BIOLOGICALLY ACTIVE NATURAL PRODUCTS: AGROCHEMICALS - CHAPTER 15 doc

15
Spectrum 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 Webster
5
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
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has 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.)
© 1999 by CRC Press LLC
of 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.)
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Extending 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,
saturated γ- 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
spores ml
–1
were added to 20 ml volumes of molten PDA. For each spore concentration 3 ml aliquots were
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.)
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judged 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.)
© 1999 by CRC Press LLC
15.4 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).
28
In the preparation of this methyl substituted analog difficulty in prepara-
tion of the esterified reagent is mitigated, and lactonization 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.
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FIGURE 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 10
6
spores ml
–1
, coarse shading — Botrytis cinerea at 10
5
spores ml
–1
, fine
shading — Monilinia fructicola at 10
4
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.
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6-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.
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