Methods and compositions for treating septoria infections
FIELD OF THE INVENTION 
     The invention is in the field of agricultural fungicides. 
     BACKGROUND OF THE INVENTION 
     Numerous fungal species are capable of acting as pathogens on plants of economic significance. In order to prevent and reduce diseases caused by fungal pathogens, numerous compounds having fungicidal or fungistatic activity have been developed. 
     Plant fungal pathogens of particular interest are species belonging to the genus Septoria. Pathogenic Septoria species includes S. agropyrina, S. apiicola, S. azaleae, S. chrysanthemi, S. cornicola, S. elymi, S. lycopersici and S. glycines. Septoria species generally produce leaf spots and blights. Plants affected by Septoria pathogens include lettuce, tomato, chrysanthemum, celery, beets, and carrots. The invention described herein pertains to novel classes of compounds for use in inhibiting the growth of pathogenic fungi, particularly Septoria species, on plants of interest. 
     Two embodiments of compounds for use in the subject methods of controlling Septoria infections, and infections of similar fungal species, are pentamidine and netropsin. Pentamidine has been used to treat several animal parasites; however, the use of pentamidine to treat or prevent fungal infections of plants are unprecedented. Similarly, netropsin has been used to treat bacterial and viral infections in animals; however, the use of netropsin to treat or prevent fungal infections of plants is unprecedented. 
     SUMMARY OF THE INVENTION 
     The invention comprises compounds having the general formula: 
     
         X.sub.1 -A.sub.1 -B.sub.1 -C-B.sub.2 -A.sub.2 -X.sub.2     (I) 
    
     wherein X 1  is an amidine, a guanidine, or an imidazole group. 
     A 1  is either a five-membered aromatic ring, a six-membered aromatic ring, a fusion of two five-membered aromatic rings, a fusion of 2 six-membered aromatic rings, or a fusion of a six-membered aromatic ring and a five-membered aromatic ring; 
     B 1  is either O (oxygen) or absent; 
     C is a straight chain alkyl comprising 1-8 carbons; 
     B 2  is either O (oxygen) or absent; 
     A 2  is either a five-membered aromatic ring, a six-membered aromatic ring, a fusion of two five-membered aromatic rings, a fusion of 2 six-membered aromatic rings, or a fusion of a six-membered aromatic ring and a five-membered aromatic ring; and 
     X 2  is an amidine, a guanidine, or an imidazole. 
     A preferred embodiment of the compounds of formula (I) for use in inhibiting fungal growth is pentamidine. 
     The invention also provides compounds having the general formula: ##STR1## where X 1  is an amidine group, a guanidine group, or an imidazole; where Y is 1-5; 
     where Z is 1-2; and 
     where X 2  is an amidine group, a guanidine group, or an imidazole; 
     A preferred embodiment of compounds of formula (II) is netropsin. 
     Another aspect of the invention is to provide methods for reducing fungal growth by applying an effective amount of a compound of formula (I) or (II). 
     The subject methods of reducing fungal growth may be employed either prophylactically or to reduce the growth of fungi already present in the area to be treated. Preferably, the subject methods are used to reduce or prevent the growth of fungal plant pathogens on plants; however, the subject methods may be used to inhibit fungal growth in a variety of non-agricultural applications, e.g. to reduce weathering damage to wood, paint, and the like. 
     Another aspect of the invention is to provide formulations comprising either a compound of formula (I) or (II) for use in exposing plants to either compounds of formula (I) or (II), respectively. The formulations of the invention comprise an inert carrier and either a compound of formula (I) or (II). 
     DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
     The invention described herein provides novel compounds for the reduction of fungal growth, particularly the growth of the plant pathogen, Septoria. The invention also provides methods of treating fungal infections of plants by applying the compounds of the invention. Another aspect of the invention is to provide novel formulations for application of fungal growth reducing compounds of the invention. 
     The invention comprises compounds having the general formula: 
     
         X.sub.1 -A.sub.1 -B.sub.1 -C-B.sub.2 -A.sub.2 -X.sub.2     (I) 
    
     wherein X 1  is an amidine, a guanidine, or an imidazole group. 
     A 1  is either a five-membered aromatic ring, a six-membered aromatic ring, a fusion of two five-membered aromatic rings, a fusion of 2 six-membered aromatic rings, or a fusion of a six-membered aromatic ring and a five-membered aromatic ring; 
     B 1  is either O (oxygen) or absent; 
     C is a straight chain alkyl comprising 1-8 carbons; 
     B 2  is either O (oxygen) or absent; 
     A 2  is either a five-membered aromatic ring, a six-membered aromatic ring, a fusion of two five-membered aromatic rings, a fusion of 2 six-membered aromatic rings, or a fusion of a six-membered aromatic ring and a five-membered aromatic ring; and 
     X 2  is an amidine, a guanidine, or an imidazole. 
     Preferred embodiments of the compound of formula I are capable of specifically binding the minor groove on a double-stranded DNA helix. A particularly preferred embodiment of the compounds of formula (I) for use in inhibiting fungal growth is pentamidine. 
     The preferred embodiment of the compound of formula (I) is pentamidine. Methods for the synthesis of pentamidine are well known. For example, pentamidine may be synthesized as described in U.K. Pat. No. 567,565, Ashley et al. J. Chem. Soc., 1942, 103, and U.S. Pat. No. 2,394,003 and by numerous variations of these synthesis methods that would be obvious to persons of ordinary skill in the art of organic chemistry. In addition to being able to synthesize pentamidine, the person of ordinary skill in the art may readily synthesize the full range of compounds within the scope of formula (I). The details on such syntheses may be obtained by performing searches of chemical abstracts and through the review of standard texts such as a Vogel&#39;s Textbook of Practical Organic Chemistry 5th ed, Tatchell et al., John Wiley and Sons, NY, N.Y. (1989), March, Advanced Organic Chemistry: Reactions, Mechanisms and Structures 4th ed, John Wiley and Sons, NY, N.Y. (1993), and the like. 
     The invention also provide compounds having the general formula: ##STR2## where X 1  is an amidine group., a guanidine group, or an imidazole; where Y is 1-5; 
     where Z is 1-2; and 
     where X 2  is an amidine group, a guanidine group, or an imidazole. 
     Preferred embodiments of the compound of formula II are capable of specifically binding the minor groove on a double-stranded DNA helix. A particularly preferred embodiment of the compounds of formula (II) is the compound netropsin. Netropsin may be isolated from the bacterium Streptomyces netropsis. The preparation of netropsin is described in Finlay et al., J. Am. Chem. Soc 73, 341 (1951). The preparation of netropsin and various minor groove binding derivatives thereof is described in, among other places, Wade et al. J. A. Chem. Soc. 114: 8783-8794 (1992). A person of ordinary skill in the art of organic chemistry may prepare netropsin as well as other compounds of formula (II) using well known organic synthesis methods. 
     Many compounds capable of specifically binding to the minor groove of double-stranded DNA, preferably AT rich regions, may have fungicidal activity, particularly against Septoria species. In addition to the compounds of formulae I and II, the subject invention specifically contemplates the use of other minor groove DNA binding compounds to treat and/or prevent fungal infections of plants and also contemplates formulations for treating plants that comprise such minor groove DNA binding compounds. Other DNA minor groove binding compounds that can be used in the methods and formulation of the invention include berenil, bis-benzamide, distamycin A, and DAPI (4&#39;,6-diamidino-2-phenylindole or 2-[4&#39;-guanyl-phenyl]-6-guanylinole). 
     The term &#34;minor groove DNA binding&#34; as used herein, refers to the property of preferentially binding to the minor groove of a double-stranded DNA molecule (B form), as opposed to binding to other portions of a double-stranded DNA molecule. A compound having a minor groove DNA binding property may preferentially bind to portions of a DNA molecule comprising particular nucleotide bases, i.e., sequence specific binding, or the minor groove binding may be non-sequence specific. Preferred compounds for use in the treatment and/or prevention of fungal infection have minor groove DNA binding properties similar to that of pentamidine or netropsin. 
     Dicationic molecules such as pentamidine, Berenil, DAPI, and netropsin bind to DNA targets by selectively interacting with regions of the minor groove. AT-rich regions of the minor groove are preferred binding sites for bis-benzamidines due to the protrusion of the bulky 2-amino group of guanine residues into the minor groove of GC-rich DNA. AT-rich regions of the minor groove have the highest negative electrostatic potential and hence the greatest avidity for dicationic ligands. In addition, van der Waals contacts between aromatic polyamidines and the floor of the minor groove are maximized in AT-rich regions due to the narrowness of AT-rich motifs which increases the strength of van der Waals interactions with aromatic systems. Hydrogen bonding interactions with bases on the floor of the groove are also important to binding affinity. The complimentary of the curvature of dicationic bis-.benzimidazoles with that of the DNA minor groove is also of considerable importance. Molecules with a curvature which closely fits the curvature of the minor groove have the highest target affinity. The above explanation of DNA binding should not be construed as a limitation of the invention. The interaction of aromatic polyamidines (and other molecules) with DNA can be measured by the increase in thermal melting temperature (ΔTm) following complex formation with synthetic copolymers such as poly dA-dT (Wilson, W. D., Ratmeyer, L., Zhao, M., Strkowski, W. and D. W. Boykin. 1993. Biochemistry 32: 4098-4104). 
     Formulation 
     The compounds of this invention will generally be used in formulation with a liquid or solid diluent or with an organic solvent. The invention specifically provides for numerous formulation comprising either compounds of formula (I) or formula (II) and an inert carrier, such as a diluent. The term &#34;inert&#34; is used to indicate that the carrier does not have significant fungicidal activity. The formulations of the inventions comprise either a compound according to formula (I) or formula (II) and a diluent or surfactant, which may not act as an inert carrier. The formulations may further comprise additional compounds that have fungicidal activity. Useful formulations of the compounds of formula (I) and formula (II) can be prepared in conventional ways. They include dusts, granules, pellets, solutions, emulsions, wettable powders, emulsifiable concentrates and the like. Many of these may be applied directly. Sprayable formulations can be extended in suitable media and used at spray volumes of from about one to several hundred liters per hectare. High strength compositions are primarily used as intermediates for further formulation. The formulations, broadly, contain about 1% to 99% by weight of active ingredient(s) and at least one of a) about 0.1% to 35% surfactant(s) and b) about 5% to 99% solid or liquid inert diluent(s). More specifically, they will contain these ingredients in the following approximate proportions: 
     
         ______________________________________                                    
            Active  Percent by Weight                                     
            Ingredient                                                    
                    Diluent(s)                                            
                              Surfactant(s)                               
______________________________________                                    
Wettable Powders                                                          
              20-90      0-74     1-10                                    
Oil Suspensions,                                                          
               5-50     40-95     0-35                                    
Emulsions, Solutions,                                                     
(including Emulsifiable                                                   
Concentrates)                                                             
Aqueous Suspensions                                                       
              10-50     40-84     1-20                                    
Dusts          1-25     70-99     0-5                                     
Granules and Pellets                                                      
               1-95      5-99     0-15                                    
High Strength 90-99      0-10     0-2                                     
Compositions                                                              
______________________________________                                    
 
    
     Lower or higher levels of active ingredient can, of course, be present depending on the intended use and the physical properties of the compound. Higher ratios of surfactant to active ingredient are sometimes desirable, and are achieved by incorporation into the formulation or by tank mixing. 
     Typical solid diluents are described in Watkins et al., &#34;Handbook of Insecticide Dust Diluents and Carriers,&#34; 2nd Ed., Dorland Books, Caldwell, N.J. The more absorptive diluents are preferred for the wettable powders and the denser ones for dusts. Typical liquid diluents and solvents are described in Marsden, &#34;Solvents Guide,&#34; 2nd Ed., Interscience, New York, N.Y., 1950. Solubility under 0.1% is preferred for suspension concentrates; solution concentrates are preferably stable against phase separation at 0° C. &#34;McCutcheon&#39;s Detergents and Emulsifiers Annual,&#34; MC Publishing Corp., Ridgewood, N.J., as well as Sisely and Wood, &#34;Encyclopedia of Surface Active Agents,&#34; Chemical Publ. Co., Inc., New York, N.Y., 1964, list surfactants and recommended uses. All formulations can contain minor amounts of additives to reduce foam, caking, corrosion, microbiological growth, etc. Additives to protect the active compounds against light induced degradation, e.g., photoprotectants, UV screening compounds, and the like are also preferably included in the subject formulations. Preferably, ingredients should be approved by the U.S. Environmental Protection Agency for the use intended. 
     The methods of making such compositions are well known. Solutions are prepared by simply mixing the ingredients. Fine solid compositions are made by blending and, usually, grinding as in a hammer or fluid energy mill. Suspensions are prepared by wet milling (see, for example, Littler, U.S. Pat. No. 3,060,084). Granules and pellets may be made by spraying the active material upon prefoamed granular carriers or by agglomeration techniques. See J. E. Browning, &#34;Agglomeration,&#34; Chemical Engineering, Dec. 4, 1967, pp. 147ff and &#34;Perry&#39;s Chemical Engineer&#39;s Handbook,&#34; 4th Ed., McGraw-Hill, New York, N.Y., 1963, pp. 8-59ff. 
     For more information regarding the art of formulation, see for example: 
     H. M. Loux, U.S. Pat. No. 3,235,361, Feb. 15, 1966, Column 6, Line 16 through Column 7, Line 19 and Examples 10 through 41. 
     R. W. Luckenbaugh, U.S. Pat. No. 3,309,192, Mar. 14, 1967, Column 5, line 43 through Column 7, Line 62 and Examples 8, 12, 15, 39, 41, 52, 53, 58, 132, 138-140, 162-164, 166, 167, 169-182. 
     H. Gysin and E. Knusli, U.S. Pat. No. 2,891,855, Jun. 23, 1959, Column 3, Line 66 through Column 5, Line 17 and Examples 1-4. 
     G. C. Klingman, &#34;Weed Control as a Science,&#34; John Wiley and Sons, Inc., New York, N.Y., 1961, pp. 81-96. 
     J. D. Fryer and S. A. Evans, &#34;Weed Control Handbook,&#34; 5th Ed. Blackwell Scientific Publications, Oxford, 1968, pp. 101-103. 
     Methods of Use 
     The invention provides for novel methods of inhibiting the growth of plant pathogenic fungi, particularly fungi that are species of the genus Septoria. The methods of the invention comprise the step of applying a compound of either formula (I) or formula (II) to a plant of interest. The compound may be applied to a plant of interest by a variety of means such as spraying a liquid, dusting a powder and the like, well known to the person of ordinary skill in the art of crop protection. The particular method of application selected will be dependent upon a number of factors such as the type of plant, the formulation selected, the arrangement of plants in the field, weather conditions, and the like. The actual amount of fungal growth inhibiting compound applied to each plant may be varied so as to achieve the desired degree of growth inhibition. Optimal dosage for a given plant for a given pathogen, under a given set of environmental conditions may be determined through routine experimentation in which the dosage is systematically varied. 
    
    
     The invention may be better understood by referring to the following examples. The following examples are offered for the purpose of illustrating the invention and should not be interpreted as a limitation of the invention. 
     EXAMPLES 
     Example 1 
     Several compounds were tested for their fungicidal activity against a variety of fungi that are plant pathogens. The compounds were dissolved in a 5% solution of polyethylene glycol and sprayed onto plants and dried at 24° C. The plants were then inoculated with the indicated pathogen one or two days later. After a period of five: to eleven days, the presence of disease was assessed. The extent of protection is indicated on a linear scale 0 (no protection) to 10 (100% protection). Pentamidine and netropsin were the most effective of the compounds tested. Concentration is given in parts per million (ppm). 
     
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                B. cinerea                                                
                      B. cinerea                                          
                            C. arachidicola                               
                                     P. oryzae                            
                                           V. inaequalia                  
                                                   S. nodorum             
                                                          P. teres        
                apple grape peanut   rice  apple   wheat  barley          
Compound                                                                  
       Concentration                                                      
                fruit foliar                                              
                            foliar   foliar                               
                                           foliar  foliar foliar          
__________________________________________________________________________
Pentamidine                                                               
       200      9     9     3        0     7       8      8               
       60       9     8     0        0     3       6      7               
       20       8     7     0        0     0       4      3               
       6        7     6     0        0     0       0      0               
       2        0     1     0        0     0       0      0               
Netropsin                                                                 
       200      10    0     0        0     0       3      0               
       60       9     0     0        0     0       0      0               
       20       8     0     0        0     0       0      0               
       6        6     0     0        0     0       0      0               
       2        0     0     0        0     0       0      0               
__________________________________________________________________________
 
    
     Incorporation by Reference 
     All patents, patents applications, and publications cited are incorporated herein by reference. 
     Equivalints 
     The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. Indeed, various modifications of the above-described modes for carrying out the invention which are obvious to those skilled in the field of organic chemistry or related fields are intended to be within the scope of the following claims.