Note: Descriptions are shown in the official language in which they were submitted.
CA 02230102 1998-02-20
W O 97/48279 PCT~B97/007S0
AGENTS WlTtJ ANTIFUNGAL ACTIVITY AND METHODS OF IJSF
THEREOF
This application claims priority from provisionai application Serial
No. 60/020,035, filed June 21, 1996.
5 Background Of the Inventiion
This invention relates to compounds that have antifun~al activity.
These compounds reduce or inhibit the growth of fungi from a variety of
genera and species.
Trembling aspen or aspen (Populus tremuloides Michx.) is an
10 important source of pulp for the paper-making industry. Aspen is also used
for making particle board and solid wood products. Decay and stain
caused by several fungi have been identified as the two most important
factors limiting the use of aspen (Hiratsuka et al. 199û, 1995~. Recently,
the present inventors reported on the inhibition of the growth of aspen
15 decay and stain fungi using certain antagonistic fungi (Chakravarty and
Hiratsuka 1992, 1994; Chakravarty et al. 1994; Hutchison et al. 1994). The
growth of aspen decay fungus Phellinus tremulae ~Bond.) Bond. et Borisov
was inhibited by metabolites of another decay fungus Peniophora
polygonia (Pers.:Fr.) Bou. et Gal. (Trifonov et al. 1992). The antagonistic
20 activity of the coprophilous fungus Sporormiella simiJis Khan and Cain,
frequently found on aspen black gali, as well as the activity of its
metabolites were also studied (Chakravarty et al. 1994). It was shown that
S. similis inhibits the in vltro growth as well as aspen wood chip
colonization by blue stain fungi. The present invention is an extension of
25 work reported on the antifungal activity of metabolites of Peniophora
poJygonia (Trifonov et al. 1992) and SporormielJa similis (Chakravarty et
al. 1994) against aspen pathogens.
CtlNrlnl\1~10N COPY
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W O 97/48279 PCT~B97/00750
Summary of the Invention
The inventors extracted a variety of compounds ~rom decay fungus
Peniophora polygonia and found that isobutyric and isovaleric acid were
amongst the most active compounds. Following this lead, they tested 4-
S phenyl-3-butenoic acid - a structural analog of the above acids. 4-phenyl-
3-butenoic acid is known as a potent mechanism-based inhibitor of
peptidylglycine amidating monooxygenase (Bradbury et al. 1990; Rhodes
and Honsinger 1993), as a modulator of the activlty of y-glutamic acid
transpeptidase (Thompson and Meister 1980), as well as an antibacterial
10 compound (Khristov 1973).
The objective of this invention is to provide an antifungal composition and
methods of use against a wide range of fungi. Specifically, the invention is directed
to methods of re~ cing or inhibiting fungal growth by applying the conll)o~ulds of
the invention to a region to be treated. The antifungal compositions of the
15 invention can be used against a wide variety of fi ngi, and it is well within the skill
of an ohlhl~ worker in the art to screen particular fungi against which the
compositions of the invention are effective.
The objective of part of this invention was to determine the activity of
homologs and analogs of 4-phenyl-3-butenoic acid against P. tremulae and two
20 species of blue stain fungi, Ophiostoma piliferum (Fr.) H. and P. Sydow and O.
crassivaginatum (~.D. Griffin) T.C. ~rnngtcln For cc ,npa~iso~ purposes,
phomalone (Ayer and Jimenez 1994), pentachlorophenol, copper 8-oxyquinolinate,
Zn salt of 1o-lln~lpcenoic acid, as well as lauric acid were included in this study.
The inventors have also found that tiglic acid and senecioic acids, minor
2~ metabolites of S. similis, significantly inhibited the growth of the blue stain fimgus
Ophiostoma piliferum, even at concentrations as low as 1 ~lg/ml. The inventors
therefore synthPsi7P~ derivatives and analogs of these two leads to test their
activity against the aspen decay fungus Phellinus tremulae and the aspen blue stain
-- 2 -
,
CA 02230102 1998-02-20
WO 97/48Z79 PCT~B971007~0
fi~ngi Ophios~oma crassivaginatum and 0. piliferum.
Selected com~ ds from the derivatives, homologs and analogs of tiglic
acid, senecioic acid and 4-phenyl-3-butenoic acid were also tested against the
conifer decay fimgi Phellinus pini and ~aematostereum sanguinolentum and the
- 5 conifer blue stain fimgi Ophiostoma clavigerum and 0. ips. Finally, select
compounds were tested against the fairy ring f~ngus Marasmius oreades.
Thus, ~e l~lc;s~llLly-disclosed compounds include deliv~ s, homologs
and analogs of tiglic acid, senecioic acid and 4-phenyl-3-butenoic acid which are
~;Live ~ntifim~l compounds. These compounds can be used in ~lli
compositions, and methods of reduc;ng or inhibiting fungal growth.
More specifically, the present invention is directed to an antifungal
co,~ o~i~on compri~in~ at least one compound selec~ed from the group con.~i~t;n~of:
(a) at least one compound of formula I
R- ~3
R C C (CH2)n COOR6
R4 R5
_ h
1 5 wherein
R~ is selecte~ from the group con~i~ting of (1) H, (2) Cl-C~5 alkyl, (3) a
group R~
R~
- 3 -
CA 02230102 1998-02-20
W O 97/48279 PCT~B97/00750
wherein R7 and R8 are Cl-CIs alkyl or, together with the carbon ...
atoms to which they are bound, form a 3-8 membered carbocyclic ring, and (4) a
group Ar, wht;~ Ar is an aromatic ring;
R2 through Rs are each independently H or C~-C~5 alkyl, or R2 and R3
5 together are a bond thus creating a double bond between the carbon atoms to which
they are bound, or RZ and R3 together, and R4 and Rs together, are bonds thus
creating a triple bond between the carbon atoms to which they are bound, or
two of R' through R5, together with the carbon atoms to which they are
bound, fo~n part of a 3-8 membered carbocyclic ring,
with the proviso that when R2 through Rs are each H, then Rl is only
Ar;
R6 is selected from the group consisting of (l) H, (2) Cl-C~5 alkyl, (3)
phenyl, (4) a group N(CH3)2(CloH2,)2, (5) a group N(CI-Cl5 alkyl~4, wherein the
alkyl groups are the same or different, (6) NHm~X)4 ,l" wherein each X is~5 independently selected from Cl-CI5 alkyl and 2-hydroxyethyl, and m is 0-4,
with the proviso that when Rl is Ar, then R6 is other than phenyl;
nisO-lOandn'isOor l;and
(b) at least one compound of formula II
R9~ R~'
R
.
--4-
CA 02230102 1998-02-20
W O 97/48279 PCTnB97100750
Wil~
R9 is seiected from the group con.~i~ting of H, OH and C,-C,5 alkyl;
R'~ is H or OH; and
Rl' and Rl2 are each indepen~7P-ntly H or C,-CIs alkyl,
or an alkali or alkali earth salt thereof, and
a carrier or diluent therefor.
It is ~l~r~lc;d that Ar is s~lecte~ from the group consict;ng of
10 wherein each of the rings may have non~ fe,hlg substihlpnt~ thereon.
The present invention also re~ates to a method of re~ cing or inhibiting
fungal growth in a region to be treated, comrri~;ng applying to the region a fungal
rerl~lr-ing- or inhibiting-e~cli~ amount of at least one compound selected f~om
the group con~ictin~ of:
(a) at least one conlpoLIlld of fiorml7~,7 I, wherein formula I is as defined
above; and
(b) at least one compound of formula II, wherein formula II is as defined
above,
or an alkali or alkali earth salt thereof.
CA 02230102 1998-02-20
W 097/48279 PCT~B97/00750
.
The more ~fer~lled embonim~nt~ of the present invention are disclosed in
the Detailed Description of the Invention, as follows.
Dt,~ tion of the ~r~f~. I ed Embodiments
In a ~irst aspect, ~e present invention is di~ ,L~d to an ~ntifimg~l
5 co~ o~ilion co,1~ g at least one compound selected from the group Con~i~ting
of:
(a) at least one compound of fonm
~- R3
Rl C C (CH,)~, .COOR6
R4 R~ ~
wl~
R' is select~d from the group COn~ lg of (1) H, (2) C,-C,5 ~ 1, (3) a
1 0 group
R\
wherein R7 and Rg are Cl-C,5a~ or, together with the carbon
CA 02230102 1998-02-20
W O 97/48279 PCT~B97100750atoms to which tbey are bound, f~rm a 3-8 membered carbocyclic ring, and (4) a
group Ar, wherein Ar is an aromatic ring;
R2 through R5 are each independently H or C,-C15 alkyl, or R2 and R3 are a
bond thus creating a double bond between the carbon atoms to which they are
5 bound, or R2 and R3 and R4 and R5 are bonds thus creating a triple bond bc;l~
the carbon atoms to which they are bound, or
two of R~ thrt~ugh R5, together with the carbon atorns to which they are
bound, folm part of a 3-8 ~.~I)eled carbocyclic ring,
with the proviso that when R2 through ~5 are each H, t~en R~ is only
10 Ar;
R6 is s~l~cte~ om the group cn...~ o~(1) H, (2) C,-C15 alkyl, (3)
phenyl, (4) a group N~C~I3)2(C",H2,)2, (5) a group N(C,-CI5 allcyl)4, wherein the
alkyl groups are the sarne or ~Iirr~G~lt, (6) NHm(X)~,I" wherein each X is
independently selected from Cl-C~s alkyl arld 2-hydroxyethyl, and m is 0-4,
1~ with the proviso that when R' is Ar, then R6 is other than pheny~;
n is O-lO and n' is O or l; and
(b) at least one compound of for~nula II
R9 ~ < Rl'
wherein
- R~ is selected i~om the group consisting of ~, OH and C,-C~s allcyl;
R'~ is H or OH; and
CA 02230102 1998-02-20
W 097/48279 PCT~B97/007S0
Rl l and Rl2 are each independently H or (~,-C,5allcyl,
or an alkali or alkali earth salt thereof, and
a carrier or diluent therefor.
Unless otherwise spe~ifie~l ~r is preferably selected from the group
~o~ Lil~g of
wherein each of the rings may have non-interfering substit~lPntc thereon. Non-
Ç~"illg substituents can include, for exarnple, but are not limited to, lower all~yl
10 groups, lower alkoxy groups, lower acyl groups, am;no groups, hydroxyl groups,
etc. The terrn "lower" is u~derstood as meaning 1-6 brarlched or unbranched
carbon atoms. Usually, monocyclic rings may have 0-2 substitnpntc~ and bicyclic
rings may have 0-4 substi~l~nt~ but this number is not critical.
There are generally four classes of p.~ .d compounds. A first class is
1~ cc,.l.~ou.lds of forrnula III
Rl ~ C~O~ 6
wherein
R~3 through R'5 are each independently H or C~-C ~5 alkyl, or two of R'3
CA 02230102 1998-02-20
W O 97/48279 PCT~B97/00750
through R'5, toget~er with the carbon atoms to which they are bound, form a 3-8
membered carbocyclic ring, and
R~ is as defined above.
A second class of ~Ic~l~ed compounds are co.u~ou.lds of forrn
R~
Q~
~; W~ 1G~1
R6 through R8 and n are as defined above.
A third class of ~lG~lled compounds are compounds of forrnula V
RJ~ ~18
2)n--COO~LI
R'~ R~
wherein
Rl6 is a group Ar, WLC1~ Ar is as defined above;
R'7 through R20 are each H, or R'' and Rl8 are a bond thus creating a double
bond between the carbon atoms to which they are bourld, or Rl' and Rl8 and Rl9
- and R2~ are bonds thus creating a triple bond between the carbon atoms to which
they are bound;
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W 097/48279 PCT~B97/00750
R2' is selected ~om the group con~i~tin~ of (1) EI, (2) C,-C,5 alkyl, (3) a
group N(CH3)~(C~oH7~)2~ (4~ a group N(C,-C,s alkyl)4, wherein the alkyl groups are
the same or difr~rc.ll, (5) NH",(X)~m, wherein each X is independently selected
from C,-CI5 alkyl and 2-hy~l.v~y~lyl, and m is û-4; and
n is as defined above. ''
The fourth class of ~lef~ ,1 compounds are cor~oul~ds of fonn~
R
R9 ~) Ç~ Rl'
R~
wherein R9 through Rl2 are as defLlled above.
In another aspect, the present invention is directed to a method of reducing
or inhibiting fungal growth in a region to be treated, comrri~ing applying to the
10 region a fungal re~ ring- or inhibiting-effeetive amount of at least one compound
selected from the group consi~ting of:
(a) at least one compound of forrnula I, wherein forrnula I is as defiIled
above, and
(b) at least one colllpou,ld of formula II, wherein formula II is as defined
1 ~ above,
or an alkali or alkali earth salt thereof.
- 10-
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W 097148279 PCT~B97/00750
The term "antifimgal" as used herein includes, but is not limited to,
compounds that reduce, destroy or '.cill fungi (fungicidal) or innibit the growth of
fungi (filngi~t~tic). The terrn "antifimgal composition" as used herein includes not
only the compounds of the present invention in a conventional diluent and/or
solvent, but also inrlll~es the compounds of the present invention in other carriers
and/or fi~al ~le~ ;on~, such as paints, stains, wood stains, elt ~n~rs,
tli~ii"re~ , rinses and wood or lawn Lr~i~lr~
The term "wood" as used herein inrhl~ec, but is not lir~uted to, trees, logs,
woody plants, wood products, wood chips, processed wood, saw dust or items
10 made completely or partially of wood.
The term "wood tre~trnent" includes, but is not limited to, washes. rinses,
stains, pigrnented fini~hec, natural fini~he~, exterior fini~h~, interior fini~htos,
ru~ c fini~he~, filling, washcoating, sealing, topco~ing, cleaners, polishes or
fire ~eL~L~d~lL~. Preferred tre~trnent includes oil based or water based l~ni~h~s
The term "region" refers not only to the surface of the area to be treated, but
also to areas lmcl~rn.~th the surface, if the surface is permeable. For example, the
term "region" applies not only to the surface of soil having plants thereon, but also
to areas lm~rne~th the soil surface. Similarly, the term "region" includes the inner
portion of wood below the surface of the wood. However, the term "region" is also
meant to include the surface of non-permeable substrates, such as bathroom tile or
kitchen coLl~ Lops, or non-permeable painted s-lrf~s.
Herein, the term "alkyl" is a radical co..li.;..i..~ only carbon and hydrogen,
and lacking double or triple bonds. In this invention, unless stated otherwise, alkyl
radicals contain 1 to 15 carbons and, unless stated otherwise, the alkyl radicals can
25 be branched or unbranched.
The antifungal compositions of the present invention are effective against a
CA 02230102 1998-02-20
W O 97148279 PCT~B97/00750variety of genera ~nd species of fungi. Preferred fungi against which the present
compositions are effective include the genera Phellinus, Ophiostoma
fCeratocystis), Haematostereum and ll~arasmius. Preferred species include P.
tremulae, P. pini, O. crassivaginatum, O. piliferum, O. clavigerum, O. ips, H.
5 sanguinolentum and M. oreades.
Preferred woods to be treated include aspen and conifers.
This invention also in-~.h~ c the use of the present compounds in a method
for the tre~tm~llt of wood or wood products to reduce or inhibit decay a~d/or
st~ining comrri.~in~ applying to a region to be treated a fungal-decay andlor
1 () fungal-st~inin~ re~ ring- or inhibiting-erf~ilivt: amount of at least one coll~o~ld
selected from the group co~ lhlg of:
(a) at least one compound of formula I, wherein formula I is as defined
above; and
(b) at least one compound of formula II, wherein forrnula II is as defined
1 5 above,
or an alkali or alkali earth salt thereof.
.
This invention also incll~les the use of these compounds in a method of
treating fairy rings comprising applying to a region to be treated a fairy-ring fungal
reducin~- or inhibiting-effective amount of at least one compound selected from
the group con~i~tin~ of:
(a) at least one compound of formula I, wherein formula I is as defined
above; and
(b) at least one compound of formula II, wherein formula II is as defined
above,
or an alkali or alkali earth salt thereof.
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W O 97/48279 PCTAB97/00750
It is l3lcr~ d to use these compounds to reduce or inhibit t~e grow~h of
fairy rings on grassy regions (e.g., golf courses and other grassy lawns).
The compounds of the present invention can be used in pa~nts, stains, wood
stains, cleaners, disil~cL~lL~, rinses or other wood k~ .CntC The compounds can
5 aiso be u~sed for other plants besides woody plants and ~accec Although ~
for wood, grasses and other plants are preferred, ~e compounds of the present
invention can also be ~ced on non-wood ~-- r~C~s as an active agent of çl~nf~rs,di~ fe~ or rinses, such as ~athroom or kitchen c3e~nt?r~, cleaners for non-
wood ~e.g., ~ ll;lllllll or vinyl) house siding or other ~ ning products or
1 0 disinfectants where reducing or inhibiting fungal grow~ is desired.
With reference to the numerical listings of vanous co~ ou~ds of the
invention in Tables 1-7, the following compounds are ~re~.-ed ~or the following
uses:
~ The use of one or more of the following compounds in a me~od for
1 5 reduction or inhibition of fungal growth: la, le, lf, 2, 4a, 4b, 6a, 6d, 7, 8a, 8c, 8f,
9a, 9b, 13, 14, 15,20, 21a, 21d, 21e,22, 24,25d, 31, 32, 33, 34, 26a, 26b, 26c,
26d, 26e, 26f, 26g, 28, 29, 23a and 35. The use of at least one of the followingcompounds is especially preferred: la,2, 4b, 7, 8f, 13, 14, 20,21a, 26c, 26d, 26f,
28, 31 and 32.
~ The use of one or more of the folIowing compounds in a method for
reducing decay and/or staining of wood: la, 2, 4b, 7, 8c, 8f, 13, 14, 3:~, 34, 20,
21a, 21e, 24, 26b, 26c, 26d, 26f, 28, 31 and 32.
~ A method for reducing wood staining using one or more of the following
compounds: la, 2, 4a, 4b, 6a, 7, 8a,8c, 8f, 9a, 9b, 13, 14, 20, 21a, 21d, 21e, 22,
24, 26b, 26c, 26d, 26f, 28, 31 and 32 .
,
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W O 97t48279 PCTAB97/00750
~ A method for re~ ing wood decay using one or more of the followin~
compounds: 1~, le, 2, ~b, 7, 8c, 8f, 13, 14, 15, 33,34, 20, 21a, 21e, 24, Z~d, 26c,
26d~ 26f, 26g, 28, 31 and 32.
.,
~ A method for reducing or inhibiting growth of fairy ring fungus growth
using one or more of the following compounds: la, 8f, 13, 14, 21a, 26d, 32 and 35.
An ~ntifi-ngsll composition in accol~ce with the present invention
ef~,.ably contains at least one of the following compounds: la, le, lf, 2, 4a, 4b,
6a, 6d, 7, 8a, 8c, 8f, 9a, 9b, 12, 13, 14, 15, 33, 34, 20, 21a, 21d, 21e, 22, 23a, 24,
25d, 26a, 26b, 26c, 26d, 26e, 26f, 26g,28, 29, 31, 32 and 35. More ~ d are
1 0 at least one of the following compounds: la, le, lf,2, 4a, 4b, 7, 8c, 8f, 12, 13, 14,
20, 21a, 26b, 26c, 26d, 26f,28,31,32, 33, and 34, and most preferred are at least
one of the following compounds: la, 2, 4b, 7, 8c and 8f.
An antifungal composition and method of use for re~ ing or inhibiting
fungal growth preferably inc~ two or more compounds to increase the bread~h
1 5 of OL~ inhibited or reduced, or to increase the potency of the compounds. It
is particularly preferred to use at least one compound of the present invention,along with benzoic acid and/or salicylic acid. Preferred compositions in this regard
are compositions cont~inin~ benzoic acid and at least one of the following
compounds of the invention: 8f, la, 13 and 14. It is ~l~r~l,ed to apply the
20 co.l,~o~lds in 50:50 ratios (ben~oic acid:compound(s) of the invention). However,
a range of 25:75 to 75:25 is suitable.
Formulations
The present compounds are soluble in at least one of acetone, alcohol and
water. These compounds can also be dissolved in a variety of other solutions or
25 solvents. The compounds can also be directly added to a final ~ ~dlion (e.g.,paint, stain, wood or lawn treatrnent, cleaner or disinfectant). Tf the antifungal
- 14-
.
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W O 97/48279 PCT~B97/007S0
compound will not readily dissolve in the final ~)lcy<~ ;OIt~ then the c~n{ o~ d
must be first dissolved in an ~u~-iate organic solvent and then diluted, as known
in the art. The ~y~v~l;ate solvent must dissolve the compound and be able to rnix
with the final p,.,~dlion. A worker skilled in the art can readily ~let~rrnine the
5 ~ .~liatesolventwithoutundue~ nt~tion. Thedesiredamountofthe
~ntifimF~;~1 compounds in the composition is between l and 50 times the amount
which inhibited 95 to 100% of fungal growth ~accordill~ to ;.- rO. . . ~;~I;on from
Tabies l to7,fore~mple). Thecu..~ ionofthe ~..1;r,."~,.l co~l~oulldsinthe
composition is not critical. Normally, however, the ch~ ion range will be
from 0.1% to 50%, more ~r~r~ d 1% to 30%. It is well within the Ol lhl~ y skill
of a worker in the art to (let~-rrnin~ suitable amounts and con~ ~ntr~ti~ ns of the
specific ~ 1 composition to be form~ tt?ll, dc~illg on the treatmPnt
strategy.
One or more of the present compounds can be applied to wood using a
15 variety of methods in~ in~ but not limited to using a brush Ll~ t, dipping,
seeping, diffusion, spraying, dusting, ~c;s~ processes or ground line tre?~trnt~nt~
The basic forrnulations for wood tre~tments, cleaners and disil~ ~ are
known to those skilled in the art.
Formulations for other applications may include but are not limited to
20 solutions, emulsions, suspensions, dusting powders, lyoI~hi1i7~d solutions,
liposome form~ tions or slow release form~ tions. These forrn~ tions can
include a variet,v of additives including solvents, lipids, b1~ffering chemicals,
d~Le.~e~ , fertilizers or other nutrients. Pl~ d dLion of specific fnrrn~ tion~ is
well within the skill of an ordinarv worker in the art, and will depend on the
25 particular L~edLme.lt strategy employed.
~,Y~ mrl~c
-
CA 02230102 1998-02-20
WO 97/48279 PCT~B97/00750The effect of, 8 homologs and analogs of 4-phenyl-3-butenoic acid against
Phellinus tremulae. Ophiosroma crassivaginatum, and 0. piliferum was studied.
These compounds showed varied inhibition of the in viko growth and the wood
chip colonization by these fungi (Table 1). Six of the compounds tested prevented
the development of blue stain caused by 0. ~ ivaginatum and 0. piliferum in
wood chips at conr~nlT~tions of lO llg/rnl (Table 3).
The effect of d~,i iv~lives and analogs of tiglic acid and senecioic acids,
minor metabolites of S. similis, were also studied. Activity was tested against the
aspen decay fungus Phellinus tremulae and ~e aspen blue stain fungi Ophiostoma
crassivaginatum and 0. piliferum (Table 2).
The effect of selected compounds against the conifer decay fungi P~ellinus
pini and Haematostereum sanguinolentum and the conifer blue stain fungi
Ophiostoma clavigerum and O. ips were also studied (Tables 4 and j).
Table 6 shows the effect of selected con~ ds on the in vitro growth of
fairy ring fungus l~arasmius oreades. Ta~le 7 shows the effect of ber~70ic acid
alone and in combination with selected compounds on the in vitro growth of M.
oreades.
E~ample 1. Org~lniem~
Phellinus tremulae (P. tremulae, NOF 1464 = UAMH 7005), an aspen
decay fungus and two species of blue stain fungi, O. crassivaginatum (NOF 1509
- Ceratocystiopsis crassivaginata), and 0. piliferum ~OF 1772) isolated from
decayed and stained wood of P. tremuloides were used in this study. Two conifer
decay fungi Phellinus pini (NOF 2231) and Haematostereum sanguinolentum
(NOF 1 l 8) and two species of conifer blue stain fungi Ophiostoma cZavigerum
(NOF 838) and 0. ips (NOF 684), isolated from conifers were also used in this
study. These fungi were m~int~inecl at 5~C on 2% malt extract agar in McCartney
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'
CA 02230102 1998-02-20
W O 97/48279 PCTAB97/007~0
bottles until used and a~e deposited in the fimgal culture collechon at the Northern
Forestry Centre, C.ln~ n Forest Service~ Edmonton, Alberta (NOF) under the
listed accession numbers.
Marasmius oreades (~OF 2255) was obtained from a fairy ring and
5 ~ on malt extract agar.
le 2. ChPmi'!~
The mo~lifi~ti~n~ of the ~l.u~;LLue of the lead-compound la included:
Pst~ifir~tion (Ib), ~ub~iLulion of the phenyl group ~lc,d), replacement of the
phenyl group by a naphthyl group (le,f ), introduction of a triple bond (2) and an
10 allenic group (3), reduction (4a,b), introduction of a cycloL).o~le ring (~), chain
shul~el~ g (6, 7, 8), and chain elongation (9-12). Two dicarboxylic acids (10, 11)
were also included in the study. The hydro~y~ ...ic acids 6b and 6c,
phenyl~.o~ylloic acid (7), and hydro~inn~mic acid (8a) were considered as
promising candidates because of their low toxicity and enzyme inhibitory activity
15 (Hodgins 1971, Kubata et al. 1974).
The methyl ester of 4-phenyl-3-butenoic acid was prepared by tre~ nt of
the acid with diazomethane. The naphthyl analogs le and If (Table 1~ were
synthe~i7e~1 from 1- or 2-naphthyl ~et~kl~hyde and malonic acid (Ment~er &
Pillon, 1950). l'he acetylenic acid 2 and the allene carboxylic acid 3 were
20 synthesi7~1 according to Shevchenko et al. 1974, Mansfield and Whiting, 1956,and Maer~l 1961. The saturated acids 4a, 8d, 8e, 11, and 12 were obtained by
catalytic hydrogenation of the colle~ol,ding ullsdluLdL~d acids la, 6e, 6f, 9b, and
10 over Pd/C at 1 atm. The dicarboxylic acid 10 and the phenyl analog of sorbic
- acid 9b were ~y~ ii7~(1 following the method of Stuart et al. (1886).
The identity of the synthetic compounds was based on three independent
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criteria (usually m.p. proton NMR spect~n and IR spectrum).
Compounds la, 4b, 4c, 6a, 6b, 6c, 6d, 6e, 6f, 6g, 7, 8a, 8b, 8f, 9a, 13, 14,
16, 17, 18, 19, 33, 34, and 35 are commercially available and were purchased from
Aldrich.
The modifications of the lead co~ ou,lds tiglic (20) and senecioic (21a)
acids incl~ formation of metal and lt~ 01~;11l l l salts (21b-e), synthesis of esters
(23b, 24) and amides (25), replArPm~t of the OH group with ~ul)~liLuled phenyl
groups (senecioph-?none~ 26) removal of the methyl groups (acrylophenone 28
and its precursor 27, as well as the ~b~I; L~ r1 acrylophenones 29 and 30), and
1 0 repl~ ment of the methyl groups by a cyclohexane ring (31 32).
Compounds 20, 21a, and 22 are cornmercially available and were
purchased from Aldrich. The Zn salt of senecioic acid was pl~cLL_d by stirring asuspension of ZnO in a dilute solution of senecioic acid. The amrnonium salt 21dwas plcpaL~ed by lL. cLL~ L of 21a with 1 equivalent of diethanolamine while for1 5 the ~ LLalion of the ammonium salt 21e the sodium salt of senecioic acid was
treated with didecyldimethylamrnonium bromide. Tr~o~tn~nt of 23a (Nie~ki et al.
1887) with senecioic anhydride afforded the diester 23b (Keh~n~nn 1895). The
ester 24 was obtained from phenol and 21a in the attempt to syntheci7~ 26b
(Bragole et al. 1960). The amides 25a,b,c were obtained by treAtTn~nt of senecioyl
chloride with the coL,e~oLlding amine, while 25d was prepared by acylation of
A~ lAllilicacidwithsenecioicanhydride(~I.r...A~ 1909).Seneciophenone
(26a), and its deL;vcLliv~s 26b, 26d, and 26e were ~yll~ ed by Friedel-Krafts
reaction with b~n7t-n~, anisole (afforded a mixture of 26b and 26c) toluene, or
isopropylbenzene (Smith 1949). Compounds 26c and 26f were prepared following
the procedures of Dev (1956) and Sowrnithr m (1985~ respectively. Methylation of26f with di~omethane afforded compound 26g. Compounds 27 and 28 were
prepared as described (Mzmr~ich et al. 1922). Con~n~tion of p- -
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WO 97/48Z79 PCT~B97/00750methylacetoph~none with ffirfural or cinnamic aldehyde afforded compounds 29
(Kostanecki 1896) and 30 (Scholtz, 1903).1-Cyclohexene-l-carboxylic acid (31)
was obtained by ~Ik~lin~ hydrolysis of the cornmercial methyl ester. The
unsaturated acid 32 was prepared by Wittig reaction of cyclohexanone (G. Fodor,
5 1961) followed by ~Ik~line hydrolysis ofthe ester.
The identity of all compounds was based on three criteria (usually m.p., 'H-
nmr, and IR spectra).
The co~ r-;ial wood pL~ sel ~L~es 22 and 23a were also included in this
study for colnp~ on purposes.
F~ rle 3. Improved ~y~h~ . of 4-phenyl-3-butynoic acid
Metallic lithium (0.77g, 0.11 M) was added to a mixture of dry benzene (15
ml) and hexamethylphosphoric triamide (HMPT) (45 ml) under nitrogen at room
tt;lll~ dlule and stirred until dissolved (approximately 2 h). A solution of
phenylacetylene (10.2 g, 0.10M) in benzene (20 ml) was added dropwise and the
15 mixture was stirred overnight at room temperature. The reaction mixture was
cooled to 0~C and a solution of ethylene oxide (5.72 g, 0.1 3 M) in dry bell~;elle ( I S
ml) was added dropwise. A~ter stirring for 24 hours at room temperature, the
reaction mixture was q~nch~(l with ice and ~ a~ d with ether (3 x 150 ml). The
ether extract was washed with water (3 x 150 ml), dried over sodium sulfate,
20 filtered and the solvent removed under vacuum. The residue was passed through a
short silica gel column with petroleum ether:ether equals 1:1 to give pure 4-phenyl-
3-butynol (7.2 g, 50%) as an oil. 'H n.m.r. (CDC13):7.45-7.38, (m,2H,aromatic),
7.33-7.25 (m, 3H, aromatic), 3.81 (t,J=6.5, 2H, H-C(l)), 2.80 (t, J=6.5, 2~, H-
C(2)), 2.88 (br. s, lH, OH~.
A solution of CrO3 (2.67 g) in 8 ml of 30% H2SO4 was added dropwise to a
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solution of 4- phenyl-3-butynol (lg, 0.0068 M) in acetone (35 ml) at 0~C. The
reaction ~ ule was stirred for 30 minntes at 0~C and for 4 hours at room
t~nl~ Lule. Saturated solution of NaCl was added and the organic m~teri~l was
extracted with ether. The ether extract was dried over sodium sulfate, the solvent
5 removed and the residue recryst~lli7ç~1 from petroleum ether:benzene to give 4-
phenyl-3-butynoic acid (0.3 g). The mother liquor was sub~ected to column
chromatography with methylene chloride:ethanol equals 95:5 to provide ~ ition:~
amount of the acid (0.14g). Total yield 40~/O. M.p. 74.0-?5.5~C (Mansfield and
Whiting, 1956; m.p. 71~C). I.r. ~CHC13): 3400-2600, 3051, 1690, 1428, 1087 cm~l.1 0 'H n.m.r. (CDCl3): 7.45 (dd, J=8.0, 2.0, 2~I, arom.), 7.38-7.30 (m, 3H, arom.), 3.62
(s,2H, H-C(2)).
Example 4. Bioass..y~
A. Agar cultures.
To test the effect of the above compounds on the in vitro growth of P.
15 tremulae, O. crassivaginatum, and O. piliferum, these fungi were grown on
MULTIWELLTM Tissue culture plates (1.~. cm x 1.5 crh, rli~net~r x length,
individual well; m~nllf~-~tured by Becton Dickinson Labware, NJ, U.~.A.).
MULTIWELLTM culture plates were prepared by adding 2 ml of st-?rili7~1 carrot
agar into each well. Twenty five ~ll of each of the compounds at 1, lO, 100, and20 1000 ,ug/ml in acetone was added s~;~.al~Lt;ly to the surface of the agar in each well.
In the case of the controls, 25 ~11 acetone was added. All the MULTIWELLTM
plates were kept in a laminar flow hood for two mintlt~s to allow the acetone toevaporate. Each agar well was then individually inoc~ tecl with S mm agar plugs
of P. tremulae, O.crassivaginatum, and O. piliferum. The MULTIWELL~M plates
25 were then wrapped with parafilm and inrllb~t~d at 22~C in the dark. The colony
diameter was measured and mycelia were observed under a microscope.
The same method was used to screen for inhibition of Marasmius oreades
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except the agar was malt extract agar.
Many of these compounds also may have antibacterial or other
~ntimicrobial activity. The breadth of each compound's activity can be ~l~t~rmin~d
using a similar assay. For testing other fungi, b~-t~ or other microbes the same5 procedure can be used, but the nl~tri~nt media used will be based on the nlltrient
requirements of the u~ ll to be tested. Some examples include blood agar
plates, nutrient agar or another media or agar selected based on the organism being
screened. An individual skilled in the art would be famili~r with which agar or
media is suitable for a specific organism for this assay.
B. Aspen wood chips
Two hundred grams of aspen wood chips were soaked in water in each of
seventy-six 250 ml flaslcs. After one hour excess of water was drained and the
flasks were autoclaved for one hour at 121~C. When the wood chips were cooled,
the flasks were treated s~aLely with S ml of 1, 10, and 100 ,ug/ml ofthe most
active compounds listed in Table 1, namely: la, 2, 4b, 7, 8c, and 8f, and then
inoculated separately with P tremulae, O. crassivaginatum, and O. piliferum. Four
flasks were used for each tre~tm~tlt In the case of the controls, flasks were treated
with 2 ml sterile distilled water and five sterile agar plugs without inoculum. The
flasks were kept in the inrub~tor in the dark at 22~C and were shaken periodically
20 to fragment the growing mycelia in the wood chips. After 60 days of incubation,
wood chips were removed from flasks and photographed. The ~ u...ical
structures of the wood chips were also studied by fixing wood chips in formalin-acetic acid-ethanol (FAA). After ten days, the chips were rinsed with 50% ethanol,
dehydrated through a n-butanol series under vacuum, and infiltered with
25 PARAPLAST X-TRATM (Monoject Scientific, St. Louis, Missouri) under vacuum
at ~6~C. Transverse and radial or tangential sections were made using a rotary
microtome. The sections were then observed under a microscope. Data were
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subjected to analysis of variance. The individual means were conlL,~ed using
Scheffe's test for nnllltirle co~ on using SAS so~ware (S~S Tn~itllte ~nc.
1990).
A visual ~ses~m~nt of colony growth can also be perforrned and used as an
indicator of the potency of a colllp~ d. Table 3 ~1lllllll~l ;~es the effects of some
congeners of 4-phenyl-3-butenoic acid on col~ni7~ti~-n of aspen chips by P.
tremulae, O. crassivaginatum and O. piliferum.
Toxicity
The toxicity of many of these compounds is relatively iow, for examp}e
1 0 senecioic acid has an LD5D intr~g~trically of 3130 mg/kg in rats (Chemical
Abstracts 103, 33135w; Gurova, A.I., Smolyar, N.Y., Dr~7h7hin~ N.A., Gig. Tr.
Prof. Zabol. 1985 et al, 1985) and tiglic acid has an LDfr (Amount of chemical
ingested during the Food Reduction test which killed or did not kill more than
50% of the test mice) equal to 1150 mg/kg/day (Chemical Abstracts 103: 1655) or
1 5 LD50 equals 111 mg/kg acute oral toxicity (Chemical abstracts 99:65537).
Other org~ni~
Some of these compounds have antibacterial activity or inhibit the growth
of other microor~;~ni~m~, for example, senecioic acid inhibits Clostridium
botulinum (Hnht~nen et al, 1985).
The same procedures described above can be used to test the activit,v of the
present compounds against other microorg~ni~m~ (including other fungi of
interest) or other types of wood including hardwoods or softwoods, conifers or
deciduous trees.
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Example ~. Activity against aspen decay and stain fun~
A. Co..~ of 4-phenyl -3-hutenoic acid (Table 1)
Thirty eight compounds related to 4-phenyl-3-butenoic acid were tested
against P. tremulae, O.crassivaginatum, and O. piliferum and varied inhibition of
these fimgi was observed. E4-Phenyl-3-butenoic acid (la3, E4-(1-naphthyl)-3-
butenoic acid (le), 4-phenyl-3 -butynoic acid (2), 4-cyclohexylbutanoic acid (4b),
ph~l~yl~ noic acid (7), 3-cycloh~yl~opalloic acid (8c), 3-(3-indolyl)propanoic
acid (8~, cyclok~ c~. boxylic acid (13), cyclohexylacetic acid (14),
pt?nt~ lorophenol (16), copper 8-oxyquinolinate (18), and the zinc salt of 10-
llntl~-c~noic acid (~9) completely inhibited the in vitro growth of P. tremulae at
100 and 1000 llg/ml (Table 1). Except for compounds le, 7, and 19, the other nine
compounds were inhibitory to P. tremulae even at 1 llg/ml.
Compounds If, 8a, and lS showed inhibition of P. tremulae only at 1000
llg/ml, while the ~ ;llg 22 compounds were inact*e.
For O. crassivaginatum, compounds 2, 4b, 8c, 8f, 14, 16, and 18 were
highly toxic and showed an inhibitory effect even at 1 llg/ml. Compounds 7,13,
and 19 did not show any inhibitory effect against O. crassivaginatum at 1 ,ug/ml,
however, at 10-1000 llg/ml the growth was signific~ntIy reduced. The acids la, le,
9a, and 9b showed inhibitory effect against O. crassivaginatum at 100-1000 ~lg/ml.
The acids lf and 6d showed growth reduction by 25% and 18%, respectively, at
1000 ,ug/ml. The rem~inin~ 22 col.~oullds did not show any inhibitory effect
against O. crassivaginatum (Table 1).
For O. piliferum, compounds 2,13, and 14, as well as compounds 16, 18,
and 19 completely inhibited the in vitro growth at 10 ~g/ml, while co.l.3.oullds la,
4a, 4b, 8a, and 8c were inhibitory at 1-1000 ~lglml. The acids le, 6a, 7, 8~, and
sorbic acid (9a) inhibited the growth of O. piliferum at 100 to 1000 llg/ml. Therem~ining 22 compounds did not show any inhibition at 1-1000 ,ug/ml.
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W O 97/48279 PCT~B97/00750
Six of the most active compounds, namely la, 2, 4b, 7, 8c, and 8f, were
chosen for tre~tmen1 of aspen wood chips. The organic co-solvent used for these
expe~ IG-ll~ was acetone. All six compounds prevented colonization of aspen by P.
tremulae, O. crassivaginatum, and O. piliferum even at 10 llg/ml (Table 3).
P. tremulae is a serious pathogen causing ~2~LeI~si~e decay of aspen.
Previously, the inventors reported growth inhibition of P. tremulae by the
metabolites of an antagonistic fimgus Peniophora polygonia (Per.:Fr.) Boud. et
Galzin (Trifonov et al. 1992). The redIlctinn of growth and colonization of aspen
wood chips by blue stain fungi (O. crassivaginatum and O. piliferum) was also
observed when treated with metabolites of the antagonistic hypomyceteous fungi
Lecythophora hoffmannii (van Beyma) W. Gams and McGinnis, Stachybotrys
cylindrospora C.N.Jensen, and Sporormiella similis Khan and Cain (HildL~ d et
al. 1994, Chahav~Ly and Hiratsuka 1994, and Chahdv~ly et al. 19943.
Growth of P. tremulae, O. crassivaginatum, and O. piliferum is
significant}y reduced when these are treated with congeners of the enzyme
inhibitor 4-phenyl-3-butenoic acid (la).
Of the compounds which retain the chain length of the lead compound la
(group C4) the naphthyl analog le showed coll.p~dble activity against P.
tremulae. 4-Cyclohexylbutanoic acid 14b~, which is the product of complete
reduction of la, showed the same activity against P. tremulae as la, and was also
very active against the blue-stain fungi. The most active product in the C4 group of
derivatives is 4-phenyl-3-butynoic acid (2), which inhibited the growth of all three
fungi by appro~im~tely 50% at 1 ,ug/ml and by 100% at 10 ~Lglml, thus proving tobe a more active and more general fimgicide than la. The methyl ester of la, thederivatives with para-bromo (lc) or methoxy (Id) substit~ nt~> as well as the
cyclo~.Iopa~le analog ~ were completely inactive even at the highest concentration
tested (Table l).
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The chain shortening of la leading to group C3 was less effective. Only the
acids with a triple bond (7), cyclohexyl (8c), or 3-indolyl (8f) substituent exhibited
activity comparable to that of la.
The chain elongation (group C5) proved to be least effective. Sorbic acid
(ga) and its phenyl analog 9b showed only a weak inhibition of O.
crassivaginatum and O. piliferum. Lauric acid (17), although lcnown as filngici~(Byrde, 1969), showed no activity against the three fu~gi even at 1000 ~lg/ml.
Cyclohe~necarboxylic acid (13~ and especially cyclohexylacetic acid (14) were
the most active of the compounds tested. The latter inhibited the growth of all three
fungi by at least 50% at I ,uglml and is superior to pentachlorophenol against P.
tremulae.
The activity of these congeners was co~ ~kd with the activity of the
known fungicides pentachlorophenol (16), copper 8-oxyquinolinate (18), and the
zinc salt of 10-~1n~lec~noic acid (19). Several of the compounds tested showed
activity similar to that of the cornmercial filngicidçs The high toxicity and
pereiet~nce of pentachlorophenol in the en-vho~ cnt for a long period of time has
resulted in its withdrawal from the mar~et. The copper complex 18 and the zinc
salt 19 have sirnilar characteristics. Our results show that some of the congeners of
the enzyme inhibitor la can be used to control P. tremulae, O. crassivaginatum,
and O. piliferum since their efficacy is very similar to that of pentachlorophenol.
These congeners are less toxic, do not contain heavy metals or covalently bound
chlorine, and do not persist in the ellvilullllle~lt as does pentachlorophenol, which
makes them good candidates for use as antifungal agen~s.
B. D~ ali~es of tiglic acid and senecioic acid (Table 2)
Twenty seven compounds which are related to tiglic acid or senecioic acid
were tested against P tremulae, O. crassivaginatum, and O. piliferum. The growth
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of P. tremu/ae was completely inhibited by tiglic acid (20), and the cyclic analogs
31 and 32 at 10 ,ug/ml. Of the p-substituted seneciophenones the methyl derivative
26d showed the highest activity - complete inhibition at 100 llg/ml and 47%
inhibition at 10 ~lg/ml. The ie~t~ g seneciophenones 26~,h~c~ç,f and g, the
~mmorlium salts 21d,e, the wood preservative 22, phenyl senecioate (24), p-
methylacrylophenon~ (28), and compound 29 showed moderate activity at
concentrations 1-1000 llg/ml, while the rlom~inin~ compounds were inactive.
Tiglic acid (20), senecioic acid (21a), and their cyclohexyl analogs 31 and
32 were the most active compounds against O. crassivaginatum (complete
inhibition at 10 ~g/ml), followed by the 4~t~ mmonium salt 21e, the
substituted seneciophenones 26d,f, and p-methylacrylophenone (28), which had
the same efficacy at lO0 llg/ml. Compounds 22, 24, 26c, 26e, and 29 were weakly
active at lO0-1000 ,ug/ml, while the rem~ining co~ ou~lds were inactive.
For O. piliferum 100% inhibition was observed with the parent acids 20 and
1 5 21a, with the substituted seneciophenones 26b,c,f, as well as with compounds 31
and 32 at lO ,ug/ml. For p-methylseneciophenone (26d) the inhibition
concentration was 100 ~g/ml, while conl~ou~lds 21d, 21e, 22, 24, 26a, 26e, 26g,
and 28 exhibited weaker activity at concentrations 1-1000 ~lg/ml. The metal salts
21b, 21c, benzophenone oxime (23a), its ester 23b, the senecioic acid amides 25a,
25b, 25c, the Mannich base 27, as well as colll~ounds 29 and 30 were completely
inactive at 1000 ~lg/ml.
The most active compounds reported in Table 2 include the acids 20, 21a,
31, and 32 (against all three fiungi), and the p-sllb~l;L~-Ie~1 seneciophenones 26d and
~6f (against the blue stain fungi O. crassivaginatum and O. piliferum), and 26b
(against O. piliferum) which show efficacy superior to tbat of the commercial
fungicides 22 and 23a. In addition the above compounds have a low ms-mm~ n
toxicity (Gurova et al. 1985) which makes them good candidates for chemiç~l
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W O 97/48279 PCTnB97/00750
protection of aspen wood.
Example 6. Activity against conifer decay and stain fungi (Tables 4 and
5~
Selected most active against aspen pathogens compounds showed varied
inhibitory effect on the in vitro growth of two conifer decay fungi Haematostereum
sanguinolentum and Phellinus pini. The most active compounds against ~1:
sanguinolentum were la, 4b, 8c, 14, 20, 21a, 22, and 26b (Table 4). Complete
inhibition at 100 llglml was observed for 8c, while la, 4b, 14, 20, 21a, 26b, and
the commçrcial fim~ici~e 22 showed 45-65% inhibition at this concentration. For
1 O P. pini only compounds 4b, 8c, 8f, 20, and 21a showed activity comparable to that
of the cornmercial fungicide 18 (Table 4). These compounds were also inhibitory
against two species of conifer blue stain fungi Ophiostoma clavigerum and 0. ips.
For 0. clavigerum the growth was completely arrested by compounds la, 14, 18,
and 22 at 100 ,ug/ml, while 4b, 8c, 8f, 20, and 21a, showed 50-80% inhibition atthe same concentration. For 0. ips compounds la and 8c (10û% inhibition at 100
,ugtml), as well as 4b, 14, 20, and 21a (25-75% inhibition at the same
concentration) were superior to the commercial fungicides 18 and 22 (Table 5).
lh.Y~lnrle 7. Activity against fairy ring fungus Mur~~,.i~ oreades
(Tables 6 and 7)
Several of the compounds described in Tables 1 and 2 were also tested
against the fairy ring fungus Marasmius oreades (Table 6). Of the compounds
tested for their ability to inhibit the growth of Marasmius oreades the most potent
inhibitors were structures la, 21a, 14, 13, 32, 8f, and 35. Structures 19 and 26d
were also inhibitory but for this o~ sln they were not as potent.
Combinations of compounds were also tested for their ability to ;nhibit
Marasmius oreades (Table 7). The combinations of equal amounts of benzoic acid
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W O 97/48279 PCT~B97/00750
(35) with la, 21a, 13, 14 or 8f resulted in the inhibition of growth more than or
equal to the inhibition of la, 21a, ~3, 14 or 8f alone and significantly more
inhibition than 35 alone. There was a potentiation observed when the compounds
were combined~
5 l~eferences:
Ayer, W.A., Jim~n~7 L.D., 1994, Phomalone, an antifungal metabolite of Phoma
etheridgei, Can.J.Chem. 73, 2326-2332.
Bradbury, A.F., Mistry, J., Roos, B.A., Smyth, D.G., 1990, 4-Phenyl-3-butenoic
acid, an in vivo inhibitor of peptidylglycine hydrolase (peptide amidating
1 0 enzymes), Eur.J.~iochem. 189(2), 363-370.
Bragole, l~.A., Shepard, R.A., 1960, Pl~ ~dlion of certain 4-acylphenols, J.Org.Chem., 25, 1230-1232.
Byrde, R.J.W., 1969, Non~.,~ Lic organics, in Fungicides, Ed. D.C.Torgeson, v.
2, p.531, 568, ~c~d~rnic Press.
1 5 Chakravarty, P., Hiratsuka, Y., 1994, Evaluation of Lecythophora hoffinannii as a
potential biological control compound against a blue stain fungus on Populus
tremuloides, Zeit. Pfl~n7~nkr Pfl~qn7.~n ,101, 74-79.
Chakravarty, P.,Trifonov, L., Hutchison, L.J., ~ir~t~llk~ Y., Ayer, W.A., 1994,
Role of Sporormiella similis as a potential bioprotectant of Populus
20 tremuloides wood against the blue-stain fungus Ophiostoma piliferum,
Can.J.For.Res., 24, 2235-2239.
Chak,dv~ Ly, P, Hiratsuka, Y., I 992, Antagonism of two decay fungi, Peniophora
polygonia and Phellinus tremulae associated with Populus tremllloides,
Eur.J.For.Path., 22, 354- 361.
25 Dev, S., 1956, Organic reactions with polyphosphoric acid. Intermolecular
acylation, J.Indian Chem.Soc., 33, 703-708.
Fodor, G., Tomoskosi, I., 1961, The reaction of carbetoxyme~ylenetriphenyl-
phosphorane with ketones. Tetr.Letters, 579-582.
-28 -
.
CA 02230102 1998-02-20
W 097/48279 PCT~B97/00750
Geiger, W.B., 1948, Antibacterial tmsaturated ketones and their mode of action,
Arch.
Biochem. 423435.
Gurova, A.I., Smolyar, N.Y., Dro7h7hin~, N.A., 1985, Toxicological
5 characteristicsand substantiationofm~ x ~ pP~ni~ihleconcentrationsof
- di~ ylacrylic acid and ethyl methacrylate in workplace air,
Gir.Tr.Pro~Zabol., 4, 52-53 (Chemical Abstracts: 103: 33135w).
~ir~tmk~, Y., Chakravarty, P., Miao, S., Ayer, W.A., 1994, Potential ~or biological
protection against blue stain in Populus tremuloides with a hyphomycetous
10 fiJngus, Stachybotrys cylindrospora, Can.J.For.Res., 24, 174-179.
Hiratsuka, Y., Gibbard, D.A., Bakowsky, O., Maier, G.B. 1990, Classification andmea~ enl of aspen decay and stain in Alberta. Northern For. Cent., Can. For.,
Canada. Info. Report NOR-X-314, pp29.
Hiratsuka, Y., Stokes, T., Chah~ y, P., Morgan, D., 1995, A field guide to
1 5 classify and measure aspen decay and stain. Can. For. Service, Northern For. Cent. C~n~ Special Report4, pp. 27.
Hodgins, D.S., 1971, Yeast Phenylalanine arnmonia-lyase. Purif~lcation, properties
and the id~ntific~tion of catalytically f~ss~nti~ dehydro~l~nine, J.Biol.Chem.
246(9), 2977-2985.
20 ~tlht~n~-n, C.N., Trenchard, H., Milnes-McCaffrey, L.,1985, Inhibition of
Clostridiurn bot~llin--m in cc,.l....;r~ l bacon by Short-Chain Alkynoic and
Alkenoic Acids and Esters.J. Food Protection 48 (7), 570-573.
Hutchison, L.J., Chakl~v~Ly, P., Kawchuk, L.M., Hiratsuka, Y., 1994, Phoma
etheridgei sp. nov. from black galls and cankers of trembling aspen (Populus
25 tremuloides) and its potential role as a bioprotectant against the aspen decay
pathogen Phellinus tremulae, Can.J.Bot., 72,1424-1431.
..r.,.~..,., A., 1909, ~ber Acylieren von Aminen und Phenolen, ~3erichte, 42,
3480-3483.
- K~hrrn~nn, F., 1895, Uber raumisomere Esters von Chinondioximen, Berichte, 28,
30 340-343.
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Khristov, K, lg73, Effect of ~nti~minoacids on Trichomonas vaginalis,
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CA 02230102 1998-02-20
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CA 02230102 1998-02-20
W O 97/48279 PCT~B97/00750
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CA 02230102 1998-02-20
WO 97/48279 PCT~B97/00750
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-39-
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PCT~B97/00750
Table 6: Ef~ect of compounds on the in v~ro growth of M~rLb"~iuS oreades
# Compound name ~mcen~r~tinn ~llg/mL)'
100 1000
la ~Phenyl-3-butenoic acid 15.0b 100.0a100.0a100.0a
8f 3-(3-Indolyl)propanoic acid 20.0c 57.0b100.0a100.0a
13 ~yolf~b ~ -lJu~ylic acid - 17.5b 100.0a
14 (~cloh~yla~ ic acid 12.5c 35.0100.0a 100.0a
lg l0-Untl~c~noir ~id Zn salt - - - 27.5
21a Seneciûic acid 37.5b 100.0a100.0a100.0a
21c S~ eioic acid Zn salt
26d p-M~ yl~ e~io~hf .-on~ - 10.0b 45.0a
26f 2,~Dihydroxyseneciophenone
32 Cyclohexyli~lf ~ ic acid 5.5c 12.0b100.0a100.0a
R.o.n7:0ic. ~id - 17.540.5b 10.0aa
' Values are means of 10 replicates. l:)ata analyzed by one-way ANOVA and Scheffe's test for
m~ iple COIl~y~iSOI~ after Arscine Tran~rul~LLon. Values in each row followed by t~e sarne
letter do not differ ~i~nifc~ntly (p=o~o5)~
~o--
CA 02230102 1998-02-20
W O 97/48279 PCT~B97/00750
Table 7: Effect of Benzoic acid alone and in cQ~ t~ with other compounds on the in
r~ro growfh of MUr~~ L~S oreades
Co~l~uul.d name cQl~re~ n (,ug/mL)~
100 500 1000
la ~Phenyl-3-butenoic acid 25c 35b lOOa lOOa lOOa lOOa lOOa
+35 ~ Benzoic acid
21a Senecioic ~id 45c 65b lOOa lOQa lOOa lOOa lOOa
+35 + Benzoic acid
14 Cyclohexylacetic acid lSd 45c 68b lOOa lOOa lOOa lOOa
+35 + Benzoic ~id
13 Cyrloh.ox~,.r~ ~,boxylic acid lOe 21d 45c 75b lOOa lOOa lOOa
+35 + Benzoic acid
8f 3-(3-Indolyl)propanoic acid 35d 65c 75b lOOa lOOa lOOa lOOa
+35 ~ Benzoic acid
la ~Phenyl-3-butenoic acid 14c 65b lOOa lOOa lOOa lOOa lOOa
21a Senecioic acid 37c 75b lOOa lOOa lOOa lOOa lOOa
14 Cyclohexylacetic acid 13c 35b 35b lOOa lOOa lOOa lOOa
13 Cy~loh~-x~.~rc;1,bo,~ylic acid Sd lOc llc 75b lOOa lOOa lOOa
8f 3-(3-Indolyl)propanoic acid 20e SOd 56c 88b lOOa lOOa lOOa
Benzoic acid - - lSc 4~b lOOa lOOa lOOa
' Values are means of 10 replicates. Data analyzed by one-way ANOVA and Scheffe's test for
multiple coll~L)~ison after Arscine Transforrnation. Values in each row followed by the same
letter do not differ significantly (P=0.05).
Concentrations refer to that of the ~lrst coll~u..ent in the two component mixtures.
Benzoic acid is added in equal amounts.
The media was malt extract.
--41--
