Fungicidal activity of β-thujaplicin analogues

Article abstract of DOI:10.1002/ps.379

The fungicidal activity of analogues of β-thujaplicin, a natural product responsible for the durability of heartwood of several cupressaceous trees, was investigated in vitro on the growth of different white and brown rot fungi involved in wood biodegradation, Coriolus versicolor, Phanerochaete chrysosporium, Poria placenta and Gloephyllum trabeum. The study shows that 2-hydroxycyclohepta-2,4,6-trienone (tropolone), easily prepared according to a literature procedure, possesses interesting fungicidal activity when compared to β-thujaplicin, azaconazole, tebuconazole and copper oxine, which suggests this compound should be examined further as a potential biocide for wood preservation.

Full text of DOI:10.1002/ps.379

Pest Management Science
Pest Manag Sci 57:833±838 0online: 2001)
DOI: 10.1002/ps.379
Fungicidal activity of b-thujaplicin analogues
1
1
2
1
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Mounir Baya, Patrice Soulounganga, Eric Gelhaye and Philippe Gerardin *
1
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Laboratoire d’Etudes et de Recherches sur le Materiau Bois, UMR INRA 1093, Equipe de Chimie Organique et Microbiologie, Universite
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Henri Poincare, Nancy I, Faculte des Sciences, BP 239, 54506 Vandoeuvre-les-Nancy, France
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UMR INRA 1136, Equipe Interactions Arbres Micro-organismes, Universite Henri Poincare, Nancy I, Faculte des Sciences, BP 239,
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54506 Vandoeuvre-les-Nancy, France
Abstract: The fungicidal activity of analogues of b-thujaplicin, a natural product responsible for the
durability of heartwood of several cupressaceous trees, was investigated in vitro on the growth of
different white and brown rot fungi involved in wood biodegradation, Coriolus versicolor,
Phanerochaete chrysosporium, Poria placenta and Gloephyllum trabeum. The study shows that 2-
hydroxycyclohepta-2,4,6-trienone tropolone), easily prepared according to a literature procedure,
possesses interesting fungicidal activity when compared to b-thujaplicin, azaconazole, tebuconazole
and copper oxine, which suggests this compound should be examined further as a potential biocide for
wood preservation.
# 2001 Society of Chemical Industry
Keywords: b-thujaplicin; tropolone; fungicide; wood preservation
1
INTRODUCTION
b-Thujaplicin is a naturally occuring compound
responsible for the resistance to fungal decay and
insect attack on heartwood of several cupressaceous
trees such as Thuja plicata Donn, Thujopsis dolobrata
Sieb and Zucc and Thuja standishii Carr.1±3 In addition
to these insecticidal and fungicidal activities, b-
thujaplicin also shows antibacterial4 and plant-growth
regulating activity,5 cytotoxic effects on mammalian
cells,6 scavenging activity against active oxygen spe-
cies7 and inhibitory activity against various
enzymes.8±11 Several studies have described the
extraction of b-thujaplicin, also known as hinokitiol
in Japan, from different cupressaceous plants, starting
either from wood1,2 or cell culture.12 However such
methods are dependent on the yields of extraction, so
that chemically synthetized b-thujaplicin is now used
predominantly.A variety of synthetic approaches have
been reported in the literature.13 The cycloaddition of
1-isopropylcyclopentadiene or cyclopentadiene with
dichloroketene is one of the most convenient methods
due to its simplicity; only two steps are needed, and the
availability of the starting materials allows large-scale
preparation of product 0Fig 1).14,15
Figure 1. Cycloaddition process.
enone 02b), 2-benzyloxycyclohepta-2,4,6-trienone
02c) and cyclohepta-2,4,6-trienone 02d) 0Fig 2).
In order to evaluate the potential of our compounds,
we have compared them in our biological trials with
tebuconazole, azaconazole and oxine copper, three
commercially available fungicides in current use as
wood preservatives 0Fig 3).
2
MATERIALS AND METHODS
2.1 Chemicals
Commercially available products including tropone
02d) and b-thujaplicin 01) were purchased from
Aldrich or Lancaster.The triazoles and oxine copper
were generously furnished by Xylochimie 0Albi,
However, while several studies have been reported
on the potential fungicidal activity of naturally occur-
ing b-thujaplicin,12,16 to our knowledge, no studies
have described the use of synthetic tropolone deriva-
tives as wood-preservative products.
This paper deals with structure/activity relationships
of the b-thujaplicin analogues 2-hydroxycyclohepta-
2,4,6-trienone 02a), 2-methoxycyclohepta-2,4,6-tri-
Figure 2. b-Thujaplicin and its analogues.
´
´
* Correspondence to: Philippe Gerardin, Laboratoire d’Etudes et de Recherches sur le Materiau Bois, UMR INRA 1093, Equipe de Chimie
´
´
´
`
Organique et Microbiologie, Universite Henri Poincare, Nancy I, Faculte des Sciences, BP 239, 54506 Vandoeuvre-les-Nancy, France
E-mail: Philippe.Gerardin@lermab.uhp-nancy.fr
Contract/grant sponsor: Agence Nationale de Valorisation de la Recherche; contract/grant number: J0003023LJJ-LERMAB
(Received 30 January 2001; revised version received 26 April 2001; accepted 31 May 2001)
# 2001 Society of Chemical Industry. Pest Manag Sci 1526±498X/2001/$30.00
833

M Baya et al
15h.The combined organic layers were evaporated
and the resulting orange liquid distilled under reduced
pressure to give 2a as a yellow solid, bp: 70°C
03mmHg), in 77% yield. 1H NMR 0400MHz,
deuterochloroform): 9.20 0broads, 1H), 7.40±7.30
0m, 4H), 7.05 0tt, J =9Hz, J1=1.3Hz, 1H).
Figure 3. Structures of commercially available fungicides.
2.1.2 Synthesis of 2-methoxycyclohepta-2,4,6-trienone
!2b)
Â
France) and La Quinoleine 0Oissel, France).NMR
Iodomethane 07.1g, 50mmol) was added to a stirred
mixture of tropolone 01.22g, 10mmol), anhydrous
potassium carbonate 04.15g, 30mmol) and dicyclo-
hexyl-18-crown-6 0375mg, 1mmol) in dry acetonitrile
050ml).The mixture was heated to re¯ux during 10h.
After cooling to room temperature, the solvent was
evaporated and the residue dissolved in methylene
dichloride.The organic layer was washed with
potassium carbonate solution 02M) to remove un-
reacted tropolone, dried over anhydrous magnesium
sulfate and concentrated to give 2b as an oil which was
puri®ed by column chromatography on silica gel using
ethylacetate‡hexane 01‡3 by volume) as eluent, mp:
40±41°C in 70% yield. 1H NMR 0400MHz, deutero-
chloroform): 7.28±7.20 0m, 2H), 7.11 0dd, J =J1 =
10.5Hz, 1H), 6.88 0m, 1H), 6.74 0d, J =10Hz, 1H),
3.95 0s, 3H).
spectra were recorded on a Bruker AM 400 spectro-
meter.IR spectra were recorded as thin ®lms between
sodium chloride plates or potassium bromide discs on
a Mattson Genesis Series FTIR instrument.Column
chromatographic separations were performed on
Kieselgel 60 070±230 mesh ASTM) purchased from
Macherey Nagel.Thin-layer chromatography was
performed using precoated Macherey Nagel plates,
silica gel 60 SIL G/UV254.All organic solvents were
appropriately dried and puri®ed before use.Melting
points were determined with a BuÈchi Tottoli apparatus
and are reported uncorrected.
2.1.1 Synthesis of 2-hydroxycyclohepta-2,4,6-trienone
!2a)
7,7-Dichlorobicyclo[3.2.0]hept-2-en-6-one.
Dichloro-
acetyl chloride 030.88g; 209mmol) and cyclopenta-
diene 052.5ml; 618mmol) were mixed in pentane
0200ml) in a 500-ml round-bottomed ¯ask ®tted with
an addition funnel and a re¯ux condenser.The
solution was heated to re¯ux under nitrogen while a
solution of triethylamine 021.9g; 216mmol) dissolved
in pentane 0100ml) was added dropwise during about
1h.After 4h under re¯ux, the mixture was cooled to
room temperature and water 075ml) added.The
organic layer was separated and the aqueous layer
extracted with pentane 02Â50ml).The combined
organic layers were dried over anhydrous magnesium
sulfate and the solvent evaporated.The resulting
brown liquid was distilled under reduced pressure to
give 7,7-dichlorobicyclo[3.2.0]hept-2-en-6-one as a
colourless liquid, bp: 61±62°C 01mmHg), in 88%
2.1.3 Synthesis of 2-benzyloxycyclohepta-2,4,6-trienone
!2c)
2a 01.0g, 8.19mmol) was treated with aqueous
tetrabutylammonium hydroxide 0400g kgÀ1ꢀ1.54M;
5.36ml, 8.19mmol) at room temperature for 1h under
nitrogen. Benzyl bromide 01.4g, 8.19mmol) dissolved
in methylene dichloride 020ml) was added and the
reaction monitored by TLC until the disappearance of
the starting materials.Water 025ml) was added and
the mixture vigorously stirred for 10min.The organic
layer was separated and the aqueous layer extracted
with methylene dichloride 03Â30ml).The combined
organic layers were dried over anhydrous magnesium
sulfate, concentrated, and the residue puri®ed by
column chromatography on silica gel using methylene
dichloride‡ethyl acetate 07‡3 by volume) as eluent,
mp: 82°C, in 65% yield. 1H NMR 0400MHz,
deuterochloroform): 7.48±7.10 0m, 7H), 6.99 0dd,
J =J1 =10Hz, 1H), 6.88±6.76 0m, 2H), 5.27 0s, 2H).
1
yield. H NMR 0400MHz, deuterochloroform): 6.06
0m, 1H), 5.82 0m, 1H), 4.28 0dd, J =J1 =9Hz, 1H),
4.09 0m, 1H), 2.81 0d, J =16Hz, 1H), 2.63 0m, 1H).
2-Hydroxycyclohepta-2,4,6-trienone. Sodium hydroxide
030g) was mixed cautiously with glacial acetic acid
0150ml) in a 500-ml round-bottomed ¯ask.After the
pellets were totally dissolved, 7,7-dichlorobicy-
clo[3.2.0]hept-2-en-6-one 032.6g) was added and
the solution maintained at re¯ux under nitrogen
during 8h.The solution was cooled to room tempera-
ture and concentrated hydrochloric acid added until
the pH of the mixture was about 1.After addition of
toluene 030ml), the solid sodium chloride which
precipited was ®ltered off and washed with further
toluene 030ml).The organic layer was separated and
the aqueous layer transferred into a continuous liquid±
liquid extractor and extracted with toluene during
2.2 Fungicidal activity
2.2.1 Microorganisms
Two white rot fungi, coriolus versicolor 0L) Quelet
0strain CTB 863A), Phanerochaete chrysosporium Bued-
sall 0strain CBS 246.84) and two brown rot fungi,
Poria placenta 0strain FPRL 280) and Gloeophyllum
trabeum 0strain BAM Ebw 109), were used throughout
this study.Stock cultures of fungi were maintained on
malt-agar slants, which were kept at 4°C before use.
2.2.2 Growth inhibition
Mycelium was grown in 10-cm Petri dishes ®lled with
20ml of malt-agar medium containing different
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Fungicidal activity of b-thujaplicin analogues
concentrations of growth inhibitors added aseptically
from ®lter-sterilized 1M ethanolic solution.Plates were
inoculated by placing a 10-mm diameter plug, cut
from the edge of a starting colony growing on malt-
agar medium, in the centre of each plate.The cultures
0two replicates for each experiment) were kept 15 days
in a growth chamber at 25°C.Growth was evaluated
every two or three days by measuring two perpendi-
cular diameters of the colony and expressed as a
percentage of the room available for growth, ie the
diameter of the dish.
rotting fungi are reported in Fig 4.As reported
previously,12,16 b-thujaplicin 01) shows strong anti-
fungal activity against P placenta, G trabeum, C
versicolor and P chrysosporium.For the last fungus, the
most ef®cient b-thujaplicin analogue was tropolone
02a), in contrast to 2-methoxycyclohepta-2,4,6-trien-
one 02b) and 2-benzyloxycyclohepta-2,4,6-trienone
02c), which inhibited growth more weakly, while
cyclohepta-2,4,6-trienone 02d) had no effect.These
results demonstrate the importance of the hydroxyl
group of tropolone or b-thujaplicin, since when it was
replaced with a methoxy or a benzyloxy group, the
derivative obtained was less active as growth inhibitor.
b-Thujaplicin 01) and tropolone 02a) exhibit similar
strong growth inhibitory effects again tested wood-
rotting fungi.This suggests that the isopropyl group
present in 1 is probably not involved in the growth-
inhibition process.
3
RESULTS AND DISCUSSION
3.1 Effect of b-thujaplicin analogues on growth in
vitro of brown and white rot fungi mycelia
The inhibitory effects of b-thujaplicin and its ana-
logues 0tested at 100mM) on growth of several wood
Figure 4. Effects of b-thujaplicin and its analogues at 100mM on growth of white and brown rot fungi. (&) Control, (~) b-thujaplicin, (1); (*) tropolone, (2a);
(&) 2b; (*) 2c; (~) 2d.
Pest Manag Sci 57:833±838 0online: 2001)
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M Baya et al
The ability of 1 and 2a to form stable chelate
complexes with metal ions has been reported in the
literature to be due to the ketoenolic function.17 When
the hydroxyl group of 2a was replaced by hydrogen,
methoxy or benzyloxy groups, the derivative was less
active as a growth inhibitor, suggesting that chelating
properties are important in the process of inhibition.
This suggests that one possible mechanism for the
inhibition of fungal growth by 1 and 2a could be the
chelation of metal ions present in the culture medium.
Furthermore, these two compounds have been shown
previously to inhibit the activity of metalloenzymes
such as laccase, tyrosinase and lipoxygenase, chelating
catalytically essential metal ions.9±11
inhibition was observed for the four fungi using 50mM
of either compound after 4 days.
3.2 Comparison with commercial products
Compounds 1 and 2a have been compared to the
commercial fungicides azaconazole, tebuconazole and
oxine copper.Figure 6 shows the growth of the tested
fungi over 15 days in the presence of the different
biocides at 10mM.Tebuconazole was clearly the more
ef®cient inhibitor since the growth of all the tested
fungi was totally inhibited in presence of this product.
In comparison, depending of the tested fungus, 1 and
2a exhibited comparable or weaker ef®ciency, but
both compounds were in most cases more ef®cient
than oxine copper or azaconazole.
Figure 5 shows the effects of various concentrations
of 1 and 2a on the growth of C versicolor and P
chrysosporium 0Fig 5A) and P placenta and G trabeum
0Fig 5B) after 4 days.
4
CONCLUSIONS
The extent of growth inhibition induced by 1 and 2a
depends on the fungus.In these conditions 0four
incubation days), the ef®ciencies of both compounds
are quite similar whatever the tested fungus.Complete
From these investigations, we can conclude that, in
vitro, tropolone 02a) possesses similar activity to
commercially available fungicides currently used for
wood preservation.Slightly less active than tebucona-
Figure 5. Effect of various
concentrations of b-thujaplicin (1) or
tropolone (2a) on growth of (A) white
and (B) brown rot fungi after 4 days.
Coriolus versicolor (Cv),
Phanerochaete chrysosporium (Pc),
Poria placenta (Pp), Gloephyllum
trabeum (Gt).
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Fungicidal activity of b-thujaplicin analogues
Figure 6. Comparison between b-thujaplicin, tropolone and commercial fungicides at 10mM on growth of different white and brown rot fungi. (&) Control, (~) b-
thujaplicin (1), (*) tropolone (2a), (~) azaconazole (3), (&) tebuconazole (4), (*) copper oxine (5).
REFERENCES
zole, 2a is more ef®cient than azaconazole or copper
oxine in preventing the growth of wood-rotting fungi.
Easily prepared in two steps from low-cost starting
materials, tropolone appears to have potential as a
fungicide.The related naturally occuring b-thujaplicin
01) has been widely used as an antimicrobial agent in
cosmetics.18,19
1 Ohira T, Terauchi F and Yatagai M, Tropolones extracted from
the wood of western red cedar by supercritical carbon dioxide.
Holzforschung, pp 308±312 01994).
2 Ohira T, Yatagai M, Itoya Y and Nakamura S, Ef®cient extrac-
tion of hinokitiol from the wood of Hiba with supercritical
carbon dioxide. Mokuzai Gakkaishi 42:1006±1012 01996).
3 Sakai K, Yamaguchi T and Itose R, Biosynthesis of a heartwood
tropolone, b-thujaplicin. Mokuzai Gakkaishi 43:696±698
01997).
However, these results are only initial laboratory
®ndings which need further investigation.In particu-
lar, studies on the ability to protect treated wood
blocks, on leachability, and on the mechanism of
action of these compounds are needed to evaluate their
potential as industrial wood preservatives.
4 Okabe T, Saito K, Fukui T and Iinuma K, Antibacterial activity
of hinokitiol against methicillin-resistant Staphylococcus aureus
0MRSA). Mokuzai Gakkaishi 40:1233±1238 01994).
5 Inamori Y, Nishiguchi K, Matsuo N, Tsujibo H, Baba K and
Ishida N, Phytogrowth-inhibitory activities of tropolone and
hinokitiol. Chem Pharm Bull 39:2378±2381 01991).
6 Saito K, Okabe T, Fukui T and Inamori Y, Antibacterial activity
of hinokitiol against methicillin-resistant Staphylococcus aureus
0MRSA).II.Applications to the disinfections of hospital
environments. Mokuzai Gakkaishi 43:882±891 01997).
7 Arima Y, Hatanaka A, Tsukihara S, Fujimoto K, Fukuda K and
Sakurai H, Scavenging activities of a-, b- and g-thujaplicins
ACKNOWLEDGEMENTS
This research was supported by a grant from the
Agence Nationale de Valorisation de la Recherche 0No
J0003023LJJ-LERMAB).
Pest Manag Sci 57:833±838 0online: 2001)
837

M Baya et al
against active oxygen species. Chem Pharm Bull 45:1881±1886
01997).
14 Tanaka K and Yoshikoshi A, Cycloaddition of isopropylcyclo-
pentadienes and dichloroketene, and the solvolysis of the
adducts. Tetrahedron 27:4889±4899 01971).
8 Belleau B and Burba J, Occupancy of adrenergic receptors and
inhibition of catechol O-methyl transferase by tropolones. J
Med Chem 6:755±759 01963).
15 Minns RA, Tropolone. Org Synth 57:117±121 01977).
16 Inamori Y, Sakagami Y, Morita Y, Shibata M, Sugiura M,
Kumeda Y, Okabe T, Tsujibo H and Ishida N, Antifungal
activity of hinokitiol-related compounds on wood-rotting fungi
and their insecticidal activities. Biol Pharm Bull 8:995±997
02000).
9 Suzuki H, Ueda T, JuraÂnek I, Yamamoto S, Katoh T, Node M
and Suzuki T, Hinokitiol, a selective inhibitor of the platelet-
type isozyme of arachidonate 12-lipoxygenase. Biochem Bio-
phys Res Commun 275:885±889 02000).
10 Fernandez E, Sanchez-Amat A and Solano F, Location and
catalytic characteristics of a multipotent bacterial polyphenol
oxidase. Pigment Cell Res 12:331±339 01999).
17 Pietra F, Seven-membered conjugated carbo- and heterocyclic
compounds and their homoconjugated analogs and metal
complexes.Synthesis, biosynthesis, structure, and reactivity.
Chem Rev 73:293±364 01973).
11 Poma A, Pacioni G, Colafarina S and Miranda M, Effect of
tyrosinase inhibitors on Tuber borchii mycelium growth in vitro.
FEMS Microbiol Lett 180:69±75 01999).
18 Saeki Y, Ito Y, Shibata M, Sato Y, Okuda K and Takazoe I,
Antimicrobial action of natural substances on oral bacteria.
Bull Tokyo Dent Coll 30:129±135 01989).
12 Yamaguchi T, Fujita K and Sakai K, Biological activity of
extracts from Cupressus lusitanica cell culture. J Wood Sci
45:170±173 01999).
19 Fujisaki Y, Oyama M, Hideko W and Ikeda E, Cosmetics con-
taining microbicidal preservatives, Jap patent: JP 89-62293
01991).
13 Miyashita M, Hara S and Yoshikoshi A, Regiospeci®c synthesis
of b-thujaplicin 0hinokitiol) from 2-isopropylphenol. J Org
Chem 52:2602±2604 01987).
838
Pest Manag Sci 57:833±838 0online: 2001)