Cyclic peptide antifungal agents

BACKGROUND OF THE INVENTION 
This invention relates to semi-synthetic cyclic peptide compounds which are 
useful as antifungal and antiparasitic agents and which have improved 
stability and water solubility. In particular, it relates to derivatives 
of the echinocandin class of cyclic peptides; to methods for treating 
fungal and parasitic infections, and to formulations useful in the 
methods. 
The compounds provided by this invention are semi-synthetic compounds 
derived from cyclic peptides which are produced by culturing various 
microorganisms. A number of cyclic peptides are known in the art including 
echinocandin B (A30912A), aculeacin, mulundocandin, sporiofungin, 
L-671,329, and S31794/F1. 
In general, these cyclic peptides may be structurally characterized as a 
cyclic hexapeptide core (or nucleus) with an acylated amino group on one 
of the core amino acids. The amino group is typically acylated with a 
fatty acid group forming a side chain off the nucleus. For example, 
echinocandin B has a linoleoyl side chain while aculeacin has a palmitoyl 
side chain. 
The fatty acid side chains may be removed from the cyclic peptide core to 
provide an amino nucleus (for example, a compound of formula I, below, 
where R.sub.2 is hydrogen). The amino group may then be re-acylated to 
provide semi-synthetic compounds such as those claimed in the present 
application. 
The echinocandin B nucleus has been re-acylated with certain non-naturally 
occurring side chain moieties to provide a number of antifungal agents 
(see, Debono, U.S. Pat. No. 4,293,489). Among such antifungal agents is 
cilofungin which is represented by a compound of formula I where R', R", 
and R'" are methyl; R.sup.x1, R.sup.x2, R.sup.y1, R.sup.y2, R.sup.y3, 
R.sup.y4 and R.sup.0 is hydroxy and R.sup.2 is p-(octyloxy)benzoyl. 
SUMMARY OF THE INVENTION 
The present invention provides a compound of the formula: 
##STR4## 
wherein: 
R' is hydrogen, methyl or --CH.sub.2 C(O)NH.sub.2 ; 
R" and R'" are independently methyl or hydrogen; 
R.sup.x1 is hydrogen, hydroxy or --O--R; 
R is C.sub.1 -C.sub.6 alkyl, benzyl, --(CH.sub.2).sub.2 Si(CH.sub.3).sub.3, 
--CH.sub.2 CHOHCH.sub.2 OH, --CH.sub.2 CH.dbd.CH.sub.2, --(CH.sub.2).sub.a 
COOH, --(CH.sub.2).sub.b NR.sup.z1 R.sup.z2, --(CH.sub.2).sub.c POR.sup.z3 
R.sup.z4 or --[(CH.sub.2).sub.2 O].sub.d --(C.sub.1 -C.sub.6)alkyl; 
a, b and c are independently 1, 2, 3, 4, 5 or 6; 
R.sup.z1 and R.sup.z2 are independently hydrogen, C.sub.1 -C.sub.6 alkyl, 
or R.sup.z1 and R.sup.z2 combine to form --CH.sub.2 (CH.sub.2).sub.e 
CH.sub.2 --; 
R.sup.z3 and R.sup.z4 are independently hydroxy or C.sub.1 -C.sub.6 alkoxy; 
d is 1 or 2; 
e is 1, 2 or 3; 
R.sup.x2, R.sup.y1, R.sup.y2, R.sup.y3 and R.sup.y4 are independently 
hydroxy or hydrogen; 
R.sup.0 is hydroxy, --OP(O) (OH).sub.2 or a group of the formulae: 
##STR5## 
R.sup.1 is C.sub.1 -C.sub.6 alkyl, phenyl, p-halo-phenyl, p-nitrophenyl, 
benzyl, p-halo-benzyl or p-nitro-benzyl; 
R.sup.2 is 
##STR6## 
R.sup.3 is 
##STR7## 
R.sup.3a, R.sup.3b, R.sup.3c and R.sup.3d are independently hydrogen, 
C.sub.1 -C.sub.12 alkyl, C.sub.2 -C.sub.12 alkynyl, C.sub.1 -C.sub.12 
alkoxy, C.sub.1 -C.sub.12 alkylthio, halo, or --O--(CH.sub.2).sub.m --[O 
--(CH.sub.2).sub.n ].sub.p --O--(C.sub.1 -C.sub.12 alkyl) or 
--O--(CH.sub.2).sub.q --X--R.sup.4 ; 
m is 2, 3 or 4; 
n is 2, 3 or 4; 
p is 0 or 1; 
q is 2, 3 or 4; 
X is pyrrolidino, piperidino or piperazino; and 
R.sup.4 is hydrogen, C.sub.1 -C.sub.12 alkyl, C.sub.3 -C.sub.12 cycloalkyl, 
benzyl or C.sub.3 -C.sub.12 cycloalkylmethyl; 
or a pharmaceutically acceptable salt thereof. 
Also provided are pharmaceutical formulations, methods for inhibiting 
parasitic or fungal activity and methods of treating fungal or parasitic 
infections which employ the compounds of the invention.

DETAILED DESCRIPTION 
As used herein, the term "C.sub.1 -C.sub.12 alkyl" refers to a straight or 
branched alkyl chain having from one to twelve carbon atoms. Typical 
C.sub.1 -C.sub.12 alkyl groups include methyl, ethyl, propyl, isopropyl, 
butyl, sec-butyl, t-butyl, pentyl, 5-methylpentyl, hexyl, heptyl, 
3,3-dimethylheptyl, octyl, 2-methyl-octyl, nonyl, decyl, undecyl, dodecyl 
and the like. The term "C.sub.1 -C.sub.12 alkyl" includes within its 
definition the terms "C.sub.1 -C.sub.6 alkyl" and "C.sub.1 -C.sub.4 alkyl. 
" 
The term "C.sub.2 -C.sub.12 alkynyl" refers to a straight or branched 
alkynyl chain having from two to twelve carbon atoms. Typical C.sub.2 
-C.sub.12 alkynyl groups include ethynyl, 1-propyn-1-yl, 1-propyn-2-yl, 
1-butyn-1-yl, 1-butyn-3-yl, 1-pentyn-3-yl, 4-pentyn-2-yl, 1-hexyn-3-yl, 
3-hexyn-1-yl, 5-methyl-3-hexyn-1-yl, 5-octyn-1-yl, 7-octyn-1-yl, 
4-decyn-1-yl, 6-decyn-1-yl and the like. 
The term "halo" refers to chloro, fluoro, bromo or iodo. 
The term "C.sub.1 -C.sub.12 alkylthio" refers to a straight or branched 
alkyl chain having from one to twelve carbon atoms attached to a sulfur 
atom. Typical C.sub.1 -C.sub.12 alkylthio groups include methylthio, 
ethylthio, propylthio, isopropylthio, butylthio, 3-methyl-heptylthio, 
octylthio, 5,5-dimethyl-hexylthio and the like. 
The term "C.sub.1 -C.sub.12 alkoxy" refers to a straight or branched alkyl 
chain having from one to twelve carbon atoms attached to an oxygen atom. 
Typical C.sub.1 -C.sub.12 alkoxy groups include methoxy, ethoxy, propoxy, 
butoxy, sec-butoxy, pentoxy, 5-methyl-hexoxy, heptoxy, octyloxy, decyloxy 
dodecyloxy and the like. The term "C.sub.1 -C.sub.12 alkyl" includes 
within its definition the terms "C.sub.1 -C.sub.6 alkoxy" and "C.sub.1 
-C.sub.4 alkoxy." 
The term "C.sub.3 -C.sub.12 cycloalkyl" refers a saturated hydrocarbon ring 
structure having from three to twelve carbon atoms. Typical C.sub.3 
-C.sub.12 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, 
cyclohexyl and cycloheptyl, cyclooctyl and the like. 
The term "hydroxy protecting group" refers to a substituent of an hydroxy 
group that is commonly employed to block or protect the hydroxy 
functionality while reactions are carried out on other functional groups 
on the compound. Examples of such hydroxy protecting groups include 
tetrahydropyranyl, 2-methoxyprop-2-yl, 1-ethoxyeth-1-yl, methoxymethyl, 
.beta.-methoxyethoxymethyl, methylthiomethyl, t-butyl, t-amyl, trityl, 
4-methoxytrityl, 4,4'-dimethoxytrityl, 4,4',4"-trimethoxytrityl, benzyl, 
allyl, trimethylsilyl, (t-butyl)dimethylsilyl, and 
2,2,2-trichloroethoxycarbonyl and the like. The species of hydroxy 
protecting group is not critical so long as the derivatized hydroxy group 
is stable to the conditions of the subsequent reaction(s) and can be 
removed at the appropriate point without disrupting the remainder of the 
molecule. A preferred hydroxy protecting group is trimethylsilyl. Further 
examples of hydroxy protecting groups are described in T. W. Greene, 
"Protective Groups in Organic Synthesis," John Wiley and Sons, New York, 
N.Y., (2nd ed., 1991) chapters 2 and 3. The term "protected hydroxy" 
refers to a hydroxy group bonded to one of the above hydroxy protecting 
groups. 
The term "amino protecting group" as used in the specification refers to 
substituents of the amino group commonly employed to block or protect the 
amino functionality while reacting other functional groups on the 
compound. Examples of such amino protecting groups include formyl, trityl, 
phthalimido, trichloroacetyl, chloroacetyl, bromoacetyl, iodoacetyl 
groups, or urethane-type blocking groups such as benzyloxycarbonyl, 
4-phenylbenzyloxycarbonyl, 2-methylbenzyloxycarbonyl, 
4-methoxybenzyloxycarbonyl, 4-fluorobenzyloxycarbonyl, 
4-chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl, 
2-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 
4-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl, 
4-nitrobenzyloxycarbonyl, 4-cyanobenzyloxycarbonyl, t-butoxycarbonyl, 
2-(4-xenyl)isopropoxycarbonyl, 1,1-diphenyleth-1-yloxycarbonyl, 
1,1-diphenylprop-1-yloxycarbonyl, 2-phenylprop-2-yloxycarbonyl, 
2-(p-toluyl)-prop-2-yloxycarbonyl, cyclopentanyloxycarbonyl, 
1-methylcyclopentanyloxycarbonyl, cyclohexanyloxycarbonyl, 
1-methylcyclohexanyloxycarbonyl, 2-methylcyclohexanyloxycarbonyl, 
2-4-toluylsulfonyl)ethoxycarbonyl, 2-(methylsulfonyl)ethoxycarbonyl, 
2-(triphenylphosphino)-ethoxycarbonyl, fluorenylmethoxycarbonyl ("FMOC"), 
2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, 
1-(trimethyisilylmethyl)prop-1-enyloxycarbonyl, 
5-benzisoxalylmethoxycarbonyl, 4-acetoxybenzyloxycarbonyl, 
2,2,2-trichloroethoxycarbonyl, 2-ethynyl-2-propoxycarbonyl, 
cyclopropylmethoxycarbonyl, 4-decyloxy)benzyloxycarbonyl, 
isobornyloxycarbonyl, 1-piperidyloxycarbonyl and the like; 
benzoylmethylsulfonyl, 2-nitrophenylsulfenyl, diphenylphosphine oxide and 
like amino protecting groups. The species of amino protecting group 
employed is not critical so long as the derivatized amino group is stable 
to the condition of subsequent reaction(s) on other positions of the 
intermediate molecule and can be selectively removed at the appropriate 
point without disrupting the remainder of the molecule including any other 
amino protecting group(s). Preferred amino protecting groups are 
t-butoxycarbonyl (t-Boc), allyloxycarbonyl and benzyloxycarbonyl (CbZ). 
Further examples of groups referred to by the above terms are described by 
J. W. Barton, "Protective Groups in Organic Chemistry", J. G. W. McOmie, 
Ed., Plenum Press, New York, N.Y., 1973, Chapter 2, and T. W. Greene, 
"Protective Groups in Organic Synthesis", John Wiley and sons, New York, 
N.Y., 1981, Chapter 7. 
The term "inhibiting", i.e. a method of inhibiting parasitic or fungal 
activity, includes stopping, retarding or prophylactically hindering or 
preventing the growth or any attending characteristics and results from 
the existence of a parasite or fungus. 
The term "contacting", i.e. contacting a compound of the invention with a 
parasite or fungus, includes a union or junction, or apparent touching or 
mutual tangency of a compound of the invention with a parasite or fungus. 
However, the term does not imply any further limitations to the process, 
such as by mechanism of inhibition, and the methods are defined to 
encompass the spirit of the invention, which is to inhibit parasitic and 
fungal activity by the action of the compounds and their inherent 
antiparasitic and antifungal properties, or in other words, the compounds, 
used in the claimed methods are the causative agent for such inhibition. 
The term "pharmaceutically acceptable salt" as used herein, refers to salts 
of the compounds of the above formula which are substantially non-toxic to 
living organisms. Typical pharmaceutically acceptable salts include those 
salts prepared by reaction of the compounds of the present invention with 
a mineral or organic acid or an inorganic base. Such salts are known as 
acid addition and base addition salts. 
Acids commonly employed to form acid addition salts are mineral acids such 
as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, 
phosphoric acid and the like, and organic acids such as p-toluenesulfonic, 
methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic 
acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. 
Examples of such pharmaceutically acceptable salts are the sulfate, 
pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, 
monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, 
chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, 
acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, 
malonate, succinate, suberate, sebacate, fumarate, maleate, 
butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, 
methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, 
phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, 
phenylbutyrate, citrate, lactate, .lambda.-hydroxybutyrate, glycollate, 
tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, 
napththalene-2-sulfonate, mandelate and the like. Preferred 
pharmaceutically acceptable acid addition salts are those formed with 
mineral acids such as hydrochloric acid and hydrobromic acid, and those 
formed with organic acids such as maleic acid and methanesulfonic acid. 
Base addition salts include those derived from inorganic bases, such as 
ammonium or alkali or alkaline earth metal hydroxides, carbonames, 
bicarbonates, and the like. Such bases useful in preparing the salts of 
this invention thus include sodium hydroxide, potassium hydroxide, 
ammonium hydroxide, potassium carbonate, sodium carbonate, sodium 
bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, 
and the like. The potassium and sodium salt forms are particularly 
preferred. 
It should be recognized that the particular counterion forming a part of 
any salt of this invention is not of a critical nature, so long as the 
salt as a whole is pharmacologically acceptable and as long as the 
counterion does not contribute undesired qualities to the salt as a whole. 
Preferred compounds of this invention are those compounds of formula I 
where: 
R', R" and R'" are each methyl; 
R.sup.y1, R.sup.y2, R.sup.y3 and R.sup.y4 are each hydroxy; 
R.sup.x1 is hydrogen, hydroxy or --O--R; 
R is methyl, benzyl, --CH.sub.2 CHOHCH.sub.2 OH, --(CH.sub.2).sub.b 
NR.sup.z1 R.sup.z2 or --(CH.sub.2).sub.2 POR.sup.z3 R.sup.z4 ; 
b is 2, 3, 4, 5or 6; 
R.sup.z1 and R.sup.z2 are independently hydrogen or C.sub.1 -C.sub.4 alkyl; 
R.sup.z3 and R.sup.z4 are independently hydroxy or methoxy; 
R.sup.x2 is hydrogen or hydroxy; 
R.sup.0 is hydroxy, or a group of the formulae: 
##STR8## 
R.sup.1 is methyl; 
or a pharmaceutically acceptable salt thereof. 
Of these compounds, more preferred are those compounds of formula I where: 
R.sup.x1 is hydroxy; 
R.sup.x2 is hydroxy; 
R.sup.0 is hydroxy; 
R.sup.2 is a group of the formula: 
##STR9## 
R.sup.3c and R.sup.3d are independently hydrogen, C.sub.2 -C.sub.12 
alkynyl, C.sub.1 -C.sub.12 alkoxy or --O--(CH.sub.2).sub.m 
--[O--(CH.sub.2).sub.n ].sub.p --O--(C.sub.1 -C.sub.12 alkyl); or a 
pharmaceutically acceptable salt thereof. 
Of these compounds, the most preferred are those compounds where 
R.sup.2 is 
##STR10## 
or 
R.sup.2 is 
##STR11## 
or a pharmaceutically acceptable salt thereof. 
##STR12## 
wherein: 
R.sup.nat is a naturally occurring cyclic peptide sidechain; and 
R', R", R'", R.sup.x1, R.sup.x2, R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4, 
R.sup.0 and R.sup.2 are as defined above. 
Reaction scheme I, above, is accomplished by carrying out reactions A-C, in 
order. Once a reaction is complete, the intermediate compound may be 
isolated by procedures well-known in the arm, for example, the compound 
may be crystallized or precipitated and then collected by filtration, or 
the reaction solvent may be removed by extraction, evaporation or 
decantation. The intermediate compound may be further purified, if 
desired, by common techniques such as crystallization or precipitation, or 
chromatography over solid supports such as silica gel, alumina and the 
like, before carrying out the next step of the reaction scheme. 
In reaction IA, a naturally occurring cyclic peptide of the formula IA is 
deacylated using procedures known in the art to provide an amino nucleus 
of formula IB. This reaction is typically carried out using enzymatic 
deacylation by exposing the naturally occurring cyclic peptide to a 
deacylase enzyme. The deacylase enzyme may be obtained from the 
microorganism Actinoplanes utahensis and used substantially as described 
in U.S. Pat. Nos. 4,293,482 and 4,304,716, herein incorporated by 
reference. The deacylase enzyme may also be obtained from the Pseudomonas 
species. Deacylation may be accomplished using whole cells of Actinoplanes 
utahensis or Pseudomonas or the crude or purified enzyme thereof or using 
an immobilized form of the enzyme. See European Patent Application No. 0 
460 882 (Dec. 11, 1991). Examples of naturally occurring cyclic peptides 
which may be used as starting materials include aculeacin (palmitoyl side 
chain), tetrahydroechinocandin B (stearoyl side chain), mulundocandin 
(branched C.sub.15 side chain), L-671,329 (C.sub.16 branched side chain), 
S 31794/F1 (tetradecanoyl side chain), sporiofungin (C.sub.15 branched 
side chain), FR901379 (palmitoyl side chain) and the like. A preferred 
naturally occurring cyclic peptide is echinocandin B (a compound of 
formula IA where R', R" and R'" are each methyl, R.sup.x1, R.sup.x2, 
R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4 and R.sup.0 are each hydroxy and 
R.sup.2 is linoleoyl). 
In Reaction IB, the resulting amino nucleus is N-alkylated using reductive 
amination to provide a compound of formula I where R.sup.2 is as defined 
hereinabove. The reaction is typically carried out by reacting the amino 
nucleus of formula IB with an appropriately substituted aldehyde of the 
formula R.sup.2 -COH in the presence of a reducing agent such as sodium 
cyanoborohydride. The reaction is typically carried out for one to sixty 
five hours at a temperature of from about 20.degree. C. to about 
100.degree. C. in a mutual inert solvent. Typical solvents for this 
reaction include dimethylformamide, methanol or a mixture of such 
solvents. Solvent choice is not critical so long as the solvent employed 
is inert to the ongoing reaction and the reactants are sufficiently 
solubilized to effect the desired reaction. The aldehyde reactant is 
generally employed in a slight excess relative to the amino nucleus. 
The compounds of formula I where R.sup.x1 is hydroxy may be reacted with an 
appropriately substituted alcohol in the presence of an acid to provide a 
compound of formula I where R.sup.x1 is --O--R, where R is C.sub.1 
-C.sub.6 alkyl, benzyl, --(CH.sub.2).sub.2 Si(CH.sub.3).sub.3, --CH.sub.2 
CH.dbd.CH.sub.2, --(CH.sub.2).sub.a COOH, --(CH.sub.2).sub.b NR.sup.z1 
R.sup.z2, --(CH.sub.2).sub.c POR.sup.z3 R.sup.z4 or --[(CH.sub.2).sub.2 
O].sub.d --(C.sub.1 -C.sub.6) alkyl. The reaction is typically carried out 
in a polar aprotic solvent such as dioxane or dimethylsulfoxide at a 
temperature of from about 0.degree. C. to about 35.degree. C., preferably 
at about room temperature. Solvent choice is not critical so long as the 
solvent employed is inert to the ongoing reaction and the reactants are 
sufficiently solubilized to effect the desired reaction. Preferred acids 
include p-toluenesulfonic acid, hydrochloric acid and camphorsulfonic 
acid. 
The compounds of formula I where R.sup.x1 is --(CH.sub.2).sub.b NR.sup.z1 
R.sup.z2 where R.sup.z1 and R.sup.z2 are hydrogen may be prepared via a 
protected compound wherein R.sup.x1 is --(CH.sub.2).sub.b NHR.sup.a where 
R.sup.a is an amino protecting group. The resultant protected compound is 
then deprotected according to procedures known in the art. 
The compounds of formula I where R.sup.x1 is --CH.sub.2 CHOHCH.sub.2 OH may 
be prepared by hydroxylating a compound of formula I where R.sup.x1 is 
--CH.sub.2 CH.dbd.CH.sub.2 with osmium tetroxide in the presence of a 
catalyst at a temperature in the range of from about 0.degree. C. to about 
40.degree. C. for about one to twenty four hours in a organic/aqueous 
solvent mixture, for example dioxane/water. Suitable catalysts include 
N-methylmorpholine N-oxide (NMO) and the like. Typical solvents suitable 
for use in this reaction include dimethylformamide, tetrahydrofuran, 
acetone and dioxane. Solvent choice is not critical so long as the solvent 
employed is inert to the ongoing reaction and the reactants are 
sufficiently solubilized to effect the desired reaction. The reaction is 
preferably conducted at a temperature in the range of from about 
20.degree. C. to about 30.degree. C. for about eighteen to twenty four 
hours. 
The compounds of formula I where R.sup.0 is hydroxy may be phosphorylated 
by reaction with an appropriately substituted alkyl or phenyl phosphate to 
provide a compound of formula I where R.sup.0 is --0--P(O)OH--R.sup.1 
where R.sup.1 is C.sub.1 -C.sub.6 alkoxy or phenoxy, or by reaction with 
an appropriately substituted alkyl or phenyl phosphonic acid to provide a 
compound of formula I where R.sup.0 is --O--P(O)OH--R.sup.1 where R.sup.1 
is C.sub.1 -C.sub.6 alkyl, or an appropriately substituted phenyl or 
benzyl moiety, to provide a compound of formula I where R.sup.0 is a group 
of the formula --OP/O)OH--R.sup.1. The phosphonic acid is typically used 
in an activated form, for example as a phosphonic halide, preferably a 
phosphonic chloride. The reaction is carried out in the presence of a base 
such as lithium trimethylsilanolate (LiOTMS), lithium 
bis(trimethylsilyl)amide (LHMDS), pyridine and the like. The reaction is 
typically carried out for up to one hour at a temperature from about 
-30.degree. C. to about 0.degree. C. in an aprotic solvent such as 
tetrahydrofuran and dimethylformamide. The reaction is generally complete 
in about fifteen minutes when carried out under these conditions. The 
phosphate or phosphonate reactant is generally employed in equimolar 
proportions to about a one mole excess relative to the amino nucleus in 
the presence of an equimolar or slight excess of the base. Phosphorylation 
of an amino nucleus with unprotected aminal hydroxy groups is typically 
carried out at lower temperatures, for example from about -30.degree. C. 
to about -15.degree. C. 
Alternatively, the aminal hydroxy moieties on the compound of formula I are 
optionally protected with an hydroxy protecting group using procedures 
known in the art. For example, the reaction is typically carried out by 
combining the compound of formula I with a suitable hydroxy protecting 
group in the presence of a catalyst at a temperature in the range of from 
about 0.degree. C. to about 40.degree. C. for about one to five hours in a 
mutual inert solvent. The hydroxy protecting group is generally employed 
in an amount ranging from about equimolar proportions to about a 100 molar 
excess relative to the compound of formula I, preferably in a large molar 
excess. Suitable catalysts include strong acids such as p-toluenesulfonic 
acid, camphorsulfonic acid (CSA), hydrochloric acid, sulfuric acid, 
trifluoroacetic acid and the like. Typical solvents suitable for use in 
this reaction include any organic solvent such as dioxane. Solvent choice 
is not critical so long as the solvent employed is inert to the ongoing 
reaction and the reactants are sufficiently solubilized to effect the 
desired reaction. The reaction is preferably conducted at a temperature in 
the range of from about 20.degree. C. to about 30.degree. C. for about two 
to four hours. The protected compound of formula I is then phosphorylated 
as described above. The hydroxy protecting group(s) are then removed 
according to procedures known in the art to provide a phosphorylated 
compound of formula I. For example, the protecting groups can be removed 
by reaction with a Lewis acid in a mutual inert organic solvent such as 
methylene chloride. Examples of Lewis acids include trimethylsilylbromide, 
boron trifluoride etherate and the like. The reaction is typically carried 
out at a temperature of from about 0.degree. C. to about 40.degree. C., 
preferably at a temperature of from about 20.degree. C. to about 
30.degree. C. A preferred Lewis acid is boron trifluoride etherate. 
The dideoxy compounds of formula I are prepared by removing the benzylic 
and aminal hydroxy groups (R.sup.x2 and R.sup.x1, respectively). The 
hydroxy groups may be removed by subjecting a non-dideoxy compound of 
formula I (where R.sub.2 is hydrogen or acyl) to a strong acid and a 
reducing agent at a temperature of between -5.degree. C. and 70.degree. 
C., in a suitable solvent. Typical strong acids include trichloroacetic 
acid, trifluoroacetic acid or borontrifluoride etherate. A preferred 
strong acid is trifluoroacetic acid. Typical reducing agents include 
sodium cyanoborohydride or triethylsilane. A preferred reducing agent is 
triethylsilane. Suitable solvents include methylene chloride, chloroform 
or acetic acid, preferably methylene chloride. The strong acid should be 
present in an amount of from 2 to 80 mol per mol of substrate, and the 
reducing agent should be present in an amount of 2 to 80 mol per mol of 
substrate. This process affords selective removal of the aminal and 
benzylic hydroxy groups. 
The cyclic peptides used to make the compounds of the present invention may 
be prepared by fermentation of known microorganisms. For example, the 
cyclic peptide of formula IB where R', R" and R'" are methyl, R.sup.x1, 
R.sup.x2, R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4 are hydroxy and R.sup.0 
is hydroxy (cyclic nucleus corresponding to A-30912A) may be prepared 
using the procedure detailed in Abbott et al., U.S. Pat. No. 4,293,482, 
which is herein incorporated by reference. The cyclic peptide of formula 
IB where R', R" and R'" are methyl, R.sup.x1 is hydroxy, R.sup.x2 is 
hydrogen, R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4 and R.sup.0 is hydroxy 
(cyclic nucleus corresponding to A-30912B) may be prepared using the 
procedure detailed in Abbott et al., U.S. Pat. No. 4,299,763, which is 
herein incorporated by reference. Aculeacin may be prepared using the 
procedure detailed in Mizuno et al., U.S. Pat. No. 3,978,210 which is 
herein incorporated by reference. The cyclic peptide of formula IB where 
R' is --CH.sub.2 C(O)NH.sub.2, R" is methyl, R'" is hydrogen, R.sup.x1, 
R.sup.x2, R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4 and R.sup.0 is hydroxy 
may be prepared by deacylating the cyclic peptide prepared using the 
procedure detailed in Chen et al., U.S. Pat. No. 5,198,421, which is 
herein incorporated by reference. 
The aldehydes of the formula R.sup.2 --COH, used in the reductive 
aminiation, may be obtained commercially or prepared according to 
procedures known in the art. For example, an appropriately substituted 
phenyl boronic acid or biphenyl boronic acid reactant may be reacted with 
a p-halobenzaldehyde reactant in the presence of a catalyst such as 
tetrakis(triphenylphosphine)palladium and an inorganic base such as 
potassium carbonate in a mutual inert organic solvent such as toluene at a 
temperature of from about 20.degree. C. to the reflux temperature of the 
reaction mixture to provide the corresponding biphenyl aldehydes and 
terphenyl aldehydes used to prepare the compounds of formula I. The 
reaction is typically carried out with equimolar proportions of the 
boronic acid reactant and the p-benzaldehyde reactant, or a slight molar 
excess of the p-benzaldehyde reactant relative to the boronic acid 
reactant, and a 1-2 molar excess of the inorganic base. The reaction is 
generally complete after about four to about ten hours when carried out at 
reflux temperature in toluene. 
The boronic acid reactant may be prepared by reacting an appropriately 
substituted halophenyl or halobiphenyl reactant with two equivalents of 
triisopropyl borate in the presence of an alkyl lithium, for example 
sec-butyl lithium, in a mutual inert solvent such as tetrahydrofuran. The 
alkyl lithium is typically employed in a slight molar excess relative to 
the halophenyl or halobiphenyl reactant. The alkyl lithium is typically 
combined with the solvent by dropwise addition at reduced temperatures 
(&lt;-70.degree. C.) and allowed to stir for approximately thirty minutes 
before the addition of the triisopropyl borate. The reaction is typically 
carried out initially at a temperature of from about -100.degree. C. to 
about -50.degree. C., preferably from about -75.degree. C. to about 
-85.degree. C. for thirty minutes to two hours and then warmed to room 
temperature and reacted for an additional one to three hours. The reaction 
is generally complete in from several minutes to about four hours. When 
the reaction is substantially complete, the boronic acid moiety is formed 
by the addition of an acid. A preferred acid is a 1N hydrochloric acid 
solution. 
The R.sup.2 -COH aldehydes having an acetylene moiety may be prepared by 
reacting an appropriately substituted acetylene reactant with an 
appropriately substituted phenyl or biphenyl reactant of the formula 
##STR13## 
where L is a suitable leaving group such as bromo, iodo, methanesulfonate, 
toluenesulfonate, trifluoromethanesulfonate and the like, in the presence 
of a catalyst and preferably in the presence of an acid scavenger in a 
mutual inert solvent such as acetonitrile. Examples of acid scavengers 
include triethylamine and pyridine, preferably triethylamine. A preferred 
catalyst is formed in situ from palladium (II) chloride, 
triphenylphosphine and copper (I) iodide. The reaction is typically 
carried out for thirty minutes to twenty one hours at a temperature from 
about room temperature to the reflux temperature of reaction mixture. The 
reaction is generally complete after about two to about six hours when 
carried out at reflux temperature. 
Alternatively, a suitably substituted phenyl reactant of the formula 
##STR14## 
may be reacted with an appropriately substituted acetylene reactant as 
described above to provide, for example, a compound of the formula 
##STR15## 
which can be coupled with a phenyl boronic acid reactant as described 
above. 
The following Preparations and Examples further describe how to synthesize 
the compounds of the present invention. The terms melting point, proton 
nuclear magnetic resonance spectra, mass spectra, infrared spectra, 
ultraviolet spectra, elemental analysis, high performance liquid 
chromatography, and thin layer chromatography are abbreviated "m.p.", 
"NMR", "MS", "IR", "UV", "Analysis", "HPLC", and "TLC", respectively. In 
addition, the absorption maxima listed for the IR spectra are only those 
of interest and not all of the maxima observed. 
Preparation 1 
4-octyloxybenzaldehyde 
A solution containing 3.053 g (25 mmol) of 4-formylphenol, 6.48 ml (3705 
mmol) of 1-bromooctane and 6.9 mg (50 mmol) of potassium carbonate in 100 
ml of acetone was refluxed overnight. When the reaction was substantially 
complete, as indicated by thin layer chromatagraphy (TLC), the reaction 
was quenched by the addition of 100 ml of water. The desired compound was 
extracted from the reaction mixture using two 100 ml portions of diethyl 
ether. The resultant solution was dried over magnesium sulfate, filtered 
and then concentrated in vacuo to provide a liquid which was purified 
using HPLC (eluent of 10 ethyl acetate in hexane) to provide the desired 
compound. 
MS(FAB) : 235.2 (M+H). 
Preparation 2 
##STR16## 
To a cold (-78C) solution of 10.0 mg (42.9 mmol) of 
1-bromo-4-phenylbenzene, was added 42.9 ml of a 1.3M solution of 
sec-butyllithium in tetrahydrofuran (55.8 mmol), dropwise. To the 
resultant mixture was added 14.85 ml (64.35 mmol) of triisopropyl borate, 
dropwise. The resultant reaction mixture was stirred for approximately 
thirty minutes and then warmed to room temperature and allowed to react 
for approximately two hours. The reaction was then quenched by the 
addition of approximately 50 ml of 1N hydrochloric acid and the resultant 
mixture was concentrated in vacuo to provide a residue. This residue was 
redissolved in diethyl ether, filtered and dried in vacuo to provide 1.58 
g of the desired subtitled compound. 
##STR17## 
A 2M solution of sodium carbonate was added to a solution of 2.970 g (15 
mmol) of the compound of Preparation 2A in 120 ml of toluene. After 
degassing the resultant mixture, 3.470 g (18.75 mmol) of 
1-bromo-4-formylbenzene and 1.713 g (1.5 mmol) of 
tetrakis(triphenylphosphine)palladium were added to the above solution and 
the resultant reaction mixture was refluxed overnight. When the reaction 
was substantially complete, as indicated by TLC, the reaction mixture was 
cooled to room temperature and concentrated in vacuo to provide a residue. 
This residue was redissolved in methylene chloride and washed with two 30 
ml portions of brine. The organic portion was then filtered and dried in 
vacuo to provide a solid. 
Preparation 3 
##STR18## 
A solution containing 50 g (200 mmol) of 4-bromophenol, 33.5 g (298 mmol) 
of potassium t-butoxide and 40 ml (298 mmol) of 1-iodopentane in 1000 ml 
of tetrahydrofuran was reacted at reflux temperature for approximately 
twenty four hours. When the reaction was substantially complete, as 
indicated by TLC, the reaction was filtered. The resultant filtrate was 
concentrated in vacuo to provide a purple solid. This solid was 
redissolved in a water/diethyl ether mixture to provide a yellow solution. 
This solution was washed sequentially with 200 ml of water (twice), 100 ml 
of 2N sodium hydroxide (twice) and 200 ml of brine (twice), dried over 
sodium sulfate and then concentrated in vacuo to provide a yellow powder. 
This solid was recrystallized from hot hexanes to provide a white powder. 
Yield: 45.8 mg (72%). 
##STR19## 
To a cold (-78.degree. C.) solution of 10.0 mg (42.9 mmol) of 29 g (90.8 
mmol) of the compound of Preparation 1A, was added 91 ml of 
sec-butyllithium in 1000 ml of tetrahydrofuran (118 mmol), dropwise. To 
the resulting mixture was added 41.9 ml (181.7 mmol) of triisopropyl 
borate, dropwise. The resultant reaction mixture was stirred for 
approximately thirty minutes and then warmed to room temperature and 
allowed to react for approximately two hours. The reaction was then 
quenched by the addition of 1N hydrochloric acid. The resultant mixture 
was concentrated in vacuo to provide a residue. This residue was 
redissolved in diethyl ether, filtered and dried to provide the desired 
subtitled compound. 
Yield: 
##STR20## 
A solution of 4.87 mg (26.2 mmol) of 1-bromo-4-formyl benzene in methanol 
was added to a solution containing 6 g (21 mmol) of the compound of 
Preparation 3B, 60 ml of 2M sodium carbonate and 2.5 g (2.1 mmol) of 
tetrakis(triphenylphosphine)palladium in 120 ml of toluene. The resultant 
reaction mixture was allowed to react at reflux temperature for 
approximately five hours. When the reaction was substantially complete, as 
indicated by TLC, the biphasic mixture was separated and the organic layer 
was washed sequentially with water and brine, dried over magnesium 
sulfate, filtered and concentrated in vacuo to provide a solid. This solid 
was recrystallized from hot hexanes. 
MS(FD) : 344(M.sup.+). 
Preparation 4 
##STR21## 
To a solution of 2.5 g (13 mmol) of p-bromo benzaldehyde in 16 ml of 
acetonitrile, was added 1.5 g (14 mmol) of phenyl acetylene, 0.55 g (0.52 
mmol) of palladium-on-copper, 0.54 g (2 mmol) of triphenylphosphine, 0.1 g 
(0.52 mmol) of copper (I) iodide and 32.5 ml of triethylamine. The 
resultant reaction mixture was degassed in vacuo and flushed with argon 
(three times). After the reaction mixture was refluxed, under argon, for 
twenty four hours, the mixure was cooled to room temperature and 
concentrated in vacuo to provide a residue. This residue was purified 
using flash chromatography (silica gel; eluent of 20% ethyl acetete in 
hexanes) to provide 1 g of a white powder. 
Yield: 37%. 
.sup.1 H NMR (CDCl.sub.3, 300 MHz): 
.delta. 7.4 (m, 3H), 7.6 (m, 2H), 7.7 (d, J=7.68 Hz, 2H), 7.85 (d, J=7.68 
Hz, 2H), 10.02 (s, 1H) . 
Preparation 5 
##STR22## 
To a solution containing 5 g (21.2 mmol) of 1,4-dibromobenzene, 18.8 mg 
(0.106 mmol) of palladium (II) chloride, 55.6 mg (0.212 mmol) of 
triphenylphosphine and 5.91 ml (0.726 mmol) of triethylamine in 300 ml of 
acetonitrile, was added 2.327 g (21.2 mmol) of phenyl acetylene and 40.0 
mg (0.212 mmol) of copper (I) iodide. The resultant reaction mixture was 
allowed to react at room temperature for approximately two days. The crude 
material was purified using HPLC (eluent of hexane) to provide 660 mg of a 
white solid. 
##STR23## 
The desired subtitled compound was prepared substantially in accordance 
with the procedure detailed in Preparation 2B, using 3.07 g (11.9 mmol) of 
the subtitled compound of Preparation 5A and 1.78 g 11.9 mmol) of 
1-boronic acid-4-formylbenzene, 60 ml of 2M sodium carbonate and 1.360 g 
(1.19 mmol) of tetrakis(triphenylphosphine)palladium in 90 ml of toluene. 
MS (FAB) : 283.1 (M+H). 
EXAMPLE 1 
Preparation of the compound of formula I where R', R" nd R'" are each 
methyl, R.sup.x1, R.sup.x2, R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4 and 
R.sup.0 are each hydroxy and R.sup.2 is 4-octyloxybenzyl 
A solution containing 1.5 g (1.88 mmol) of the (A-30912A) nucleus (compound 
of formula IB where R', R" and R'" are each methyl, R.sup.x1, R.sup.x2, 
R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4 and R.sup.0 are each hydroxy), 697 
mg (2.07 mmol) of the compound of Preparation 1, and 130 mg (2.07 mmol) of 
sodium cyanoborohydride in a 1:1 dimethylformamide/methanol mixture was 
heated at 70.degree. C. overnight. When the reaction was substantially 
complete, as indicated by TLC, the reaction mixture was concentrated in 
vacuo the desired compound was isolated using HPLC (eluent of 40% aqueous 
acetonitrile; 60 ml/min.; 280 nm). The fractions containing the desired 
compound were combined and concentrated in vacuo to provide crude 
material. This material was purified using HPLC (eluent of 50% aqueous 
acetonitrile; 50 ml/min.; 280 nm). 
Yield: 19 mg. 
MS(FAB) for C.sub.49 H.sub.72 N.sub.7 O.sub.15 : 
Calcd: 998.5086 (M--H.sub.2 O); 
Found: 998.5076. 
EXAMPLE 2 
Preparation of the compound of formula I where R', R" and R'" are each 
methyl, R.sup.x1, R.sup.x2, R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4 and 
R.sup.0 are each hydroxy and 
R.sup.2 is 
##STR24## 
The desired compound was prepared substantially in accordance with the 
procedure detailed in Example 1 using 1.5 g (1.88 mmol) of the (A-30912A) 
nucleus (compound of formula IB where R', R" and R'" are each methyl, 
R.sup.x1, R.sup.x2, R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4 and R.sup.0 is 
hydroxy), 533.5 mg (2.068 mmol) of the compound of Preparation 2B, and 130 
mg (2.07 mmol) of sodium cyanoborohydride in 100 ml of a 1:1 
dimethylformamide/methanol mixture with the exception that the reaction 
was substantially complete after approximately twelve hours. The crude 
material was purified using HPLC (eluent of 50% aqueous acetonitrile; 60 
ml/min.; 280 nm). 
Yield: 24 mg. 
MS(FAB) for C.sub.53 H.sub.65 N.sub.7 O.sub.15 : 
Calcd: 1040.4617 (M+H); 
Found: 1040.4636. 
EXAMPLE 3 
Preparation of the compound of formula I where R', R" and R'" are each 
methyl, R.sup.x1, R.sup.x2, R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4 and 
R.sup.0 are each hydroxy and 
R.sup.2 is 
##STR25## 
The desired compound was prepared substantially in accordance with the 
procedure detailed in Example 1 using 1 g (1.25 mmol) of the (A-30912A) 
nucleus (compound of formula IB where R', R" and R'" are each methyl, 
R.sup.x1, R.sup.x2, R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4 and R.sup.0 is 
hydroxy), 474.0 mg (1.38 mmol) of the compound of Preparation 3C, and 86.7 
mg (1.38 mmol) of sodium cyanoborohydride in 100 ml of a 3:1 
merthanol/dimethylformamide mixture, with the exception that the reaction 
was substantially complete after approximately six hours. After isolating 
the crude material using HPLC (eluent of 50% aqueous acetonitrile; 60 
ml/min.; 280 nm), the fractions containing the desired compound were 
combined, concentrated in vacuo and lyophilized. 
MS(FAB) : 1132.5 (M+Li). 
EXAMPLE 4A 
Preparation of the compound of formula I where R', R" and R'" are each 
methyl, R.sup.x2, R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4 and R.sup.0 are 
each hydroxy, R.sup.x1 is hydrogen, and 
R.sup.2 is 
##STR26## 
A solution of 203.0 mg (0.253 mmol) of the (A-30912A) nucleus (compound of 
formula IB where R', R" and R'" are each methyl, R.sup.x1, R.sup.x2, 
R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4 and R.sup.0 is hydroxy) and 83.0 mg 
(0.455 mmol) of 4-phenylbenzaldehyde in 10 ml of methanol was reacted at 
reflux temperature. 
Yield: 22 mg. 
MS (FAB) for C.sub.47 H.sub.61 N.sub.7 O.sub.15 : 
Calcd: 964.4348 (M+H); 
Found: 964.4304. 
EXAMPLE 4B 
Alternate Preparation of the compound of formula I where R', R" and R'" are 
each methyl, R.sup.x1, R.sup.x2, R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4 
and R.sup.0 are each hydroxy and 
R.sup.2 is 
##STR27## 
A solution of 1.5 g (1.88 mmol) of the (A-30912A) nucleus (compound of 
formula IB where R', R" and R'" are each methyl, R.sup.x1, R.sup.x2, 
R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4 and R.sup.0 are each hydroxy), 
376.8 mg (2.068 mmol) of 4-phenylbenzaldehyde and 130 mg (2.07 mmol) of 
sodium cyanoborohydride in a 100 ml of a 3:1 methanol/dimethylformamide 
mixture was allowed to react overnight at reflux temperature. The 
resultant crude material was isolated using HPLC (eluent of 50% aqueous 
acetonitrile; 60 ml/min.; 280 nm). 
Yield: 68 mg. 
MS(FAB) for C.sub.47 H.sub.62 N.sub.7 O.sub.15 : 
Calcd: 964.4304 (M+H); 
Found: 964.4348. 
EXAMPLE 5 
Preparation of the compound of formula I where R', R" and R'" are each 
methyl, R.sup.x1, R.sup.x2, R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4, and 
R.sup.0 are each hydroxy and 
R.sup.2 is 
##STR28## 
The desired compound was prepared substantially in accordance with the 
procedure detailed in Example 4A, using 375.3 mg (0.495 mmol) of the 
(A-30912A) nucleus (compound of formula IB where R', R" and R'" are each 
methyl, R.sup.x1, R.sup.x2, R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4 and 
R.sup.0 are each hydroxy) and 158.3 mg (0.767 mmol) of the compound of 
Preparation 4 in 10 ml of ethanol. 
Yield: 28 mg. 
MS (FAB) for C.sub.49 H.sub.60 N.sub.7 O.sub.14 : 
Calcd: 970.4198 (M+H-H.sub.2 O); 
Found: 970.4222. 
EXAMPLE 6 
Preparation of the compound of formula I where R', R" and R'" are each 
methyl, R.sup.x1, R.sup.x2, R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4, and 
R.sup.0 are each hydroxy and 
R.sup.2 is 
##STR29## 
The desired compound was prepared substantially in accordance with the 
procedure detailed in Example 4A, using 577.8 mg (0.649 mmol) of the 
(A-30912A) nucleus (compound of formula IB where R', R" and R'" are each 
methyl, R.sup.x1, R.sup.x2, R.sup.y1, R.sup.y2, R.sup.y3, R.sup.y4 and 
R.sup.0 is hydroxy) and 164.6 mg (0.583 mmol) of the compound of 
Preparation 5B in 10 ml of ethanol. 
Yield: 51 mg. 
MS(FAB) for C.sub.55 H.sub.64 N.sub.7 O.sub.14 : 
Calcd: 1046.4511 (M-H.sub.2 O); 
Found: 1046.4530. 
The compounds of formula I exhibit antifungal and antiparasitic activity. 
For example, the compounds of formula I inhibit the growth of various 
infectious fungi including Candida spp. such as C. albicans, C. 
parapsilosis, C. krusei, C. glabrata, or C. tropicalis, C. lusitaniae; 
Torulopus spp. such as T. glabrata; Aspergillus spp. such as A. fumigatus; 
Histoplasma spp. such as H. capsulatum; Cryptococcus spp. such as C. 
neoformans; Blastomyces spp. such as B. dermatitidis; Fusarium spp., 
Trichophyton spp., Pseudallescheria boydii, Coccidioides immitis, 
Sporothrix schenckii and the like. 
Antifungal activity of a test compound is determined in vitro by obtaining 
the minimum inhibitory concentration (MIC) of the compound using a 
standard agar dilution test or a disc-diffusion test. The compound is then 
tested in vivo (in mice) to determine the effective dose of the test 
compound for controlling a systemic fungal infection. 
Accordingly, the following compounds were tested for antifungal activity 
against C. albicans. 
TABLE 5 
______________________________________ 
Minimal inhibitory concentration against C. albicans 
Example No. MIC (.mu.g/ml) 
______________________________________ 
1 0.039 
2 0.005 
3 5.0 
4 0.312 
5 20 
6 0.039 
______________________________________ 
In addition, the effective dose of the following 
compounds for controlling a systemic fungal infection 
(C. albicans) was tested in vivo (mice). 
ED.sub.50 (mouse) 
Example No. ED.sub.50 (mg/kg) 
______________________________________ 
1 63 
2 &gt;20 
3 N.T. 
4 &gt;2.5 
5 &gt;2.5 
6 &gt;2.5 
______________________________________ 
N.T. not tested 
The compounds of the invention also inhibit the growth of certain organisms 
primarily responsible for opportunistic infections in immunosuppressed 
individuals. For example the compounds of the invention inhibit the growth 
of Pneumocystis carinii the causative organism of pneumocystis pneumonia 
(PCP) in AIDS and other immunocompromised patients. Other protozoans that 
are inhibited by compounds of formula I include Plasmodium spp., 
Leishmania spp., Trypanosoma spp., Cryptosporidium spp., Isospora spp., 
Cyclospora spp., Trichomonas spp., Microsporidiosis spp. and the like. 
The compounds of formula I are active in vitro and in vivo and are useful 
in combating either systemic fungal infections or fungal skin infections. 
Accordingly, the present invention provides a method of inhibiting fungal 
activity comprising contacting a compound of formula I, or a 
pharmaceutically acceptable salt thereof, with a fungus. A preferred 
method includes inhibiting Candida albicans or Aspergillus fumigatis 
activity. The present invention further provides a method of treating a 
fungal infection which comprises administering an effective amount of a 
compound of formula I, or a pharmaceutically acceptable salt thereof, to a 
host in need of such treatment. A preferred method includes treating a 
Candida albicans or Aspergillus fumigatis infection. 
With respect to antifungal activity, the term "effective amount," means an 
amount of a compound of the present invention which is capable of 
inhibiting fungal activity. The dose administered will vary depending on 
such factors as the nature and severity of the infection, the age and 
general health of the host and the tolerance of the host to the antifungal 
agent. The particular dose regimen likewise may vary according to such 
factors and may be given in a single daily dose or in multiple doses 
during the day. The regimen may last from about 2-3 days to about 2-3 
weeks or longer. A typical daily dose (administered in single or divided 
doses) will contain a dosage level of from about 0.01 mg/kg to about 100 
mg/kg of body weight of an active compound of this invention. Preferred 
daily doses generally will be from about 0.1 mg/kg to about 60 mg/kg and 
ideally from about 2.5 mg/kg to about 40 mg/kg. 
The present invention also provides pharmaceutical formulations useful for 
administering the antifungal compounds of the invention. Accordingly, the 
present invention also provides a pharmaceutical formulation comprising 
one or more pharmaceutically acceptable carriers, diluents or excipients 
and a compound of claim 1. The active ingredient in such formulations 
comprises from 0.1% to 99.9% by weight of the formulation, more generally 
from about 10% to about 30% by weight. By "pharmaceutically acceptable" it 
is meant that the carrier, diluent or excipient is compatible with the 
other ingredients of the formulation and not deleterious to the recipient 
thereof. 
A compound of formula I may be administered parentorally, for example using 
intramuscular, sub-cutaneous, or intra-peritoneal injection, nasal, or 
oral means. In addition to these methods of administration, a compound of 
formula I may be applied topically for skin infections. 
For parentoral administration the formulation comprises a compound of 
formula I and a physiologically acceptable diluent such as deionized 
water, physiological saline, 5% dextrose and other commonly used diluents. 
The formulation may contain a solubilizing agent such as a polyethylene 
glycol or polypropylene glycol or other known solubilizing agent. Such 
formulations may be made up in sterile vials containing the antifungal and 
excipient in a dry powder or lyophilized powder form. Prior to use, a 
physiologically acceptable diluent is added and the solution withdrawn via 
syringe for administration to the patient. 
The present pharmaceutical formulations are prepared by known procedures 
using known and readily I0 available ingredients. In making the 
compositions of the present invention, the active ingredient will 
generally be admixed with a carrier, or diluted by a carrier, or enclosed 
within a carrier which may be in the form of a capsule, sachet, paper or 
other container. When the carrier serves as a diluent, it may be a solid, 
semi-solid or liquid material which acts as a vehicle, excipient or medium 
for the active ingredient. Thus, the compositions can be in the form of 
tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, 
emulsions, solutions, syrups, aerosols, (as a solid or in a liquid 
medium), ointments containing, for example, up to 10% by weight of the 
active compound, soft and hard gelatin capsules, suppositories, sterile 
injectable solutions, sterile packaged powders and the like. 
For oral administration, the antifungal compound is filled into gelatin 
capsules or formed into tablets. Such tablets may also contain a binding 
agent, a dispersant or other suitable excipients suitable for preparing a 
proper size tablet for the dosage and particular antifungal compound of 
the formula I. For pediatric or geriatric use the antifungal compound may 
be formulated into a flavored liquid suspension, solution or emulsion. A 
preferred oral formulation is linoleic acid, cremophor RH-60 and water and 
preferably in the amount (by volume) of 8% linoleic acid, 5% cremophor 
RH-60, 87% sterile water and a compound of formula I in an amount of from 
about 2.5 to about 40 mg/ml. 
For topical use the antifungal compound may be formulated with a dry powder 
for application to the skin surface or it may be formulated in a liquid 
formulation comprising a solubilizing aqueous liquid or non-aqueous 
liquid, e.g., an alcohol or glycol. 
The following formulation examples are illustrative only and are not 
intended to limit the scope of the invention in any way. The term "active 
ingredient" means a compound according to formula I or a pharmaceutically 
acceptable salt thereof. 
FORMULATION 1 
Hard gelatin capsules are prepared using the following ingredients: 
______________________________________ 
Quantity 
(mg/capsule) 
______________________________________ 
Active ingredient 250 
Starch, dried 200 
Magnesium stearate 10 
Total 460 mg 
______________________________________ 
FORMULATION 2 
A tablet is prepared using the ingredients below: 
______________________________________ 
Quantity 
(mg/capsule) 
______________________________________ 
Active ingredient 250 
Cellulose, microcrystalline 
400 
Silicon dioxide, fumed 
10 
Stearic acid 5 
Total 665 mg 
______________________________________ 
The components are blended and compressed to form tablets each weighing 665 
mg. 
FORMULATION 3 
An aerosol solution is prepared containing the following components: 
______________________________________ 
Weight 
______________________________________ 
Active ingredient 0.25 
Methanol 25.75 
Propellant 22 74.00 
(Chlorodifluoromethane) 
Total 100.00 
______________________________________ 
The active compound is mixed with ethanol and the mixture added to a 
portion of the propellant 22, cooled to -30.degree. C. and transferred to 
a filling device. The required amount is then fed to a stainless steel 
container and diluted with the remainder of the propellant. The valve 
units are then fitted to the container. 
FORMULATION 4 
Tablets, each containing 60 mg of active ingredient, are made as follows: 
______________________________________ 
Active ingredient 60 mg 
Starch 45 mg 
Microcrystalline cellulose 
35 mg 
Polyvinylpyrrolidone 4 mg 
(as 10% solution in water) 
Sodium carboxymethyl starch 
4.5 mg 
Magnesium stearate 0.5 mg 
Talc 1 mg 
Total 150 mg 
______________________________________ 
The active ingredient, starch and cellulose are passed through a No. 45 
mesh U.S. sieve and mixed thoroughly. The aqueous solution containing 
polyvinyl-pyrrolidone is mixed with the resultant powder, and the mixture 
then is passed through a No. 14 mesh U.S. sieve. The granules so produced 
are dried at 50.degree. C. and passed through a No. 18 mesh U.S. sieve. 
The sodium carboxymethyl starch, magnesium stearate and talc, previously 
passed through a No. 60 mesh U.S. sieve, are then added to the granules 
which, after mixing, are compressed on a tablet machine to yield tablets 
each weighing 150 mg. 
FORMULATION 5 
Capsules, each containing 80 mg of active ingredient, are made as follows: 
______________________________________ 
Active ingredient 80 mg 
Starch 59 mg 
Microcrystalline cellulose 
59 mg 
Magnesium stearate 2 mg 
Total 200 mg 
______________________________________ 
The active ingredient, cellulose, starch and magnesium stearate are 
blended, passed through a No. 45 mesh U.S. sieve, and filled into hard 
gelatin capsules in 200 mg quantities. 
FORMULATION 6 
Suppositories, each containing 225 mg of active ingredient, are made as 
follows: 
______________________________________ 
Active ingredient 225 mg 
Saturated fatty acid glycerides 
2,000 mg 
Total 2,225 mg 
______________________________________ 
The active ingredient is passed through a No. 60 mesh U.S. sieve and 
suspended in the saturated fatty acid glycerides previously melted using 
the minimum heat necessary. The mixture is then poured into a suppository 
mold of nominal 2 g capacity and allowed to cool. 
FORMULATION 7 
Suspensions, each containing 50 mg of active ingredient per 5 ml dose, are 
made as follows: 
______________________________________ 
Active ingredient 50 mg 
Sodium carboxymethyl cellulose 
50 mg 
Syrup 1.25 ml 
Benzoic acid solution 0.10 ml 
Flavor q.v. 
Color q.v. 
Purified water to total 5 ml 
______________________________________ 
The active ingredient is passed through a No. 45 mesh U.S. sieve and mixed 
with the sodium carboxymethyl cellulose and syrup to form a smooth paste. 
The benzoic acid solution, flavor and color are diluted with a portion of 
the water and added, with stirring. Sufficient water is then added to 
produce the required volume. 
FORMULATION 8 
An intravenous formulation may be prepared as follows: 
______________________________________ 
Active ingredient 100 mg 
Isotonic saline 1,000 ml 
______________________________________ 
The solution of the above ingredients generally is administered 
intravenously to a subject at a rate of 1 ml per minute. 
The present invention further provides a method for treating or preventing 
the onset of Pneumocystis pneumonia in a host susceptible to Pneumocystis 
pneumonia which comprises administering an effective amount of a compound 
of formula I, or a pharmaceutically acceptable salt thereof, to a host in 
need of such treatment. The compounds of formula I can be used 
prophylactically to prevent the onset of the infection which is caused by 
the organism Pneumocystis carinii, or alternatively they can be used to 
treat a host that has been infected with P. carinii. A compound of formula 
I may be administered parenterally, for example using intramuscular, 
intravenous or intra-peritoneal injection, orally or by inhaling directly 
into the airways of the lungs. A preferred mode of administration is 
inhalation of an aerosol spray formulation of a compound of formula I. 
With respect to antiparasitic activity, the term "effective amount," means 
an amount of a compound of the present invention which is capable of 
inhibiting parasitic activity. An effective amount of the compound of 
formula I is from about 3 mg/kg of patient body weight to about 100 mg/kg. 
The amount administered may be in a single daily dose or multiple doses 
of, for example, two, three or four times daily throughout the treatment 
regimen. The amount of the individual doses, the route of delivery, the 
frequency of dosing and the term of therapy will vary according to such 
factors as the intensity and extent of infection, the age and general 
health of the patient, the response of the patient to therapy and how well 
the patient tolerates the drug. It is known that Pneumocystis pneumonia 
infections in AIDS patients are highly refractory owing to the nature of 
the infection. For example, in severe, advanced infections the lumenal 
surface of the air passages becomes clogged with infectious matter and 
extensive parasite development occurs in lung tissue. A patient with an 
advanced infection will accordingly require higher doses for longer 
periods of time. In contrast, immune deficient patients who are not 
severely infected and who are susceptible to Pneumocystis pneumonia can be 
treated with lower and less frequent prophylactic doses.