PSEUDOMYCIN AMIDE AND ESTER ANALOGS

ANALOGUES AMIDES ET ESTERS DE PSEUDOMYCINE

English Abstract

Acid-modification of the aspartic acid and/or hydroxyaspartic acid units of
naturally occurring or semi-synthetic pseudomycin compounds is described as
well as methods of treatment against fungal activities.

French Abstract

Cette invention concerne la modification acide d'unités d'acide aspartique et/ou d'acide hydro-aspartique d'un composé de pseudomycine naturel ou semi-synthétique ainsi que des méthodes de traitement antifongiques.

Claims

WE CLAIM:
1. A pseudomycin compound having the following
structure I
<IMG>
wherein
R is
<IMG>
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
53

double bond, or taken together with Rc forms a
six-membered aromatic ring;
Rb and Rb' are independently hydrogen,
halogen, or methyl, or either Rb or Rb' is amino,
alkylamino, a-acetoacetate, methoxy, or hydroxy;
Rc is hydrogen, hydroxy, C1-C4 alkoxy,
hydroxy(C1-C4)alkoxy, or taken together with Re
forms a 6-membered aromatic ring or C5-C6
cycloalkyl ring;
Re is hydrogen, or taken together with Rf is
a six-membered aromatic ring, C5-C14 alkoxy
substituted six-membered aromatic ring, or C5-C14
alkyl substituted six-membered aromatic ring, and
Rf is C8-C18 alkyl, or C5-C11 alkoxy;
R is
<IMG>
where
Rg is hydrogen, or C1-C13 alkyl, and
Rh is C1-C15 alkyl, C4-C15 alkoxy, (C1-C10)
alkyl)phenyl, -(CH2)n-aryl, or -(CH2)n-(C5-C6
cycloalkyl), where n = 1 or 2; or
R is
54

<IMG>
where
R1 is a hydrogen, halogen, or C5-C8 alkoxy,
and m is 1, 2 or 3;
R is
<IMG>
where
Rj is C5-C14 alkoxy or C5-C14 alkyl, and
p = 0, 1 or 2;
R is
<IMG>
where
Rk is C5-C14 alkoxy; or
R is -(CH2)-NRm-(C13-C18 alkyl), where Rm is H, -CH3 or
-C(O)CH3;
R1 is independently -NH2 or -NHp-Pg, where p is 0 or 1;
R2 and R3 are independently -OR2a, or -N(R2b) (R2c),
where
55

R2a and R2b are independently hydrogen, C1-C10
alkyl, C3-C6 cycloalkyl, hydroxy(C1-C10)alkyl,
alkoxy (C1-C10)alkyl, C2-C10 alkenyl, amino(C1-
C10)alkyl, mono- or di-alkylamino(C1-C10)alkyl,
aryl(C1-C10)alkyl, heteroaryl(C1-C10)alkyl, or
cycloheteroalkyl (C1-C10)alkyl, or
R2b is an alkyl carboxylate residue of an
aminoacid alkyl ester, and
R2c is hydrogen or C1-C6 alkyl,
provided that both R2 and R3 are not -OH; and
pharmaceutically acceptable salts and solvates thereof.
2. A pseudomycin prodrug having the following
structure
<IMG>
wherein
R is
56

<IMG>
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 Rc forms a
six-membered aromatic ring;
Rb and Rb' are independently hydrogen,
halogen, or methyl, or either Rb or Rb' is amino,
alkylamino, .alpha.-acetoacetate, methoxy, or hydroxy;
Rc is hydrogen, hydroxy, C1-C4 alkoxy,
hydroxy(C1-C4)alkoxy, or taken together with Re
forms a 6-membered aromatic ring or C5-C6
cycloalkyl ring;
Re is hydrogen, or taken together with Rf is
a six-membered aromatic ring, C5-C14 alkoxy
substituted six-membered aromatic ring, or C5-C14
alkyl substituted six-membered aromatic ring, and
Rf is C8-C18 alkyl, C5-C11 alkoxy or biphenyl;
R is
57

<IMG>
where
Rg is hydrogen, or C1-C13 alkyl, and
Rh is C1-C15 alkyl, C4-C15 alkoxy, (C1-C10
alkyl)phenyl, -(CH2)n-aryl, or -(CH2)n-(C5-C6
cycloalkyl), where n = 1 or 2; or
R is
<IMG>
where
R1 is a hydrogen, halogen, or C5-C8 alkoxy,
and m is 1, 2 or 3;
R is
<IMG>
where
Rj is C5-C14 alkoxy or C5-C14 alkyl, and
p = 0, 1 or 2;
R is
58

<IMG>
where
Rk is C5-C14 alkoxy; or
R is -(CH2)-NRm-(C13-C18 alkyl), where Rm is H, -CH3 or
-C(O)CH3;
R1 is independently -NH2 or -NHp-Pg, where p is 0 or 1;
R2 and R3 are -OR2a, where R2a is C1-C3 alkyl; and
pharmaceutically acceptable salts and solvates thereof.
3. A 3-amido derivative of a pseudomycin compound
prepared by the steps of
(i) providing a pseudomycin compound having the
following structure
<IMG>
59

wherein
R is
<IMG>
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 Rc 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;
Rc is hydrogen, hydroxy, C1-C4 alkoxy,
hydroxy(C1-C4)alkoxy, or taken together with
Re forms a 6-membered aromatic ring or C5-C6
cycloalkyl ring;
Re is hydrogen, or taken together with
Rf is a six-membered aromatic ring, C5-C14
alkoxy substituted six-membered aromatic
60

ring, or C5-C14 alkyl substituted six-membered
aromatic ring, and
Rf is C6-C18 alkyl, C5-C11 alkoxy or
biphenyl;
R is
<IMG>
where
Rg is hydrogen, or C1-C13 alkyl, and
Rh is C1-C15 alkyl, C4-C15 alkoxy, (C1-C10
alkyl)phenyl, -(CH2)n-aryl, or -(CH2)n-(C5-C6
cycloalkyl), where n = 1 or 2; or
R is
<IMG>
where
R1 is a hydrogen, halogen, or C5-C8
alkoxy, and m is 1, 2 or 3;
R is
61

<IMG>
where
Rj is C5-C14 alkoxy or C5-C14 alkyl, and
p = 0, 1 or 2;
R is
<IMG>
where
Rk is C5-C14 alkoxy; or
R i s -(CH2) -NRm-(C13-C18 alkyl), where Rm i s H, -CH3
or -C(C)CH3;
R1 is -NH2;
R2 and R3 are -OH; and
pharmaceutically acceptable salts and solvates
thereof;
(ii) protecting the amino groups, R1, at positions 2, 4
and 5 with an amino-protecting group;
(iii)forming an amide linkage at position 3 using o-
benzotriazol-1-yl-N,N,N',N'-tetramethyluronium
tetrafluoroborate or 2(1H-benzotriazole-1-yl)-
1,1,3,3,-tetramethyluronium hexafluorophosphate as
a coupling agent;
62

(iv) removing said amino-protecting groups.
4. The 3-amido derivative of Claim 3 wherein step
(iii) forming an amide linkage is accomplished in the
presence of a bulky amine.
5. The 3-amido derivative of Claim 3 wherein step
(iii) forming an amide linkage is accomplished in the
presence of a bulky amine and at a temperature between about
0°C and -20°C .
6. An 8-amido derivative of a pseudomycin compound
prepared by the steps of
(i) providing a pseudomycin compound having the
following structure
<IMG>
63

I
wherein
R is
<IMG>
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 Rc forms a six-membered
aromatic ring;
Rb and Rb' are independently hydrogen,
halogen, or methyl, or either Rb or Rb' is
amino, alkylamino, .alpha.-acetoacetate, methoxy,
or hydroxy;
Rb is hydrogen, hydroxy, C1-C4 alkoxy,
hydroxy(C1-C4)alkoxy, or taken together with
Re forms a 6-membered aromatic ring or C5-C6
cycloalkyl ring;
Re is hydrogen, or taken together with
Rf is a six-membered aromatic ring, C5-C14
64

alkoxy substituted six-membered aromatic
ring, or C5-C14 alkyl substituted six-membered
aromatic ring, and
Rf is C6-C18 alkyl, C5-C11 alkoxy or
biphenyl;
R is
<IMG>
where
Rg is hydrogen, or C1-C13 alkyl, and
Rh i s C1-C15 alkyl , C4-C15 alkoxy, ( C1-C10
alkyl)phenyl, -(CH2)n-aryl, or -(CH2)n-(C5-C6
cycloalkyl), where n = 1 or 2; or
R is
<IMG>
where
R1 is a hydrogen, halogen, or C5-C8
alkoxy, and m is 1, 2 or 3;
R is
65

<IMG>
where
R j is C5-C14 alkoxy or C5-C14 alkyl, and
p = 0, 1 or 2;
R is
<IMG>
where
R k is C5-C14 alkoxy; or
R is -(CH2)-NR m-(C13-C18 alkyl), where R m is H, -CH3
or -C (C)CH3;
R1 is -NH2;
R2 and R3 are -OH; and
pharmaceutically acceptable salts and solvates
thereof;
(ii) protecting the amino groups at positions 2, 4
and 5 with an amino-protecting group;
(iii) forming an amide linkage at position 8 using
benzotriazol-1-yloxy-tripyrrolidinophosphonium
hexafluorophosphate as a coupling agent;
(iv) removing said amino-protecting groups.
66

7. The use of a compound as claimed in any one of the
preceding claims in the preparation of a medicament for use
in combating either systemic fungal infections or fungal
skin infections.
8. A process for making a 3-amido derivative of a
pseudomycin compound comprising the steps of
(i) providing a pseudomycin compound having the
following structure
<IMG>
wherein
R is
67

<IMG>
where
R a and R a' are independently hydrogen or
methyl, or either R a or R a' is alkyl amino,
taken together with R b or R b' forms a six-
membered cycloalkyl ring, a six-membered
aromatic ring or a double bond, or taken
together with R c forms a six-membered
aromatic ring;
R b and R b' are independently hydrogen,
halogen, or methyl, or either R b or R b' is
amino, alkylamino, .alpha.-acetoacetate, methoxy,
or hydroxy;
R c is hydrogen, hydroxy, C1-C4 alkoxy,
hydroxy(C1-C4)alkoxy, or taken together with
R e forms a 6-membered aromatic ring or C5-C6
cycloalkyl ring;
R e is hydrogen, or taken together with
R f is a six-membered aromatic ring, C5-C14
alkoxy substituted six-membered aromatic
ring, or C5-C14 alkyl substituted six-membered
aromatic ring, and
68

R f is C6-C18 alkyl, C5-C11 alkoxy or
biphenyl;
R is
<IMG>
where
R g is hydrogen, or C1-C13 alkyl, and
R h is C1-C15 alkyl, C4-C15 alkoxy, (C1-C10
alkyl)phenyl, -(CH2)n-aryl, or -(CH2)n-(C5-C6
cycloalkyl), where n = 1 or 2; or
R is
<IMG>
where
R i is a hydrogen, halogen, or C5-C8
alkoxy, and m is 1, 2 or 3;
R is
<IMG>
where
69

R j is C5-C14 alkoxy or C5-C14 alkyl, and
p = 0, 1 or 2;
R is
<IMG>
where
R k is C5-C14 alkoxy; or
R is -(CH2)-NR m-(C13-C18 alkyl), where R m is H, -CH3
or -C(C)CH3;
R1 is -NH2;
R2 and R3 are -OH; and
pharmaceutically acceptable salts and solvates
thereof;
(ii) protecting the amino groups, R1, at positions 2, 4
and 5 with an amino-protecting group;
(iii)forming an amide linkage at position 3 using o-
benzotriazol-1-yl-N,N,N',N'-tetramethyluronium
tetrafluoroborate or 2(1H-benzotriazole-1-yl)-
1,1,3,3,-tetramethyluronium hexafluorophosphate as
a coupling agent in the presence of a bulky amine
and at a temperature between about 0°C and -20°C;
(iv) removing said amino-protecting groups.
70

9. A process for making an 8-amido derivative of a
pseudomycin compound comprising the steps of
(ii) providing a pseudomycin compound having the
following structure
<IMG>
wherein
R is
<IMG>
where
R a and R a' are independently hydrogen or
methyl, or either R a or R a' is alkyl amino,
taken together with R b or R b' forms a six-
71

membered cycloalkyl ring, a six-membered
aromatic ring or a double bond, or taken
together with R c forms a six-membered
aromatic ring;
R b and R b' are independently hydrogen,
halogen, or methyl, or either R b or R b' is
amino, alkylamino, .alpha.-acetoacetate, methoxy,
or hydroxy;
R c is hydrogen, hydroxy, C1-C4 alkoxy,
hydroxy(C1-C4)alkoxy, or taken together with
R e forms a 6-membered aromatic ring or C5-C6
cycloalkyl ring;
R e is hydrogen, or taken together with
R f is a six-membered aromatic ring, C5-C14
alkoxy substituted six-membered aromatic
ring, or C5-C14 alkyl substituted six-membered
aromatic ring, and
R f is C6-C18 alkyl, C5-C11 alkoxy or
biphenyl;
R is
<IMG>
where
72

R g is hydrogen, or C1-C13 alkyl, and
R h is C1-C15 alkyl, C4-C15 alkoxy, (C1-C10
alkyl)phenyl, -(CH2)n-aryl, or -(CH2)n-(C5-C6
cycloalkyl), where n = 1 or 2; or
R is
<IMG>
where
R i is a hydrogen, halogen, or C5-C8
alkoxy, and m is 1, 2 or 3;
R is
<IMG>
where
R j is C5-C14 alkoxy or C5-C14 alkyl, and
p = 0, 1 or 2;
R is
<IMG>
where
R k is C5-C14 alkoxy; or
73

R is -(CH2)-NR m-(C13-C18 alkyl), where R m is H, -CH3
or -C(C)CH3;
R1 is -NH2;
R2 and R3 are -OH; and
pharmaceutically acceptable salts and solvates
thereof;
(ii) protecting the amino groups at positions 2, 4
and 5 with an amino-protecting group;
(iii) forming an amide linkage at position 8 using
benzotriazol-1-yloxy-tripyrrolidinophosphonium
hexafluorophosphate as a coupling agent;
(iv) removing said amino-protecting groups.
10. A pharmaceutical formulation comprising a compound
of Claim 1 and a pharmaceutically acceptable carrier.
11. A pharmaceutical formulation comprising a prodrug
of Claim 2 and a pharmaceutically acceptable carrier.
12. A method for treating an antifungal infection in
an aminal in need thereof, which comprises administering to
said animal a pseudomycin compound of Claim 1.
74

13. A method for treating an antifungal infection in
an animal in need thereof, which comprises administering to
said animal a prodrug of Claim 2.
75

Description

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PSEUDOMYCIN AMIDE & ESTER ANALOGS
FIELD OF THE INVENTION
The present invention relates to pseudomycin compounds,
in particular, acid-modified, semi-synthetic 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-C1) with the terminal carboxyl group
closing a macrocyclic ring on the OH group of the N-terminal

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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
this class of compounds for other potential compounds that
may be useful as antifungal agents having less adverse side
affects.
2

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BRIEF SLTt~lARY OF THE INVENTION
The present invention provides 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
R' O O O
NH
O O O N~R
H
N NH
O
RZ R'
R' O
I
wherein R is
b b~ d
R R R
r
R
a a,
~ ~
R R c e
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-
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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, a-
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 C5-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 CS-C14 alkyl substituted
six-membered aromatic ring, and
Rf is C6-C18 alkyl, C5-C11 alkoxy, or biphenyl;
R is
R9
Rn
where
Rg is hydrogen, or C1-C13 alkyl, and
Rh is C1-C15 alkyl, C4-C15 alkoxy, (C1-Clo
alkyl ) phenyl , - ( CH2 ) n-aryl , or - ( CHZ ) n- ( C5-Cs
cycloalkyl), where n = 1 or 2; or
R is
4

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R
m
where
R1 is a hydrogen, halogen, or CS-C8 alkoxy, and
m is 1, 2 or 3;
R is
OH
p R'
where
R' is C5-C14 alkoxy or C5-C14 alkyl, and p = 0, 1 or
2;
R is
-N
Rk
where
Rk is CS-C14 alkoxy; or
R is - (CHZ) -NRm- (C13-C1$ alkyl) , where Rm is H, -CH3 or
-C ( 0 ) CH3 ;
R1 is independently -NHz or -NHp-Pg, where p is 0 or 1;
RZ and R3 are independently -ORza, or -N(R2b) (R2~) ,
where
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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, n-amyl, i-amyl, n-hexyl, n-
heptyl, n-octyl, n-nonanyl, n-decyl, etc.), C3_C6
cycloalkyl (e. g., cyclopropyl, cyclobutyl, cyclopentyl,
cyclopentylmethyl, methylcyclopentyl, cyclohexyl, etc.)
haloalkyl ( a . g . , CF3CH2- ) , hydroxy ( C1-Clo ) alkyl ,
alkoxy ( C1-C1o ) alkyl ( a . g, methoxyethyl ) , al lyl , CZ-Clo
alkenyl , amino ( C1-Clo ) alkyl , mono- or di-alkylamino ( C1-
Clo ) alkyl , aryl ( C1-Clo ) alkyl ( a . 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
R2b is an alkyl carboxylate residue of an
aminoacid alkyl ester ( a . g . , -CHZCOZCH3 ,
-CH ( COzCH3 ) CH ( CH3 ) 2 , -CH ( C02CH3 ) CH ( phenyl ) ,
-CH ( COzCH3 ) CH20H, -CH ( COZCH3 ) CH2 (p-hydroxyphenyl ) ,
-CH ( COZCH3 ) CH2SH, -CH ( COZCH3 ) CHZ ( CHZ ) 3NH2 ,
-CH (COZCH3 ) CH2 (4- or 5-imidazole) , -CH (COZCH3 ) CHZCOzCH3,
-CH ( COZCH3 ) CH2C02NH2 , and the 1 ike ) , and
RZ° is hydrogen or C1-C6 alkyl,
provided that both Rz and R3 are not -OH; and
pharmaceutically acceptable salts and solvates thereof.
6

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In another embodiment of the present invention, a
prodrug of a pseudomycin compound is provided having
structure I represented above wherein Rz and R3 are
represented by -OR2a, where R2a is C1-C3 alkyl.
In yet another embodiment of the present invention, a
3-amido derivative of a pseudomycin compound is provided
where the compound is prepared by the steps of (i) providing
a compound having structure I above wherein R1 is -NHz and R2
and R3 are both -OH; (ii) protecting the amino groups, R1,
at positions 2, 4 and 5 with an amino-protecting group;
(iii) forming an amide linkage at position 3 using an o-
Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium
tetrafluoroborate as a coupling agent; and (iv) removing the
amino-protecting groups. An 8-amido derivative is also
provided where the derivative is prepared using the steps
described above except using benzotriazol-1-yloxy-
tripyrrolidinophosphonium hexafluorophosphate as the
coupling agent.
In another embodiment of the present invention, a
pharmaceutical formulation is provided which includes the
pseudomycin compound represented by structure I above and a
pharmaceutically acceptable carrier.
In yet another embodiment of the present invention, a
method is provided for treating an antifungal infection in
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an animal in need thereof, which comprises administering to
the animal the pseudomycin compound I described above.
Definitions
As used herein, the term "alkyl" refers to a
hydrocarbon radical of the general formula CnH2n+i 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, alkenoyl
or alkenoate group has the same definition as above.
The term "alkynyl" refers to an acyclic hydrocarbon
containing at least one carbon carbon triple bond. The
alkyne radical may be straight, or branched. The alkyne
radical may be substituted or unsubstituted. The alkynyl
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portion of an alkynoxy, alkynoyl or alkynoate 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.
The term "heteroaryl" refers to aromatic moieties
containing at least one heteratom within the aromatic ring
system (e. g., pyrrole, pyridine, indole, thiophene, furan,
benzofuran, imidazole, oxazine, pyrimidine, purine,
benzimidazole, quinoline, etc.). The aromatic moiety may
consist of a single or fused ring system. The heteroaryl
groups may be substituted or unsubstituted.
"NHp-Pg" and °'amino protecting group" refer to a
substituent of the amino group (Pg) commonly employed to
block or protect the amino functionality while reacting
other functional groups on the compound. When p is 0, the
amino protecting group, when taken with the nitrogen to
which it is attached, forms a cyclic imide, e.g.,
phthalimido and tetrachlorophthalimido. When p is 1, the
protecting group, when taken with the nitrogen to which it
is attached, can form a carbamate, e.g., methyl, ethyl, and
9-fluorenylmethylcarbamate; or an amide, e.g., N-formyl and
N-acetylamide.
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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
di-alkyl amino, quaternary ammonium salts, aminoalkoxy,
hydroxyalkylamino, aminoalkylthio, carbamyl, carbonyl,
carboxy, glycolyl, glycyl, hydrazino, guanyl, and
combinations thereof.
The term "solvate" refers to an aggregate that
comprises one or more molecules of the solute, such as a
compound of structure I, with one or more molecules of a

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pharmaceutical solvent, such as water, ethanol, and the
like.
The term "pharmaceutically acceptable salt" refers to
organic or inorganic salts of the compounds represented by
structure I that are substantially non-toxic to the
recipient at the doses administered.
The term "prodrug" refers to a class of drugs which
result in pharmacological action due to conversion by
metabolic processes within the body (i.e.,
biotransformation). In the present invention, the
pseudomycin prodrug compounds contain ester functionalities
that can be cleaved by esterases in the plasma to produce
the active drug.
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
acid functionality attached to the hydroxyaspartic acid
and/or aspartic acid units of a 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.
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fumigatus. Some bis-esters have been shown to act as a
prodrug; therefore, these particular compounds have reduced
in vitro activity but show in vivo efficacy.
Scheme I below illustrates the general procedures for
synthesizing Compound I from any one of the naturally
occurring pseudomycins or N-acyl modified derivatives. In
general, three synthetic steps are used to produce Compound
I: (1) selective amino protection; (2) condensation with the
appropriate alcohol or amine to produce the respective ester
or amide; and (4) deprotection of the amino groups.
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HO O
O OH HO
O OH
$ N OH O NH H N OH
HO,~
/ -NH O ,-,~CI (1~ HO~H O CI
_ ~ O
H PgHN ~O O
H
~N R O O H 0~~~~N R
N N~NH
H
NHZ
OH NHPg
H2N U PgHN O
(2)
3 O
R
O OH p OH
O NH H N OH O~H N OH
HO~ O CI HO~ NH O CI
~NH O~ ~ (3) _ ~ O
H N~O O ~ PgHN~O O
NH O O "'N R O NH O H O ~~~N R
O
H
H N~--!NH O
H N~-!NH O
~NH2 RZ ~NHPg
H2N O PgHN O
Scheme I
The pendant amino groups at residues 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 conditions
of subsequent reactions) on other positions of the
intermediate molecule and the protecting group can be
selectively removed at the appropriate point without
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disrupting the remainder of the molecule including any other
amino protecting group(s). Preferred amino protecting
groups are t-butoxycarbonyl (t-Boc), allyloxycarbonyl,
phthalimido, and benzyloxycarbonyl (CBZ). Most preferred is
allyloxycarbonyl (Alloc) and benzyloxycarbonyl (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.
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 pseudomycin
compound in the appropriate alcohol in the presence of a
protic acid (e. g., HC1, TFA, p-toluenesulfonic acid, 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 under
anhydrous conditions).
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
14

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to isolate pure mono-amides at residue 8 and (in some cases)
pure bis amides simultaneously. Whereas, coupling agents
such as o-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TBTU) and 2(1H-benzotriazole-1-yl)-
1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU) favor
formation of monoamides at residue 3.
Applicants also discovered that the addition of a bulky
amine enhances the ratio of monoamides at residue 3. The
ratio of amidation at residue 3 vs. residue 8 increased from
about 1:1 to about 6:1 and the amount of bis-amides was
reduced through the addition of a bulky amine. The term
"bulky amine" refers to an amine having multiple and/or
large substituents on the nitrogen atom. Any tertiary amine
may be used that is compatible with the reaction conditions.
Preferred bulky amines include N,N-diisopropylethylamine
(DIEA) and N-ethyldicyclohexylamine. The amount of bulky
amine added is generally from about 1 to 10 equivalents,
preferably 3 to 8 equivalents, more preferably 5 to 6
equivalents. The reaction is generally ran at temperatures
from about room temperature (25°C) to about -20°C. However,
Applicants discovered that lower temperatures (from about
0°C to about -20°C) further enhance the formation of
monoamides at residue 3. The ratio of amidation at residue
3 vs. residue 8 increased as much as 20:1 by adding a bulky
amine and lowering the temperature of the reaction.

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However, it will be understood by those skilled in the art
that the lower temperature limit will depend upon the
solubility of the reactive components.
Once the acid groups) are modified, then the amino
protecting groups (at positions 2, 4 and 5) may be removed
using standard procedures appropriate for the specific
protecting group used. For example, CBZ groups are removed
by hydrogenation in the presence of a hydrogenation catalyst
(e.g., 10o Pd/C). When the amino protecting group is
allyloxycarbonyl, then the protecting group may be removed
using tributyltinhydride and triphenylphosphine palladium
dichloride. This particular protection/deprotection scheme
has the advantage of reducing the potential for
hydrogenating the vinyl group of the Z-Dhb unit of the
pseudomycin structure.
As discussed earlier, pseudomycins are natural products
isolated from the bacterium Pseudomonas syringae that have
been characterized as lipodepsinonapetpides 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 below and described in more detail in PCT Patent
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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
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
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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 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
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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-
s 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 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
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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.
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

CA 02379321 2002-O1-14
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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 occux 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 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
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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.
Each pseudomycin, pseudomycin intermediate 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 activity in a 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.
Alternatively, the amido or ester derivative can be
formed from an N-acyl semi-synthetic compound. Semi-
synthetic pseudomycin compounds may be synthesized by
exchanging the N-acyl group 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
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different side-chain; and (4) deprotection of the amino
groups. The aspartic acid and/or hydroxyaspartic acid units
can be modified prior to deprotecting the amino groups.
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 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
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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,
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
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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.
The acid-modification of the protected N-acyl semi-
synthetic compound is then accomplished by reacting at least
one of the pendant carboxyl groups attached to. the aspartic
or hydroxyaspartic peptide units of the N-acyl modified
semi-synthetic pseudomycin compound to form the desired
amide or ester linkage(s). The protecting groups are then
removed as described earlier.
The pseudomycin compound 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, 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,

CA 02379321 2002-O1-14
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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.,
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 compound in the appropriate solvent with heat
and slowing cooling to generate an amorphous or crystalline
solvate form.
The active ingredient (i.e., pseudomycin compound) 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 0.10
to 99.90 by weight of active ingredient, more generally from
about 10o 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
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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
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.
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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
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 5% 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
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infections or fungal skin infections. Accordingly, a method
is provided for inhibiting fungal activity comprising
contacting the pseudomycin compound of the present invention
with a fungus. A 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 an
animal 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 species of the host. The particular
dose regimen likewise may vary according to these factors.
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The medicament 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)
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 may be any 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
Unless indicated otherwise, all chemicals can be
acquired from Aldrich Chemical (Milwaukee, WI). 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-0-C(0)-
2 5 CBZ = benzyloxycarbonyl , C6HSCH2-O-C ( O ) -
PyBOP = benzotriazol-1-yloxy-tripyrrolidinophosphonium
hexafluorophosphate
TBTU = o-Benzotriazol-1-yl-N,N,N',N'-tetramethyluronium

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tetrafluoroborate
DIEA = N,N-diisopropylethylamine
HPLC Conditions
Unless indicated otherwise, analytical reverse-phase
HPLC work was done using the Waters 600E systems equipped
with Waters ~,Bondapak (C18, 3.9 X 300 mm) column. The
eluent used was 65:35 acetonitrile/0.1o aqueous TFA solvent
system to 100% acetonitrile over 20 minutes with a flow rate
of 1.5 ml/minute and using UV detection at 230 nm.
Preparative HPLC work was performed with a Waters Prep
2000 system using Dynamax 60 angstrom C18 column and
identical solvent systems as used in the analytical HPLC
system but with a flow rate of 40 ml/min.
Biological Analysis
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
~,1) causes 10-12 mm diameter zones of inhibition on C.
albicans x657 seeded agar plates.
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Tail Vein Toxicity:
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).
General Procedures
CBZ-Protected Pseudomycin: General procedures used to
protect the pendant amino groups at positions 2, 4 and 5 of
Pseudomycin A, A' , B, B' , C or C' wi th CBZ.
Dissolve/suspend pseudomycin compound (Rl=H) in DMF (20
mg/ml, Aldrich Sure Seal). V~hile stirring at room
temperature add N-(Benzyloxycarbonyloxy)succinimide (6 eq).
Allow to stir at room temperature for 32 hours. Monitor
reaction by HPLC (4.6x50 mm, 3.5 E,tm, 300-SB, C8, Zorbax
column). Concentrate reaction to 10 ml on high vacuum
rotovap at room temperature. Put material in freezer until
ready to prep by chromatography. Reverse phase preparative
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HPLC yields an amorphous, white solid after lyophilization
(R1 - CBZ in structure II below).
Alloc-Protected Pseudomycin: General procedures used to
protect the pendant amino groups at positions 2, 4 and 5 of
Pseudomycin A, A', B, B', C or C' with Alloc.
Diallyl pyrocarbonate (558 mg, 3.0 mmol) was added to a
solution of Pseudomycin A (1.22 g, 1.0 mmol) ixi 600 ml DMF.
The reaction was stirred at room temperature overnight. The
solvent was removed in vacuo to afford an oily residue which
was washed with ether three times. The oily residue was
redissolved in a mixture of water and ACN (~1:1) and
lyophilized to provide an alloc-protected psuedomycin A
compound in 90o yield.
The alloc-protected pseudomycin B compound was prepared
using the same procedures in 90o yield (R1 - alloc in
structure II below).
General procedures used to remove CBZ protecting groups at
position 2, 4 and 5 by hydrogenation.
Dissolve CBZ-protected acylated-derivative in a cold 10
to 10o acetic/methanol solution (5 mg/ml) and add an
equivalent amount of 10% Pd/C. Charge the reaction with
hydrogen by degassing reaction and replacing volume with HZ
,4-7 times. Allow reaction to proceed at room temperature.
33

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Monitor the reaction by HPLC every hour until starting
material is consumed. When the reaction is complete, remove
balloon and filter reaction with 0.45 ~,m filter disk
(Acrodisk GHP, GF by Gelman). Concentrate to about 1/l0th
volume and prep by HPLC. Lyophilize fractions containing
product.
General procedures used to remove Alloc protecting groups at
position 2, 4 and 5 with tributyltinhydride and
triphenylphosphine palladium dichloride.
Acetic acid (1 ml) was added to a suspension of alloc-
protected pseudomycin B (0.05 mmol) in 5 ml methylene
chloride. After degassing under vacuum, the solution was
treated with 6.0 mg PdCl2(PPH3)2 (0.008 mmol) and 0.40 ml
tri-n-butyltin hydride (1.5 mmol)at room temperature for 2
hours. The solvent was evaporated in vacuo and the residue
dissolved in water/ACN (~1:1) and filtered. The resulting
solution was purified by preparative HPLC to afford the
desired pseudomycin B compound in 93o yield. Alternatively,
5 ml tetrahydrofuran and 0.1 ml acetic acid may be used as
the solvent instead of 5 ml methylene chloride and 1.0 ml
acetic acid.
The following structure II will be used to describe the
products observed in Examples 1 through 27. Although a
specific pseudomycin natural product (pseudomycin B) was
34

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used in the Examples below, those skilled in the art will
appreciate that other pseudomycin natural products or semi-
synthetic derivatives may be used as starting materials.
0
r, ~n OH
H N _
R O O
O ...H~(CHzyoCHa
H
1NHR'
II
Examples 1-3 illustrate the formation of bis-esters at
residues 3 and 8.
Example 1
Synthesis of Bis-Ethyl ester 1-1:
R1 - H
RZ - -OCHZCH3
R3 - -OCHZCH3
1-1
A 50 ml round bottom flask was charged with 10 ml of
absolute ethanol and CBZ-protected pseudomycin B(251.7 mg,
0.156 mmol). To this mixture was added ~ 1 ml of acidified
ethanol (previously acidified using HC1 gas) and the
reaction was allowed to stir at room temperature overnight.
HO,. / O

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The solvent was then removed in vacuo and the residue was
carried on to the next step without further purification by
dissolving it in a solution of 10 ml MeOH/1.5 ml glacial
AcOH. Standard hydrogenolysis using 249.7 mg of 10o Pd/C
for 30 minutes, removal of the catalyst via filtration and
purification via preparatory HPLC led to Compound 1-1 (120.9
mg) after lyophilization. MS (Ionspray) calcd for
C55H96C1N1zOlg (M+H)+ 1264.89, found 1264.3.
The mono-esters may be isolated by following the
reaction carefully by HPLC. The reaction is stopped at the
appropriate time when the ratio of starting material: mono
ester(s): bis ester is greatest. The methodology remains
the same. The resulting mixture of mono esters is isolated
where some ester is formed on the aspartic acid residue and
some on the hydroxy aspartic acid residue. This mixture of
CBZ-protected, mono esters is hydrogenated using standard
methodology to yield a mixture of mono ethyl esters of
Pseudomycin B.
Compounds 1-2 and 1-3 were synthesized using the same
procedures described above.
R = -H R = -H
R2 - -OCH3 Rz - -OCH ( CH3 ) 2
R3 = -OCH3 R3 - -OCH ( CH3 ) z
1-2 1-3
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Example 2 illustrates the synthesize of bis-esters
using basic conditions.
Example 2
Synthesis of Bis-propyl ester 2-1:
R = -H
R2 - -OCHzCH2CH3
R3 - -OCH2CHzCH3
2-1
CBZ-protected pseudomycin B (247.3 mg, 0..154 mmol) was
dissolved in 5 ml DMF. A large excess of propyl iodide and
an excess of NaHC03 were then added. The reaction was
allowed to stir for 10 h at room temperature. Purification
via preparatory HPLC followed by lyophilization provided
147.6 mg of the protected bis ester. Hydrogenolyis of this
compound under standard condition using 149.3 mg of 10% Pd/C
yielded 78.9 mg of Compound 2-1 after HPLC purification and
lyophilization.
Exantpl a 3
R = -H R = -H
2 0 RZ - -0 ( CHz ) 4CH3 RZ - -OH
R3 - -OH R3 - -O ( CHZ ) 4CH3
3-1 3-2
CBZ-protected pseudomycin B (282.3 mg, 0.175 mmol) was
dissolved in 5 ml DMF. A large excess of n-pentyl iodide
and an excess of NaHC03 were then added. The reaction was
allowed to stir for 10 h at room temperature. Purification
via preparatory HPLC followed by lyophilization provided
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49.1 mg of the mixture of protected mono pentyl esters.
Hydrogenolyis of this mixture under standard condition using
47.3 mg of 10o Pd/C yielded 30.6 mg of Compounds 3-1 and 3-2
after HPLC purification and lyophilization.
R = -H R = -H R = -H
R2 - -O ( CHZ ) 3CH3 RZ= -O ( CHz ) 3CH3 Rz - -OH
R3 - -O ( CHZ ) 3CH3 R3 - -OH R3 - -0 ( CHZ ) 3CH3
3-3 3-4 3-5
Substitution of the propyl iodide with n-butyl iodide
afforded the bis-butyl ester (3-3), a mixture of mono esters
(3-4 + 3-5) and a mixture of mono ester + the following
cyclic imide compound 3-6:
o O O
O O NH H ~ OH
O ~CI
,~~ O O
HZN~O
NH
O O H O~"'H (CH2)~oCH3
N NH
O
OR NH2
H2N O
3-6
Example 4
Synthesis of cyclopentylmethyl ester 4-1:
R = -H
Rz - -OCHZ(cyclopentyl)
R3 - -OH
4-1
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CBZ-protected pseudomycin B, a large excess of p-
toluenesulfonic acid and cyclopentanemethanol are mixed and
allowed to stir overnight. An additional 10 equivalents of
alcohol was added the next day. The CBZ-protected ester was
isolated via preparatory HPLC and then hydrogenated using
standard methodology to produce Compound 4-1.
Each of the compounds synthesized in Examples 1-4
showed measurable activity against Candida Albicans,
Cryptococcus neoformans, Aspergillus Fumigatus, Candida
Parapsilosis, or Histoplasma capsulatum. However, the
following basic trends in activity were observed based on
the compounds synthesized. Simple esters (bis-methyl, bis-
ethyl and mono-ethyl) were active and efficacious; however,
the larger esters exhibited less efficacy (e. g., propyl
esters and larger). ADME has shown that Compounds 1-1 and
2-1 quickly cleave to the parent pseudomycin B compound.
Examples 5-11 illustrate the synthesis of amide
derivatives at residue 3.
Exasnpl a 5
Synthesis of Compound 5-1:
R = -H
Rz _ -NHz
R3 - -OH
5-1
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CBZ-protected pseudomycin B (1.12 g) and 224 mg TBTU,
0.56 ml DIEA and 1.0 g deprotected rink amide resin (4-
(2',4'-dimethoxyphenyl-aminomethyl)-phenoxy resin, available
from Advance ChemTech, Inc., Louisville, KY) were mixed for
3 days. The mixture was filtered and the resin washed 3x
with DMF and 3x with dichloromethane. The resin was treated
with 5o water in 1:1 TFA/CHZC12 for 3 hours. The mixture
was filtered and the resin washed 3x with TFA. The filtrate
was collected and concentrated in vacuo. Upon purification
by HPLC, 60 mg (5.30) of the CBZ-protected amido product was
isolated.
The protected amido compound (60 mg) was dissolved in 6
ml of 1o AcOH in methanol and 60 mg of 10o Pd/C was added.
The mixture was stirred for 30 minutes under hydrogen at
room temperature. After filtering, the solution was
concentrated in vacuo. The residue was dissolved in 500
ACN/water and lyophilized to yield 45 mg (900) yield of
Compound 5-1.
Example 6
Synthesis of Compound 6-1:
R = -H
R2 - -NH(cyclopropyl)
R3 - -OH
6-1
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CBZ-protected pseudomycin B (400 mg, 0.25 mmol) is
dissolved in 4 ml dry DMF. TBTU (79 mg, 0.25 mmol), DIEA
(200 ~,1, 6 equivalents) and cyclopropylamine (14.2 mg, 0.25
mmol) were added sequentially. The reaction was stirred at
room temperature under nitrogen while being monitored by
HPLC. Upon completion the reaction was concentrated in
vacuo. The crude product purified by preparative HPLC.
Lyophilization yielded 209.2 mg (51.10) of a colorless
powder.
The 3-amido compound (279.1 mg, 0.169 mmol) was
hydrogenated under hydrogen balloon catalyzed by 10% Pd/C in
1o HOAc/MeOH for 45 minutes. The reaction was filtered and
concentrated in vacuo. The residue was picked up in a 1:1
mixture of water:ACN and then lyophilized to give 208.3 mg
(98.60) of a colorless powder. The structure was verified
by Hl-NMR.
Compound 6-1 can also be made from the Alloc-protected
pseudomycin B using the following procedures.
1-Hydroxybenzotriazole hydrate (136 mg, 1.0 mmol) and
EDCI (211 mg, 1.1 mmol) was added to a solution of alloc-
protected pseudomycin B (730 mg, 0.50 mmol) in 7 ml of DMF.
After stirring overnight, cyclopropylamine (85.6 mg, 1.5
mmol) was added. The progress of the reaction was monitored
by HPLC. Upon completion, the alloc-protected pseudomycin
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derivative (334 mg, 50o yield) was isolated via preparative
HPLC and lyophilization.
The alloc-protected intermediate (117 mg, 0.078 mmol)
was dissolved in 15 ml of methylene chloride and 1 ml of
acetic acid. After degassing the reaction mixture with dry
nitrogen, 30 mg of (PPh3)ZPdCl2 and 1 ml of
tributyltinhydride was added to the mixture. The progress
of the reaction was monitored by HPLC. Upon completion, the
reaction mixture was purified by reverse phase preparative
HPLC to provide 88 mg (91o yield) of Compound 6-1.
Table I below lists other 3-amido derivatives that were
synthesized using the same general procedures described
above using the appropriate corresponding amine starting
material.
Table I
Example # R R R
6-2 -H -NHCH3 -OH
6-3 -H -NHCHzCH3 -OH
6-4 -H -NHCH2CF3 -OH
6-5 -H -NH (CHZ) zCH3 -OH
6 - 6 -H -NHCH2 ( CH3 ) 2 -OH
6-6 -H -NH(cyclopropyl) -OH
6-7 -H -NHCHZCH=CHz -OH
6-8 -H -NH (CHz ) 4CH3 -OH
6-9 -H -NHCH ( CH3 ) ( CHZ -OH
) ZCH3
6-10 -H -NH (CHZ) SCH3 -OH
6-11 -H -NH(cyclohexyl) -OH
6-12 -H -NH(CHZ)6CH3 -OH
6-13 -H -NH (CHZ) 7CH3 -OH
6-14 -H -NH (CHZ) 8CH3 -OH
6 -15 -H -NH ( CHZ ) 9CH3 -OH
6-16 -H -OH
~N
H
6-17 -H -NH ( CHZ ) ZN ( CH3 -OH
) 2
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6-18 -H -NH ( CH2 ) 2N ( CHZCH3-OH
) 2
6-19 -H -NH ( CHz ) 3N ( CH3 -OH
) 2
6-2 0 -H -NH ( CH2 ) 3N ( CHZCH3-OH
) z
6-21 -H -NH ( CH2 ) 4N ( CH3 -OH
) 2
6-22 -H -NH ( CH2 ) 6N ( CH3 -OH
) 2
6-2 3 -H -NH ( CH2 ) 7N ( CH3 -OH
) 2
6-24 -H -OH
'~N~N~
H
6-25 -H -OH
H
~N //~
Exampl a 7
Synthesis of 3-amido compound 7-1:
R = -H
R2 - '~N \
H ~.~~~~
i
N
R3 - -OH
7-1
In a 500 mL oven dried round bottom flask, CBZ-
protected Pseudomycin B(0.5 g, 0.311mmo1) was dissolved in
25 mL of DMF. To this solution was added TBTU(0.2 g, 0.622
mmol), 3-(aminomethyl)pyridine(0.067 g, 0.622mmo1), and N-
ethyldicyclohexylamine(0.391 g, 1.87 mmol). The solution
was stirred for three hours and then concentrated down. The
product was isolated by reverse-phase preparatory HPLC, and
lyophilized to yield, (96 mg, 18o yield) CBZ-protected
amide. The deprotection of the CBZ groups was performed by
adding slowly an equivalent mass of lOoPd/C to a cold 10
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acetic/methanol solution of CBZ-protected amide. The
solution was allowed to warm to rt and stirred rapidly for
3.5 hours under 1 atm H2. After removal of the catalyst via
filtration, purification on reverse phase HPLC and
lyophilization yielded 40 mg , 55o yield of Compound 7-1.
MS data Calculated for C57 H93 Cl N14 018 Mol. Wt. - 1296.6
Found ES+ 1297.15, ES- 1294.95
Example 8
Synthesis of 3-amido compound 8-1:
R = -H
Rz = ~N~N~
H
R3 - -OH
8-1
The same general procedures as described in Example 7
may be used. When no base is added, a mixture of 8 and 3
amido substituted compounds are observed.
Example 9
Synthesis of 3-amido compound 9-1:
R = -H R = -H
Rz - -NH(benzyl) RZ - -NH(benzyl)
R3 - -OH R3 - -NH(benzyl)
9-1 9-2
The same general procedures as described in Example 7
may be used. When no base is added, a mixture of Compounds
9-1 and 9-2 are observed.
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Example 10
Synthesis of 3-amido compound 10-1:
R = -H
~NH
R2 = ~N~N~
H
R3 - -OH
10-1
The same general procedures as described in Example 7
are used to synthesize Compound 10-1 using the appropriate
corresponding amine starting material.
Example Z1
Synthesis of 3-amido compound I1-2:
R = -H
Rz = ~N \
H
iN
R3 - -OH
11-1
The same general procedures as described in Example 7
are used to synthesize Compound 11-1 using 4-(aminomethyl)
pyridine as the amine starting material.
Example Z2
Synthesis of 3-amido Compound 12-1:
R = -H
Rz - -N ( CH3 ) 2
R3 - -OH
12-1

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CBZ-protected pseudomycin B (260 mg, 0.16 mmol), 51.8
mg TBTU and 152 ~l DIEA were dissolved in 3 ml DMF and 320
ml dimethylamine (0.16 mmol) in THF (2 molar solution). The
reaction was stirred at room temperature for 20 minutes and
the then purified via HPLC. The product was lyophilized to
give 172,mg (66% yield) of the desired CBZ-protected amide.
The CBZ-protected amide was hydrogenated using the
general procedure described above to provide Compound 12-1.
Example 13 illustrates the synthesis of pseudomycin
compounds where the carboxylic acid group is reacted with a
variety of amino acid alkyl esters.
Example 13
Synthesis of 3-amido Compound 13-1:
R = -H
Rz - -NHCH (COZCH3 ) CHZCHZCHzCH2NHz
R3 - -OH
13-1
CBZ-protected Lysine methyl ester (164 mg, 0.49 mmol)
was added to a solution of CBZ-protected pseudomycin B (800
mg, 0.49 mmol), TBTU (158 mg, 0.49 mmol) and 400 ml DIEA
(2.51 mmol) in 8 ml DMF. The reaction was allowed to stir
at room temperature for 20 minutes and then purified via
HPLC to yield 260 mg (32o yield) of the CBZ-protected amide.
The CBZ-protected amide was hydrogenated using the
general procedures described above to produce Compound 13-1.
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The compounds 13-2 through 13-4 listed in Table II may
be synthesized using the same general procedures as
described above using the appropriate corresponding
aminoacid ester.
Table II
Example # R R R
13-2 -H -NHCH2COzCH3 -OH
13-3 -H O O,CH -OH
3
~N
H
13 - 4 -H O 0 , CH -OH
3
~N
H
OH
Examples 14-16 illustrate the synthesis of amide
derivatives at residue 8.
Example 14
Synthesis of 8-amido Compound 14-1:
R = -H
R2 - -OH
R3 _ -NH2
14-1
Compound 14-1 is synthesized using the same procedures
as described for compound 6-1 using a rink amide resin with
the exception that PyBOP is used as the coupling agent
instead of TBTU.
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Example 15
Synthesis of 8-amido Compound 15-1:
R = -H
R2 - -OH
R3 - -NH ( CHZ ) 3CH3
15-1
n-Butyl amine (45.4 mg, 0.62 mmol) was added to a
solution of CBZ-protected pseudomycin B (1000 mg, 0.62 mmol)
and PyBop (323 mg, 0.62 mmol) dissolved in 10 ml of DMF.
The reaction was stirred at room temperature for 1 hour and
then purified via HPLC. The product was lyophilized to give
280 mg (27% yield) of the CBZ-protected amide.
The CBZ-protected amide (280 mg, 0.17 mmol) was
hydrogenated under hydrogen catalyzed by 10o Pd/C in 10
acetic acid/methanol for 45 minutes. The reaction mixture
was filtered and the solvent removed in vacuo. The residue
was dissolved in 50o ACN in water and lyophilized to give
189 mg (89o yield) of Compound 15-1.
The 8-amido compounds listed in Table III may be
synthesized using the same general procedures described
above using the appropriate corresponding amine starting
material.
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Table III
Example # R R R
15-2 -H -OH -NHCH3
15-3 -H -OH -NHCHZCH3
15-4 -H -OH -NH (CHZ ) ZCH3
15-5 -H -OH -NH(cyclopropyl)
15-6 -H -OH -NH(cyclobutyl)
15-7 -H -OH -NHCHZCHZOH
15 - 8 -H -OH -NHCHZCH2N ( CH3
) 2
15-9 -H -OH -NHCHzCH2CH2N (
CH-~ )
Example 16
Synthesis of 8-amido Compound 26-1:
R = -H
Rz - -OH
R3._ ~N~N~
H
16-1
In a 100 ml round bottom flask, alloc-protected
Pseudomycin B(0.25 g, 0.171 mmol) was dissolved in 25 ml of
DMF. To this solution was added Pybop (0.0898, 0.171
mmol)and 4-(2-Aminoethyl)morpholine (0.022 g, 0.171 mmol).
The solution was stirred rapidly overnight under 1 atm N2,
The solution was concentrated down, and the product was
isolated by reverse-phase HPLC, and lyophilized to yield
(140 mg, 0.089 mmol, 520) alloc-protected Psuedomycin B
Morpholine derivative. The deprotection of the alloc groups
was performed by adding Bu3SnH(0.648 g, 2.23 mmol), and
(Ph3P)ZPdCl2(0.009g, 0.013 mmol) to a 1o acetic/
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dichloromethane solution of alloc-protected Psuedomycin B
Morpholine derivative (10 mg/mL). Reaction time was 30
minutes. Reaction was monitored by HPLC. The solution was
concentrated down, and the product was isolated by reverse
phase HPLC prep, and lyophilized to yield 38 mg, 320 of
Compound 16-1. MS data: Calculated for C57 H99 C1 N14 019
Mol. Wt. 1318.7 Found ES+ 1320.0, ES- 1318.0
The 8-amido compounds listed in Table IV were
synthesized using the same general procedures described
above using the appropriate corresponding amine starting
material.
Table IV
Example # R R R
16-2 -H -OH -NH(benzyl)
16-3 -H -OH ~NH
'~N~N~
H
16-4 -H -OH
N
H
~
N
Each of the compounds synthesized in Examples 5-16
showed measurable activity against Candida Albicans,
Cryptococcus neoformans, Aspergillus Fumigatus, Candida
Parapsilosis, or Histoplasma capsulatum. However, the
following basic trends in activity were observed based on
the compounds synthesized.

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When the 8-amido derivatives were assayed against C.
albicans, several trends were apparent from the data. The
in vitro potency decreases in the following order of R3
substitution: -NHz > -NHCH3 > -NHCHZCH3 > -NH (CHZ) 2CH3 >
-NH ( CH2 ) 3CH3 ; -NHCH2CHZN ( CH3 ) 2 > -NH ( CH2 ) 3IV ( CH3 ) 2 ; and
-NH(GIyOMe) > -NH(PheOMe). In general, better activities
were realized with amido groups having smaller alkyl groups.
The free amide group was found to be the most active of the
series. In addition, the cycloalkyl amides demonstrated
better activity than the corresponding straight chain alkyl
groups. Alkyl groups having a polar substitution on the end
of the alkyl chain showed less activity than the
corresponding natural product. Unlike the parent natural
product, none of the 8-amido derivatives showed tail vein
irritation.
The 3-amido derivatives demonstrated a similar trend as
observed with the 8-amido derivatives in comparison with the
parent natural product (e.g., amide substituents at R2
having shorter alkyl chains were more active than longer
alkyl chains). Unlike the 8-amido derivatives, the 3-amido
derivatives did not show a significant decrease in in vitro
activity against C. albicans until the alkyl chain reached
7-carbons or longer (3-amido PSB compound where RZ -
-NH(CHz)6CH3 had a MIC = 20 ~,g/ml) versus 4-carbons or longer
for the 8-amido derivatives (8-amido PSB compound where R3 -
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-NH(CHz)3CH3 had a MIC = 20 ~g/ml). Most of the 3-amido
derivatives tested showed an improvement in tail vein
irritation. The exceptions being Rz - -NH(iso-amyl), -NH(n-
hexyl ) , -NH ( CHz ) zN ( CHZCH3 ) z , and -NH ( CHz ) 3N ( CH3 ) z .
Although formation of an amide bond at residues 3 and 8
demonstrated an improved toxicity profile in comparison with
the corresponding natural product (Pseudomycin B), in vivo
efficacy generally decreased.
52