J. Antibiot.

58(1): 61–64, 2005

THE JOURNAL OF
ORIGINAL ARTICLE ANTIBIOTICS

Favolon B, a New Triterpenoid Isolated from the Chilean

Mycena sp. Strain 96180
Pedro Aqueveque, Timm Anke, Heidrun Anke, Olov Sterner, José Becerra, Mario Silva

Received: September 11, 2004 / Accepted: December 10, 2004

© Japan Antibiotics Research Association

Abstract A new biologically active triterpenoid, favolon Contulmo, near to Concepción, Chile. Mycelial cultures
B (1), was isolated from fermentation broths of Mycena sp. were produced from spore prints of fruiting bodies, which
strain 96180. Favolon B showed antifungal activities were then grown in a YMG medium composed of yeast
towards Botrytis cinerea, Mucor miehei, Paecilomyces extract 0.4%, malt extract 1%, glucose 0.4%, and agar
variotii and Penicillium notatum. No activities were 1.5%, pH 5.5. Small sections of the stock culture were cut
observed against bacteria and yeasts. The structure of under sterile conditions and transferred to a 5-liter
favolon B was elucidated by spectroscopic techniques. Erlenmeyer flask containing 2500 ml of YMG. This flask
was incubated at 22°C on a rotary shaker (120 rpm). After
Keywords terpenoids, fungi, basidiomycetes, bioactive 20 days of fermentation, the culture broth was extracted
metabolites with EtOAc (2.5 liters). Evaporation of the organic phase
yielded a crude extract (915 mg), which was applied onto a
column containing silica gel (Merck 60, 0.063
0.2 m m;
Introduction column 3
30 cm). Elution with ethyl acetate : methanol
(3 : 7) yielded 33 mg of an enriched product containing the
In our search of novel and biologically active metabolites active compound 1.
from Chilean basidiomycetes, we previously reported four Final purification was achieved by preparative HPLC
new bioactive compounds, the himanimides, from Serpula (Jasco model PU-980 with a diode array detector; column:
himantoides [1]. In the course of further screenings, a new Macherey and Nagel, 250
21.2 mm containing 7 m m
antifungal and cytotoxic triterpenoid, favolon B (1), related
to favolon (2) [2] (see Figure 1 for structures), was isolated
from fermentations of Mycena sp. strain 96180. This paper
describes the fermentation of Mycena sp. strain 96180 and
the isolation and chemical as well as biological
characterization of favolon B.

Results and Discussion

1 2
Mycena 96180 was found on endemic forest soil from Fig. 1 Relative structures of favolon B (1) and favolon (2).

P. Aqueveque, J. Becerra, M. Silva (Corresponding author): T. Anke, H. Anke: Lehrbereich Biotechnologie der Universität,
Departamento de Botánica. Facultad de Ciencias Naturales y Paul-Ehrlich-Strase 23, D-67663 Kaiserslautern, Germany
Oceanográficas. Universidad de Concepción, Casilla 160-C,
O. Sterner: Division of Organic and Bioorganic Chemistry, Lund
Concepción, Chile, E-mail: mjsilva@udec.cl
University, P.O. Box 124, S-221 00 Lund, Sweden
62

1
Table 1 H (500 MHz) and 13C (125 MHz) NMR data of favolon B

1 13 1 13
C H (C6D6) C (C6D6) H (CDCl3) C (CDCl3)

1a 1.28 (m) 30.9 (t) 2.02 (m) 30.7 (t)

1b 0.98 (m) 1.66 (m)
2a 1.77 (m) 27.4 (t) 2.11 (m) 26.8 (t)
2b 1.47 (m) 1.90 (m)
3 4.63 (m) 75.4 (d) 4.85 (m) 75.2 (d)
4a 2.10 (dd, J4.7, 11.6 Hz) 37.4 (t) 2.63 (m) 37.3 (t)
4b 2.18 (ddd, J1.3, 11.4, 11.6 Hz) 2.67 (m)
5 – 156.4 (s) – 156.9 (s)
6 5.65 (d, J1.3 Hz) 127.2 (d) 5.81 (s) 126.6 (d)
7 – 197.2 (s) – 197.6 (s)
8 2.86 (d, J7.3 Hz) 44.1 (d) 3.35 (m) 43.6 (d)
9 – 64.6 (s) – 64.4 (s)
10 – 43.8 (s) – 43.6 (s)
11 2.81 (d, J2.6 Hz) 56.9 (d) 3.36 (m) 56.9 (d)
12a 1.11 (d, J14.7 Hz) 40.3 (t) 1.69 (m) 39.8 (t)
12b 2.02 (dd, J2.6, 14.7 Hz) 2.40 (dd, J2.3, 14.9 Hz)
13 – 42.5 (s) – 42.0 (s)
14 1.01 (m) 47.3 (d) 1.48 (m) 46.9 (d)
15a 1.45 (m) 22.5 (t) 1.50 (m) 21.7 (t)
15b 2.81 (m) 2.54 (m)
16a 1.56 (m) 27.8 (t) 1.59 (m) 27.0 (t)
16b 1.27 (m) 1.30 (m)
17 1.44 (m) 55.6 (d) 1.53 (m) 55.0 (d)
18 2.60 (dq, J10.2, 6.7 Hz) 44.3 (d) 2.68 (m) 43.6 (d)
19 – 217.0 (s) – 216.9 (s)
20 4.41(s) 80.4 (d) 4.31 (s) 79.8 (d)
21 1.58 (m) 41.7 (d) 1.63 (m) 41.0 (d)
22 1.85 (m) 32.2 (d) 1.76 (m) 31.4 (d)
23 1.09 (d, J6.9 Hz) 21.3 (q) 1.07 (d, J6.9 Hz) 20.9 (q)
24 0.99 (d, J6.9 Hz) 21.1 (q) 0.99 (d, J6.9 Hz) 20.7 (q)
25 0.79 (d, J6.8 Hz) 11.7 (q) 0.70 (d, J6.7 Hz) 11.1 (q)
26 1.02 (d, J6.7 Hz) 16.3 (q) 1.10 (d, J6.9 Hz) 15.7 (q)
27 1.10 (s) 16.3 (q) 0.99 (s) 15.7 (q)
28 0.73 (s) 16.4 (q) 1.08 (s) 16.3 (q)
29 – 173.1 (s) – 172.3 (s)
30 3.92 (s) 78.0 (d) 3.97 (s) 77.1 (d)
31 – 72.4 (s) – 72.1 (s)
32 1.32 (s) 26.6 (q) 1.32 (s) 25.9 (q)
33 1.26 (s) 25.9 (q) 1.24 (s) 25.0 (q)

Nucleosil C18, flow rate: 5 ml/minute). Elution with water - IR (KBr): 3433, 2961, 2925, 1701, 1652, 1617, 1380, 1200,
methanol gradient 23 : 77 v/v yielded 11 mg of compound 1144 and 1094 cm1. UV (MeOH), l max 235 nm. NMR
(1). data are given in Table 1.
The structure was elucidated by spectra of 1H NMR The elemental composition suggested by the high
(500 MHz, CDCl3, C6D6) and 13C NMR (125 MHz, CDCl3, resolution MS data show that the molecule has 9
C6D6). EIMS. (70 eV), m/z (rel. int.): 574 (100%, M), 556 unsaturations, and with one carbon - carbon double bond
(40%) 440 (60%) 423 (20%). HRFABMS observed and three carbonyl groups (suggested by the 1D NMR data)
575.3589 for MH (calculated for C33H51O8 575.3584). the compound should consequently be pentacyclic. The
 63

Table 2 Antifungal activity of favolon B in the agar diffusion assay

Diameter of inhibition zone (mm)

Organism
m g/disc*
0.1 1 10

Absidia glauca (+) – – –

A. glauca (–) – – –
Alternaria porri – 10di 26di
Aspergillus ochraceus 8di 15di 20di
Botrytis cinerea 20 24 42
Cladosporium cladosporoides – – –
Epicoccum purpurascens – – –
Fusarium fujikuroi – – –
F. oxysporum – – –
Mucor miehei 15 17 22
Nematospora coryli – – –
Paecilomyces varioti 20 25 30
Penicillium islandicum – – –
P. notatum 10 12 18
Pythium ultimum – – –
Rhodotorula glutinis – – –
Saccharomyces cerevisiae is 1** – – –
S. cereviseae a 288c – – –
Ustilago nuda – 9 16
Zygorhynchus moelleri – – –
Bacillus brevis ATCC 9999 – – –
B. subtilis ATCC 6633 – – –
Enterobacter dissolvens – – –
Sarcina lutea – – –

* diameter6 mm; –no inhibition zone; didiffuse inhibition zone; **Gift from
Prof. Lacrouter, Strasbourg, France.

basic steroid structure could be established by the COSY showing that it is alfa, and the closeness of the two protons
and HMBC correlations (in C6D6), for example the HMBC is revealed by a Dreiding model of the suggested structure,
correlations from 28-H3 to C-1, C-5, C-9 and C-10, from and in addition to 14-H. 27-H3 give NOESY correlations to
27-H3 to C-12, C-13, C-14 and C-17, from 26-H3 to C-17, 15-Hb and 18-H, while 17-H correlates to 12-Ha , 16-Ha
C-18 and C-19, from 6-H, 8-H and 14-H to C-7, from and 26-H3. The correlations between 18-H and 16-Ha as
12-H2 to C-9 and C-11, and from 8-H to C-9. The position well as 21-H, together with those already mentioned,
of the ester group is shown by the HMBC correlation from demonstrate the configuration of C-18. 20-H correlates to
3-H to C-29. The epoxide function is supported by the 16-Hb and 18-H, but not to 25-H3, and must consequently
chemical shifts as well as the correlations observed, and in be on the upper side of the molecule. A favourable
addition by the coupling constant between 11-H and C-11 conformations allowing for an intermolecular hydrogen
(171 Hz). The relative configuration of favolon B was bond between 20-OH and 19O, the NOESY correlations
suggested by the correlations observed in NOESY for 20-H discussed above and the NOESY correlation
experiments (in C6D6). The C-10 methyl group gives between 18-H and 21-H is obtained if the configurations of
NOESY correlations with the axial protons 2-Hb and C-20 and C-21 are as suggested in Figure 1, but not in other
4-Hb , as well as to 11-H. 3-H correlates with 2-Ha and diastereomers. 30-H only gives NOESY correlations to
4-Ha , and to the axial 1-Ha , and 1-Hb gives as expected a 32-H3 and 33-H3, and it is not possible to propose the
correlation to 11-H. 8-H gives a strong NOESY to 1-Ha , relative configuration of C-30 based on NMR data.
64

reduced spectrum of sensitive fungal strains and positive

cytotoxic activities.
It is interesting to note that the only active compound
produced by Mycena sp. strain 96180 was favolon B and
not the antifungal compounds strobilurins and/or
oudemansins, the typical active substances of the Mycena
genus [6].
This compound is probably ecologically significant in
nature, where the Mycena species uses the isopentyl
pyrophosphate pathway, an alternative pathway to shikimic
acid, thereby producing favolon B, an antifungal compound
Fig. 2 Inhibitory effect of favolon B on incorporation of that gives the species competitive advantages against
radio-labelled precursors into macromolecules HL-60 cells.
potential competitors [7].
[14C] thymidine into DNA, control 61267 cpm; [14C] uridine
into RNA, control 124829 cpm; [14C] leucine into proteins,
Acknowledgement We acknowledge the financial support from
control 18837 cpm.
DAAD, FONDECYT-Chile (Grand number 1040445), Escuela de
Graduados and Dirección de Investigación of the Universidad de
Concepción.
The assays for antimicrobial [3] and cytotoxic activities
[4] were carried out towards HL-60 (ATCC CCL 240) and
Colon 320 (DSMZ ACC-144) cells. The incorporation of References
the precursors [2-14C thymidine into DNA, 2-14C uridine
1. Aqueveque P, Anke T, Sterner O. The Himanimides, New
into RNA, and 1-14C leucine into proteins] was assayed
Bioactive Compounds from Serpula himantoides. Zeitschrift
with HL-60 cells [5].
Naturforschung 57c: 257–262 (2002)
The compound’s antifungal and antibacterial activities
2. Anke T, Werle A, Zapf S. Favolon, a new antifungal
are compared in Table 2. Flavolon B (1) exhibits higher triterpenoid from Favolaschia species. J Antibiot 48:
antifungal activities towards Botrytis cinerea, Mucor 725–726 (1995)
miehei, Paecilomyces variotii, and Penicillium notatum. No 3. Anke H, Bergendorff O, Sterner O. Assays of the biological
antibiotic activities were observed against bacteria and activities of guaiane sesquiterpenoids isolated from the fruit
yeasts. bodies of edible Lactarius species. Food Chem Toxicol 27:
Favolon B (1) also showed moderate cytotoxic activity 393–397, 1989
towards HL-60 (ATCC CCL 240), with IC50’s of 10 m g/ml, 4. Zapf S, Hoßfeld M, Anke H, Velten R, Steglich W.
whereas lower cytotoxic effects (25 m g/ml) were observed Darlucins A and B, new isocyanide antibiotics from
with Colon 320 (DSMZ ACC-144) cells. The incorporation Sphaerellopsis filum (Darluca filum). J Antibiot 48: 36–41
(1995)
of 14C-labelled precursors into macromolecules in HL-60
5. Becker U, Anke T. A novel halogenated compound
showed a preferential inhibition of RNA and protein
possessing antibiotic and cytotoxic activities isolated from
biosynthesis starting from 5
10 m g/ml (Fig. 2). the fungus Resinicium pinicola (J. Erikss.) Erikss. & Hjortst.
Favolon B (1) differs from favolon (2) due to the Z. Naturforschung 49c: 772–774 (1994)
presence of a double bond in the B ring, which replaces the 6. Lorenzen K, Anke T. Basidiomycetes as a source for new
original favolon’s epoxide-group. The structure of favolon bioactive natural products. Current Organic Chemistry 2:
B is confirmed by the presence of the proton C-6 in the 329–364, 1998
1
H NMR as a singlet at 5.65. 7. Turner W, Aldridge D. In Fungal Metabolites. Academic
This difference apparently confers a selective and Press London, New York, Paris, 1983