Methods and compositions for treating septoria infections

The invention provides fungicidal compounds having the general formula: EQU X.sub.1 -A.sub.1 -B.sub.1 -C-B.sub.2 -A.sub.2 -X.sub.2 (I) PA1 wherein X.sub.1 is an amidine, a guanidine, or an imidazole group. PA1 A.sub.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; PA1 B.sub.1 is either O (oxygen) or absent; C is a straight chain alkyl comprising 1-8 carbons; B.sub.2 is either O (oxygen) or absent; A.sub.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.sub.2 is an amidine, a guanidine, or an imidazole.

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: 
EQU X.sub.1 -A.sub.1 -B.sub.1 -C-B.sub.2 -A.sub.2 -X.sub.2 (I) 
wherein X.sub.1 is an amidine, a guanidine, or an imidazole group. 
A.sub.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.sub.1 is either O (oxygen) or absent; 
C is a straight chain alkyl comprising 1-8 carbons; 
B.sub.2 is either O (oxygen) or absent; 
A.sub.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.sub.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.sub.1 is an amidine group, a guanidine group, or an imidazole; 
where Y is 1-5; 
where Z is 1-2; and 
where X.sub.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: 
EQU X.sub.1 -A.sub.1 -B.sub.1 -C-B.sub.2 -A.sub.2 -X.sub.2 (I) 
wherein X.sub.1 is an amidine, a guanidine, or an imidazole group. 
A.sub.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.sub.1 is either O (oxygen) or absent; 
C is a straight chain alkyl comprising 1-8 carbons; 
B.sub.2 is either O (oxygen) or absent; 
A.sub.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.sub.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'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.sub.1 is an amidine group., a guanidine group, or an imidazole; 
where Y is 1-5; 
where Z is 1-2; and 
where X.sub.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',6-diamidino-2-phenylindole or 
2-[4'-guanyl-phenyl]-6-guanylinole). 
The term "minor groove DNA binding" 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 (.DELTA.Tm) 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 "inert" 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., "Handbook of 
Insecticide Dust Diluents and Carriers," 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, "Solvents Guide," 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.degree. C. "McCutcheon's Detergents and Emulsifiers 
Annual," MC Publishing Corp., Ridgewood, N.J., as well as Sisely and Wood, 
"Encyclopedia of Surface Active Agents," 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, "Agglomeration," Chemical Engineering, 
Dec. 4, 1967, pp. 147ff and "Perry's Chemical Engineer's Handbook," 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, "Weed Control as a Science," John Wiley and Sons, Inc., New 
York, N.Y., 1961, pp. 81-96. 
J. D. Fryer and S. A. Evans, "Weed Control Handbook," 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.degree. 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). 
__________________________________________________________________________ 
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.