MXPA96001913A - Fungicide agents ll-15g256-gamma, ll-15g256-delta and ll-15g256-epsilon produced by ll-15g256hypoxylon oceanic - Google Patents

Abstract

This invention provides fungicidal compounds of formula I or II: wherein R, R1 and R2 are defined in the specification produced by culture LL-15G256 (Hypoxylon oceanicum), and their production by fermentation and use as fungicide agents

Description

FUNGICIDE AGENTS LL-15G256-GAMMA, LL-15G256-DELTA and LL-15G256-EPSILON PRODUCED BY L-15G256 (HYPOXYLON OCEANICUM) «FIELD AND BACKGROUND OF THE INVENTION This invention relates to the fungicidal compounds produced by the culture LL-15G256 (Hypoxylon oceanicum), and to its production by fermentation. The isolation and purification of the active compounds produced by the culture have produced fungicidal compounds designated LL-15G256-gamma, LL-15G256-delta, LLL 5G256-epsilon as well as other minor components.

BRIEF DESCRIPTION OF THE INVENTION The invention provides the technique with fungicidal compounds of Formula I and II: Formula I Formula II where n is 3 to 10; R is hydrogen, alkyl of 1 to 4 carbon atoms or a pharmaceutically acceptable salt cation selected from the group consisting of sodium, potassium, calcium, lithium, magnesium, ammonium and tetra- (alkyl of 1 to 4 carbon atoms) ) -ammonium; R is hydrogen or methyl; and 2 R is hydrogen or methyl. This invention also provides a process for the production of fungicidal compounds of Formula I and II by aerobic fermentation of the fungus LL-15G256; a biologically pure culture of the fungus LL-15G256; the composition containing the compounds of Formula I and II, and methods for the protection of plants against pathogenic fungi for plants.

DRAWINGS Figure 1: Ultraviolet absorption spectrum for LL-15G256-gamma, LL-15G256-delta, and LL-15G256-epsilon. Figure 2: Proton nuclear magnetic resonance spectrum for LL-15G256-gamma. Figure 3: Proton nuclear magnetic resonance spectrum for LL-15G256-delta. Figure 4: Proton nuclear magnetic resonance spectrum for LL-15G256-epsilon.

DETAILED DESCRIPTION OF THE INVENTION Of particular interest are four depsipeptides designated LL-15G256-gamma, LL-15G256-delta, LL-15G256-epsilon and methyl ester of LL-15G256-gamma, which have the following physicochemical characteristics: LL-15G256-gamma a) Apparent molecular formula: C 'H ,, N, 0q; b) Molecular weight: MS (FAB) = m / z 643 (M + H) + HRMS calculated for C33H46N4 ° g = m z 643.3343 HRMS observed = m / z 643.3349; ? mmu = 0.6; c) Specific Rotation: [alpha] = +22.9 +1, (c = 1.07%, MeOH); d) Ultraviolet Absorption Spectrum: as shown in Figure 1; Amax nm (e) (MeOH) = 206 (41,710), 245 (16,820), 264 (12,275), 307 (16,100); e) Infrared Absorption Spectrum: 3335, 3121, 2957, 2927 (s), 2855 (s), 1723 (s), 1681 (s), 1664 (s), 1645 (s), 1534, 1518 (s), 1459, 1430, 1376 (s), 1319, 1266, 1245, 1227, 1194, 1115, 748 cm "1; f) Magnetic Resonance Spectrum of Protoons: as shown in Figure 2 (300 MHz, d, -DMS0) g) Nuclear Magnetic Resonance Spectrum of carbon 13 (300 MHz, d, -DMS0, low field ppm from TMS) The significant peaks are listed below: 184.9 174.9 173.5 171.9 169.0 167.4 136.9 136.6 125.5 123.3 122.4 121.1 113.4 112.4 78.39 62.04 58.83 54.92 51.89 41.21 33.36 33.15 31.23 29.93 9.14 28.96 28.64 26.67 4.91 22.04 16.08 13.89 3.42 h) Analysis calculated for C 33H4.6, N4.0 ~ 9; Theory C - 61.68; H = 7.17; N = 8.72 Found: C-61.70; H - 7.83; N - 7.37 15G256-delta a) Apparent Molecular Formula: '-'33 ^ 6 ^ ^ 10' b) Molecular Weight: MS (FAB) = m / z 659.3 (M + H) +; MS (FAB) = m / z 681.3 (M + Na) + HRMS calculated for C33H46N4 ° 10Na = m ^ z 681.3112 HRMS observed = m / z 681.3119; ? mu = -0.7 mmu; c) Ultraviolet Absorption Spectrum: as shown in Figure 1; ? max nm (e) MeOH = 206 (33,720), 246 (13,600), 262 (9,925), 310 (13,100); d) Proton Magnetic Resonance Spectrum: as shown in Figure 1 (300 MHz, dft-DMS0); e) Carbon 13 Nuclear Magnetic Resonance Spectrum: (300 MHz, dft-DMS0, low field ppm from TMS), the significant peaks are listed below: 185.9 177.0 173.6 172.0 169.2 166.8 137.1 136.6 125.6 123.2 122.2 121.1 114.2 112.4 76.16 64.97 61.78 60.35 54.55 52.76 41.21 40.93 31.26 30.61 30.10 29.46 28.97 28.70 26.34 25.59 22.05 15.51 13.90 1 G256-epsi lon a) Apparent Molecular Weight: C-.H.-N.Ogj b) Molecular Weight: MS (FAB) = m / z 615.2 (M + H) + MS (FAB) = m / z 637.3 (M + Na) + HRMS calculated for C ^ H ^ N ^ OgNa = m / z 637.2849 HRMS observed = m / z 637.2840; ? mmu = +0.9 mmu; c) Spectrum of Ultraviolet Absorption: as seen in Figure 1; ? max nm (=) MeOH = 206 (33,720), 246 (13,600), 262 (9,925), 310 (13,100); d) Infrared Absorption Spectrum: 3350, 3121, 2956 (s), 2928 (s), 1645 (s), 1518 (s), 1455, 1432, 1375 (s), 1339, 1316, 1245, 1192 cm "1 e) Proton Magnetic Resonance Spectrum: as shown in Figure 4 (300 MHz, d, -acetone) f) Nuclear Magnetic Resonance Spectrum of carbon 13 (300 MHz, d, -acetone, ppm of low field from TMS.) Significant peaks are listed below: 185. 3 176.4 174.0 172.1 170.3 168.4 138.0 137.8 127.0 124.4 123.4 122.6 114.8 113.1 79.95 63.25 61. 23 55.89 53.81 43.13 . 14 34.56 32.55 30.40 . 15 27.89 25.99 23.27 16. 77 14.30 14.08 LL-15G256-gamma methyl ester a) Apparent Molecular Weight: C-34, H4,8oN4,0n9: b) Ultraviolet Absorption Spectrum:? max nm (e) (MeOH) = 206 (33,720), 246 (13,600), 262 (9,925), 310 (13,100); c) 13 C Nuclear Magnetic Resonance Spectrum (300 MHz, d ^ -acetonitrile, low field ppm from TMS). Significant peaks are listed below 185.6 176.6 174.1 172.4 170. 6 168.6 138.1 137.8 126. 9 124.9 123.9 122.6 114. 8 113.4 80.8 63.4 61. 6 55.7 54.3 52.3 43. 3 35.7 34.5 32.7 . 9 30.6 30.4 30.1 27. 9 26.0 23.4 17.0 14. 43 14.38 These compounds are a class of depsipeptides which contain a branched-chain beta-hydroxy fatty acid residue and a beta-keto-tryptophan moiety as distinguishing features. The fungicidal compounds LL-15G256-gamma, LL-15G256-delta, and LL-15G256-epsilon, as well as other minor components, are produced by aerobic fermentation of LL-15G256 (Hypoxylon oceanicum). It will be appreciated by those skilled in the art that the present invention includes within its scope the fungicidal compounds of Formula I and II, as well as any other minor components produced. Such compounds can be obtained in diluted form, as crude concentrates, as a complex of all the components or in pure form as individual components. The LL-15G256 in culture has been taxonomically characterized based on the teleomorphic morphology as a strain of Hypoxylon oceanicum. The colonies of this culture grow on oatmeal agar, cover the surface of the medium in the Petri dish in 4 weeks. The growth is initially white, velvety, imprisoned, irregularly marked areas, with full and dense margins, then darkens over the zoning lines. The back side of the culture box is colorless. On discarded wood, the stromae of this crop are superficially settled, occasionally embedded at the base, hemispherically pulverulent, 0.4 - 0.8 mm in diameter, alone or coalesce several of them, linear to suborbicular, with carbonaceous surface, with the inner part with leather appearance. When young, the stroma is covered with a layer of whitish hyphae, at maturity, it presents black peritectial projections in general non-conspicuous, with prominent pores with subglobose perithecia, 0.4 to 0.6 mm in diameter. The toilets have 8 sori, from 177-219 mra sp. and 112-140 mm p., 37-79 mm stipes, dark blue apical apparatus in the Meizer reagent, cylindrical that tapers with the distinctive apical margin, (.7-) 5.6-6. ? > X 4.2-4.7 mm, uniseriate ascospores at obliquely uniseriate or partially biseriate at the upper end of the asea. The ascospores are opaque brown to olive brown, more or less unilaterally ellipsoid, with open sides varying in degree of convex curvature, with a broadly rounded upper end, and slightly dotted lower end, of (17.9-) 18.8-21.6 (-22.6) X 8.5-9.9 mm (average 20.7 X 9.3 mm, n / 20), bigutu-ladas. The wall of the ascospores is soft and relatively thick, without appendages or loose episporas, the germination by division is clearly observed in a usual way on the straight, conspicuous dorsal side, the total length of the spore is 1/2 - 3/4 , the paraphyses are long as thread, remotely septate with droplets. It should be understood that for the production of the novel fungicidal compounds LL-15G256-gamma, LL-15G256-delta, and LL-15G256-epsilon, the present invention is not limited to this particular organism or to organisms that fully respond to the previous growth and microscopic characteristics, which are given for illustrative purposes only. In fact it is desired and intended to include the use of mutants produced from this organism by various means such as exposure to X-ray radiation, ultraviolet radiation, N'-methyl-N-nitroso-guanidine and the like.

BIOLOGICAL ACTIVITY The LL-15G256 gamma and epsilon were tested for fungicidal activity by the micro-dilution method (Table 1). The serial dilutions of the compounds are mixed with the test organisms in a liquid growth medium in the wells of the microtiter plate. After incubation at 28 ° C for 20 hours, the wells are observed for turbidity, the absence of which indicates inhibition of growth.

Table 1 Fungicidal activity (MIC, μg / ml) of 15G256-gamma and 15G256-epsi Ion Organism 15G256-gamma 15G256- Candida albicans 54 > 256 > 256 Saccharomyces cerevisiae 427 > 256 > 256 Ustilago maydis 106 16 32 Table 1 (continued) Organism 15G256-gamma t 15G256-delta Rhodotorula rubra 388 128 128 Candida neoformans 4414 256 256 Neurospora crassa (OS-1) 256 256 Method of dilution in microcalde, medium: YMB, inoculum: 4 approximately 10 colony forming units / ml, incubation at 28 ° C for 20 hours.

In Vivo Evaluation as Fungicide Agents The compounds are dissolved in a 50/50 (v / v) mixture of methanol and acetone, diluted to the desired concentration with water and surfactant, and sprayed onto the test plants. After drying, the test plants are treated with the inoculum of the fungus. When the development of the symptom of the disease is optimal, the plants are qualified to control the disease. Untreated plants inoculated, plants treated with solvent / surfactant and plants treated with a reference standard, are used for comparison.

Test Organism HEADER: COMMON NAME: SCIENTIFIC NAME: AS Venturia apple crust. inaequalis GDM Downy mildew of the grape Plasmopara vitícola PB Botrytis of pepper Botrytis cinerea RB Bore of rice Pyricularia oryzae SBC Cercospora of the remoCercospora beticola lacha azurrera TEB Early tomato rust Alternaria solani WSN Septoria nodorum of the wheat Leptosphaeria nodurum WPM Dusty powdery mildew Erysiphe graminis f.sp. tritici The compounds are qualified for the control of each disease according to the scale shown below:% Disease Control Rating 0 0 1 1 - - 1 4 2 1 5 - -29 3 30- -44 4 45- -59 5 60- -74 6 75- -89 7 90- -95 Rating% of Disease Control 8 96-99 9 100 The results are reported in Table 2.

Table 2 In Vivo Selection Data: CONTROL OF DISEASE BY OBJECTIVE COMP. DOSES AS GDN PB RB SBC TEB WPM WSN ppm 156G256-gamma 500.0 6 9 0 4 7 2 6 0 125.0 3 6 5 0 6 2 0 0 31.3 0 0 0 0 3 0 0 0 The fungicidal compounds of the present invention are effective to control and / or prevent phytopathogenic fungi, when employed in effective amounts. This will vary somewhat with the virulence of the fungus in question and with other factors such as the environment in which the treatment is conducted. These compounds are especially useful for the control of fungi that are the causative agents for grape villous mold and late rust of potato and tomato. Certain compounds of the invention may not only be used to control fungi that have infected the plants, but may also be applied to healthy plants or seeds or to the soil in which the plant is to be planted, in order to prevent infestation. To protect plants against phytopathogenic fungi, the compounds of this invention are applied to the foliage of the plant, to the seed of the plants, or to the soil in which the plant develops or will develop, in the form of a liquid, preferably an aqueous spray, or powder, or granular formation. Solutions or suspensions containing from about 20 ppm to about 1,000 ppm, and preferably from 50 ppm to 500 ppm, of the compounds of this invention, are generally effective for this use. The compounds of the invention can be formulated as emulsifiable concentrates, concentrates capable of flowing, or wettable powders that are diluted with water or another appropriate polar solvent, in general if situ, and then applied as a diluted spray. Said compounds can also be formulated in dry compacted granules, in granular formulations, powders, powder concentrate, suspension concentrates, microemulsions and the like, all of which lend themselves to applications to seeds, soil, water and / or foliage, for Provide the required plant protection. Such formulations include the compounds of the invention mixed with inert, pharmacologically acceptable solid or liquid diluents. For example, wettable powders, powders and concentrated powder formulations can be prepared by grinding and blending together from about 25% to about 85% of the compound of this invention and about 75% to about 15% by weight of a solid diluent such as bentonite, diatomaceous earth, kaolin, atapulguite, or the like, 1% to 5% by weight of a dispersing agent such as sodium lignosulfonate, and about 1% to 5% by weight of a nonionic surfactant, such as octylphenoxy-ethanol , nonylphenoxy-polyethoxy-ethanol or the like. A typical flowable liquid can be prepared by mixing about 30% to 90% by weight of the active ingredient with about 1% to 3% by weight of a gelling agent such as bentonite, 2% to 5% by weight of a dispersing agent such as sodium lignosulfonate, about 1% by weight of polyethylene glycol, and about 40% to 60% by weight of water. A typical emulsifiable concentrate can be prepared by dissolving from about 15% to 70% by weight of the active ingredient in about 85% to 30% by weight of a solvent such as isophorone, toluene, butyl cellosolve, methyl acetate, ether. mono-methyl propylene glycol, or the like, and dispersing therein about 1% to 5% by weight of a nonionic surfactant such as an alkylphenoxy polyethoxy alcohol. The application of the material is done by adding a predetermined amount of the formulated product, as described above, to the desired volume of water or other suitable solvent, alone or in combination with other agricultural chemicals, for simultaneous use. It is understood that the compounds of the present invention can be applied alone or in combination with one or more other fungicidal compounds, such application being carried out either in combination of the fungicidal compounds or their formulations in a common container before use, or by sequential application of the active fungicidal compounds or their formulations to the host harvest or to their environment.

Compounds which can be combined with the compounds of the invention include, but are not limited to the following: 4,6-dinitro-o-cresol, benalaxyl, beno-myl, captafol, captan, carbendazim, chlorothalonil, copper, cymoxanil, diclobutrazol , diclofuanid, diet-fencarb, diphenconazole, dimetromorf, diniconazole, dino-cap, dithianone, fenarimol, fentin acetate, ferbam, flusilazole, folpet, fosetil, hexaconazole, imazalil, iprodione, mancobre, mancozeb, maneb, mepronil, mercuric oxide, metalaxyl, metiram, myclobutanil, muarinol, ofurace, oxadixil, penconazole, penciuron, phosphorous acid, procymidone, propineb, pirifenox, quintozene, sodium arsenite, sulfur, thiabendazole, methyl tofanate, thiram, tolclofosmethyl, triadimefon, triadimenol, triforine, vinclozolin , zineb, and / or ziram. The cultivation of the fungus LL-15G256 can be carried out in a wide variety of solid and liquid culture media. Means that are useful for the production of antibiotics include an assimilable source of carbon, such as potato starch, dextrose, maltose, malt extract, etc., and an assimilable source of nitrogen such as peptone or malt or potato extract. . The inorganic elements are supplied from vegetable or animal extracts, complexes, used in the preparation of the medium. The aeration is supplied either by shaking in a flask or by forcing air through the fermentation. Additional agitation is provided by a mechanical device. Antifoaming agent such as silicone oil may be added as necessary.

INSULATION OF LL-15G256 The recovery of fungicidal agents LL-15G256 from the fermentation broth is achieved by extraction of the complete broth with an immiscible organic solvent such as butanol or ethyl acetate, the separation of the phases and the isolation of the 15G256 component of the organic phase Alternatively, fungicidal agents LL-15G256 can be obtained from the complete broth first by the addition of one-half the volume of fermentation of an appropriate water-miscible solvent, such as methyl alcohol or acetonitrile, and thereafter any solid material by filtration, then the filtrate obtained is diluted with water to twice its volume, and the liquid is passed over a resin such as HP-20 or XAD-2 to adsorb the fungicidal compounds. The LL-15G256 components are eluted from the resin by the addition of an organic solvent, such as methyl alcohol, acetone or acetonitrile. The crude product obtained by the above extractions represents a complex of fungicidal agents LL-15G256 from which the simple compounds can be separated by chromatography. The purification of the individual components is achieved by a succession of liquid chromatographic steps on silica gel and / or reverse phase resins with a variety of elution conditions. The invention will be further described in conjunction with the following non-limiting examples.

Example 1 Inoculum The culture is maintained on a piece of corn flour / seawater agar, prepared by dissolving 17 g of bacto corn flour agar in one liter of natural seawater (Carolina Biological Supply Company), adjusting the pH to 6.0 and then aliquoting in appropriate containers, and sterilizing by autoclaving for 20 minutes. After the inoculation of the piece of agar, the culture develops at 22 ° C. A stopper of this culture is added to a 25 x 150 mm capped culture tube, containing 10 ml of potato dextrose broth, prepared by the addition of 24 g of dehydrated papa-dextrose broth, Difco, to 1 liter of distilled water which was then sterilized by autoclaving for 20 minutes at 121 ° C, and two 6 mm glass spheres. This tube is incubated at 22 ° C with shaking at 170 rpm and a two inch game or stroke. After a period, usually between 4 and 5 days, vigorous growth is observed. Ten ml of this culture are transferred to a 250 ml Erlenmeyer flask containing 50 ml of potato-dextrose broth. The flask containing the broth is incubated at 22 ° C with shaking at 200 rpm with a two-inch stroke or set, until vigorous growth is observed, which usually occurs after 3 days of incubation.Fermentation Two and a half ml of the culture produced in Example 1 are transferred to a 250 ml Erlenraeyer flask containing 50 ml of Sabaroud maltose medium, prepared by dissolving 10 g of Neopeptone Difco and 40 g of Bacto maltose in one liter of distilled water, and adjusting the pH to 5.6. This mixture is incubated at 22 ° C with agitation at 200 rpm with a game or run of two inches for 7 days.

Progressive Increase to Five Liters Fifty flasks were prepared as described above and their contents were combined.

Example 2 Progressive Increase to 300 Liters Sowing Media FM-3 Potato-dextrose powder 2.4% Tap water 1 liter Production Medium FM-7P: Ingredients% Maltose 4.0 Neo-peptone 1.0 CaC03 0.1% Antifoam DF40-P 0.3% Water of the tap 1 liter Sowing SI A 500 ml flask with 100 ml of seed medium is inoculated with frozen mycelial suspension (-95 ° C). The flask is allowed to develop for 5 days at 28 ° C on a rotary shaker at 200 rpm.

Sowing S2 A 4-liter tank containing 3.0 liters of planting medium is inoculated with 100 ral of sowing growth S2. The tank is allowed to develop for 3 days at 22 ° C. Aeration is adjusted to 3 liters per minute and agitation at 200 rpm.

Sowing S3 A 41-liter tank containing 30 liters of planting medium is inoculated with 3.0 liters of S2 seed. The aeration is adjusted to 30 liters per minute, the temperature is 28 ° C, and the agitation is 300 rpm. After 3 days of growth, the planting is inoculated in the production state.

Production A 410 liter tank containing 300 liters of sterile production medium is inoculated with the growth content of the S3 sowing tank. The temperature is adjusted to 28 ° C, the agitation to 250 rpm, and the air flow is 250 liters per minute. The back pressure is initially adjusted to 0.56 kg / cm2 (8 psi). The fermentation is allowed to proceed for 99 hours, after which time it is harvested.

Example 3 Isolation and Purification of LL-15G256 The whole mixture (5 liters) is separated into a button and a supernatant by centrifugation at 3,000 rpm for 30 minutes. The button is extracted with 2 liters of > 85% aqueous acetone while the supernatant is extracted with 2 liters of ethyl acetate. The organic extract of the supernatant and the button extracts are processed separately under the following conditions. After evaporation of the solvent, the remaining solids (approximately 2 grams from the button and approximately 1.2 grams from the supernatant) are resuspended in methylene chloride and loaded onto self-packed silica gel columns (21 x 300 mm). The compounds are eluted with increasing concentrations of ethyl acetate (10, 25, 50 and 100%) in methylene chloride, followed by a final wash of the column with 100% methyl alcohol. Six fractions are collected from the button extract as indicated below. 0 Fraction: Weight: Composition of the Eluent: 1. 33 mg Methylene chloride 2. 1 14 mg 10-25% EtOAc / CH2Cl2 3. 423 mg 25-50% Et: 0Ac / CH2Cl2 5 44 .. 112255 mmgg 50-100 Et: 0Ac / CH2Cl2 5. 52 mg 100% EtOAc 6. 467 mg 100% MeOH Fraction 5 contains essentially pure LL-15G256-Q gamma. Fraction 6 was further purified by reverse phase high performance liquid chromatography to produce additional LL-.L5G256-gamma. The residue of fraction 6 is dissolved in 2 ml of dimethylsulfoxide and charged onto a column of reverse phase high resolution liquid chromatography of C-8 (2 x 50 cm). The column is eluted with a mixture of 75% methyl alcohol / water containing 0.2% trifluoroacetic acid, at a flow rate of 9.9 ml / minute. The LL-15G256-gamma is eluted from the column between 20-25 minutes. The compound is recovered from the solvent mixture by concentration in vacuo to remove most of the methyl alcohol and then extracted with ethyl acetate. Evaporation of the ethyl acetate yields 140 mg of LL-15G256-gamma. The material obtained from the silica gel chromatography of the supernatant extract is handled in a similar manner.

Example 4 Isolation and Purification of LL-15G256-gamma, LL-15G256-delta and LL-15G256-epsilon The whole mixture (300 liters) is mixed with 1.5% toluene and stirred for 30 minutes, followed by filtration through a ceramic microfilter using tangential flow. The filtered and retained fractions are processed separately to produce the LL-15G256 compounds. The retentate is resuspended in methyl alcohol (100 liters) and filtered. The extract is concentrated in vacuo to remove most of the methyl alcohol and the resulting aqueous suspension is extracted with ethyl acetate. Concentration of the ethyl acetate under vacuum produces an oily residue, which upon trituration with hexane (2 liters) results in the formation of a precipitate. The precipitate is suspended with 300 g of silica gel in 500 ml of acetone for 20 minutes. The acetone is evaporated and the resulting powder is transferred to a column of partially charged silica gel, to produce a final column of 10 x 15 cm [diameter]. The column is eluted sequentially with 1 liter of methylene chloride, 2 liters of 50% ethyl acetate / methylene chloride, and 6 liters of ethyl acetate, followed by 2 liters of washing with methyl alcohol. The ethyl acetate fraction produces about 10 g of LL-15G256-gamma. The filtrate is mixed with half a volume of ethyl acetate for three hours. The ethyl acetate phase is separated and concentrated in vacuo to yield a crude mixture. To this mixture (containing the precipitated material), ethyl acetate (12 liters) is added and the mixture is washed with water. The organic phase is concentrated under vacuum to produce an oily residue which is triturated with hexane (2 liters) to produce a precipitate. The precipitate is mixed with 10 g of silica gel and acetone. The residue is collected after evaporation of acetone and loaded onto a column of silica gel (5 [diameter] x 10 cm) which is then eluted with a gradient in steps (about 250 ml each) of ethyl acetate in methylene chloride (5, 10, 25, 50, 75% and 3 x 100%). Fractions 7 and 8 (eluted with 100% ethyl acetate) are cotranscentrated to yield 4.5 g of solid material. The solid is dissolved in 30 ml of methyl alcohol and subjected to chromatography in three aliquots on a column C.1 o0 (5 x 30 cm) using 55% acetonitrile / trifluoroacetic acid 0.1 M at a flow rate of 40 ml / minute. The effluent is checked periodically by a variable wavelength detector at 300 nm. The fractions containing LL-15G256-gamma, LL-15G256-delta and LL-15G256-eps? Lon are eluted at 13 minutes, 15 minutes and 30 minutes, respectively. Ren-dimiento of the pure components: LL-15G256-gamma 62 mg, LL-15G256-delta 14 mg, and LL-15G256-epsilon 2 mg.

Methyl Ester of 15G256-gamma By keeping crude or purified 15G256-gamma as a methanolic solution, it slowly produces insoluble quantities of methyl ester of 15G256-gamma which is separated from 15G256-gamma by reverse phase chromatography under the conditions described below. This method provides 45 mg of methyl ester of 15G256-gamma from a reversed phase separation. The 15G256-gamma (30 mg) is dissolved in about 30 ml of methyl alcohol, and 4 ml of a solution of diethyl ether / diazomethane are added while stirring the reaction vessel. The reaction mixture is then left to stand for 10 minutes. A high resolution liquid chromatography analysis using an analytical Cfi Whatman reversed phase column, eluted with 75% methanol / 10% 0.1 M trifluoroacetic acid / 15% water, reveals that the original peak of 15G256-gamma at a Retention time of 4.4 minutes has disappeared, and a new single peak is produced instead of a 6.0 minute retention time. The reaction mixture is evaporated to dryness, redissolved in methyl alcohol twice, and prepared for nuclear magnetic resonance analysis. The product (32 mg) is identified as the methyl ester of 15G256-gamma.

It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the one, which is clear from the present description and the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (11)

1. A selected fungicidal compound of the formula I of formula II: characterized in that in formula I and II, n is 3 to 10; R is hydrogen, alkyl of 1 to 4 carbon atoms or a cation of the pharmaceutically acceptable salt, selected from the group consisting of sodium, potassium, calcium, lithium, magnesium, ammonium, and tetra ^ alkyl of 1 to 4 carbon atoms ) -ammonium; and R is hydrogen or methyl, and 2 in the formula I, R is hydrogen or methyl.
2. 2. The compound according to formula I of claim 1:
3. 3 «The compound according to the formula II of claim 1:
4. 4. The compound according to the formula I of claim 1:
5. 5. The compound according to formula I of claim 1:
6. 6. A process for the production of a fungicidal compound of the formula I: or of formula II: where n is 3 to 10; R is hydrogen, alkyl of 1 to 4 carbon atoms or a cation of the pharmaceutically acceptable salt thereof, selected from the group consisting of sodium, potassium, calcium, lithium, magnesium, ammonium and tetra (alkyl of 1 to 4 carbon atoms) car bonus) -ammonium; R 1 is hydrogen or methyl; and R2 is hydrogen or methyl, the process is characterized in that it comprises: the aerobic fermentation of the fungus LL-15G256 or the mutants thereof, which produce the fungicidal compounds, in a liquid medium containing assimilable sources of carbon, nitrogen and inorganic salts, until the substantial fungicidal activity is imparted to said medium; and the fungicidal compound thereof is recovered.
7. 7. A process according to claim 6, characterized in that it is for the production of a fungicide compound selected from the group consisting of LL-15G256-gamma, LL-15G256-delta, LL-15G256-epsilon and methyl ester of LL-15G256- gamma.
8. 8. A biologically pure culture, characterized in that it is derived from a newly discovered and previously uncultivated fungus, LL-15G256.
9. 9. A complex of fungicidal compounds, characterized in that it is produced by the aerobic fermentation of a mushroom culture LL-15G256 in a liquid culture medium containing assimilable sources of carbon, nitrogen and inorganic salts, until the activity is imparted fungicide substantial to said medium, and then the complex of the compounds thereof is recovered.
10. 10- A method for the protection of plants against the effects of plant pathogenic fungi, characterized in the method because it comprises the application to the locus of the plant or to the hgos, of a fungicidally effective amount of a compound according to claim 1 or by the process according to claim 6
11. 11. A fungicidal composition, characterized in that it comprises a solid or inert liquid diluent and a fungicidally effective amount of a compound according to claim 1, or prepared by the process according to claim 6.