G. Spectral Data IR;

(CHC13) 3493, 1743,1706, 1637, 1600, 1175, 1081 cm4. 1H NMR: (CDC13) 7.57(dd, 1H, J=ll.5, 15.5Hz, H-8'), 6.60(dd, 1H, J=ll.5, ll.5Hz, H-9'), 5.80(dd, 1H, J=5.7, 15.5Hz, H-7'), 5.76(d, 1H, J=l 1.5Hz, H-10'), 5.53(dd, 1H, J=4.5, 8.0Hz, H-4), 5.39(br d, 1H, J=5.0Hz, H- 10), 5.09 and 3.47(AB, 1H each, J=12.3Hz, H-15), 4.72 and 4.42(AB, 1H each, J=18.4Hz, H-5'), 3.79(d, 1H, J=4.8Hz, H-2),

482

17.

Miotoxins

3.73(m, 2H, H-6', H-13'), 3.53(br d, 1H, J=5.0Hz, H-11), 3.11 and 2.80(AB, 1H each, J=4.0Hz, H-13), 2.94 and 2.59(AB, 1H each, J=17.6Hz, H-2'), 2.57(dd, 1H, J=8.0, 15.4Hz, H-3a), 2.07(ddd, 1H, J=4.5, 4.8, 15.4Hz, H-3b), 1.80-2.00(m, 4H, H-7, H-8), 1.71(s, 3H, H-16), 1.27(s, 3H, H-12')l.15(d, 3H, J=5.9Hz, H-14'), and 0.71ppm (s, 3H, H-14). 13CNMR: (CDC13) 79.0, C-2; 36.7, C-3; 76.4, C-4; 48.8, C-5; 44.0, C-6; 20.5, C-7; 28.0, C-8; 141.3, C-9; 119.6, C-10; 67.2, C-11; 65.1, C-12; 48.0, C-13; 7.5, C-14; 63.6, C-15; 23.2, C-16; 172.3, C-I'; 43.0, C-2'; 77.0, C-3'; 211.0, C-4'; 69.2, C-5'; 85.7, C-6'; 135.9, C-7'; 130.7, C-8'; 141.6, C-9'; 118.0, C-10'; 166.4, C-11'; 26.0, C-12'; 70.2, C13'; and 18.7ppm, C-14'. Reference B. B. Jarvis, S. Wang, C. Cox, M. M. Rao, V. Philip, M. S. Varaschin, and C. S. Barros; Brazilian Baccharis toxins: livestock poisoning and the isolation of macrocyclic trichothecene glucosides; Natural Toxins, Volume 4, pp. 58-71(1996).

17.

Miotoxins

483

Common/Systematic Name Miotoxin F Molecular Formula/Molecular Weight C29H40010; MW = 548.26215 ~o H

I"OH

12

H

~

HL

H O " ~ ~ 14'

General Characteristics An oil; [t~]D+29 ~ (c=0.87, in CHC13). Plant Source Female Baccharis coridifolia. Isolation/Purification The 70-100% ethyl acetate in hexane and 10% methanol-methylene chloride fractions from the initial column chromatography of the crude extract (see description under verrucarin A ]3-glucoside) were subjected to flash chromatography (silica, 80g, methanolmethylene chloride) to yield various trichothecene-containing fractions which were combined, based on TLC analysis, and subjected to further chromatographic procedures including preparative TLC, filtration reversed phase chromatography, preparative HPLC, and countercurrent chromatography (CCC). From these operations were isolated miotoxins E, F, and G. Spectral Data IR: (CHC13) 3475, 1712, 1636, 1600, 1418, 1375, 1181, and 1081cm"l. IH NMR: (CDC13) 7.78(dd, 1H, J=l 1.3, 15.6 Hz, H-8'), 6.66(dd, 1H, J=l 1.3, 11.3, H-9'); 5.98(dd, 1H, J=2.9, 15.6 Hz, H-7'); 5.81(dd, 1H, J=4.5, 8.1 Hz, H-4); 5.76(d, 1H, J=l 1.3 Hz, H-10'); 5.40(br d, lI-I, J=4.8 Hz, H-10), 4.58 and 3.97(AB, 1H each, J=12.3 Hz, H-15), 4.08(dd, 1H, J=2.0, 9.5 Hz, H-4'), 3.83(d, 1H, J=5.0 Hz, H-2),

484

17.

Miotoxins

3.66(m, 1H, H-6'), 3.62(dq, 1H, J=6.0, 6.9 Hz, H-13'), 3.59(br d, 1H, J=4.8 Hz, H-11), 3.59(dd, 1H, J=2.0, 9.5 Hz, H-5'B); 3.48(dd, 1H, J=-9.5, 9.5 I-~ H-5'A), 3.11 and 2.79(AB, 1H each, J=4.0 Hz, H-13), 2.65 and 2.44(AB, 1H each, J=15.7 Hz, H-2'), 2.41(dd, 1H, J=8.1, 15.4 Hz, H-3a), 2.15(ddd, 1H, J=4.5, 5.0, 15.4 Hz, H-3b), 1.802.00(m, 4H, n-7, H-8), 1.70(s, 3H, H-16); 1.19(d, 3H, J=6.0 Hz, H-14'), 1.12(s, 3H, H-12'); and 0.72ppm (s, 3H, H-14). 13C NMR: (CDC13) 79.1, C-2; 34.9, C-3; 74.6, C-4; 49.1, C-5; 43.9, C-6; 20.6, C-7; 27.8, C-8; 140.8, C-9; 118.2, C-10; 67.3, C-11; 65.2, C-12; 48.0, C-13; 7.2, C-14; 64.4, C-15; 23.3, C-16; 171.9, C-I'; 43:9, C-2'; 72.4, C-3'; 74.4, C-4'; 70.9, C-5'; 85.3, C-6'; 138.8, C-7'; 126.2, C-8'; 144.1, C-9'; 117.5, C-10'; 166.8, C-11'; 22.7, C-12'; 71.4, C-13'; and 18.4ppm, C-14'. Reference B. B. Jarvis, S. Wang, C. Cox, M. M. Rao, V. Philip, M. S. Varaschin, and C. S. Barros; Brazilian Baccharis toxins: livestock poisoning and the isolation of macrocyclic trichothecene glucosides; Natural Toxins, Volume 4, pp. 58-71(1996).

17. Miotoxins

485

Common/Systematic Name Miotoxin G Molecular Formula/Molecular Weight C29H4009; MW = 532.26723

~oH 0 . ~ H2

~6",,,~~/~

3

I, ':1 "-.J...~ I -

l~

12'; , ~ a , 0//'~0

o

' "~o~'~

HI

....

HO%

General Characteristics An oil; [a]D +31 o (C=0.27, in CHCI3). Plant Source Female Baccharis coridifolia. Isolation/Purification The 70-100% ethyl acetate in hexane and 10% methanol-methylene chloride fractions from the initial column chromatography of the crude extract (see description under verrucarin A 13-glucoside) were subjected to flash chromatography (silica, 80g, methanolmethylene chloride) to yield various trichothecene-containing fractions which were combined, based on TLC analysis, and subjected to further chromatographic procedures including preparative TLC, filtration reversed phase chromatography, preparative HPLC, and countercurrent chromatography (CCC). From these operations were isolated miotoxins E, F, and G. Spectral Data IR:

(CHC13) 3481, 1781, 1700, 1643, 1606, 1187, and 1081cm"1. 1H N]VIR:

(CDC13) 7.59(dd, 1H, J-11.3, 15.5 Hz, H=8'); 6.60(dd, 1H, J-11.3,11.3 Hz, H-9)); 6.10(dd, 1H, J=3.8, 8.0 Hz, H-4); 5.83(dd, 1H, J=7.5, 15.5 Hz, H-7'); 5.77(d, II-L d-11.3 Hz, H-10'); 5.47(br d, lI-I, J=5.0 Hz, H-10); 4.16(ddd, lI-I, J=3.3, 5.2, 6.8 Hz, H-4'); 3.89(br d, 1H, ,/=5.0 Hz, H-11); 3.83 and 3.65(AB, 1H each, J=12.5 Hz, H-15);

486

17.

Miotoxins

3.82(d, 1H, J=5.2 Hz, H-2); 3.72(dd, 1H, J=3.3, 11.0 Hz, H-5'B); 3.71(m, 1H, H-13'); 3.66(m, 1H, H-6'); 3.51(dd, 1H, J=5.2, 11.0 Hz, H-5'A); 3.11 and 2.80(AB, 1H each, J=4.0 Hz, H-13); 2.73(dd, 1H, J=8.5, 17.5 Hz, H-2'B); 2.48(dd, 1H, J=8.0, 15.3 Hz, H3a); 2.43(m, 1H, H-3'); 2.17(dd, 1H, J=8.4, 17.5 Hz, H-2'A); 2.07(ddd, 1H, J=3.8, 5.2, 15.3 Hz, H-ab); 1.92-1.99(m, 3 H, n-7a, n-8); 1.70(s, 3H, n-16); 1.54(m, 1n, H-7A); 1.15(d, 3H, J-6.8 Hz, H-12'); 1.13(d, 3H, J=6.2 Hz, H-14'); and 0.81ppm (s, 3H, H14). 13C NMR: (CDC13) 79.0, C-2; 36.0, C-3; 75.4, C-4; 48.9, C-5; 44.2, C-6; 21.2, C-7; 28.0, C-8; 140.6, C-9; 118.7, C-10; 66.8, C-11; 65.6, C-12; 48.1, C-13; 6.8, C-14; 62.8, C-15; 23.3, C-16; 176.3, C-I'; 36.7, C-2'; 32.1, C-3'; 85.8, C-4'; 69.4, C-5'; 85.5, C-6'; 139.0, C-7'; 130.8, C-8'; 143.7, C-9'; 118.7, C-10'; 166.8, C-11'; 18.4, C-12'; 69.7, C-13'; and 18.2ppm, C- 14'. Reference B. B. Jarvis, S. Wang, C. Cox, M. M. Rao, V. Philip, M. S. Varaschin, and C. S. Barros; Brazilian Baccharis Toxins: Livestock Poisoning and the Isolation of Macrocyclic Trichothecene Glucosides; Natural Toxins, Volume 4, pp. 58-71(1996).

Roritoxins Roritoxin Roritoxin Roritoxin Roritoxin

A B C D

487


18.

Roritoxins

489

Common/Systematic Name Roritoxin A Molecular Formula/Molecular Weight C29H340]0, MW = 542.21520 ~o H

H

O-x~OH OH General Characteristics Crystals; mp., 220-225~

[0[,]D 25 -at- 16~ (c = 0.51, in chloroform).

Fungal Source Myrothecium roridum (CL-514; ATCC 20605).

Isolation/Purification At the end of 3 weeks of rice fermentation with M. roridum the culture was extracted with methanol in a sonicator. The methanol extracts were combined, washed with hexane, and concentrated in vacuo to give an aqueous solution which was extracted with ethyl acetate. The ethyl acetate extract was dried and solvent was removed by rotary evaporation to give a brown gum. The crude extract was subjected to filtration chromatography (silica gel) with methanol in dichloromethane as the eluting solvent. Fractions obtained with 1-5% methanol in dichloromethane were combined to give a yellow material. This fraction was subjected to flash chromatography with a step gradient ofisopropyl alcohol in dichloromethane as eluting solvent to obtain five fractions. Fractions 2, 3, and 4 obtained with 3-10% isopropyl alcohol were shown to contain trichothecenes by TLC analysis. These were subjected to further purification. Fraction 2 was triturated with dichloromethane and filtered. The white solid precipitate was recrystallized from methanol to give roritoxin D. The mother liquor from the recrystallization and the filtrate from the previous step were combined and purifed on the Chromatotron (eluting solvent 0-3% methanol in dichloromethane). An additional amount of roritoxin D was obtained, and fractions which were collected after the roritoxin D band were added to fraction 3 for further purification. Fraction 3 was triturated with dichloromethane and filtered. The

490

18.

Roritoxins

dichloromethane insoluble white solid obtained was recrystallized from methanol to yield crystals of a mixture of roritoxin C and D. The filtrate of the dichloromethane solution and the mother liquor of the recrystallization were combined and purified on the Chromatotron, (0-5% methanol in dichloromethane). The roritoxin C containing fraction was recrystallized from ethyl acetate to yield pure roritoxin C. The fractions which, by TLC, contained trichothecenes more polar than roritoxin C were combined with fraction 4. Fraction 4 was crystallized from 95% ethanol to yield roritoxin B. The mother liquor was purified on the Chromatotron (0-7% methanol in dichloromethane as eluting solvent). The roritoxin B containing fraction was recrystallized from ethyl acetate to give pure roritoxin B. Roritoxin A was isolated from a more polar fraction from this chromatography. Biological Activity The roritoxins were acutely cytotoxic against L1210 leukemia cells and they exhibited the following IDs0's: roritoxin A, 0.0011 lag/ml; roritoxin B, 0.00171ag/ml; roritoxin C, 0.00421ag/ml; and roritoxin D, 0.00211xg/ml. Roritoxin B was toxic at 801xg~g in mice against P388 leukemia cells; roritixin B also exhibited toxicity (LDs0 ca. 1001.tg&g) when tested against B 16 melanoma in mice. Spectral Data UW:

X ~"

231 (log e= 4.35) and 260nm (log e= 4.70).

1H NMR: (CDCI3) 3.57(1H, J=5.1Hz, H-2); 2.46(1H, dd, J=8.4, 15.4Hz, H-3ct);- 5.9(1H, H4 ) ; - 1.9(1H, n-7); - 1.9(2H, H-8); 5.45(1H, J=4.8Hz, H-10); 3.85(1H, J=4.8Hz, H11); 3.14(1H, J=a.9Hz, H-13A); 2.83(1H, J=3.9Hz, n-13a); 0.80(3H, s, n-14); 4.50(1H, J=12.4Hz, H-15A);- 3.9(1H, n-15a); 1.73(3H, s, n-16);-5.9(1H, n-2'); 2.2(2H, n-4'); 3.9(2H, n-5'); 6.06(1H, J=16.6Hz, H-7'); 7.34(1H, J=10.6, 16.6Hz, n8'); 6.60(1H, d=10.6, 10.6Hz, H-9'); 5.98(1H, J=10.6Hz, U-10'); 4.48(1H, s, H- 12');5.9(1H, H-13'); and 5.22ppm (1H, J=5.0Hz, H-14'). 13C NMR: (CDC13) 79.2, C-2; 34.6, C-3; 74.4, C-4; 49.1, C-5; 43.5, C-6; 20.4, C-7; 27.7, C-8; 140.2, C-9; 119.0, C-10; 68.1, C-11; 65.4, C-12; 48.0, C-13; 7.5, C-14; 65.3, C-15; 23.2, C-16; 165.5, C-I'; 120.8, C-2'; 151.3, C-3'; 25.7, C-4'; 64.0, C-5'; 81.8, C-6'; 133.4, C-7'; 129.0, C-8'; 141.4, C-9'; 121.9, C-10'; 166.9, C-11'; 81.8, C-12'; 81.0, C13'; and 96.7ppm, C-14'. Mass Spectrum: HREIMS: 542.2145role (M § calcd. 542.2151). Reference B. B. Jarvis and C. S. Yatawara; Roritoxins, New Macrocyclic Trichothecenes from Myrothecium roridum; J. Org. Chem., Vol. 51, pp. 2906-2910(1986).

18.

Roritoxins

491

Common/Systematic Name Roritoxin B Molecular Formula/Molecular Weight C29H34Oll, M W = 558.21011 lo

H ---

0

H 2

3

Io,, O~OH OH General Characteristics Crystals, mp. 262-265~ (dec.); [0I,]D 25 + 2.6~ (c=0.60, in chloroform). Fungal Source Myrothecium roridum (CL-514; ATCC 20605). Isolation/Purification See roritoxin A. Biological Activity See roritoxin A. Spectral Data UV:

L maxMO ' H254nm(log e =4.99). IH NMR: (CDC13) 3.84(1H,J=4.8Hz, H-Z); 2.45(1H, dd, J=8.5, 15.0Hz, H-3a); --~5.9(1H, H4); ~ 1.9(1H, H-7); ~ 1.9(2H, H-8); 5.41(1H, J=5.5Hz, H-10); 3.57(1H, J=5.5Hz, H11); 3.14(1H, J=3.9Hz, H-13A); 2.81(1H, J=3.9Hz, H-13B); 0.81(3H, s, H-14); 4.02(1H, J=12.0Hz, H-15A); 3.94(1H, J=12.0Hz, H-15B); 1.71(3H, s, n-16); 3.3 l(1n, s, n-2'); -~ 2.2(2H, n-4');-3.9(2H, n-5'); 5.87(1H, J=16.4Hz, n-7'); 7.01(1H, J=8.3, 16.4Hz, n-8'); 6.67(1H, J=8.3, 12.2Hz, n-9'); 5.96(1H, J=12.2Hz, H10'); 4.25(1H, s, H-12'); 5.22(1H, J=5.0Hz, H-13'); and 5.93ppm (1H, J=5.0Hz, H-

492

18.

Roritoxins

14'). 13CNM~: (CDC13) 78.4, C-2; 33.9, C-3; 73.9, C-4; 48.7, C-5; 42.8, C-6; 19.2, C-7; 26.8, C-8; 138.8, C-9; 118.9, C-10; 66.6, C-11; 65.3, C-12; 47.3, C-13; 7.7, C-14; 64.2, C-15; 22.8, C-16; 167.3, C-I'; 56.4, C-2'; 62.0, C-3'; 22.8, C-4'; 60.1, C-5'; 82.2, C-6'; 131.2, C-7'; 129.7, C-8'; 143.3, C-9', 120.1, C-10'; 166.5, C-11'; 81.9, C-12'; 78.1, C-13'; and 100.4ppm, C-14'. Mass Spectrum: HREIMS: 558.2106m/e (M +calcd. 558.2139). Reference B. B. Jarvis and C. S. Yatawara; Roritoxins, New Macrocyclic Trichothecenes from Myrothecium roridum, J. Org. Chem., Vol. 51, pp. 2906-2910(1986).

18.

Roritoxins

493

Common/Systematic Name Roritoxin C Molecular Formula/Molecular Weight C29H32012; M W = 572.18938

~

,. . . . .

H2C,

t

~ J]'"'

O~/~--OH 0 General Characteristics Crystals; mp., 288-290~ (dec.); [0l]D25 + 8.9~ (c= 0.40, in chloroform). Fungal Source Myrothecium roridum (CL-514, ATCC 20605). Isolation/Purification See roritoxin A. Biological Activity See roritoxin A. Spectral Data UV:

~, maxMeOH254nm (log e = 4.97). 1H NMR: (CDCI3) 3.93(1H, J=4.8Hz, H-Z),-- 2.4(1H, H-3~);-- 6.1(1H, H-4),-- 1.9(1H, H-V), --~1.9(2H, H-8); 3.06(1H, J=5.0Hz, H-10), 3.56(1H, J=5.0Hz, H-11); 3.14(1H, J=3.8Hz, H-laA); 2.81(1H, J=a.8Hz, H-laB); 0.82(3H, s, H-14); 4.41(1H, J=12.6Hz, H-15A); 4.32(1H, J=12.6Hz, H-15B); 1.55(3H, s, n-16); 3.36(1H, s, n-2'),- 2.4(2H, n-4');-~a.9(En, n-5'); 5.82(1H, J=16.0Hz, n-7'); 7.10(1H, J-8.0, 16.0Hz, n-8'); 6.68(1H, J=8.0, 11.0Hz, H-9'); 5.97(1H, J=l 1.0Hz, H-10'); 4.24(1H, s, H-12'); and

494

18.

Roritoxins

5.40ppm (1H, s, H- 13'). 13C NMR: (Roritoxin C Acetate) (CDC13) 78.8, C-2; 34.2, C-3; 73.1, C-4; 49.0, C-5; 42.9, C-6; 17.4, C-7; 26.2, C-8; 57.4, C-9; 57.7, C-10; 68.2, C-11; 64.8, C-12; 48.0, C-13; 8.0, C-14; 64.7, C-15; 22.2, C-16; 166.8, C-I'; 57.2, C-2'; 60.7, C-3'; 22.3, C-4'; 61.4, C-5'; 80.6, C-6'; 132.5, C-7'; 127.3, C-8', 142.2, C-9'; 122.0, C-10'; 166.6, C-11'; 79.5, C-12'; 74.1, C-13'; 169.7, C14'; 20.4, CH3COO-; and 168.6ppm, CH3COO-.

Mass Spectrum: HREIMS: 572.1955m/e (M +calcd. 572.1974). Reference B. B. Jarvis and C. S. Yatawara; Roritoxins, New Macrocyclic Trichothecenes from Myrothecium roridum; J. Org. Chem.; Vol. 51, pp. 2906-2910(1986).

18.

Roritoxins

495

Common/Systematic Name Roritoxin D Molecular Formula/Molecular Weight C29H32Oll, MW = 556.19446 v

~o H

H 0

o'"

IS O,~OH 0

General Characteristics Crystals; mp., 294-297~ (dec.); [G/,]D25 Jr- 3 0 . 0 ~ ( C - - 0 . 1 0 , in chloroform). Fungal Source Myrothecium roridum (CL-514; ATCC 20605). Isolation/Purification See roritoxin A. Biological Activity See roritoxin A. Spectral Data UV: ~,maxM~~ 253nm (log e = 4.98). IH N]VIR: (CDC13) 3.84(1H, J=4.6Hz, H-2); 2.45(1H, J-8.6, 15.2Hz, H-3a); ~5.8(1H, H- 4); ~2.0(1H, H-7); ~2.0(2H, H-8); 5.41(1H, J-5.0Hz, H-10); 3.57(1H, J-5.0Hz, H-11); 3.14(1H, J-3.9Hz, H-13A); 2.81(1H, J=3.9Hz, H-13B); 0.82(3H, s, H-14); 4.32(1H, J=12.5Hz, H-15A), 4.20(1H, J=12.5Hz, H-15B); 1.71(3H, s, H-16); 3.36(1H, s, H-2'); ~2.0(2H, H-4'); ~4.0(2H, H-5'); 5.88(1H, J=16.4Hz, H- 7'); 7.10(1H, J=8.2, 16.4Hz, H-8'); 6.67(1H, J-8.2, 11.1Hz, H-9'); 6.01(1H, J=l 1.1Hz, H-10'); 3.87(1H, s, H-12');

496

18.

Roritoxins

and 5.27ppm (1H, s, H-13'). 13CNMR: (Roritoxin D Acetate) (CDCI3) 79.1, C-2; 34.3, C-3; 73.2, C-4; 49.3, C-5; 43.3, C-6; 20.4, C-7; 27.4, C-8, 140.5, C-9; 118.5, C-10; 67.8, C-11, 65.3, C-12; 48.0, C-13; 7.9, C-14, 65.2, C-15, 22.2, C-16; 166.7, C-I'; 57.3, C-2'; 60.4, C-3'; 23.3, C-4'; 61.3, C-5'; 80.5, C-6'; 132.5, C-7'; 127.0, C-8'; 142.0, C-9'; 122.1, C-10'; 166.4, C-11'; 79.4, C-12'; 74.0, C-13'; 169.6, C- 14'; 20.0, CH3COO-; and 168.8ppm, CH3COO-. Mass Spectrum: HREIMS: 556.1940m/e (M +calcd. 556.1980). Reference B. B. Jarvis and C. S. Yatawara; Roritoxins, New Macrocyclic Trichothecenes from Myrothecium roridum; J. Org. Chem., Vol. 51, pp. 2906-2910(1986).

Myrotoxins and Mytoxins Myrotoxin A Myrotoxin B Myrotoxin C Myrotoxin C Hydrate Myrotoxin D Myrotoxin D Hydrate Mytoxin A Mytoxin B Mytoxin C

497


19. Myrotoxins and Mytoxins

499

Common/Systematic Name Myrotoxin A Molecular formula./Molecular Weight C27H3209; ~

~o H

-- 5 0 0 . 2 0 4 6 3

H

O.,J

I

~

,,:o I o

OH General Characteristics Colorless solid; mp., 220-222~

[(~]D +122.0 (c-0.50, in CH2C12).

Fungal Source

Myrothecium roridum (ATCC 60379).

Isolation/Purification An aqueous shake culture ofMyrothecium roridum (ATCC 60379) was filtered and supernatant extracted with ethyl acetate and the mycelium with acetone. The extracts were combined, concentrated, and subjected to filtration chromatography (silica gel, ethyl acetate-hexane) followed by MPLC (ethyl acetate-hexane through 10% MeOH/CH2C12). The 50% ethyl acetate-hexane fraction was subjected to MPLC (silica gel, 1-5% MeOH/CH2C12) followed by PTLC of a latter fraction (silica gel, 20-40% ethyl acetate/hexane followed by alumina, 20-60% ethyl acetatel-hexane) giving myrotoxins A and B. Biological Activity Cytotoxic: IDs0 = 5 x 10"4 pg/mL, L-1200 cells. Spectral Data IR~

(CHEC12) 3590(OH), 1760, and 1715cm1 (C=O's). 1H NMR:

(CDCI~) 0.80(3H, s, H-14); 1.sa(aH, s, H-16); 1.90(2H, m, H-4'B); 2.0 - 2.1(4H, m,

500


H-7 and H-8); 2.17(1H, ddd, J=3.8, 5.0, 15.5Hz, H-3); 2.48(1H, dd, J=8.2, 15.5Hz, H3); 2.60(1H, ddd, 3'=2.0, 9.0, 14.6Hz, H-8q3); 2.84(2H, m, H-4'A); 3.36(1H, s, H-2'); 3.63[1H, s(atter D20 exchange], H-12'); 3.69(1H, d, 3"=4.2Hz, H-11); 3.85(1H, d, 3"=5.0Hz, H-2); 2.79 and 3.13(1H each, AB,3"=4.0Hz,H-13); 3.85 and 4.68(1H each, AB, 3'=12.0Hz, H-15), 4.00(1H, m, H-5'B); 4.12,(1H, dddd, 3'=1.5, 8.8, 9.8, 14.6Hz, H-8'A); 4.25(1H, m, H-5'A); 4.65(1H, dd, 3"=2.0, 9.8Hz, H-7'); 5.42(1H, d, 3"=4.2Hz, H-10), 5.85(1H, dd, 3"=3.8 and 8.2Hz, H-4); 5.93(1H, dd, ,/=1.5, 11.2Hz, H-10); and 6.59ppm (1H, ddd, 3"=8.8, 9.0, 11.2Hz, H-9'). laC NMR: (CDCI3) 79.1, C-2; 35.1, C-3; 74.8, C-4; 49.5, C-5; 43.2, C-6; 20.2, C-7; 27.4, C-8; 140.6, C-9; 118.5, C-10; 67.4, C-11; 65.4, C-12; 47.6, C-13; 7.8, C-14; 64.2, C-15; 23.3, C-16; 164.7, C-I'; 57.1, C-2'; 65.3, C-3'; 23.5, C-4'; 67.2, C-5'; 150.9, C-6'; 109.8, C-7'; 24.3, C-8'; 148.7, C-9'; 121.9, C-10'; 167.1, C-11'; and 72.1ppm, C-12'. References B. B. Jarvis, F. T. Comezoglu, Y.-W. Lee, J. L. Flippen-Anderson, R. D. Gilardi, and C. F. George; Novel Macrocyclie Trichothecenes from Myrotheciumroridum;Bull. Soe.Chim. Belg., 95, pp. 681-697(1986). B. B. Jarvis, Y.-W. Lee, F. T. Comezoglu, S. N. Comezoglu, and G. A. Bean; Myrotoxins: A New Class ofMacrocyclic Trichothecenes; Tetrahedron Lett., Vol. 26, pp4859-4862(1985).

19.

Myrotoxins and Mytoxins

501

Common/Systematic Name Myrotoxin B Molecular Formula/Molecular Weight C29H34Oll; M W = 558.21011

16

10 H

H

AcO,,,~,~"2"04 I H2(X, j

.O ~ k L 11'

~

HO' ""~ General Characteristics Colorless solid; mp., 195-197~

[c~]D = +10117~ (c=0.60 in CH2C12).

Fungal Source Myrothecium roridum (ATCC 60379 and ATCC 52485). Isolation/Purification An aqueous shake culture ofMyrothecium roridum (ATCC 60379) was filtered and the supematant extracted with ethyl acetate and the mycelium extracted with acetone. The extracts were combined, concentrated, and subjected to filtration chromatography (silica gel, ethyl acetate-hexane) followed by MPLC (ethyl acetate-hexane through 10% methanol-methylene chloride). The 50% ethyl acetate-hexane fraction was subjected to MPLC (silica gel, 1-5% methanol-methylene chloride) followed by PTLC of a latter fraction (silica gel, 20-40% ethyl acetate-hexane followed by alumina, 20-60% ethyl acetate-hexane) giving myrotoxins A and B. Biological Activity Immunotoxirg cytotoxic, LDs0 in mice = 1.0mg/kg, inhibition ofblastogenesis; LDs0 = 1l~g/rat intracerebrally. Spectral Data IR~

(CH2CI2) 3600 (br, OH), 1710, 1730, and 1755cm~ (C=O's).

502


1H NMR: (CDC13) 0.79(3H, s, H-14); 1.76(3H, s, H-16); 1.89(2H, m, H-4'B); 2.1(2H, m, H-7); 2.17(1H, ddd, J=3.6, 5.2, and 15.7Hz, H-313);2.49(1H, dd, J=8.1 and 15.7Hz, H-3a); 2.61(1H, ddd, J=2.0, 9.0, and 14.6Hz, H-8'B); 2.86(2H, m, H- 4'A); 3.32(11-1, s, H-2'); 3.63(1H, s(atier D20 exchange), H-12'); 3.68(1H, d, J=5.4Hz, H-11); 3.85(1H, d, J=5.2 Hz, H-2); 2.80 and 3.12(1H each, AB, J=4.0 Hz, H-13); 3.92 and 4.89(1H each, AB, J=12.0Hz, H-15); 3.98(1H, m, H-5'B); 4.11(1H, dddd, J=l.6, 8.7, 9.8, and 14.6Hz, H-8'A); 4.28(1H, m, H-5'A); 4.65(1H, dd, J=2.0 and 9.8Hz, H-7'); 5.28(1H, d, J=5.2Hz, H-8); 5.66(1H, d, J=5.4Hz, H-10); 5.83(1H, dd, J=3.6 and 8.1Hz, H-4); 5.92(1H, dd, J=l.6 and 11.2Hz, H-10'); 6.60ppm (1H, ddd, J=8.7, 9.0, and 11.2Hz, H9'). 13CNMR: (CDCI3) 78.9, C-2; 35.0, C-3; 74.8, C-4; 49.5, C-5; 42.0, C-6; 23.4, C-7; 68.1, C-8; 136.7, C-9; 164.5, C-I'; 56.7, C-2'; 65.2, C-3'; 23.2, C-4'; 67.1, C-5'; 150.9, C-6'; 109.7, C-7'; 26.7, C-8'; 149.0, C-9'; 121.9, C-10'; 166.8, C-11'; 72.1, C-12'; 20.5, acetate methyl; and 170.8ppm, acetate C=O. References B. B. Jarvis, F. T. Comezoglu, Y.-W. Lee, J. L. Flippen-Anderson, R. D. Gilardi, and C. F. George; Novel Macrocyclic Trichothecenes from Myrotheciumroridum;Bull. Soc.Chim. Belg.,Vol. 95, pp. 681-697(1986). B. B. Jarvis, Y.-W. Lee, F. T. Comezoglu, S. N. Comezoglu, and G. A. Bean; Myrotoxins: A New Class of Macrocyclic Trichothecenes; Tetrahedron Lett., Vol. 26, pp4859-4862(1985).


503

Common/Systematic Name Myrotoxin C Molecular Formula/Molecular Weight C27H3209; M W -- 5 0 0 . 2 0 4 6 3

~~

~o H

H

~

~~)~,,;,,0 H2(~

l'

0

4~ J'L'"

OH General Characteristics Colorless solid; mp., 240~ (dec.);

[~]D =

+124.5 ~ (c=0.98, in CH2C12).

Fungal Source Myrothecium roridum (ATCC 60379). Isolation/Purification An acetone extract of a rice culture ofMyrothecium roridum (ATCC 60379) was concentrated and partitioned between aqueous NaHCO3 and ethyl acetate; the ethyl acetate solubles were subjected to filtration chromatography (silical gel, ethyl acetatehexane) followed by MPLC (ethyl acetate-hexane through 10% MeOH-CH2C12). The 50% ethyl acetate-hexane fraction was subjected to MPLC (silica gel, 1-5% MeOH-CH2C12) followed by PTLC of a latter fraction (silica gel, 20-40% ethyl acetate-hexane) to give myrotoxin C. A later MPLC fraction gave myrotoxin D upon PTLC (alumina, 20-60% ethyl acetate-hexane). Biological Activity Cytotoxic. Spectral Data IR~

(CH2C12) 3580, 2900, 1750, 1715, 1642, 1405, 1190, 1160, 1110, 1095, 1035, 992, 970, and 670 cm~

504


:H NMR: (CDCI3) 0.Sl(3H, s, H-14); 1.73(3H, s, H-16); 1.8(1H, m, H-SB), 1.9(2H, m, H- 7); 2.0(2H, m, H-SA and H-4~B); 2.15(1H, ddd, J=3.9, 5.2, 15.5Hz, H-3A); 2.36(1H, dd, J=8.2, 15.SHz, H-3); 2.57(1H, m, H-8~B); 2.80, 3.13(1H each, AB, J=4.1Hz, H-13); 3.57(1H, d, J--4.1Hz, H-11); 3.79(1H, s, H-2H); 3.84(1H, J=12.1Hz, n-15a); 3.85(1I-I, d, J=5.2Hz, n-2); 3.93(1H, ddd, J=2.4, 11.1, 13.1Hz, H-5~B); 4.15(1H, m, H-5'A); 4.20(1H, m, H-8'A); 4.43(1H, s after D20 exchange, H-12'); 4.58(1H, d, J=12.1Hz, H-15A); 4.84(1H, ddd, J=-l.8, 1.8, 10.0Hz, H-7'); 5.41(1H, d, J=4.1Hz, H10); 5.86(1H, dd, J=3.9, 8.2Hz, H-4), 5.91(1H, dd, J=l.5, 11.2Hz, n-10'), 6.62ppm (1H, ddd, J=8.9, 8.9, 11.2, H-9'). 13CNMR: (CDCla) 79.1, C-2; 35.0, C-3; 72.5, C-4; 49.5, C-5; 43.1, C-6; 20.1, C-7; 27.4, C-8; 140.6, C-9; 118.4, C-10; 67.4, C-11; 65.3, C-12; 47.6, C-13; 7.7, C-14; 63.9, C-15; 23.2, C-16; 164.7, C-I'; 52.3, C-2'; 66.5, C-3'; 23.9, C-4'; 66.8, C-5'; 151.1, C-6'; 104.1, C-7'; 28.5, C-8'; 149.4, C-9'; 121.4, C-10'; 168.4, C-11'; and 67.Sppm C-12'. Reference B. B. Jarvis, F. T. Comezoglu, Y.-W. Lee, J. L. Flippen-Anderson, R. D. Gilardi, and C. F. George; Novel Macrocyclic Trichothecenes from Myrotheciumroridum;Bull. Soc. Chim. Belg., Vol. 95, pp. 681-697(1986).


505

Common/Systematic Name Myrotoxin C hydrate Molecular Formula/Molecular Weight C27H34010; M W -- 5 1 8 . 2 1 5 2 0

0=4

No~ OH

~' OH

General Characteristics Colorless solidi mp., 178-182~

[a]D =

+29.5 ~ (c=0.44, in CH2C12).

Fungal Source Myrothecium roridum (ATCC 60379 and ATCC 52485). Isolation/Purification A fraction from a rice culture ofM. roridum (ATCC 60379) rich in myrotoxins C and D was allowed to stand at room temperature in a mixture of acetone and chloroform. The solvent was removed, and the mixture subjected to MPLC (150g of 13-25gt silica gel, elution with 40-60% ethyl acetate-hexane) followed by PTLC (2mm alumina plate, elution with 40-70% ethyl acetate-hexane) to give 80mg of myrotoxin C hydrate and 60mg of myrotoxin D hydrate. Spectral Data IR:

(CHECI2) 3590(OH), 1750, and 1710cml(c=O's). 1H ~ : (CDCl3) 0.82(3H, s, H-14); 1.71(3H, s, H-16); 1.6-2.4(10R m, H-313, H-7, H-8, H4', H-7', H-8'A); 2.43(1H, dd, J=8.5 and 15.4Hz, H-3A); 3.00(1H, m, H-8'B); 2.80 and 3.12(1H each, AB, J=4.0Hz, H-13); 3.54(1H, d, ,/--5.4 Hz, H-11); 3.80(1H, s alter D20 exchange, H-12'); 3.83(1H, s, H-2'); 3.85(2H, m, H-5'); 3.85(1H, d, J=5.3Hz, H2); 4.03 and 4.56(1H each, AB, J=12.3Hz, n-15); 5.40(1H, d, J=5.4Hz, H-10); 5.90(1H, dd, ,/--1.8 and 11.4Hz, H-10'); 5.93(1I-I, dd, J=4.5 and 8.5Hz, H-4); and 6.60ppm (1H, ddd, J=8.8, 8.8, and 11.4Hz, H-8').

506


13CNMR.: (CDCI3) 8.0, C-14; 19.8, C-7'; 20.1, C-7; 23.2, C-16; 25.7, C-4'; 27.4, C-8; 27.7, C8'); 34.4, C-3; 43.2, C-6; 47.8, C-13; 49.8, C-5; 53.5, C-2'; 60.3, C-5'; 64.1, C-15; 64.3, C-3; 65.2, C-12; 67.3, C- 11; 72.8, C-4; 74.8, C-12'; 79.1, C-2; 100.6, C-6', 118.3, C-10; 120.8, C-10'; 140.5, C-9; 150.9, C-9'; 166.5, C-I'; and 168.2ppm C-11'. References B. B. Jarvis, F. T. Comezoglu, S. Wang, and H. L. Ammon; Mycotoxins from a Plant Pathogenic Isolate ofMyrotheciumroridum;Mycotoxin Res., Vol. 7, pp. 73-78(1991). F. T. Comezoglu; Triehothecenes: The Isolation of Myrotoxins and Mytoxins; Mechanistic Studies of Rearrangement and Biosynthesis; Ph.D. Thesis, University of Maryland, College Park, MD.

19.


507

Common/Systematic Name Myrotoxin D Molecular formula/Molecular Weight C29H34Oll; M W -- 558.21011

~0.,,~ 2 3 Ac ~'~"04~ H~, 16

10

H

H

'

ON General Characteristics Colorless solid; mp., 232-236~

[~]D--

+ 106.0~ (C=0.10, in CHC13).

Fungal Source Myrothecium roridum (ATCC 60379). Isolation/Purification An acetone extract of a rice culture ofMyrothecium roridum (ATCC 60379) was concentrated and partitioned between aqueous NaHCO3 and ethyl acetate; the ethyl acetate solubles were subjected to filtration chromatography (silical gel, ethyl acetatehexane) followed by MPLC (ethyl acetate-hexane through 10% methanol-methylene chloride). The 50% ethyl acetate-hexane fraction was subjected to MPLC (silica gel, 1-5% methanol-methylene chloride) followed by PTLC of a latter fraction (silica gel, 20-40% ethyl acetate-hexane) to give myrotoxin C. A later MPLC fraction gave myrotoxin D upon PTLC (alumina, 20-60% ethyl acetate-hexane). Biological Activity Cytotoxic. Spectral Data IR:

(CHC13) 3580,2900, 1750, 1710, 1640, 1370, 1160, 1115, 1095, 1000, and 975cm1. 1H NIVIR: (CDC13) 0.78(3H, s, H-14); 1.73(3H, s, H-16); 1.95(3H, s, CH3-acetate); 2.06(1H, m,

508


H-4~B); 2.07(1H, m, H-7B); 2.17(1H, m, H-7A); 2.15(1H, m, H-313); 2.32(1H, m, H4'A); 2.46(1H, dd, J=8.1 and 15.6Hz, n-3tt); 2.55(1H, m, H-8~B); 2.79(1H, d, J=4.0Hz, H-13B); 3.08(1H, d, J--4.0Hz, H-13A); 3.65(1H, d, J=5.4Hz, H-11); 3.74(1H, s, H-2'); 3.82(1H, d, J=5.3Hz, H-2); 3.86, 4.87(1H each, AB, J=l 1.9Hz, H15); 3.87(1H, m, H-5~B);4.12(2H, m, H-5'A and H-8'A); 4.40(1H, s after D20 exchange, H-12'); 4.80(1H, ddd, J=1.9, 1.9, and 9.9Hz, H-7'); 5.25(1H, d, J=5.0Hz, H8); 5.63(1H, d, J=-5.4Hz, n-10); 5.80(1H, dd, J=-3.6 and 8.1Hz, H-4); 5.88(1H, dd, ,/=1.6 and 11.2Hz, H-10'); 6.60ppm (lI-I, ddd, J=8.8, 8.8, and 11.2Hz, H-9'). 13C NMR: (CDCI3) 79.0, C-2; 35.0, C-3; 72.0, C-4; 49.5, C-5; 42.0, C-6; 24.0, C-7, 68.1, C-8; 136.8, C-9; 123.7, C-10; 66.8, C-11; 65.2, C-12; 47.5, C-13; 7.7, C-14; 65.9, C-15; 21.1, C-16; 164.6, C-I'; 51.9, C-2'; 66.9, C-3'; 26.8, C-4'; 66.9, C-5'; 151.2, C-6'; 104.2, C-7'; 28.2, C-8'; 149.7, C-9'; 121.5, C-10'; 166.2, C-11'; 67.9, C-12'; 20.5, acetate Me; and 170.8ppm, acetate C=O.

Reference B. B. Jarvis, F. T. Comezoglu, Y.-W. Lee, J. L. Flippen-Anderson, R. D. Gilardi, and C. F. George; Novel Macrocyclic Trichothecenes from Myrothecium roridum;Bull. Soc. Chim. Belg., Vol. 95, pp. 681-697(1986).

19.


509

Common/Systematic Name Myrotoxin D hydrate Molecular Formula/Molecular Weight C29H36012; M W -- 5 7 6 . 2 2 0 6 8

6

10 H

H

AcO,,.~"2"O I H2C I

ol,,

O kLl"

General Characteristics Colorless solidi mp., 225-229~

[a]D

--

+31.5~ (c=0.92, in CH2C12).

Fungal Source Myrothecium roridum (ATCC 60379 and ATCC 52485). Isolation/Purification A fraction from a rice culture ofM. roridum (ATCC 60379) rich in myrotoxins C and D was allowed to stand at room temperature in a mixture of acetone and chloroform. The solvent was removed, and the mixture subjected to MPLC (150g of 13-251x silica gel, elution with 40-60% ethyl aeetate-hexane) followed by PTLC (2mm alumina plate, elution with 40-70% ethyl aeetate-hexane) to give 80mg of myrotoxin C hydrate and 60mg of myrotoxin D hydrate. Spectral Data IR:

(CH2C12) 3590 (OH), 1750, 1730, and 1710cm1 (C=O's).

IH ~ : (CDC13) 0.80(1H, s, H-14); 1.75(3H, s, H-16); 1.93(3H, s, acetate CH3), 1.6-2.4(9H, m, H-313, H-7, H-4', H-7', and H-8A); 2.45(1H, dd, J=8.3 and 15.3H_z, H-3a); 2.97(1H, m, H-8~B); 2.81, 3.10(1H each, AB, J=4.0Hz, H-13); 3.65(1H, d, J=5.4I-Iz, H-11); 3.80(1H, s after D20 exchange, H-12'); 3.80(1H, s, H-2'); 3.85(1H, m, H-5'); 3.83(1H, d, J=-5.3Hz, H-2); 4.09, 4.82(1H each, AB, J=12.3Hz, H-15); 5.30(1H, d, J=4.2Hz, H-8); 5.66(1H, d, J=5.4 Hz, H-10); 5.89(1H, dd, J=l.8 and 11.4Hz, H-10'); 5.93(1H, dd, J=4.5 and 8.3H~ H-4); and 6.60ppm (1H, ddd, Js.9,s.9=l1.4Hz, H-9').

510


13C NMR: (CDC13) 8.0, C- 14; 20.2, C-7'; 20.4, acetate CH3; 20.9, C- 16; 25.8, C-7; 26.4, C-4'; 27.4, C-8'; 34.5, C-3; 42.0, C-6; 47.7, C-13; 49.8, C-5; 53.0, C-2'; 60.6, C-5'; 64.3, C3'; 65.1, C-12; 66.0, C-15; 66.9, C-11; 68.2, C-8; 72.5, C-4; 75.4, C-12'; 79.1, C-2; 100.5, C-6'; 120.9, C-10'; 123.7, C-10; 136.7, C-9; 150.9, C-9'; 166.5, C-I'; 167.9, C11'; and 170.7ppm, acetate C=O. References B. B. Jarvis, F. T Comezoglu, S. Wang, and H. L. Ammon; Mycotoxins from a Plant Pathogenic IsolateofMyrotheciumroridum;Mycotoxin Res., Vol. 7, pp. 73-78(1991). F. T Comezoglu; Trichothecenes: The Isolation of Myrotoxins and Mytoxins; Mechanistic Studies of Rearrangement and Biosynthesis; Ph.D. Thesis, University of Maryland, College Park, MD.


511

Common/Systematic Name Mytoxin A Molecular Formula/Molecular Weight C29H36010; M W = 544.23085

~o H

H ~,~,,0

I \

0

'11.

0

~

r

H" OH ~2-Me

General Characteristics Amorphous colorless solid; [C~]D+22.9 ~ (c--0.96, in CH2C12). .Fungal Source Myrothecium roridum (ATCC 60379). Isolation/Purification An aqueous shake culture ofMyrothecium roridum was filtered and the supernatant extracted with ethyl acetate; the mycelium extracted with acetone. The two extracts were combined, concentrated, and subjected to filtration chromatography (silica gel, ethyl acetate-hexane) followed by MPLC (ethyl acetate-hexane through 10% methanolmethylene chloride). The 50% ethyl acetate-hexane fraction was subjected to MPLC (silica gel, 1-5% methanol-methylene chloride) followed by PTLC of a latter fraction (silica gel, 20-40% ethyl acetate-hexane followed by alumina, 20-60% ethyl acetatehexane) to give mytoxins A and B. Mytoxin C was isolated by PTLC (silica gel, ethyl acetaate-hexane followed by silica gel, methanol-methylene chloride) of an earlier MPLC fraction. Biological Activity Cytotoxic.

512


Spectral Da.ta IR;

(CH2C12) 3580(OH), 1750, and 1710cm~(C=O). 1H NMR:

(CDCI3) 0.79(3H, s, H-14), 1.58(1H, m, H-8'B), 1.67(1H, m, H-7'B), 1.67-2.00(6H, m, H-7, H-8, H-4B and H-7'B); 1.71(3H, s, H-16); 2.16(1H, ddd, J=4.5, 5.0, 15.4Hz, H-3~); 2.29(3H, s, H-14'); 2.33(1H, m, H-7'A); 2.56(1H,ddd, ,/-9.6, 9.6, and 14.1Hz, H-4'A); 2.79(1H, d, J=4.0Hz, H-13B), 3.02(1H, m, H-8'A), 3.12(1H, d, J=4.0Hz, H13A); 3.23(1H, s(aiter D20 exchange), H-12'); 3.42(1H, s, H-2'); 3.54(1H, d, d=5.1Hz, H-11), 3.84(1H, d, J=5.0Hz, H-2); 4.03(2H, d, J=9.6Hz, H- 5"s), 4.04(1H, d, J=12.3Hz, H-15B); 4.54(1H, d, J=12.3Hz, H-15A), 5.40(1H, d, J=5.1Hz, H-10), 5.85(1H, dd, J=l.8, 11.5Hz, H-10'); 5.92(1H, dd, J=4.5, 8.4Hz, H-4); and 6.44 ppm (1H, ddd, J=7.6, 9.1, 11.5Hz, H-9'). 13CNMR: (CDC13) 79.0, C-2; 34.4, C-3; 72.9, C-4; 49.6, C-5; 43.2, C-6, 19.9, C-7; 27.3, C-8; 140.5, C-9; 118.3, C-10; 67.3, C-11; 65.2, C-12; 47.8, C-13; 7.9, C-14; 64.4, C-15; 23.3, C-16; 166.4, c-r; 58.7, C-2'; 63.9, C- 3'; 22.5, C-4'; 60.3, C-5'; 87.2, C-6'; 22.3, C-7'; 26.4, C-8'; 149.7, C-9'; 120.7, C-10'; 166.7, C-11'; 75.1, C-12'; 212.6, C-13'; and 28.6ppm, C-14'. Mass Spectrum: HRNICIMS: 544.2295m/e for C29H36010;calcd 544.2308. Reference B. B. Jarvis, F. T. Comezoglu, Y.-W. Lee, J. L. Flippen-Anderson, R. D. Gilardi, and C. F. George; Novel Macrocyclic Trichothecenes from Myrothecium roridum; Bull. Soc. Chim. Belg., Vol. 95, pp. 681-697(1986).

19.


513

Common/Systematic Name Mytoxin B Molecular Formula/Molecular Weight C29H3609, M W = 528.23 593 10

H -

O.J

13

~1~

~ ~

H

~

!

n

illi 1~'j

0

o=4: ,/--Xo H" OH Xl~Me 0 14'

General Characteristics Colorless solid, mp., 158-161~ [a]D25= +15.4~ (c=0.78, in CH2C12). Fungal Source Myrothecmm roridum. Isolation/Purification See mytoxin A. Biological Activity Cytotoxic. Spectral Data IR:

(CH2C12) 3600, 2925, 1720, 1655, 1640, 1355, 1230, 1180, 1165, 1085, 1055, 1030, 1000, 910, and 820cm"1. 1H ~ : (CDC13) 0.77(3H, s, H-14); 1.58(1H, ddd, J-3.3, 13.4, 13.4Hz, H-7'B), 1.70(3H, s, H-16), 1.73-2.02(5H, m, H-7, H-8 and H-8'B); 2.11(1H, ddd, J=3.6, 5.3, 15.6Hz, H313); 2.29(3H, s, H-14'), 2.30(1H, m, H-7'A), 2.45(1H, dd, J=8.1,15.6Hz, H-3t~), 2.69(1H, m, H-4~B); 2.72(1H, m, H-8'A), 2.78, 3.10(1H each, AB, J=4.0Hz, H-13); 3.57(1H, d, J=4.0 Hz, H-11); 3.63(1H, d, J-13.6Hz, H-4'A), 3.82(1H, d, J=5.3Hz, H2), 3.96, 4.21(1H each, AB, J=12.6Hz, H-15), 4.02(1H, s after D20 exchange, H-12');

514


4.04(2H, dd, J=2.0, 8.1Hz, H-5'); 5.41(1H, d, J=4.0Hz, H-10); 5.75(1H, s, H-2'); 5.76(1H, dd, J=3.6, 8.1Hz, H-4); 5.80(1H, dd, J=2.0, 11.7Hz, H-10'); and 6.37ppm (1H, ddd, J=6.6, 9.1, 11.7Hz, H-9'). 13CNMR: (CDCIa) 79.1, C-2; 35.0, C-3; 73.4, C-4, 49.5, C-5, 42.9, C-6, 20.6, C-7; 27.5, C-8, 140.3, C-9, 118.8, C-10, 67.7, C-11; 65.3, C-12, 47.6, C-13, 7.7, C-14, 64.3, C-15, 23.3, C-16, 165.9, C-I'; 117.3, C-2'; 154.3, C-3'; 25.7, C-4'; 63.2, C-5'; 87.7, C-6'; 22.0, C-7'; 27.9, C-8'; 148.7, C-9'; 121.3, C-10', 166.3, C-11', 77.3, C-12', 213.1, C13'; and 28.6ppm, C-14'. Reference B. B. Jarvis, F. T. Comezoglu, Y.-W. Lee, J. L. Flippen-Anderson, R. D. Gilardi, and C. F. George; Novel Macrocyclic Trichotheeenes from Myrothecium roridum; Bull. Soc. Chim. Belg., Vol. 95, pp. 681-697(1986).


515

Common/Systematic Name Mytoxin C Molecular Formula/Molecular Weight C29H36010; ~

-" 5 4 4 . 2 3 0 8 5

~~ ~~,,;'0 ~0

H

H

0

,/--Xo f \

0

~

r

H"OH Me 14'

General Characteristics Colorless solid; mp., 181-185~

[{g]D --

+ 4-0~ (C=0.83, in CH2C12).

Fungal Source Myrothecium roridum (ATCC 60379). Isolation/Purification See mytoxin A. Biological Activity Cytotoxic. Spectral Data IR: (CH2C12) 3510, 2900, 1750, 1710, 1650, 1360, 1200, 1170, 1100, 1090, 1040, 995, 960, 910, 860, 820, and 650crn"1. 1HNIVIR: (CDCI3) 0.81(3H, s, H-14); 1.58(1H, m, H=8'B); 1.70(3H, s, H-16); 1.85(1H, m, H= TB); 1.9=2.1(6H, m, H=7, H-8, and H=4'); 2.17(1H, ddd, J=4.5, 5.2, 15.3Hz, H-3A); 2.29(1H, m, H-7'A); 2.31(3H, s, H-14'); 2.42(1H, dd, J-8.4, 15.3Hz, H-3); 2.79, 3.11(1H each, AB, J=4.0, H=13); 3.07(1H, m, H-8A); 3.53(1H, d, J=5.2Hz, H-11); 3.82(1H, d, J-5.2Hz, H-2); 3.84(1H, s after D20 exchange, H-12'); 3.86(1H, s, H- 2'); 3.91(1H, ddd, J=l.3, 5.2, 11.8Hz, H=5'B); 4.00(1H, ddd, J=2.1, 11.8, 11.8Hz, H=5'A);

516


4.02, 4.54(1H each, AB, J=12.3Hz, H-15), 5.39(1H, d, J=-5.2H~ H-10); 5.86(1H, dd, J=l.8, 11.4Hz, H-10'); 5.92(1H, dd, J=4.5, 8.4Hz, n-4); 6.47(1H, ddd, J=7.7, 9.0, 11.4Hz, H-9'). 13CNMR: (CDC13) 79.1, C-2, 34.5, C-3, 73.0, C-4; 49.7, C-5, 43.2, C-6, 19.8, C-7, 27.4, C-8, 140.6, C-9, 118.3, C-10, 67.4, C-11, 65.3, C-12; 47.8, C-13, 8.0, C-14, 64.1, C-15, 23.2, C-16, 166.5, C-I'; 53.7, C-2', 63.3, C- 3', 23.6, C-4', 60.2, C-5', 85.7, C-6', 22.4, C-7'; 27.8, C-8', 149.9, C-9', 120.8, C-10', 168.3, C-11', 69.2, C-12', 214.2, C-13'; and 26.6ppm C 14'. Reference B. B. Jarvis, F. T. C6mezoglu, Y.-W. Lee, J. L. Flippen-Anderson, R. D. Gilardi, and C. F. George; Novel Maeroeyelie Triehotheeenes from Myrothecium roridum;Bull. Soe. Claim. Belg., Vol. 95, pp. 681-697(1986).

Baccharinoids Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baceharinoid Baccharinoid Baccharinoid Baceharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid Baccharinoid

B1 B2 B3 B4 B5 B6 B7 B8

B9 B10 B12 B13 B14 B16 B17 B20 B21 B23 B24 B25 B27

517


20.

Baccharinoids

519

Common/Systematic Name Baccharinoid B 1 Molecular Formula/Molecular Weight C29H40010; M W = 5 4 8 . 2 2 6 2 1 5

H -

o.j

H ....

HO"

v

- v

,o I

OHO..... v 0,,~ HO"-H~ General Characteristics Melting point, 158-161~

[ a ] D --

+100.0 ~ (c=1.40, in CHzCI2).

Plant Source Baccharis megapotamica. Isolation/Purification A portion (200mg) of fraction 9D-4 (see isolation ofbaccharinoid B25) was subjected to semi-preparative HPLC (amino column, 1.5% MeOH-CH2C12) to give two fractions, the first of which gave 60mg ofbaccharinoid B 1, recrystallized from ethyl acetate-hexane. The second fraction gave 100mg of B2, recrystallized from ethyl acetate-methylene chloride. Spectral Data UV: maxE~" 263nm (e=20,100). 1H NMR: (CDC13) 0.77(3H, s, H-14), 1.02(3H, d, J=6.4Hz, H-12'), 1.20(3H, d, J=6.0Hz, H14'); 1.83(3H, s, H-16); 2.20(5H, m, H-2', H-3, and H-7); 2.44(2H, m, H-3' and H-3); 2.85, 3.13(1H each, AB pattern, J=4.0Hz, H-13); 3.6(2H, m, H-5'); 3.63(1H, d, J=5.3Hz, H-11); 3.70(2H, m, H-4' and H-13'); 3.86(1H, d, J=4.5Hz, H-2); 4.01(1H, m, H-8); 3.76, 4.68(1H each, AB pattern, J=12.4Hz, H-15); 4.10(1H, m, H-6'); 5.50(1H,

520

20.

Baccharinoids

d, J=5.3Hz, H-10); 5.77(1H, m, H-4); 5.78(1H, d, J=l 1.3Hz, H-10'); 6.05(1H, dd, J=3.1 and 15.5Hz, H-7'); 6.67(1H, dd, Js,9~J~,lo~l 1.3Hz, H-9'); and 7.70ppm (1H, dd, J-11.3 and 15.5Hz, H-8'). 13CNMR: (CDCla) 79.3, C-2; 34.9, C-3; 74.1, C-4; 49.4, C-5; 45.3, C-6; 30.7, C-7; 68.3, C-8; 143.0, C-9; 120.8, C-10; 67.3, C-11, 65.3, C-12, 47.9, C-13; 7.0, C-14; 64.1, C-15; 18.8, C-16; 172.7, C-I'; 38.3, C-2'; 32.4, C-3'; 73.1, C-4'; and 74. lppm C-5'. Reference B. B. Jarvis, S. N. C6mezoglu, H. L. Ammon, C. K. Breedlove, R. F. Bryan, R. W. Miller, M. K. Woode, D. R. Streelman, A. T. Sneden, R. G. Dailey, Jr., and S. M. Kupchan; New Macrocyclic Trichothecenes from Baccharis megapotamica; J. Nat. Prod., Vol. 50, pp. 815-828(1987).

20.

Baccharinoids

521

Common/Systematic Name Baccharinoid B2 Molecular Formula/Molecular Weight C29H40010, M W = 5 4 8 . 2 2 6 2 1 5

H -

HO"

H

o~j

.

.

.

.

I

.

I

H General Characteristics Crystals; m.p., 177-180~ [IX]D -- +116.0 ~ (c=l.0, in CH2C12). Plant Source Baccharis megapotamica. Isolation/Purification See isolation of baccharinoid B 1 Spectral Data UV: X EtOHmax

263nm (e=20,100).

1H NMR: (CDCI3) 0.77(3H, s, H-14); 1.02(3H, d, J=6.4Hz, H-12'); 1.20(3H, d, J=6.0Hz, H54'); 1.83(3H, s, H-16); 2.20(5H, m, H-2', H=3, and H-7); 2.44(2H, m, H=3' and H=3); 2.85, 3.13(1H each, AB pattern, J=4.0Hz, H-13); 3.6(2H, m, H-5'); 3.63(1H, d, J=5.3Hz, H-15); 3.70(2H, m, H-4' and H-13'); 3.86(1H, d, J=4.5Hz, H-2); 4.01(1H, m, H-8); 3.76, 4.68(1H each, AB pattern, J=12.4Hz, H-15); 4.10(1H, m, H-6'); 5.50(1H, d, J=5.3Hz, H-S0); 5.77(1H, m, H-4); 5.78(1H, d, J=l 1.3Hz, H-10'), 6.05(1H, dd, J=3.1 and 15.5Hz, H-7'); 6.67(1H, dd, Js.,9~J~,lo.=l 1.3Hz, H-9'); and 7.70ppm (1H, dd, J=l 1.3 and 15.5Hz, H-8').

522

20.

Baccharinoids

13CNMR~ (CDCI3) 79.3, C-2, 34.9, C-3, 74.1, C-4, 49.4, C-5, 45.3, C-6, 30.7, C-7, 68.3, C-8, 143.0, C-9, 120.8, C-10; 67.3, C-11; 65.3, C-12; 47.9, C-13, 7.0, C-14; 64.1, C-15; 18.8, C-16, 172.7, C-I', 38.3, C-2', 32.4, C-3', 73.1, C-4', and 74. lppm C-5'. Reference B. B. Jarvis, S. N. CSmezoglu, H. L. Ammon, C. K. Bmedlove, R. F. Bryan, R. W. Miller, M. K. Woode, D. R. Strcdman, A. T. Sne,den, R. G. Dailey, Jr., and S. M. Kupchan, New Macrocyclic Tdchothecenes from Baccharis megapotamica; J. Nat. Prod., Vol. 50, pp. 815-828(1987).

20.

Baccharinoids

523

Common/Systematic Name Baceharinoid B3 Molecular Formula/Molecular Weight C29H40010; M W -- 5 4 8 . 2 6 2 1 5

H

H~ O

H

o..j

....,0

H General Characteristics Melting point, 172-180~

laiD-"

+164.0 ~ (c=0.58, in CH2C12).

Plant Source Baccharis megapotamica. Isolation/Purification See isolation ofbaccharinoid B7. The mother liquor from the first crystallization of baccharinoid B7 was subjected to PTLC (10% MeOH-CH2C12, SiO2, 2mm plates) to yield baecharinoid B3. Spectral Data UV2 ~bEtOH

max 263nm (6=20,200).

IR:

(CHCIa) 3590, 2450, 1720, 1650, and 1605cm"l. 'H NMR: (CDCI3) 0.79(3H, s, H-14); 1.12(3H, d, J=6.9Hz, H-12'-H); 1.19(3H, d,J=6.0Hz, H 14'); 1.55(2H, m, H-4'); 1.83(3H, s, H-16); 2.10(21-I, m, H-7); 2.20(1H, rn, H-313); 2.28(1H, m, H-3'); 2.44(1H, dd, J=8.4 and 15.3, H-3a); 2.85, 3.13(1H each, AB

524

20.

Baccharinoids

pattern, J=4.0 Hz, H-13); 3.30(1I-1, s, H-2'); 3.40-3.65(5H, m, H-11, H-5', H-6' and H13'); 3.87(11-1, m, H-2); 4.01(1H, dd, J=5.3 and 10.3Hz, H-8); 3.91, 4.85(1H each, AB pattern, J=12.4Hz, H-15); 4.16(1H, d, J=4.2Hz, H-2'); 5.49(1H, d, J=5.2Hz, H-10); 5.75(1H, dd, ,/--4.5 and 8.4Hz, H-4); 5.78(1H, d, J=l 1.6Hz, H-10'); 6.01(1H, dd, J=2.6 and 15.4Hz, H-7'); 6.67(1H, dd, Js-9~J~,10~l 1.6Hz, H-9'); and 7.60ppm (1H, dd, jr=l 1.6 and 15.4Hz, H-8'). 13CNMR: (CDC13) 79.3, C-2; 35.0, C-3; 74.3, C-4; 49.4, C-5; 45.7, C-6; 30.8, C-7; 68.2, C-8; 143.3, C-9; 120.6, C-10; 67.2, C-11; 65.1, C-12; 47.8, C-13; 7.1, C-14; 64.7, C-15; 18.8, C-16; 172.6, C-I'; 76.9, C-2'; 35.0, C-3'; 31.0, C-4'; 68.3, C-5'; 82.6, C-6'; 138.8, C-7'; 127.0, C-8'; 143.8, C-9'; 117.3, C-10'; 166.8, C-11'; 16.1, C-12'; 69.7, C-13'; and 18. lppm C-14'. Reference B. B. Jarvis, S. N. CSmezoglu, H. L. Ammon, C. K. Breedlove, R. F. Bryan, R. W. Miller, M. K. Woode, D. R. Streelman, A. T. Sneden, R. G. Dailey, Jr., and S. M. Kupchan; New Macrocyclic Trichothecenes from Baccharis megapotamica; J. Nat. Prod., Vol. 50, pp. 815-828(1987).

20.

Baccharinoids

525

Common/Systematic Name Baccharinoid B4 Baccharinol Molecular formulamolecular Weight C29H38Oll; M W -- 5 6 2 . 2 4 1 4 2

H H - 0., 3 HO"

v -

y

'"

J

0 HO/- x H General Characteristics Melting point, 259-263~

[aiD =

+ 165.0~ (C=0.50, in MeOH).

Plant Source Baccharis megapotamica. Isolation/Purification See isolation ofbaccharinoid B5. The 100% methanol fraction from the initial alumina column was subjected to silica gel (MeOH-CHC13) chromatography and the 10% MeOH fraction crystallized (MeOH-CHC13) and recrystallized (acetone-hexane) to give 3.5g of baceharinoid B4. Spectral Data UV: EtOH

~,m= 260nm (e=20,400). IR:

(CHCI3) 3360, 1750, 1715, 1170, 1640, and 1600cmq.

526

20.

Baccharinoids

~HNMR: (CDCI3) 0.83(3H, s, H-14); 1.18(3H, d, J=6.6Hz, H-14'); 1.59(3H, s, H-12'); 1.83(31t, s, H-16); 2.50(1H, dd, ,/--15 and 8Hz, H-3a); 2.88, 3.13(1H each, AB pattern, J=4Hz, H-13); 3.44(1H, s, H-2'); 4.24, 4.44(1H each, AB pattern, J=12Hz, H15); 5.46(1H, d, J=5Hz, H-10); 5.8(1H, rn, H-4), 5.83(1H, d, J=l 1Hz, H-10'), 6.02(1H, dd, J=3 and 15Hz, H-7'); 6.63(1H, dd, Js,,e=Je,lo~l 1Hz, H-9'); and 7.42ppm (1H, dd, J= 11 and 15Hz, H-8'). 13CNMR: (CDC13) 78.6, C-2; 34.5, C-3; 73.8, C-4; 48.9, C-5; 44.7, C-6; 29.6, C-7; 66.7, C-8; 143.2, C-9; 119.4, C-10; 66.4, C-11; 65.0, C-12; 47.2, C-13; 6.5, C-14; 64.4, C-15; 18.3, C-16; 167.3, C-I'; 55.9, C-2'; 64.8, C-3'; 74.9, C-4'; 72.0, C-5'; 85.7, C-6'; 138.1, C-7'; 125.3, C-8'; 142.2, C-9'; 117.7, C-10'; 166.2, C-11'; 11.8, C-12'; 71.0, C-13'; and 17.8ppm C-14'. Reference S. M. Kupchan, D. R. Streelman, B. B. Jarvis, R. G. Dailey, Jr., and A. T. Sneden; Isolation of Potent New Antileukemic Trichothecenes from Baccharis megapotamica; J. Org. Chem., Vol. 42, pp. 4221-4225(1977).

20. Baccharinoids

527

Common/Systematic Name Baceharinoid B5; Baccharin Molecular Formula/Molecular Weip.ht C29H38Oll; ~

,6, .... .~~

= 562.24141

_15

H

3

1~ Is H2C I .

,1

i

9'


diacetate, mp., 254-256~

[a]D = + 41.5 ~ (C=2.2, in CHCI3).

Plant Source

Baccharis megapotamica.

Isolation/Purification An ethanol extract of 54kg of plant material (leaves and twigs) was concentrated to a black tar and then partitioned between water and ethyl acetate. The ethyl acetate solubles were partitioned between hexane and 10% water in methanol. The methanol solubles were taken up in methanol-ethyl acetate (1:4, v/v) and the mixture filtered through a column of alumina (activity H-HI), washing with 6L of 20% methanol in ethyl acetate. The filtrates were combined, concentrated, and subjected to column chromatography over alumina (activity II-III, elution with increasing methanol in ethyl ether). The 10% methanol-ethyl ether (48g) was subjected to column chromatography on silica gel (methanol-chloroform) and an early 2% methanol fraction (3g) upon crystallization from methanol-chloroform gave a solid which upon recrystallization from acetone-hexane gave 1. lg ofbaccarinoid B5. Spectral Data UV:

Eto. 26Ohm.

max

528

20.

Baccharinoids

IR:

(CHC13) 3580,2880, 1755, 1720, 1170, and ll00cm q. 1H (CDCI3) 0.77(3H, s, H-14); 1.23(3H, d, J=6.5Hz, H-14'); 1.37(3H, s, H-12'); 1.66(3H, s, n-16); 2.19(1H, ddd, J2,ap=Jap,4=4.6Hz and Js~--15.3Hz, H-313); 2.42.6(3H, m, H-3 and H- 4'); 2.76, 3.17(1H each, AB pattern, J=3.9 Hz, H-13); 3.09(1H, d, J=5.4Hz, n-10); 3.30(1H, s, n-2'); 3.32-3.38(1H, m, H-5'B); 3.57(1H, d, J=5.4Hz, n-11); 3.84-3.92(1H, m, H-H-5'A); 3.94(1H, d, J=5.0I-Iz, n-2); 4.20, 4.43(1H each, AB pattern, J=12.6Hz, H-15); 5.76(1H, dd, ,/-=4.6 and 8.2Hz, H-4); 5.80(1H, d, J=l 1.2Hz, H-10'), 5.97(1H, dd, J=3.0 and 15.6Hz, H-7'); 6.60(1H, dd, Js,,a,=dr~,lo~l1.2Hz, H-9'); and 7.54ppm (1H, dd, ,/=11.2 and 15.6Hz, H-8'). 13CNMR: (CDC13) 78.1, C-2; 34.0, C-3; 73.8, C-4; 48.5, C-5; 42.3, C-6; 16.7, C-7; 25.7, C-8; 57.7, C-9; 56.9, C-10; 66.6, C-11; 65.2, C-12; 47.1, C-13; 6.5, C-14; 63.1, C-15; 21.6, C-16; 167.4, C-I'; 56.0, C-2'; 64.4, C-3'; 75.3, C-4'; 72.1, C-5'; 86.8, C-6'; 138.2, C-7'; 125.1, C-8'; 142.6, C-9', 117.4, C-10', 166.3, C-11', 11.6, C-12'; 71.0, C-13'; and 17.7ppm C-14'. Reference B. B. Jarvis, S. N. CSmezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

529

Common/Systematic Name Baccharinoid B6; Isobaccharinol Molecular Formula/Molecular Weight C29H38Oll, M W = 5 6 2 . 2 4 1 4 1

H -

H

o...I

v~"- 1 " H20 I

HOv

|

O, HC~'" " ~ 0 ~ H General Characteristics Melting poim, 249-251~

[a]D = + 149.0 ~ (C=0.66, in MeOH).

Plant Source Baccharis megapotamica. Isolation/Purification See isolation ofbaccharinoid B4. The 10% methanol fraction from which baccharinoid B4 was isolated was crystallized from methanol-chloroform followed by recrystallization from acetone-hexane to give 0.20g ofbaccharinoid B6. Spectral Data UV:

EtOH

~.~

260nm (6=20,400).

IR:

(KBr) 3420, 1750, 1720, 1170, 1650, and 1605cm"l.

530

20.

Baccharinoids

~HNMR: (CDCh) 0.83(3H, s, H-14); 1.16(3H, d, J--6.6Hz, H-14'); 1.65(3H, s, H-12'), 1.83(3H, s, H-16); 2.84, 3.13(1H each, AB pattern, J=4Hz, H-13); 3.38(1H, s, H-2'); 4.24, 4.46(1H each, AB pattern, J=12Hz, H-15); 5.52(1H, d, J=5I-Iz, H-10); 5.8(1H, m, H-4); 5.81(1H, d, J=l 1Hz, H-10'); 5.92(1H, dd, J=3 and 15Hz, H-7'); 6.59(1H, dd, Js, ~=Jv,~o~l1Hz, H-9'); and 7.40ppm (1H, dd, J=l 1 and 15Hz, H-8'). 13CNMR: (CDCI3) 78.8, C-2; 34.5, C-3; 73.9, C-4; 49.1, C-5; 44.8, C-6, 29.7, C-7, 67.0, C-8; 143.3, C-9, 119.6, C-10, 66.7, C-11; 65.4, C-12; 47.5, C-13; 6.7, C-14, 64.6, C-15; 18.5, C-16; 167.4, C-1'; 56.3, C-2', 65.0, C-3', 75.5, C-4', 72.3, C-5', 85.2, C-6', 138.4, C-7'; 125.3, C-8', 142.5, C-9', 117.5, C-10', 166.4, C-11'; 11.9, C-12'; 69.0, C-13'; and 15.Sppm C-14'. Reference S. M. Kupchan, D. R. Streelman, B. B. Jarvis, R. G. Dailey, Jr., and A. T. Sneden; Isolation of Potent New Antileukemic Trichothecenes from Baccharis megapotamica; J. Org. Chem., Vol. 42, pp. 4221-4225(1977).

20.

Baccharinoids

531

Common/Systematic Name. Baccharinoid B7 Molecular Formula/Molecular Weight C29H40010; MW = 548.26215 H

H

o.,j

H

0

0

H

General Characteristics Melting point, 229-231 ~

[ a ] D --

+ 150~ (C=0.66, in CH2C12).

Plant Source Baccharis megapotamica. Isolation/Purification See isolation ofbaccharinoid B 17. Fraction F8 from the first silica gel column of the large scale extract was taken up in methylene chloride and the solution carefully washed with 2.5% aqueous NaOH. The methylene chloride soluble material was subjected to filtration chromatography over alumina (activity III, 15 % isopropyl alcohol-hexane, 100 % isopropyl alcohol, and 100 % MeOH). Crystallization of the 100 % isopropyl alcohol fraction from ethyl acetate-hexane and recrystallization from ethanol gave baccharinoid B7. Spectral Data UVz ~ ~.~o,max 263nm (e=20,100). IR:

(KBr) 3590, 2450, 1720, 1650, and 1605cm"1. 1H NIVIR: (CDCI3) 0.78(3H, s, H-14); 1.12(3H, d, J=6.9Hz, H-12'); 1.19(3H, d, d=6.0Hz, H14'); 1.60(2H, m, n-4'); 1.83(3H, s, n-16); 2.10(2R rn, n-7); 2.22(1H, m, H-313);

532

20.

Baccharinoids

2.28(1H, m, H-3'), 2.44(1H, dd, J=8.1 and 15.2Hz, H-3t~); 2.85, 3.13(1H each, AB pattern, J=4.0Hz, H-13), 3.30(1H, s, H-2'); 3.40-3.65(5H, m, H-11, H-5', H-6' and H13'); 3.87(1H, d, J=4.0Hz, H-2), 4.01(1H, dd, J=4.9 and 9.4Hz, H-8), 3.91, 4.89(1H each, AB pattern, J=12.3Hz, H-15); 4.18(1H, d, J=4.0Hz, H-2'); 5.50(1H, d, J=5.4Hz, H-10); 5.76(1H, dd, J=4.7 and 8.1Hz, n-4); 5.79(1H, d, d=l 1.5Hz, 10'-H); 6.00(1H, dd, J=2.5 and 15.5Hz, H-7'); 6.66(1H, dd, Js,9_Jg,,lo~11.5Hz, H-9'), and 7.65ppm (1H, dd, J=l 1.5 and 15.5Hz, H-8'). 13CNMR.: (CDC13) 79.2, C-2; 35.0, C-3; 74.3, C-4, 49.4, C-5; 45.7, C-6; 30.9, C-7; 68.2, C-8; 143.3, C-9; 120.9, C-10; 67.2, C-11; 65.1, C-12; 47.8, C-13; 7.1, C-14; 64.8, C-15; 18.8, C-16; 172.5, C-I'; 76.9, C-2', 34.8, C-3'; 30.8, C-4'; 67.9, C-5'; 83.9ppm, C-6'; 139.1, C-7', 126.8, C-8', 143.7, C-9', 117.6, C-10'; 166.7, C-11'; 16.1, C-12', 70.7, C13'; and 18.3ppm, C-14'. TLC Data Rf=0.54 (5% MeOH-CH2CI2), 0.29 (EtOAc), and 0.55 (40% isopropyl alcohol-hexane). Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica, J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

533

Common/Systematic Name Baccharinoid B8; Isobaccharin Molecular Formula/Molecular Weight C29H38Oll; MW - 562.24141

|111|

3 13-

-

s

I

.

I ,0

4

14

U

,

iii I

HO,,"~

12'

O,.....6.~ r H

General Characteristics Melting poim, 228-230~

[a]D = + 42.0 ~ (C=0.36, in CHC13).

Plant Source


Isolation/Purification See baccharinoid B5. Continued elution of the silica gel column with 2% MeOH-CHC13 gave a baccharinoid B8-containi'ng fraction which upon PTLC (25% isopropyl alcoholbenzene) followed by recrystallization from acetone-hexane gave baccharinoid B8. Spectral Data UV:

X~" max 260nm (e=21,300). IR: (KBr) 3470, 1755, 1710, 1170, 1650, and 1605cm-1. 1H NIV[R:

(CDCI3) 0.76(3H, s,H-16); 1.17(3H, d, J=6.6 Hz, H-14'); 1.34(3H, s,H-16), 1.68(3H, s,H-12');2.47(IH, dd, J=16 and 8 Hz, H-3(x);2.75, 3.16(IH each, AB pattern,J=4 Hz, H-13), 3.09(IH, d, J=6 Hz, H-10); 3.35(IH, s,H-2');4.22, 4.47(IH each, AB pattern,J=12.2 Hz, H-15), 5.8(IH, m, H-4), 5.80(IH, d, J=l I Hz, H-10');

534

20.

Baccharinoids

each, AB pattern, J=12.2 Hz, H-15), 5.8(1H, m, H-4); 5.80(1I-I, d, J-11 Hz, H-10'); 5.93(1H, dd, J=3 and 15.5 Hz, n-7'); 6.60(1H, dd, Js,,~ =J~,lo. =11 Hz, n-9'); and 7.44ppm (1H, dd, J=l 1 and 15.5 Hz, 8'-H). laC NMR: (CDCI3) 78.2, C-2; 34.0, C-3, 73.9, C-4, 48.5, C-5; 42.4, C-6, 16.7, C-7, 25.8, C-8, 57.7, C-9, 57.0, C-10, 66.7, C-11, 65.4, C-12, 47.2, C-13, 6.6, C-14, 63.1, C-15, 21.6, C-16; 167.4, C-I', 56.1, C-2', 64.4, C-3', 75.6, C-4', 72.1, C-5'; 85.3, C-6'; 138.7, C-7', 125.0, C-8'; 142.7, C-9'; 117.1, C-10'; 166.3, C-11'; 11.6, C-12', 68.8, C-13'; and 15.6ppm, C-14'. Reference B. B. Jarvis, S. N. CSmezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

535

Common/Systematic Name Baeeharinoid B9 Molecular F0rmula/Mo!ecular Weight C29I-/38010; MW = 546.24650 0

H

H

I0

I

o

N General Characteristics Crystals; mp., 216-218~

[tt]D = + 2.4 ~ (C= 0.68, in CH2C12).

Plant Source


Isolation/Purification See baeeharinoid B 10 isolation. On the basis of TLC analysis, two of the later chromatography fractions from fractions E1 and E2 were combined and crystallized from ethyl ether-methylene chloride to give baecharinoid B9. Spectral Data IR"

(CHCI3) 2930, 2870, 1720, 1650, 1610, and 1095em"1. UV: ~.~

220 and 262nm.

536

20.

Baccharinoids

1HNMR:

(CDC13) 0.69(3H, s, H-14), 1.18(3H, d, J=6.0Hz, H-14'), 1.33(3H, s, H-16), 2.102.25(1H, m, H-3]3); 2.31(3H, d, J=l.2Hz, H-12'); 2.45(1H, dd, J=8.5, 15.8Hz, H3t~); 2.74, 3.14(1H each, AB pattern, J=4.0Hz, H-13); 3.07(1H, d, J=5.2Hz, H-10); 3.63-3.73(6H, m, H-4', HS', H-6', H-11, and H-13'); 3.90(1H, d, J=5.0Hz, H-2); 3.76, 4.46(1H each, AB pattern, J=12.7Hz, H-15); 5.71(1H, d, J=l 1.3Hz, H-10'); 5.87(1H, dd, J=2.5, 16.1Hz, H-7'), 5.98 (2H, m, H-2', H-4), 6.53 (1H, dd, Js,,9,=Jg,lo,=ll.3Hz, H-9'); and 7.38ppm (1H, dd, J=l 1.3, 16.1Hz, H-8'). 13CNMR: (CDC13) 78.8, C-2; 35.1, C-3; 74.1, C-4; 47.8, C-5; 42.5, C-6; 18.3, C-7; 26.3, C-8; 57.5, C-9; 57.5, C-10; 67.7, C-11; 65.0, C-12; 48.5, C-13; 6.9, C-14; 63.1, C-15; 22.3, C-16; 166.4, C-I'; 115.0, C-2'; 161.0, C-3'; 74.8, C-4'; 74.7, C-5'; 84.2, C-6'; 138.0, C7', 126.6, C-8'; 143.7, C-9'; 117.8, C-10'; 166.0, C-11'; 15.7, C-12'; 70.9, C-13'; and 18.6ppm, C-14'. TLC Data Re= 0.40 (4% methanol-methylene chloride), 0.43 (ethyl acetate), and 0.48 (30% isopropyl alcohol-hexane). Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from A Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

537

Common/Systematic Name Baccharinoid B 10 Molecular Formula/Molecular Weight C29H38010; M~V = 546.24650 0

o

H

H

oJ)

H General Characteristics Crystals; mp., 157-158~

[(g]D-- + 8.1 (C=0.62, CHC13).

Plant Source

Baccharis megapotamica (original trichothecene nucleus suspected to be of fungal origin).

Isolation/Purification See baccharinoid B 17 for general isolation procedure through the first column. Fraction 7 (1.125kg) from this column was chromatographed over SiO2 with increasing MeOH in CH2C12 to give five fractions: F7A-F7E. Fraction F7E (290g) was taken up in CH2C12 and carefully washed with 2.5% aqueous NaOH to give 12 lg of CH2C12 material which was subjected to filtration chromatography over alumina: 20% i-PrOH/hexane (E 1), 100 % i-PrOH (E2), and 100% MeOH (E3) fractions. The 20% i-PrOH/hexane fraction (80g) was subjected to filtration chromatography (A12Os) and MPLC (SiO2) and several PTLC's (Chromatotron) to give baccharinoids B1 - B5 and two additional fractions Ela and Elb that contained additional baccharinoids. Fraction E2 (100% i-PrOH) was subjected to Sephadex chromatography (CH2C12) to give four fractions. Fraction E2b (5.4g) was subjected to flash chromatography (EtOAc/hexane) and PTLC (A12Os, i-PrOH/CH2C12, Chromatotron). A fraction from this was combined with E lb and crystallized from Et20/CHC13 to give 65mg of B 10.

538

20.

Baccharinoids

Spectral Data IR;

(CHC13) 3420, 1720, 1650, 1610, and 1170cm"1. UV:

~.ma~ 220 and 260nm. 1H NMR:

(CDCI3) 0.72(3H, s, 14-8); 1.19(3H, d, J=6.4Hz, 14'-H); 1.33(3H, s, 16-H); 2.102.25(1H, m, 3~3-H); 2.33(3H, d, J-1.0Hz, 12'-H); 2.43(1H, dd, J=8.0 and 15.5Hz, 3czH); 2.74, 3.18(1H each, AB pattern, J=4.0Hz, 13-I-I); 3.08(1H, d, J-5.2I-Iz, lO-H); 3.60(1H, d, J=5.2Hz, 1l-H); 3.90(1H, d, J=4.9Hz, 2-H); 3.81, 4.45(1H each, AB pattern, J=12.7Hz, 15-H); 5.71(1H, d, J-11.4Hz, 10'-H); 5.87(1H, dd, J-3.0 and 15.6Hz, 7'-H); 5.97(1H, br s, 2'-H); 5.98(1H, dd, J-4.2 and 8.0Hz, 4-H); 6.53(1H, dd, Js.~=J~,lo~=l1.4Hz, 9'-H); and 7.34ppm (1H, cld, J=l 1.4 and 15.6Hz, 8'-H). 13CNMR: (CDC13) C-2, 78.7; C-3, 34.9; C-4, 74.2; C-5, 48.4; C-6, 42.5; C-7, 18.2; C-8, 25.2; C-9, 57.4; C-10, 57.5; C-11, 67.7; C-12, 65.0; C-13, 47.6; C-14, 6.9; C-15, 62.9; C16, 22.2; C-I', 166.3; C-2', 115.0; C-3', 161.3; C-4', 75.1; C-5', 74.3; C-6', 83.0; C-7', 137.9; C-8', 126.6; C-9', 143.5; C-10', 117.5; C-11', 166.2; C-12', 15.5; C- 13', 68.9; and C-14', 17.1ppm. TLC Data Rf = 0.40 (4% MeOH/CH2CI2), 0.36 (EtOAc), and 0.43 (30% i-PrOH/hexane). Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M.Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56 (1987).

20.

Baccharinoids

539

Common/Systematic Name Baccharinoid B 12 Molecular Formula/Molecular Weight C29H38Oll; M W -- 5 6 2 . 2 4 1 4 2

H 03

..,,OH .i,,,0

HO"

v

-v

o

I

iiii j

H

HO H Plant Source Baccharis megapotamica. Isolation/Purification See baccharinoid B 17 for general isolation procedure through the first column. Fraction 7 (1.125kg) from this column was chromatographed over silica gel with increasing methanol in methylene chloride to give five fractions: F7A-F7E. Fraction F7B (290g) was taken up in CH2C12 and carefully washed with 2.5% aqueous NaOH to give 172g of methylene chloride material which was subjected to filtration chromatography over alumina: 15% isopropanol-hexane, 100% isopropanol, and 100% methanol fractions. The 100% isopropanol fraction was triturated with ethyl ether-methylene chloride to give a precipitate which upon recrystallization from acetone-hexane gave 430mg ofbaccharinoid B12. Spectral Data UV:

~m~ H 260nm. IR: (CHC13) 3500,2990, 1730, 1715, 1180, and ll00cm 1.

540

20.

Baccharinoids

1H NMR.: (CDCI3) 0.85(3H, s, H-14); 1.19(3H, d, ,/=6.41--Iz,H-14'); 1.46(3H, s, H-12'); 1.75(3H, s, H-16); 2.79, 3.08(1H each, AB pattern, J=4.0Hz, H-13); 3.30(1H, s, H-2'); 3.4-3.5 (1H, m, H-5'B); 3.62-3.75 (5H, m, H-5'A, H-6'A, and H-13'); 4.18 (1H, d, J=5.6Hz, H-11); 4.19, 4.43(1H each, AB pattern, J=12.6Hz, H-15); 4.35 (1H, dd, J=2.7 and 5.0Hz, H-313); 5.76(1H, dd, J=4.6 and 8.2Hz, H-4); 5.80(1H, d,J=ll.2Hz, H-10'); 5.57(1H, d, J=5.6Hz, H-10); 6.02(1H, dd, J=2.7 and 15.6Hz, H-7'); 6.63(1H, dd, ffs,,9,=J~,lo,=l1.5Hz, H-9'); and 7.50ppm(1H, dd, J=l 1.5 and 15.6Hz, H-8'). 13CNMR: (CDCI3) C-2, 79.4; C-3, 75.5; C-4, 83.0; C-5, 49.2; C-6, 44.1; C-7, 20.0; C-8, 25.5; C-9, 139.0; C-10, 119.1; C-11, 67.4; C-12, 64.4; C-13, 46.3; C-14, 6.8; C-15, 63.7; C16, 23.0; C-I', 167.9; C-2', 57.8; C-3', 63.1; C-4', 39.8; C-5', 67.9; C-6', 84.7; C-7', 140.6; C-8', 125.2; C-9', 143.7; C-10', 116.7; C-11', 166.2; C-12', 17.2; C- 13', 69.1; and C- 14', 17.9ppm. Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation of Macrocyclic Trichothecenes from a Large Scale Extract of Baccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

541

Common/Systematic Name Baccharinoid B 13 Molecular Formula/Molecular Weight C29H38Olo; M ~

H

= 546.24650

H iO

H O"

v

- v

I ,

o

H General Characteristics Crystals, m.p., 218-219~

[ ~ ] D --

"+"130~ (C=0.74, in MeOH).

Plant Source Baccharis megapotamica. Isolation~urification See baccharinoid B 17 for general isolation procedure through the first column. Fraction 9 from this column was chromatographed over silica gel with increasing methanol in methylene chloride to give five fractions 9A-9E. Fraction 9D was dissolved in methylene chloride and hexane was added slowly to give a precipitate. This material was dissolved in methylene chloride and the solution washed with 2.5% aqueous NaOH, concentrated to dryness, and subjected to preparative HPLC using alumina and eluting with isopropyl alcohol-methylene chloride to give six fractions. From these fractions by successive chromatographic procedures baccharinoids B 13, B 14, B23, B24, B25, and B27 were isolated. Spectral Data UV: EtOH

max 220 and 262nm. IR:

(CHC13) 3600, 2880, 1720, and 1180cm ~

542

20.

Baccharinoids

1H NMR: (CDCI3) 0.76(3H, s, H-14); 1.21(3H, d, J=6.4Hz, H-14'); 1.81(3H, s, H-16), 2.052.15(1H, m, H-313); 2.22(3H, d, J=l.2 Hz, H-12'); 2.51(1H, dd, J=8.3, 15.7Hz, H3~); 2.83, 3.14(1H each, AB pattern, J=4.0Hz, H-13); 3.62-3.69 (3H, m, H-13', H5'B, H-6'); 3.85(1H, d, J=5.2Hz, H-2); 3.90-3.96(2H, m, H-11 and H-5'); 3.93, 4.23 (1H each, AB pattern, J=12.5Hz, H-15); 4.05(1H, br s, H-8); 4.37(1H, br s, H-4'); 5.54(1H, d, J=5.6Hz, H-10); 5.75(1H, d, J=l 1.5Hz, H-10'); 5.85(1H, dd, J=3.0, 15.7Hz, H-7'); 6.21(1H, dd, J=3.9, 8.1Hz, H-4); 6.24(1H, br s, H-2'); 6.55(1H, dd, Js,,9,=Jg,,1o,=l1.SHz, H-9'); and 7.48ppm (1H, dd, J=l.5, 15.7Hz, H-8'). 13C NMR: (CDC13) 79.3, C-2; 35.6, C-3; 73.9, C-4; 48.7, C-5; 45.2, C-6; 31.7, C-7; 68.1, C-8; 142.4, C-9; 118.0, C-10; 67.1, C-11; 65.5, C-12; 48.0, C-13; 6.8, C-14; 64.2, C-15; 18.6, C-16; 166.3, C-I'; 121.1, C-2'; 160.5, C-3'; 74.3, C-4'; 73.6, C-5'; 84.0, C-6'; 137.6, C-7'; 126.8, C-8'; 143.5, C-9'; 115.0, C-10'; 165.9, C-11'; 18.9, C-12'; 70.8, C13'; and 16.0ppm, C-14'. TLC Data Rf 0.19 (4% methanol-methylene chloride), 0.28 (ethyl acetate), and 0.43 (30% isopropyl alcohol-hexane). Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract of Baccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

543

Common/Systematic Name Baccharinoid B 14 Molecular Formula/Molecular Weight C29H3801o; M W

H

HO ~"

= 546.24650

H

--__ o,,,]

....,0

o

H General Characteristics Crystals from ethyl acetate-hexane; m.p., 149-151~

[tt]D = + 72.4 ~ (C=0.76, in MeOH).

Plant Source Baccharis megapotamica. Isolation/Purification See baccharinoid B 17 for general isolation procedure through the first column. Fraction 9 from this column was chromatographed over silica gel with increasing methanol in methylene chloride to give five fractions 9A-9E. Fraction 9D was dissolved in methylene chloride and hexane was added slowly to give a precipitate. This material was dissolved in methylene chloride and the solution washed with 2.5% aqueous NaOH, concentrated to dryness, and subjected to preparative HPLC using alumina and eluting with isopropyl alcohol-methylene chloride to give six fractions. From these fractions by successive chromatographic procedures baccharinoids B 13, B 14, B23, B24, B25, and B27 were isolated. Spectral Data UV~

~EtoH max 220 and 260nm. IRz

(CHC13) 3590, 2870, 1715, and l l80cm "1.

544

20.

Baccharinoids

1H NMR: (CDCI3) 0.79(3H, s, H-14); 1.20(3H, d, J=6.4Hz, H-14'); 1.83(3H, s, H-16); 2.052.20(1H, m, H-313); 2.27(3H, s, n-12'); 2.52(1H, dd, J=8.0, 15.4Hz, n-3ct); 2.86, 3.16(1H each, AB pattern, J=3.9Hz, H-13); 3.66, 3.77(1H each, d of AB pattern, J=3.9, J4,,S=--4.7Hz,Jgem=9.8Hz, H-5'); 3.87(1H, d, J=5.0Hz, H-2); 3.86-4.04(4H, m, H-11, H-4', H-6', and H-13'); 3.99, 4.26(1H each, AB pattern, d=12.7Hz, H- 15); 5.57(1H, d, J=6.0Hz, H-10); 5.76(1H, d, J=l 1.2Hz, H-10'); 5.89(1H, dd, J=3.0, 15.5Hz, H-7'); 6.17-6.18(2H, m, H-2' and H-4' overlapping); 6.57(1H, dd, ds,,9_Jg,,lo~l 1.5Hz, H-9'); and 7.41ppm (1H, dd, J=l 1.5, 15.4Hz, H-8'). 13CNMR: (CDC13) 79.3, C-2; 35.6, C-3; 74.0, C-4; 48.6, C-5; 45.2, C-6; 31.5, C-7; 68.1, C-8; 142.4, C-9; 117.7, C-10; 67.1, C-11; 65.5, C-12; 48.0, C-13; 6.8, C-14; 64.1, C-15; 17.1, C-16; 166.3, C-I'; 121.1, C-2'; 161.3, C-3'; 74.8, C-4'; 73.1, C-5'; 82.7, C-6'; 137.3, C-7', 126.9, C-8'; 143.5, C-9'; 114.9, C-10'; 166.3, C-11'; 19.0, C-12'; 68.5, C13'; and 15.9ppm, C-14'. TLC Data Rf=0.19 (4% methanol-methylene chloride), 0.21 (ethyl acetate), and 0.42 (30% isopropyl alcohol-hexane). Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

545

Common/Systematic Name Baccharinoid B 16 Molecular Formula/Molecular Weight C29H38010; M W -- 5 4 6 . 2 4 6 5 0

HOH2C~O,,,~ H

H

H2~ I o 6

~) o I

H General Characteristics Crystals from ethanol-hexane, mp., 160-161~

[a]D = + 63.5 ~ (C=0.47, in MeOH).

Plant Source Baccharis megapotamica. Isolation/Purification See isolation for baccharinoid B25. Fraction 9E in methylene chloride was washed with 2.5% aqueous NaOH, concentrated, and chromatographed by silica gel MPLC eluting with ethyl acetate-hexane to give two fractions. The second fraction was fractionated using preparative PTLC (60% ethyl acetate-hexane, Chromatotron) which gave baccharinoid B 16 which crystallized from methylene chloride-hexane. Spectral Data UV;

~.m~x 220 and 260nm. IR:

(CHC13) 3600, 2860, 1710, and 1180cm"1.

546

20.

Baccharinoids

1H NIVIR: (CDCI3) 0.78(3H, s, H-14); 1.16(3H, d, J=6.4Hz, H-14'); 2.15-2.20(IH, m, H-313); 2.22(3H, d, J=1.0Hz, H=I2'); 2.54(IH, dd, J=8.2, 15.5Hz, H-3a); 2.81, 3.12(IH each, AB pattern, J=4.0I-Iz, H-13); 3.62, 3.76(IH each, d of AB pattern, J=3.7, 9. IHz, H5'); 3.63(IH, d overlapping with H-5', H-I I); 3.84(IH, d, J=4.9Hz, H-2); 3.964.07(IH, m, H-6'); 4.01(2H, br s, H-16); 4.26(IH, br s, H-4'); 4.04, 4.29(IH each, AB pattern, J=12.5Hz, H-15); 5.74(IH, d, J=l 1.4Hz, H-10'); 5.82(IH, d, J= 5.4Hz, HI0); 5.86(1H, dd, J=2.8, 15.8Hz, H-7'); 6.08(1H, br s, H-2'); 6.55(1H, dd, Js,,9~19,.lo~11.4Hz,H-9'); and 7.38ppm (1H, dd, J=l 1.4, 15.8 Hz, H-8').

13CNMR: (CDC13) 79.4, C-2; 35.4, C-3; 74.5, C-4; 48.9, C-5; 43.5, C-6; 20.8, C-7; 23.2, C-8; 143.7, C-9; 117.6, C-10; 67.1, C-11; 65.7, C-12; 48.1, C-13; 6.9, C-14; 65.7, C-15; 63.5, C-16; 166.8, C-I'; 118.6, C-2'; 161.4, C-3'; 75.1, C-4'; 74.0, C-5'; 83.2, C-6'; 138.1, C-7'; 126.8, C-8'; 143.5, C-9'; 115.0, C-10'; 165.9, C-11'; 18.9, C-12'; 70.8, C13'; and 16.0ppm, C-14'. TLC Data Rf0.10 (4% methanol-methylene chloride), 0.17 (ethyl acetate), and 0.29 (30% isopropyl alcohol-hexane). Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

547

Common/Systematic Name Baccharinoid B 17 Molecular .Formula/Molecular Weight C29H38Olo, M~W -- 5 4 6 . 2 4 6 5 0

1~, .0~1o H IIIIiill

H

H2.~1~

H General Characteristics Crystals from ethyl ether-methylene chloride, m.p., >300~ CH2C12).

[a]D = + 11.0 ~ (C= 0.68, in

Plant Source Baccharis megapotamica. Isolation/Purification A large scale extract of 1800 kg of plant material was extracted with a total of 6000gal of isopropyl alcohol, which was concentrated to a black tar and partitioned between hexane and 10% water in methanol. The methanol solubles were treated by ferric gel precipitation, filtered, and the "pudding-like" insolubles washed with 50% aqueous methanol. The resulting filtrate was extracted with methylene chloride, and concentrated to give a total of 4.7kg of a dark oil. This material was chromatographed over silica gel, eluted with methylene chloride in hexane, methanol in methylene chloride, and 100% methanol to give 10 fractions (F 1 - F 10). Fraction F6 was taken up in methylene chloride and the solution carefully washed with a solution of 2.5% aqueous NaOH. The methylene chloride soluble material was subjected to filtration chromatography using alumina eluted with 10% isopropyl alcohol-hexane, 100% isopropyl alcohol, and 10% methanol in isopropyl alcohol). The 100% isopropyl alcohol fraction was chromatographed over Sephadex LH-20 eluted with methylene chloride and then chromatographed by flash chromatography over silica gel eluted with ethyl acetate-hexane). Final purification was by PTLC (Chromatotron) to give baccharinoid B 17 after recrystallization from ethyl ethermethylene chloride.

548

20.

Baccharinoids

Spectral Data UV:

~E~On max 260nm. IR:

(CHC13) 3580,2880, 1755, 1720, 1170, and ll00cm 1. 1H N/V[R:

(CDCI3) 0.77(3H, s, H-14); 1.23(3H, d, J=6.5Hz, H-14'); 1.37(3H, s, H-12'); 1.66 (3H, s, H-16); 2.19(1H, ddd, Jz3p=J3p,4=4.6Hz and Jgem=15.3Hz, H-313); 2.4- 2.6(3H, m, H-3ot and H-4'); 2.76, 3.17(1H each, AB pattern, J=3.9Hz, H-13); 3.09 (1H, d, J=5.4Hz, H-10); 3.30(1H, s, H-2'), 3.32-3.38(1H, m, H-5'B); 3.57(1H, d, J= 5.4Hz, H- 11); 3.84-3.92(1H, m, H-5'A); 3.94(1H, d, J=5.0Hz, H-2); 4.20, 4.43(1H each, AB pattern, J=12.6Hz, H-15); 5.76(1H, dd, J=4.6, 8.2Hz, H-4); 5.80(1H, d, J=l 1.2Hz, H-10'); 5.97(1H, dd, J=3.0, 15.6Hz, H-7'); 6.60(1H, dd, Js,,9,=Jg,,lo,=l1.2Hz, H-9'); and 7.54ppm (1H, dd, J= 11.2, 15.6Hz, H-8'). 13CNMR: (CDC13) 78.8, C-2; 34.8, C-3; 74.3, C-4; 49.1, C-5; 42.9, C-6; 17.6, C-7; 26.4, C-8; 57.7, C-9; 58.2, C-10; 67.5, C-11; 64.9, C-12; 47.6, C-13; 7.1, C-14; 63.7, C-15; 22.2, C-16; 168.2, C-I'; 57.4, C-2'; 63.7, C-3'; 40.1, C-4'; 67.9, C-5'; 86.0, C-6'; 138.6, C-7'; 126.3, C-8'; 143.5, C-9'; 117.9, C-10'; 166.5, C-11'; 17.4, C-12'; 70.8, C13'; 18.3, C-14'. TLC Data Rf0.66 (4% methanol-methylene chloride), 0.74 (ethyl acetate), and 0.54 (30% isopropyl alcohol-hexane). Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

549

Common/Systematic Name Baccharinoid B20 Molecular Formula/Molecular Weight C29H40010; M W -- 5 4 8 . 2 6 2 1 3

i

IIIIil, '

;

I,,'"~ J

o


[s

"-

q- 39.9 ~ (C=1.44, in CHC13).

Plant Source Baccharis megapotamica. Isolation/Purification See isolation ofbaccharinoid B9. Fractions E1 and E2 were combined and crystallized from acetone-hexane to give 210mg baccharinoid B20. Spectral Data UV:

~EtOH

max 262nm.

IR:

(CHCI3) 940, 1730, and 1370cm1. 1H NMR: (CDC13) 0.69(3H, s, H-14); 0.98(3H, d, J=6.6Hz, H-12'); 1.16(1H, d, J=6.5Hz, H-14'); 1.34(3H, s, H-16); 2.05-2.21(1H, m, H-313); 2.35-2.50(2H, m, H-3a and H-3'); 2.74, 3.14(1H each, AB pattern, J=4.0Hz, H-13); 3.06(1H, d, J=5.1Hz, H-10); 3.54(1H, d, ,/--5.1 Hz, H-11); 3.60-3.68(3H, m, H-4' and H-5'); 3.80-4.00(3H, m, H-2,

550

20.

Baccharinoids

H-6', and H-13'); 3.78, 4.64(1H each, AB pattern, J=12.3Hz, H-15), 5.74(1H, d, J=l 1.1Hz, H-10'); 5.74(1H, m, H-4); 6.05(1H, dd, J=3.4 and 16.4Hz, H-7'); 6.71(1H, dd, Js,,9,=J9,,lo~l 1.1Hz, H-9'); and 7.77ppm (1H, dd, J=l 1.1 and 16.4Hz, H-8'). 13CNMR: (CDCI3) 78.6, C-2; 34.6, C-3; 74.4, C-4; 48.8, C-5; 42.9, C-6; 17.6, C-7; 26.4, C-8; 57.4, C-9; 57.8, C-10; 67.6, C-11; 64.8, C-12; 47.6, C-13; 6.9, C-14; 63.7, C-15; 22.1, C-16; 172.7, C-I'; 38.3, C-2'; 32.5, C-3'; 73.4, C-4'; 74.2, C-5'; 84.4, C-6'; 139.6, C-7'; 126.3, C-8'; 144.1, C-9'; 117.1, C-10'; 166.7, C-11'; 14.9, C-12'; 69.8, C-13'; and 17.9ppm C- 14'.

Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

551

Common/Systematic Name Baccharinoid B21 Molecular Formula/Molecular Weight C29H38Olo; M W -- 5 4 6 . 2 4 6 5 0

H

H

.-: o~d

H2dl

....'~

HO ~- - ~ H General Characteristics Melting point, 259-260~

[{g]D =

q- 73.5 ~ (C=0.68, in CH2C12).

Plant Source Baccharis megapotamica. Isolation/Purification See baccharinoid B 17 for general isolation procedure through the second column. Fraction 7C was subjected to preparative HPLC (3.5% methanol-methylene chloride, Waters Prep. LC/500) to give 10 fractions. The third fraction was sequentially subjected to MPLC (ethyl aeetate-hexane) and PTLC (50-100% ethyl acetate-hexane, Chromatotron) to give a fraction which upon crystallization from ethyl ether-methylene chloride gave 140mg of baccharinoid B21. Spectral Data UV~ EtOH j~max 2 6 0 n m . IR;

(CHCI3) 2880, 1750, 1715, 1170, and ll00cm "1.

552

20.

Baccharinoids

1H ~ : (CDCIa) 0.82(3H, s, H-14); 1.18(3H, d, J--6.0Hz, H-14'); 1.55(3H, s, H-12'); 1.81(3H, s, n-16); 2.10-2.22(3H, m, n-3~, and n-7); 2.44(1H, dd, Ja~.4=8.3Hz and Js~=15.3Hz, H-3a); 2.82, 3.11(1H each, AB pattern, J--4.0Hz, H-13); 3.29(1H, s, H2'), 3.33-3.37(1I-I, m, H-5~B), 3.58-3.64(2H, m, H-5'A and n-11); 3.64-3.80(2H, m, H6' and H-13'); 3.85(1I-I, d, J--4.9Hz, H-2); 3.82-3.99(1H, m, n-8), 4.19, 4.40(1H each, AB pattern, J=-12.3Hz, H-15); 5.48(1H, d, J=5.3Hz, H-10); 5.75(1I-I, dd, J=4.3 and 8.3Hz, n-4); 5.78(1H, d, J=-I 1.4Hz, n-10'); 5.95(1H, dd, J=-2.9 and 15.7Hz, H-7'); 6.58(1I-I, dd, Ja. ~=J~,~o~l1.4Hz, H-9'); and 7.48ppm (1H, dd, J=l 1.4 and 15.7Hz, H8'). 13CNMR: (CDCI3) 79.2, C-2, 35.1, C-3, 74.0, C-4, 49.4, C-5, 45.2, C-6; 30.7, C-7; 67.0, C-8, 142.9, C-9; 120.8, C-10, 68.0, C-11, 65.2, C-12, 47.8, C-13; 7.0, C-14; 65.0, C-15; 18.8, C-16; 168.1, C-I', 58.2, C-2'; 63.4, C-3'; 39.7, C-4', 67.6, C-5', 85.8, C-6'; 138.5, C-7'; 126.3, C-8'; 142.8, C-9', 118.0, C-10', 166.4, C-11'; 17.4, C-12'; 70.9, C-13'; and 18.3ppm C-14'. Reference B. B. ]arvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis meg~potamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

553

Common/Systematic Name Baccharinoid B23 Molecular Formula/Molecular Weight C29H40010; M W -- 5 4 8 . 2 6 2 1 5

H H -.-- O~r_~__ ~ ....,0

HOH2C

H2 I o

HO'~ H General Characteristics Colorless glass; [a]D = + 113.5~ (C=0.90, in MeOH). Plant Source Baccharis megapotamica. Isolation/Purification See isolation procedure for baccharinoid B25. Spectral Data UV:

EtOH

~,m,x 261nm. IR:

(CH2C12) 3600, 2870, 1730, 1715, and l175crn1. :H NMR: (CDC13) 0.77(3H, s, H-14); 1.00(3H, d, J=6.4Hz, H-12'); 1.21(3H, s, H-14'); 2.102.25(3H, m, H-3~, and H-2'); 2.40-2.53(2H, m, H-3a and H-3'); 2.82, 3.12(1H each, AB pattern, J=4.0Hz, H-13); 3.60-3.75(4H, m, H-4', H-5', and H-13'); 3.73(2H, s, H16); 3.84(1H, d, J=4.SHz, H-2); 4.06(1H, br s, H-6'); 3.92, 4.67(1H each, AB pattern, J=12.SHz, H-15); 5.77-5.85(2H, m, H-10 and H-4); 5.79(1H, d, J=l 1.3Hz, H-10');

554

20.

Baccharinoids

6.06(1H, dd, J=3.3 and 15.7Hz, H-7'); 6.58(1H, dd, Jg.,9~J9.,10,=l1.3Hz, H-9'); and 7.81ppm (1H, dd, J=l 1.3 and 15.7Hz, H-8'-H). 13C NMR: (CDC13) 79.2, C-2; 34.9, C-3; 74.5, C-4; 49.2, C-5; 43.9, C-6; 20.1, C-7; 23.2, C-8; 143.6, C-9; 118.3, C-10; 66.8, C-11; 65.3, C-12; 47.9, C-13; 7.0, C-14; 66.0, C-15; 63.5, C-16; 172.8, C-I'; 38.2, C-2'; 32.4, C-3'; 73.0, C-4'; 74.3, C-5'; 86.0, C-6'; 139.8, C-7'; 126.5, C-8'; 144.0, C-9'; 117.6, C-10'; 166.8, C-11'; 15.0, C-12'; 71.0, C-13'; and 18.6ppm C-14'. Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation of Macrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

555

Common/Systematic Name Baccharinoid B24 Mol.eeul~ Formula/Molecular Weight C29H40010; M W - 548.26215 H

H

"

I

HOH2C,~.- O,,J .... ,o i

OJ) H General Characteristics Colorless glass from methylene chloride-hexane; [a]D = + 90.8 ~ (C=1.36, in MeOH). Plant Source Baccharis megapotamica. Isolation/Purification See isolation for isolation ofbaeeharinoid B25. Spectral Data UV:

~.~ 260nm. IR:

(CHCh) 3600, 2875, 1720, 1715, and llS0em "~. 1H N/VIR:

(CDCI3) 0.79(3H, s, H-14); 1.01(3H, d, J=6.5 Hz, H-12'); 1.17(3H, d,J=6.4Hz, H14'); 2.10-2.50(5H, rn, H-3, H-2', and H-3'); 2.82, 3.12(1H each, AB pattern, d=4.0 Hz, H-13); 3.62-3.74(4H, m, H-11', H-5', and H-13'); 3.72(2H, s, H-16), 3.84(1H, d, J---4.8H~ H-2); 4.04(1H, br s, H-6'); 3.94, 4.63(1H each, AB pattern, J=-12.4I-!z, H15); 5.69(1H, d, J=5.3Hz, n-10), 5.78(1H, d, J=l 1.3H~ H-10'); 5.84(1H, dd, ,/---4.5

556

20.

Baccharinoids

and 8.2Hz, H-4); 6.06(1H, dd, ,/=3.0 and 15.6Hz, H-7'); 6.69(1H, dd,

Js,,9,=J9,,1o,=l 1.3Hz, H-9'); and 7.79ppm (1H, dd, J=l 1.3 and 15.6Hz, H-8'). 13CNMR: (CDC13) 79.3, C-2; 35.0, C-3; 74.7, C-4; 49.4, C-5; 44.0, C-6; 20.2, C-7; 23.3, C-8; 143.6, C-9; 118.5, C-10; 66.9, C-11; 65.3, C-12; 47.9, C-13; 7.0, C-14; 66.1, C-15; 63.4, C-16; 172.8, C-I'; 38.4, C-2'; 32.6, C-3'; 73.2, C-4'; 74.7, C-5'; 84.7, C-6'; 139.3, C-7'; 126.8, C-8'; 144.0, C-9'; 117.5, C-10'; 166.8, C-11'; 14.8, C-12'; 70.1, C-13'; and 18.0ppm C- 14'. Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

557

Common/Systematic Name Baccharinoid B25 Molecular Formula/Molecular Weight C27H32Olo; M W = 516.19955 H

H

-

HO"

v

0

-

....,oI

y

0

0 General Characteristics Melting point, 205~

[a]D -- + 214 ~ (C=0.70, in MeOH).

Plant Source Baccharis megapotamica. Isolation/Purification See baccharinoid B 17 for general isolation procedure through the first column. Fraction 9 from this column was chromatographed over silica gel with increasing methanol in methylene chloride to give five fractions 9A-9E. Fraction 9D was dissolved in methylene chloride and hexane was added slowly to give a precipitate. This material was dissolved in methylene chloride and the solution washed with 2.5% aqueous NaOH, concentrated to dryness, and subjected to preparative HPLC using alumina and eluting with isopropyl alcohol-methylene chloride to give six fractions. From these fractions by successive chromatographic procedures baccharinoids B 13, B 14, B23, B24, B25, and B27 were isolated. Spectral Data UV: ~EtOH max

220 and 260nm.

IR:

(KBr) 1740, and 1165cm"1.

558

20.

Baccharinoids

1H NMR: (CDCI3) 0.81(3H, s, H-14); 1.83(3H, s, H-16); 2.10-2.30(1H, m, H-313); 2.53(1H, dd, J3~4=8.1Hz, Jg =15.5Hz, H-3ot); 2.86, 3.17(1H each, AB pattern, J=4.0Hz, H13); 3.88(1H, d, J=5.0Hz, H-2); 3.91(1H, d, J=5.4Hz, n-11); 4.03-4.14(3H, m, H5'A, H-8-H, and H-15B); 4.40-4.48(2H, m, H-4' and H-5'A); 4.17(1H, AB pattern, J=12.7Hz, H-15A); 5.56(1H, d, J=5.4Hz, n-10); 5.99(1H, dd, J=2.9, 15.7Hz, n-7'); 6.07(1H, br s, n-2'); 6.06-6.1 l(1n, m, n-4); 6.12(1H, d, J=l 1.3Hz, H-10'); 6.63(1H, dd, Js,,9,=Jg,,ltr=ll.3Hz, H-9'); and 8.1 lppm (1H, dd, J=l 1.3, 15.8 Hz, n-8'). 13CNMR: (CDC13) 78.3, C-2; 35.4, C-3; 75.1, C-4; 48.7, C-5; 45.4, C-6; 31.6, C-7; 68.3, C-8; 142.4, C-9; 121.2, C-10; 66.9, C-11; 65.4, C-12; 48.1, C-13; 6.8, C-14; 64.2, C-15; 18.9, C-16; 165.8, C-I'; 116.8, C-2'; 158.3, C-3'; 74.3, C-4'; 63.7, C-5'; 165.1, C-6'; 126.5, C-7'; 139.1, C-8'; 139.7, C-9'; 125.9, C-10'; 165.8, C-11'; and 13.9ppm, C-12'. TLC Data Rr 0.31 (4% methanol-methylene chloride), 0.64 (ethyl acetate), and 0.58 (30% isopropyl alcohol-hexane). Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

20.

Baccharinoids

559

Common/Systematic Name Baccharinoid B27 Molecular Formula/Molecular Weight C29I-I36010; M W = 544.23085 H

H

=- o~I

~

H General Characteristics Melting point, 165~

[tX]D= + 5.4~ (C----0.40,in methanol).

Plant Source Baccharis megapotamica. Isolation/Purification See description for isolation ofbacchafinoid B25.

Spectral Data UV"

~.~.tOH 223 and 263nm. IR:

(KBr) 3600, 2900, 1720, and 1685crn"1. 1HNMR: (CDCh) 0.76(3H, s, 1H-14); 1.18(3H, d, J--6.4Hz, H-14'); 1.83(3H, s, H-16); 2.10(1H, ddd, Jz3f4.9Hz, J~p,4~3.9Hz, Jg~=l 5.5Hz, H-313);2.24(1H, d, J=l. 1Hz, H12'); 2.60(1H, dd, J=-8.1, 15.5Hz, n-3ct); 2.42, 2.94(1H each, AB pattern, J=-16.3I~ H-7); 2.85, 3.15(1H each, AB pattern, J--4.0Hz, 13-H); 3.64, 3.82(1H each, d of AB pattern, J=4.0, 9.1I~ n-5'); 3.93(1H, d, J=4.9Hz, H-2); 4.00-4.3 l(6H, m, H-11, H15, n-4', H-6', and H-lY); 5.75(1H, d, J=l 1.5Hz, H-10'); 5.91(1H, dd, J=2.6, 15.4Hz, n-7'); 6.18(1H, br s, n-2'); 6.20(1H, dd, J=3.9, 8. lI-~ U-4); 6.57(1H, d, J=5.0Hz, H-

560

20.

Baccharinoids

10), 6.58(1H, dd, ,]8,9,=.]9,]o_11.5Hz, H-9'), and 7.48ppm (1H, dd, J=l 1.5, 15.4Hz, H8'). 13C NMR: (CDC13) 79.4, C-2; 36.1, C-3; 73.6, C-4; 48.3, C-5; 46.5, C-6; 38.8, C-7; 197.1, C-8; 136.6, C-9; 138.5, C-10; 66.4, C-11; 65.7, C-12; 47.9, C-13; 6.4, C-14; 64.6, C-15; 17.4, C-16; 166.4, C-I'; 114.6, C-2'; 161.1, C-3'; 74.6, C-4'; 71.8, C-5'; 82.3, C-6'; 137.2, C-7'; 127.3, C-8'; 143.4, C-9'; 117.7, C-10'; 165.8, C-11'; 15.5, C-12'; 68.2, C13'; and 16.1ppm, C-14'. TLC Data Rf=0.44 (4% methanol-methylene chloride), 0.44 (ethyl acetate), and 0.51 (30% isopropyl alcohol-hexane). Reference B. B. Jarvis, S. N. C6mezoglu, M. M. Rao, N. B. Pena, F. E. Boettner, T. M. Williams, G. Forsyth, and B. Epling; Isolation ofMacrocyclic Trichothecenes from a Large Scale Extract ofBaccharis megapotamica; J. Org. Chem., Vol. 52, pp. 45-56(1987).

Fumonisins, AAL Toxins, and Related Metabolites Fumonisin B1 Fumonisin B2 Fumonisin B3 Fumonisin B4 Fumonisin A1 Fumonisin A2 Fumonisin C~ Hydroxylated Fumonisin C1 Fumonisin C3 Fumonisin Ca Fumonisin AK1 AAL Toxin TA~ AAL Toxin TA2 AAL Toxin TB 1 AAL Toxin TB2 AAL Toxin TC~ AAL Toxin TC2 AAL Toxin TD~ AAL Toxin TD2 /X~L Toxin TE~ AAL Toxin TE2 Sphingofungin A Sphingofungin B Sphingofungin C Sphingofungin D Myriocin

561


21.

Fumonisins, AALToxins, and Related Metabolites

563

Common/Systematic Name Fumonisin BI, Macrofusin Molecular Formula/Molecular Weight C34I-I59NO15; MW = 721.38847 30 O H 0

o.o.

2

1 20

o.

OH General Characteristics Fumonisins are highly functionalized hydrocarbon derivatives that are readily soluble in polar solvents and nearly insoluble in nonpolar solvents. Fungal Source

Fusarium moniliforme Sheldon (M-2326), (MRC-826).

Isolation/Purification Culture material was extracted twice with ethyl acetate to remove lipid soluble materials. The residue after filtration was extracted with methanol-water (1:3, v/v). The extract was partitioned with chloroform and the aqueous phase was evaporated to dryness under vacuum at 50~ The dried extract was dissolved in methanol-water (1:3, v/v) and applied to an Amberlite XAD-2 column previously equilibrated with methanol-water (1:3, v/v). The column was eluted successively with methanol-water (1:3, v/v), methanol-water (1:1, v/v), and methanol. The fumonisins were eluted with the methanol fraction. The methanol fraction was chromatographed on a silica gel 60 column (anhydrous sodium sulfate on top of column) eluted with chloroform-methanol-acetic acid (6:3:1, v/v/v). The main fumonisin B1 fraction was again fractionated on a silica gel 60 column eluted with chloroform-methanol-water-acetic acid (55:36: 8:1, v/v/v/v) but without the anhydrous sodium sulfate on top of column. Fractions containing only fumonisin B1 were combined and finally purified using RP-C18 column using methanol-water (1:1,v/v) as eluant. The pH of the sample was adjusted to 3.5 with 1N HC1 prior to application to the column. Fractionation was achieved using a linear gradient from methanol-water (1:1, v/v) to methanol-water (4:1, v/v) to produce purified fumonisin B1.

564

21.


Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly-phytotoxic. Spectral Data 13C NMR: (D20) 17.7, C-I; 55.5, C-2; 71.8, C-3; 42.2, C-4; 69.9, C-5; 39.5, C-6; 27.7, C-7; 27.6, C-8; 39.8, C-9; 71.4, C-10; 45.1, C-11; 27.7, C-12; 37.5, C-13; 74.7, C-14; 80.6, C-15; 35.9, C-16; 34.3, C-17; 30.8, C-18; 25.1, C-19; 16.3, C-20; 17.3, C-21; 22.4, C22; 175.2, C-I'; 37.8, C-2'; 40.0, C-3'; 38.1, C-4'; 179.6, C-5'; 179.1, C-6'; 175.1, C1"; 37.8, C-2"; 39.9, C-3"; 38.0, C-4"; 178.0, C-5"; and 177.6ppm, C-6". (Hydrolysis product) 16.8, C-I; 53.8, C-2; 70.4, C-3; 42.9, C-4; 68.5, C-5; 39.3, C-6; 26.8, C-7; 26.9, C-8; 39.6, C-9; 70.7, C-10; 44.6, C-11; 26.9, C-12; 41.6, C-13; 70.4, C-14; 80.8, C-15; 35.9, C-16; 31.7, C-17; 30.6, C-18; 24.2, C-19; 14.5, C-20; 16.0, C21; and 21.4ppm, C-22. Mass Spectrum: FABMS: (M+H) § 722m/e and (M+Na) + 744m/e. References S. C. Bezuuidenhout, W. C. A. Gelderblom, C. P. Gorst-Allman, R. M. Horak, W. F. O. Marasas, G. Spiteller, and R. Vleggaar; Structure Elucidation of the Fumonisins, Mycotoxins from Fusarium moniliforme; J. Chem. Sot., Chem. Commun. pp. 743-745 (1988). D. Laurent, N. Platzer, F. Kohler, M. P. Sauviat and F. Pellegrin; Macrofusine et Micromoniline: Deux Nouvelles Mycotoxines Isolees De Mais Infeste par Fusarium moniliforme Sheld.; Microbiol, Aliments, Nutrition; Vol. 7, pp. 9-16(1989). R. G. Powell and R.D. Plattner; Fumonisins; In Alkaloids: Chemical and Biological Perspectives; ed. S. W. Pelletier; Pergamon Press; pp. 247-278(1995). M. E. Cawood, W. C. A. Gelderblom, R. Vleggaar, Y. Behrend, P. G. Thiel, and W. F. O. Marasas; Isolation of the Fumonisins: A Quantitative Approach; J. Agric. Food Chem.;Vol. 39, pp. 1958-1962(1991).

21.


565

Common/Systematic Name Fumonisin B2 Molecular Formula/Molecular Weight C34H59NO14; M W -- 7 0 5 . 3 9 3 5 6

0

OH OH NH2

OH

O/ CH3 O.,,,,~O OH

OH General Characteristics Fumonisins are highly funetionalized hydrocarbon derivatives that are readily soluble in polar solvents and nearly insoluble in nonpolar solvents. Fungal Source

Fusarium rnoniliforme Sheldon (M-2326); (MRC-826).

Isolation/Purification Culture material was extracted twice with ethyl acetate to remove lipid soluble materials. The residue alter filtration was extracted with methanol-water (1:3, v/v). The extract was partitioned with chloroform and the aqueous phase was evaporated to dryness under vacuum at 50 ~ C. The dried extract was dissolved in methanol-water (1:3, v/v) and applied to an Amberlite XAD-2 column previously equilibrated with methanol-water (1:3, v/v). The column was eluted successively with methanol-water (1:3, v/v), methanol-water (1:1, v/v), and methanol. The fumonisins were eluted with the methanol fraction. The methanol fraction was chromatographed on a silica gel 60 column (anhydrous sodium sulfate on top of column) eluted with chloroform-methanol-acetic acid (6:3:1, v/v/v). The fumonisin B2 fraction that coeluted with B1 and B3 was again fractionated on a silica gel 60 column eluting with chloroform-methanol-water-acetic acid (55:36:8:1, v/v/v/v) but without the anhydrous sodium sulfate on top of column. Fractions containing only fumonisin B2 were combined and finally purified using RP-C 18 column using methanol-water (1:1, v/v) as eluant. The pH of the sample was adjusted to 3.5 with IN HC1 prior to application to the column. Fractionation was achieved using a linear gradient from methanol-water (1:1, v/v) to methanol-water (4:1, v/v) to produce purified fumonisin B2 (92% purity).

566

21.


Biological Activity The primary animal diseases that have been caused by the fumonisins are leueoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic. Spectral Data 13CNMR: (D20) 16.0, C-l; 53.8, C-2; 70.4, C-3; 41.8, C-4; 68.7, C-5; 39.3, C-6; 27.7, C-7; 29.7, C-8; 26.7, C-9; 30.7, C-10; 36.1, C-11; 30.2, C-12; 36.2, C-13; 73.1, C-14; 78.9, C-15; 34.9, C-16; 33.2, C-17; 30.8, C-18; 23.9, C-19; 14.4, C-20; 16.0, C-21; 20.9, C22; 173.0, C-I'; 36.5, C-2'; 38.6, C-3'; 36.1, C-4'; 276.8, C-5'; 175.2, C-6'; 172.9, C1"; 36.5, C-2"; 38.5, C-3"; 36.1, C-4"; 176.6, C-5"; and 175.0ppm, C-6". (Hydrolysis product) 16.8, C-l; 53.8, C-2; 70.9, C-3; 41.9, C-4; 68.6, C-5; 39.3, C-6; 26.8, C-7; 27.9, C-8; 31.2, C-9; 30.8, C-10; 36.9, C-11; 30.5, C-12; 40.7, C-13; 70.4, C-14; 80.9, C-15; 36.0, C-16; 31.9, C-17; 30.6, C-18; 24.2, C-19; 14.5, C-20; 16.0, C21; and 21.5ppm, C-22. Mass Spectrum: FABMS (M+H)+ 706m/e and (M+Na) + 728role. References S. C. Bezuidenhout, W. C. A. Gelderblom, C. P. Gorst-AUman, R. M. Horak, W. F. O. Marasas, G. Spiteller, and R. Vleggaar; Structure Elucidation of the Fumonisins, Mycotoxins from Fusarium moniliforme; J. Chem. Sot., Chem. Commun.; pp. 743-745 (1988). D. Laurent, N. Platzer, F. Kohler, M. P. Sauviat and F. Pellegrin; Macrofusine et Micromoniline: Deux Nouvelles Mycotoxines Isolees De Mais Infeste par Fusarium moniliforme Sheld.; Microbiol, Aliments, Nutrition; Vol. 7, pp. 9-16(1989). R. G. Powell and R.D. Plattner; Fumonisins; In Alkaloids: Chemical and Biological Perspectives; ed. S. W. Pelletier; Pergamon Press; pp. 247-278(1995). M. E. Cawood, W. C. A. Gelderblom, R. Vleggaar, Y. Behrend, P. G. Thiel, and W. F. O. Marasas; Isolation of the Fumonisins: A Quantitative Approach; J. Agile. Food Chem.;Vol. 39, pp. 1958-1962(1991). B. E. Branham and R. D. Plattner; Isolation and Characterization of a New Fumonisin from Liquid Cultures ofFusarium moniliforme; J. Nat. Products; Vol. 56, pp. 1630-1633 (1993).

21.


567

Common/Systematic Name Fumonisin B3 Molecular Formula/Molecular Weight. C34H59NO14; MW

-- 7 0 5 . 3 9 3 5 6

% / OH

NH2

0/

OH

CH3

O ~

OH

i

OH General Characteristics Fumonisins are highly functionalized hydrocarbon derivatives that are readily soluble in polar solvents and nearly insoluble in nonpolar solvents. Fungal Source

Fusarium moniliforme Sheldon (M-2326); (MRC-826).

Isolation/Purification Culture material was extracted twice with ethyl acetate to remove lipid soluble materials. The residue after filtration was extracted with methanol-water (1:3, v/v). The extract was partitioned with chloroform and the aqueous phase was evaporated to dryness under vacuum at 50~ The dried extract was dissolved in methanol-water (1:3, v/v) and applied to an Amberlite XAD-2 column previously equilibrated with methanol-water (1:3, v/v). The column was eluted successively with methanol-water (1:3, v/v), methanol-water (1:1, v/v), and methanol. The fumonisins were eluted with the methanol fraction. The methanol fraction was chromatographed on a silica gel 60 column (anhydrous sodium sulfate on top of column) eluted with chloroform-methanol-acetic acid (6:3:1, v/v/v). The fumonisin B2 fraction that coeluted with B~ and B3 was fractionated on an Amberlite XAD-2 column equilibrated with methanol-water (1:1, v/v). The pH of the sample was adjusted to 3.5 with 1N HC1 prior to application to the column. The column was washed with methanolwater (1:1, v/v). Fractionation was achieved using acetonitrile-methanol (1:1, v/v) to produce purified fumonisin B3.

568

21.


Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic. Spectral Data 13C lX~iR: (D20) 15.9, C-I; 53.5, C-2; 73.1, C-3; 34.6, C-4; 26.2, C-5; 26.1, C-6; 26.8, C-7; 29.6, C-8; 39.3, C-9; 69.9, C-10; 44.5, C-11; 26.9, C-12; 36.4, C-13; 73.1, C-14; 78.8, C-15; 34.9, C-16; 33.1, C-17; 30.7, C-18; 23.8, C-19; 14.4, C-20; 16.0, C-21; 20.6, C22; 173.1, C-I'; 36.6, C-2'; 38.6, C-3'; 36.1, C-4'; 177.0, C-5'; 175.2, C-6'; 173.0, C1"; 36.6, C-2"; 38.6, C-3"; 36.1, C-4"; 176.6, C-5"; and 175.0ppm, C-6". (Hydrolysis product) 16.8, C-I; 53.5, C-2; 73.1, C-3; 34.7, C-4; 26.3, C-5; 30.7, C-6; 30.8, C-7; 26.9, C-8; 39.6, C-9; 70.1, C-10; 44.6, C-11; 27.0, C-12; 41.6, C-13; 70.4, C-14; 80.8, C-15; 35.9, C-16; 31.7, C-17; 30.7, C-18; 24.2, C-19; 14.5, C-20; 16.0, C21; and 21.4ppm, C-22. Mass Spectrum: FABMS: 706role (M+H) § and MS/MS ofM + from FABMS (706rn/e). References S. C. Bezuidenhout, W. C. A. Gelderblom, C. P. Gorst-Allman, R. M. Horak, W. F. O. Marasas, G. Spiteller, and R. Vleggaar; Structure Elucidation of the Fumonisins, Mycotoxins from Fusarium moniliforme; J. Chem. Sot., Chem. Commun.; pp. 743-745 (1988). D. Laurent, N. Platzer, F. Kohler, M. P. Sauviat and F. Pellegrin; Macrofusine et Micromoniline: Deux Nouvelles Mycotoxines Isolees De Mais Infeste par Fusarium moniliforme Sheld.; Microbiol, Aliments, Nutrition; Vol. 7, pp. 9-16(1989). R. G. Powell and R.D. Plattner; Fumonisins; In Alkaloids: Chemical and Biological Perspectives; ed. S. W. Pelletier; Pergamon Press; pp. 247-278(1995). M. E. Cawood, W. C. A. Gelderblom, R. Vleggaar, Y. Behrend, P. G. Thiel, and W. F. O. Marasas; Isolation of the Fumonisins: A Quantitative Approach; J. Agric. Food Chem.;Vol. 39, pp. 1958-1962(1991 ). B. E. Branham and R. D. Plattner; Isolation and Characterization of a New Fumonisin from Liquid Cultures ofFusarium moniliforme; J. Nat. Products; Vol. 56, pp. 1630-1633 (1993).

21.


569

Common/Systemati_c Name Fumonisin B4 Molecular Formula/Molecular Weight C34H59NO13~, U W

-- 6 8 9 . 3 9 8 6 4

0===~O 0H,~~--OH / O"

OH NH2

Me

OH

0 ~ . ~ 0

OH General Characteristics Fumonisins are highly functionalized hydrocarbon derivatives that are readily soluble in polar solvents and nearly insoluble in nonpolar solvents. Fungal Source Fusarium moniliforme Sheldon (M-2326); (MRC-826). Isolation/Purification Culture material was extracted twice with ethyl acetate to remove lipid soluble materials. The residue aider filtration was extracted with methanol-water (1:3, v/v). The extract was partitioned with chloroform and the aqueous phase was evaporated to dryness under vacuum at 50~ The dried extract was dissolved in methanol-water (1:1, v/v) and applied to an Amberlite XAD-2 column previously equilibrated with methanol-water (1:1, v/v). The column was eluted with methanol-acetonitrile (1:1, v/v). The fractions containing fumonisin B4 were further chromatographed on a silica gel 60 column eluted with chloroformmethanol-water-acetic acid (55:36: 8:1, v/v/v/v). The fumonisin B4 was finally fractionated through a Sep-Pak ClS cartridge. Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high

570

21.


levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic. References S. C. Bezuidenhout, W. C. A. Gelderblom, C. P. Gorst-Allman, R. M. Horak, W. F. O. Marasas, G. Spiteller, and R. Vleggaar; Structure Elucidation of the Fumonisins, Mycotoxins from Fusarium moniliforme; J. Chem. Sot., Chem. Commun.; pp. 743-745 (1988). D. Laurent, N. Platzer, F. Kohler, M. P. Sauviat and F. Pellegrin; Macrofusine et Micromoniline: Deux Nouvelles Mycotoxines Isolees De Mais Infeste par Fusarium moniliforme Sheld.; Microbiol, Aliments, Nutrition; Vol. 7, pp. 9-16(1989). R. G. Powell and R.D. Plattner; Fumonisins; In Alkaloids: Chemical and Biological Perspectives; ed. S. W. Pelletier; Pergamon Press; pp. 247-278(1995). M. E. Cawood, W. C. A. Gelderblom, R. Vleggaar, Y. Behrend, P. G. Thiel, and W. F. O. Marasas; Isolation of the Fumonisins: A Quantitative Approach; J. Agric. Food Chem.;Vol. 39, pp. 1958-1962(1991). B. E. Branham and R. D. Plattner; Isolation and Characterization of a New Fumonisin from Liquid Cultures ofFusarium moniliforme; J. Nat. Products; Vol. 56, pp. 1630-1633 (1993).

21.


571

Common/Systematic Name Fumonisin A1 Molecular Formula/Molecular Weight C36H61NO]6; M W = 763.39904

O

OH

OH

NHCOMe

0~~//

OH Me O~OoH O''?

OH General Characteristics Fumonisins are highly functionalized hydrocarbon derivatives that are readily soluble in polar solvents and nearly insoluble in nonpolar solvents. Fumonisin A1 was isolated as the tetramethyl derivative as a colorless oil. Funsal Source v


Isolation/Purification Culture material was extracted twice with ethyl acetate to remove lipid soluble materials. The residue after filtration was extracted with methanol-water (1:3, v/v). The extract was partitioned with chloroform and the aqueous phase was evaporated to dryness under vacuum at 50 ~ C. The dried extract was dissolved in methanol-water (1:3, v/v) and applied to an Amberlite XAD-2 column previously equilibrated with methanol-water (1:3, v/v). The column was eluted successively with methanol-water (1:3, v/v), methanol-water (1:1, v/v), and methanol. The fumonisins were eluted with the methanol fraction. The methanol fraction was chromatographed on a silica gel 60 column (anhydrous sodium sulfate on top of column) eluted with chloroform-methanol-acetic acid (6:3:1, v/v/v). Fractions containing fumonisin A1 and A2 were further purified using an Amberlite XAD-2 column. The column was eluted successively with methanol-water (1:3, v/v), methanol-water (1:1, v/v), and methanol. Fumonisins ml and A2 were further purified on a silica gel column eluted with chloroform-methanol-acetic acid-water (65:25:6:4, v/v/v/v) which resulted in separation of fumonisins A] and A2. Final purification was achieved on a RP C1, column equilibrated with methanol-water (1:1, v/v) and eluted with methanol-water (3:1, v/v).

572

21.


Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic. Spectral Data 13C NMR: Tetramethyl derivative; (CDC13) 18.03, C-I; 49.72, C-2; 71.49; C-3; 40.22, C-4; 68.83, C-5; 38.55, C-6; 25.63, C-7; 25.48, C-8; 37.22, C-9; 68.78, C-10; 43.08, C-11; 25.17, C-12; 35.39, C-13; 71.24, C-14; 77.79, C-15; 33.64, C-16; 31.84, C-17; 28.48, C-18; 22.73, C-19; 13.92, C-20; 15.36, C-21; 20.36, C-22; 175.48, C-23; 2332, C-24; 170.88, C-25; 35.27, C-26; 37.28, C-27; 35.10, C-28; 173.41, C-29; 171.69, C-30; 52.31, C-31; and 51.82ppm, C-32. Mass Spectrum: Tetramethyl derivative; FABMS: (M+H) + 820m/e and (M+Na) + 842m/e. References S. C. Bezuuidenhout, W. C. A. Gelderblom, C. P. Gorst-Allmart, R. M. Horak, W. F. O. Marasas, G. Spiteller, and R. Vleggaar; Structure Elucidation of the Fumonisins, Mycotoxins from Fusarium moniliforme; J. Chem. Soc., Chem. Commun.; pp. 743-745 (1988). R. G. Powell and R.D. Plattner; Fumonisins; In Alkaloids: Chemical and Biological Perspectives; ed. S. W. Pelletier; Pergamon Press; pp. 247-278(1995). M. E. Cawood, W. C. A. Gelderblom, R. Vleggaar, Y. Behrend, P. G. Thiel, and W. F. O. Marasas; Isolation of the Fumonisins: A Quantitative Approach; J. Agric. Food Chem.;Vol. 39, pp. 1958-1962(1991).

21.


573

Common/Systematic Name Fumonisin A2 Molecular Formula/Molecular Weight C36H61NO15, MW = 747.40412

0

OH

NHCOMe

OH

OH

0/

Me

0.,,,,,~0

L

OH

I. o OH

General Characteristics Fumonisins are highly functionalized hydrocarbon derivatives that are readily soluble in polar solvents and nearly insoluble in nonpolar solvents. Fumonisin A2 was isolated as the tetramethyl derivative as a colorless oil. Fungal Source


Isolation/Purification Culture material was extracted twice with ethyl acetate to remove lipid soluble materials. The residue after filtration was extracted with methanol-water (1:3, v/v). The extract was partitioned with chloroform and the aqueous phase was evaporated to dryness under vacuum at 50 ~ C. The dried extract was dissolved in methanol-water (1:3, v/v) and applied to an Amberlite XAD-2 column previously equilibrated with methanol-water (1:3, v/v). The column was eluted successively with methanol-water (1:3, v/v), methanol-water (1:1, v/v), and methanol. The fumonisins were eluted with the methanol fraction. The methanol fraction was chromatographed on a silica gel 60 column (anhydrous sodium sulfate on top of column) eluted with chloroform-methanol-acetic acid (6:3:1, v/v/v). Fractions containing fumonisin A1 and A2 were further purified using an Ambedite XAD-2 column. The column was eluted successively with methanol-water (1:3, v/v), methanol-water (1:1, v/v), and methanol. Fumonisins A1 and A2 were further purified on a silica gel column eluted with chloroform-methanol-acetic acid-water (65:25:6:4, v/v/v/v) which resulted in separation of fumonisins A~ and A2. Final purification was achieved on a RP C18 column equilibrated with methanol-water (1:1, v/v) and eluted with methanol-water (3:1, v/v).

574

21.


Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic. Spectral Data Mass Spectrum: Tetramethyl derivative; FABMS: (M+W) 84m/e. References S. C. Bezuuidenhout, W. C. A. Gelderblom, C. P. Gorst-Allman, R. M. Horak, W. F. O. Marasas, G. Spiteller, and R. Vleggaar; Structure Elucidation of the Fumonisins, Mycotoxins from Fusarium moniliforme; J. Chem. Sot., Chem. Commun. pp. 743-745 (1988). R. G. Powell and R.D. Plattner; Fumonisins; In Alkaloids: Chemical and Biological Perspectives; ed. S. W. Pelletier; Pergamon Press; pp. 247-278(1995). M. E. Cawood, W. C. A. Gelderblom, R. Vleggaar, Y. Behrend, P. G. Thiel, and W. F. O. Marasas; Isolation of the Fumonisins: a quantitative approach; J. Agric. Food Chem.;Vol. 39, pp. 1958-1962(1991).

21.


575

Common/Systematic Name Fumonisin C1 Molecular Formula/Molecular Weight C33H57NO15, MW = 707.37282

\

OH OH NH2

0==~

OH

0/ OH

Me

0~0

OH

OH General Characteristics Obtained as a colorless liquid. Fungal Source


Isolation/Purification Method 1. The crude culture material was separated on preparative reversed-phase HPLC using a ClS column eluted with a gradient from 100% water to 100% methanol. The fraction containing a solvent concentration of approximately methanol-water (60:40) was taken to dryness and chromatographed on a C~s column using an isocratic mobile phase of 0.05 M NaH2PO4 (adjusted to pH 3.35)-acetonitrile (72:24, v/v). Fractions containing both fumonisins B~ and C1 were rechromatographed on a C~s column using a mobile phase of 0.05 M NaH2PO4 (adjusted to pH 3.35)-acetonitrile (74:26, v/v). Fractions containing fumonisin C~ were combined, taken to dryness, and desalted on a Cls mini-column. Method 2. Solid culture material was extracted with methanol-water (3:1, v/v), filtered, and concentrated to dryness. The aqueous phase was chromatographed on an Amberlite XAD-2 column eluted successively with methanol-water (1:3, v/v), (1:1, v/v), (3:1, v/v), and finally with methanol. Fumonisin C1, hydroxylated C1, C3, and C4 were eluted in the methanol-water (3:1) and methanol fractions. These fractions were combined, concentrated to dryness and chromatographed on a silica gel column eluted with chloroform-methanol-acetic acid (6:3:1, v/v). The eluant was reduced to two fractions, F-1 and F-2. The F-1 fraction was

576

21.


chromatographed on a silica gel column eluted with ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v). The fractions containing fumonisin Ca or C4were combined separately and concentrated to dryness. Fraction F-2 was separated on a laBondapak Cis column eluted with a gradient from methanol-water-acetic acid (20: 80:1, v/v/v) to methanol-acetic acid (100:1, v/v). Fractions containing either fumonisin C~ or hydroxylated C~ were combined separately and reduced to dryness. Final purification of each of the fumonisins was performed on a strong-anion exchange (SAX) Sep-Pak cartridge successively washed with methanol-water (3:1, v/v) and methanol. The fumonisins were eluted from the column with 1% acetic acid in methanol. Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic. Spectral Data 13C NMR: (D20) 47.5, C-l; 71.2, C-2; 43.6, C-3; 67.4, C-4; 39.7, C-5; 27.6, C-6; 27.5, C-7; 39.7, C-8; 69.8, C-9; 42.5, C-10; 27.9, C-11; 37.6, C-12; 74.7, C-13; 80.5, C-14; 35.8, C-15; 34.0, C-16; 30.5, C-17; 24.9, C-18; 15.9, C-19; 17.4, C-20; 22.2, C-21; 175.4, C-I'; 38.4, C-2'; 41.0, C-3'; 39.0, C-4'; 180.4, C-5'; 180.0, C-6'; 175.4, C-I"; 38.4, C-2"; 41.0, C-3"; 38.9, C-4"; 179.0, C-5"; and 178.6ppm, C-6". (CDaOD) 46.6, C1; 70.0, C-2; 43.1, C-3; 66.3, C-4; 37.9, C-5; 26.7, C-6; 26.6, C-7; 39.0, C-8; 68.6, C9; 44.6, C-10; 26.9, C-11; 34.9, C-12; 72.8, C-13; 78.8, C-14; 34.9, C-15; 33.0, C-16; 29.7, C-17; 23.9, C-18; 14.4, C-19; 16.0, C-20; 20.8, C-21; 173.5, C-22; 37.0, C-23; 39.7, C-24; 37.6, C-25, 178.7, C-26; 178.0, C-27, 173.3, C-28; 36.8, C-29; 39.6, C-30; 37.2, C-31; 176.7, C-32; and 176.7ppm, C-33. 1H ~: (CDaOH) 5.14(IH, dt,d=11.3, 3.7Hz, H-13); 4.93(IH, dd, J=8.3, 3.9I-Iz,H-14); 4.00(IH, m, H-2); 3.79(IH,m, H-4); 3.66(IH, l'n,H-9); 3.14(2 x IH, m, H-24 and H30); 3.03(IH, dd, J=13.0, 3.4I-Iz,H-Ib); 2.80(IH, dd, J=12.9, 7.6I-Iz);2.76(IH, H-la); 2.72(IH, dd, J=7.6, 3.0I-Iz);2.72(IH, dd, 7.6,3.0Hz);2.69-2.66(3 x IH, m); 2.532.41(3 x IH, m); 1.81(IH, m, H-I I); 1.67(IH, H-15); 1.58(IH, H-12b); 1.351.54(16H,m); 1.30(IH, m, H-17a); 1.16(IH, I0~); I.II(IH, H-16a); 0.95(3H, d,J= 6.3Hz, H-21); 0.93(3H, d, J=6.81-Iz,H-20); and 0.88ppm (3H, t,J=7. II-Iz,H-19). Mass Spectrum: FABMS: (M+H) § 708role and MS/MS of 708role from FABMS 708role. 708(parent ion), 690(25), 532(7), 514(5), 374(5), 356(30), 338(100), and 320m/e(55). TFA derivative of hydrolyzed fumonisin C1 (molecular weight 981), 740(0.1), 626(6),

21.


577

568(2), 542(3), 541(3), 528(7), 512(6), 414(3), 264(4), 211(20), 180(60), 126(59), 97(100), 55(66), and 43m/e (43). TLC: Rf = 0.29 on Cls reversed phase plates developed in chloroform-methanol-water-acetic acid (55:36:8:1, v/v/v/v). Fumonisins were visualized by spraying with 0.5% panisaldehyde solution in methanol-sulfuric acid-acetic acid (85: 5:10, v/v/v) and heating at 110~ for 10 rain. References B. E. Branham and R. D. Plattner; Isolation and Characterization of a New Fumonisin from Liquid Cultures ofFusarium moniliforme; J. Nat. Products; Vol. 56, pp. 1630-1633 (1993). J-A Seo, J-C. Kim, and Y-W Lee; Isolation and Characterization of Two New Type C Fumonisins Produced by Fusarium oxysporum; J. Nat. Products; Vol. 59, pp. 1003-1005 (1996).

578

21.


Common/Systematic Name Hydroxylated Fumonisin C1 Molecular Formula/Molecular Weight C33H57NO16; M W = 723.36774

O=:~OOH~OH OH OH NH2 OH

O/ OH Me 0.~0 ?H0

,r,~ OH General Characteristics Obtained as a colorless liquid. Fungal Source Fusarium oxysporum.

Isolation/Purification Solid culture material was extracted with methanol-water (3:1, v/v), filtered, and concentrated to dryness. The aqueous phase was chromatographed on an Amberlite XAD-2 column eluted successively with methanol-water (1:3, v/v), (1:1, v/v), (3:1, v/v), and finally with methanol. Fumonisin C~, hydroxylated CI, C3, and C4 were eluted in the methanol-water (3:1) and methanol fractions. These fractions were combined, concentrated to dryness and chromatographed on a silica gel column eluted with chloroform-methanol-acetic acid (6:3:1, v/v). The eluant was reduced to two fractions, F-1 and F-2. The F-1 fraction was chromatographed on a silica gel column eluted with ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v). The fractions containing fumonisin C3 or C4 were combined separately and concentrated to dryness. Fraction F-2 was separated on a laBondapak C]s column eluted with a gradient from methanol-water-acetic acid (20: 80:1 v/v/v) to methanol-acetic acid (100:1, v/v). Fractions containing either fumonisin C1 or hydroxylated C~ were combined separately and reduced to dryness. Final purification of each of the fumonisins was performed on a strong-anion exchange (SAX) Sep-Pak cartridge successively washed with methanol-water (3:1, v/v) and methanol. The fumonisins were eluted from the column with 1% acetic acid in methanol.

21.


579

Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic. Spectral Data 1H N-IVIR: (CD3OH) 5.14(1H, dt, d=l 1.0, 3.0Hz, H-13); 4.95(1H, dd, J=8.3, 3.2Hz, H-14); 3.88(1H, td, d=8.0, 3.4Hz, H-2); 3.79(1H, td, J=7.8, 3.4Hz, H-4); 3.63(1H, m, H-9); 3.36(1H, m, H-3); 3.28(1H, dd, J=13.4, 3.4Hz, H-lb); 3.15(2 x 1H, m, H-24 and H30); 3.00(1H, dd, J=12.7, 8.3Hz, H-la); 2.78(1H, dd, J=16.6, 7.1Hz); 2.74(1H, dd, 11.8, 3.9Hz); 2.69(1H, dd, J=7.1, 4.3Hz); 2.63(1H, dd, J=7.6, 4.9Hz); 2.57 (1H, dd, J=13.9, 6.6Hz); 2.56 (1H, dd, J=l 1.2, 5.4Hz); 2.50 (1H, dd, J=l 1.7, 6.1Hz); 2.46 (1H, dd, J=10.5, 6.3Hz), 2.46 (1H, dd, J=10.5, 6.3Hz); 1.81(1H, m, H-11); 1.698(1H, H-15); 1.64(1H, H-12b), 1.35- 1.57(14H,m); 1.31(1H, m, H-17a); 1.16(1H, H-10a); 1.09(1H, H-16a); 0.96(3H, d, J=6.3Hz, H-21), 0.94(3H, d, J=6.8Hz, H-20); and 0.90ppm (3H, t, J=7.1Hz, H-19). 13CNMR: (CD3OH) 44.5, C-l; 70.9, C-2; 76.0, C-3; 69.0, C-4; 37.6, C-5; 26.9, C-6; 26.8, C-7; 39.2, C-8; 70.0, C-9; 44.0, C-10; 26.9, C-11; 34.8, C-12; 72.9, C-13; 78.7, C-14; 34.6, C-15; 34.6, C-16; 33.0, C-17; 23.9, C-18; 14.4, C-19; 16.0, C-20; 20.7, C-21; 173.4, C-22; 36.8, C-23; 39.5, C-24; 37.3, C-25; 178.6, C-26; 178.0, C-27; 173.2, C-28; 36.7, C-29; 39.5, C-30; 37.1, C-31; 176.9, C-32; and 176.5ppm, C-33. Mass Spectrum: FABMS: (M+H)§ 336m/e (4).

(100%) 706(6), 688(4), 548(9), 372(12), 354(6), and

TLC: Rf = 0.26 on C18 reversed phase plates developed in chloroform-methanol-water-acetic acid (55:36: 8:1, v/v/v/v). Fumonisins were visualized by spraying with 0.5% p-anisaldehyde solution in methanol-sulfuric acid-acetic acid (85:5:10, v/v/v) and heating at 110~ for 10 min.

580

21.


References B. E. Branham and R. D. Plattner; Isolation and Characterization of a New Fumonisin from Liquid Cultures ofFusarium moniliforme; J. Nat. Products; Vol. 56, pp. 16301633(1993). J-A Seo, J-C. Kim, and Y-W Lee; Isolation and Characterization of Two New Type C Fumonisins Produced by Fusarium oxysporum; J. Nat. Products; Vol. 59, pp. 10031005(1996).

21. Fumonisins, AALToxins, and Related Metabolites

581

Common/Systematic Name Fumonisin C3 Molecular Formula/Molecular Weight C33H57NO]4; MW = 691.37791

% / OH

NH2

0/

OH Me 0~0

?H0

OH General Characteristics Obtained as a colorless liquid. Fungal Source


Isolation/Purification Solid culture material was extracted with methanol-water (3:1, v/v), filtered, and concentrated to dryness. The aqueous phase was chromatographed on an Amberlite XAD-2 column eluted successively with methanol-water (1:3, v/v), (1" 1, v/v), (3 1, v/v), and finally with methanol. Fumonisin C1, hydroxylated C1, C3, and C4 were eluted in the methanol-water (3"1) and methanol fractions. These fractions were combined, concentrated to dryness and ehromatographed on a silica gel column eluted with chloroform-methanol-acetic acid (6:3:1, v/v/v). The eluant was reduced to two fractions, F-1 and F-2. The F-1 fraction was chromatographed on a silica gel column eluted with ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v). The fractions containing fumonisin C3 or C4 were combined separately and concentrated to dryness. Fraction F-2 was separated on a laBondapak C18 column eluted with a gradient from methanol-water-acetic acid (20:80:1, v/v/v) to methanol-acetic acid (100:1, v/v). Fractions containing either fumonisin C1 or hydroxylated C1 were combined separately and reduced to dryness. Final purification of each of the fumonisins was performed on a strong-anion exchange (SAX) Sep-Pak cartridge successively washed with methanol-water (3-1_ v/v] and methanol. The fumonisins were eluted from the column with 1% acetic acid in methanol.

582

21.


Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic. Spectral Data 1H NMR: (CD3OD) 5.13(1H, dt, d-11.3, 2.9Hz, H-13); 4.95(1H, dd, J=8.6, 2.9Hz, H-14), 3.77(1H, m, H-2), 3.61(1H, m, H-9); 3.16(2 x 1H, m, H-24 and H-30); 3.03(1H, dd, d=12.7, 2.7Hz, H-lb); 2.80(1H, dd, J=l 1.7, 7.3Hz); 2.76(1H, H-In); 2.74-2.64(3 x 1H, m); 2.61-2.52(3 x 1H, m); 2.48(1H, dd, d=16.6, 6.6Hz); 1.79(1H, m, H-11); 1.70(1H, H-15); 1.66(1H, H-12b); 1.27-1.53(19H, m), 1.17(1H, H-10a); 1.09(1H, 16a); 0.95(3H, d, d=6.6Hz, H-21); 0.94(3H, d, J=6.6Hz, H-20); and 0.88ppm (3H, t, J=6.8Hz, H-19). 13C NMR: (CD3OD) 46.1, C-l; 69.9, C-2; 39.2, C-3; 30.6, C-4; 30.5, C-5; 26.7, C-6; 26.2, C-7; 39.0, C-8; 68.7, C-9; 44.5, C-10; 26.9, C-11; 35.8, C-12; 73.0, C-13; 78.7, C-14; 34.8, C-15; 33.0, C-16; 29.5, C-17; 23.8, C-18; 14.4, C-19; 15.9, C-20; 20.6, C-21; 173.3, C-22; 36.6, C-23; 39.0, C-24; 36.8, C-25; 177.7, C-26; 177.3, C-27; 173.1, C-28; 36.6, C-29; 39.0, C-30; 36.8, C-31; 176.0, C-32; and 175.7ppm, C-33.

Mass Spectrum: FABMS: (M+H)§692(100), 674(7), 516(8), 340(15), 322(14) and 304(7), 512(6), 414(3), 264(4), 211(20), 180(60), 126(59), 97(100), 55(66), and 43role (43). TLC: Rf = 0.36 on C~s reversed phase plates developed in chloroform-methanol-water-acetic acid (55:36:8:1, v/v/v/v). Fumonisins were visualized by spraying with 0.5% panisaldehyde solution in methanol-sulfuric acid-acetic acid (85:5:10, v/v/v) and heating at 110~ for 10 rain. Reference J-A. Seo, J-C. Kim, and Y-W. Lee; Isolation and Characterization of Two New Type C Fumonisins Produced by Fusarium oxysporum; J. Nat. Products; Vol. 59, pp. 1003-1005 (1996).

21.


583

Common/Systematic Name Fumonisin C4 Molecular Formula/Molecular Weight C33H57NO13, MW = 675.38299

OH

NH2

ohm_ 0/

Me 0...~0 OH

General Characteristics Obtained as a colorless liquid. Fungal Source Fusarium oxysporum (strain CAR isolated from carnation in Korea; F. moniliforme Sheldon). Isolation/Purification Solid culture material was extracted with methanol-water (3:1, v/v), filtered, and concentrated to dryness. The aqueous phase was chromatographer on an Amberlite XAD-2 column eluted successively with methanol-water (1:3, v/v), (1:1, v/v), (3:1, v/v), and finally with methanol. Fumonisin C1, hydroxylated C1, C3, and C4 were eluted in the methanol-water (3:1) and methanol fractions. These fractions were combined, concentrated to dryness and chromatographed on a silica gel column eluted with chloroform-methanol-acetic acid (6:3:1, v/v/v). The eluant was reduced to two fractions, F-1 and F-2. The F-1 fraction was chromatographed on a silica gel column eluted with ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v). The fractions containing fumonisins C3 or Ca were combined separately and concentrated to dryness. Fraction F-2 was separated on a laBondapak C18 column eluted with a gradient from methanol-water-acetic acid (20: 80:1, v/v/v) to methanol-acetic acid (100:1, v/v). Fractions containing either fumonisin C1 or hydroxylated C1 were combined separately and reduced to dryness. Final purification of each of the fumonisins was performed on a strong-anion exchange (SAX) Sep-Pak cartridge successively washed with methanol-water (3:1, v/v) and methanol. The fumonisins were eluted from the column with 1% acetic acid in methanol.

584

21.


Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoencephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic. Spectral Data 13C NMR: (CD3OD) 46.1, C-l; 68.9, C-2; 39.0, C-3; 30.7, C-4; 30.5, C-5; 27.5, C-6; 26.3, C-7; 30.5, C-8; 30.5, C-9; 30.1, C-10; 20.9, C-11; 35.9, C-12; 73.0, C-13; 78.8, C-14; 34.9, C-15; 33.0, C-16; 29.6, C-17; 23.8, C-18; 14.4, C-19; 16.0, C-20; 20.9, C-21; 173.1, C-22; 36.3, C-23; 38.9, C-24; 36.7, C-25; 177.5, C-26; 177.3, C-27; 173.0, C-28; 36.1, C-29; 38.9, C-30; 36.6, C-31; 175.9, C-32; and 175.6ppm, C-33. 1H NMR: (CD3OD) 5.17(1H, dt, J-11.1, 3.2Hz, H-13); 4.92(1H, dd, J=8.5, 3.4Hz, H=14); 3.76(1H, m, H-2); 3.16(2 x 1H, m, H-24 and H-30); 3.03(1H, dd, J=12.7, 2.9Hz, Hlb); 2.80(1H, dd, J-12.7, 7.1Hz); 2.76(1H, H-l,); 2.74-2.64(3 x 1H, m); 2.59(1H, dd, ,/=10.3, 6.4Hz); 2.56-2.472(3 x 1H, m); 1.71(1H, H-15); 1.62(1H, H-12b); 1.141.54(23H, m); 1.06(1H, 16,); 0.93(3H, d, J=7.1Hz, H-21); 0.91(3H, d, J-6.4Hz, H20); and 0.89ppm (3H, t, J-7.1Hz, H-19). Mass Spectrum: FABMS: (M+H)+ 676(100), 658 (3), 500 (5), 324 (30), and 306m/e (11). TLC: Rf = 0.44 on C~s reversed phase plates developed in chloroform-methanol-water-acetic acid (55:36:8:1, v/v/v/v). Fumonisins were visualized by spraying with 0.5% panisaldehyde solution in methanol-sulfuric acid-acetic acid (85:5:10, v/v/v) and heating at 110~ for 10 min. References J.-A. Seo, J.-C. Kim, and Y.-W. Lee; Isolation and Characterization of Two New Type C Fumonisins Produced by Fusarium oxysporum; J. Nat. Products; Vol. 59, pp. 1003-1005 (1996). B. E. Branham and R. D. Plattner; Isolation and Characterization of a New Fumonisin from Liquid Cultures ofFusarium moniliforme; J. Nat. Products; Vol. 56, pp. 1630-1633 (1993).

21.


585

Common/Systematic Name Fumonisin AK1 Mole.cular Formula/Molecular Weight C3oi-I45NO11; M W --- 6 0 4 . 3 6 9 6 9

OH

OH

MeC~NH2 II O

0

OH

Me

O..

tq

_..

O

OH General Characteristics Fumonisins are highly functionalized hydrocarbon derivatives that are readily soluble in polar solvents and nearly insoluble in non-polar solvents. Fungal Source v

Fusarium proliferatum (M- 1597).

Isolation/Purification Solid culture material was extracted with methanol-water (75:25, v/v) and filtered. The filtrate was diluted with water to a final methanol concentration of 30%. The diluted solution was chromatographed on a Bondapak Cls preparative column washed with water, methanol-water (50:50, v/v), and eluted with methanol-water (70:30, v/v). The methanol was removed from this fraction and the aqueous portion was applied to a Dynamax Cls column (Rainin Corp.) washed with acetonitrile-water (25:75, v/v). The fumonisin containing fractions were eluted with acetonitrile-water (70:30, v/v). The acetonitrile was removed in vacuo and the aqueous fraction was applied to a pBondapak cyano columrL The fumonisins were eluted with 1% pyridine in water. Final purification and separation of fumonisin B1 and fumonisin AK~ was achieved on a Cs column using a gradient from acetonitrile containing 0.1% acetic acid-water (20: 80, v/v) to aeetonitrile-water (45:55, v/v). Biological Activity The primary animal diseases that have been caused by the fumonisins are leucoeneephalomalacia in equine and porcine pulmonary edema. Acute liver and kidney toxicity has also been reported from feeding culture materials containing high levels of the fumonisins. The mode of action of the fumonisins involves disruption of sphingolipid metabolism which, in turn, may lead to disruption of normal cellular regulation. Fumonisins are carcinogenic and also highly phytotoxic.

586

21.


Spectral Data 13C NMR: (D20) 18.4, C-I; 52.3, C-2; 72.5, C-3; 42.1, C-4; 70.3, C-5; 39.2, C-6; 27.1, C-7; 27.3, C-8; 27.1, C-9; 71.4, C-10; 44.6, C-11; 28.6, C-12; 39.0, C-13; 79.4, C-14; 219.5, C-15; 44.8, C-16; 33.9, C-17; 31.3, C-18; 24.4, C-19; 15.6, C-20; 21.6, C-21; 19.7, C-22; 181.6, C-23; 38.2, C-24; 41.4, C-25; 39.3, C-26; 179.1, C-27; 176.2, C28; 175.9, C-33; and 24.5ppm, C-34'. Mass Spectrum: Negative-ion ESMS: Showed only a molecular anion at 603 daltons. References S. M. Musser, R. M. Eppley, E. P. Mazzola, C. E. Hadden, J. P. Shockcor, and G. E. Martin; Identification of an N-Acetyl Keto Derivative of Fumonisin B 1 in Corn Cultures ofFusarium proliferatum; J. Natural Prod., Vol. 58, pp. 1392-1397(1995).

21.


587

Common/Systematic Name AAL Toxin TAI Molecular Formula/Molecular Weight C2sHaTNO10; MW = 521.32000

OH OH

OH

1

NH2

OH

Me O~o O ~ ~ ~H

OH General Characteristics AAL toxin TAI was ninhydrin positive. Fungal Source Alternaria alternata f. sp. lycopersici. Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness m vacuo, dissolved in ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system. After 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biological Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay.

588

21.


Spectral Data 1H NMR: (CD3OD) 3.05(1H, H-l); 2.82(1H, n-l'); 4.01(1H, H-2); 1.72(1H, n-3); 1.51(1H, H-3'); 3.67(1H, H-4); 3.44(1H, H-5), 1.36(1H, H-6); 1.68(1H, H-12), 5.12(1H, dd, J=3.5, 3.THz, H-13); 3.89(1H, dd, J=3.5, 3.THz, H-13); and 1.34ppm (1H, H-15). 13CNMR: (CD3OD) 47.9, C-l; 64.97, C-2; 70.46, C-4; 74.70, C-5; 74.46, C-13; and 76.30ppm C-14. Mass Spectrum: FABMS: (M+H)§ 522m/e. References A.T. Bottini, J. R. Bowen, and D. G. Gilchrist; Phytotoxins. II. Characterization of A Phytotoxic Fraction from Alternaria alternata F. sp. Lycopersici; Tetrahedron Letters, Vol. 22, pp. 2723-2726(1981). E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilchrist; Structural Characterization of Three New AALToxins Produced by Alternaria alternata f. sp. Lycopersici; J. Agric. Food Chem., Vol. 42, pp. 327-333(1994).


589

Common/Systematic Name AAL Toxin TA2 Molecular Formula/Molecular Weight C25I-I47NO10; MW = 521.32000

OH

NH2

O~ \

OH

O

OH

Me

OH

OH

/

Me

General Characteristics AAL toxin TA2 was ninhydrin positive. All AAL toxin pairs are regioisomers at C-13 and C-14. Fungal Source Alternaria alternata f. sp. lycopersici. Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness in vacuo, dissolved in ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system. After 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biological Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay.

590

21.


Spectral Data 1H NMR: (CD3OD) 3.05(1H, H-l), 2.82(1H, H-I'); 4.01(1H, H-2); 1.72(1H, 5.2Hz, H-3); 1.51(1H, n-3'), 3.67(1H, n-4); 3.44(1H, n-5); 1.36(1H, n-6); 1.81(1H, H-12); 3.75,(1H, dd, J=3.5, 3.7Hz, H-13); 4.76(1H, dd, J=2.9, 8.0Hz, H-14); and 1.77ppm (1H, n-15). 13CNMR" (CD3OD) 47.9, C-l, 64.97, C-2, 70.46, C-4, 74.70, C-5, 69.16, C-13, and 81.72ppm C-14. Mass Spectrum: FABMS: (M+H)+ 522m/e. References A.T. Bottini, J. R. Bowen, and D. G. Gilchrist; Phytotoxins. II. Characterization of A Phytotoxic Fraction from Alternaria alternata F. sp. Lycopersici; Tetrahedron Letters, Vol. 22, pp. 2723-2726(1981). E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilchrist; Structural Characterization of Three New AALToxins Produced by Alternaria alternata f. sp. Lycopersici; J. Agric. Food Chem., Vol. 42, pp. 327-333(1994).

21. Fumonisins,AALToxins, and RelatedMetabolites

591

Common/Systematic Name AAL Toxin TB1 Molecular Formula/Molecular Weight C25H47NO9; MW = 505.32508

OH

NH2

OH

OH

Me 0 . . ~ 0

OH General Characteristics AAL toxin TB1 was ninhydrin positive. All five AAL toxin pairs are regioisomers at C-13 and C-14. Fungal Source Alternaria alternata f. sp. lycopersici. Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness in vacuo, dissolved in ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system. Atter 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biological Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay.

592

21.


Spectral Data 1H NMR: (CD3OD) 3.03(1H, H-I); 2.82(1H, H-I'); 4.02(1H, H-2); 1.57(1H, H-3); 1.44(1H, H-3'); 3.79(1H, H-4); 1.58(1H, H-5); 1.68(1H, H-12); 5.11(1H, dd, J=3.5, 3.7Hz, H13); 3.39(1H, dd, J=3.5, 3.7I-Iz, H-14); and 1.37ppm (1H, H-15). 13CNMR: (CD3OD) 46.50, C-l, 66.27, C-2, 68.58, C-4; 74.62, C-13; and 77.30ppm C-14. Mass Spectrum: FABMS: (M+H) § 506m/e. References A.T. Bottini, J. R. Bowen, and D. G. Gilchrist; Phytotoxins. II. Characterization of A Phytotoxic Fraction from Alternaria alternata F. sp. Lycopersici; Tetrahedron Letters, Vol. 22, pp. 2723-2726(1981). E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilchrist; Structural Characterization of Three New AALToxins Produced by Alternaria alternata f. sp. Lycopersici; J. Agric. Food Chem., Vol. 42, pp. 327-333(1994).

21.


593

Common/Systematic Name AAL Toxin TB2 Molecular Formula/Molecular Weight CzsH47NO9; MW = 505.32508

\ OH

NH2

OH

0

Me

OH

/

/

Me

General Characteristics AAL toxin TB2 was ninhydrin positive. All AAL toxin pairs are regioisomers at C-13 and C-14. Fungal Source Alternaria alternata f. sp. lycopersici. Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness in vacuo, dissolved in ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system. A_~er 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biological Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay.

594

21.


Spectral Dat.a. 1H NMR: (CD3OD) 3.03(1H, H-I); 2.82(1H, H-I'); 4.02(1H, H-2); 1.57(1H, H-3); 1.44(1H, H-3'); 3.79(1H, H-4); 1.58(1H, H-5); 1.81(1H, H-12); 3.76(1H, dd, J-3.5, 3.7Hz, H13); 4.77(1H, t, J=5.7I-Iz, H-14); and 1.74ppm (1H, H-15). 13CNMR: (CD3OD) 46.50, C-l 66.27, C-2; 68.58, C-4; 69.75, C-13; and 82.12ppm, C-14. Mass Spectrum: FABMS: (M+H)§ 506m/e. References A.T. Bottini, J. R. Bowen, and D. G. Gilchrist; Phytotoxins. II. Characterization of A Phytotoxic Fraction from Alternaria alternata F. sp. Lycopersici; Tetrahedron Letters, Vol. 22, pp. 2723-2726(1981). E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilchrist; Structural Characterization of Three New AALT0xins Produced by Alternaria alternata f. sp. Lycopersici, J. Agric. Food Chem., Vol. 42, pp. 327-333(1994).


595

Common/Systematic Name AAL Toxin TC1 Molecular Formula/Molecular Weight C25I--I4708; M W -- 4 8 9 . 3 3 0 1 7

OH

NH2

OH

Me

0

~

0

OH General Characteristics AAL toxin TC1 was not ninhydrin positive. All AAL toxin pairs are regioisomers at C-13 and C-14. Fungal Source Alternaria alternata f. sp. lycopersici. Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness in vacuo, dissolved in ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system. After 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biological Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay.

596

21.


Spectral Data 1H ~ : (CD3OD) 3.01(1H, H-l); 2.75(1H, H-I'); 3.77(1H, H-2); 1.70(1H, H-3); 1.46(1H, H-3'); 1.67(1H, H-12); 3.39(1H, dd, .]=3.5, 3.7Hz, H-13); 5.02(1H, dd, J=3.5Hz, H14); and 1.37ppm (1H, H-15). Mass Spectrum: FABMS: (M+H)§ 490m/e. Reference E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilchrist; Structural Characterization of Three New AALToxins Produced by Alternaria alternata f. sp. Lycopersici; J. Agile. Food Chem., Vol. 42, pp. 327-333(1994).


597

Common/Systematic Name AAL Toxin TC2

0 = = ~ \

Molecular Formula/Molecular Weight C25I--I47N08; M W -- 4 8 9 . 3 3 0 1 7

OH

NH2

OH

0/

Me

OH

Me

General Characteristics AAL toxin TC2 was not ninhydrin positive. All five AAL toxin pairs are regioisomers at C-13 and C-14. Fungal Source Alternaria alternata f. sp. lycopersici Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness in vacuo, dissolved in ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system. After 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biological Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay. Spectral Data 1H NMR: (CD3OD) 3.01(1H, H-I); 2.75(1H, H-I'); 3.77(1H, H-2); 1.70(1H, H-3); 1.46(1H, H-3'); 1.81(1H, H-12); 3.74(1H, H-13); 4.75(1H, t, J-5.7Hz, H-14); and 1.75ppm (1H, H-15).

598

21.


Mass Spectrum: FABMS: (M+H) + 490m/e. Reference E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilchrist; Structural Characterization of Three New AAI_Toxins Produced by Alternaria alternata f. sp. Lycopersici; J. Agile. Food Chem., vol. 42, pp. 327-333(1994).

21.


599

Common/Systematic Name AAL Toxin TD1 Molecular Formula/Molecular Weight C27Ha9NO10; MW = 547.33565

OH OH NHCMe II 0

OH Me O ~ j ~ O

OH General Characteristics AAL toxin TD1 was not ninhydrin positive. All AAL toxin pairs are regioisomers at C-13 and C-14. Fungal Source Alternaria alternata f. sp.. lycopersici. Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness in vacuo, dissolved in ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system. After 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biological Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay.

600

21.


Spectral Data 1H NMR: (CD3OD) 3.01(1H, H-l); 2.75(1H, n-l'); 3.77(1H, H-2); 1.70(1n, H-3); 1.46(1H, H-3'); 1.67(1H, H-12); 5.02(1H, dd, J=3.5, 3.7Hz, H-13); 3.39(1H, dd, .]=3.5, 3.7Hz, H-14); and 1.37ppm (1H, H-15). Mass Spectrum: FABMS: (M+H) § 548m/e. Reference E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilchrist; Structural Characterization of Three New AAL Toxins Produced by Alternaria alternata f. sp. Lycopersici; J. Agric. Food Chem., vol. 42, pp. 327-333(1994).

21.


601

Common/Systematic Name AAL Toxin TD2 Molecular Formula/Molecular Weight C27H49NO10; M W -- 5 4 7 . 3 3 5 6 5

ON

OH

NHCMe II 0

OH

0

Me

OH

Me

General Characteristics AAL toxin TD2 w a s not ninhydrin positive. All AAL toxin pairs are regioisomers at C-13 and C-14. Fungal Source A lternaria alternata f. sp. Lycopersici. Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness in vacuo, dissolved in ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system. After 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biologica/. Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay.

602

21.


Spectral Data 1H NMR: (CD3OD) 3.25(1H, dd, J=4.8, 5.2Hz,H-1); 3.12(1H, H-I'); 3.82(1H, m, J=2.2 2.8Hz, H-2); 1.48(1H, H-3); 1.46(1H, H-3'), 3.76(1H, H-4); 1.39(1H, H-5), 1.31(1H, H-12); 8.78(1H, H-13); 4.77(1H, t, J=5.7Hz, H-14); and 1.74ppm (1H, H-15). 13CNMR: (CD3OD) 46.13, C-l; 68.37, C-2; 69.49, C-4; 68.94, C-13; and 82.24ppm C-14. Mass Spectrum: FABMS: (M+H)+ 548m/e. Reference E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilehrist; Structural Characterization of Three New AALToxins Produced by Alternaria alternata s sp. Lycopersici; J. Agile. Food Chem., vol. 42, pp. 327-333(1994).


603

Common/Systematic Name AAL Toxin TEl Molecular Formula/Molecular Weight C27H49NO9; MW-- 531.34073

OH

NHCMe

II 0

OH

Me

0...,~0

OH 0

OH General Characteristics AAL toxin TEl was not ninhydrin positive. All AAL toxin pairs are regioisomers at C-13 and C-14. Fungal Source Alternaria alternata f sp. lycopersici. Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness in vacuo, dissolved in ethyl acetate-acetic acid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system. After 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biological Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay. Spectral Data 1H NMR:

604

21.


(CDaOD) 3.26(1H, dd, J=4.0, 4.5Hz, H-l); 3.06(1H, H-I'), 3.60(1H, dd, J=2.9, 8.0Hz, H-2); 1.36(1H, H-3); 1.38(1H, n-3'); 1.60(1H, n-12); 5.1 l(1n, n-13), 3.36(1H, dd, J=3.5, 3.7Hz, H-14); and 1.36ppm (1H, H-15). 13CNMR: (CD3OD) 46.71, C-l; 68.49, C-2; 74.89, C-13, and 77.58ppm C-14. Mass Spectrum: FABMS: (M+H) § 532m/e. Reference E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilchrist; Structural Characterization of Three New AAL Toxins Produced by Alternaria alternata f. sp. Lycopersici; J. Agile. Food Chem., Vol. 42, pp. 327-333(1994).


605

.Common/Systematic Name AAL Toxin TE2 Molecular Formula/Molecular Weight C27H49NO9; M W -- 5 3 1 . 3 4 0 7 3

\

0=~

OH)OH

/

OH

0II

NHCMe

0

Me

OH

Me

General Characteristics AAL toxin TE2 was not ninhydrin positive. All AAL toxin pairs are regioisomers at C-13 and C-14. Fungal Source Alternaria alternata f. sp. lycopersici. Isolation/Purification XAD-2 was added to culture filtrates and stirred for 4-6 hours, the adsorbed XAD beads were washed with water, and the toxins eluted with methanol. The extract was evaporated to dryness in vacuo, dissolved in ethyl acetate-acetic aeid-hexane-water (6:2:2:1, v/v/v/v), and fractionated on a silica gel column by flash chromatography using the same solvent system.After 20 fractions were collected the solvent system was changed to ethyl acetateacetic acid-water (6:3:1, v/v/v). Fractions were evaluated by TLC using the solvent system ethyl acetate-acetic acid-water (6:8:1, v/v/v). Toxins were detected by spraying plates with p-anisaldehyde. Final purification was performed on a SAX column washed successively with 0.1 N NaOH, water to pH 7.0, and equilibrated with methanol at pH 7.7. The selected fractions were dried, dissolved in methanol at pH 7.7, applied to a SAX column, washed with methanol, and eluted with 1% acetic acid in methanol. Biological Activity The AAL toxins are responsible for expression of Alternaria stem canker disease of tomato. All five congers were toxic to leaf tissue of the asc/asc isoline of tomato in the standard detached leaflet assay.

606

21.


Soectral Data 1H NMR:

(CD3OD) 3.26(IH, dd, ,/=4.0,4.5I--Iz,H-I); 3.06(IH, H-I'); 3.60(IH, dd, J=2.9,

8.0Hz, H-2); 1.36(1H, H-3); 1.38(1H, H-3'); 1.81(1H, H-12); 3.77(1H, H-13), 4.78(1H, dd, J=2.9, 3.0Hz, H-14); and 1.74ppm (1H, H-15). 13CNMR: (CD3OD) 46.71, C-l; 68.49, C-2; 71.33, C-13 and 82.30ppm C-14. Mass Spectrum: FABMS: (M+H) + 532role. Reference E. D. Caldas, A. D. Jones, B. Ward, C. K. Winter, and D. G. Gilchrist; Structural Characterization of Three New AAL toxins Produced by Alternaria alternata f. sp. Lycopersici; J. Agric. Food Chem., vol. 42, pp. 327-333(1994).


607

Common/Systematic Name Sphingofungin A Molecular Formula/Molecular Weight C21H41N306; M W = 431.29954

OH _

OH

_

HOOC

I

OH

OH

NHO(NH)NH2 Fungal Source

Aspergillusfumigatus Fres. (ATCC 20857 = MF 5038) was isolated from soil collected from a pasture in Young, Departmento Rio Negro, Uruguay.

Isolation/Purification The fermentation broth was extracted with methanol, filtered, concentrated, and purified by various chromatographic techniques including Amberlite XAD-2, silica gel, and Sephadex LH-20. Final separation of the four sphingofungins was accomplished with reversed phase C~s HPLC chromatography. For larger liquid fermentations a five step isolation procedure was developed. After extraction with methanol and removal of the solids by centrifugation, the aqueous filtrate was adsorbed onto a SP207 column. After elution of sphingofungins B and C with methanol, the eluate was diluted with water, and the pH was adjusted to 6.0. The solution was passed through a column ofDowex 1 (CI) resin. The Dowex 1 eluant, containing sphingofungins B and C was adsorbed onto Diaion HP-20 and eluted with a stepwise gradient of aqueous methanol. Lipophilic contaminants were removed by washing the aqueous methanol solution with hexane-ethyl acetate. The methanol and water were removed to obtain a mixture of sphingofungins B and C, which were separated by HPLC chromatography. Biological Activity The sphingofungins A, B, and C are potent antifungal agents especially against Cryptococcus neoformans. They also show selective activity against various Candida species and are essentially inactive against filamentous fungi and bacteria. In general sphingofungin D is much less potent than the other three sphingofungins. References F. VanMiddlesworth, R. A. Giacobbe, M. Lopez, G. Garrity, J. A. Bland, K. Bartizal, R. A. Fromtling, J. Polishook, M Zweerink, A. M. Edison, W. Rozdilsky, K. E. Wilson and R. L. Monaghan; Sphingofungins A, B, C, and D; A New Family of Antifungal Agents. 1. Fermentation, Isolation, and Biological Activity; Journal of Antibiotics, Vol. 45, pp. 861867(1992).

608

21.


F. VanMiddlesworth, C. Dufresne, R. T. Moslely, and K. E. Wilson; Determination of the Relative and Absolute Stereochemistry of Sphingofungins A, B, C, and D; Tetrahedron Letters, pp. 297-300(1992).

21.


609

Common/Systematic Name Sphingofungin B Molecular Formula/Molecular Weight C20H39NO6; M W -- 3 8 9 . 2 7 7 7 4

OH _

OH

_

HOOC

_

NH2 OH

OH

Fungal Source


Isolation/Purification The fermentation broth was extracted with methanol, filtered, concentrated, and purified by various chromatographic techniques including Amberlite XAD-2, silica gel, and Sephadex LH-20. Final separation of the four sphingofungins was accomplished with reversed phase ClS HPLC chromatography. For larger liquid fermentations a five step isolation procedure was developed. After extraction with methanol and removal of the solids by centrifugation, the aqueous filtrate was adsorbed onto a SP207 column. Alter elution of sphingofungins B and C with methanol, the eluate was diluted with water, and the pH was adjusted to 6.0. The solution was passed through a column ofDowex 1 (CI) resin. The Dowex 1 eluant, containing sphingofungins B and C was adsorbed onto Diaion HP-20 and eluted with a stepwise gradient of aqueous methanol. Lipophilic contaminants were removed by washing the aqueous methanol solution with hexane-ethyl acetate. The methanol and water were removed to obtain a mixture of sphingofungins B and C, which were separated by HPLC chromatography. Biological Activity The sphingofungins A, B, and C are potent antifungal agents especially against Cryptococcus neoformans. They also show selective activity against various Candida species and are essentially inactive against filamentous fungi and bacteria. In general sphingofungin D is much less potent than the other three sphingofungins. References F. VanMiddlesworth, R. A. Giacobbe, M. Lopez, G. Garrity, J. A. Bland, K. Bartizal, R. A. Fromtling, J. Polishook, M Zweerink, A. M. Edison, W. Rozdilsky, K. E. Wilson and R. L. Monaghan; Sphingofungins A, B, C, and D; A New Family of Antifungal Agents. 1. Fermentation, Isolation, and Biological Activity; Journal of Antibiotics, Vol. 45, pp. 861867(1992). F. VanMiddlesworth, C. Dufresne, R. T. Moslely, and K. E. Wilson; Determination of the Relative and Absolute Stereochemistry of Sphingofungins A, B, C, and D; Tetrahedron Letters, pp. 297-300(1992).

610

21.


Common/Systematic Name Sphingofungin C Molecular Formula/Molecular Weight C22H43NOs; ~

OH

-- 4 0 1 . 3 1 4 1 2

OAc

_

_

NH2

OH

OH

Fungal Source


Isolation/Purification The fermentation broth was extracted with methanol, filtered, concentrated, and purified by various chromatographic techniques including Amberlite XAD-2, silica gel, and Sephadex LH-20. Final separation of the four sphingofungins was accomplished with reversed phase ClS HPLC chromatography. For larger liquid fermentations a five step isolation procedure was developed. After extraction with methanol and removal of the solids by centrifugation, the aqueous filtrate was adsorbed onto a SP207 column. After elution of sphingofungins B and C with methanol, the eluate was diluted with water, and the pH was adjusted to 6.0. The solution was passed through a column of Dowex 1 (C1-) resin. The Dowex 1 eluant, containing sphingofungins B and C, was adsorbed onto Diaion HP-20 and eluted with a stepwise gradient of aqueous methanol. Lipophilic contaminants were removed by washing the aqueous methanol solution with hexane-ethyl acetate. The methanol and water were removed to obtain a mixture of sphingofungins B and C, which were separated by HPLC chromatography. Biological Activity The sphingofungins A, B, and C are potent antifungal agents especially against Cryptococcus neoformans. They also show selective activity against various Candida species and are essentially inactive against filamentous fungi and bacteria. In general, sphingofungin D is much less potent than the other three sphingofungins. References F. VanMiddlesworth, R. A. Giacobbe, M. Lopez, G. Garrity, J. A. Bland, K. Bartizal, R. A. Fromtling, J. Polishook, M Zweerink, A. M. Edison, W. Rozdilsky, K. E. Wilson and R. L. Monaghan; Sphingofungins A, B, C, and D; ANew Family of Antifungal Agents. 1. Fermentation, Isolation, and Biological Activity; Journal of Antibiotics, Vol. 45, pp. 861867(1992). F. VanMiddlesworth, C. Dufresne, R. T. Moslely, and K. E. Wilson; Determination of the Relative and Absolute Stereochemistry of Sphingofungins A, B, C, and D; Tetrahedron Letters, pp. 297-300(1992).

21.

Fumonisins, AAL Toxins, and Related Metabolites

611

Common/Systematic Name Sphingofungin D Molecular Formula/Molecular Weight CzzH41NOs; MW = 447.28322

OH

OH _

HOOC

"

OH NHAc Fungal Source


Isolation/Purification The fermentation broth was extracted with methanol, filtered, concentrated, and purified by various chromatographic techniques including Amberlite XAD-2, silica gel, and Sephadex LH-20. Final separation of the four sphingofungins was accomplished with reversed phase Cls HPLC chromatography. For larger liquid fermentations a five step isolation procedure was developed. After extraction with methanol and removal of the solids by centrifugation, the aqueous filtrate was adsorbed onto a SP207 column. After elution of sphingofungins B and C with methanol, the eluate was diluted with water, and the pH was adjusted to 6.0. The solution was passed through a column ofDowex 1 (CI) resin. The Dowex 1 eluant, containing sphingofungins B and C,was adsorbed onto Diaion HP-20 and eluted with a stepwise gradient of aqueous methanol. Lipophilic contaminants were removed by washing the aqueous methanol solution with hexane-ethyl acetate. The methanol and water were removed to obtain a mixture of sphingofungins B and C, which were separated by HPLC chromatography. Biological Activity The sphingofungins A, B, and C are potent antifungal agents especially against Cryptococcus neoformans. They also show selective activity against various Candida species and are essentially inactive against filamentous fungi and bacteria. In general, sphingofungin D is much less potent than the other three sphingofungins. References F. VanMiddlesworth, R. A. Giacobbe, M. Lopez, G. Garrity, J. A. Bland, K. Bartizal, R. A. Fromtling, J. Polishook, M Zweerink, A. M. Edison, W. Rozdilsky, K. E. Wilson and R. L. Monaghan; Sphingofungins A, B, C, and D; A New Family of Antifungal Agents. 1. Fermentation, Isolation, and Biological Activity; Joumal of Antibiotics, Vol. 45, pp. 861867(1992). F. VanMiddlesworth, C. Dufresne, R. T. Moslely, and K. E. Wilson; Determination of the Relative and Absolute Stereochemistry of Sphingofungins A, B, C, and D; Tetrahedron Letters, pp. 297-300(1992).

612

21.

Fumonisins, AAL Toxins, and Related Metabolites

Common/Systematic Name Myriocin Molecular Formula/Molecul~ Weight C21H39NO6; M W -~- 401.27774

.oo 2 A 1

OH

HOH2C~ /

28

NH2 OH

O

General Characteristics Crystals from methanol; mp., 180-181~ ninhydrin test.

[a]D 24 W 10.3~ (c=0.386,

in MeOH). Positive

Fungal Source Myriococcum albomyces. Isolation/Purification See D. Kluepfel et. al., Journal of Antibiotics, Vol. 25, p. 109 (1972). Biological Activity Antifungal activity. Spectral Data IR:

(Nujol) Broad band in OH region, 1702, 1665, and 962cm"1. 1H NMR: (CDCI3) (Tetraacetate derivative) 6.30(1H, NH); 4.51(s); 5.79(d); 4.74(m); 5.5(q) and 0.88ppm. Mass Spectrum: LREIMS: 383(M + - 18) and 256m/e (base peak, M § - [ 127 + 18]). Reference J. F. Bagli, D. Kluepfel, and M. St-Jacques; Elucidation of Structure and Stereochemistry ofMyriocin. A Novel Antifungal Antibiotic; J. Organ. Chem., Vol. 38, pp. 1253-1260 (1973).

Ochratoxins and Related Metabolites Ochratoxin A Ochratoxin B 4-Hydroxyochratoxin A Ochratoxin C Mellein 4-Hydroxymellein

613


22.

Ochratoxins and Related Metabolites

615

Common/Systematic Name Ochratoxin A 7-Carb•xy-5-ch••r•-8-hydr•xy-3•4-dihydr•-3•-methy•is•c•umarin-7-L-•3-pheny•a•anine Molecular FormulaJMolecular Weight C20H]sO6NC1, MW = 403.0822

~ 17

22

COOH I 0

OH

N"~"~ ,'~ l 6L I.,L H "~1o~

CI General Characteristics Crystals from benzene; mp., 94-96~ (c = 1.1, in chloroform).

0 "O ~ 3..L'"'Me "~H

Crystals from xylene; mp., 169~

[~]D25 -118 ~

Fungal Source Aspergillus ochraceus (NRRL 3174); A. sulphureus (NRRL 4077); A. melleus (NRRL 3519; 3520); Penicillium viridicatum (ATCC 18411). Biological Activity LDs0 in weanling rats dosed orally was 22mg/kg, in trout IP, 3.0mg/kg. Spectral Data UV: Em~ 215 (36,800) and 333nm (6,400). 1H NMR:

(CDC13) 4.76 (1H, H-3), 2.97 (2H, H-4); 8.07 (1H, H-6); 8.58 (1H, d, J= 7.3Hz, NH12); 4.76 (1H, H-13), 3.18 (2H, H-14); 7.25 (5H, H-16-H-20); and 1.46ppm (3H, d, J= 6.1Hz, H-21). 13CNMR: (CDC13) 169.1, s, C-l; 76.2, d, C-3; 32.4, t, C-4; 122.3, s, C-5; 136.8, d, C-6; 120.9, s, C-7; 163.5, s, C-8; 112.1, s, C-9; 142.6, s, C-10; 159.1, s, C-11; 54.3, d, C-13; 37.4, t, C-14; 137.7, s, C-15; 129.1, d, C-16, C-20; 130.0, d, C-17, C-19, 127.4, d, C18; 20.8, q, C-21; and 173.1ppm, s, C-22. Mass Spectrum: LREIMS: 256m/e (M) +. TLC Data Silica gel; benzene-acetic acid(3:1, v/v), Rf = 0.50; detected as a green fluorescent spot under UV light.

616

22.


References P. S. Steyn; Ochratoxin and Other Dihydroisocoumarins; In Microbial Toxins; A. Ciegler, S. Kadis and S. J. Ajil, eds. Academic Press, New York, N.Y., Vol. VI, pp. 179-203 (1971). R. J. Cole and R. H. Cox, Ochratoxins; In Handbook of Toxic Fungal Metabolites, Academic Press, Inc., New York, N.Y., pp. 137-140 (1981).

22.


617

Common/Systematic Name Ochratoxin B 7-Carboxy-8-hydroxy-3,4-dihydro-3R-methylisocoumarin-7-L-13-phenylalanine Molecular Formula/Molecular Weight C20H1906N; ~

-- 3 6 9 . 1 2 1 2 4 22

1,

COOH I

,-----,

H

0

OH

0

~ , o ~

General Characteristics Crystals from acidic methanol; mp., 221 ~ (c=0.29, in MeOH).

~H [•]D25 -35 ~

(c=0.15, in EtOH);

[a]D 25 - 5 6 ~

Fungal Source

Aspergi llus ochraceus.

Biological Activity Ochratoxin B is considerably less toxic than ochratoxins A or C. The LDs0 in day-old chicks was dosed orally was 54mg/kg while ochratoxin A was 3.3-3.9mg/kg in the same assay. Spectral Data UV: EtOH max

218(37,200) and 318nm (6,900).

1H N-IV[R:

(CDC13) 4.90(1H, H-3); 3.10(2H, H-4); 8.21(d, J=6.8Hz, H-5); 7.02(1H, d, J=6.0Hz, H-6); 8.64(1H, d, J=6.1Hz, NH-12); 4.90(1H, H-13); 3.10(2H, H-14); 7.28(5H, H-16H-20); and 1.45ppm (3H, d, J=5.4Hz, H-21). 13C NMR: (CDCI3) 168.7, s, C-l; 75.1, d, C-3; 33.1, t, C-4; 117.3, d, C-5; 135.5, d, C-6; 117.9, s, C-7; 162.0, s, C-8; 107.7, s, C-9; 142.7, s, C-10; 158.9, s, C-11; 52.8, d, C-13; 36.3, t, C-14; 137.0, s, C-15; 128.2, d, C-16, C-20; 127.0, d, C-17, C-19; 125.5, d, C-18; 19.3, q, C-21; and 171.6ppm, s, C-22. Mass Spectrum: LREIMS: 369role (M) +.

618

22.


TLC Data Silica gel; benzene-acetic acid (4:1, v/v), Re = 0.35, detected as a blue fluorescent spot under UV light. References P. S. Steyn; Ochratoxin and Other Dihydroisocoumarins; In Microbial Toxins; A. Ciegler, Kadis and S. J. Ajil, eds. Academic Press, New York, N.Y., Vol. VI, pp. 179-203 (1971). R. J. Cole and R. H. Cox; Ochratoxins; In Handbook of Toxic Fungal Metabolites; Press, Inc., New York, N.Y., pp. 137-140 (1981).

22. Ochratoxins and Related Metabolites

619

Common/Systematic Name 4-Hydroxyochratoxin A 7-Carboxy-5-chloro-4,8-dihydroxy-3,4-dihydro-3R-methylisocoumarin-7-L-13phenylalanine Molecular Formula/Molecular Weight C20HlaOTNC1, MW = 419.07718 22

17

COOH

is

13

12 0 l

8iHO ,L,

I..L Cl

o

3~.,,,,Me OH

General Characteristics Colorless crystals from benzene; mp., 216-218~ Fungal Source Penicillium viridicatum (ATCC 18411).

Biological Activity 4-Hydroxyochratoxin A was excreted in urine of male Wister rats dosed with ochratoxin A (IP). 4-Hydroxyochratoxin A had no effect when dosed to rats at a level of 40mg/kg; ochratoxin A at this level caused 100% mortality. Therefore, 4-hydroxyochratoxin A may be a detoxification product in animals dosed with ochratoxin A Spectral Data UV: ~k EtOH max

213 (32,500) and 334nm (6,400).

IR:

(CH3C1) 3380, 3000, 2500, 1723, 1678, 1655, and 1535cm-1. 1H NMR:

(CDC13) 4.80(1H, d, J=7.2Hz, H-3); 5.11(1H, d, J=2.0Hz, H-4); and 8.70ppm (1H, H-6). Mass Spectrum: LREIMS: 419m/e (M) +. TLC Data Silica gel, benzene-acetic acid (3:1, v/v), Re= 0.25; detected as a green fluorescent spot under UV light.

620

22.


References P. S. Steyn; Ochratoxin and Other Dihydroisocoumarins; In Microbial Toxins; A. Ciegler, Kadis and S. J. Ajil, eds. Academic Press, New York, N.Y., Vol. VI, pp. 179-203 (1971). R. J. Cole and R. H. Cox; Ochratoxins; In Handbook of Toxic Fungal Metabolites; Press, Inc., New York, N.Y., pp. 137-140 (1981).

22.


621

Common/Systematic Name Ochratoxin C Molecular Formula/Molecu!ar W~/ight C22H2206NCI; M'W = 431.11357 22

COOC2H5 '~

is

13

12 0

8OH

1 oL E

H

y~o~

0

~H

Cl General Characteristics Amorphous compound. Fungal Source Aspergillus ochraceus. Biological Activity Originally thought to be relatively nontoxic; however, it was later reported to be comparable to ochratoxin A. Spectral Data UV:

~

EtOH max

214 (30,000), 333 (7,000) and 378nm (2,050).

IR:

(CH3C1) 1730 and 1680em1. Mass Spectrum: LREIMS: 43 lm/e (M) +. TLC Data Silica gel; benzene-acetic acid (25:1, v/v), Re = 0.55; detected as a light green fluorescent spot under UV light. References P. S. Steyn; Ochratoxin and Other Dihydroisocoumarins; In Microbial Toxins; A. Ciegler, Kadis and S. J. Ajil, eds. Academic Press, New York, N.Y., Vol. VI, pp. 179-203 (1971). R. J. Cole and R. H. Cox; Ochratoxins; In Handbook of Toxic Fungal Metabolites; Press, Inc., New York, N.Y., pp. 137-140 (1981).

622

22.


Common/Systematic Name Mellein; Ochracin (-)3,4-Dihydro-8-hydroxy-3-methylisocoumarin Molecular Formula/Molecular Weight CloHloO3; MW

OH 8

~

-- 1 7 8 . 0 6 2 9 9

0

9

0 g

,, lo g

A,

H

General Characteristics Crystals (subl.); mp., 54-55~ Fungal Source Aspergillus melleus; A. ochraceus. Biological Activity Biological activity unknown. Spectral Data UV: Ef~ 212 (20,000), 246 (6,500) and 314nm (4,100). 1H NMR: (CDCI3) 4.76(1H, dd, J-7.0, 7.0Hz, H-3); 2.95 (2H, d, J=7.0Hz, H-4); 6.72 (1H, d, J-7.5Hz, H-5). 7.44 (1H, dd, J-7.5, 8.0Hz, H-6); 6.91 (1H, d, J=8.0Hz, H-7); 1.55 (d, J-7.0Hz, H-11); and 11.07ppm (1H, OH-8). ~3CNMR: (CDC13) 169.8, C-I; 76.1, C-3; 34.7, C-4; 117.8, C-5; 136.0, C-6; 116.2, C-7; 162.1, C-8; 108.2, C-9; 139.2, C-10; and 20.8ppm, C-11. Mass Spectrum: LREIMS: 178(M)+ (100%), 161,149, and 134m/e. TLC Data Silica gel G-HR; toluene-ethyl acetate-formic acid (5:4:1, v/v/v), Rf = 0.82; detected as a blue fluorescent spot under UV light. References P. S. Steyn; Ochratoxin and Other Dihydroisocoumarins; In Microbial Toxins; A. Ciegler, Kadis and S. J. Ajil, eds. Academic Press, New York, N.Y., Vol. VI, pp. 179-203 (1971). R. J. Cole and R. H. Cox; Ochratoxins; In Handbook of Toxic Fungal Metabolites; Press, Inc., New York, N.Y., pp. 137-140 ( 1981).

22.


623

Common/Systematic Name 4-Hydroxymellein (-)3,4-Dihydro-4,8-dihydroxy-3-methylisocoumarin Molecular Formula/Molecular Weight CloHloO4;

OH

C

~

L

MW'

= 194.05791

0

0 -

~.,,,,Me

ox. ". General Characteristics Crystals from chloroform-methanol; mp., 131-132~

[ a ] D 25 -

40~ (c: 1.0, in CHC13).

Fungal Source Aspergillus melleus; A. ochraceus; A. oniki; Lasiodiplodia theobromae (cis-4hydroxymellein); Apiospora camptospora. Biological Activity Biological activity unknown. Spectral Data UV: EtOH ~ max

247(5,300) and 315nm (4,200).

1H NMR: (CDC13) 4.63(1H, H-3); 4.63(1H, H-4), 7.04(1H, d, J=7.5Hz, H-5), 7.57(1H, H-6); 7.02(1H, d, J=8.2Hz, H-7); 1.52(d, J=6.5Hz, H-11); 2.16(1H, H-4OH); and 11.01ppm (1H, H=8OH). 13CNMR: (CDC13) 168.1, C-I; 79.7, C-3; 69.1, C-4; 117.7, C-5; 136.4, C-6; 115.9, C-7; 161.7, C-8; 106.5, C-9; 140.9, C-10; and 29.6ppm, C-11.

Mass Spectrum:

LREIMS: 194(M)+ (100%), 161,149, and 134m/e.

TLC Data Silica gel G-HR; toluene-ethyl acetate-formic acid (5:4:1, v/v/v), Re = 0.74; detected as a blue fluorescent spot under UV light.

624

22.


References P. S. Steyn; Ochratoxin and Other Dihydroisocoumarins; In Microbial Toxins; A. Ciegler, Kadis and S. J. Ajil, eds. Academic Press, New York, N.Y., Vol. VI, pp. 179-203 (1971). R. J. Cole and R. H. Cox, Ochratoxins; In Handbook of Toxic Fungal Metabolites, Press, Inc., New York, N.Y., pp. 137-140 (1981).

Miscellaneous Metabolites

4,5,10,11-Tetrahydroxybisboline 24-Ethyllanosta-8,24(24')-diene-313,22~-diol 25-Methylpisolactone 24-Methyllanosta-8,24'-diene-313,22~-diol 24-Methyllanosta-8,24(24')-diene-313,22~-diol 3a-Acetoxylanosta-8,24-dien-21-oic acid 21-Hydroxylanosta-8,24-dien-3-one 3a-(4-Carboxymethyl-3-hydroxy-3-methylbutanoyloxy)lanosta-8,24-dien-21-oicacid Isofunicone Xylobovatin Deacetyl-19,20-epoxycytochalasin Q 19,20-Epoxycytochalasin Q Deacetyl-19,20-epoxycytochalasin C 19,20-Epoxycytochalasin C Fusaproliferin Retigeranic Acid Moniliformin Cyclopiazonic Acid

625


23.


627

Common/Systematic Name 4,5,10,11-Tetrahydroxybisboline Molecular Formula/Molecular Weight

H~

C15H2804; M W -- 272.19876

OH

.........

OH "*"Hil~~o H

General Characteristics Colorless glass. Isolation/Purification Fungal cultures were extracted with chloroform-acetone (85:15, v/v), allowed to sit overnight, filtered, and the solid residue re-extracted with acetone. The acetone extract was combined with the chloroform-acetone extract, concentrated under vacuum, and the nonpolar materials removed by hexane drip. The solvent was decanted, concentrated, and chromatographed by flash chromatography with toluene, toluene-acetone 4:1, 2:1, 1:1 (v/v), acetone, and acetone-methanol, 1:1 (v/v). The toluene-acetone (1:1, v/v) fraction was further purified by reversed-phase TLC using methanol-water, 7:3 (v/v), which resulted in purified 4,5,10,11-tetrahydroxybisboline. Fungal Source


Soectral Data UV:

~, maxA~176 195nm (e=5,000, ~ to ~* transition). IR:

(film) 3376(OH), 1370, 1158, 1124, 1048, and 1050cm-1 (-C-O-C-). 1H NMR: (CDC13) 1.17(3H, s, H-12), 1.22(3H, s, H-13), 1.27(3H, s H-14), 1.47(1H, m, H-9a), 1.57(1H, m, H-3a), 1.59(1H, m, 2a), 1.64(1H, m, H-9b), 1.69(1H, m, H-6a), 1.74(1H, m, H-2b), 1.78(1H, m, H-3b), 1.92(11-1, m, H-6b), 2.10(1H, m, H-8a), 2.30(1H, m, H-l); 2.36(1H, m, H-8b); 3.38(1H, dd, J=l.7, 10.4Hz, H-10), 3.64(1H, b t, J=3.0Hz, H-5); 4.79(1H, b m, H-15a); and 4.84ppm (1H, m, H-15b).

628

23.


13CNMR: (acetone-d6) 25.1 (q, C-12); 25.9(q, C-13); 27.7(t, C-2); 28.1(q, C-14); 31.0(t, C-6); (t, C-8), 34.6(t, C-3), 35.8(t, C-9), 37.1(t, C-l); 70.8(s, C-4), 72.8(s, C-11), 74.1(d, El0); 78.7(d, C-5); 107.2(t, C-15); and 156.1ppm (s, C-7). Mass Data: LREIMS: 254(M + - H20, 1%), 239(2), 236(3), 218(2), 125(21), 108(64), 81(89), and 43m/e (100); exact mass for C~5H2603 (M + - 1-120); calcd 254.188; found 254.190m/e. Reference D. R. Sanson, D. G. Codey, C. L. Barnes, S. Searles, E. O. Schlemper, M. S. Tempesta, and G. E. Rottinghaus; New Mycotoxins from Fusarium sambucmum; J. Org. Chem., Vol. 54, pp. 4313-4318(1989).

23.


629

Common/Systematic Name 24-Ethyllanosta-8,24(24')-diene- 313,22~-diol Molecular Formula/Molecular Weight C32H5402; M W = 470.41238

OH

24'

21 .......

s HO

/28

29


[ a i D 24 + 3 9 . 2 7 ~

(c=0.45, in CHC13).

Fungal Source

Pisofithus tinctorius, a commercially important ectomycorrhizal fungus that is considered

to have a broad host range. Isolation/Purification Freeze-dried P. tinctorius myeelia from submerged cultures were powdered in liquid N2 and extracted three times with ethyl ether. The combined extracts were evaporated to dryness yielding a pale yellow solid containing mainly a mixture of triterpenoids. The solid was redissolved in 2-propanol and centrifuged. Separation and purification of the triterpenoids was carried out by repeated chromatography on a reversed-phase (Cs) HPLC column (PrepPack Bondapak 25 x 200 mm) using 80% aqueous methanol (adjusted to pH 3 with formic acid) as eluting solvent at a flow rate of 10ml/minute and detection at 210nm. Spectral Data UV:

End absorption.

630

23.


1H NE/IR: (CDC13) 1.72, 1.23(2H, H-I); 1.67, 1.58(2H, H-2), 3.24(1H, dd, J=l 1.6, 4.6Hz, H3); 1.05(1H, H-5); 1.68, 1.50(2H, H-6); 2.04, 2.04(2H, H-7), 2.01, 2.01(2H, H-11); 1.78, 1.67(2H, n-12); 1.61, 1.20(2H, n-15); 2.00, 1.35(2H, n-16); 1.92(1H, n-17), 0.70(3H, s, n-18); 0.99(3H, s, n-19); 1.42(1H, H-20); 0.91(3H, d, J=6.6Hz, n-21); 3.77(1H, ddd, J=8.6, 4.6, 1.SHz, H-22); 2.05, 2.05(2H, H-23); 2.85(1H, septet, J=7.0Hz, H-25), 1.06(3H, d, J=7.0Hz, H26"); 1.00(3H, d, J=6.9Hz, H-27"); 5.25(1H, q, J=6.8Hz, H-24'); 1.00(3H, s, H-28); 0.81(3H, s, H-29); 0.91(3H, s, H-30); and 1.65ppm (3H, d, J=6.8Hz). * Assignmems may be reversed. 13CNMR: (CDC13) 35.6, C-l; 27.9, C-2; 79.0, C-3; 38.9, C-4; 50.4, C-5; 18.3, C-6; 26.5, C-7; 134.4, C-8; 134.5, C-9; 37.0, C-10; 21.0, C-11; 21.0, C-12; 44.5, C-13; 49.9, C-14; 30.8, C-15; 27.7, C-16; 47.2, C-17; 15.6, C-18; 19.1, C-19; 41.3, C-20; 12.0, C-21; 71.0, C-22; 38.7, C-23; 142.4, C-24; 28.6, C-25; 21.2, C-26; 21.0, C-27; 120.6, C-24'; 28.0, C-28, 15.4, C-29; 24.4, C-30; and 12.9ppm C-31. Mass Spectrum: HREIMS: 470.4096(M+, calcd for C32H5402,470.4124, 16%), 455[M - Me]+ (6%), 437[M - Me - H20] + (8), 419(3), 372.3037[calcd for C25H4002,372.3029] (36), 357.2782[calcd for C2,I-I3702, 357.2794] (100), 344(8), 339.2664[calc for C24I-I350, 339.2688] (32), 329(5), 321(6), 314(9), 311(11), 299(12), 281(9), 215(6), 187(8), 161(7), and 109role (9). Reference A. Baumbert, B. Schumann, A. Porzel, J. Schmidt, and D. Strack; Triterpenoids from Pisolithus tinctorium Isolates and Ectomycorrhizas; Phytochemistry, Vol. 45, pp. 499504(1997).

23.


631

Common/Systematic Name 25-Methylpisolactone (22S)-24-Methyllanosta-8-ene-22,24'-epoxy-313-01-24'-one Molecular Formula/Molecular Weight C32H5203; M W -- 484.39165 0

0

27 18 .....

" 26

28

29

General Characteristics Colorless needles; mp., 313-316~

[a]D 24 +38.7 ~ (c=0.23, in CHC13).

Fungal Source

Pisolithus tinctorius, a commercially important ectomycorrhizal fungus that is considered

to have a broad host range. Isolation/Purification Freeze-dried P. tinctorius mycelia from submerged cultures were powdered in liquid N2 and extracted three times with ethyl ether. The combined extracts were evaporated to dryness yielding a pale yellow solid containing mainly a mixture oftriterpenoids. The solid was redissolved in 2-propanol and centrifuged. Separation and purification of the triterpenoids was carried out by repeated chromatography on a reversed-phase (Cs) HPLC column PrepPack Bondapak (25 x 200 mm) using 80% aqueous methanol (adjusted to pH 3 with formic acid) as eluting solvent at a flow rate of 10ml/minute and detection at 210nm. Spectral Data UV~

End absorption.

632

23.


~HNMR: (CDCI3) 1.72, 1.23(2H, H-I); 1.68, 1.58(21-1,H-2); 3.24(11-1, dd, J=l 1.4, 4.6Hz, H3); 1.05(1H, H-5), 1.68, 1.50(2H, H-6); 2.03, 2.03(2H, H-7), 2.03, 2.02(2H, H-11); 1.77, 1.65(2H, H-12); 1.61, 1.23(2H, H-15); 2.01, 1.34(2H, H-16); 1.96(1H, H-17); 0.70(3H, s, H-18); 0.98(3H, s, n-19); 1.58(1H, H-E0); 0.94(3H, d, J=6.7Hz, H-E1), 4.39(1H, ddd, J=10.7, 6.1, 1.5Hz, H-22); 2.01, 1.92(2H, H-23); 2.46(1H, dd, J=12.6, 8.9Hz, H-24); 1.07(3H, s, H-26"); 1.07(3H, s, H-27"); 1.00(3H, s, H-28); 0.81(3H, s, H-29); 0.89(3H, s, H-30); and 1.07ppm (3H, s, H-31). * Assignments may be reversed. 13CNMR: (CDC13) 35.6, C-I; 27.9, C-2; 78.9, C-3; 38.9, C-4; 50.4, C-5; 18.2, C-6; 26.5, C-7; 134.2, C-8; 134.7, C-9; 37.0, C-10; 21.0, C-11; 30.9, C-12; 44.6, C-13; 49.8, C-14; 30.8, C-15; 27.9, C-16; 47.4, C-17; 15.6, C-18; 19.1, C-19; 39.8, C-20; 12.2, C-21; 79.4, C-22; 28.5, C-23; 50.6, C-24; 31.8, C-25; 29.6, C-26; 29.6, C-27; 177.2, C-24'; 28.0, C-28; 15.4, C-29; 24.2, C-30, and 29.6ppm, C-31. Mass Spectrum: HREIMS: 484.3905[calcd for C32H5203,484.3917] (28), 469[M - Me] + (80), 451.3542[M-Me- H20] § [calcd for C3~H4702,451.3576] (100), 329(7), 299(9), 281(14), 227(9), 215(8), 213(8), 187(10), 161(10), 159(9), 147(6), 135(17), 121(10), 95(15), and 57m/e (18). Reference A. Baumbert, B. Schumann, A. Porzel, J. Schmidt, and D. Strack; Triterpenoids from Pisolithus tinctorium Isolates and Ectomycorrhizas; Phytochemistry, Vol. 45, pp. 499504(1997).

23.


633

Common/Systematic Name 24-Methyllanosta-8,24(24 ')-diene-313,22~-diol Molecular Formula/Molecular Weight C31H5202, M W ' = 456.39673 24'

21 ....

28

OH ZZ~

j

27

2g

General Characteristics Crystals from isopropanol, mp. 177-179~

[ a ] D 24 + 4 9 . 7 ~

(c=0.20, in CHCI3).

Fungal Source

Pisolithus tinctorius, a commercially important ectomycorrhizal fungus that is considered

to have a broad host range. Isolation/Purification Freeze-dried P. tinctorius mycelia from submerged cultures were powdered in liquid N2 and extracted three times with ethyl ether. The combined extracts were evaporated to dryness yielding a pale yellow solid containing mainly a mixture oftriterpenoids. The solid was redissolved in 2-propanol and centrifuged. Separation and purification of the triterpenoids was carried out by repeated chromatography on a reversed-phase (Cs) HPLC PrepPack Bondapak column (25 x 200 mm) using 80% aqueous methanol (adjusted to pH 3 with formic acid) as eluting solvent at a flow rate of 10ml/minute and detection at 210nm. Spectral Data UV:

End absorption.

634

23.


1H N1VIR: (CDC13) 1.72, 1.23(2H, H-I); 1.66, 1.58(2H, H-2); 3.239(1H, dd, J=l 1.7, 4.5Hz, H3), 1.04(1H, H-5), 1.68, 1.49(2H, H-6); 2.03, 2.03(2H, H-7); 2.03, 2.01(2H, H-11); 1.79, 1.68(2H, H-12); 1.61, 1.21(2H, H-15), 2.00, 1.36(2H, H-16); 1.91(1H, H-17); 0.70(3H, s, H-18); 0.99(3H, s, H-19); 1.43(1H, H-20), 0.92(3H, d, J=6.6Hz, H-21); 3.81(1H, ddd, J=9.4, 3.9, 1.4Hz, H-22); 2.26(1H, dd, J=14.3, 9.4Hz, H-23); 2.10(1H, dd, J=14.3, 3.9Hz, H-23); 2.24(1H, septet, J=6.8Hz, H-25); 1.06(3H, d, J=6.8Hz, H26"); 1.04(3H, d, J=6.9Hz, H-27"); 4.89(1H, dd, ,/=1.3, 1.3Hz, H-24'); 4.79(1H, dd, J=2.4, 1.3Hz, H-24'); 1.00(3H; s, H-28); 0.81(3H, s, H-29); and 0.92ppm (3H, s, H-

30). * Assignments may be reversed. 13CNMR: (CDC13) 35.6, C-I; 27.9, C-2; 79.0, C-3; 38.9, C-4; 50.4, C-5; 18.3, C-6; 26.5, C-7; 134.4, C-8; 134.6, C-9; 37.0, C-10; 21.0, C-11; 31.0, C-12; 44.5, C-13; 49.9, C-14; 30.8, C-15; 27.7, C-16; 47.1, C-17; 15.6, C-18; 19.1, C-19; 41.1, C-20; 12.0, C-21; 70.8, C-22; 41.2, C-23; 153.4, C-24; 33.5, C-25; 21.7, C-26; 22.0, C-27; 109.2, C-24'; 28.0, C-28; 15.4, C-29; and 24.3ppm C-30. Mass Spectrum: LREIMS: 456[M] § (32%), 441[M-Me] § (5), 423[M-Me-H20] § (9), 405(4), 372(8), 357(100), 344(6), 339(35), 329(5), 321(9), 314(6), 311(13), 299(11), 281(10), 215(6), 187(7), 161(6), 135(6), and 95role (8). Reference A. Baumbert, B. Schumann, A. Porzel, J. Schmidt, and D. Strack, Triterpenoids from Pisolithus tinctorium Isolates and Ectomycorrhizas; Phytochemistry, Vol. 45, pp. 499504(1997).

23.


635

Common/Systematic Name 3 t~-Acetoxylanosta-8,24-dien-21-oic acid Molecular Formula/Molecular Weight C32H5004; MW = 498.37091

HO0

:

AcO

General Characteristics White powder; mp., 192-194~

[~]D 21 + 1 4 ~

(c=l.0, in CHC13).

Fungal Source Fomitopsispinicola, a wood-rotting fungus growing on coniferous and broad-leafed trees in Europe. Isolation/Purification Lyophilized fruiting bodies of F. pinicola were extracted successively with methylene chloride and methanol (3x). The methylene chloride extract was separated on a silica gel column Si 60 with a step-gradient of petrol-ethyl acetate (95:5-9:1-7:1-5:1-3:1-1:1, v/v), ethyl acetate and finally methanol to give 19 fractions (A-S). Fraction I was submitted to low pressure liquid chromatography (LPLC) on silica gel with chloroformisopropanol (98:2, v/v) giving two fractions (I1 and 12). Fraction I1 was further purified on a Sephadex LH20 column with chloroform-methanol (1:1, v/v) to yield the non-UV absorbing metabolite 3a-acetoxylanosta-8,24-dien-21-oic acid. Fraction H was submitted to LPLC with petrol-ethyl acetate (5:1, v/v) which gave five fractions (H1-H5). Further separation of fraction H4 on a Sephadex LH20 column with chloroform-methanol (1:1, v/v) afforded 21-hydroxylanosta-8,24-dien-3-one. Fraction N was submitted to medium pressure liquid chromatography on DIOL with a stepwise gradient of n-hexanechloroform (2:1-1:1, v/v) affording 3 fractions (N1-N3). A further purification of fraction N3 on a Sephadex LH20 column with chloroform-methanol (1:1, v/v) yielded 3tt-(4carboxymethyl-3-hydroxy-3-methylbutanoyloxy)lanosta-8,24-dien-21-oic acid as a white non-UV absorbing powder. Biological Activity Antimicrobial activity against Bacillus subtilis in a TLC bioassay.

636

23.


Spectral Data IR:

3600-2750, 2920, 1715, and 1680cmq. 1H NMR:

(CDC13) 0.74(3H, s, H-18); 0.83(3H, s, H-29); 0.88(3H, s, H-28); 0.92(3H, s, H-30); 0.95(3H, s, H-19); 1.56, 1.65(each 3H, s, H-26, H-27); 2.04(3H, s, acetoxy); 4.65(1H, br s, H-3); and 5.07ppm (1H, t, H-24). 13C NMR: (CDC13) 30.3, C-l; 23.4, C-2; 77.9, C-3; 36.8, C-4; 45.2, C-5; 17.9, C-6; 26.9, C-7; 134.4, C-8; 133.8, C-9; 36.9, C-10; 20.8, C-11; 28.9, C-12; 44.2, C-13; 49.5, C-14; 30.8, C-15; 27.0, C-16; 47.1, C-17; 16.0, C-18; 18.8, C-19; 47.7, C-20; 183.3, C-21; 32.4, C-22; 25.9, C-23; 123.6, C-24; 132.2, C-25; 17.6, C-26; 25.7, C-27; 21.8, C-28; 27.5, C-29; 24.3, C-30; 171.0, CH3C=O; and 21.3ppm, CH3C=O. Mass Spectrum: LREIMS: 498(M) + (45%), 483(18), 437(17), 423(100), 281(27), 187(23), and 69m/e (23). D/CI-MS: 516[M + NH4]§ (100%), 499[M + H] § (6), and 472m/e (21). TLC Data Ethyl acetate-petrol (1:1, v/v), Rf= 0.54; ethyl acetate-petrol (1:3, v/v), Re = 0.24. Reference A. C. Keller, M. P. Maillard, and K. Hostettmann; Antimicrobial Steroids from the Fungus Fomitopsis pinicola; Phytochemistry, Vol. 41, pp. 1041-1046(1996).

23.


637

Common/Systematic Name 21-Hydroxylanosta-8,24-dien-3-one Molecular Formula/Molecular Weight C30H4sO2; MW = 440.36543 HOH2C .....

General Characteristics White powder; mp., 92-96~

[a]D 21 + 5 9 ~

(c=l.0, in CHC13).

Fungal Source

Fomitopsis pinicola, a wood-rotting fungus growing on coniferous and broad-leafed trees in Europe.

Isolation/Purification Lyophilized fruiting bodies ofF. pinicola were extracted successively with methylene chloride and methanol (3x). The methylene chloride extract was separated on a silica gel column Si 60 with a step-gradient of petrol-ethyl acetate (95:5-9:1-7:1-5:1-3:1-1:1, v/v), ethyl acetate and finally methanol to give 19 fractions (A-S). Fraction I was submitted to low pressure liquid chromatography (LPLC) on silica gel with chloroformisopropanol (98:2, v/v) giving two fractions (I1 and 12). Fraction I1 was further purified on a Sephadex LH20 column with chloroform-methanol (1:1, v/v) to yield the non-UV absorbing metabolite 3a-acetoxylanosta-8,24-dien-21-oic acid. Fraction H was submitted to LPLC with petrol-ethyl acetate (5:1,v/v),which gave five fractions (H1-H5). Further separation of fraction H4 on a Sephadex LH20 column with chloroform-methanol (1:1, v/v) afforded 21-hydroxylanosta-8,24-dien-3-one. Fraction N was submitted to medium pressure liquid chromatography on DIOL with a stepwise gradient of n-hexanechloroform (2:1-1:1, v/v) affording 3 fractions (N l-N3). A further purification of fraction N3 on a Sephadex LH20 column with chloroform-methanol (1:1, v/v) yielded 3a-(4carboxymethyl-3-hydroxy-3-methylbutanoyloxy)lanosta-8,24-dien-21-oic acid as a white non-UV absorbing powder. Biological Activity Antimicrobial activity against Bacillus subtilis in a TLC bioassay.

638

23.


Spectral Data IR:

3250, 2905, and 1685cm"1. 1H NMR: (CDC13) 0.69(3H, s, H-18); 0.86(3H, s, H-30), 1.01(3H, s, H-28), 1.04(3H, s, H-29); 1.07(3H, s, H-19); 1.56, 1.63(each 3H, s, H-26, H-27); 3.65(2H, m,H-21); and 5.07ppm (1H, t, H-24). 13CNMR: (CDC13) 35.9, C-l; 34.4, C-2; 217.0, C-3; 47.2, C-4; 51.0, C-5; 19.3, C-6; 26.2, C-7; 133.0, C-8; 135.1, C-9; 36.8, C-10; 20.9, C-11; 29.6, C-12; 44.1, C-13; 49.8, C-14; 30.7, C-15; 27.5, C-16; 44.2, C-17; 16.0, C-18; 18.6, C-19; 42.7, C-20; 62.3, C-21; 30.3, C-22, 24.8, C-23; 124.8, C-24; 132.2, C-25; 17.6, C-26; 25.6, C-27; 21.2, C-28, 26.0, C-29; and 24.3ppm, C-30. Mass Spectrum: LREIMS: 440[M]+ (100%), 425(61), 407(54), 271(16), 257(19), 245(25), 109(63), and 69m/e (23). D/CI-MS: 458[M + NH4]+ (100). TLC Data Ethyl acetate-petrol (1:3, v/v), Re= 0.25. Reference A. C. Keller, M. P. Maillard, and K. Hostettmann; Antimicrobial Steroids from the Fungus Fomitopsis pinicola; Phytochemistry, Vol. 41, pp. 1041-1046(1996).

23.


639

Common/Systematic Name 3 tt-(4-Carboxymethyl-3-hydroxy-3-methylbutanoyloxy)lanosta-8,24-dien-21 -oic acid Molecular Formula/Molecular Weight C37H5807; M - ~ = 614.41825

HOOC .....

.0 Me

OH .0

M e O ~ ~ ~ ' ~ O

General Characteristics White powder; mp., 147~

,.....

[~]D 21 + 5 ~

(c=l.0, in CHC13).

Fungal Source

Fomitopsispinicola, a wood-rotting fungus growing on coniferous and broad-leafed trees in Europe.

Isolation/Purification Lyophilized fruiting bodies ofF. pinicola were extracted successively with methylene chloride and methanol (3x). The methylene chloride extract was separated on a silica gel column Si 60 with a step-gradient of petrol-ethyl acetate (95:5-9:1-7:1-5:1-3:1-1:1, v/v), ethyl acetate and finally methanol to give 19 fractions (A-S). Fraction I was submitted to low pressure liquid chromatography (LPLC) on silica gel with ehloroformisopropanol (98:2, v/v) giving two fractions (I1 and I2). Fraction Ilwas further purified on a Sephadex LH20 column with chloroform-methanol (1:1, v/v) to yield the non-UV absorbing metabolite 3t~-acetoxylanosta-8,24-dien-21-oic acid. Fraction H was submitted to LPLC with petrol-ethyl acetate (5:1, v/v) which gave five fractions (H1-H5). Further separation of fraction H4 on a Sephadex LH20 column with chloroform-methanol (1:1, v/v) afforded 21-hydroxylanosta-8,24-dien-3-one. Fraction N was submitted to medium pressure liquid chromatography on DIOL with a stepwise gradient of n-hexanechloroform (2:1-1:1 v/v) affording 3 fractions (N1-N3). A further purification of fraction N3 on a Sephadex LH20 column with chloroform-methanol (1:1, v/v) yielded 3t~-(4carboxymethyl-3-hydroxy-3-methylbutanoyloxy)lanosta-8,24-dien-21-oic acid as a white non-UV absorbing powder. Biological Activity Antimicrobial activity against Bacillus subtilis in a TLC bioassay.

640

23.


Spectral Data 1HNMR: (pyridine-ds) 0.86(3H, s, H-18); 0.91(3H, s, H-29); 0.95(3H, s, H-28); 0.99(3H, s, H30); 1.05(3H, s, H-19); 1.62, 1.66(each 3H, s, H-26, H-27); 1.66(3H, s, 3'-methyl); 2.97(2H, s); 3.01(d, J=13.9Hz); 3.61(3H, s, 5'-methoxy); 4.93(1H, s, H-3); and 5.32ppm (1H, t, H-24).

13C NMR: (pyridine-ds) 30.7, C-l; 23.5, C-2; 78.1, C-3; 36.8, C-4; 45.8, C-5; 18.2, C-6; 26.7, C-7; 134.2, C-8; 134.1, C-9;.37.1, C-10; 21.1, C-11; 29.2, C-12; 44.8, C-13; 49.8, C14; 30.9, C-15; 27.4, C-16; 47.6, C-17; 16.3, C-18; 19.0, C-19; 49.0, C-20; 178.6, C21; 33.2, C-22; 26.2, C-23; 124.8, C-24; 131.7, C- 25; 17.6, C-26; 25.7, C-27; 21.8, C-28; 27.5, C-29; 24.2, C-30; 171.8, C-I'; 45.9, C-2'; 69.8, C-3'; 28.4, C-3' methyl; 46.3, C-5'; 171.2, C-6'; and 51.2ppm C-5'-O-methyl. Mass Spectrum: LREIMS: 614[M] + (0.4%), 540(1.6), 438(56), 423(100), 296(32), 281(24), 187(29), 177(60), 159(39), 117(30), and 69m/e (17). D/CI-MS: 632[M + NH4]+ (50%), 615[M + H] + (16), 474[M-side chain + NH4]+ (17), 458(22), 439[M-side chain + H] § (68), 194[side chain + NH4]§ (100). TLC Data Chloroform-isopropanol (9:1 v/v), Re = 0.60. Reference A. C. Keller, M. P. Maillard, and K. Hostettmann; Antimicrobial Steroids from the Fungus Fomitopsis pmicola; Phytochemistry, Vol. 41, pp. 1041-1046(1996).

23.


641

Common/Systematic Name Isofunicone (E)•3•Meth•xy•2•pr•peny•-5-(2'-carb•meth•xy-4'-hydr•xy-6'-meth•xybenz•y•)-4-pyr•ne Molecular Formula/Molecular Weight C19HIsOs; MW = 374.10017 8'

O~

OMe r

0

0

10

z

HO 4

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