FUNGICIDAL COMPOUNDS (B)

Abstract: Use of dunnione of formula (I) as a fungicide is provided. Fungicidal compositions containing dunnione are also provided.

Description: FUNGICIDAL COMPOUND? (B) This invention relates to the use of dunnione as a fungicide and processes for the preparation of compositions containing dunnione suitable for such use. The ability of fungi to develop resistance to fungicidal agents means that there is an ongoing need for new agents for their control. It is known that certain napthoquinones and their derivatives have fungicidal activity eg US 4 970 328 and US 4 929 642. Furanyl derivatives, (A) and (B), of 1 ,4-Napthoquinones with one or two alkyl substituents at the carbon ortho- to the furanyl oxygen were prepared and investigated by Jacobsen and Wengel (Pestie. Sci., 1986, 17, 686-690). The dialkyi derivative (B) was found to have intermediate fungicidal activity and the mono- alkylated derivative (A) was inactive; it is apparent that both these compounds are not as fungicidally active as some of the non- furanyl compounds investigated by Jacobsen and Wengel. (A) (B) The present inventors have now determined that a naturally occurring fungicidal derivative of napthoquinone, dunnione, may be used as a fungicidal agent having superior fungicidal activity, especially against strains resistant to presently used agents. The attendant advantages of a naturally occurring agent include natural product sourcing and a potentially safer product. A first aspect ofthe present invention provides the use of dunnione as a fungicidal agent. The structure of dunnione is given as formula I. It should also be appreciated that dunnione is capable of existing as different enantiomers and the invention includes both the individual enantiomers and mixtures of such enantiomers. Dunnione may be extracted from the aerial parts of the plants Streptocarpus dunnii or Calceolaria Integrifolia or prepared from either II or III by acid catalysed cyclisation reactions. Ill is formed from IV using an aqueous solution of methanol and potassium hydroxide. (II) (III) (IV) A second aspect of the invention provides a fungicidal composition which comprises a carrier and, as active ingredient, dunnione. This aspect necessarily includes a method of making such a composition by bringing dunnione into association with at least one carrier. It is also envisaged that different enantiomers or mixtures of enantiomers of dunnione may have different levels or spectra of activity and thus compositions may comprise individual enantiomers or mixtures of enantiomers. The compositions ofthe invention may contain from 0.0001 to 0.1% by weight of the active ingredient of formula I. Preferably the compositions contain from 0.0002 to 0.075% by weight ofthe active ingredient when they are in ready-to-use form. However, higher concentrations, for instance, up to 95%, may be present in compositions to be sold as concentrates for dilution before use. To prepare the compositions ofthe invention dunnione may be mixed with a variety of appropriate inert carriers such as solvents, diluents and/or surface-active agents to form dusts, granular solids, wettable powders or other solid preparations or emulsions, emulsifiable concentrates, sprays, aerosols or other liquid preparations. Suitable solvents and diluents include water, aliphatic and aromatic hydrocarbons such as xylene or other petroleum fractions and alcohols such as ethanol. Surface-active agents may be of an anionic, cationic or non-ionic type. Anti-oxidants or other stabilisers may also be included as well as perfumes and colourings. These inert carriers may be of the type and in proportions such as are conventionally used in fungicidal compositions. In addition to these inert carriers, the compositions of the invention may also contain one or more further active ingredients. These further active ingredients may be other compounds which exhibit fungicidal activity and these other compounds may exhibit a synergistic effect with the compounds ofthe present invention. A third aspect of the invention provides a method of combatting fungal pests comprising treating a locus subject to attack by said fungal pests with dunnione or a composition as defined above. Preferably, the locus comprises the fungus or fungi per se or environments subject to or subjected to attack by these. The invention is further illustrated by the following examples. Example 1 - Preparation of dunnione The preparation of dunnione has been described by Cooke, (Nature, (1948), 163. 178). 2-[l,l-dimethyl-2-propenyl]-3-hydroxy-l,4-napthoquinone (500 mg, 2.06 mmol) prepared according to WO 95/32176 was dissolved in concentrated sulphuric acid (10 ml), stirred for 10 minutes at room temperature and poured onto ice in water (100 ml). The aqueous mixture was extracted with ether (3 x 50 ml) and the combined ether layers were washed with water (2 x 50 ml), saturated sodium chloride (50 ml) and dried over magnesium sulphate. Filtration of the resulting solution, removal of the solvent by evaporation and recrystallisation ofthe product from petroleum ether (60 - 80°C) yielded dunnione (476 mg) as orange-red needles, mp 98-99°C. Example 2 - Extraction of dunnione The extraction of dunnione from Calceolaria Integrifolia was carried out according to the method of Rϋedi and Evgster (Helvetica Chimica Acta, 6_Q_, 945 - 947, (1977)). Extraction of dunnione from streptocarpus dunnii was carried out according to the method of Price and Robinson (J. Chem. Soc, (1939), 1522). Examples 3 and 4 - Determination ofthe Anti-Fungal Activity of dunnione The anti-fungal activity of dunnione on fungi grown under in vitro conditions and on 14 day old seedlings was investigated. The species investigated in Example 3 were Septoria tritici (wheat leaf blotch), Rhvnchosporium secalis (barley leaf blotch), Ustilago maydis (maize smut), Neurospora crassa. Botrytis cinerea (grey mould), Fusarium gram earu > Pseudocercosporella herpotrichoides (eyespot) and Ervsiphe graminis f.sp hordei (powdery mildew). The effects that the dosage levels of the compounds under investigation had on the colony size of the above mentioned species was observed and conclusions were drawn regarding their anti-fungal activities. Example 3 - In Vitro Assays Fungal colonies were incubated and grown on potato dextrose agar (PDA), which was prepared from stock solutions (20 mg ml " 1 ) in ethano acetone (1 :1), autoclaved and treated with the compound under investigation. A four fold dilution series was prepared for each compound with doses ranging from 20 g ml "1 to 0.08 μg ml'l . For fungi which grow only as yeast-like colonies the growth was measured as the Minimum Inhibitory Concentration (MIC); for other species the growth was measured as colony diameter and the Effective Dose^ Q (ED^Q) values were calculated from the dose response relationship. In one series of experiments Czapek-Dox plus mycological peptone (1% w/v CDM) was used instead of PDA. Each fungus was incubated at the optimum temperature for their growth. The anti-fungal activity of dunnione was compared with that of known anti-fungal compounds as a control. The results, shown in Tables 1(a) to (g) below indicate that the anti-fungal activity of the compound tested was dependent on the nature of the fungal strains used, dunnione was active against Septoria tritici. Rhynchosporium secalis and Ustilago maydis: the levels of activity against these pathogens were similar, or better, than the azole fungicides currently in use. It also exhibited some activity against Botrvtis cinerea and Fusarium graminearum: these two pathogens are difficult to control using existing azole fungicides. Significant activity against Neurospora crassa was observed. Ta le 1 (a) Septoria tritici Sensitivity MIC (μg 1 ml) Isolate Flutrifol dunnione S27 40 5 RL2 1 5 Table \(b) Rhvnchosporium secalis Sensitivity MIC (μg 1 ml) Carbendazim Triadimenol Propiconazole dunnione Isolate CDM agar 81 0.1 >50 2.0 5 86 0.1 3.2 0.4 >5 97 0.1 >50 >10 5 101 0.1 >50 >10 >5 110 0.1 >50 2.0 >5 115 0.1 1.6 0.08 20 117 0.1 12.5 2.0 5 118 0.1 >50 >10 20 120 100 >50 >10 20 PDA 810 0.1 >50 >10 20 Table lfc 1 Ustilago mavdis ED 50 (μg / ml) Isolate dunnione ATCC- 14826 wild-type 3.0 Table lfdl Neurospora crassa ED 50 Oug / ml) Isolate dunnione Am 132a 2 Table 1(e) Bptrytis cinerea Sensitivity ED50 (μg 1 ml) Isolate Dicarboximide Benzimidazole dunnione R3B6 R R > 20 RW10 S S > 20 S = Susceptible; R = Resistant Table 1(f) Fusarium graminearum Sensitivity ED50 (μg 1 ml) Isolate Benzimidazole dunnione JF18 R 20 R = Resistant Table \(ς) Pseudocercosporella herpotrichoides Sensitivity ED50 (μg 1 ml) Isolate Benzimidazole dunnione CR03A R 17 I = Resistant Example 4 - Further In Vitro Assays The fungicidal activity of dunnione to isolates of Aspergillus niger. Pyricularia oryzae and Rhizoctonia solani was tested using the procedure described in Example 3. Each fungus was tested on agar in four petri dishes per treatment, with three replicate fungal colonies per plate (one for R. solamΥ A. niger and R. solani were incubated for 4 days at 20 - 25 °C; P. oryzae was incubated for 7 days. Increase in colony diameter was then used to measure colony activity. Dunnione was also tested on four isolates of Pseudocercosporella herpotrichoides: two slow growing R-types and two faster growing W-type isolates. One isolate of each type was sensitive to fungicides such as carbendazi and prochloraz and the other was fully resistant. Colony diameters were measured after two weeks at 20°C. Dunnione was also tested against four isolates of the potato tuber pathogens Helminthosporium solani and Fusarium sulphureum. Two isolates of each were sensitive to the fungicide thiabendazole (TBZ) and two had some degree of resistance. The same procedures as those used for P. herpotrichoides were followed except that the replicates were grown on malt extract agar and growth was measured on two occasions. The results, shown in Tables 2(a) to 2(f) below, indicate that dunnione is active against Aspergillus niger. Pyricularia oryzae and Rhizoctonia solani. Its activity against Pseudocercosporella herpotrichoides was inconsistent. There was no evidence of cross- resistance with carbendazim (Table 2(d)) or with thiabendazole in H. solani or I sulphureum (Tables 2(e) and (f))- Table 2(a1 - Activity of dunnione against Aspergillus niger Compound % inhibition of growth in colony diameter 100 mg/1 20 mg/1 4 mg/1 l mg/1 dunnione 100 99 30 Prochloraz 97.1 Table 2fb - Activity of dunnione against Pvricularia orvzae Compound % inhibition of growth in colony diameter 100 mg/1 20 mg/1 4 mg/1 l mg/1 dunnione 100 99 75 Carbendazim 99.4 14.7 Table 2(c 1 - Activity of dunnione against Rhizoctonia solani Compound % inhibition of growth in colony diameter 100 mg/1 20 mg/1 4 mg/1 l mg/1 dunnione 100 93 37 Carbendazim 81.2 3.3 Table 2(ά) - Activity of dunnione against W-tvpe and R-tvpe isolates of Pseudocercosporella herpotrichoides Fungus Type Sensitivity to Activity at 5 mg/1 Carbendazim Carbendazim dunnione W-type Sensitive 100 50 W-type Resistant 5 30 R-type Sensitive 100 -15 R-type Resistant -14 4 Table 2(e) -Activity of dunnione against isolates of Helminthosporium solani with different levels of resistance to thiabendazole (TBZ') Isolate Activity at 5 mg/1 8 days 14 days TBZ dunnione TBZ dunnione SI 100 65 100 53 S2 100 63 100 86 Rl 43 57 20 87 R2 100 70 100 56 S 1 , S2 = isolates : sensitive to TBZ; 11, R2 = isolates resistant to TBZ. Table 2(f) - Activity of dunnione against isolates of Fusarium sulphureum with different levels of resistance to thiabendazole Isolate Activity at 5 mg/1 8 days 14 days TBZ dunnione TBZ dunnione SI 100 22 100 nd S2 100 11 100 5 Rl 60 4 64 6 R2 70 16 70 12 S 1 , S2 = isolates sensitive to TBZ; Rl , R2 = isolates resistant to TBZ. nd = no data; the fungal colonies on the control medium had reached the edges of the culture plates. Example 5 - 14 day Old Seedlings 14 day old seedlings were sprayed to run off 24 hours prior to inoculation with conidia of Erysiphe graminis f.sp hordei. Solutions of dunnione, prepared in dilute Tween 20 solutions (1 drop Tween 20 per 100 ml water), were sprayed onto the seedlings; the final concentrations ofthe dunnione in the solutions used were 0.75 mg ml "1 and 0.075 mg ml"l , the former being equivalent to the field application rate of a triazole fungicide. Control seedlings were sprayed with a solution of Tween 20 only. The growth of barley powdery mildew was assessed 7 days after inoculation as a percentage of leaf area infected. The results shown in Table 3 indicate that dosage levels below those currently used for triazole fungicides are fungicidally effective and produce no noticeable phytotoxic effects. It may be preferred to use concentrations of less than 0.75 mg ml " 1 when treating certain species of plant as higher concentrations may induce some phytotoxic effects. Table 3 - Effect of dunnione on the Growth of Ervsiphe graminis f.sp horedi % Disease Control Isolate Triadimenol dunnione mg/ml 0.75 0.075 23D5 S 100 45 212 R 100 50 S = Sensitive; R = Resistant

Claims: CLAIMS Use of dunnione of formula (I) as a fungicide. 2. Use of enantiomers or mixtures of enantiomers of dunnione as fungicides. 3. A fungicidal composition comprising an inert carrier and dunnione. 4. A fungicidal composition according to claim 3 wherein the inert carrier is selected from the group comprising water, aliphatic or aromatic hydrocarbons and alcohols. 5. A fungicidal composition according to claim 4 wherein the aliphatic or aromatic hydrocarbons are xylene and other petroleum fractions. 6. A fungicidal composition according to claim 4 wherein the alcohol is ethanol. 7. A fungicidal composition according to any one of claims 3 to 6 which also further comprises one or more surface-active agents. 8. A fungicidal composition according to any one of claims 3 to 7 which further comprises a stabiliser. 9. A fungicidal composition according to any one of claims 3 to 8 which further comprises one or more additional fungicidally active ingredients. 10. A method of preparing a fungicidal composition which comprises the steps of admixing dunnione with a suitable carrier. 11. A method of controlling or preventing fungal infestation which comprises the steps of contacting a fungicidal composition according to claim 9 with fungi. 12. Material treated with dunnione according to any one of claims 1 to 9. 13. Material according to claim 12 wherein the material treated is plant material. 14. Use of a plant extract as a fungicide, characterised in that the plant extract comprises dunnione of formula (I). (I) 15. Use according to Claim 14 wherein the plant extract is obtainable from Strepocarpus dunnii. 16. Use according to Claim 14 wherein the plant extract is obtainable from Calceolaria Integrifolia.

Inventors: BATTY DUNCAN, KHAMBAY BHUPINDER PALL SINGH, BATTY, DUNCAN, KHAMBAY, BHUPINDER, PALL, SINGH
Applicants: BATTY DUNCAN, BRITISH TECH GROUP, KHAMBAY BHUPINDER PALL SINGH, BATTY, DUNCAN, BRITISH TECHNOLOGY GROUP LIMITED, KHAMBAY, BHUPINDER, PALL, SINGH
Application year: 1996
Main classification: A01N43
IPC codes: A01N43, A01N65, A01P3