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 © 1999 by CRC Press LLC 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.) © 1999 by CRC Press LLC 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.) © 1999 by CRC Press LLC 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. © 1999 by CRC Press LLC 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. © 1999 by CRC Press LLC 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. 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. © 1999 by CRC Press LLC 17. 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. © 1999 by CRC Press LLC . 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. 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) Press LLC 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