Note: Descriptions are shown in the official language in which they were submitted.
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AMINE-MODIFIED PSEUDOMYCIN 'COMPOUNDS
FIELD OF THE INVENTION
The present invention relates to pseudomycin
compounds, in particular, amine-modified, pseudomycin
compounds.
BACKGROUND OF THE INVENTION
Pseudomycins are natural products isolated from liquid
cultures of Pseudomonas syringae (plant-associated
bacterium) and have been shown to have antifungal
activities. (see i.e., Harrison, L., et al.,
"Pseudomycins, a family of novel peptides from Pseudomonas
syringae possessing broad-spectrum antifungal activity," J.
Gen. Microbiology, 137(12), 2857-65 (1991) and US Patent
Nos. 5,576,298 and 5,837,685) Unlike the previously
described
antimycotics from P. syringae (e. g., syringomycins,
syringotoxins and syringostatins), pseudomycins A-C contain
hydroxyaspartic acid, aspartic acid, serine,
dehydroaminobutyric acid, lysine and diaminobutyric acid.
The peptide moiety for pseudomycins A, A', B, B', C,
C' corresponds to L-Ser-D-Dab-L-Asp-L-Lys-L-Dab-L-aThr-Z-
Dhb-L-Asp(3-OH)-L-Thr(4-Cl) with the terminal carboxyl
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group closing a macrocyclic ring on the OH group of the N-
terminal Ser. The analogs are distinguished by the N-acyl
side
chain, i.e., pseudomycin A is N-acylated by
3,4-dihydroxytetradeconoyl, pseudomycin A' by
3,4-dihydroxypentadecanoyl, pseudomycin B by
3-hydroxytetradecanoyl, pseudomycin B' by
3-hydroxydodecanoyl, pseudomycin C by
3,4-dihydroxyhexadecanoyl and pseudomycin C' by
3-hydroxyhexadecanoyl. (see i.e., Ballio, A., et al.,
"Novel bioactive lipodepsipeptides from Pseudomonas
syringae: the pseudomycins," FEBS Letters, 355(1), 96-100,
(1994) and Coiro, V.M., et al., "Solution conformation of
the Pseudomonas syringae MSU 16H phytotoxic
lipodepsipeptide Pseudomycin A determined by computer
simulations using distance geometry and molecular dynamics
from NMR data," Eur. J. Biochem., 257(2), 449-456 (1998).)
Pseudomycins are known to have certain adverse
biological effects. For example, destruction of the
endothelium of the vein, destruction of tissue,
inflammation, and local toxicity to host tissues have been
observed when pseudomycin is administered intraveneously.
Since the pseudomycins have proven antifungal activity and
relatively unexplored chemistry, there is a need to explore
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this class of compounds for other potential compounds that
may be useful as antifungal agents having less adverse side
affects.
BRIEF SUMMARY OF THE INVENTION
The present invention provides amine-modified
pseudomycin compounds represented by the following
structure which are useful as antifungal agents or in the
design of antifungal agents.
O
R3
O
OH
O~N H
NH H N OH
HO O
NH SCI
O
O O
R'HN O
NH
O O O H R
N NH
O
R2 NHR'
R' HN O
wherein R is
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Rb~ Rd
Rf
Ra Ra~ ~c ~a
R R
where
Ra and Ra~ are independently hydrogen or methyl,
or either Ra or Ra~ is alkyl amino, taken together with
Rb or Rb~ forms a six-membered cycloalkyl'ring, a six-
membered aromatic ring or a double bond, or taken
together with R~ forms a six-membered aromatic ring;
Rb and Rb~ are independently hydrogen, halogen, or
methyl, or either Rb or Rb~ is amino, alkylamino, oc-
acetoacetate, methoxy, or hydroxy;
R° is hydrogen, hydroxy, C1-C4 alkoxy, hydroxy (C1-
C4)alkoxy, or taken together with Re forms a 6-membered
aromatic ring or CS-C6 cycloalkyl ring;
Re is hydrogen, or taken together with Rf is a
six-membered aromatic ring, CS-C14 alkoxy substituted
six-membered aromatic ring, or C5-C14 alkyl substituted
six-membered aromatic ring, and
Rf is C$-C18 alkyl , C5-C11 alkoxy, or biphenyl ;
R is
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Rg
Rn
O
where
Rg is hydrogen, or C1-C13 alkyl, and
Rh is C1-C15 alkyl, C4-C15 alkoxy, (C1-Clo
alkyl ) phenyl , - ( CHZ ) n-aryl , or - ( CH2 ) n- ( CS-Cs
cycloalkyl), where n = 1 or 2; or
R is
R~
m
where
Ri is a hydrogen, halogen, or C5-C8 alkoxy, and
m is 1, 2 or 3;
R is
OH
a R'
where
R' is C5-C14 alkoxy or CS-C14 alkyl , and p = 0 , 1 or
2;
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R is
-N
Rk
where
Rk is CS-C14 alkoxy; or
R is - (CH2) -NRm- (C13-C18 alkyl) , where Rm is H, -CH3 or
-C (O) CH3;
R1 is independently hydrogen, formyl, an acylalkyl (e.g., -
C ( 0 ) CH3 , -C ( 0 ) CH2CH3 , -C ( O ) CH ( CH3 ) 2 , and -C ( O ) C ( CH3 )
3 ) , an
acylalkylamine (e. g., -C(0)CH(NH2)CH3), an acylazaalkyl
(e. g., -C(O)NHCH3 and -C(O)NHCH(CH3)2), an acyloxyalkene
( a . g . , -C ( 0 ) OCH2CH=CH2 ) , an acyloxyaryl ( a . g . , -C ( O ) OC6H5
) ,
or an acylmethylenecarbamate (e. g., compounds 1(a) depicted
below)
O
N O
~R~a
R' b O
1 (a)
Rla i s C1-Clo alkyl , C1-C1o alkenyl , benzyl , or aryl
and R1b is hydrogen or methyl,
provided that at least one R1 is not hydrogen;
RZ and R3 are independently -OR2a, or -N(RZb) (R2°) ,
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where
RZa and R2b are independently hydrogen, C1-Clo alkyl
(e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-
butyl, s-butyl, t-butyl, etc.), C3_C6 cycloalkyl (e. g.,
cyclopropyl, cyclobutyl, cyclopentyl,
cyclopentylmethylene, methylcyclopentyl, cyclohexyl,
etc . ) hydroxy ( C1-Clo ) alkyl , alkoxy ( C1-Clo ) alkyl ( a . g,
methoxyethyl ) , or C2-Clo alkenyl , amino ( C1-Clo ) alkyl ,
mono- or di-alkylamino ( C1-Clo ) alkyl , aryl ( C1-Clo ) alkyl
(e.g., benzyl), heteroaryl(C1-Clo)alkyl (e.g., 3-
pyridylmethyl, 4-pyridylmethyl), or
cycloheteroalkyl(C1-Clo)alkyl (e. g., N-tetrahydro-1,4-
oxazinylethyl and N-piperazinylethyl), or
RZb is an alkyl carboxylate residue of an
aminoacid alkyl ester (e.g. , -CHzCO2CH3,
-CH ( COzCH3 ) CH ( CH3 ) 2 , -CH ( COZCH3 ) CH ( phenyl ) ,
-CH ( COZCH3 ) CH20H, -CH ( C02CH3 ) CH2 (p-hydroxyphenyl ) ,
-CH ( COZCH3 ) CHzSH, -CH ( C02CH3 ) CHz ( CHZ ) 3NH2 ,
-CH ( COZCH3 ) CHZ ( 4- or 5-imidazole ) , -CH ( COZCH3 ) CHzCOzCH3,
-CH ( COZCH3 ) CHZC02NH2 , and the 1 i ke ) , and
R2~ is hydrogen or C1-C6 alkyl; and
pharmaceutically acceptable salts and solvates thereof.
In another embodiment of the present invention, a
pharmaceutical formulation is provided which includes the
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pseudomycin compound described above and a pharmaceutically
acceptable carrier.
In yet another embodiment of the present invention, a
method is provided for treating an antifungal infection in
an animal in need thereof, which comprises administering to
the animal the pseudomycin compound described above.
Definitions
As used herein, the term "alkyl" refers to a
hydrocarbon radical of the general formula CnH2n+1 containing
from 1 to 30 carbon atoms unless otherwise indicated. The
alkane radical may be straight (e. g. methyl, ethyl, propyl,
butyl, etc.), branched (e. g., isopropyl, isobutyl, tertiary
butyl, neopentyl, etc.), cyclic (e. g., cyclopropyl,
cyclobutyl, cyclopentyl, methylcyclopentyl, cyclohexyl,
etc.), or multi-cyclic (e. g., bicyclo[2.2.1]heptane,
spiro[2.2]pentane, etc.). The alkane radical may be
substituted or unsubstituted. Similarly, the alkyl portion
of an alkoxy group, alkanoyl, or alkanoate have the same
definition as above.
The term "alkenyl" refers to an acyclic hydrocarbon
containing at least one carbon carbon double bond. The
alkene radical may be straight, branched, cyclic, or multi-
cyclic. The alkene radical may be substituted or
unsubstituted. The alkenyl portion of an alkenoxy,
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alkenoyl or alkenoate group has the same definition as
above.
The term "aryl" refers to aromatic moieties having
single (e. g., phenyl) or fused ring systems (e. g.,
naphthalene, anthracene, phenanthrene, etc.). The aryl
groups may be substituted or unsubstituted.
Within the field of organic chemistry and particularly
within the field of organic biochemistry, it is widely
understood that significant substitution of compounds is
tolerated or even useful. In the present invention, for
example, the term alkyl group allows for substitutents
which is a classic alkyl, such as methyl, ethyl, propyl,
hexyl, isooctyl, dodecyl, stearyl, etc. The term "group"
specifically envisions and allows for substitutions on
alkyls which are common in the art, such as hydroxy,
halogen, alkoxy, carbonyl, keto, ester, carbamato, etc., as
well as including the unsubstituted alkyl moiety. However,
it is generally understood by those skilled in the art that
the substituents should be selected so as to not adversely
affect the pharmacological characteristics of the compound
or adversely interfere with the use of the medicament.
Suitable substituents for any of the groups defined above
include alkyl, alkenyl, alkynyl, aryl, halo, hydroxy,
alkoxy, aryloxy, mercapto, alkylthio, arylthio, mono- and
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di-alkyl amino, quaternary ammonium salts, aminoalkoxy,
hydroxyalkylamino, aminoalkylthio, carbamyl, carbonyl,
carboxy, glycolyl, glycyl, hydrazino, guanyl, and
combinations thereof.
The term "animal" refers to humans, companion animals
(e. g., dogs, cats and horses), food-source animals (e. g.,
cows, pigs, sheep and poultry), zoo animals, marine
animals, birds and other similar animal species.
DETAILED DESCRIPTION OF THE INVENTION
Applicants have discovered that modification of the
pendant amino groups attached to the lysine or 2,4-
diaminobutyric acid peptide units in the pseudomycin
natural product or semi-synthetic derivative provides
compounds having in vitro indications which suggest that
the new compounds may be active against C. albican, C,
neoformans, and/or A. fumigatus. The amino groups are
modified using an acylating agent containing a suitable
leaving group such that an amide, carbamate, urea or imide
linkage with the pendant amino group on the pseudomycin
structure can be formed. Suitable leaving groups are well
known to those skilled in the art and include groups such
as p-nitrophenoxy and N-oxysuccinimide.
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Amide linkages are synthesized using conventional
chemistry well-known to those skilled in the art. Suitable
acylating agents include derivatives of the desired
carboxylic acid to produce a pseudomycin compound where R1 -
acylalkyl or amino acid to produce a pseudomycin compound
where R1 - acylalkylamine. Typically, the acylating agent
is formed by replacing the -OH of the carboxylic acid group
with a leaving group (e.g., N-oxysuccinimde). When an
amino acid acylating agent is used, the amino group is
protected prior to condensation using any conventional
amino-protecting group known to those skilled in the art
(e.g., benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-
bromobenzyloxycarbonyl, p-methoxybenxyloxycarbonyl, p-
methoxyphenylazobenzyloxycarbonyl, p-
phenylazobenzyloxycarbonyl, t-butyloxycarbonyl or
cyclopentyloxycarbonyl). After the amide linkage is
formed, then the amino-protecting group is removed using
standard hydrogenation chemistry (e.g., Pd/C under a
hydrogen atmosphere). See the Examples below for a more
detailed description for forming pseudomycin amide
derivatives from amino acids.
As discussed earlier, pseudomycins are natural
products isolated from the bacterium Pseudomonas syringae
that have been characterized as lipodepsinonapetpides
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containing a cyclic peptide portion closed by a lactone
bond and including the unusual amino acids 4-
chlorothreonine (ClThr), 3-hydroxyaspartic acid (HOAsp),
2,3-dehydro-2-aminobutyric acid (Dhb), and 2,4-
diaminobutyric acid (Dab). Methods for growth of various
strains of P. syringae to produce the different pseudomycin
analogs (A, A', B, B', C, and C') are described in PCT
Patent Application Serial No. PCT/US00/08728 filed by
Hilton, et al. on April 14, 2000 entitled "Pseudomycin
Production by Pseudomonas Syringae," incorporated herein by
reference, PCT Patent Application Serial No. PCT/US00/08727
filed by Kulanthaivel, et al. on April 14, 2000 entitled
"Pseudomycin Natural Products," incorporated herein by
reference, and U.S. Patent Nos. 5,576,298 and 5,837,685,
each of which are incorporated herein by reference.
Isolated strains of P. syringae that produce one or
more pseudomycins are known in the art. Wild type strain
MSU 174 and a mutant of this strain generated by transposon
mutagenesis, MSU 16H are described in U.S. Patent Nos.
5,576,298 and 5,837,685; Harrison, et al., "Pseudomycins, a
family of novel peptides from Pseudomonas syringae
possessing broad-spectrum antifungal activity," J. Gen.
Microbiology, 137, 2857-2865 (1991); and Lamb et al.,
"Transposon mutagenesis and tagging of fluorescent
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pseudomonas: Antimycotic production is necessary for
control of Dutch elm disease," Proc. Natl. Acad. Sci. USA,
84, 6447-6451 (1987).
A strain of P. syringae that is suitable for
production of one or more pseudomycins can be isolated from
environmental sources including plants (e. g., barley
plants, citrus plants, and lilac plants) as well as,
sources such as soil, water, air, and dust. A preferred
stain is isolated from plants. Strains of P. syringae that
are isolated from environmental sources can be referred to
as wild type. As used herein, "wild type" refers to a
dominant genotype which naturally occurs in the normal
population of P. syringae (e.g., strains or isolates of P.
syringae that are found in nature and not produced by
laboratory manipulation). Like most organisms, the
characteristics of the pseudomycin- producing cultures
employed (P. syringae strains such as MSU 174, MSU 16H, MSU
206, 25-B1, 7H9-1) are subject to variation. Hence,
progeny of these strains (e.g., recombinants, mutants and
variants) may be obtained by methods known in the art.
P. syringae MSU 16H is publicly available from the
American Type Culture Collection, Parklawn Drive,
Rockville, MD, USA as Accession No. ATCC 67028. P.
syringae strains 25-B1, 7H9-1, and 67 H1 were deposited
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with the American Type Culture Collection on March 23, 2000
and were assigned the following Accession Nos.:
25-B1 Accession No. PTA-1622
7H9-1 Accession No. PTA-1623
67 H1 Accession No. PTA-1621
Mutant strains of P. syringae are also suitable for
production of one or more pseudomycins. As used herein,
"mutant" refers to a sudden heritable change in the
phenotype of a strain, which can be spontaneous or induced
by known mutagenic agents, such as radiation (e. g.,
ultraviolet radiation or x-rays), chemical mutagens (e. g.,
ethyl methanesulfonate (EMS), diepoxyoctane, N-methyl-N-
nitro-N'-nitrosoguanine (NTG), and nitrous acid), site-
specific mutagenesis, and transposon mediated mutagenesis.
Pseudomycin-producing mutants of P. syringae can be
produced by treating the bacteria with an amount of a
mutagenic agent effective to produce mutants that
overproduce one or more pseudomycins, that produce one
pseudomycin (e. g., pseudomycin B) in excess over other
pseudomycins, or that produce one or more pseudomycins
under advantageous growth conditions. While the type and
amount of mutagenic agent to be used can vary, a preferred
method is to serially dilute NTG to levels ranging from 1
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to 100 ~g/ml. Preferred mutants are those that overproduce
pseudomycin B and grow in minimal defined media.
Environmental isolates, mutant strains, and other
desirable strains of P. syringae can be subjected to
selection for desirable traits of growth habit, growth
medium nutrient source, carbon source, growth conditions,
amino acid requirements, and the like. Preferably, a
pseudomycin producing strain of P. syringae is selected for
growth on minimal defined medium such as N21 medium and/or
for production of one or more pseudomycins at levels
greater than about 10 ~,g/ml. Preferred strains exhibit the
characteristic of producing one or more pseudomycins when
grown on a medium including three or fewer amino acids and
optionally, either a lipid, a potato product or combination
thereof.
Recombinant strains can be developed by transforming
the P. syringae strains, using procedures known in the art.
Through the use of recombinant DNA technology, the P.
syringae strains can be transformed to express a variety of
gene products in addition to the antibiotics these strains
produce. For example, one can modify the strains to
introduce multiple copies of the endogenous pseudomycin-
biosynthesis genes to achieve greater pseudomycin yield.
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To produce one or more pseudomycins from a wild type
or mutant strain of P. syringae, the organism is cultured
with agitation in an aqueous nutrient medium including an
effective amount of three or fewer amino acids, preferably
glutamic acid, glycine, histidine, or a combination
thereof. Alternatively, glycine is combined with one or
more of a potato product and a lipid. Culturing is
conducted under conditions effective for growth of P.
syringae and production of the desired pseudomycin or
pseudomycins. Effective conditions include temperatures
from about 22°-C to about 27°-C, and a duration of about 36
hours to about 96 hours. Controlling the concentration of
oxygen in the medium during culturing of P. syringae is
advantageous for production of a pseudomycin. Preferably,
oxygen levels are maintained at about 5 to 50o saturation,
more preferably about 30o saturation. Sparging with air,
pure oxygen, or gas mixtures including oxygen can regulate
the concentration of oxygen in the medium.
Controlling the pH of the medium during culturing of
P. syringae is also advantageous. Pseudomycins are labile
at basic pH, and significant degradation can occur if the
pH of the culture medium is above about 6 for more than
about 12 hours. Preferably, the pH of the culture medium
is maintained between 6 and 4. P. syringae can produce one
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or more pseudomycins when grown in batch culture. However,
fed-bath or semi-continuous feed of glucose and optionally,
an acid or base (e.g., ammonium hydroxide) to control pH,
enhances production. Pseudomycin production can be further
enhanced by using continuous culture methods in which
glucose and ammonium hydroxide are fed automatically.
Choice of P. syringae strain can affect the amount and
distribution of pseudomycin or pseudomycins produced. For
example, strains MSU 16H and 67 H1 each produce
predominantly pseudomycin A, but also produce pseudomycin B
and C, typically in ratios of 4:2:1. Strain 67 H1
typically produces levels of pseudomycins about three to
five fold larger than are produced by strain MSU 16H.
Compared to strains MSU 16H and 67 H1, strain 25-B1
produces more pseudomycin B and less pseudomycin C. Strain
7H9-1 are distinctive in producing predominantly
pseudomycin B and larger amount of pseudomycin B than other
strains. For example, this strain can produce pseudomycin
B in at least a ten fold excess over either pseudomycin A
or C.
Alternatively, the amine-modified pseudomycin
compounds of the present invention can be formed from an N-
acyl semi-synthetic compound. Semi-synthetic pseudomycin
compounds may be synthesized by exchanging the N-acyl group
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on the L-serine unit. Examples of various N-acyl
derivatives are described in PCT Patent Application Serial
No. , Belvo, et al., filed evendate herewith
entitled "Pseudomycin N-Acyl Side-Chain Analogs" and
incorporated herein by reference. In general, four
synthetic steps are used to produce the semi-synthetic
compounds from naturally occurring pseudomycin compounds:
(1) selective amino protection; (2) chemical or enzymatic
deacylation of the N-acyl side-chain; (3) reacylation with
a different side-chain; and (4) deprotection of the amino
groups.
The pendant amino groups at positions 2, 4 and 5 may
be protected using any standard means known to those
skilled in the art for amino protection. The exact genus
and species of amino protecting group employed is not
critical so long as the derivatized amino group is stable
to the condition of subsequent reactions) on other
positions of the intermediate molecule and the protecting
group can be selectively removed at the appropriate point
without disrupting the remainder of the molecule including
any other amino protecting group(s). Suitable amino-
protecting groups include benzyloxycarbonyl, p-
nitrobenzyloxycarbonyl,
p-bromobenzyloxycarbonyl, p-methoxybenxyloxycarbonyl,
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p-methoxyphenylazobenzyloxycarbonyl,
p-phenylazobenzyloxycarbonyl, t-butyloxycarbonyl,
cyclopentyloxycarbonyl, and phthalimido. Preferred amino
protecting groups are t-butoxycarbonyl (t-Boc),
allyloxycarbonyl (Alloc), phthalimido, and
benzyloxycarbonyl (CbZ or CBZ). Further examples of
suitable protecting groups are described in T.W. Greene,
"Protective Groups in Organic Synthesis," John Wiley and
Sons, New York, N.Y., (2nd ed., 1991), at chapter 7.
The deacylation of an N-acyl group having a gamma or
delta hydroxylated side chain (e. g., 3,4-dihydroxytetra-
deconoate) may be accomplished by treating the amino-
protected pseudomycin compound with acid in an aqueous
solvent. Suitable acids include acetic acid and
trifluoroacetic acid. A preferred acid is trifluoroacetic
acid. If trifluoroacetic acid is used, the reaction may be
accomplished at or near room temperature. However, when
acetic acid is used the reaction is generally ran at about
40°C. Suitable aqueous solvent systems include
acetonitrile, water, and mixtures thereof. Organic
solvents accelerate the reaction; however, the addition of
an organic solvent may lead to other by-products.
Pseudomycin compounds lacking a delta or gamma hydroxy
group on the side chain (e.g., Pseudomycin B and C') may be
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deacylated enzymatically. Suitable deacylase enzymes
include Polymyxin Acylase (164-16081 Fatty Acylase (crude)
or 161-16091 Fatty Acylase (pure) available from Wako Pure
Chemical Industries, Ltd.), or ECB deacylase. The
enzymatic deacylation may be accomplished using standard
deacylation procedures well known to those skilled in the
art. For example, general procedures for using polymyxin
acylase may be found in Yasuda, N., et al, Agric. Biol.
Chem., 53, 3245 (1989) and Kimura, Y., et al., Agric. Biol.
Chem., 53, 497 (1989).
The deacylated product (also known as the pseudomycin
nucleus) is reacylated using the corresponding acid of the
desired acyl group in the presence of a carbonyl activating
agent. "Carbonyl activating group" refers to a substituent
of a carbonyl that promotes nucleophilic addition reactions
at that carbonyl. Suitable activating substituents are
those which have a net electron withdrawing effect on the
carbonyl. Such groups include, but are not limited to,
alkoxy, aryloxy, nitrogen containing aromatic heterocycles,
or amino groups (e. g., oxybenzotriazole, imidazolyl,
nitrophenoxy, pentachlorophenoxy, N-oxysuccinimide, N,N'-
dicyclohexylisoure-O-yl, and N-hydroxy-N-methoxyamino);
acetates; formates; sulfonates (e. g., methanesulfonate,
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ethanesulfonate, benzenesulfonate, and p-tolylsulfonate);
and halides (e. g., chloride, bromide, and iodide).
A variety of acids may be used in the acylation
process. Suitable acids include aliphatic acids containing
one or more pendant aryl, alkyl, amino(including primary,
secondary and tertiary amines), hydroxy, alkoxy, and amido
groups; aliphatic acids containing nitrogen or oxygen
within the aliphatic chain; aromatic acids substituted with
alkyl, hydroxy, alkoxy and/or alkyl amino groups; and
heteroaromatic acids substituted with alkyl, hydroxy,
alkoxy and/or alkyl amino groups.
Alternatively, a solid phase synthesis may be used
where a hydroxybenzotriazole-resin (HOBt-resin) serves as
the coupling agent for the acylation reaction.
Once the amino group is deacylated and reacylated
(described above), then the amino protecting groups (at
positions 2, 4 and 5) can be removed by hydrogenation in
the presence of a hydrogenation catalyst (e. g., 10o Pd/C).
Tn~hen the amino protecting group is allyloxycarbonyl, then
the protecting group can be removed using
tributyltinhydride and triphenylphosphine palladium
dichloride. This particular protection/deprotection scheme
has the advantage of reducing the potential for
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hydrogenating the vinyl group of the Z-Dhb unit of the
pseudomycin structure.
The amine-modification of the N-acyl semi-synthetic
compound is then accomplished by acylating at least one of
the pendant amino groups attached to the lysine or 2,4-
diaminobutyric acid peptide units of the N-acyl modified
semi-synthetic pseudomycin compound to form the desired
amide, urea, carbamate or imide linkage.
The amine-modified pseudomycin compounds may be
further modified by amidation or esterification of the
pendant carboxylic acid group of the aspartic acid and/or
hydroxyaspartic acid units of the pseudomycin ring.
Examples of various acid-modified derivatives are described
in PCT Patent Application Serial No. , Chen, et al.,
filed evendate herewith entitled "Pseudomycin Amide & Ester
Analogs" and incorporated herein by reference. The acid-
modified derivatives may be formed by condensing any of the
previously described amine-modified pseudomycin compounds
with the appropriate alcohol or amine to produce the
respective ester or amide.
Formation of the ester groups may be accomplished
using standard esterification procedures well-known to
those skilled in the art. Esterification under acidic
conditions typically includes dissolving or suspending the
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pseudomycin compound in the appropriate alcohol in the
presence of a protic acid (e. g., HCl, TFA, etc.). Under
basic conditions, the pseudomycin compound is generally
reacted with the appropriate alkyl halide in the presence
of a weak base (e. g., sodium bicarbonate and potassium
carbonate).
Formation of the amide groups may be accomplished
using standard amidation procedures well-known to those
skilled in the art. However, the choice of coupling agents
provides selective modification of the acid groups. For
example, the use of benzotriazol-1-yloxy-
tripyrrolidinophosphonium hexafluorophosphate(PyBOP) as the
coupling agent allows one to isolate pure mono-amides at
residue 8 and (in some cases) pure bis amides
simultaneously. Whereas, the use of o-benzotriazol-1-yl-
N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) as
the coupling agent favors formation of monoamides at
residue 3.
The pseudomycin amide derivatives may be isolated and
used per se or in the form of its pharmaceutically
acceptable salt or solvate. The term "pharmaceutically
acceptable salt" refers to non-toxic acid addition salts
derived from inorganic and organic acids. Suitable salt
derivatives include halides, thiocyanates, sulfates,
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bisulfates, sulfites, bisulfites, arylsulfonates,
alkylsulfates, phosphonates, monohydrogen-phosphates,
dihydrogenphosphates, metaphosphates, pyrophosphonates,
alkanoates, cycloalkylalkanoates, arylalkonates, adipates,
alginates, aspartates, benzoates, fumarates,
glucoheptanoates, glycerophosphates, lactates, maleates,
nicotinates, oxalates, palmitates, pectinates, picrates,
pivalates, succinates, tartarates, citrates, camphorates,
camphorsulfonates, digluconates, trifluoroacetates, and the
like.
The term "solvate" refers to an aggregate that
comprises one or more molecules of the solute (i.e., amine-
modified pseudomycin compound) with one or more molecules
of a pharmaceutical solvent, such as water, ethanol, and
the like. When the solvent is water, then the aggregate is
referred to as a hydrate. Solvates are generally formed by
dissolving the pseudomycin derivative in the appropriate
solvent with heat and slowing cooling to generate an
amorphous or crystalline solvate form.
Each pseudomycin compound, semi-synthetic pseudomycin
derivatives, and mixtures can be detected, determined,
isolated, and/or purified by any variety of methods known
to those skilled in the art. For example, the level of
pseudomycin or amine-modified pseudomycin activity in a
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broth or in an isolate or purified composition can be
determined by antifungal action against a fungus such as
Candida and can be isolated and purified by high
performance liquid chromatography.
The active ingredient (i.e., pseudomycin compound of
the present invention) is typically formulated into
pharmaceutical dosage forms to provide an easily
controllable dosage of the drug and to give the patient,
physician, or veterinarian an elegant and easy to handle
product. Formulations may comprise from 0.1o to 99.90 by
weight of active ingredient, more generally from about 100
to about 30o by weight.
As used herein, the term "unit dose" or "unit dosage"
refers to physically discrete units that contain a
predetermined quantity of active ingredient calculated to
produce a desired therapeutic effect. When a unit dose is
administered orally or parenterally, it is typically
provided in the form of a tablet, capsule, pill, powder
packet, topical composition, suppository, wafer, measured
units in ampoules or in multidose containers, etc.
Alternatively, a unit dose may be administered in the form
of a dry or liquid aerosol which may be inhaled or sprayed.
The dosage to be administered may vary depending upon
the physical characteristics of the animal, the severity of
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the animal's symptoms, the means used to administer the
drug and the animal species. The specific dose for a given
animal is usually set by the judgment of the attending
physician or veterinarian.
Suitable carriers, diluents and excipients are well
known to those skilled in the art and include materials
such as carbohydrates, waxes, water soluble and/or
swellable polymers, hydrophilic or hydrophobic materials,
gelatin, oils, solvents, water, and the like. The
particular carrier, diluent or excipient used will depend
upon the means and purpose for which the active ingredient
is being applied. The formulations may also include
wetting agents, lubricating agents, surfactants, buffers,
tonicity agents, bulking agents, stabilizers, emulsifiers,
suspending agents, preservatives, sweeteners, perfuming
agents, flavoring agents and combinations thereof.
A pharmaceutical composition may be administered using
a variety of methods. Suitable methods include topical
(e.g., ointments or sprays), oral, injection and
inhalation. The particular treatment method used will
depend upon the type of infection being addressed.
In parenteral iv applications, the formulations are
typically diluted or reconstituted (if freeze-dried) and
further diluted if necessary, prior to administration. An
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example of reconstitution instructions for the freeze-dried
product are to add ten ml of water for injection (WFI) to
the vial and gently agitate to dissolve. Typical
reconstitution times are less than one minute. The
resulting solution is then further diluted in an infusion
solution such as dextrose 5o in water (D5W), prior to
administration.
Pseudomycin compounds have been shown to exhibit
antifungal activity such as growth inhibition of various
infectious fungi including Candida spp. (i.e., C. albicans,
C. parapsilosis, C. krusei, C. glabrata, C. tropicalis, or
C. lusitaniaw); Torulopus spp.(i.e., T. glabrata);
Aspergillus spp. (i.e., A. fumigatus); Histoplasma spp.
(i.e., H. capsulatum); Cryptococcus spp. (i.e., C.
neoformans); Blastomyces spp. (i.e., B. dermatitidis);
Fusarium spp.; Trichophyton spp., Pseudallescheria boydii,
Coccidioides immits, Sporothrix schenckii, etc.
Consequently, the compounds and formulations of the
present invention are useful in the preparation of
medicaments for use in combating either systemic fungal
infections or fungal skin infections. Accordingly, a
method is provided for inhibiting fungal activity
comprising contacting the amine-modified pseudomycin
compound of the present invention with a fungus. A
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preferred method includes inhibiting Candida albicans or
Aspergillus fumigatus activity. The term "contacting"
includes a union or junction, or apparent touching or
mutual tangency of a compound of the invention with a
fungus. The term does not imply any further limitations to
the process, such as by mechanism of inhibition. The
methods are defined to encompass the inhibition of fungal
activity by the action of the compounds and their inherent
antifungal properties.
A method for treating a fungal infection which
comprises administering an effective amount of a
pharmaceutical formulation of the present invention to a
host in need of such treatment is also provided. A
preferred method includes treating a Candida albicans or
Aspergillus fumigatus infection. The term "effective
amount" refers to an amount of active compound 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, the tolerance of the host to the
antifungal agent and the species of the host. The
particular dose regimen likewise may vary according to
these factors. The medicament may be given in a single
daily dose or in multiple doses during the day. The
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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) contains a dosage level between about 0.01
mg/kg to 100 mg/kg of body weight of an active compound.
Preferred daily doses are generally between about 0.1 mg/kg
to 60 mg/kg and more preferably between about 2.5 mg/kg to
40 mg/kg. The host is generally an animal including
humans, companion animals (e. g., dogs, cats and horses),
food-source animals (e. g., cows, pigs, sheep and poultry),
zoo animals, marine animals, birds and other similar animal
species.
EXAMPLES
The following abbreviations are used through out the
examples to represent the respective listed materials:
ACN - acetonitrile
TFA - trifluoroacetic acid
DMF - dimethylformamide
EDCI - 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride
BOC = t-butoxycarbonyl, (CH3)3C-O-C(O)-
CBZ = benzyloxycarbonyl , C6HSCH2-O-C ( 0 ) -
PyBOP = benzotriazol-1-yloxy-tripyrrolidinophosphonium
hexafluorophosphate
TBTU = o-Benzotriazol-1-yl-N,N,N',N'-
tetramethyluronium
tetrafluoroborate
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DIEA = N,N-diisopropylethylamine
The following structure II will be used to describe the
products observed in Examples 1 through 7.
R'.~.HN
N
O O O ,,.N n
N N N
O '
Rz NHR'
R'~~HN O
II
Detection and Quantification of An ti fungal Activity:
Antifungal activity was determined in vitro by
obtaining the minimum inhibitory concentration (MIC) of the
compound using a standard agar dilution test or a disc-
diffusion test. A typical fungus employed in testing
antifungal activity is Candida albicans. Antifungal
activity is considered significant when the test sample (50
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~,l) causes 10-12 mm diameter zones of inhibition on C.
albicans x657 seeded agar plates.
Tail Vein Toxi ci ty:
Mice were treated intravenously (IV) through the
lateral tail vein with 0.1 ml of testing compound (20
mg/kg) at 0, 24, 48 and 72 hours. Two mice were included
in each group. Compounds were formulated in 5.0o dextrose
and sterile water for injection. The mice were monitored
for 7 days following the first treatment and observed
closely for signs of irritation including erythema,
swelling, discoloration, necrosis, tail loss and any other
signs of adverse effects indicating toxicity.
The mice used in the study were outbred, male ICR mice
having an average weight between 18-20 g (available from
Harlan Sprangue Dawley, Indianapolis, IN).
Preparations
Compound 1a-2:
O O
/ O N
O-N
O
O
1a-1
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Compound 1a-1 is commercially available from Novabiochem
(San Diego, CA).
Preparation of Compound 2a-1:
O
N~N~O
N-N
2a-1
Compound 2a-1 is prepared using the procedures
described in Admiak, R.W., et al., Tetrahedron Lett., No.
22, 1935-1936 (1997).
In each of the following Examples a specific
pseudomycin compound is used as the starting material;
however, those skilled in art in the art will recognize
that other N-acyl derivatives may be synthesized using the
same procedures except starting with a pseudomycin compound
having a different N-acyl group.
Example 1
Example 1 illustrates the formation of acylalkylamine
derivatives of pseudomycin B (n = 10, Rz and R3 - -OH).
Synthesis of Compound 1-2:
R1. ~ Ri , , and R1 . , , _ _ C ( O ) CH2NH2
1-1
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A 50 ml round bottom flask was charged with 10 ml of
anhydrous DMF, pseudomycin B (250.6 mg, 0.181 mmol) and the
acylating agent la-1 (343.0 mg, 1.12 mmol). The reaction
was allowed to stir at room temperature for 24 hours. The
solvent was then removed in vacuo and the residue was taken
up in ACN and purified via preparatory HPLC to yield 172.5
mg of the tri-substituted protected amine after
lyophilization. 134.4 mg of tri-substituted protected
amine was dissolved in a solution of 10 ml MeOH/1.5 ml
glacial AcOH. Standard hydrogenolysis using 129.6 mg of
10o Pd/C for 20 minutes, removal of the catalyst via
filtration and purification via preparatory HPLC yielded
74.8 mg of Compound 1-1 after lyophilization. MS
(Ionspray) calcd for C57H97C1N15O22 (M+H)+ 1378.65, found
1378.9.
The following pseudomycin amide derivatives (1-2 and
1-3) may be synthesized using the same procedures as
described above and the appropriate amino acid to form the
acylating agent.
O
NH2
Rl . ~ Rl , . ~ and Ri , . ,
1-2
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O
NH2
Rl , ~ Rl , . ~ and Rl , , .
1-3
Exempla 2
Example 2 illustrates the synthesis of acyloxyaryl
derivatives of pseudomycin B(n = 10, RZ and R3 - -OH).
Synthesis of Compound 2-1:
O
R1, ~ R1. . ~ and Ri . , . - ~O
2-1
Compound 2-1 is synthesized using the same procedures
as described in Example 1 except Compound 2a-1 is used as
the acylating agent.
Compound 2-1 may be alternatively synthesized by
adding phenyl chloroformate (389 mg, 2.48 mmol) to a
solution of HOBT (37.5 mg, 2.48 mmol) and DIEA (322.8 mg,
399 ml, 2.48 mmol) at 0-4°C. The mixture is diluted with
100 ml DMF and pseudomycin B (1.0 g, 0.83 mmol) is added.
The mixture is allowed to stir overnight. The solvent is
then removed in vacuo and the residue purified by HPLC to
yield 430 mg (33% yield) of Compound 2-1.
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Example 3
Example 3 illustrates the synthesis of acylalkyl
derivatives of pseudomycin B(n = 10, Rz and R3 - -OH).
Synthesis of Compound 3-2:
O
R1, ~ R1, , ~ and Ri , . . -
_CH
3
3-1
Compound 3-1 is synthesized using the same procedures
as described in Example 1 except acetic anhydride is used
as the acylating agent.
Synthesis of Compound 3-2:
O
R1. ~ Rl . . ~ and Rl . , , - ~
_C ( CH )
3 3
3-2
Compound 3-2 is synthesized using the same procedures
as described in Example 1 except trimethylacetic anydride
is used as the acylating agent.
Example 4
Example 4 illustrates the synthesis of acylazaalkyl
(i.e., urea linkage) derivatives of pseudomycin B(n = 10, RZ
and R3 - -OH ) .
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Synthesis of Compound 4-2:
O
R1, ~ R1, . ~ and R1, , . - ~
" _NHCH
3
4-1
Compound 4-1 is synthesized using the same procedures
as described in Example 1 except methyl isocyanate is used
as the acylating agent.
example 5
Example 5 illustrates the synthesis of a formyl
derivative of pseudomycin B(n = 10, Rz and R3 - -OH).
O
R1, ~ R1, . ~ and R1, , . -
H
5-1
Compound 5-1 is synthesized using the same procedures
as described in Example 1 except 4-nitrophenylformate is
used as the acylating agent.
Example 6
Example 6 illustrates the synthesis of an
acyloxyalkenyl derivative of pseudomycin B(n = 10, Rz and R3
- -OH ) .
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O
Rl ' , Rl ' ' , and R1, .
O
6-1
Compound 6-1 is synthesized using the same procedures
as described in Example 1 except diallylpyrocarbonate is
used as the acylating agent. Yield 770
37
