PENTACYCLIC TRITERPENES

Abstract: The present invention relates to fungicidally effective compositions containing at least one pentacyclic triterpene compound in an agriculturally acceptable carrier.

Description: PENTACYCLIC TRITERPENES FIELD OF THE INVENTION The invention relates to plant protection compositions including pentacychc triterpenes and methods for use thereof BACKGROUND Outer layers of plants such as leave cuticle, fruit peels, as well as bark protect the plant against abrasion, prevent water loss, and also protect against pathogenic microorganisms. The breaking through the plant cuticle is a prerequisite foi a pathogen to be able to enter the plant's internal tissue. The mechanism, bv which plants naturally defend themselves against this early stage of pathogenesis has not been fully understood. The initial process of fungal propagule b attaching to a host plant is essential to the successful establishment of pathogenesis The established facts that aerial fungal pathogens bind strongly to very hydrophobic surfaces suggests that hydrophobic forces are involved in the attachment processes The attachment of aerial fungal pathogens involves an active process of secretion of extracellular mucilages or adhesives, which may start within minutes after contact with the host. Other reported components in the adhesive secretions include enzymes, among them esterases and cutinases. The erosion of cuticular waxes adjacent to and underlying the conidium may became observable wrthin 20 mm of liquid release. The growth of the appressoπal germ tube appears to be limited to the zone of deposition of the liquid film Some studies have suggested that preparation of the infection court involves active dissolution of the host cuticle by foliar pathogen. It is also believed that this dissolution is the purpose of these enzymes. Nicholson, R.L., and L. In: The Fungal Spore and Disease Initiation in Plants and Animals.. Eds. Cole, G. TΛ, and Hoch, H. C.,1991 Plenum Press, New York, 3-23. Thus, the cuticle has to be penetrated by the attacking pathogen before the sequential steps of disease development occur. Some fungal spores help themselves with mechanical force exerted by the infection structure in addition to the enzymatic degradation (Koller, W. in: The Fungal Spore and Disease Initiation n Plants and Animals.. Eds. Cole, G. T., and Hoch, H. C, 1991 Plenum Press, New Yoik, 219-246 Pentacychc triterpenes (PT) are among the most common plant secondary metabolites, but their function in plants have not been understood. They are usually concentrated in the outermost layers such as plant cuticle, fruit peel and bark. In some cases, these layers contain very high concentration of pentacychc triterpenes. For example, an apple peel contains about 0.1 grams of ursolic acid per fruit, and the outer bark of white birch species contains up to 40% w/w of betulin. The amount of betulin obtainable from the birch bark waste in the wood-working industry in Finland is estimated at about 150,000 tons per annum. At present, waste bark is used as a low value fuel for energy production. Jaaskelainen, P. (1981) Pap. Puu 63, 599-603. Literature supplies numerous examples of enzymes that can be inhibited by PT, indicating the ability of PT to act broadly in a non-specific mode on multiple targets. See for example, (a.) Bύchler et al. (1991) Biochem. Biophys. Acta 1075, 206-212, Inhibition of rat renal 11^ -hydroxysteroid dehydrogenase by steroidal compounds and triterpenoids; structure/function relationship; (b.) Koch et al. (1994) Phytother. Res. 8, 109-111. In vitro inhibition of adenosine deaminase by a group of steroid and triterpenoid compounds.; (c.) Najid et al. (1992) FEBS 299, 213-217, Characterization of ursolic acid as a lipoxygenase and cyclooxygenase inhibitor using macrophages, platelets and differentiated HL60 leukemic cells.; (d.) Pengsuparp et al. (1994) J. Nat. Prod. 57, 415-418, Pentacychc triterpenes derived from Maprounea africana are potent inhibitors of HIV-1 reverse transcriptase.; (e.) Simon et al. (1992) Biochem. Biophys. Acta 1125, 68-72, Inhibition of lipoxygenase activity and HL60 leukemic cell proliferation by ursolic acid isolated from heather flowers (Calluna vulgarise; (f.) Ying et al. (1991) Biochem. J. 277, 521-526 Inhibition of human leucocyte elastase by ursolic acid. Evidence for a binding site for pentacychc triterpenes. The disclosures of each of these references is herein incorporated by reference. In plant tissue cultures, stress induced by inactivated fungi or fungal enzymes has been used to enhance production of biologically active secondary metabolites. In several instances it has been reported that this fungal elicitation led to overproduction of pentacychc triterpenes instead of some other expected metabolites. Suitable example is given by Van der Heijden et al., (1988) Plant Cell Rep. 7, 51-54, where tissue cultures of Tabernaemontana spp., normally producing indole alkaloids, were subjected to stress induced by either fungi, bacteria, or enzyme cellulase or pectinase. When stressed, however, the culture produced up to 3.3 times the normal rate of the ursane-type pentacychc triterpenes (2% of dry mass) but no increase in the production of indole alkaloids occurred. Other experiments with Tabernaemontana divaricata treated with Candida albicans elicitor led to production of a series of pentacychc triterpenes of the ursane and oleane types, and was accompanied by inhibition of both growth and indole alkaloid accumulation (Van der Heijden et al, (1989) Phytochemistry 28, 2981-1988). Also, the tissue culture of Triptervgium wilfordii. normally a source of potent cytotoxic diterpenes, stressed by fungal Botrytis elicitor dramatically enhanced production of oleane-type pentacychc triterpenes but not the diterpenes, prompting a conclusion that only triterpenes are inducible anti-microbial phytoalexins. Kutney, et al., (1993) Anti- inflammatory oleane triterpenes from Triptervgium wilfordii cell suspension cultures by fungal elicitation. Plant Cell Rep. 12, 356-359. The conventional treatments for leafy and grassy plants that have been attacked by fungi and bacteria are usually exercised after an outbreak occurs. The affected plants are then treated with one or more of the commercial synthetic contact antimicrobial sprays at apphcation rates that do not pose phytotoxicity concerns. Effective treatment rates must be balanced against the risks of harming the treated plant with chemicals that are structurally unrelated to those in the plant physiology. It would be desirable to have a protective agent that is effective against microbial pathogens which would also work in a manner analogous to the plant's natural defense mechanisms to reduce the risk of phytotoxicity. One of the problems associated with treating hydrophobic leaf surfaces is an effective apphcation of the material. Current spraying techniques result in a portion of the sprayed material falling to the ground direcdy or after being washed off from rain occurring shordy after apphcation. Either event increases concerns for environmental contamination. Protective agents that are applied by spraying should remain on the plant surface for a time sufficient to serve their intended function. Stability against ultraviolet and visible hght is, therefore, a concern for foliar treatments. It would be desirable to develop fungicides for foliar apphcation that resist degradation by exposure to ultraviolet as well as visible hght. Moreover, invading organisms have been known to evade the effects of a treatment agent by mutation and propagation of resistant strains. Such developed resistance is economically detrimental because it forces the discovery of new treatments. It would be helpful to provide plant anti-infective agents which cannot be evaded by mutating pathogens. SUMMARY OF THE INVENTION It is an object of the invention to provide a composition and process for use thereof for protecting plant surfaces against microbial pathogens with ingredients that are compatible with natural plant defense mechanisms. It is another object of the invention to provide a composition and method of use that poses httle risk to the environment, humans, or beneficial insects. It is another object of the invention to provide plant anti-infective agents, which cannot be evaded by developing resistance by mutating pathogens. It is yet another object of the invention to provide a composition having good resistance to degradation by ultraviolet exposure, long storage times, and good inherent sticking ability to plant surfaces. In accordance with these and other objects of the invention, which will become apparent from the description herein, a composition according to the invention comprises: a pentacychc triterpene or a mixture of pentacychc triterpenes, including some plant extracts that are particularly rich in pentacychc triterpenes, having as main components compounds described by any of formulas I, II, or III. A method according to the invention comprises applying to plant surfaces an effective amount of a composition comprising a pentacychc triterpene or a mixture of pentacychc triterpenes exhibiting any of formulas I, II or III. Formulas I, II, and III are: wherein: R l = Me, CH 2 OH, CH 2 OY\ CH 2 0-X-OH, CH 2 0-X-OY\ CH z O-X-Y 2 , CH 2 0-X-Y\ CH^HY 1 , CH 2 NY 1 2 ,CH 2 Y 3 , CH 2 NH-X-OH, CH 2 NH-X-Y 2 , CH 2 NH-X-Y 3 , CH 2 NH-X-OY\ CH 2 OC(0)-OY\ CH 2 0-X-OY\ C0 2 N, COY\ COY 2 , CHO, CH=N(CH 2 ) m (0(CH 2 ) m )„R 4 , or CH=N(CH 2 ) m (0(CH 2 ) J n Y 2 ; R 2 , R 3 H, OH, OY 1 , O-X-OH, O-X-OY 1 , O-X-Y 2 , Y\ NHY 1 , NY 1 * Y\ NH-X-OH, NH-X-Y 2 , NH-X-Y 3 , NH-X-OY 1 , NY'-X-OH, NY'-X-Y 2 , NY'-X-Y 3 , or NY'-X- OY 1 ; provided that one of R 2 and R 3 is H or that R 2 and R 3 together denote carbonyl oxygen; R 4 = H, OH, OY\ or Y 3 ; Y , = H, alkyl of 1-30 carbon atoms, straight chain or branched, cycloalkyl of 3-30 carbon atoms, alkanyl of 3-30 carbon atoms, oxyalkyl of 4-30 carbon atoms, phenylalkyl of 7-30 carbon atoms, or phenoxyalkyl of 7-30 carbon atoms; γ2= NH 2 , NHY 1 , or NY 1 2 ; Y 3 = -(O(CH 2 )J n R 4 or -(0(CH 2 ) n Y 2 , where m=2-4 and n= 1-230; X= -O CH^ p CO- where p=l-22. The present invention provides a composition and method of use that are particularly effective in preventing outbreaks of airborne fungal and bacterial diseases on treated plant surfaces. The pentacychc triterpenes of the composition are apphed to form a film over the plant surface. The film of material prevents the prerequisite attachment of aerial fungal pathogen and penetration of the plant cuticle. In addition, the compounds are not phytotoxic and are compatible with natural plant defenses. DETAILED DESCRIPTION The present invention relates to compositions containing and methods of us t pentacychc triterpenes having the following formulas I, II, or III: wherein: R' = Me, CH 2 OH, CH 2 OY\ CH 2 0-X-OH, CH.O-X-OY 1 , CH 2 0-X-Y 2 , CH 2 0-X-Y\ CH 2 NHY', CH 2 NH-X-OH, CH 2 NH-X-Y 2 , CH 2 NH-X-Y 3 , C^NH-X-OY 1 , CH 2 OC(0)-OY 1 , CH 2 0-X-OY\ CON 1 , COY 3 , COY 2 , CHO, CH=Ν(CH 2 ) m (0(CH 2 ) m ) n R 4 , or CH=N(CH 2 ) m (0(CH 2 ) J n Y 2 ; R 2 2 , τR> 3- H, OH, ON, O-X-OH, O-X-OY 1 , O-X-Y 2 , Y 3 , ΝHY 1 , ΝY' 2 , Y 3 , NH-X-OH, NH-X-Y 2 , NH-X-Y 3 , NH-X-OY 1 , NY^X-OH, NY'-X-Y 2 , NN-X-Y 3 , or NN-X- OY 1 ; provided that one of R 2 or R 3 is H or that R 2 and R 3 together denote carbonyl oxygen; R 4 = H, OH, OY 1 , or Y 3 ; H, alkyl of 1-30 carbon atoms, straight chain or branched, cycloalkyl of 3-30 carbon atoms, alkanyl of 3-30 carbon atoms, oxyalkyl of 4-30 carbon atoms, phenylalkyl of 7-30 carbon atoms, or phenoxyalkyl of 7-30 carbon atoms; Y ' : 2— NH 2 , NHY 1 , or NY ] 2 ; Y 3 = -(O CHa) J n R 4 or -(0(0^ J n Y 2 , where m=2-4 and n= 1-230, X= -O CH^ p CO- where p=l-22. The preferred pentacychc triterpenes include betuhn, betuhmc acid, ursolic acid, oleanohc acid, betu n mono- and di-succinate or glutarate, as well as polyethylene glycol derivatives of thereof. Particularly useful are those pentacychc triterpenes exhibiting an IC 5(1 value against human leucocyte elastase at concentration less than about 15 micromolai (μM), more preferably an IC 5ϋ value of less than about 10, and most pieferably an IC 5 value at less than about 8 micromolar. The IC 50 value represents the concentration of an inhibitor, which can be expressed in micromoles per hter, at which activity of an enzyme is reduced by 50% Thus, lower IC 5ϋ values suggest higher levels of enzyme inhibitory activity The pentacychc triterpenes of the invention are the same as, derived from, synthesized, or otherwise related to those found naturally in the outer surfaces of plants leaves, fruits, bark, and are subjected to pathogenesis involving enzymatic degradation of cuticle. For the present invention, the pentacychc triterpenes or their derivatives are apphed to the exposed outer plant surfaces in conjunction with a suitable carrier such as water, an aqueous fihn-forming solution, detergents, emulsion forming additives, suitable polymers to enhance physical properties of the sprayed layer. Pentacychc triterpenes can be obtained by extracting the pentacychc triterpene- contaming plant tissues with one or more organic solvents suitable for the triterpenes Preferred plant tissue sources for PT include bark from white birch trees, apple peels, and the leaves of plants belonging to Vaccinium and Myristica spp. Useful solvents for the extraction include ethyl acetate, acetone, methyl ethyl ketone, ethanol, propanol, isopropanol, methanol, methylene chloride, chloroform, or their mixtures. The pentacychc triterpenes or their polyethylene glycol derivatives of the invention should be formed in a non-crystalhne state into a well mixed colloidal suspension for apphcation as a uniform coating on the treated plant surfaces. The uniform coating helps to ensure that plant surfaces are well protected against pathogens with the exception of under surfaces or secluded regions unable to be reached by conventional spraying equipment for liquid formulations. The pentacychc triterpene compounds of the invention are, however, crystalline sohds of hydrophobic character. The sohds can be dissolved in a number of solvents suitable for agricultural use. If desired, the solutions or colloidal concentrates of pentacychc triterpenes can be prepared for shipping and storing. This concentrate can then be further diluted for use by a formulator or applicator. A preferred solvent for pentacychc triterpene sohds contains about 1 -25 wt% acetone, about 0-10 wt% dimethylsulfoxide (DMSO), 0-35% polyethyleneglycol ester of an aliphatic acid, and about 0-25 wt% of a surfactant such as commercially available detergents like Tween 80™ or Palmolive™ dishwashing detergent. Generally, this solvent mixture may carry a concentration of a PT being 1500-4000%, by weight, of that needed for apphcation to the plants. The concentrate can be further diluted with 100-4000%, preferably 300-1000% by weight of an agriculturally acceptable carrier, preferably water, to make a sprayable composition according to the invention. Particularly useful concentrations are within the range from about 7-30 grams per gallon of water. Adequate rnixing requires only low to moderate shearing to ensure adequate mixing of the concentrate during dilution. For example, metering the concentrate into a reservoir attached to a venturi mixer would provide adequate shear to completely mix the concentrate with additional water. Pentacychc triterpenes or their polyethylene glycol derivatives according to the invention are apphed at a rate sufficient to prevent pathogenic infections. The inhibitor}' properties of PT are utilized by the plants to inactivate the enzymes excreted by the fungal spore in order to degrade the plant cuticle. Suitable apphcation rates for effective protection include rates within the range from about 0.1-1000 kg/h. Preferably, the apphcation rate is within the range from about 0.1-100 kg/h. The specific apphcation rate that is best for a particular type of plant in a particular region is readily determined by the apphcation of the ordinary skill in the art. The apphcation should, however, be designed to fall on and cover the exposed leaf surfaces of the plants being treated such as it occurs with conventional fohar treatments using conventional fohar spraying equipment. If desired, the PT or their polyethylene glycol derivatives may be apphed in conjunction with one or more inert or active ingredients. Exemplary materials include dyes, additives affecting stability of the concentrate and additives affecting physical properties of the sprayed layer, fohar fertilizers, fungicides, and insecticides. Virtually any plant that may get infected through the waxy cuticle layer can be beneficially treated with compositions according to the present invention. Commercial plants that would benefit include grain grasses (e.g., rye, wheat, and barley), tomato, bean, pepper, wheat, and peanut plants. Grain grasses may benefit in particular from the present invention. These plants do not generally produce sufficient levels of pentacychc triterpenes, if at all, to inhibit enzymatic attacks by invading microorganisms. Natural pathogens of these plants are generally not adapted to produce sufficient amounts of enzymes to overcome the inhibiting effects of the externally apphed pentacychc triterpene compounds. Plants susceptible to smaU insect (e.g., aphids) infestation also benefit from treatment according to the invention. Ursohc acid showed toxicity and feeding deterrent effects towards the mites and their survival. The mites' reproductive indexes decreased in direct proportion to ursohc acid content in the diet, and in addition, ingestion time on diet containing ursohc acid was reduced about 30% (Varanda et al., 1992). These observations imply a possibility that the insect's digestive enzymes were compromised by ursohc acid. The mode of inhibition of enzymes by PT is non-specific and is based primarily on hydrophobic interaction with an enzyme's hydrophobic domain. This property suggests that the likelihood for developing microbial pathogen resistance through mutation is remote. Pathogens had to deal with the presence of PT in the plant cuticle for as long as the plants existed and it could be assumed that microbial resistance to the PT has been already optimized. The PT based formulations for plant protection should be advantageous against the pathogenic microorganisms that use enzymes to break through the cuticle, such as fungi, bacteria, nematodes and viruses. These formulations should be also advantageous against small insects, acting inhibitory on their digestive enzymes. By covering the leafy surface with a film containing pentacychc triterpenes, the passive defensive properties of the cuticle are enhanced, which decreases or entirely prevents successful pathogenic penetration. The following examples are included to assist in an understanding of the invention and are not intended to limit the scope of the attached claims. EXAMPLES Commercially available compounds were acquired either from Extrasyntheses (France), Aldrich, Sigma or ICN Pharmaceuticals. Melting points were obtained using a Reichert Thermovar melting point apparatus and are corrected. Hnmr spectra were acquired in C 5 D 5 N with TMS as an internal standard ( "" ^O.O ppm) using a Bruker WM-250 or 270 NMR spectrometer. Mass spectra were acquired using a Finnigan 4023 EI/CI GCMS mass spectrometer with Super-INCOS software and direct insertion probe. Examples la and lb - Synthesis of 28-hemisuccinylbetulin and 3,28-Di-hemisuccinylbetuhn. Example la. A reaction mixture is prepared by dissolving imidazole (4 eq) and succinic anhydride (1.08 eq) in N-methylpyrrohdone. The volume of the solvent is about 4.5 times the weight of betulin. When the two reagents are dissolved, betulin (1 eq) is added. The reaction mixture is left at room temperature with occasional shaking. After approximately 48 hrs, about 92-95 % of the betulin has been converted into 28-hemisuccinilbetulin, with 2-4 % as unconverted betulin and diacylbetulin. The 28-hemisuccinilbetulin is purified by pouring the reaction mixture into a 20X volume of water with slow stirring. The water is decanted from the sohd residue and the washing is repeated with several new portions of water. The sohd is separated, dried and dissolved in a mixture of chloroform and isopropanol. The chloroform is removed by evaporation enabling 28-hemisuccinylbetulin to form and crystallize. The 28- hemisuccinylbetulin crystals are white with a melting point of 242-244°C. Calculations for C 34 H 54 O 5 , C, 75.22%, H, 10.03; found C, 75.0, H, 9.91. The 'Hnmr (DMSO-d 6 ) gave peaks at: 4.86 and 4.72 (1H, s, each, CH 2 =C), 4.57 and 4.14 (AB pattern, J=12 Hz, OCH 2 O), 3.44 (1H, t, J=8.6 Hz), 2.95 (4H, s, CHOCH 2 CH 2 CO), 1.71, 1.22, 1.02, 0.98, 0.98, 0.84 (s, each, Me); eims (m/z, rel. int.): 524 (20, [M-H 2 0]+), 424 (15), 411 (27), 207 (38), 203 (41), 189 (100), 135 (70), 119 (61), 107 (62), 95 (81), 81 (79), 55(90%); cims: 543 (11, M+), 525(64, [MH-H 2 0]+), 425(80), 407 (100), 217 (91), 203 (90), 191 (48), and 189 (52%). Example lb. Betulin, 443 mg (1 mmole), imidazole, 227 mg (4 mmoles), succinyl anhydride, 400 mg (4 mmoles) and 0.6 ml of methylpyrrohdone were combined in a vial. The reaction mixture was maintained at 70°C for 20 hrs. The reaction mixture was then added dropwise to 100 ml of water and stirred for half an hour. The resulting precipitate was filtered off, re- suspended and agitated in another portion of water in order to remove the remaining reactants. This procedure was repeated several times until no more of the reactants were washed out. The sohd was coUected, dried and dissolved in acetone. The acetone solution was filtered and evaporated to give 470 mg of 3,28-dihemisuccinylbetulin. Calculated for: C 38 H 58 0 8 • V* H 2 0, MW 642.84; C, 70.5%, H, 9.1%; found C 70.43%, H 9.15%. 1H NMR: 4.70 and 4.61 (1H, s, each, =CH2), 4.52 (1H, dd, J, = 5.4 Hz, J 2 = 10.0 Hz), 4.33 and 3.90 (1H, d, each, AB pattern, J = 11.1 Hz, OCH j ), 2.63 - 2.71 (8H, m, succinyl chain), 1.70, 1.05, 0.99, 0.87, 0.86, and 0.85 (Me, s, each). MALDI-TOF-MS gave m/z 665 (M + Na + ) and also m/z 542 and 525. Example 2 - Synthesis of acetylbetulinic acid. Betulinic acid, 100 mg (0.22 mmoles) was added to a solution containing 4 mg of 4-dime ylaminopyridine (DMAP) and 0.5 ml of (CH,CO) 2 0 in 5 ml of CH 2 C1 2 . After 2 hrs CH 2 C1 2 was removed in vac o, and the remaining residue stirred with 25 ml of water. During the stirring enough of K 2 C0 3 was added to decompose an excess of acetic anhydride, after which the reaction mixture was extracted with 25 ml of CH 2 C1 2 . The extract was evaporated to dryness and the resulting sohd crystallized from MeOH to give 104 mg of acetylbetulinic acid as white crystals, m.p. 285° C. Calculations for C 32 H 50 O 4 , C, 77.06, H, 10.10; found C, 77.02, H, 10.08%. The 'Hnmr (DMSO-d 6 ) gave peaks at: 4.93 and 4.76 (1H, s, each, CH 2 =C), 4.67 (1H, dd, C3-H, J=5.0, 10.0 Hz), 2.05 (s, CH 3 CO), 1.78, 1.06, 1.00, 0.87, 0.84 and 0.73 (each, s, CH 3 ); eims (m/z, relative intensity): 499 (1, MH+), 452 (2), 438 (38), 395 (26), 189 (40), 43 (100%); cims (m/z, rel. intensity): 499 (37, MH+), 440 (27), 439 (100), 437 (18), 393 (20), 203 (15), 191 (24%). Example 3 - Synthesis of 3-methanesulfonylbetuhnic acid. Betulinic acid, 100 mg (0.22 mM) was dissolved in 5 ml of pyridine and treated with 100 mg (0.88 mmoles) of CH 3 S0 2 C1. After 16 hrs, the pyridine was removed in vacuo and the resulting residue suspended in 10 ml of water. Excess of MeS0 2 Cl was decomposed with an aqueous NaHC0 3 solution, and the reaction mixture extracted with 10 ml of CH 2 C1 2 . Evaporation of the solvent gave a crude product containing 3- rnethanesulfonylbetulinic acid, which was purified by column chromatography over 3 g of silica gel using CH 2 C1 2 with 0 to 5% gradient of MeOH. The chromatography afforded 3- methanesulfonylbetulinic acid, which on crystallization from MeOH gave 62 mg of white crystals, m.p. 210-212° C. Calculations for C 31 H 50 O 5 S: C, 69.62, H, 9.42, S, 6.00; found C, 69.78, H, 9.46, S, 5.90%. Ηnmr (DMSO-d 6 ): 4.95 and 4.78 (1H, each, s, CH 2 =C), 4.50 (1H, dd, J 4.6, 11.8 Hz), 3.30 (3H, s, CH^O,), 1.79, 1.07, 1.06, 1.00, 0.82, 0.72 (each, s, CH 3 ); eims (m/z, relative intensity): 438 (9), 423 (11), 395 (72), 259 (12), 161 (26), 135 (62), 121 (100), 107 (60), 93 (61), 79 (54%); eims (m/z, rel. intensity): 535 (11, MH+), 439 (91), 423 (25), 395 (52), 249 (12), 203 (28), 191 (34), 97 (100%). Example 4 - Synthesis of the methyl ester of 3-methanesulfonylbetulinic acid. Betulinic acid methyl ester, 500 mg (1.06 mmoles), and 320 mg (3.3 mmoles) of Me 3 N were dissolved in 10 ml of CH 2 C1 2 . The solution was cooled in an ice-bath and treated with 190 mg (1.7 mmoles) of MeS0 2 Cl. The reaction mixture was allowed then to warm up to room temperature and left overnight. Evaporation of the solvent in vacuo left a residue, which was dissolved in 10 ml of Et 2 0 and washed 3 times with 5 ml H 2 0. The etheral layer was dried over MgS0 4 , filtered, and evaporated to give 582 mg of the methyl ester of 3-methanesulfonylbetulinic acid. Recrystalhzation from EtOH gave crystals with a melting point of 190° C. Calculation for C 32 H 52 0 5 S: C, 70.03, H, 9.55, S, 5.84; found C, 69.85, H, 9.50, S, 5.65%. 'Hnmr (DMSO-d 6 ): 4.89 and 4.73 (1H, each, CH 2 =C), 4.47 (1H, dd, C3-H, J=2.5, 12.5 Hz), 3.70 (s, OCH 3 ), 3.29 (s, CT SO,), 1.72, 1.04, 0.99, 0.92, 0.81, 0.72 (s, each, CH 3 ); eims (m/z, relative intensity): 452 (9), 409 (14), 341 (8), 273 (15), 255 (12), 189 (100), 175 (47), 121 (72), 107 (72), 93 (76), 79 (85%); eims (m/z, rel. intensity): 549 (16%, MH+), 453 (100), 393 (19), 203 (13), 189 (12), 97 (80%). Examples 5-24 - Elastase inhibition Studies, performed with human leucocyte elastase (HLE) indicated that many common naturally occurring PTs with lupane, oleane, and ursane skeletons inhibit HLE at low micromolar concentrations. Several derivatives of these PTs were prepared using synthetic methodology to explore structure-activity relationship and on testing they also inhibited HLE. The values of inhibitory constants, IC 50 's obtained from HLE inhibition, for the tested compounds, are given in Table 1. Stock solutions were mixed to yield a reaction mixture consisting of 0.5 mM MeO-Suc-Ala-Ala-Pro-Nal-pNA (Sigma) and 0.7 μmg/ml human leucocyte elastase (Elastin Products) in 0.1 M Tris, 0.5 M NaCl and 3% DMSO (v/v) at a pH of 7.5. Test compounds were solubihzed in DMSO. Equal volumes of enzyme and inhibitor were mixed and allowed to incubate for 15 minutes at room temperature. The reaction was started by the addition of a third equal volume containing substrate. Reactions were carried out in microhter plates. Appearance of p-nitroanihne was monitored at 405 nm in a Dynatech microelisa Reader M600. Initial rates in the presence and absence of test compound were compared to determine IC 50 values which are reported in Table 1. Table 1 Ex Compound Name IC Bn (μM) 5 Lupeol 8 4 6 Lupeol acetate 14 9 7 Betu n 5 0 8 28-Succιnylbetulιn 6 3 9 Betulinic acid 3 3 10 Methyl ester of betulinic acid 5 7 1 1 Acetylbetulinic acid 7 0 12 3-Ketobetulιnιc acid 1 1 0 13 Methanesulfonylbetu nic acid 1 1 2 14 Methyl ester of 3 methanesulfonylbetuhnic acid 7 3 15 β-Amyπn 5 4 16 Uvaol 4 5 17 Urso c acid 7 7 18 Ursolic acid. Me ester 4 9 19 α-Amyπn 21 1 20 Oleanohc acid 18 7 21 Oleanohc acid, Me ester 8 5 22 Echinocystic acid 21 1 23 Hederagenin 16 8 24 Caulophyllogenin 0% @ 21 ° Further experiments, carried out with ursohc acid and oleanohc acid, indicated that these compounds can inhibit also other enzymes, such as plasmin and urokinase. The values of inhibition, measured at 22 μM in the plasmin assay were 43% for ursohc acid and 25% for oleanohc acid, while in the urokinase assay were 83% for ursohc acid and 40% for oleanohc acid. For betuhnic acid, in the following assays, IC 5ϋ values were determined to be thrombin 2 3, trypsin 8.5, plasmin 6.2, and urokinase 5.0 μM. The apparent lack of specificity within the tested enzymes and an observation that the inhibition ceases in the presence of 0.1% of albumin suggested that the inhibition results from a non-specific binding to proteins. Thus, PTs appear to bind non-covalendy to hydrophobic domains of enzymes and either block an access to the enzyme active site or cause conformational change, both resulting in the enzyme's inability to perform its function. The most potent inhibitors of the investigated group of PTs contain one to three oxygenated substituents, such as hydroxy or carboxy groups. These moieties contribute to the overall inhibitory effect of the compounds. Examples 25- - Fungicidal activity Tomato, bean, pepper, wheat, and peanut plants are grown for one to three weeks (depending upon species) in the greenhouse. Two pots, which represent two replicates, of each plant species are placed into a flat such that each flat contains all the plants to be sprayed by one compound. The plants in each flat are sprayed to runoff with the desired spraying solution or with fungicide standard. As a control, check plants are sprayed with water. The plants are allowed to air-dry two to three hours. After the plants are dry, they are sorted and grouped by plant species. Table 2 identifies the compositions of examples 25-28 that were tested for their ability to protect hydrophobic plant surfaces: Table 2 Example Material 25 Control vehicle containing acetone 18 ml, dimethylsulfoxide 4 ml, and Palmolive™ detergent 200 mg. Tested as 3% aqueous concentration. 26 40 mg betulin dissolved in 1.5 ml of vehicle and added to 98.5 ml of water. 27 80 mg betulin, 3.0 ml of vehicle, and 97 ml of water. 28 40 mg of ursolic acid, 2.0 ml of vehicle, and 98 ml of water. The plant pathogenic fungi Phytophthora infestans (Pi), Alternaria solani (As), Botrytis cinerea (Be), and Cercospora arachidicola (Ca) were grown in the laboratory on appropriate media. Inoculvim from each fungus was harvested and concentrations adjusted to predetermined levels. The obhgate plant pathogenic fungi (Erysiphe graminis f.sp. tritici, Puccinia recondita f.sp. tritici are harvested from their hosts in the greenhouse and concentration are adjusted to predetermined levels. The plants previously treated with test compounds are sprayed with fungal inoculum then placed in humidity chambers for a period of time which is found to be optimum for development of each disease. After incubation, the plants are moved to the greenhouse, symptoms allowed to develop, and the plants evaluated for disease intensity. Table 3 reports the percent disease control as the average of two rephcates. Table 3 Disease (% control) Example Pi Pv As Be Sn Pr Eq 25 8 phytotoxic 5 12 0 0 0 26 0 0 17 3 0 0 0 27 40 0 3 7 0 0 0 28 65 phytotoxic 8 33 0 0 0 29 33 0 12 12 0 0 0 Pi: Phvtophthora infestans; Late bliαht tomato Pv: Plasmopara viticola; Grape mildew As: Alternaria solani; Early bliαht tomato Be: Botrvtis cinerea; Gray mold Sn: : Septoria nodorum Pr: Puccinia recondita; Wheat leaf rust Eh: : Erysiphe qraminis; Powdery mildew wheat Following review of the control achieved by examples 25-29, the concentration of pentacychc triterpene and carrier vehicle were changed. Table 4 identifies the compositions of examples 30-35, and Table 5 shows the percent control of plant diseases following a protective apphcation of materials in examples 30-35. The results in table 5 are the mean of three rephcates. Table 4 Example Composition 30 Carrier vehicle containing 0.35% solution of Tween 80™ in water. 31 200 mg betulin in 100 ml of carrier. 32 400 mg betulin in 100 ml of carrier. 33 800 mg betulin in 100 ml of carrier. 34 400 mg ursolic acid in 100 ml of carrier. Table 5 Disease (% control) Ex. Pi Pv As Be Sn Pr Eg Sm-p Sm-t 30 7 0 43 94 0 0 0 50 30 31 10 0 47 100 0 0 0 60 20 32 7 37 53 82 0 0 0 65 30 33 60 0 63 97 0 0 0 50 30 34 0 0 0 60 0 0 0 40 60 Pi: Phvtophthora infestans; Late blight tomato Pv: Plasmopara viticola: Grape mildew As: Alternaria solani; Early blight tomato Be: Botrvtis cinerea: Gray mold Sn: Septoria nodorum Pr: Puccinia recoπdita: Wheat leaf rust Eh: Erysiphe graminis: Powdery mildew wheat Sm-p: Sclerotinia minor; Sclerotinia blight (on peanut) Sm-t: Sclerotinia minor; Sclerotinia blight (on tomato) As seen from Table 5, an increase in the amount of apphed betulin generally increased control in susceptible fungus.

Claims: CLAIMS 1. A composition useful for inhibiting enzyme-based attacks on plant surfaces comprising: an agriculturally acceptable carrier and a pentacychc triterpene compound exhibiting a structure of any of formulas I, II, or III: wherein: R 1 Me, CH 2 OH, CH 2 OY\ CH 2 0-X-OH, CH 2 0-X-OY', CH 2 0-X-Y 2 , CH 2 0-X-Y 3 , CH 2 NHY\ C^NY^CHN 3 , CH 2 NH-X-OH, CH 2 NH-X- Y 2 , CH 2 NH-X-Y 3 , CH^H-X-OY 1 , CH 2 OC(0)-OY\ CH 2 0-X-OY\ C0 2 Y\ COY 3 , COY 2 , CHO, CH=N(CH 2 ) m (0(CH 2 ) J n R 4 , or CH=N(CH 2 ) m (0(CH 2 ) n Y 2 ; R 2 , R 3 = H, OH, OY 1 , O-X-OH, O-X-OY 1 , O-X-Y 2 , Y 3 , NHY 1 , NY' 2 , Y 3 , NH-X- OH, NH-X-Y 2 , NH-X-Y 3 , NH-X-OY 1 , NY'-X-OH, NY'-X-Y 2 , NY ] -X- Y 3 , or NY^X-OY 1 ; provided that one of R 2 or R 3 is H or that R 2 and R 3 together denote carbonyl oxygen; R 4 = H, OH, OY\ or Y 3 ; Y 1 = H, alkyl of 1-30 carbon atoms, straight chain or branched, cycloalkyl of 3- 30 carbon atoms, alkanyl of 3-30 carbon atoms, oxyalkyl of 4-30 carbon atoms, phenylalkyl of 7-30 carbon atoms, or phenoxyalkyl of 7-30 carbon atoms; Y 2 = NH^ NHY^ or NY 1 ,; Y 3 = -(O(CHj) J n R 4 or -(O(CH 2 ) J,N 2 , where m=2-4 and n= 1 -230; X= -OC(CH 2 ) p CO- where p= 1-22. 2. A composition as in claim 1 wherein said pentacychc triterpene exhibits the structure of formula I. 3. A composition as in claim 1 wherein said pentacychc triterpene exhibits the structure of formula II. 4. A composition as in claim 1 wherein said pentacychc triterpene exhibits the structure of formula III. 5. A composition as in claim 1 wherein said pentacychc triterpene is selected from the group consisting of lupeol, betulin, 28-hemisuccinylbetulin, betulinic acid, <• - amyrin, uvaol, ursohc acid, and methyl esters thereof. 6. A composition as in claim 1 wherein said pentacychc triterpene compound is selected from the group consisting of lupeol, betulin, 28-succinylbetulin, and the methyl ester of 28-succinylbetulin. 7. A composition as in claim 1 wherein said pentacychc triterpene is betulin. 8. A composition as in claim 1 wherein said pentacychc triterpene is ursohc acid. 9. A composition as in claim 1 wherein said pentacychc triterpene is selected from the group consisting of betulinic acid ester of polypropylene glycol; ursohc acid ester of polypropylene glycol, oleanohc acid ester of polypropylene glycol; 28- hemisuccinylbetulin ester of polypropylene glycol; 3, 28-dihemisuccinylbetulin diester of polypropylene glycol; 28-polyρropylene glycol-betulin ether; and 3, 28- di(polypropylene glycol)-betulin diether. 10. A method for protecting plants against fungus attack by a process comprising: applying to exposed plant surfaces a carrier and an effective amount of a composition containing: a pentacychc triterpene compound exhibiting a structure according to formulas I, II, or III: wherein: R 1 = Me, CH 2 OH, CH 2 ON , CH 2 0-X-OH, CH 2 0-X-OY' , CH 2 0-X-Y 2 , CH 2 0-X-Y 3 , CH 2 NHN, CH 2 NN 2 ,CH 2 Y 3 , CH 2 NH-X-OH, CH 2 NH-X- Y 2 , CH 2 NH-X-Y 3 , CH^H-X-OY 1 , CH 2 OC(0)-OY\ CH 2 0-X-ON, C0 2 Y ] , COY 3 , COY 2 , CHO, CH=N(CH 2 ) m (0(CH 2 ) J n R 4 , or CH=N(CH 2 ) m (0(CH 2 ) m )N 2 ; R 2 , R 3 = H, OH, OY 1 , O-X-OH, O-X-OY 1 , O-X-Y 2 , Y 3 , NHY 1 , NY 1 ,, Ϋ 3 , NH-X- OH, NH-X-Y 2 , NH-X-Y 3 , NH-X-OY 1 , NY^X-OH, NY^X-Y 2 , NY'-X- Y 3 , or NY 1 -X-OY 1 ; provided that one of R 2 and R 3 is H or that R 2 and R 3 together denote carbonyl oxygen; R 4 = H, OH, ON, or Y 3 ; Y 1= H, alkyl of 1-30 carbon atoms, straight chain or branched, cycloalkyl of 3- 30 carbon atoms, alkanyl of 3-30 carbon atoms, oxyalkyl of 4-30 carbon atoms, phenylalkyl of 7-30 carbon atoms, or phenoxyalkyl of 7-30 carbon atoms; Y 2 = NH 2 , NHY 1 , or NY 1 ,; Y 3 = -(0(CH 2 ) n R 4 or - O^H , where m=2-4 and n=l-230; X= -OC^H^ p CO- where p=l-22. 11. A method as in claim 10 wherein the applying step comprises applying a composition containing a pentacychc triterpene compound having the structure of formula I. 12. A method as in claim 10 wherein the applying step comprises applying a composition containing a pentacychc triteφene compound having the structure of formula II. 13. A method as in claim 10 wherein the applying step comprises applying a composition containing a pentacychc triteφene compound selected from the group consisting of lupeol, betulin, 28-succinylbetulin, betulinic acid, methanesulfonylbetuhnic acid, ^ -amyrin, uvaol, ursohc acid, and methyl esters thereof. 14. A method as in claim 10 wherein the applying step comprises applying a composition containing a pentacychc triteφene compound selected from the group consisting of lupeol, betulin, 28-succinylbetulin, and methyl esters thereof. 15. A method as in claim 10 wherein the applying step comprises applying a composition containing betulin. 16. A method as in claim 10 wherein the applying step comprises applying a composition containing ursohc acid. 17. A method as in claim 10 wherein the applying step comprises applying said composition to surfaces of grain grasses.

Inventors: BRANLY KEITH, GLINSKI JAN, BRANLY, KEITH, GLINSKI, JAN
Applicants: BOEHRINGER INGELHEIM PHARMA, BRANLY KEITH, GLINSKI JAN, MICRO FLO CO, BOEHRINGER INGELHEIM PHARMACEUTICALS, INC., BRANLY, KEITH, GLINSKI, JAN, MICRO FLO COMPANY
Application year: 1999
Main classification: A61K31
IPC codes: A01N37, A01N41, A01N45, C07C69, C07C309