Spectrum of Activity of Antifungal Natural Products and Their Analogs Stephen R.. Cutler CONTENTS 15.1 Introduction 15.2 The Synthesis of 6-pentyl-2H-pyran-2-one 15.3 Structure–Activity
Trang 1Spectrum of Activity of Antifungal Natural
Products and Their Analogs
Stephen R Parker, Robert A Hill, and Horace G Cutler
CONTENTS
15.1 Introduction
15.2 The Synthesis of 6-pentyl-2H-pyran-2-one
15.3 Structure–Activity Relationships of Natural Analogs
15.4 Synthetic Analogs
15.5 Closing Remarks
Acknowledgments
References
ABSTRACT Synthetic and naturally occurring analogs of the Trichoderma metabolite 6-pentyl-2H-pyran-2-one have been tested for their activity against a range of filamentous
fungi Candidates for development as “natural” or “soft” fungicides have been identified
15.1 Introduction
The Trichoderma metabolite 6-pentyl-2H-pyran-2-one (I) is a deceptively simple molecule
(Figure 15.1) Interestingly, its chemical synthesis was achieved before it was identified as
a natural product In 1969 Nobuhara reported its synthesis in one of a series of papers examining the organoleptic properties of γ- and δ-lactones.1-4 The synthetic compound is available today as a “nature identical” product supplied by certain flavor and fragrance manufacturers It is used as a food additive for modifying flavor/aroma The Flavor and
Extract Manufacturers’ Association (FEMA) monograph for I (FEMA 3696) cites its use in
a wide range of food stuffs, including baked goods, cheese, and confectionery The com-pound has an aroma described variously as similar to coconut or mushroom
In 1971 Denis and Webster5 demonstrated the production of volatile antibiotics by
Tricho-derma isolates The authors reported that the active isolates “were all characterized by a
def-inite ‘coconut’ smell.” However, they also noted that not all the isolates that produced this aroma had antagonistic activity by “vapor action,” and the antibiotic activity was
tenta-tively assigned to the production of acetaldehyde A year later, I was identified as a major
aroma constituent of Trichoderma viride.6 A direct assessment of the antifungal activity of the compound was not performed However, the temporal proximity of these two publications
Trang 2has led to confusion as to whether or not I is a volatile antifungal agent Direct investigations
of the vapor action of I have been performed, and vapor mediated phytotoxicity has been
observed in vitro.7,8 It is worth noting that the vapor pressure of I is around 0.006 mmHg at
20°C Claydon et al (1987) questioned whether the phytotoxicity observed in vitro would
be of significance in the soil environment
A direct demonstration of the antifungal activity of I was first referred to in 1983.9 The compound was tested in a standard agar diffusion assay following its purification from
cultures of a Trichoderma harzianum isolate observed to be growing profusely over the sur-face of Slash Pine (Pinus elliottii Engelm.) logs.10 Interestingly the compound was initially isolated by bioassay directed fractionation on the basis of its plant growth regulatory activ-ity in the etiolated wheat coleoptile assay.11 The purified metabolite was subsequently assayed for antifungal activity As an aside, in 1984 a European patent application was filed
for the use of Trichoderma harzianum, and/or the products of its culture, as biocontrol agents
for the control of plant pathogens.12 Cited in its claims was the use of I as a phytosanitary
product This application subsequently lapsed
The natural occurrence of I is now widely recognized and it has been identified as a
com-ponent of fruit volatiles such as nectarines,13,14 peaches,15 and plums.16 Its production also
has been noted for other genera of fungi including Aspergillus.17 It is unlikely that its
anti-fungal activity is of any significance at the concentrations of I observed in fruit However, the natural occurrence of I, its established use as a food additive, and its relatively simple
chemical structure make it an attractive candidate for development as a “natural fungi-cide” However, as alluded to above, appearances can be deceptive
15.2 The Synthesis of 6-pentyl-2H-pyran-2-one
The original synthetic route of Nobuhara is laborious.3 A number of alternative synthetic routes have since been published.18-20 The route proposed by Pittet and Klaiber (1975) is a
two-step procedure for the preparation of I (Figure 15.2).18 Consideration of the synthetic
pathway illustrates why the synthesis of I is problematic Although the route is simple, the
preparation of one of the key reagents, methyl 3-butenoate, is difficult due to conjugation
FIGURE 15.1
Structure of 6-pentyl-2H-pyran-2-one (I) and its analogs: massoialactone (II),γ-decalactone (III), δ-decalactone (IV),γ-dodecalactone (V), and δ-dodecalactone (VI) (From Parker, S.R., J Agric Food Chem., 45, 2774-2776, 1997.
With permission.)
Trang 3of the carbonyl and olefinic bonds being favored Although vinyl acetic acid is readily available, its esterification under standard conditions of alcohol and acid will permit migration of the terminal olefinic bond Therefore, other methods for the preparation of methyl 3-butenoate need to be employed.21-23
The employment of a “nonstandard” method for lactonization of the mixed keto-acids obtained from the Friedel-Crafts acylation may be related to the structural requirements of
an acyclic intermediate for the synthesis of I The olefinic bonds of such an acyclic
precur-sor are required to be in a cis-trans configuration Whereas, if a trans-trans configuration is
adopted, the lactonization will not occur (It is interesting to consider how such a step is
achieved biosynthetically by Trichoderma where it would be reasonable to assume that I is
derived from a single acyclic precursor molecule.) By comparison the lactonization of 5-hydroxydecanoic acid to form the corresponding δ-decalactone is spontaneous in the presence of acid
Recognizing that these difficulties in the preparation of I might represent obstacles to its
commercial development as a natural fungicide, we were prompted to consider what other structurally related candidates could be examined for this application We sought
com-pounds that shared the favorable attributes of I, but were readily available and less costly.8
15.3 Structure–Activity Relationships of Natural Analogs
Earlier structure–activity relationships determining the antifungal activity (by agar
diffu-sion) of a range of synthetic analogs of I demonstrated that the structural requirements for
activity appeared to be stringent.24 Shortening of the 6-alkyl substituent resulted in a marked loss of activity, as did saturation of the ∆2-bond of the pyrone ring (Figure 15.3)
The 6-pent-1-enyl substituted analog of I,25 which is frequently observed as a co-metabolite
of I in Trichoderma cultures, had activity comparable with that of I.
FIGURE 15.2
Synthetic scheme for the preparation of 6-pentyl-2H-pyran-2-one (I) (Adapted from Pittet, A.O and Klaiber,
E.M., J Agric Food Chem., 23, 1189, 1975.)
Trang 4Extending these studies to a group of compounds that were all available commercially and used as food flavoring compounds, we were surprised to observe the greater
antifun-gal activity of massoialactone (II) relative to I (Figure 15.4) Unlike the synthetic and racemic,
saturatedγ- and δ-lactones (III-VI) tested, II is a purified botanical extract obtained from
the bark of the tree Cryptocaria massoia Like I, it has a potent flavor and its use is cited in a
similar range of processed foods (FEMA 3744) The compound is the main component (as
FIGURE 15.3
Summary of structure activity relationships for selected compounds in an agar diffusion-based antifungal assay (Adapted from Dickinson, J.M., Ph.D thesis, University of Sussex, U.K., 1988.)
FIGURE 15.4
Antifungal activity of 6-pentyl-2H-pyran-2-one (I) and its analogs (II–VI) in an agar diffusion assay Suspensions
of Penicillium spores (Solid — P digitatum, coarse shading — P expansum, fine shading — P italicum) were
prepared by washing PDA slopes with two 5 ml volumes of aqueous sterile 0.1% (v/v) Tween 80 The spore density of the combined volumes was determined for a 20-fold dilution using an improved Neubauer hemocy-tometer The calculated volumes of spore suspension required to yield final spore densities of 10 5 , 10 6 , and 10 7
transferred to each of the wells of a six-well microtitre plate (Nunc) and allowed to solidify Solutions of test compounds were prepared in acetone at a concentration of 25 mg ml –1 Twenty microliters of each test compound solution, containing 500 nl (c 500 µg) of each test compound, was applied to a 5 mm diameter sterile filter paper (Whatman No.1) disc After allowing the solvent to evaporate, the impregnated filter paper disc for each of the test compounds was placed at the center of each of a well Plates were incubated for 48 h at 20°C The diameters
(d) of the resulting zones of total inhibition were measured and recorded (From Parker, S.R., J Agric Food Chem.,
45:7, 2775, 1997 With permission.)
Trang 5judged by gas chromatography) of massoia bark oil (FEMA 3747) More recently, the micro-bial production of this metabolite has been reported in yields anticipated to make the bio-synthetic production of this chiral molecule economical.26,27 The compound therefore has
all the favorable attributes of I, with greater in vitro antifungal activity and the potential for
economical production as a “natural”
Early trials of II alerted us to the potential phytotoxicity of this compound When applied
to leaf surfaces as a 1.0% (v/v) aqueous emulsion, localized tissue necrosis was observed within 24 h of application However, it was noted that the same effects were observed with
each of the lactones (I, III-VI) when applied in this manner over the same concentration
range The phytotoxicity was not systemic as judged by the continued healthy growth of untreated parts of the plant This “nonspecific” mode of phytotoxicity contrasted with the relative activity of the compounds in both the etiolated wheat coleoptile assay and the let-tuce seed germination assay (Figure 15.5)
Seeking an application where the potential phytotoxicity of II would not be an issue, we
evaluated the compound for its ability to control sapstain in sawn timber (Pinus radiata).
Sapstain, as its name implies, is a staining of the sap wood of sawn timber It is caused by
a heterogeneous collection of fungi that grow through the wood and become pigmented, thus degrading its visual appearance Marked differences were observed between the
rel-ative ability of I, II and δ-decalactone (IV) to control the development of sapstain in a
lab-oratory based trial (Figure 15.6) These results are particularly striking when one considers that each compound in the series differs only in its degree of desaturation
FIGURE 15.5
Assessments of phytotoxicity of 6-pentyl-2H-pyran-2-one (I), massoialactone (II),γ-decalactone (III), δ-decalac-tone (IV),γ-dodecalactone (V), and δ-dodecalactone (VI) (From Parker, S.R., J Agric Food Chem., 45:7, 2776,
1997 With permission.)
Trang 615.4 Synthetic Analogs
An alternative approach to examining the structure–activity relationships of naturally
occurring analogs of I, was to assess the synthetic obstacles to the economical production of
I and determine what, if any, synthetic analogs could be prepared more readily The 4-methyl
substituted analog of I; 4-methyl-6-pentyl-2H-pyran-2-one (VII), may be prepared by a
route analogous to that proposed for the synthesis of I by Pittet and Klaiber (1975)
(Figure 15.7).28In the preparation of this methyl substituted analog difficulty in prepara-tion of the esterified reagent is mitigated, and lactonizaprepara-tion of the mixed keto-acids formed
by the Friedel-Crafts acylation of hexanoyl chloride proceeds under standard conditions
The ease with which VII could be prepared was confirmed and the vacuum distilled
product tested for antifungal activity The in vitro activity of VII was comparable with that
of I (Figure 15.8) Recognizing that although innate biodegradability is an attractive aspect
of natural products for use as agrochemicals, too short a biological half-life may render
their use impractical Structural modification of the “lead compound” (I) in this manner
may yield compounds of practical use in the field, both in terms of their relative cost and rate of biodegradation
15.5 Closing Remarks
The research reviewed here serves to underscore the adage that bioactive natural products
serve as “lead compounds” for discovery From the identification of I as a “natural
fungi-cide” two promising candidates for further development have been identified Along the way we are generating data that will help us understand the key structural requirements that define antifungal activity for this family of compounds However, we should be cau-tious not to be too simplistic in our approach Although a simple molecule, the behavior of
FIGURE 15.6
Control of sapstain by 6-pentyl-2H-pyran-2-one (I),
mas-soialactone (II), and δ-decalactone (IV) Freshly sawn wood
blocks (50 × 50 × 7 mm) were sterilized by γ-irradiation.
Blocks were dipped individually in a 1% (v/v) emulsion of
test compound prepared in sterile 0.1% (v/v) Tween 80.
Each block was dipped for 30 seconds with gentle agitation
and then placed on edge and allowed to drain Single blocks
were inoculated with 200 µl of a spore suspension (c 10 6
spores ml –1 ) of sapstaining organisms FK64 and FK150 and
placed in 500 ml glass jars Each glass jar contained a filter
paper disc moistened with 2 ml sterile distilled water and
was sealed Wood blocks were not in direct contact with the
filter paper discs Ten wood blocks were employed per
treat-ment set The wood blocks were incubated at 25°C for 7 to
10 days and scored for the presence or absence of sapstain.
Trang 7FIGURE 15.7
Synthetic scheme for the preparation of 4-methyl-6-pentyl-2H-pyran-2-one (VII) (cf.Figure 15.2 ) (Adapted from
Lohaus, G et al., Chem Ber., 100, 658, 1967.)
FIGURE 15.8
Spore suspensions were prepared by washing sporulating plates or slopes of the test organism with 10 ml sterile 0.1% (v/) Tween 80 The spore density of the aspirated volume was determined using an improved Neubauer hemocytometer The spore suspension was used to inoculate molten potato dextrose agar (PDA) maintained at
45°C (Solid — Penicillium digitatum at 106 spores ml –1, coarse shading — Botrytis cinerea at 105 spores ml –1 , fine
shading — Monilinia fructicola at 104 spores ml –1 ) Ten milliliters of the inoculated PDA was poured over the surface of a petri dish (90 mm dia.) containing a uniform base layer of 10 ml 1% (w/v) water agar and allowed
to solidify Solutions of test compounds (6-pentyl-2H-pyran-2-one (I), 4-methyl-6-pentyl-2H-pyran-2-one (VII),
or 4,6-dimethyl-2H-pyran-2-one (VIII)) were prepared in acetone and applied to sterile 6 mm diameter filter
paper discs (Whatman No 3) After allowing the solvent to evaporate, the impregnated filter paper discs were placed on the surface of the solidified agar Three discs were used per plate placed equidistant from each other and the center of the plate Plates were incubated at 25°C for 24 h and the diameters (d) of the resulting zones
of inhibition measured.
Trang 86-pentyl-2H-pyran-2-one is complex Understanding its mode of action may prove more challenging than one might anticipate, if indeed 6-pentyl-2H-pyran-2-one is the
physiolog-ically relevant species and not simply an artifact of our extraction methods
ACKNOWLEDGMENTS: The authors wish to thank Dr George Majetich and Paul Spearing
of the University of Georgia, Athens, for providing samples of 6-methyl-, 6-propyl-, and
6-hexyl-2H-pyran-2-one for testing Technical assistance was provided by Philip Sale The research was
funded in part by the Foundation for Research, Science and Technology, Wellington, New Zealand.
References
1 Nobuhara, A., Syntheses of unsaturated lactones I Some lactones of 5-substituted-5-
hydroxy-2-enoic acids as a synthetic butter or butter cake flavor, Agric Biol Chem., 32(8), 1016, 1968.
2 Nobuhara, A., Synthesis of unsaturated lactones II Flavorous nature of some 4- and
5-substituted 5-hydroxy-2-enoic acid lactones, Agric Biol Chem., 33(2), 225, 1969.
3 Nobuhara, A., Unsaturated lactones III Flavorous nature of some δ-decalactones having the
double bond at various sites Agric Biol Chem., 33(9), 1264, 1969.
4 Nobuhara, A., Syntheses of unsaturated lactones IV Flavorous nature of some aliphatic
γ-lactones Agric Biol Chem., 34(11), 1745, 1970.
5 Denis, C and Webster, J., Antagonistic properties of species-groups of Trichoderma II Produc-tion of volatile antibiotics, Trans Br Mycol Soc., 57(1), 41, 1971.
6 Collins, R.P and Halim, A.F., Characterization of the major aroma constituent of the fungus
Trichoderma viride (Pers.) J Agric Food Chem., 20(2), 437, 1972.
7 Claydon, N., Allan, M., Hanson, J.R., and Avent, A.G., Antifungal alkyl pyrones of Trichoderma
harzianum, Trans Br Mycol Soc., 88(4), 503, 1987.
8 Parker, S.R., Cutler, H.G., Jacyno, J.M., and Hill, R.A., The biological activity of 6-pentyl-2H-pyran-2-one and its analogs, J Agric Food Chem., 47(7), 2774, 1997.
9 Cutler, H.G., Biologically active natural products from fungi: templates for tomorrow’s
pes-ticides, in Bioregulators, Chemistry and Uses, Ory, R.L and Rittig, F.R., Ed., American Chemical
Society, Washington, D.C., 1984.
10 Cutler, H.G., Cox, R.H., Crumley, F.G., and Cole, P.D., 6-Pentyl-α-pyrone from Trichoderma
harzianum: its plant growth inhibitory and antimicrobial properties, Agric Biol Chem., 50(11),
2943, 1986.
11 Cutler, H.G., A fresh look at the wheat coleoptile bioassay, in Proc 11th Annual Meeting of the
Plant Growth Regulator Society of America, Boston, 1984.
12 Merlier, O.A.M., Boirie, M.J., Pons, B.J., and Renaud, C.M., European Patent Application
EP84-400545, 1984.
13 Engel, K.-H., Flath, R.A., Buttery, R.G., Mon, T R., Ramming, D.W., and Teranishi, R., Inves-tigation of volatile constituents in nectarines 1 Analytical and sensory characterization of
aroma components in some nectarine cultivars., J Agric Food Chem., 36, 549, 1988.
14 Engel, K.-H., Ramming, D.W., Flath, R.A., and Teranishi, R., Investigation of volatile
constit-uents in nectarines 2 Changes in aroma composition during nectarine maturation., J Agric.
Food Chem., 36, 1003, 1988.
15 Horvat, R.J., Chapman, G.W., Robertson, J.A., Meredith, F.I., Scorza, R., Callahan, A.M., and Morgens, P., Comparison of the volatile compounds from several commercial peach cultivars,
J Agric Food Chem., 38, 234, 1990.
16 Horvat, R.J., Chapman, G.W., Jr., Senter, S.D., Robertson, J.A., Okie, W.R., and Norton, J.D.,
Comparison of the volatile compounds from several commercial plum cultivars, J Sci Food
Agric., 60(1), 21, 1992.
Trang 917 Kikuchi, T., Mimura, T., Harimaya, K., Yano, H., Arimoto, T., Masada, Y., and Inoue, T., Volatile metabolite of aquatic fungi Identification of 6-pentyl-α-pyrone from Trichoderma and
Aspergil-lus species, Chem Pharm Bull., 22(8), 1946, 1974.
18 Pittet, A.O and Klaiber, E.M., Synthesis and flavor properties of some alkyl-substituted α-pyrone
derivatives, J Agric Food Chem., 23, 1189, 1975.
19 Dieter, R.K and Fishpaugh, J.R., Synthesis of α-pyrones from vinylogous thiol esters and α-oxo
ketene dithioacetals, J Org Chem., 53, 2031, 1988.
20 Zhang, C., Wang, X.C., Zhang, F.N., and Pan, X.F., A facile total synthesis of 6-
pentyl-α-pyrone, Chin Chem Lett., 7(4), 317, 1996.
21 Matsamura, J., Japanese Patent 43-29,924, 1968.
22 Montino, F., Ger Offen 1936725, 1970.
23 Scarborough, R.M., Jr and Smith, A.B., III, An efficient general synthesis of o-olefinic methyl
esters, Tetrahedron Lett., 50, 4361, 1977.
24 Dickinson, J.M., Ph.D thesis, University of Sussex, U.K., 1988.
25 Moss, M.O., Jackson, R.M., and Rogers, D., The characterization of 6-(pent-1-enyl)-α- pyrone
from Trichoderma viride, Phytochemistry, 14, 2706, 1975.
26 Kurosawa, T., Sakai, K., Nakahara, T., Oshima, Y., and Tabuchi, T., Extracellular accumulation
of the polyol lipids, 3,5-dihydroxydecanoyl and 5-hydroxy-2-decenoyl esters of arabitol and
mannitol, by Aureobasidium sp., Biosci Biotech Biochem., 58(11), 2057, 1994.
27 Hiroyuki, O., Nobuhisa, S., Hiroshi, H., and Junko, T., Japanese Patent Application 07212318, 1997.
28 Lohaus, G., Friedrich, W., and Jeschke, J.P., Aufbaureaktionen mit β.β-dialkyl-acrylsäureestern,
Chem Ber., 100, 658, 1967.
29 Kurtz, T.E., Link, R.F., Tukey, J.W., and Wallace, D.L., Short-cut multiple comparisons for
balanced single and double classification: part 1, results, Technometrics, 7, 95, 1965.