Note: Descriptions are shown in the official language in which they were submitted.
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CYCLIC PEPTIDE ANTIFUNGAL AGENTS
FIELD OF THE INVENTION
The present invention relates to anti-fungal/anti-parasitic agents, in
particular,
derivatives of Echinocandin compounds and their use in the treatment of fungal
and
parasitic infections.
BACKGROUND ART
A number of naturally occurnng cyclic peptides are known in the art including
echinocandin B (A30912A), aculeacin, mulundocandin, sporiofungin, L-671,329,
and
S3 i 794/F1. In general, these cyclic peptides can be structurally
characterized as a cyclic
hexapeptide core (or nucleus) with an acylated amino group on one of the core
amino
acids. This acyl group is typically a fatty acid moiety foaming a side chain
off the
nucleus. For example, echinocandin B has a linoleoyl side chain while
aculeacin has a
palmitoyl side chain.
These natural products have limited inherent antifungal and antiparasitic
properties. The natural compounds can be structurally modified in order to
enhance these
properties or improve the compound's stability and/or water solubility. Turner
et al. Cur.
Pharm. Des. 2:209 (1996). For example, the fatty acid side chain can be
removed from
the cyclic peptide core to yield an amino nucleus which can be re-acylated to
yield semi-
2 0 synthetic compounds.
DISCLOSURE OF THE INVENTION
A compound represented by structure I is provided
R
R1 O
z N
R ~ ,R
R'
i
RS
R'
N
R1
O
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where R is an alkyl group, an alkenyl group, an alkynyl group, an aryl group,
or
heteroaryl group; R' is independently -H, -OH or -O-Pg; R-' is -H, -CH,, -NH2,
or -NH-Pg;
R' is -H, -CH3, -CH,CONH" -CHzCONH-Pg, -CHZCH,NH,, or -CHZCH,NH-Pg; RS is -
OH, -OSO,H, or -OPO,HRa, where Ra is hydroxy, C1-C6 alkyl, C1-C6 alkoxy,
phenyl,
phenoxy, p-halophenyl, p-halophenoxy, p-nitrophenyl, p-nitrophenoxy, benzyl,
benzyloxy, p-halobenzyl, p-halobenzyloxy, p-nitrobenzyl, or p-nitrobenzyloxy;
R6 is -H, -
OH, or -OS03H; R' is -H or -CH3; R" and R8 are independently, hydrogen, or
hydroxy and
at least one of R~ and R8 is a sugar moiety of the formula
R9 O O p~ R9 O O O
Rs~R9
R9 Rs Rs
or
where R9 is independently -H, -OH, -N3, -O-Pg, -NHz, -NH-Pg, -OPOzRe, or a
second
sugar moiety containing one to three sugar units of
9c
s;
R
O
a
O
s
O O
9a
R9b R9a Rsa
R9a
R9a
' and mixtures
thereof, where R9' is -H, -OH, -N" -NHz, -O-Pg, or -NH-Pg, R9b is -OPO~Ra, -
OS03H, -H,
-NH2, -OH, -O-P~, or -NH-Pg, R9' is -CH3, -CHZOH, -CH,N3, -CH,OS03H, -CH,NH-
Pg, -
CH,O-Pg, -CO,H, or -CO,-Pg, where Ra is as defined above, and no more than one
R9 is
represented by said second sugar moiety; Pg is a protecting group ( i.e., -O-
Pg is a
hydroxy protecting group, -NH-Pg is an amino protecting group, -CH,CONH-Pg is
an
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amido protecting group and -CO,-Pg is a carboxy protecting group); and
pharmaceutically
acceptable salts, esters, hydrates or solvates thereof.
The invention encompasses a pharmaceutical formulation is containing one or
more pharmaceutical carriers, diluents or excipients and a Compound I
described above.
The invention encompasses a method for inhibiting fungal and parasitic
activity
by administering an effective amount of Compound I to a recipient in need
thereof.
"Alkyl" is a hydrocarbon radical of the general formula C"HZ"+, containing
from 1
to 30 carbon atoms unless otherwise indicated. The alkane radical can be
straight,
branched, cyclic, or multi-cyclic. The alkane radical can be substituted or
unsubstituted.
The alkyl portion of an alkoxy group, alkylthio group or alkanoate have the
same
definition as above.
"Cl-C12 alkyl" is a straight or branched saturated alkyl chain of from one to
twelve carbon atoms. Cl-C12 alkyl groups include, but are not limited to,
methyl, ethyl,
propyl, isopropyl, butyl, sec-butyl, t-butyl, pentyl, 5-methylpentyl, hexyl,
heptyl, 3,3-
dimethylheptyl, octyl, 2-methyl-octyl, nonyl, decyl, undecyl and dodecyl. "C1-
C12
alkyl" includes "C1-C( alkyl", "Cl-C4 alkyl", and "C3-C12 cycloalkyl".
"C3-C12 cycloalkyl" is a cyclic saturated alkyl chain of from 3 to 12 carbon
atoms. Moreover, the term "C3-C12 cycloalkyl" includes "C3-C~ cycloalkyl",
i.e.,
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. "C 1-C 12
alkoxy"
2 0 refers to a C 1-C 12 alkyl group attached through an oxygen atom. C 1-C 12
alkoxy groups
include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, sec-butoxy,
n-pentoxy,
5-methyl-hexoxy, heptoxy, octyloxy, decyloxy and dodecyloxy. "C 1-C 12 alkoxy"
includes "Cl-C( alkoxy", "C3-C~ alkoxy", and "Cl-C~ alkoxy".
"C 1-C 12 alkylthio" is a C 1-C 12 alkyl group attached through a sulfur atom.
C 1-
C12 alkylthio groups include, but are not limited to, methylthio, ethylthio,
propylthio,
isopropylthio, butylthio, 3-methyl-heptylthio, octylthio and S,5-dimethyl-
hexylthio. "Cl-
C 12 alkylthio" includes "C 1-C(, alkylthio" and "C 1-C4 alkylthio."
"Alkenyl" is an acyclic hydrocarbon containing at least one carbon-carbon
double
bond. The alkene radical can be straight, branched, cyclic, or multi-cyclic,
substituted or
3 0 unsubstituted.
"Alkynyl" is an acyclic hydrocarbon containing at least one carbon-carbon
triple
bond. The alkyne radical can be straight, or branched, substituted or
unsubstituted.
"C2-C1? alkynyl" is a straight or branched mono-alkynyl chain having from two
to twelve carbon atoms. C2-C 12 alkynyl groups include, but are not limited
to, ethynyl,
1-propy-1-yl, 1-propyn-2-yl, 1-butyn-1-yl, 1-butyn-3-yl, 1-pentyn-3-yl, 4-
pentyn-2-yl, 1-
3
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hexyn-3-yl, 3-hexyn-1-yl, 5-methyl-3-hexyn-I-yl, 5-octyn-1-yl, 7-octyn-I-yl
and 4-
decyn-1-yl, 6-decyn-1-yl.
"Aryl" is aromatic moieties having single (e.g., phenyl) or fused ring systems
(e.g., naphthalene, anthracene, phenanthrene, etc.). The aryl groups can be
substituted or
unsubstituted. Substituted aryl groups include a chain of aromatic moieties
(e.g.,
biphenyl, terphenyl, phenylnaphthalyl, etc.).
"Heteroaryl" is an aromatic moiety containing at least one heteratom within
the
aromatic ring system (e.g., pyn ole, pyridine, indole, thiophene, furan,
benzofuran,
imidazole, pyrimidine, purine, benzimidazole, quinoline, etc.). The aromatic
moiety can
consist of a single or fused ring system. The heteroaryl groups can 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. Alkyl group allows for substituents which is a
classic alkyl, such
as methyl, ethyl, propyl, n-butyl, i-butyl, t-butyl, hexyl, isooctyl, dodecyl,
stearyl, etc.
The term group includes 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. The substituents should not adversely affect the
pharmacological characteristics of the compound or adversely interfere with
the use of the
2 0 medicament. The same is true for each of the other groups (i.e., aryl,
alkynyl, alkenyl,
heteroaryl). 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,
2 5 hydrazino, guanyl, and combinations thereof.
"Halo" refers to chloro, fluoro, bromo and iodo.
"O-Pg" and "hydroxy protecting group" refer to a substituent of a hydroxy
group
commonly employed to block or protect the hydroxy functionality while
reactions are
carried out on other functional groups on the compound. This substituent, when
taken
30 with the oxygen to which it is attached, can form an ether, e.g., methyl,
methoxymethyl,
and benzyloxymethyl ether, a silyl ether, an ester, e.g. acetoxy, or a
sulfonate moiety, e.g.
methane and p-toluenesulfonate. The exact genus and species of hydroxy
protecting
group is not critical so long as the derivatized hydroxy group is stable to
the conditions of
subsequent reactions} and the protecting group can be removed at the
appropriate point
3 5 without disrupting the remainder of the molecule. A preferred hydroxy
protecting group
is acetyl. Specific examples of hydroxy protecting groups are described in
Greene,
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"Protective Groups in Organic Synthesis," John Wiley and Sons, New York, N.Y.,
(2nd
ed., 1991 ), (Greene) chapters 2 and 3 and Preparations and Examples sections
herein.
"NHp-Pg" and "amino protecting group" are a substituent of the amino group
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-forrnyl and N-acetylamide. 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). Preferred amino protecting
groups are t-
butoxycarbonyl (t-Boc), allyloxycarbonyl, phthalimido, and benzyIoxycarbonyl
(CbZ).
See, Greene at chapter 7.
"-COZ Pg" and "carboxy protecting group" are a substituent of a carbonyl
commonly employed to block or protect the carboxy functionality while
reactions are
carried out on other functional groups on the compound. This substituent, when
taken
with the carbonyl to which it is attached, can form an ester, e.g., CI-C6
alkyl, substituted
C I -C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, benzyI, substituted
benzyl,
benzhydryl, substituted benzhydryl, trityl, substituted trityl, and
trialkylsilyl ester. The
exact species of carboxy protecting group is not critical so long as the
derivatized carboxy
group is stable to the conditions of subsequent reactions) and the protecting
group can be
2 5 removed at the appropriate point without disrupting the remainder of the
molecule. Other
examples of groups referred to by the above terms are described in Greene, at
chapter 5.
"C(O)NH-Pg" and "amido protecting group" are a substituent of an amide
commonly employed to block or protect the amino portion while reacting other
functional
groups on the compound. This protecting group, when taken with the nitrogen to
which it
is attached, can form an amide, e.g. N-allyl, N-methoxymethyl, and N-
benzyloxyrnethyl
amide. The exact species of amido protecting group employed is not critical so
long as
the derivatized amido 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
3 5 including any other amido protecting group(s). Other examples of groups
referred to by
the above terms are described in Greene, chapter 7, pg. 397.
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"Carbonyl activating group" is a substituent of a carbonyl that promotes
nucleophilic addition reactions at that carbonyl. Suitable activating
substituents 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 such as
oxybenzotriazole, imidazolyl, nitrophenoxy, pentachlorophenoxy, N-
oxysuccinimide,
N,N'-dicyclohexylisoure-O-yl, N-hydroxy-N-methoxyamino; acetates, formates,
sulfonates such as methanesulfonate, ethanesulfonate, benzenesulfonate, or p-
tolylsulfonate; and halides such as chloride, bromide, or iodide.
"Pharmaceutical" or "pharmaceutically acceptable" are substances substantially
non-toxic and substantially non-deleterious to the recipient. "Pharmaceutical
formulations" are those in which the carrier, solvent, excipients and salt are
compatible
with the active ingredient of the formulation (i.e., Compound I).
"Pharmaceutical salt" or "pharmaceutically acceptable salt" are salts of the
compounds represented by structure I that are substantially non-toxic to the
recipient at
the doses administered. Typical pharmaceutical salts include those prepared by
reaction
of the compounds of the present invention with a mineral or organic acid or
inorganic
base. Such salts are known as acid addition and base addition salts. For
further
exemplification of pharmaceutical salts, see e.g. Berge et al., J. Pharm.
Sci., 66:1 ( 1977).
"Solvate'' represents an aggregate that comprises one or more molecules of the
2 0 solute, such as a formula I compound, with one or more molecules of a
pharmaceutical
solvent, such as water, ethanol, and the like. "Suitable solvent" is any
solvent, or mixture
of solvents, inert to the ongoing reaction that sufficiently solubilizes the
reactants to
afford a medium within which to effect the desired reaction.
"Thermod}mamic base" is a base which provides a reversible deprotonation of an
2 5 acidic substrate or is a proton trap for those protons that can be
produced as byproducts of
a given reaction. and is reactive enough to effect the desired reaction
without significantly
effecting any undesired reactions. Examples of thermodynamic bases include,
but are not
limited to, acetates, acetate dihydrates, carbonates, bicarbonates, C 1-C4
alkoxides, and
hydroxides (e.g. silver, lithium, sodium, or potassium acetate, acetate
dihydrate,
30 carbonate, bicarbonate, methoxide, or hydroxide), tri-(C1-C4 alkyl)amines,
or aromatic
nitrogen containing heterocycles (e.g. imidazole and pyridine).
"Inhibiting" includes prohibiting, stopping, retarding, alleviating,
ameliorating,
halting, restraining, slowing or reversing the progression, or reducing the
severity of the
growth or any attending characteristics, symptoms, and results from the
existence of a
3 5 parasite or fungus. These methods include both medical therapeutic (acute)
and/or
prophylactic (prevention) administration as appropriate.
6
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"Effective amount" refers to an amount of a compound of formula I which is
capable of inhibiting fungal and/or parasitic activity.
"Recipient" includes mammals, preferably, humans.
DETAILED DESCRIPTION
It has now been found that compounds represented by structure I are useful as
antifungal and antiparasitic agents or as an intermediate thereof. The most
convenient
means of producing compounds represented by structure I is by modifying
naturally
occurring compounds.
For illustrative purposes, Scheme I (below) starts with a specific
echinocandin
derivative. However, one can begin with any natural product, semi-synthetic or
synthetic
Echinocandin-type compound containing one or more hydroxy groups that are
capable of
being derivatized with one of the sugar moiety represented below:
R9 O O O~ R9 O O O
Rs~Rs
R9 R9 R9
or
R9 is defined as described above.
The term "echinocandin-type compounds" refers to compounds having the
following general structure including any simple derivatives thereof
Re R1
O
N ' Fi R
N
I O
Rs
R'
wherein R is an alkyl group, an alkenyl group, an alkynyl group, an aryl
group, or
heteroaryl group; R' is independently -H or -OH; R' is -H or -CH3; R3 is -H, -
CH3, -
2 0 CH~CO~I, or -CH,CH~NHz; R4 is -H or -OH; R' is -OH, -OP03Hz, -OPO,HCH3,
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OPO,HCH,, or -OSO,H; R6 is -H, -OH, or -OS03H; R' is -H or -CH3; Rg is -H or -
OH;
and pharmaceutically acceptable salts, esters, hydrates or solvates thereof.
"Natural product" refers to those secondary metabolites, usually of relatively
complex structure, which are of more restricted distribution and more
characteristic of a
specific source in nature. Suitable natural product starting materials of the
Echinocandin
cyclopeptide family include Echinocandin B, Echinocandin C, Aculeacin Ay,
Mulundocandin, Sporiofungin A, Pneumocandin Ao, WF1 I899A, and Pneumocandin
Bo.
The cyclic peptides used in the present invention can be produced by culturing
various microorganisms. In general, the cyclic peptides can be characterized
as a cyclic
hexapeptide nucleus with an acylated amino group on one of the amino acids.
The amino
group on the naturally-occurring cyclic peptide is typically acylated with a
fatty acid
group forming a side chain off the nucleus. Naturally-occurnng acyl groups
include, but
are not limited to, linoleoyl (Echinocandin B, C and D), palmitoyl (Aculeacin
Ay and
WF 11899A}, stearoyl, 12-methylmyristoyl (Mulundocandin), 10,12-
dimethylmyristoyl
(Sporiofungin A and Pneumocandin Ao}.
Semi-synthetic derivatives can be generally prepared by removing the fatty
acid
side chain from the cyclic peptide nucleus to produce a free amino group
(i.e., no pendant
acyl group -C(O)R). The free amine is then reacylated with a suitable acyl
group. For
example, the echinocandin B nucleus has been re-acylated with nonnaturally
occurring
2 0 side chain moieties to yield a number of antifungal agents. U.S. Patent
No. 4,293,489.
The N-acyl side chain includes a variety of side chain moieties known in the
art. Suitable
side chain moieties include substituted and unsubstituted alkyl groups,
alkenyl groups,
alkynyl groups, aryl groups, heteroaryl groups and combinations thereof.
Preferably, the
side chain contains both a linearly rigid section and a flexible alkyl section
to maximize
2 5 antifungal potency. Representative examples of preferred acyl side chains
include R
groups having the following structures:
\ /
/ \ - \ / H,
/ \ ~ / - \ /
or
/ \ / \
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where A, B, C and D are independently hydrogen, C~-C~, alkyl, C,-C~z alkynyl,
C~-C~2
alkoxy, C~-C~, alkylthio, halo, or -O-(CHZ)m-[O-(CHZ)~]p O-(C~-C~z alkyl) or -
O-(CHz)q X-
E; m is 2, 3 or 4; n is 2, 3 or 4; p is 0 or 1; q is 2, 3 or 4; X is
pyrrolidino, piperidino or
piperazino; and E is hydrogen, C1-C12 alkyl, C3-C12 cycloalkyl, benzyl or C3-
C12
cycloalkylmethyl.
Scheme I illustrates the general semi-synthetic route above where a natural
product (Compound II(a)) is modified to provide an acylated intermediate
(Compound
II(c)) which is modified to provide a Compound of structure I as in Scheme II.
R1 O H R1 O H
H
R2 N N.Rnat R2 N NH2
1 \\\~~H 1
\~~~ H
3 N O R HN O 3 N O R HN O
R ~ O R' Deacylate R >--~ O R'
HO
HO NH H N OH NH H 'N~ OH
O N~~~R1 O N~~~R1
HO / ~ O HO / I O
w ~R1 w ~4 ~R1
R4 R
II (a) II (b)
R1 O H H
R2~~'~~N N~R
R3 N H R1HN~070
O
Re-acylate ~ O R
HO
NH H\tt~H
O N ~R1
HO / I O
W ' 1
4 R
R
II (c)
Scheme I
The cyclic peptides of structure II(a) can be prepared by fermentation of
known
microorganisms. The cyclic peptide II(a) where R' and R4 are each hydroxy, R',
R3 and
R' are each methyl(cyclic nucleus corresponding to A-30912A) can be prepared
by the
procedure in U.S. Patent No. 4,293,482. The cyclic peptide II(a) where R' is
hydroxy, R2,
R3 and R' are each methyl, and R4 is hydrogen (cyclic nucleus corresponding to
A-
30912B) can be prepared by the procedure in U.S. Patent No. 4,299,763.
Aculeacin can
be prepared by the procedure in U.S. Patent No. 3,978,210. The cyclic peptide
II(a)
9
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where R' is CH2C(O)NH2, R' is methyl, RZ is hydrogen, and R' and R~ are
hydroxy can
be prepared by the procedure in U.S. Patent No. 5,198,421.
The naturally occurring cyclic peptide II(a) can be deacylated using
procedures
known in the art to provide an amino nucleus represented by structure II(b).
This reaction
is typically carried out enzymatically by exposing the naturally occurring
cyclic peptide to
a deacylase enzyme. The deacylase enzyme can be obtained from the
microorganism
Actinoplanes utahensis and used substantially in U.S. Patent Nos. 4,293,482
and
4,304,716. The deacylase enzyme can also be obtained from the Pseudomonas
species.
Deacylation can be accomplished using whole cells of A. utahensis or
Pseudomonas or
the crude or purified enzyme thereof or using an immobilized form of the
enzyme. See
European Patent Application No. 0 460 882. Examples of naturally occurring
cyclic
peptides useful as starting materials include aculeacin (palmitoyl side
chain),
tetrahydroechinocandin B (stearoyl side chain), Mulundocandin (branched C 15
side
chain), L-671,329 (C16 branched side chain), S 31794/F1 (tetradecanoyl side
chain),
sporiofungin (C15 branched side chain), FR901379 (palmitoyl side chain) and
the like. A
preferred naturally occurnng cyclic peptide is echinocandin B (Compound II(a)
where R',
R4 and R8 are each hydroxy, R2, R3 and R' are each methyl, and R'~' is
linoleoyl).
The amino nucleus II(b) can be re-acylated, as in U.S. Patent Nos. 5,646,11 l,
and
5,693,611, to yield compounds represented by structure II(c). See Preparation
12 for an
2 0 example of this transformation and U.S. Patent Nas. 5,646,111 and
5,693,611 for
preparation of the acyl groups at R. Cyclic peptides II(c) where R contains 1
or more
heterocyclic rings can be prepared as in U.S. Patent No. 5,693,611.
Compound I can be prepared from Compound II(c) as illustrated in Scheme 2
where R; and Rg are independently hydrogen or hydroxy provided at least one of
R' or R8
is a hydroxy group and R, Rl, R2, R3, R5, R' and R9 are as defined above.
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s O O OH
R /~\\~~
Rl O RB H R9 R9
R2~N NCR
N H RJ~ O III
R3~0 ~ or _
O R7
HO NH H~Hl
s ~ N R R9 O O OH
R \~ O
~Rl R9 R9
R4 R9
II (c) IV
R4 and RB is -H or -OH
Rl O Re H
R2~N~N1~R
N H RHN 00
R3~0 O~R7
HO NH H NN OrH
Rs / O N~~Rl
O
1
4 R
R
Scheme 2
A mixture of mono- and bis-coupled compounds represented by structure I can be
prepared by adding a protected compound of III or IV to Compound II(c)
dissolved or
suspended in a suitable solvent in the presence of a suitable acid. A
convenient and
preferred solvent for the reaction is 1,4-dioxane while a convenient and
preferred acid is
p-toluenesulfonic acid. The reaction can be performed at from OoC to the
reflux
temperature of the mixture but is typically performed at ambient temperatures
for about 4
hours. See Example I below for further instruction on reaction conditions.
Each mono
and bis isomer can be separated and deprotected separately.
Compounds III and IV can be prepared from Compounds V and VI, respectively,
as illustrated in Scheme 3 below where R9 is as described above.
11
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O C1 or Br
Rs s O OH
OH OH R
Rs Rs Rs Rs
Rs Rs
VI IV
O C1 or Br O-g pH O
O OH
R9 Rs R9 ERs
V III
Scheme 3
2-Butene-1,4-diol can be added to a chloro or bromo sugar of formula VI,
preferably one that is protected, dissolved in a suitable solvent, in the
presence of a
thermodynamic base, preferably silver carbonate or silver triflate, to form a
compound of
formula IV. Typically, the 1,4-diol can serve as the solvent and is used in a
large molar
excess. Furthermore, the silver carbonate is also used in a molar excess
relative to the
compound VI, typically on the order of about 2 equivalents. The reaction is
typically
performed at ambient temperature for about 18 hours. For further instruction
on this
conversion, see Preparation 11 below. Compound III can be prepared in an
analogous
manner from Compound V.
Compounds V and VI are known in the art and to the extent not commercially
available can be synthesized by techniques well known in the synthetic
chemical arts.
See, CoIlins and Ferrier, "Monosaccharides: Their Chemistry and Their Roles in
Natural
Products," John Wiley and Sons, New York, NY, 1995, and "Methods in
Carbohydrate
Chemistry", Vol VI, Academic Press, New York, N.Y., 1980.
For example, Compound VI (Compound V by analogy) can be prepared as
illustrated in Scheme 4 below where R9 is as described above.
12
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R9 O O-H or -Pg 9 O C1 or Br
R
CI- or Br-
R9 ~ wR9 R9 R9
Acetic Acid
R9 Rs
VI (a) VI
Scheme 4
Compound VI(a), dissolved or suspended in a suitable solvent, can be treated
with
a source of chloride or bromide ion, to provide Compound VI. Suitable sources
of ion
include acetyl chloride, hydrochloric acid, hydrobromic acid, mixtures
thereof, and the
like. A preferred solvent is the source of ion i.e. a preferred method of
performing the
reaction is to run it neat. See Preparations 8 and 9 below.
Compound VI(a) where R9 is hydroxy at each occurrence are known as
carbohydrates or monosaccharides (sugars). These sugars can be modified by
replacing
one or more hydroxy groups with hydrogen, azide, or amino to provide the rest
of the
compounds having structure VI(a) including disaccharides (or polysaccharide)
where R9
is a second sugar moiety (see, Examples 10 and 11 ). Such compounds can be
prepared as
illustrated in Scheme 5 below where Lg is an activated hydroxy leaving group.
O O-Pg O O-Pg
OH Lg
VII VIII
O O-Pg O O-Pg
- N3 ors Reduce
I-
N3 or I NHZ or H
IX X
Scheme S
A commercially available Compound VII can have its hydroxy groups) activated
for nucleophilic displacement by standard techniques known in the art. For
example, the
hydroxy group can be sulfonylated with methane-, benzene-, or p-toluene-
sulfonyl
2 0 chloride (or bromide) to provide Compound VIII where Lg is -OS02Me, -OS02-
phenyl,
or -OS02 p-toluenyl. An example of this transformation is illustrated in
Preparation 1
13
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below. At this point, the leaving group can be displaced by azide ion, e.g.,
from sodium
or potassium azide as in Preparation 2. Alternatively, the leaving group can
be displaced
by iodide ion from, e.g., sodium or potassium iodide as in Preparation 3. The
resulting
Compound IX can be reduced to form Compound X where one or more of R9 is amino
or
hydrogen by catalytic hydrogenation, as described in Preparation 4, or with a
reducing
agent such as nickel chloride hexahydrate. It is preferred that when an amino
group is
desired in the final product Compound I, that any azido groups are converted
to amino
groups after coupling to Compound II(a).
Compound I, where any of R9 is an amino group can be formed from the a
Compound 1 where R9 is azido as described by analogous procedures well known
in the
art. See, e.g., Larock, "Comprehensive Organic Transformations," pg. 409, VCH
Publishers, New York, N.Y., 1989.
Compound I where R5, R9, R98, R96, and/or R9' is a hydroxy group, can be
phosphorylated or phosphonylated by reaction with an appropriately substituted
dichloro
phosphate or phosphonic acid of formula V
R \0
Cl~P'C1
V
in the presence of a suitable base to provide, following an aqueous work-up,
to produce
2 0 Compound I where R5, R9, R9', R9b, and/or R°' are moieties of the
formula
O Ra R ~O
O OOH ~d HO~
respectively.
Suitable bases include lithium trimethylsilanolate (LiOTMS), and lithium
bis(trimethylsilyl)amide (LHMDS). A convenient and preferred solvent is an
aprotic
solvent such as tetrahydrofuran and/or dimethylformamide. For further
instruction on
such a transformation, see U.S. Patent No. 5,693,611, incorporated herein by
reference.
Alternati<<ely, the compounds represented by structure I where R9b is hydroxy
and/or R9' is hydroxymethyl can be sulfated by reaction with a suitable
sulfation reagent
by the procedures taught in Guiseley et al., J. Org. Chem., 26:1248 ( 1961 ).
Compound I having protecting groups can have its protecting groups) removed to
3 0 form a deprotected Compound I. Initial choices of protecting groups, and
methods for
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their removal, are well known in the art. See, e.g., Greene. Preferred choices
and
methods can be found in the Examples section which follows, e.g., Example 8.
Pharmaceutical salts are typically formed by reacting Compound 1 with an
equimolar or excess amount of acid or base. The reactants are generally
combined in a
mutual solvent such as diethylether, tetrahydrofuran, methanol, ethanol,
isopropanol,
benzene, and the like for acid addition salts, or water, an alcohol or a
chlorinated solvent
such as methylene chloride for base addition salts. The salts normally
precipitate out of
solution within about one hour to about ten days and can be isolated by
filtration or other
conventional methods.
Acids commonly employed to fonm acid addition salts are inorganic acids such
as
hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid,
phosphoric acid, and
the like, and organic acids such asp-toluenesulfonic, methanesulfonic acid,
ethanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid,
succinic
acid, citric acid, tartaric acid, benzoic acid, acetic acid, and the like.
Base addition salts include those derived from inorganic bases, such as
ammonium or alkali or alkaline earth metal hydroxides, carbonates,
bicarbonates, and the
like. Such bases useful in preparing the salts of this invention thus include
sodium
hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate,
sodium
carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide,
calcium
2 0 carbonate, and the like.
The particular counterion forming a part of any salt of this invention is not
of a
critical nature, so long as the salt as a whole is pharmacologically
acceptable and as long
as the counterion does not contribute undesired qualities to the salt as a
whole.
Preferred pharmaceutical acid addition salts are those formed with mineral
acids
2 5 such as hydrochloric acid and sulfuric acid, and those formed with organic
acids such as
malefic acid, tartaric acid, and methanesulfonic acid. Preferred
phanmaceutical base
addition salts are the potassium and sodium salt forms.
The optimal time for performing the reactions of Schemes 1 - 5 can be
determined
by monitoring the progress of the reaction by conventional chromatographic
techniques.
3 0 Choice of reaction solvent is generally not critical so long as the
solvent employed is inert
to the ongoing reaction and sufficiently solubilizes the reactants to afford a
medium
within which to effect the desired reaction. Unless otherwise indicated, all
of the
reactions described herein are preferably conducted under an inert atmosphere.
A
preferred inert atmosphere is nitrogen. Once a reaction is complete, the
intermediate
3 5 compound can be isolated by procedures well-known in the art, for example,
the
compound can be crystallized or precipitated and then collected by filtration,
or the
CA 02354056 2001-06-07
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reaction solvent can be removed by extraction, evaporation or decantation. The
intermediate compound can be further purified, if desired, by common
techniques such as
crystallization, precipitation, or chromatography over solid supports such as
silica gel,
alumina and the like, before carrying out the next step of the reaction
scheme.
Preferred compounds of the present invention are those compounds represented
by
structure I wherein R' is hydroxy at each occurrence; RZ, R3 and R7 are each
methyl; R is a
moiety of the formula:
\ / \ / \' / D / \ \ ~ \ /
or ;
R' is hydroxy; and Ra is Cl-C4 alkyl or Cl-C4 alkoxy; or
a pharmaceutically acceptable salt or solvate thereof.
More preferable are those compounds wherein RS is hydroxy; R is a moiety of
the
formula
\ / \ / \ / D
R" is a moiety of the formula
O O
R
Rs wRs
Rs
D is hydrogen or C3-C~ alkoxy; R° is independently hydrogen, hydroxy,
amino, or a
moiety of the formula
Rs
9a
n
where R9b is -OPOzRa, -OS03H, -H, -NH,, -OH, -O-Pg, or -NH-Pg and n is 1, 2,
or 3; or a
2 0 pharmaceutically acceptable salt thereof.
Even more preferable are those compounds wherein D is n-pentoxy; R9 is
independently -OH, -NH, or -OPO,Ra; or a pharmaceutical salt or solvate
thereof.
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Most preferred are those compounds wherein R9 is hydroxy at each occurrence;
and R9'' is -OPO,Ra, where Ra is methyl or methoxy; or a pharmaceutically
acceptable salt
or solvate thereof.
The following Preparations and Examples further describe how to synthesize the
compounds of the present invention but do not limit the invention. All
references cited
herein are hereby incorporated by reference. The terms fast atom bombardment
mass
spectroscopy and high performance liquid chromatography are abbreviated
"MS(FAB}"
and "HPLC" respectively. The following acronyms represent the corresponding
chemical
moieties: Ms = methanesulfonyl; Ac = acetyl; Me = methyl; and Tos = tosyl (or
p-
toluenesulfonyl).
Preparation I
1,2,3,4-Tetra-O-Acetyl-6-Deoxy-6-Methanesulfonyl-(3-D-Glucopyranose
Ms
O OAc
,,
~OAc
Ac OAc
In a 100 mL round bottom flask containing 50 mL dichloromethane at
0°C was
placed 1,2,3,4-tetra-O-acetyl-(3-D-glucopyranose (4.62 g, 13.26 mmol). To this
solution
was added triethylamine (2.77 mL, 19.90 mmol) followed by dropwise addition of
methanesulfonyl chloride (1.23 mL, 15.9 mmol). The reaction was then warmed to
room
temperature and stirred for 3 hours at which time the reaction was diluted
with 100 mL
dichloromethane. The organic layer was then washed two times each with 50 mL
of
water, 1N aqueous hydrochloric acid, saturated aqueous sodium bicarbonate and
brine.
The organic layer was dried over magnesium sulfate, filtered and the solvent
removed in
vacuo to yield 4.4~ g of crude title compound as a white solid which was used
directly in
Preparation 2. (79%).
Preparation 2
2 5 1,2,3,4-Tetra-O-Acetyl-6-Azido-6-Deoxy-(3-D-Glucopyranose
N3
O OAc
,,
Q~~~ ~OAc
Ac OAc
A 100 mL round bottom flask was charged with 40 mL anhydrous
dimethylformamide. sodium azide (2.19 g, 33.6 mmol), and crude 1,2,3,4-tetra-O-
acetyl-
6-deoxy-6-methanesulfonyl-(3-D-glucopyranose (1.9374 g, 4.54 mmol). The
resulting
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homogeneous solution was heated to 70°C and allowed to react for 10
hours. The
reaction was then diluted with 200 mL of ethyl acetate and washed with copious
amounts
of water. The organic layer was dried over magnesium sulfate, filtered and the
solvent
removed in vacuo. The resulting brown solid was purified by column
chromatography
over silica gel to yield 668.3 mg of the title compound (39.5%). MS(FAB)
calculated for
C14H19N3O9 (M - OCOCH3) 314.1, found 314.1.
Preparation 3
1,2,3,4-Tetra-O-Acetyl-6-Deoxy-6-Iodo-(3-D-glucopyranose
I
O OAc
~~'OAc
Ac OAc
A 1 L round bottom flask containing 500 mL of methyl ethyl ketone was charged
with 1,2,3,4-tetra-O-acetyl-6-deoxy-6-methanesulfonyl- (3-D-glucopyranose
(4.45 g,
10.44 mmol) and sodium iodide (15.73 g, 104.9 mmol). The reaction was heated
at reflux
for 24 hours. The solvent was removed in vacuo and the resulting residue was
taken up in
250 mL dichloromethane. The organic layer was washed with sodium thiosulfate
(2 x
100 mL), water (2 x 100 mL) and once with 100 mL of brine. The organic layer
was
dried over magnesium sulfate, filtered and the solvent removed in vacuo to
yield crude
1,2,3,4-tetra-O-acetyl-6-deoxy-6-iodo-[3-D-glucopyranose as a white solid
(4.99 g) which
was used directly in Preparation 4.
Preparation 4
2 0 1,2,3,4-Tetra-O-Acetyl-6-Deoxy-(3-D-Glucopyranose
Me O OAc
Q~ ~ ~~~'OAc
Ac OAc
1,2,3,4-Tetra-O-acetyl-6-deoxy-6-iodo-~i-D-glucopyranose (251.6 mg, 0.549
mmol) was dissol~~ed in 20 mL of ethanol. To this solution was added 1 mL of
triethylamine and ~% palladium on carbon (50.0 mg). The reaction mixture was
exposed
2 5 to 60 psi of hydrogen in a Parr apparatus at room temperature for 5 hours.
The palladium
on carbon was filtered off and the ethanol was removed in vacuo to yield a
white solid.
Purification via column chromatography over silica gel yielded 82.3 mg of the
title
compound as a white solid. (45%). MS(FAB) calculated for C14H20O9 (M+): 332.1.
Found: 331.1.
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Preparation S
1,2,5,6-Diacetone-4-p-Toluenesulfonyl-D-Allofuranose
~O
O ,,, O ..,0' \
TosO O
A 500 mL round bottom flask containing 160 mL of pyridine was charged with
1,2,5,6-diacetone-D-allofuranose (40.72 g, 156.44 mmol) andp-toluenesulfonyl
chloride
(45.67 g, 239.53 mmol). The reaction was allowed to stir at room temperature
for 27
hours. The reaction mixture was poured into 1.5 L of ice water and, upon
melting, was
filtered and dried in a vacuum oven at 30°C to yield 56.74 g of the
title compound which
was used crude in Preparation 6.
Preparation 6
1,2,5,6-Diacetone-4-p-Azido-D-Allofuranose
~O
--~O O ,.O~.
N .,~0~
3
In a 2 L round bottom flask containing 1 L of dimethylformamide was added
1,2,5,6-diacetone-4-p-toluenesulfonyl-D-allofuranose (56.44 g, 136.17 mmol)
and sodium
azide (142.12 g, 2.186 mol). The reaction mixture was heated to reflux and
allowed to
react for 20 hours. The reaction was cooled to room temperature and the
dimethylformamide was removed in vacuo. The resulting residue was partitioned
between 250 mL ethyl acetate and 250 mL water. The organic layer was washed
with 300
of water and brine. The organic layer was dried over magnesium sulfate,
filtered and the
2 0 solvent removed in vauco to obtain a crude brown oil. Purification via
column
chromatography over silica gel ( 10% ethyl acetate/hexanes) yielded the title
compound.
Preparation 7
HO
0 ,.OH
HO~~~ ~ ~'OH
N3
1,2,5,6-Diacetone-4-p-azido-D-allofuranose was suspended in 50 mL of water in
a
2 5 X00 mL round bottom flask. To this suspension was added Dowex 50 X 8-100
acidic
resin (20 g), the reaction mixture was heated at 60°C for 16 hours. The
resin was filtered
and the filtrate was lyophilized to yield 9.92 g of the title compound as a
white solid.
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Preparation 8
a-D-Acetochlororhamnose
Me,,~ O Cl
~~~OAc
Q
Ac OAc
In a 25 mL round bottom flask containing 10 mL of acetyl chloride was placed
1.0117 g of L-rhamnose. The reaction was stirred for 48 h at room temperature.
The
reaction was diluted with 100 mL of dichloromethane and washed with 50 mL ice
water
and then SO mL of cold saturated aqueous sodium bicarbonate. The organic layer
was
dried over magnesium sulfate and filtered. The solvent was removed in vacuo
and the
product used without further purification.
The following compounds were prepared by the procedure of Preparation 8:
Ac
o cl o cZ o cl
~ ~ ,,.
~~~~OAc Q ~~~N ~ ,,, ''~pAc
Ac OAc Ac OAcH I ~ Ac OAc
and
Preparation 9
2,3,4-Tri-O-Acetyl-6-Deoxy-(3-D-Glucopyranosyl Bromide:
Br
N~Ac
Ac OAcH
In a 10 mL round bottom flask containing 10 mL of glacial acetic acid was
placed
6-deoxyglucose (332.3 mg) and the reaction was cooled to 0°C.
Hydrobromic acid in
glacial acetic acid (5 mL of a 30 wt. % solution) was added dropwise. The
reaction was
stirred for 4 hours. The reaction was diluted with 100 mL of dichloromethane
and
washed with 50 mL ice water and then 50 mL of cold saturated aqueous sodium
2 0 bicarbonate. The organic layer was dried over magnesium sulfate and
filtered. The
solvent was removed in vacuo to yield the title compound as a yellow solid
(636.5 mg,
85.4%) and the product was used without further purification. MS(FAB)
calculated for
C12H1707Br (M - Br) 273.1, found 273.1.
The following compounds were prepared by the procedure of Preparation 9:
Ac
O
~~,
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N3 Ac
O Br Me O Br O Br
~~'OAc
Q~~~ ~~'OAc Q°~ ~~'OAc
Ac OAc ~ Ac OAc ~ and Ac N3
Preparation 10
3,4,6-Tri-O-Acetyl-2-Azido-2-Deoxy-(3-D-Glucopyranosyl Bromide
Ac
O Br
Q,,, ,,,N
3
Ac OAc
A 1 L flask containing 400 mL of acetonitrile was charged with sodium azide
(7.75 g, 119.2 mmol) and ceric ammonium nitrate (120.7 g, 219.4 mmol). The
resulting
suspension was cooled to -30°C and a solution of tri-O-acetyl-D-glucal
(20.75 g, 76.22
mmol) in 100 mL acetonitrile was added to it dropwise. The reaction mixture
was stirred
at -30°C for 20 hours and then warmed to room temperature, taken up in
800 mL of
diethyl ether and washed with water (3 x 250 mL). The organics were dried over
magnesium sulfate, filtered, and the solvent removed in vacuo to yield an oil.
This oil
was placed in a 1 L flask containing 400 mL of acetonitrile and lithium
bromide (33.53 g,
386.1 mmol) and stirred at room temperature for at least 4 hours. The solvent
was
removed in vacuo and the resulting residue was taken up in 400 mL of
dichloromethane.
The organic layer was washed with water (2 x 250 mL), dried over magnesium
sulfate
and filtered. The solvent was removed in vacuo to yield the title compound as
a dark
yellow oil which eras used directly without further purification.
The following compounds were prepared by the procedure of Preparation 10:
Ac
O Br Me,,, O Br
"N ~N
3 Q 3
Ac OAc ~d Ac OAc
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Preparation Il
4-Acetoglucosyl-2-Butenol
Ac
O O OH
~~'OAC
Ac OAc
A flask was charged with acetobromoglucose (10.05 g, 24.44 mmol), silver
carbonate ( 13.64 g, 49.47 mmol} and 200 mL of 2-butene-1,4-diol. The reaction
mixture
was allowed to stir for 18 hours at room temperature. The crude reaction
mixture was
filtered over a celite pad to remove silver salts and washed several times
with ethyl
acetate. The organics were then washed several times with copious amounts of
water to
removed unreacted 2-butene-1,4-diol. The organic layer was then dried with
magnesium
sulfate and concentrated in vacuo to yield a yellow oil. Silica gel column
chromatography of the oil eluting with 70% ethyl acetate in hexanes yielded
2.37 g of the
title compound. (23.2%).
The following compounds were prepared by the procedure of Preparation 11:
_ Ac _
Ac ~ O Ac
O O OH Ac-0,,, p O OH
O
Q ~~'OAc Ac-O ~°O°~ ~~'OAc
Ac OAc ~d OAc OAc
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Preparation 12
n-pentyl
OH
The A-30912A nucleus (60.2 mmol) and the 2,4,5-trichlorophenol ester of [[(4"-
pentyloxy)-1,1':4',1"-terphenyl]-4-carboxylic acid (26.0 g, 48.2 mmol) were
combined in
8.5 L of dimethylformamide. The resultant reaction mixture was stirred for
approximately 48 hours at room temperature and then the solvent was removed in
vacuo
to provide a residue. This residue was slurned in ether, collected by
filtration, washed
with methylene chloride and then dissolved in methanol or a 1:1 (v/v)
acetonitrile/water
mixture. The resultant solution is subjected to reverse phase HPLC (C18;
eluent of 20-
40% aqueous acetonitrile containing 0.5% monobasic ammonium phosphate (w/v);
20
mL/min.; 230 nm). After removing the unreacted A30912A nucleus, the desired
product
is eluted from the column using an eluent of aqueous acetonitrile. The
fractions
containing the desired product are combined and then concentrated in vacuo or
lyophilized to provide 18 g of the title compound. MS(FAB):
1140.5103 (M+1).
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Examples 1-14
Examples 1 - 14 have the following base structure:
-pentyl
N
8
OH
.... N
O
HN O
O~,CH3
HO NH H N OHl1
N .""OH
HO / O
w ~ .~,"OHO
R4
Example 1
Ac
O O O
~,,
OAc HO,,, ~~~~I'
Ra = Ac OAc R4 -
The compound of Preparation 12 (269.8 mg, 0.237 mmol), the first compound of
Preparation I 1 (508.1 mg, 1.214 mmol), p-toluenesulfonic acid (100.8 mg,
0.530 mmol)
and 15 mL of 1,4-dioxane were placed in a flask and stirred at room
temperature for 4
hours. The crude reaction mixture was filtered and purified via HPLC eluting
with 40%
water in acetonitrile at 60 mL/minute using a 3 x 40 x 100 mm Novapak CIg
column to
afford 12.4 mg of the title compound. (3.4%). MS(FAB) (m/e): 1562.7 (M+Na)
Examples 2 - 7 were prepared by the procedure of Example 1.
Example 2
OAC
O O O
Q~~~ Y ~°OAc Hp,, ~L,I,
Rs - Ac oAc R4 =
MS(FAB) (m/e): 1563.5 (M+Na)
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Example 3
OMe
O O O
O
Q~~'OAc HO,,,
Rs = Ac oAc
Example 4
OMe
O O O
~'\O
Q~~'OAc HO,,,
Ra - Ac OAc
MS(FAB) (m/e): 1549.0 (M+Na)
Example S
Ac
O O O
Q ~ ~~~OAc Hp,,, ,,''..
Rs - Ac OAc Ra -
MS(FAB) (m/e): 1562.7 (M+Na)
Example 6
Ac
O O O
Q ~ ~~~OAc HO,,, ,..,..
Rs = Ac OAc R4 =
MS(FAB) (m/e): 163.7 (M+Na)
Example 7
Ac ~ Ac
O O O~ O O O..I
C?~ ~ Y~~~'OAc Q~,, ~''OAc
Rs - Ac OAc Ra - Ac OAc
MS(FAB ) (m/e): 1940.8 (M+); MS(FA.B) (m/e): 1964.0 (M+Na)
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Example 8
OMe ~ OMe
O O O O~ O O O O
°~OAc Q''' '~~OAC
Re = Ac OAc Ra ! Ac OAc
MS(FAB) (m/e): 1935.2 (M+Na); MS(FAB) (m/e): 1935.1 (M+Na}
Example 9
OAc ~ Ac
O O O~ O O O
~~°OAc Q ~~'OAc
Rg - Ac OAc R., ~ Ac OAc
MS(FAB) (m/e): 1963.9 (M+Na); MS(FAB) (m/e): 1963.9 (M+Na}
Example 10
Ac
O
Ac
Ac-0,,, p O O
~O
Ac O . ~--O''' ~''OAc Hp," ,,,III
Rs - OAc OAc R4 =
Example Il
Ac
O
Ac
Ac-0.,, O O O
O
Ac O - ~-'O''' ~~'OAc Hp,,, ,,II,.
R$ = oAc OAc R4 -
Example 12
H
O O O
HO''' ~''O ~H
Rg - OH R4 - HO.,, ",,I'
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The compound of Example 1 (9.3 mg, 0.0060 mmol), potassium carbonate (5.3
mg, 0.0038 mmol), and 4 mL of a SO% mixture of methanol in water were combined
and
stirred for 30 minutes at room temperature. The crude reaction mixture was
filtered and
purified via HPLC eluting with 40% water in acetonitrile at 60 mL/minute using
a 3 x 40
x 100 mm Novapak C 1 g column to afford 5.1 mg of the title compound. (62%)
MS(FAB) (m/e): 1373.7 (M+).
Examples 13 - 22 were prepared by the procedure of Example 1.
Example 13
H
O O O
HO~~~ ~'OH HO,, ,II
Rs - OH Ra ~
Example 14
OMe
O O O
0
HO~~~ ~~'OH
HO.,, ,,,,,.
Re - OH R4 =
MS(FAB) (m/e): 1427.5 (M+Na)
Example ~5
OMe
O O O
O
HO~~'OH
HO.,, ,",,,
Rs - OH R4 -
Example 16
H
p O O
HO Y.~~'OH HO,, ,,Ir'
Rs - OH Ra -
MS(FAB) (m/e): I372 (M+).
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Example 17
H
O O O
HO Y~~'OH HO''' ~~.~.
Ra - OH R4 =
MS(FAB) (m/e): 1372 (M+).
Example 18
H ~ H
O O O~ O O O
HO~~~ ~~'OH HO~~~ ~~'OH
R8 = OH Ra = OH
Example 19
OMe ~ OMe
O O O O~ O O O O
HO~~~ ~~'OH HO~~~ ~~'OH
~, a = OH Ra ' OH
Example 20
H ~ H
O O O~ O O O
HO Y~~'OH ~'\ HO ~~'OH
R8 = OH Ra = OH
MS(FAB) (m/e): 1605.8 (M+).
Example 21
HO
H
HO,,, O O O
O
HO _ '~.0,,, -,,OH HO,,, ~~.,,.
R: - off OH- Ra -
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Example 22
HO
H
HO ,, p O O
O
HO _ ~~.0,,, ,,,OH HO',' ~,,L,
Rs = OH OH Ra
Representative examples of Compound I exhibit antifungal and antiparasitic
activity. For example, Compound I inhibits growth of various infectious fungi
including
Candida spp. such as C. albicans, C. parapsilosis, C. krusei, C. glabrata, or
C. tropicalis,
G lusitaniae; Torulopus spp. such as T. glabrata; Aspergillus spp. such as A.
fumigatus;
Histoplasma spp. such as H. capsulatum; Cryptococcus spp. such as C.
neoformans;
Blastomyces spp. such as B. dermatitidis; Fusarium spp., Trichophyton spp.,
Pseudallescheria boydii, Coccidioides immitis, Sporothrix schenckii and the
like.
Antifungal activity of a test compound is determined in vitro by obtaining the
minimum inhibitory concentration (MIC) of the compound using a standard agar
dilution
test or a disc-diffusion test. The compound is then tested in vivo (in mice)
to determine
the effective dose of the test compound for controlling a systemic fungal
infection.
Accordingly, representative compounds of the present invention were tested
for,
and displayed, antifungal activity against at least one of the following
fungii: G albicans,
C. parapsilosis, C. neoformans, Histoplasma spp, and A. fumigatus.
The compounds of the invention also inhibit the growth of certain organisms
primarily responsible for opportunistic infections in immunosuppressed
individuals. For
example, the compounds of the invention inhibit the growth of Pneumocystis
carinii the
2 0 causative organism of pneumocystis pneumonia (PCP) in AIDS and other
immunocompromised recipients. "Topley and Wilson's Microbiology and Microbial
Infections," Vol. 5, Ch. 22, Oxford University Press, Inc., New York, N.Y.,
1998. Other
protozoans that are inhibited by compounds of formula I include Plasmodium
spp.,
Leishmania spp., Ti~panosoma spp., Cryptosporidium spp., Isospora spp.,
Cyclospora
2 5 spp., Ti-ichomonas spp., Microsporidiosis spp. and the like.
The dose of Compound I administered varies depending on such factors as the
nature and severity of the infection, the age and general health of the
recipient and the
tolerance of the recipient to the active ingredient. The particular dose
regimen likewise
can vary according to such factors and can be given in a single daily dose or
in multiple
30 doses during the day. The regimen can 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
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WO 00135944 PCT/US99/29914
level of from about 0.01 mg/kg to about 100 mg/kg of body weight of the active
compound of this invention. Preferred daily doses are generally from about 0.1
mg/kg to
about 60 mg/kg, more preferably from about 2.5 mg/kg to about 40 rng/kg.
Compound I can be administered parenterally, for example using intramuscular,
sub-cutaneous, or intra-peritoneal injection, nasal, or oral means. In
addition to these
methods of administration, Compound I can be applied topically for skin
infections.
The present invention also provides pharmaceutical formulations useful for
administering the compounds of the invention. The active ingredient in such
formulations
comprises from 0.1% to 99.9% by weight of the formulation, more generally from
about
10% to about 30% by weight.
For parenteral administration, the formulation comprises Compound I and a
physiologically acceptable diluent such as deionized water, physiological
saline, 5%
dextrose and other commonly used diluents. The formulation can contain a
solubilizing
agent such as a polyethylene glycol or polypropylene glycol or other known
solubilizing
agent. Such formulations can be made up in sterile vials containing the active
ingredient
and one or more excipients in a dry powder or lyophilized powder fore. Prior
to use, a
physiologically acceptable diluent is added and the solution withdrawn via
syringe for
administration to the recipient.
The present pharmaceutical formulations are prepared by known procedures using
2 0 known and readily available ingredients. In making the compositions of the
invention,
the active ingredient will generally be admixed with a carrier, ar diluted by
a carrier, or
enclosed within a carrier which can be in the form of a capsule, sachet, paper
or other
container. When the Garner serves as a diluent, it can be a solid, semi-solid
or liquid
material which acts as a vehicle, excipient or medium for the active
ingredient. Thus, the
2 5 compositions can be in the form of tablets, pills, powders, lozenges,
sachets, cachets,
elixirs, suspensions, emulsions, solutions, syrups, aerosols, (as a solid or
in a liquid
medium), ointments containing, for example, up to 10% by weight of the active
ingredient, soft and hard gelatin capsules, suppositories, sterile injectable
solutions, sterile
packaged powders and the like.
3 0 For oral administration, the active ingredient is filled into gelatin
capsules or
formed into tablets. Such tablets can also contain a binding agent, a
dispersant or other
suitable excipients suitable for preparing a proper size tablet for the dosage
and particular
Compound represented by structure I. For pediatric or geriatric use the active
ingredient
can be formulated into a flavored liquid suspension, solution or emulsion. A
preferred
35 oral formulation is linoleic acid, cremophor RH-60 and water and preferably
in the
CA 02354056 2001-06-07
WO 00/35944 PCT/US99/29914
amount (by volume) of 8% linoleic acid, 5% cremophor RH-60, 87% sterile water
and
Compound I in an amount of from about 2.5 to about 40 mg/mL.
For topical use the active ingredient can be formulated with a dry powder for
application to the skin surface or it can be formulated in a liquid
formulation comprising a
solubilizing aqueous liquid or non-aqueous liquid, e.g., an alcohol or glycol.
Formulations
The following formulation examples are illustrative only and are not intended
to
limit the scope of the invention in any way. The term "active ingredient"
refers to a
compound of structure I or a pharmaceutically acceptable salt or solvate
thereof.
Formulation Example 1
Hard gelatin capsules are prepared using the following ingredients:
Quantity
(mg/capsule)
Active ingredient 250
Starch, dried 200
Magnesium stearate 10
Total 460 mg
Formulation Example 2
A tablet is prepared using the ingredients below.
2 0 Quantity
(mg/capsule)
Active ingredient 250
Cellulose, microcrystalline 400
Silicon dioxide, fumed 10
2 5 Stearic acid
Total 665 mg
The components are blended and compressed to form tablets each weighing 665 mg
Formulation Example 3
An aerosol solution is prepared containing the following components:
3 0 W ei ht
Active ingredient 0.25
Ethanol 25.75
Propellant 22
(Chlorodifluoromethane) 74.00
3 5 Total 100.00
The active compound is mixed with ethanol and the mixture added to a portion
of
the propellant 22, cooled to -30°C and transferred to a filling device.
The required
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WO 00/35944 PCTNS99/29914
amount is then fed to a stainless steel container and diluted with the
remainder of the
propellant. The valve units are then fitted to the container.
Formulation Example 4
Tablets, each containing 60 mg of active ingredient, are made as follows:
Active ingredient 60 mg
Starch 45 mg
Microcrystalline cellulose 35 mg
Polyvinylpyrrolidone (as 10% solution in water) 4 mg
Sodium carboxymethyl starch 4.5 mg
Magnesium stearate 0.5 mg
Talc 1 mg
Total 150 mg
The active ingredient, starch and cellulose are passed through a No. 45 mesh
U.S.
sieve and mixed thoroughly. The aqueous solution containing polyvinyl-
pyn:olidone is
mixed with the resultant powder, and the mixture then is passed through a No.
14 mesh
U.S. sieve. The granules so produced are dried at 50°C and passed
through a No. 18
mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate and talc,
previously passed through a No. 60 mesh U.S. sieve, are added to the granules
which,
after mixing, are compressed on a tablet machine to yield tablets each
weighing 150 mg.
2 0 Formulation Example S
Capsules, each containing 80 mg of active ingredient, are made as follows:
Active ingredient 80 mg
Starch 59 mg
Microcrystalline cellulose 59 mg
2 5 Magnesium stearate 2 mg
Total 200 mg
The active ingredient, cellulose, starch and magnesium stearate are blended,
passed through a No. 45 mesh U.S. sieve, and filled into hard gelatin capsules
in 200 mg
quantities.
3 0 Formulation Example 6
Suppositories, each containing 225 mg of active ingredient, are made as
follows:
Active ingredient 225 mg
Saturated fatty acid glycerides 2,000 mg
Total 2,225 mg
35 The active ingredient is passed through a No. 60 mesh U.S. sieve and
suspended
in the saturated fatty acid glycerides previously melted using the minimum
heat
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WO 00/35944 PCT/US99/Z9914
necessary. The mixture is then poured into a suppository mold of nominal 2 g
capacity
and allowed to cool.
Formulation Example 7
Suspensions, each containing 50 mg of active ingredient per 5 mL dose, are
made
as follows:
Active ingredient 50 mg
Sodium carboxymethyl cellulose 50 mg
SY~P 1.25 mL
Benzoic acid solution 0.10 mL
Flavor q,v.
Color
q.v.
Purified water to total 5 mL
The active ingredient is passed through a No. 45 mesh U.S. sieve and mixed
with
the sodium carboxymethyl cellulose and syrup to form a smooth paste. The
benzoic acid
solution, flavor and color are diluted with a portion of the water and added,
with stirring.
Sufficient water is then added to produce the required volume.
Formulation Example 8
An intravenous formulation can be prepared as follows:
Active ingredient 100 mg
2 0 Isotonic saline 1,000 mL
The solution of the above ingredients generally is administered intravenously
to a subject
at a rate of 1 mL per minute.
33
