Total synthesis of bioactive frustulosin and frustulosinol

Article abstract of DOI:10.1016/j.tetlet.2005.11.150

Stereum hirsutum is a pathogenic fungus involved in 'Esca', a trunk wood disease of grapevine. Among the metabolites isolated from the culture medium of this fungus, frustulosin (2a) shows a high phytotoxicity. A new simple and efficient method for the sy

Full text of DOI:10.1016/j.tetlet.2005.11.150

Tetrahedron
Letters
Tetrahedron Letters 47 (2006) 909–911
Total synthesis of bioactive frustulosin and frustulosinol
*
`
Mary-Lorene Goddard and Raffaele Tabacchi
Institut de Chimie de l’Universite´ de Neuchatel, Avenue de Bellevaux 51, CH-2000 Neuchatel, Switzerland
Received 16 October 2005; revised 27 November 2005; accepted 30 November 2005
Available online 20 December 2005
Abstract—Stereum hirsutum is a pathogenic fungus involved in ÔEscaÕ, a trunk wood disease of grapevine. Among the metabolites
isolated from the culture medium of this fungus, frustulosin (2a) shows a high phytotoxicity. A new simple and efficient method for
the synthesis of frustulosin (2a) and frustulosinol (2b) was developed. Antibacterial activity and phytotoxicity are reported.
Ó 2005 Elsevier Ltd. All rights reserved.
Different fungi infect grapevines and cause severe dis-
eases leading to important economical losses. One of
the oldest known is Esca.1 This disease, progressing
worldwide, is associated with a group of fungi that
includes Stereum hirsutum.2 To find an alternative con-
trol method against Esca, as efficient as the highly toxic
sodium arsenite treatment, now forbidden, the search
for the pathogenically active secondary metabolites
appears to be necessary. S. hirsutum seems to play an
important role in the wood deterioration process.2 In a
previous work,3 we described the isolation and the iden-
tification of seven secondary metabolites from the culture
medium, and the synthesis of two phytotoxic acetylenic
compounds, sterehirsutinal (1a) and sterehirsutinol (1b)
(Scheme 1).4 Frustulosin (2a) and frustulosinol (2b) were
also isolated from the same toxic medium. These com-
pounds were previously described as metabolites of Ster-
eum frustulosum and exhibited antimicrobial activity at
modest concentrations, particularly against Staphylococ-
cus aureus, several Bacilli and Vibrio cholera.5
specific biological assays. We thus undertook the synthe-
sis of these two compounds 2a and 2b. Two different
pathways were already described for the synthesis of fru-
stulosin (2a).6,7 Herein, we report a simpler and more
efficient procedure.
Ronald and co-workers described the preparation of 2-
iodo-3,6-dihydroxybenzaldehyde in five steps as inter-
mediate in the frustulosin (2a) synthesis.7 Then, the
key step was the coupling reaction between this iodo
derivative and appropriate alkyne to lead to frustulosin
(2a). Thus, we decided to use bromo analogue 4 as key
synthon, obtained in one step from commercially avail-
able 2,5-dihydroxybenzaldehyde (3) (Scheme 2).8 This
OH
OH
OH
OH
CHO
CHO
CHO
Br
a
b
97%
97%
Br
OH
OTBDMS
3
4
5
In order to understand the role played by compounds 2a
and 2b, large amount of material is required for more
d
75%
55%
c
OMOM
CHO
OH
HO
R
HO
R
CHO
Br
OMOM
OH
OH
O
1a : Sterehirsutinal (R = CHO)
1b : Sterehirsutinol (R = CH2OH)
2a : Frustulosin (R= CHO)
2b : Frustulosinol (R = CH2OH)
7
6
Scheme 2. Reagents and conditions: (a) Br2, CHCl3, rt, 3 h; (b)
TBDMSCl, imidazole, CH2Cl2, rt, 15 min; (c) 2-methylbut-1-en-3-yne,
CuI, [PdCl2(PPh3)2], Et3N, DMF, 110 °C, 3 h; (d) MOMCl, iPr2EtN,
CH2Cl2, rt, 1 h.
Scheme 1. Phytotoxins extracted from Stereum hirsutum.
*
Corresponding author. E-mail: raphael.tabacchi@unine.ch
0040-4039/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.11.150

910
M.-L. Goddard, R. Tabacchi / Tetrahedron Letters 47 (2006) 909–911
bromination reaction afforded exclusively 2-bromo-3,6-
OH
OH
OH
dihydroxybenzaldehyde (4) in 97% yield.
CHO
CHO
Br
CHO
Br
b
a
79%
80%
Ronald and co-workers achieved the substitution of the
iodide by the alkenynyl chain thanks to copper(I) iso-
propenylacetylide in DMF. A low conversion was
observed and a large amount of starting material was
recovered. A mixture of frustulosin (2a) (less than 10%
yield) and benzofuran derivative 7 was obtained. Using
the same procedure, we attempted this transformation
with the same poor results. We investigated the cross-
coupling reaction between 2-methylbut-1-en-3-yne and
the aryl bromide 4 by the Sonogashira reaction9 in the
presence of cuprous iodide, bis(triphenylphosphine)-
palladium dichloride and triethylamine in DMF.
Unfortunately, the results were similar to the cuprate
pathway and we obtained mainly the benzofuran deriv-
ative 7.
OMOM
OH
OMOM
8
9
4
OH
OH
CHO
c
d
OH
60%
62%
OH
OH
2a
2b
Scheme 3. Reagents and conditions: (a) MOMCl, iPr2EtN, CH2Cl2, rt,
1 h; (b) 2-methylbut-1-en-3-yne, CuI, [PdCl2(PPh3)2], Et3N, DMF,
60 °C, 3 h; (c) 10:90 TFA/CH2Cl2 (v/v), rt, 5 h; (d) NaBH4, 1% (w/v)
aq NaOH soln, rt, 2 h.
In order to avoid the formation of 7, the hydroxyl group
in meta position of the aldehyde had to be protected.
TBDMS protecting group was chosen and introduced
according to the Hu procedure.8 This method allowed a
regioselective silylation of the most reactive hydroxyl
group of 4, the hydroxyl group in ortho position of bro-
mide. Indeed, the other hydroxyl group is less acidic be-
cause of an intramolecular H-bond with aldehyde
function that stabilizes the hydroxyl group. Thus, com-
pound 5 was obtained in 97% yield (Scheme 2).
different concentrations. The same test was realized
simultaneously with eutypine as reference, another fun-
gal toxin of grapevine, previously isolated in our labora-
tory that shows a high phytotoxic activity.15
Necroses already appeared at different levels on leaves
before 18 h at 25 °C the concentrations of 500, 250,
100 and 50 mg/L for frustulosin (2a). At the concentra-
tion of 25 mg/L, necroses were observed only between
18 and 24 h. The appearance of necroses caused by euty-
pine was less important and no phytotoxicity was ob-
served at the concentration of 25 mg/L. Frustulosinol
(2b) was less toxic, necroses were only observed at 250
and 500 mg/L.
At this step, the 2-methylbut-1-en-3-ynyle chain had to
be introduced into compound 5. No reaction occurred
when the Sonogashira cross-coupling was realized at
70 °C. But beyond this temperature, the silyl ether was
cleaved and the benzofuran derivative 7 formed as the
major product (Scheme 2). Tentatively, the reaction
using the cuprate6 was also tested but unsuccessfully.
Compounds were also tested on grapevine callus (kindly
supplied by Roustan, INRA, Toulouse, France). Callus
cultures of Vitis vinifera cv. Gamay were grown on a
synthetic medium, 12 h daylight for 28 days at 28 °C.
Calli were weighed before and after incubation with dif-
ferent concentrations of 2a, 2b and eutypine and percent
growth was calculated. Frustulosin (2a) and frustulosi-
nol (2b) inhibit 100% callus growth at 20 mg/L and
50 mg/L, respectively.
The MOM group was used to protect hydroxyl groups
as in the syntheses of siccayne,10 eutypine,11 and stere-
hirsutinal,4 that include a Sonogashira reaction for
the acetylenic side chain introduction. Thus, we had
obtained the protected compound 6 in 75% yield
(Scheme 2). No reaction occurred in Sonogashira condi-
tions between 6 and acetylenic derivative.
In addition, antibacterial tests were realized with frustul-
osin (2a). Activities were measured on TLC by bioau-
tography on thin-layer plates. Test solutions (10 lL)
were applied as small spots on TLC plates (Silicagel
G) to give a concentration series of 0.1–30 lg/applica-
tion zone. The organic solvent was evaporated by steam
air and plates were eluted by the adequate solvent to ob-
tain different Rfs. The solvent was evaporated once
again and the plates homogeneously sprayed with
10 mL of nutrient agar infected with 1 ml of the nutrient
broth containing bacteria (108–109 CFU/ml). Plates
were incubated for 2 days at 30 and 37 °C in the dark.
The appearance of a blank after spraying the plate with
a solution of thiazolyl blue tetrazolium bromide, indi-
cated antibacterial activity. Minimum inhibitory con-
centrations (MIC) against Bacillus subtilis and
Escherichia coli were determined from the lowest tested
compound concentration causing recognizable bacterial
growth inhibition. Chloramphenicol was used as posi-
The regioselective protection of the most nucleophilic
hydroxy group of 4 was achieved with 1.05 equiv of
MOMCl affording 8 in 80% yield (Scheme 3). The Sono-
gashira reaction between bromide 8 and 2-methylbut-1-
en-3-yne led to the protected frustulosin 9 in 79%
yield.12 In this case, the presence of MOM group in meta
position of the bromine atom is deactivating for the
Sonogashira reaction. Finally, frustulosin (2a) was ob-
tained after MOM cleavage using a solution of 10%
TFA in CH2Cl2 (v/v) in 60% yield.13 Biologically inter-
esting frustulosinol (2b) was obtained in 62% yield by
chemioselective reduction of frustulosin (2a) with
NaBH4 in aqueous medium.14
Preliminary phytotoxic tests were carried out on grape-
vine leaf discs. Solutions of frustulosin (2a) and frustu-
losinol (2b) in 98:2 H2O/ethanol were prepared in

M.-L. Goddard, R. Tabacchi / Tetrahedron Letters 47 (2006) 909–911
911
4.53 mmol, 25 mol %), Et3N (10.1 mL, 72.5 mmol, 4.0
equiv) and 2-methylbut-1-en-3-yne (6.9 mL, 72.5 mmol,
4.0 equiv) in 72 mL of DMF was heated at 60 °C under N2
atmosphere for 3 h. The mixture was then diluted with
Et2O, washed twice with 5% aq HCl soln and satd aq
NaCl soln, dried over MgSO4, and concentrated. Purifi-
cation by flash chromatography on silica gel (eluant: n-
hexane/acetone 97:3) provided 3.52 g (79%) of 9 as an
orange oil: IR (film) 2924, 1652, 1466, 1272, 1154, 1016;
1H NMR d (CDCl3, 400 MHz): 11.40 (s, 1H, C6-OH),
10.42 (s, 1H, CHO), 7.33 (d, J = 9.1 Hz, 1H, C4-H), 6.89
tive control and acetone as negative. Antibacterial activ-
ity for frustulosin (2a) was observed at 3 and 10 lg,
respectively, against B. subtilis and E. coli.
In conclusion, this new synthetic route supplies rapidly a
large amount of frustulosin (2a) with a total yield of
37% in only four steps. Frustulosinol (2b) is also ob-
tained in good yield. This procedure can be applied to
the synthesis of other phytotoxins belonging to the same
family, that is, sterehirsutinal, isolated from S. hirsutum.
Biological activities of synthesized compounds have
been checked. Phytotoxicity has been exhibited on
grapevine and the antibacterial activity has been shown
against B. subtilis and E. coli. The efficiency of the method
allows the large scale synthesis of such compounds in
order to investigate more advanced biological tests.
(d, J = 9.1 Hz, 1H, C5-H), 5.48 (qtapp
,
2J = 4J = 1.0 Hz,
1H, CH2@), 5.40 (qtapp
,
2J = 4J = 1.6 Hz, 1H, CH2@),
5.19 (s, 2H, OCH2O), 3.56 (s, 3H, OCH3), 2.03 (dd,
4
4Jciso¨ıde = 1.1 Hz, Jtranso¨ıde = 1.4 Hz, 3H, CH3); 13C
NMR d (CDCl3, 100 MHz): 197.2, 158.2, 151.6, 127.1,
126.7, 123.8, 119.5, 119.0, 117.6, 102.3, 96.8, 80.1, 56.8,
23.5; MS (ESIÀ) m/z = 245.1 [MÀH]À.
13. Deprotection of compound 9: 2,5-Dihydroxy-6-(3-meth-
ylbut-3-en-1-ynyl)benzaldehyde (frustulosin) 2a: To
a
solution of 9 (1.85 g, 7.51 mmol, 1.0 equiv) in 67.5 mL of
CH2Cl2 was added 7.5 mL of TFA at 0 °C. The reaction
mixture was stirred for 6 h and then TFA was removed in
vacuo. Purification by flash chromatography on silica gel
(eluant: n-hexane/EtOAc 95:5) provided 915 mg (60%) of
frustulosin 2a as a yellow solid: IR (KBr disc) 3281, 2923,
1641, 1464, 1280, 1149; 1H NMR d (CDCl3, 400 MHz):
Acknowledgements
We thank Dr. Eliane Abou-Mansour and Sabine Unter-
naehrer for the biological tests and The Swiss National
Science Foundation for their financial support (project
no. 20-67972-02).
5
11.25 (s, 1H, C6–OH), 10.33 (d, J = 0.5 Hz, 1H, CHO),
7.19 (d, J = 9.1 Hz, 1H, C4–H), 6.92 (dd, 3J = 9.1 Hz,
5J = 0.5 Hz, 1H, C5–H), 5.58 (s, 1H, C3–OH), 5.54 (qtapp
,
References and notes
2J = 4J = 1.0 Hz, 1H, CH2@), 5.40 (qtapp, 2J = 4J = 1.6 Hz,
4
4
1H, CH2@), 2.06 (dd, Jciso¨ıde = 1.1 Hz, Jtranso¨ıde
=
1. Chiarappa, L. Phytopathology 1959, 49, 510.
2. Larignon, P.; Dubos, B. Eur. J. Plant Pathol. 1997, 103,
147–157.
3. Dubin, G.-M.; Fkyerat, A.; Tabacchi, R. Phytochemistry
2000, 53, 571–574.
4. Fkyerat, A.; Dubin, G.-M.; Tabacchi, R. Helv. Chim. Acta
1999, 82, 1419–1422.
5. (a) Nair, M. S. R.; Anchel, M. Tetrahedron Lett. 1975, 16,
2641–2642; (b) Nair, M. S. R.; Anchel, M. Phytochemistry
1977, 16, 390–392.
6. Orr, A. F. J. Chem. Soc., Chem. Commun. 1979, 40–41.
7. Ronald, R. C.; Lansinger, J. M.; Lillie, T. S.; Wheeler, C.
J. J. Org. Chem. 1982, 47, 2541–2549.
8. Hu, Y.; Li, C.; Kulkarni, B. A.; Strobel, G.; Lobkovsky,
E.; Torczynski, R. M.; Porco, J. A. Org. Lett. 2001, 3,
1649–1652.
9. Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron
Lett. 1975, 16, 4467–4470.
10. Pinault, M.; Frangin, Y.; Genet, J.-P.; Zamarlik, H.
Synthesis 1990, 10, 935–937.
1.5 Hz, 3H, CH3); d (CDCl3, 100 MHz): 196.3, 157.1,
150.6, 125.9, 124.9, 124.8, 120.0, 118.3, 110.7, 104.4, 78.3,
23.6; MS (ESIÀ) m/z = 201.5 [MÀH]À.
14. Reduction of frustulosin (2a): 2-Hydroxymethyl-3-(3-
methylbut-3-en-1-ynyl)benzene-1,4-diol (frustulosinol) 2b:
To 2a (85 mg, 0.42 mmol, 1.0 equiv) in 1% aq NaOH soln
(10 mL) was added dropwise 10% aq NaOH soln (1 mL)
of NaBH4 (21 mg, 0.55 mmol, 1.3 equiv). After 2 h, the
reaction mixture was diluted with EtOAc and acidified
with 5% aq HCl soln. The organic layer was then washed
with aq NaHCO3 soln, dried over MgSO4 and concen-
trated in vacuo. The residue was purified by flash
chromatography on silica gel (eluant: n-hexane/EtOAc
80:20) to give frustulosinol 2 (86 mg, 62%) as a yellow
solid: IR (KBr disc) 3371, 2937, 1473, 1237, 1169; 1H
NMR d (CDCl3, 400 MHz): 7.61 (s, 1H, OH), 6.78 (s, 2H,
C5–H, C6–H), 5.52 (s, 1H, OH), 5.44 (qtapp,
2J = 4J =
0.8 Hz, 1H, CH2@), 5.38 (qtapp
,
2J = 4J = 1.5 Hz, 1H,
4
CH2@), 2.94 (s, 1H, OH), 2.02 (dd, Jciso¨ıde = 0.8 Hz,
4Jtranso¨ıde=1.4 Hz, 3H, CH3); 13C NMR
d (CDCl3,
11. Defranq, E.; Zesiger, T.; Tabacchi, R. Helv. Chim. Acta
1993, 76, 425–430.
12. Sonogashira reaction procedure: 2-Hydroxy-5-(meth-
oxymethyloxy)-6-(3-methylbut-3-en-1-ynyl)benzaldehyde
9. A solution of 8 (4.73 g, 18.1 mmol, 1.0 equiv), CuI
(1.72 g, 9.06 mmol, 0.5 equiv), [PdCl2(PPh3)2] (3.18 g,
100 MHz): 150.2, 149.5, 125.9, 124.8, 123.2, 118.6, 114.8,
107.8, 101.7, 79.9, 62.6, 23.4; MS (ESIÀ) m/z = 203.6
[MÀH]À.
15. Renaud, J.-M.; Tsoupras, G.; Tabacchi, R. Helv. Chim.
Acta 1989, 72, 929–932.