The synthesis of analogues and derivatives of two o-hydroxyphenylacetylenes, eutypine and sterehirsutinal,
the main phytotoxins isolated from the culture medium of Eutypa lata and Stereum hirsutum, is reported. Two
means of synthesis are described, based on cyclisation, oxidation, oxidative decarboxylation or reduction reactions,
and producing o-hydroxyphenylacetylene or benzofuran derivatives. Some of these synthetic compounds were tested
on grapevine callus in order to compare their toxicity with the natural analogues

Abou-Mansour, E.

Perrin-Cherioux, S.

Tabacchi, R.

Firenze University Press: E-Journals

83
Phytopathol. Mediterr. (2004) 43, 83–86
Introduction
Eutypiose and esca are two serious diseases of
grape wood. They are responsible for considerable
losses in yield and are one of the main causes of a
shortened productive life of vineyards (Moller et
al., 1981; Munkwold et al., 1994).
As an alternative to control with sodium arsen-
ite, a highly efficient, but very toxic agent that was
recently banned, it was decided to look for patho-
genically active secondary fungal metabolites to
combat this disease.
The ascomycete Eutypa lata is the causal agent
of eutypiose (also known as “dead arm”) and it also
occurs in esca-infected wood. From the culture
medium of this fungus, the main phytotoxic com-
pound, eutypine, was isolated in our laboratory
(Renaud et al., 1989) .
Stereum hirsutum is a basidiomycete involved
Synthesis and activity of grape wood phytotoxins
and related compounds
SANDRINE PERRIN-CHERIOUX, ELIANE ABOU-MANSOUR and RAFFAELE TABACCHI
Institut de Chimie, Université de Neuchâtel, Av. de Bellevaux, 51, CH-2007 Neuchâtel, Switzerland
Summary. The synthesis of analogues and derivatives of two o-hydroxyphenylacetylenes, eutypine and sterehirsutinal,
the main phytotoxins isolated from the culture medium of Eutypa lata and Stereum hirsutum, is reported. Two
means of synthesis are described, based on cyclisation, oxidation, oxidative decarboxylation or reduction reactions,
and producing o-hydroxyphenylacetylene or benzofuran derivatives. Some of these synthetic compounds were tested
on grapevine callus in order to compare their toxicity with the natural analogues.
Key words: synthesis, o-hydroxyphenylacetylene, benzofuran, calli.
Corresponding author: R. Tabacchi
Fax: +41 32 7182511
E-mail: raphael.tabacchi@unine.ch
in the last stage of esca (Larignon et al., 1997). The
chemical structure of sterehirsutinal, the main tox-
in isolated from it, is very close to eutypine (Fig. 1;
Dubin et al., 2000). In addition to an aldehyde sub-
stituent that is probably responsible for its biologi-
cal activity, sterehirsutinal contains two vinylacety-
lenic chains and two o-substituted hydroxy groups,
which facilitate cylisation to a benzofuranic form.
Several biogenetically related analogues of
these two toxins were also isolated from the cul-
ture media, but not in sufficient amount to carry
out a large array of biological activity tests. The
synthesis of these compounds is nevertheless im-
portant in order to establish the toxicity of the dif-
ferent secondary metabolites, and to investigate
the detoxification process by specific enzymes or
micro-organisms (Christen et al., 2003).
Results and discussion
The isolated analogues were essentially o-hy-
droxyphenylacetylene and benzofuran-substituted
compounds. All can be considered derivatives of eu-
84 Phytopathologia Mediterranea
S. Perrin-Cherioux et al.
typine. Indeed the occurrence of the hydroxy group
in the ortho position on the acetylenic chain leads
easily to a benzofuran derivative by cyclisation.
We followed two ways to synthesise all the com-
pounds. The structures were confirmed by spec-
troscopic analysis and compared with the natural
analogues. The cyclisation of 1 (Renaud et al., 1989,
Zesiger, 1991 ) was very easy. Treatment with Cu2O
(Fig.2) in pyridine  led to the cyclisized product.
Oxidation or reduction reactions of eutypine
(Defrancq et al, 1993) and of the benzofuran deriv-
ative 3 gave 4, 5, 6 (eutypine) and 7, 8 ,9 (benzo-
furan) with good yields.
Similar reactions are also possible starting from
sterehirsutinal (Fig. 3), but this is more difficult
because the synthesis of this toxin (Fkyerat et al,
1999) is tricky. The yield of the overall synthesis
was low so that and we had only a small amount of
material to work with. We noticed that treatment
with Cu2O in pyridine was needed for benzofuran
formation. 2, 11 and 12 led to a double cyclisation
due to the presence of two vinyl-acetylenic chains
in the ortho position of each hydroxy function.
Dibenzofuran 10 has been isolated and character-
ised; it was found to be less stable than 3 at room
temperature (Perrin-Cherioux et al., 2003)
To compare the phytotoxicity of the synthetic
derivatives with eutypine and sterehirsutinal we
CHO
OH
1 Eutypine
OH
OH
CHO
2 Sterehirsutinal
Fig. 1 Chemical structure of eutypine and sterehirsutinal.
CHO
OH
CHO
O
7 R = CH2OH
8 R = OH
9 R = CO2H
Cyclisation
R
O
R
OH
1 Eutypine
Cyclisation
3
Oxidation
or Reduction
Oxidation
or Reduction
4 R = CH2OH
5 R = OH
6 R = CO2H
Way A
Way B
Fig. 2. Synthesis of eutypine derivatives.
85Vol. 43, No. 1, April 2004
Synthesis and activity of grape wood phytotoxins
OH
OH
O
Cyclisation
Cyclisation
CHO
OH
OH
R
CHO
O
O
R
O
2 Sterehirsutinal
Oxidation 
or Reduction
Oxidation 
or Reduction
Way A
Way B
11 R=CH2OH
12 R=CO2H
13 R=CH2OH
14 R=CO2H
10
Fig. 3. Synthesis of sterehirsutinal derivatives.
0
10
20
30
40
50
60
70
80
90
100
C
a
ll
u
s
 w
e
ig
h
t 
re
d
u
c
ti
o
n
 (
%
)
Eutypine 1 Sterehirsutinal 2 Benzofuran 3 Eutypinol 4 Eutypinic ac id 5 Siccayne 6
Control 0.1 mM 0.25 mM 0.5 mM 1 mM
C
a
ll
u
s
 w
e
ig
h
t 
re
d
u
c
ti
o
n
 (
%
)
Fig. 4. Growth inhibition of Gamay callus by the synthetic eutypine derivatives compared with eutypine and stere-
hirsutinal.
86 Phytopathologia Mediterranea
S. Perrin-Cherioux et al.
carried out a simple biossay based on the growth
inhibition of Gamay callus. These bioassays (Fig.
4) showed that eutypinol (4), eutypinic acid (5) and
siccayne (6) were not phytotoxic to grape callus in
vitro, but that they seemed to be growth promoters
at concentrations lower than 0.1 mM. Eutypine (1)
sterehirsutinal (2) and the benzofuranic form of
eutypine (3) strongly inhibited growth. Similar re-
sults were observed with the derivative of sterehir-
sutinal (10-14) but quantification was not possible
because of the small amount of available material.
The presence of other metabolites in the cul-
ture medium of E. lata and S. hirsutum suggested
that eutypine and sterehirsutinal were probably
not the only agents of phytotoxicity. Mahoney et al
(2003), recently confirmed the phytoxoxicity of oth-
er metabolites from different strains of E. lata.  The
diseases caused may have resulted from several
compounds produced by the fungus, and a syner-
gistic effect was also possible.
In the infected vegetable material (branches,
leaves, inflorescences, necrotised wood) only eu-
typine and sterehirsutinal were found in our labo-
ratory and identified by tandem mass spectrome-
try (They-Ruhl, 1991; Dubin, 1998). In vivo eu-
typine can be reduced, in ‘Merlot’, by an NADPH-
dependent aldehyde reductase (ERE) to the non-
toxic eutypinol (Colrat et al., 1999a, b). Detoxifica-
tion by micro-organisms or specific enzymes is be-
ing investigated (Christen et al., 2003).
Conclusions
Two synthetic pathways were developed starting
from eutypine (1) and sterehirsutinal (2), by oxida-
tion, oxidative decarboxylation, reduction (reactions
frequently observed in biological systems) and cycli-
sation, yielding to potential bioactive derivatives.
Chemical cyclisation of all compounds was performed
because the benzofuran derivative might play a cru-
cial role in the resistance or detoxification process.
The toxicity of sterehirsutinal, eutypine and
its cyclisation products was confirmed on grape-
vine callus.
Acknowledgements
We thank the Swiss National Science Founda-
tion for financial support (project 20-61879.00).
Literature cited
Christen D., E. Abou-Mansour, M. Tharin, R. Tabacchi and
G. Défago, 2003. Microbial degradation of toxins as new
biocontrol mechanism. Case study: Eutypa dieback and
Esca disease of grapevines. In: Phytopathologia Medi-
terranea 43, 1, XXX (abstract).
Colrat S., C. Deswarte, A. Latché, A. Klaébè, M. Bouzayen,
J. Fallot and J.-P. Roustand, 1999. Enzymatic detoxifi-
cation of eutypine, a toxin from Eutypa lata, by Vitis
vinifera cells: partial purification of an NADPH-depend-
ent aldehyde reductase. Planta 544–550.
Colrat S., A. Latché, M. Guis, J.-C. Pech, M. Bouzayen, J.
Fallot and J.-P. Roustand, 1999. Purification and char-
acterization of a NADPH-dependent aldehyde reduct-
ase from Mung Bean that detoxifies eutipyne, a toxin
from Eutypa lata. Plant Physiology 119, 621–626.
Defrancq E., T. Zesiger and R. Tabacchi, 1993. The synthe-
sis of natural acetylenic compounds from Eutypa lata
(Pers.:Fr.) Tul. Helvetica Chimica Acta 76, 425–429.
Dubin G.-M, 1998. Isolement et Identification de Métabo-
lites Secondaires Phytotoxiques de Stereum hirsutum,
un des Champignons Impliqués dans une Maladie la
Vigne, l’Esca. PhD Thesis, University of Neuchâtel, Neu-
châtel, Switzerland.
Dubin G.-M., A. Fkyerat and R. Tabacchi, 2000. Acetylenic
aromatic compounds from Stereum hirsutum. Phyto-
chemistry 53, 571–574.
Fkyerat A., G.M. Dubin and R. Tabacchi, 1999. The syn-
thesis of natural acetylenic compounds from Stereum
hirsutum. Helvetica Chimica Acta 82, 1418–1422.
Larignon P. and B. Dubos, 1997. Fungi associated with esca
disease in grapevine. European Journal of Plant Pa-
thology 103, 147–157
Mahoney N., R. Lardner, R.J. Molyneux, E.S. Scott, L.R.
Smith and T.K. Scott, 2003. Phenolic and heterocyclic
metabolite profile of the grapevine pathogen Eutypa
lata. Phytochemistry 64, 475–484
Moller M.J. and N. Kasimatis, 1981. Further evidence that
Eutypa armeniacae – not Phomopsis viticola – incites dead
arm symptoms on grape. Plant Disease 65, 429–431.
Munkvold G.P., J.A. Duthie and J.J. Marois, 1994. Reduc-
tions in yield and vegetative growth of grapevines due
to Eutypa dieback. Phytopathologia 84, 186–192.
Perrin-Cherioux S., 2003, Synthesis of Sterehirsutinal de-
rivatives, Internal report FNRS, University of Neuchâ-
tel, Neuchâtel, Switzerland.
Renaud J.-M., G. Tsoupras and R. Tabacchi, 1989. Biologi-
cally active natural acetylenic compounds from Eutypa
lata (Pers.: Fr.) Tul. Helvetica Chimica Acta 72, 929–932.
They-Ruhl P., I. Philippe, J-M. Renaud, G. Tsoupras, P. De
Angelis, J. Fallot and R. Tabacchi, 1991. Eutypine, a phy-
totoxin produced by Eutyta lata, the casual agent of dy-
ing-arm disease of grapevine. Phytochemistry 30, 471–473
Zesiger T., 1991, Synthèse de métabolites phénylacétyléniques
et d’analogues benzofuraniques produits par Eutypa lata
(Pers.: Fr.), champignon parasite de la vigne. PhD Thesis,
University of Neuchâtel, Neuchâtel, Switzerland.
Accepted for publication: November 27, 2003


Synthesis and Activity of Grape Wood Phytotoxins and Related Compounds

S. Perrin-Cherioux

E. Abou-Mansour

R. Tabacchi

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83Phytopathol. Mediterr. (2004) 43, 83–86IntroductionEutypiose and esca are two serious diseases ofgrape wood. They are responsible for considerablelosses in yield and are one of the main causes of ashortened productive life of vineyards (Moller etal., 1981; Munkwold et al., 1994).As an alternative to control with sodium arsen-ite, a highly efficient, but very toxic agent that wasrecently banned, it was decided to look for patho-genically active secondary fungal metabolites tocombat this disease.The ascomycete Eutypa lata is the causal agentof eutypiose (also known as “dead arm”) and it alsooccurs in esca-infected wood. From the culturemedium of this fungus, the main phytotoxic com-pound, eutypine, was isolated in our laboratory(Renaud et al., 1989) .Stereum hirsutum is a basidiomycete involvedSynthesis and activity of grape wood phytotoxinsand related compoundsSANDRINE PERRIN-CHERIOUX, ELIANE ABOU-MANSOUR and RAFFAELE TABACCHIInstitut de Chimie, Université de Neuchâtel, Av. de Bellevaux, 51, CH-2007 Neuchâtel, SwitzerlandSummary. The synthesis of analogues and derivatives of two o-hydroxyphenylacetylenes, eutypine and sterehirsutinal,the main phytotoxins isolated from the culture medium of Eutypa lata and Stereum hirsutum, is reported. Twomeans of synthesis are described, based on cyclisation, oxidation, oxidative decarboxylation or reduction reactions,and producing o-hydroxyphenylacetylene or benzofuran derivatives. Some of these synthetic compounds were testedon grapevine callus in order to compare their toxicity with the natural analogues.Key words: synthesis, o-hydroxyphenylacetylene, benzofuran, calli.Corresponding author: R. TabacchiFax: +41 32 7182511E-mail: raphael.tabacchi@unine.chin the last stage of esca (Larignon et al., 1997). Thechemical structure of sterehirsutinal, the main tox-in isolated from it, is very close to eutypine (Fig. 1;Dubin et al., 2000). In addition to an aldehyde sub-stituent that is probably responsible for its biologi-cal activity, sterehirsutinal contains two vinylacety-lenic chains and two o-substituted hydroxy groups,which facilitate cylisation to a benzofuranic form.Several biogenetically related analogues ofthese two toxins were also isolated from the cul-ture media, but not in sufficient amount to carryout a large array of biological activity tests. Thesynthesis of these compounds is nevertheless im-portant in order to establish the toxicity of the dif-ferent secondary metabolites, and to investigatethe detoxification process by specific enzymes ormicro-organisms (Christen et al., 2003).Results and discussionThe isolated analogues were essentially o-hy-droxyphenylacetylene and benzofuran-substitutedcompounds. All can be considered derivatives of eu-84 Phytopathologia MediterraneaS. Perrin-Cherioux et al.typine. Indeed the occurrence of the hydroxy groupin the ortho position on the acetylenic chain leadseasily to a benzofuran derivative by cyclisation.We followed two ways to synthesise all the com-pounds. The structures were confirmed by spec-troscopic analysis and compared with the naturalanalogues. The cyclisation of 1 (Renaud et al., 1989,Zesiger, 1991 ) was very easy. Treatment with Cu2O(Fig.2) in pyridine  led to the cyclisized product.Oxidation or reduction reactions of eutypine(Defrancq et al, 1993) and of the benzofuran deriv-ative 3 gave 4, 5, 6 (eutypine) and 7, 8 ,9 (benzo-furan) with good yields.Similar reactions are also possible starting fromsterehirsutinal (Fig. 3), but this is more difficultbecause the synthesis of this toxin (Fkyerat et al,1999) is tricky. The yield of the overall synthesiswas low so that and we had only a small amount ofmaterial to work with. We noticed that treatmentwith Cu2O in pyridine was needed for benzofuranformation. 2, 11 and 12 led to a double cyclisationdue to the presence of two vinyl-acetylenic chainsin the ortho position of each hydroxy function.Dibenzofuran 10 has been isolated and character-ised; it was found to be less stable than 3 at roomtemperature (Perrin-Cherioux et al., 2003)To compare the phytotoxicity of the syntheticderivatives with eutypine and sterehirsutinal weCHOOH1 EutypineOHOHCHO2 SterehirsutinalFig. 1 Chemical structure of eutypine and sterehirsutinal.CHOOHCHOO7 R = CH2OH8 R = OH9 R = CO2HCyclisationROROH1 EutypineCyclisation3Oxidationor ReductionOxidationor Reduction4 R = CH2OH5 R = OH6 R = CO2HWay AWay BFig. 2. Synthesis of eutypine derivatives.85Vol. 43, No. 1, April 2004Synthesis and activity of grape wood phytotoxinsOHOHOCyclisationCyclisationCHOOHOHRCHOOORO2 SterehirsutinalOxidation or ReductionOxidation or ReductionWay AWay B11 R=CH2OH12 R=CO2H13 R=CH2OH14 R=CO2H10Fig. 3. Synthesis of sterehirsutinal derivatives.0102030405060708090100Callus weight reduction (%)Eutypine 1 Sterehirsutinal 2 Benzofuran 3 Eutypinol 4 Eutypinic ac id 5 Siccayne 6Control 0.1 mM 0.25 mM 0.5 mM 1 mMCallus weight reduction (%)Fig. 4. Growth inhibition of Gamay callus by the synthetic eutypine derivatives compared with eutypine and stere-hirsutinal.86 Phytopathologia MediterraneaS. Perrin-Cherioux et al.carried out a simple biossay based on the growthinhibition of Gamay callus. These bioassays (Fig.4) showed that eutypinol (4), eutypinic acid (5) andsiccayne (6) were not phytotoxic to grape callus invitro, but that they seemed to be growth promotersat concentrations lower than 0.1 mM. Eutypine (1)sterehirsutinal (2) and the benzofuranic form ofeutypine (3) strongly inhibited growth. Similar re-sults were observed with the derivative of sterehir-sutinal (10-14) but quantification was not possiblebecause of the small amount of available material.The presence of other metabolites in the cul-ture medium of E. lata and S. hirsutum suggestedthat eutypine and sterehirsutinal were probablynot the only agents of phytotoxicity. Mahoney et al(2003), recently confirmed the phytoxoxicity of oth-er metabolites from different strains of E. lata.  Thediseases caused may have resulted from severalcompounds produced by the fungus, and a syner-gistic effect was also possible.In the infected vegetable material (branches,leaves, inflorescences, necrotised wood) only eu-typine and sterehirsutinal were found in our labo-ratory and identified by tandem mass spectrome-try (They-Ruhl, 1991; Dubin, 1998). In vivo eu-typine can be reduced, in ‘Merlot’, by an NADPH-dependent aldehyde reductase (ERE) to the non-toxic eutypinol (Colrat et al., 1999a, b). Detoxifica-tion by micro-organisms or specific enzymes is be-ing investigated (Christen et al., 2003).ConclusionsTwo synthetic pathways were developed startingfrom eutypine (1) and sterehirsutinal (2), by oxida-tion, oxidative decarboxylation, reduction (reactionsfrequently observed in biological systems) and cycli-sation, yielding to potential bioactive derivatives.Chemical cyclisation of all compounds was performedbecause the benzofuran derivative might play a cru-cial role in the resistance or detoxification process.The toxicity of sterehirsutinal, eutypine andits cyclisation products was confirmed on grape-vine callus.AcknowledgementsWe thank the Swiss National Science Founda-tion for financial support (project 20-61879.00).Literature citedChristen D., E. Abou-Mansour, M. Tharin, R. Tabacchi andG. Défago, 2003. Microbial degradation of toxins as newbiocontrol mechanism. Case study: Eutypa dieback andEsca disease of grapevines. In: Phytopathologia Medi-terranea 43, 1, XXX (abstract).Colrat S., C. Deswarte, A. Latché, A. Klaébè, M. Bouzayen,J. Fallot and J.-P. Roustand, 1999. Enzymatic detoxifi-cation of eutypine, a toxin from Eutypa lata, by Vitisvinifera cells: partial purification of an NADPH-depend-ent aldehyde reductase. Planta 544–550.Colrat S., A. Latché, M. Guis, J.-C. Pech, M. Bouzayen, J.Fallot and J.-P. Roustand, 1999. Purification and char-acterization of a NADPH-dependent aldehyde reduct-ase from Mung Bean that detoxifies eutipyne, a toxinfrom Eutypa lata. Plant Physiology 119, 621–626.Defrancq E., T. Zesiger and R. Tabacchi, 1993. The synthe-sis of natural acetylenic compounds from Eutypa lata(Pers.:Fr.) Tul. Helvetica Chimica Acta 76, 425–429.Dubin G.-M, 1998. Isolement et Identification de Métabo-lites Secondaires Phytotoxiques de Stereum hirsutum,un des Champignons Impliqués dans une Maladie laVigne, l’Esca. PhD Thesis, University of Neuchâtel, Neu-châtel, Switzerland.Dubin G.-M., A. Fkyerat and R. Tabacchi, 2000. Acetylenicaromatic compounds from Stereum hirsutum. Phyto-chemistry 53, 571–574.Fkyerat A., G.M. Dubin and R. Tabacchi, 1999. The syn-thesis of natural acetylenic compounds from Stereumhirsutum. Helvetica Chimica Acta 82, 1418–1422.Larignon P. and B. Dubos, 1997. Fungi associated with escadisease in grapevine. European Journal of Plant Pa-thology 103, 147–157Mahoney N., R. Lardner, R.J. Molyneux, E.S. Scott, L.R.Smith and T.K. Scott, 2003. Phenolic and heterocyclicmetabolite profile of the grapevine pathogen Eutypalata. Phytochemistry 64, 475–484Moller M.J. and N. Kasimatis, 1981. Further evidence thatEutypa armeniacae – not Phomopsis viticola – incites deadarm symptoms on grape. Plant Disease 65, 429–431.Munkvold G.P., J.A. Duthie and J.J. Marois, 1994. Reduc-tions in yield and vegetative growth of grapevines dueto Eutypa dieback. Phytopathologia 84, 186–192.Perrin-Cherioux S., 2003, Synthesis of Sterehirsutinal de-rivatives, Internal report FNRS, University of Neuchâ-tel, Neuchâtel, Switzerland.Renaud J.-M., G. Tsoupras and R. Tabacchi, 1989. Biologi-cally active natural acetylenic compounds from Eutypalata (Pers.: Fr.) Tul. Helvetica Chimica Acta 72, 929–932.They-Ruhl P., I. Philippe, J-M. Renaud, G. Tsoupras, P. DeAngelis, J. Fallot and R. Tabacchi, 1991. Eutypine, a phy-totoxin produced by Eutyta lata, the casual agent of dy-ing-arm disease of grapevine. Phytochemistry 30, 471–473Zesiger T., 1991, Synthèse de métabolites phénylacétyléniqueset d’analogues benzofuraniques produits par Eutypa lata(Pers.: Fr.), champignon parasite de la vigne. PhD Thesis,University of Neuchâtel, Neuchâtel, Switzerland.Accepted for publication: November 27, 2003

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