Novel antifungal agents having the formula: ##STR1## wherein R is hydrogen or a radical of the formula ##STR2## and pharmaceutically acceptable prodrugs thereof, as well as (i) pharmaceutical compositions comprising such compounds, (ii) methods of treatment using such compounds, and (iv) methods and fungal cultures useful in making the same.

TECHNICAL FIELD 
The present invention relates to novel fungal isolates of potential 
medicinal value. More particularly, the invention relates to compounds 
isolated from cultures of the Fusarium genus, herein designated 
"fusacandins", which possesses antifungal activity, as well as to methods 
and cultures of microorganisms useful for the preparation of fusacandins, 
pharmaceutical compositions containing such compounds, and the use thereof 
in treating fungal infections. 
BACKGROUND OF THE INVENTION 
The compounds of the present invention are related to those of the 
papulacandin class, described in J. Antibiotics 33(9):967-977 (1980). 
Papulacandins include BE-29602, disclosed in a published Japanese patent 
application of Banyu Pharmaceutical Co. (No. JP05170784-A, published Jul. 
9, 1993) and isolated from a Fusarium species of fungus, and 
chaetiacandin, disclosed in J. Antibiotics 38(4):455-459 (1985) and J. 
Antibiotics 38(4):544-546 (1985). The fusacandins are distinct from the 
papulacandin compounds, however, in that they contain three sugar moieties 
not previously described in connection with other members of this class. 
SUMMARY OF THE INVENTION 
It has now been found that novel antifungal agents of the papulacandin 
class, herein designated "fusacandins", may be obtained by the 
fermentation of certain cultures belonging to the fungal strain Fusarium 
sp. AB 1900A-1314. 
Accordingly, in one aspect of the present invention are disclosed compounds 
of the formula: 
##STR3## 
as well as pharmaceutically acceptable prodrugs thereof. 
In the above formula (I), R may be a radical of the formula 
##STR4## 
in which instance the compound is designated fusacandin A. Alternatively, 
the radical R may be hydrogen, in which instance the compound is 
designated fusacandin B. 
In another aspect of the present invention are disclosed pharmaceutical 
compositions which comprise a compound of the invention in combination 
with a pharmaceutically acceptable carrier. 
In a further aspect of the invention is disclosed a method of suppressing 
or inhibiting a fungal infection in a patient in need of such treatment, 
comprising administering to the patient a therapeutically effective amount 
of a compound of the invention. 
In yet another aspect of the invention, a process for preparing the 
compounds of the invention is disclosed which comprises the steps of (a) 
culturing a microorganism having substantially all the characteristics of 
Fusarium species AB 1900A-1314 under suitable conditions in a fermentation 
medium containing assimilable sources of carbon and nitrogen; (b) allowing 
the desired compound to accumulate in the fermentation medium; and (c) 
isolating the compound from the fermentation medium. Preferably, the 
microorganism to be cultured is Fusarium strain NRRL 21252 or a mutant or 
derivative thereof. 
Similarly, in an additional aspect of the present invention is disclosed a 
biologically pure culture of a microorganism capable of producing the 
compounds of the invention, namely, a microorganism having substantially 
all the characteristics of Fusarium species AB 1900A-1314. Preferably, the 
microorganism is Fusarium strain NRRL 21252 or a mutant or derivative 
thereof.

DETAILED DESCRIPTION OF THE INVENTION 
As used throughout this specification and in the appended claims, the 
following terms have the meanings specified: 
The term "biologically pure" as used herein refers to fungal cultures which 
are substantially free from biologically active contaminants. 
The term "mutant or derivative" as used herein refers to fungal strains 
which are obtained by mutagenization or genetic modification of Fusarium 
species strain NRRL 21252 by techniques readily known in the art. 
The term "pharmaceutically acceptable prodrug" as used herein refers to 
those prodrugs of the compounds of the present invention which are, within 
the scope of sound medical judgement, suitable for use in contact with the 
tissues of humans and lower animals without undue toxicity, irritation, 
allergic response, and the like, commensurate With a reasonable 
benefit/risk ratio, and which are effective for their intended use. 
The term "prodrug" refers to compounds that are rapidly transformed in vivo 
to yield the parent compound of the above formula, for example by 
hydrolysis in blood. A thorough discussion is provided in T. Higuchi and 
V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. 
Symposium Series, and in Edward B. Roche, ed., Bioreversible Carders in 
Drug Design, American Pharmaceutical Association and Pergamon Press 
(1987). (Both here and throughout the specification, it is intended that 
citations to the literature are expressly incorporated by reference.) 
Where appropriate, prodrugs of derivatives of compounds of the present 
invention may be prepared by any suitable method. For those compounds in 
which the prodrug moiety is an amino acid or peptide functionality, the 
condensation of the amino group with amino acids and peptides may be 
effected in accordance with conventional condensation methods such as the 
azide method, the mixed acid anhydride method, the DCC 
(dicyclohexyl-carbodiimide) method, the active ester method (p-nitrophenyl 
ester method, N-hydroxy-succinic acid imide ester method, cyanomethyl 
ester method and the like), the Woodward reagent K method, the DCC-HOBT 
(1-hydroxy-benzotriazole) method and the like. Classical methods for amino 
acid condensation reactions are described in M. Bodansky, Y. S. Klausner 
and M. A. Ondetti, Peptide Synthesis, Second Edition (New York, 1976). 
Asymmetric centers may exist in the compounds of the present invention. 
Except where otherwise noted, the present invention contemplates the 
various stereoisomers and mixtures thereof. 
The compounds of the invention exhibit in vitro activity as antifungal 
agents against a variety of fungal organisms and inhibit 
(1,3)-.beta.-glucan synthetase. They are therefore expected to be useful 
in the treatment of fungal infections in mammals. When used in such 
treatment, a therapeutically effective amount of the compound of the 
present invention may be employed in pure form or, where such forms exist, 
in pharmaceutically acceptable salt, ester or prodrug form. Alternatively, 
the compound may be administered as pharmaceutical compositions containing 
the compound of interest in combination with one or more pharmaceutically 
acceptable excipients. By a "therapeutically effective amount" of the 
compound of the invention is meant a sufficient amount of the compound to 
treat the targeted disorder, at a reasonable benefit/risk ratio applicable 
to any medical treatment, which is administered in such quantities and 
such a period of time as is necessary to obtain the desired therapeutic 
effect. It will be understood, however, that the total daily usage of the 
compounds and compositions of the present invention will be decided by the 
attending physician within the scope of sound medical judgement. The 
specific therapeutically effective dose level for any particular patient 
will depend upon a variety of factors including the disorder being treated 
and the severity of the disorder; activity of the specific compound 
employed; the specific composition employed; the age, body weight, general 
health, sex and diet of the patient; the time of administration, route of 
administration, and rate of excretion of the specific compound employed; 
the duration of the treatment; drugs used in combination or coincidental 
with the specific compound employed; and like factors well known in the 
medical arts. For example, it is well within the skill of the art to start 
doses of the compound at levels lower than required to achieve the desired 
therapeutic effect and to gradually increase the dosage until the desired 
effect is achieved. 
The total daily dose of the compound of this invention administered to a 
human or lower animal may range from about 0.1 to about 100 mg/kg/day. For 
purposes of oral administration, doses may be in the range of from about 1 
to about 100 mg/kg/day or, more preferably, of from about 10 to about 20 
mg/kg/day. If desired, the effective daily dose may be divided into 
multiple doses for purposes of administration; consequently, single dose 
compositions may contain such amounts or submultiples thereof as make up 
the daily dose. 
The pharmaceutical compositions of the present invention comprise a 
compound of the invention and a pharmaceutically acceptable carrier or 
excipient, which may be administered orally, rectally, parenterally, 
intracisternally, intravaginally, intraperitoneally, topically (as by 
powders, ointments, or drops), bucally, or as an oral or nasal spray. By 
"pharmaceutically acceptable carrier" is meant a non-toxic solid, 
semi-solid or liquid filler, diluent, encapsulating material or 
formulation auxiliary of any type. The term "parenteral" as used herein 
refers to modes of administration which include intravenous, 
intramuscular, intraperitoneal, intrasternal, subcutaneous and 
intraarticular injection and infusion. 
Pharmaceutical compositions of this invention for parenteral injection 
include pharmaceutically acceptable sterile nonaqueous solutions or 
aqueous dispersions, suspensions or emulsions as well as sterile powders 
for reconstitution into sterile injectable solutions or dispersions just 
prior to use. Examples of suitable aqueous and nonaqueous carriers, 
diluents, solvents or vehicles include water, ethanol, polyols (such as 
glycerol, propylene glycol, polyethylene glycol, and the like), 
carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such 
as olive oil), and injectable organic esters such as ethyl oleate. Proper 
fluidity can be maintained, for example, by the use of coating materials 
such as lecithin, by the maintenance of the required particle size in the 
case of dispersions, and by the use of surfactants. 
These compositions may also contain adjuvants such as preservative, wetting 
agents, emulsifying agents, and dispersing agents. Prevention of the 
action of microorganisms may be ensured by the inclusion of various 
antibacterial and antifungal agents, for example, paraben, chlorobutanol, 
phenol sorbic acid, and the like. It may also be deskable to include 
isotonic agents such as sugars, sodium chloride, and the like. Prolonged 
absorption of the injectable pharmaceutical form may be brought about by 
the inclusion of agents which delay absorption such as aluminum 
monostearate and gelatin. 
In some cases, in order to prolong the effect of the drug, it is desirable 
to slow the absorption of the drug from subcutaneous or intramuscular 
injection. This may be accomplished by the use of a liquid suspension of 
crystalline or amorphous material with poor water solubility. The rate of 
absorption of the drug then depends upon its rate of dissolution which, in 
turn, may depend upon crystal size and crystalline form. Alternatively, 
delayed absorption of a parenterally administered drug form is 
accomplished by dissolving or suspending the drug in an oil vehicle. 
Injectable depot forms are made by forming microencapsule matrices of the 
drug in biodegradable polymers such as polylactide-polyglycolide. 
Depending upon the ratio of drug to polymer and the nature of the 
particular polymer employed, the rate of drug release can be controlled. 
Examples of other biodegradable polymers include poly(orthoesters) and 
poly(anhydrides) Depot injectable formulations are also prepared by 
entrapping the drug in liposomes or microemulsions which are compatible 
with body tissues. 
The injectable formulations can be sterilized, for example, by filtration 
through a bacterial-retaining filter, or by incorporating sterilizing 
agents in the form of sterile solid compositions which can be dissolved or 
dispersed in sterile water or other sterile injectable medium just prior 
to use. 
Solid dosage forms for oral administration include capsules, tablets, 
pills, powders, and granules. In such solid dosage forms, the active 
compound is mixed with at least one inert, pharmaceutically acceptable 
excipient or carrier such as sodium citrate or dicalcium phosphate and/or 
a) fillers or extenders such as starches, lactose, sucrose, glucose, 
mannitol, and silicic acid, b) binders such as, for example, 
carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose 
and acacia, c) humectants such as glycerol, d) disintegrating agents such 
as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, 
certain silicates and sodium carbonate, e) solution retarding agents such 
as paraffin, f) absorption accelerators such as quaternary ammonium 
compounds, g) wetting agents such as, for example, cetyl alcohol and 
glycerol monostearate, h) absorbents such as kaolin and bentonite clay, 
and i) lubricants such as talc, calcium stearate, magnesium stearate, 
solid polyethylene glycols and sodium lauryl sulfate, and mixtures 
thereof. In the case of capsules, tablets and pills, the dosage form may 
also comprise buffering agents. 
Solid compositions of a similar type may also be employed as fillers in 
soft and hard-filled gelatin capsules using such excipients as lactose or 
milk sugar as well as high molecular weight polyethylene glycols and the 
like. 
The solid dosage forms of tablets, dragees, capsules, pills, and granules 
can be prepared with coatings and shells such as enteric coatings and 
other coatings well known in the pharmaceutical formulating art. They may 
optionally contain opacifying agents and can also be of a composition that 
they release the active ingredient(s) only, or preferentially, in a 
certain part of the intestinal tract, optionally, in a delayed manner. 
Examples of embedding compositions which can be used include polymeric 
substances and waxes. 
The active compounds can also be in micro-encapsulated form, if 
appropriate, with one or more of the above-mentioned excipients. 
Liquid dosage forms for oral administration include pharmaceutically 
acceptable emulsions, solutions, suspensions, syrups and elixirs. In 
addition to the active compounds, the liquid dosage forms may contain 
inert diluents commonly used in the art such as, for example, water or 
other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, 
isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl 
benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils 
(in particular, cottonseed, groundnut, corn, olive, castor and sesame 
oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and 
fatty acid esters of sorbitan, and mixtures thereof. 
Besides inert diluents, the oral compositions can also include adjuvants 
such as wetting agents, emulsifying and suspending agents, and sweetening, 
flavoring and perfuming agents. 
Suspensions, in addition to the active compounds, may contain suspending 
agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene 
sorbitol and sorbitan esters, microcrystalline cellulose, aluminum 
metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof. 
Topical administration includes administration to the skin or mucosa, 
including surfaces of the lung and eye. Compositions for topical 
administration, including those for inhalation, may be prepared as a dry 
powder which may be pressurized or non-pressurized. In non-pressurized 
powder compositions, the active ingredient in finely divided form may be 
used in admixture with a larger-sized pharmaceutically acceptable inert 
carrier comprising particles having a size, for example, of up to 100 
micrometers in diameter. Suitable inert carders include sugars such as 
lactose. Desirably, at least 95% by weight of the particles of the active 
ingredient have an effective particle size in the range of 0.01 to 10 
micrometers. 
Alternatively, the composition may be pressurized and contain a compressed 
gas, such as nitrogen or a liquified gas propellant. The liquified 
propellant medium and indeed the total composition is preferably such that 
the active ingredient does not dissolve therein to any substantial extent. 
The pressurized composition may also contain a surface active agent. The 
surface active agent may be a liquid or solid non-ionic surface active 
agent or may be a solid anionic surface active agent. It is preferred to 
use the solid anionic surface active agent in the form of a sodium salt. 
Compositions for rectal or vaginal administration are preferably 
suppositories which can be prepared by mixing the compounds of this 
invention with suitable non-irritating excipients or carriers such as 
cocoa butter, polyethylene glycol or a suppository wax which are solid at 
room temperature but liquid at body temperature and therefore melt in the 
rectum or vaginal cavity and release the active compound. 
The compound of the present invention can also be administered in the form 
of liposomes. As is known in the art, liposomes are generally derived from 
phospholipids or other lipid substances. Liposomes are formed by mono- or 
multi-lamellar hydrated liquid crystals that are dispersed in an aqueous 
medium. Any non-toxic, physiologically acceptable and metabolizable lipid 
capable of forming liposomes can be used. The present compositions in 
liposome form can contain, in addition to a compound of the present 
invention, stabilizers, preservatives, excipients, and the like. The 
preferred lipids are the phospholipids and the phosphatidyl cholines 
(lecithins), both natural and synthetic. Methods to form liposomes are 
known in the art. See, for example, Prescott, Ed., Methods in Cell 
Biology, Volume XIV, Academic Press, New York, N.Y., 1976, p. 33 et seq. 
The compounds of the present invention may be produced by culturing, in 
appropriate media, fungal microorganisms which are capable of producing 
fusacandins. The compounds are produced when the culture is grown in a 
stationary fermentation with a culture medium containing a source of 
carbon and a source of nitrogen. Media which are useful include an 
assimilable source of carbon such as starch, sugar, molasses, glycerol, a 
combination of glucose plus molasses, etc.; an assimilable source of 
nitrogen such as protein, protein hydrolysate, polypeptides, amino acids, 
peptone plus yeast extract or whole yeast, etc.; and other optional 
organic and inorganic ingredients which can be added to stimulate 
production of the fusacandin compounds. For example, inorganic anions and 
cations including potassium, magnesium, calcium, ammonium, sulfate, 
carbonate, phosphate, chloride, etc. may be added to the medium. Further, 
buffers such as calcium carbonate can be added to aid in controlling the 
pH of the fermentation medium. The stationary fermentation may include a 
solid support to increase the surface area available for fungal growth. 
Suitable supports include Spoon Size Shredded Wheat, rolled oats, barley, 
cracked corn, flee, millet, corn bran, wheat bran, oat bran, vermiculite, 
etc. The culture may be incubated in stationary vessel (without movement) 
or in a cylindrical or other vessel which is rolled or agitated to 
increase aeration. Other culture methods, such as a liquid, submerged, 
agitated culture process are feasible. In these cases, aeration may be 
provided by forcing sterile air through the fermentation medium. Agitation 
can be provided by shaking the container or by stirring the culture, for 
example, with a mechanical stirrer. The fermentation is generally carried 
out in a temperature range of from about 15.degree. C. to about 35.degree. 
C. The pH of the fermentation is preferably maintained between 3 and 9. 
The compound is produced and accumulated between 3 and 28 days after 
inoculation of the fermentation medium. 
Subsequent to the fermentation process, the fusacandin compounds can be 
extracted from the fermentation broth as for example with ethyl 
acetate/acetone solvent mixtures. Partial purification of the active 
components can be achieved by sequential trituration of the organic 
extract with organic solvents such as ethyl acetate, ethanol and methanol 
in order to selectively remove the desired organic compounds. The extracts 
may be further purified by use of various partitioning solvent systems 
such as, for example, chloroform/methanol/water, hexane/ethyl 
acetate/methanol/water, or ethanol/ethyl acetate/water. Further 
purification and separation of individual components can be achieved by 
countercurrent chromatography in solvent systems such as, for example, 
ethyl acetate/ethanol/water, chloroform/methanol/water, or 
chloroform/carbon tetrachloride/methanol/water, and/or by adsorption onto 
silica gel and subsequent elution with organic solvents and solvent 
mixtures such as ethyl acetate, chloroform and methanol. Size exclusion 
chromatography on resin such as SEPHADEX LH-20, developed in a solvent 
such as methanol, affords the pure compound. 
The compounds, processes and uses of the present invention will be better 
understood in connection with the following examples, which are intended 
as an illustration of and not a limitation upon the scope of the 
invention. The following abbreviations were used: EtOAc for ethyl acetate, 
EtOH for ethanol, MeOH for methanol and TLC for thin layer chromatography. 
EXAMPLE 1 
Identification and Characterization of of the Fusacandin-Producing Strain 
Fusarium Sp. Strain AB 1900A-1314 
The compounds of the present invention, "fusacandins", were first obtained 
from a fungus isolated from a conk (fruiting body of wood-attacking 
fungus) collected in Piatt County, Illinois. The culture, which was 
designated strain AB 1900A-1314, is a Fusarium species as indicated by the 
production of characteristic macroconidia. A subculture of this 
microorganism was deposited in the permanent collection of the National 
Center for Agricultural Utilization Research, United States Department of 
Agriculture, 1815 North University Street, Peoria, Ill. 61604, U.S.A., 
under the terms of the Budapest Treaty, and accorded accession number NRRL 
21252. 
Strain AB 1900A-1314 was examined at the Pennsylvania State University, 
Fusarium Research Center, and identified as a strain of Fusarium 
sambucinum. The morphological and cultural characteristics of this strain 
grown on two media at 25.degree. C. for seven days were as described 
below. The colors and numbers shown in parenthesis were assigned based on 
the Inter-Society Color Council-National Bureau of Standards (ISCC-NBS) 
Centroid Color Charts, U.S. Dept. of Commerce supplement to NBS Cir. 553, 
Washington D.C., 1976. 
Colonies of strain AB 1900A-1314 on Potato Dextrose agar (Difco) were 
dense, floccose, medium yellow-pink (29), grew rapidly to 75-80 mm in 
diameter and produced a clear exudate. Aerial mycelium was pale pink in 
color (7) and the reverse was light orange (52) to medium orange (53). 
Spore structures were not found on this medium at seven days. As the 
culture aged, many raised, round, light orange (52) structures, 1-3 mm in 
diameter, developed on the agar surface. These aggregates were hard but 
could be broken apart by moderate pressure with an inoculating loop. 
Fragments of the structures under light microscopy consisted of flattened, 
irregular cells mixed with a few strands of mycelia. The aggregates appear 
to be similar to the perithecia-bearing stromata which Fusarium sambucinum 
(Gibberella pulicaris) can form on woody host tissue (Booth, C.: The Genus 
Fusarium. pp. 168-172, Commonwealth Mycological Institute, Kew, Surrey, 
England, 1971). Perithecia or asci, however, were not observed. 
Colonies grew rapidly on Cornmeal agar (Difco) and attained a diameter of 
75-80 mm in seven days. The culture produced characteristic four to six 
septate, sickle-shaped macroconidia and one to two septate microconidia on 
this medium. The colonies had white (263), wispy aerial mycelia and the 
reverse was colorless. Macroconidia were produced abundantly, measuring 
37.5-70.times.5-7 .mu.m while microconidia measured 20-37.5.times.3.75-7.5 
.mu.m. The culture developed medium orange (53) sporodochia after 14 days 
incubation on Commeal agar. 
EXAMPLE 2 
Growth of Fusarium Sp. Strain AB 1900A-1314 in Stationary Culture 
The fusacandin-producing culture, Fusarium sp. AB 1900A-1314, was 
maintained as a frozen inoculum stock by freezing a portion of the 
original inoculum and storing at -70.degree. C. The medium S18 (Table 1) 
was used for seed growth and the medium F9 (Table 2) was used for 
stationary fermentations. 
TABLE 1 
______________________________________ 
Seed Medium S18 
Ingredients grams/liter 
______________________________________ 
Corn steep powder (Roquette Corp., Gurnee, IL) 
2.5 
Glucose monohydrate 10.0 
Oat flour (National Oats Co., Cedar Rapids, IA) 
10.0 
Tomato paste (made by Contadina Foods, Inc. Los 
40.0 
Angeles, CA) 
CaCl.sub.2.2H.sub.2 O 10.0 
Trace element solution 10 mL/L 
______________________________________ 
Distilled water was added to achieve a volume of 1 liter. The pH was 
adjusted to pH 6.8. Reference: Goetz et al., J. of Antibiotics 38: 
1633-1637 (1985). 
______________________________________ 
Trace Element Solution 
Ingredients grams/liter 
______________________________________ 
FeSO.sub.4.7H.sub.2 O 
1.0 
MnCl.sub.2.4H.sub.2 O 
1.0 
CuCl.sub.2.2H.sub.2 O 
0.025 
CaCl.sub.2.2H.sub.2 O 
0.1 
H.sub.3 BO.sub.3 0.56 
(NH.sub.4).sub.6 MoO.sub.2.4H.sub.2 O 
0.019 
ZnSO.sub.4.7H.sub.2 O 
0.2 
______________________________________ 
Distilled water was added to achieve a volume of 1 liter. 
TABLE 2 
______________________________________ 
Fermentation Medium F9 
Ingredients grams/liter 
______________________________________ 
Lactose 24.0 
Peptone (made by Difco Laboratories, Detroit, MI) 
16.0 
MgSO.sub.4.7H.sub.2 O 0.4 
KH.sub.2 PO.sub.4 2.08 
NaNO.sub.3 1.28 
ZnSO.sub.4.7H.sub.2 O 0.004 
______________________________________ 
Distilled water was added to achieve a volume of 1 liter without pH 
adjustment. Spoon Size Shredded Wheat (Nabisco Brands, Inc., East Hanover, 
N.J.) was used as solid growth support, following separate sterilization. 
The seed flasks were prepared by dispensing 100 mL of the seed medium 
(Table 1 ) into 500 mL Erlenmeyer flasks. The flasks were sterilized for 
30 minutes at 121.degree. C., 15 psi. Inoculum for the fermentation was 
prepared by inoculating 1% of the frozen inoculum into each of several 
seed flasks. The seed flasks were incubated for 72 hours at 28.degree. C. 
C on a rotary shaker, operated at 225 rpm, with a stroke of 2 inches 
(approximately 5 cm). 
The fermentation was conducted in 3 glass 20-liter carboys. Each carboy, 
containing 300 grams of Spoon Size Shredded Wheat, was sterilized for 45 
minutes at 121.degree. C., 15 psi. The F9 fermentation medium was 
sterilized in 3 batches of 360 mL in 2-liter Erlenmeyer flasks. 
Sterilization was at 121.degree. C., 15 psi. 
The 360 mL of liquid medium was inoculated with 60 mL of 72 hour seed 
growth. The combination was mixed and added aseptically to a carboy 
containing 300 grams of Shredded Wheat. The mixture was again thoroughly 
mixed to distribute the inoculum. The carboys were incubated in an upright 
position at 20.degree. C. for 21 days. Three carboys were prepared in this 
manner. 
EXAMPLE 3 
Growth of Fusarium Sp. Strain AB 1900A- 1314 in Submerged Culture 
The seed flasks were prepared by dispensing 600 mL of the seed medium 
(Table 3) into 2-liter Erlenmeyer flasks. The flasks were sterilized for 
30 minutes at 121.degree. C., 15 psi. Inoculum for the fermentation was 
prepared by inoculating 1% of the frozen inoculum into each of 3 seed 
flasks. The seed flasks were incubated for 72 hours at 28.degree. C. on a 
rotary shaker, operated at 225 rpm, with a stroke of 2 inches 
(approximately 5 cm). 
Thirty liters of production medium (Table 4) were prepared in a 42-liter, 
stainless steel, stirred fermentor (LH Fermentation) and sterilized at 
121.degree. C. and 15 psi for 1 hour. The antifoam agent XFO-371 (Ivanhoe 
Chemical Co,. Mundelein, Ill.) was added initially at 0.01%, and then as 
needed. The fermentor was inoculated with 1500 mL of the seed flask 
growth. The temperature was controlled at 28.degree. C. The agitation rate 
was 250 rpm and aeration was 1.5 vol/vol/min. The head pressure was 
maintained at 5 psi. The fermentation was terminated at seven days, with a 
harvest volume of about 13 liters. 
TABLE 3 
______________________________________ 
Seed medium for submerged fermentation 
Ingredient grams/liter 
______________________________________ 
Mannitol 20.0 
Soy flour 20.0 
Distilled water 
1 liter 
______________________________________ 
Reference: Traxler et at., J. Antibiotics 30:289-296 (1977). 
TABLE 4 
______________________________________ 
Submerged fermentation medium 
Ingredient grams/liter 
______________________________________ 
Glucose monohydrate 55.0 
Mannitol 10.0 
Glycine 2.0 
Dried lard water (Inland Molasses, Dubuque, IA) 
5.0 
Soybean meal (Archer Daniels Midland Co., 
5.0 
Decatur, IL) 
Sodium citrate 2.0 
KH.sub.2 PO.sub.4 2.0 
CoCl.sub.2.6H.sub.2 O 0.01 
______________________________________ 
Distilled water was added to achieve a volume of 1 liter without pH 
adjustment. Reference: VanMiddlesworth et al., J. Antibiotics 44:45-51 
(1991). 
EXAMPLE 4 
Isolation of Fusacandin A from Stationary Culture 
To 3 carboys containing stationary culture were added 3 liters of acetone. 
The resulting mixture was allowed to soak for 18 hours. This acetone 
extract was removed and an additional 6 liters of acetone added to the 
stationary culture, left to soak for 1 hour and removed. This procedure 
was repeated two additional times. The combined acetone extracts were 
concentrated to afford 29.7 grams of brown oil. This oil was triturated 
sequentially with 2 liters each of hexane, EtOAc, EtOH, MeOH and distilled 
water. The ethanol soluble material was concentrated to afford 400 mg of 
tan oil which was subjected to silica gel chromatography on 200 grams of 
VARIAN 40 .mu. silica gel eluted sequentially with 500 mL each of EtOAc, 
5% MeOH in EtOAc, 10% MeOH in EtOAc, 25% MeOH in EtOAc, 50% MeOH in EtOAc, 
and 100% MeOH. The material which eluted with 10% MeOH in EtOAc was 
concentrated to afford 130 mg of a pale oil which was subjected to 
countercurrent chromatography on an Ito multi-layered coil planet 
centrifuge in a solvent system of CHCl.sub.3 /MeOH/H.sub.2 O (1:1:1), 
lower layer stationary. Fractions of 5mL each were collected from this 
countercurrent chromatography with a solvent front at fraction 19, and 
fractions 43-45 were combined to yield 3.0 mg of a clear oil. This oil was 
subjected to size exclusion chromatography on a SEPHADEX LH-20 resin 
column developed in MeOH. The active fractions from this column were 
combined and concentrated to yield 1.8 mg of pure fusacandin A. 
EXAMPLE 5 
Isolation of Fusacandin A from Submerged Fermentation 
15 liters of whole culture broth were added to 8 liters of acetone and the 
mixture was agitated for 1 hour, after which 15 liters of EtOAc was added, 
the mixture was agitated and the upper layer was removed. An additional 
two 8-liter extractions were made, combined with the first and 
concentrated under reduced pressure to yield 15 grams of brown oil. This 
oil was triturated sequentially with 2 liters each of hexane, EtOAc and 
MeOH. The MeOH soluble material was concentrated in vacuo to yield 780 mg 
of brown oil which was subjected to silica gel chromatography on 500 grams 
of VARIAN 40 .mu. silica gel eluting sequentially with 1 liter each of 
EtOAc, 2% MeOH in EtOAc, 5% MeOH in EtOAc, 10% MeOH in EtOAc, 20% MeOH in 
EtOAc, 50% MeOH in EtOAc and 100% MeOH. The material which eluted with 50% 
MeOH in EtOAc was subjected to size exclusion chromatography on a SEPHADEX 
LH-20 resin column developed in MeOH. Active fractions from this column 
were combined to yield 160 mg of pure fusacandin A. 
EXAMPLE 6 
Isolation of Fusacandin B from Submerged Fermentation 
To 4900 liters of whole broth were added 3350 liters of acetone and 3700 
liters of ethyl acetate. The resulting mixture was agitated for 
approximately 12 hours after which time the upper layer was removed, 
concentrated under reduced pressure, and deposited onto 10 kg of silica 
gel. This was loaded onto the top of a 240 kg silica gel column developed 
sequentially with 300 liters of EtOAc 300 liters of 25% MeOH in EtOAc, 300 
liter of 50% MeOH in EtOAc, 300 liters of 75% MeOH in EtOAc and finally 
300 liters of MeOH. A portion (25 g) of the material which eluted with 25% 
MeOH in EtOAc was partitioned between 3:1:2 EtOAc/EtOH/H.sub.2 O, and the 
upper layer from this partition was concentrated under reduced pressure to 
an oily solid residue. This residue was subjected to size exclusion 
chromatography on a SEPHADEX LH-20 resin column developed in MeOH. The 
active fractions from this column were combined based upon their behavior 
on thin layer chromatograph to yield fusacandin A (2.65 g) and fusacandin 
B (62 mg). 
EXAMPLE 7 
Physico-Chemical Characterization of the Fusacandins 
Fusacandin A was characterized using IR, UV, .sup.1 H and .sup.13 C NMR 
spectroscopy. The resulting infrared, proton and carbon spectra are shown 
in FIGS. 1, 2 and 3. Fusacandin A has a molecular weight of 1020 (C.sub.51 
H.sub.71 O.sub.21) and is a clear oil. [.alpha.].sub.D =+58.degree. 
(c=0.67, MeOH). TLC characterization on Merck silica gel plates: Rf=0.00 
in EtOAc, R.sub.f =0.71 in 1:1 MeOH-EtOAc, R.sub.f =0.52 in acetone, and 
R.sub.f 0.40 in 3:2 CHCl.sub.3 -MeOH. An ultraviolet spectrum of 
fusacandin A acquired in MeOH/0.01M NaOH contained a band at 
.delta..sub.max =254 nm (.epsilon.=35,000) and end absorption. An infrared 
spectrum of fusacandin A acquired in microscope mode contained bands at 
3372, 2955, 2927, 2858, 1702, 1634, 1459, 1411, 1375, 1335, 1396, 1267, 
1146, 1076, 1049, and 1003 cm.sup.-1. 
Fusacandin B was characterized using IR, UV, .sup.1 H and .sup.13 C NMR 
spectroscopy. The resulting infrared, proton and carbon spectra are shown 
in FIGS. 4, 5 and 6. Fusacandin B has a molecular weight of 872 (C.sub.41 
H.sub.60 O.sub.20) and is a white solid. m.p. 42-45.degree. C. 
[.alpha.].sub.D =+1.degree. (c=0.4, MeOH). TLC characterization on Merck 
silica gel plates: R.sub.f =0.00 in EtOAc, R.sub.f =0.60 in 1:1 
MeOH/EtOAc, R.sub.f =0.46 in acetone and R.sub.f =0.19 in 3:2 CHCl.sub.3 
/MeOH. An ultraviolet spectrum of fusacandin B acquired in MeOH or 
MeOH/0.01M HCl contained a band at .delta..sub.max =263 nm 
(.epsilon.=18,000), 231 (21,000) and end absorption. An ultraviolet 
spectrum of fusacandin B acquired in MeOH/0.01M NaOH contained a band at 
.delta..sub.max =256 nm (.epsilon.=22,000), and end absorption. An 
infrared spectrum of fusacandin B acquired in microscope mode contained 
bands at: 3305, 3040, 3005, 2880, 2850, 1708, 1645, 1625, 1570, 1465, 
1410, 1380, 1315, 1265, 1155, 1080 and 1055 cm.sup.-1. 
EXAMPLE 8 
In Vitro Assay of Antifungal Activity 
Minimal inhibitory concentrations (MICs) were determined by an agar 
dilution method. The test compounds were serially diluted in MeOH and 0.2 
mL portions were mixed with 20 mL of molten, cooled Sabouraud dextrose 
agar (Difco). Yeast cell inoculum was prepared by growing cultures on 
Sabouraud dextrose agar for 18 hours at 32.degree. C. and suspending the 
cells in phosphate buffered saline. Filamentous fungi were grown under the 
same conditions for 4 days to obtain spores. The inoculum level for all 
cultures was adjusted to 10.sup.4 cells using a Petroff-Hauser cell 
counter. The glutarimide antifungal compounds cycloheximide or 
amphotericin B were used as a control. Inoculated test plates were 
incubated at 32.degree. C. and examined after 20 hours. The results, shown 
in Tables 5a and 5b, demonstrate that the compounds of the present 
invention possess significant antifungal activity. 
TABLE 5a 
______________________________________ 
In Vitro Antifungal Activity of Fusacandin A 
MIC (.mu.g/ml) 
Fusacandin 
Microorganism A Cycloheximide 
______________________________________ 
Candida albicans CCH 442 
6.26 &gt;100 
Candida albicans ATCC997 
12.5 &gt;100 
Candida albicans ATCC 623 
6.25 &gt;100 
Candida tropicalis NRRL-Y-1 
6.25 0.4 
Candida kefyr ATTC 288 
6.25 
Torulopsis glabrata ATCC 155 
3.12 0.4 
Saccharomyces cereviseae GS 1-36 
3.12 &lt;0.05 
Aspergillus niger ATCC 164 
6.25 1.6 
Nocardia asteroides ATTC 9970 
12.5 1.6 
Streptococcus pyrogenes EES61 
6.25 50 
Streptococcus bovis A-5169 
12.5 25 
Staphylococcus aureus ATTC 6538p 
50 0.8 
______________________________________ 
TABLE 5b 
______________________________________ 
In Vitro Antifungal Activity of Fusacandin B 
MIC (.mu.g/ml) 
Fusacandin 
Microorganism B Amphotericin B 
______________________________________ 
Cryptococcus albidus ATCC 341 
&gt;100 3.12 
Saccharomyces cereviseae GS 1-36 
50 1.56 
Aspergillus niger ATCC 164 
&gt;100 1.56 
Candida albicans ATCC 102 
50 1.56 
Candida albicans 579a 
50 1.56 
Candida albicans CCH 442 
50 1.56 
Candida albicans ATCC 382 
&gt;100 50 
Candida albicans ATCC 623 
100 1.56 
Candida tropicalis NRRL Y-1 
50 1.56 
Candida kefyr ATCC 288 
100 1.56 
Torulopsis glabrata ATCC 155 
100 1.56 
______________________________________ 
EXAMPLE 9 
In Vitro Inhibition of (1,3)-.beta.-Glucan Synthase Activity 
The fungal cell wall serves as a protective barrier and is essential for 
viability in a hypotonic environment. (1,3)-.beta.-Glucan is a component 
of the Candida albicans cell wall, and the enzyme that biosynthesizes this 
polymer, glucan synthase, is not present in higher eukaryotes. (Glucan 
synthase is an integral plasma membrane protein that catalyzes 
polymerization of uridine diphosphate-glucose (UDP-Glc) into 
.beta.-glucan.) Accordingly, glucan synthase represents an ideal target 
for the development of antifungal agents. 
A microtiter screen was established to detect inhibitors of 
(1,3)-.beta.-glucan formation in C. albicans cell free extracts. 
Microsomes isolated from mid-log phase grown yeast were incubated with 
[.sup.14 C]UDP-Glc, effectors and test compound (fusacandin A). The 
formation of the water-insoluble .beta.-glucan product was measured on a 
filter after removing the substrate with water washes. The IC.sub.50 for 
fusacandin A was shown to be 20.5 .mu.g/mL compared to 3.6 .mu.g/mL for 
papulacandin B. The minimum inhibitory concentration (MIC) for fusacandin 
was 0.5 .mu.g/mL compared to 1.0 .mu.g/mL for papulacandin B. 
It is understood that the foregoing detailed description and accompanying 
examples are merely illustrative and are not to be taken as limitations 
upon the scope of the invention, which is defined solely by the appended 
claims and their equivalents. Various changes and modifications to the 
disclosed embodiments will be apparent to those skilled in the art, and 
may be made without departing from the spirit and scope thereof.