Note: Descriptions are shown in the official language in which they were submitted.
CA 02379851 2002-02-O1
WO 01/05814 PCT/US00/15017
PSEUDOMYCIN N-ACYL SIDE-CHAIN ANALOGS
FIELD OF THE INVENTION
The present invention relates to pseudomycin compounds,
in particular, semi-synthetic pseudomycin compounds having
novel N-acyl side-chains.
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
WO 01/05814 CA 02379851 2002-02-O1 PCT/US00/15017
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.
Therefore, there is a need to identify compounds within this
class that are useful for treating fungal infections without
the currently observed adverse side effects.
2
WO 01/05814 cA 02379851 2002-02-0l pCT/jJS00/15017
BRIEF SUMMARY 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
HO
O
OH
O~ N H
NH H N OH
HO O
NH SCI
O
H2N \O O O
NH ~
O O O N' _R
H
N NH
O
OH NH2
HZN O
I
wherein R is
Rb Rb~ Ra
,Rf
Ra~ Ic ~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-
3
W~ 01/05814 CA 02379851 2002-02-O1 PCT/US00/15017
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 provided that Rb~ is
not hydroxy when Ra, Rb, Rd, Re are hydrogen, R~ is
hydrogen and Rf is n-hexyl, n-octyl or n-decyl, or Ra,
Rb, Rd, Re are hydrogen, R~ is hydroxy and Rf is n-
octyl, n-nonyl, or n-decyl;
R~ is hydrogen, hydroxy, C1-C4 alkoxy,
hydroxyalkoxy, or taken together with Re forms a 6-
membered aromatic ring or CS-C6 cycloalkyl ring;
Re is hydrogen, or taken together with Rf is a
six-membered aromatic ring, C5-C14 alkoxy substituted
six-membered aromatic ring, or CS-C14 alkyl substituted
six-membered aromatic ring, and
Rf is C$-C18 alkyl, CS-C11 alkoxy, or biphenyl; or
R is
R9
Rn
O
where
Rg is hydrogen, or C1-C13 alkyl, and
4
WO 01/05814 CA 02379851 2002-o2-O1 pCT/US00/15017
Rh i s C1-C15 alkyl , C4-C15 alkoxy, ( C1-Clo
alkyl ) phenyl , - ( CHZ ) n-aryl , or - ( CHZ ) n- ( CS-C6
cycloalkyl), where n = 1 or 2; or
R is
R
m
,
where
Rl is a hydrogen, halogen, or C5-C8 alkoxy, and
m is 1, 2 or 3;
R is
OH
where
R~ i s C5-C14 alkoxy or CS-C14 alkyl , and p = 0 , 1 or
2;
R is
-N
Rk
where
Rk is C5-C14 alkoxy; or
R is - (CH2) -NRm- (C13-C18 alkyl) , where Rm is H, -CH3 or
5
WO X1/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
-C ( 0 ) CH3 ; and
pharmaceutically acceptable salts and solvates thereof.
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
an animal in need thereof, which comprises administering to
the animal the pseudomycin compound I described above.
In yet another embodiment of the present invention, a
process is provided for producing the free amine nucleus of
a pseudomycin compound which may be acylated to form the
compounds represented by structure I above. The process
includes the steps of treating a pseudomycin compound which
contains an N-aryl alkyl side-chain having at least one
gamma or delta hydroxyl group (e.g., pseudomycin A, A' or C)
with trifluoroacetic acid or acetic acid.
Definitions
As used herein, the term "free amine pseudomycin
nucleus" or "pseudomycin nucleus" refers to the structure
I-A below:
6
WO X1/05814 CA 02379851 2002-02-O1 PCZ'/jJS00/15017
O
HO
O
OH
O~ N H
NH H N OH
HO O
NH SCI
O
R' O O
NH
O O O NH2
N N NH
H
O
OH R
R' O
I-A
wherein R' is -NH2 or -NHp-Pg where Pg is an amino
protecting group and p is 0 or 1.
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.
7
WO 01/05814 CA 02379851 2002-02-O1 pC't/[JS00/15017
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
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, 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 commonly employed to block or
8
WO 01/05814 CA 02379851 2002-02-O1 PCT/US00/15017
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.
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
9
WO 01/05814 CA 02379851 2002-02-O1 PCT/US00/15017
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
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 "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 deacylation of the N-
acyl group of the L-serine unit of a pseudomycin compound
followed by reacylation with a new N-acyl group provides
compounds having in vitro indications which suggest that the
W~ 01/05814 CA 02379851 2002-02-O1 pCT/[JS00/15017
new compounds may be active against C. albican, C,
neoformans, and/or Aspergillus fumigatus.
Scheme I below illustrates the general procedures for
synthesizing Compound I from any one of the naturally
occurring pseudomycins. Although a naturally occurring
pseudomycin compound is depicted in scheme I, those skilled
in the art will understand that side-chain modification of
semi-synthetic derivatives of the naturally occurring
pseudomycin compounds may be accomplished in a similar
manner. In. general, four synthetic steps are used to
produce Compound I: (1) selective amino protection; (2)
chemical or enzymatic deacylation of the N-acyl side-chain;
(3) reacylation with a different side-chain; and (4)
deprotection of the amino groups.
11
WO 01/05814 CA 02379851 2002-02-O1 PCT/US00/15017
O O
HO HO
OH O OH
O NH H $ N OH HO O NH H N OH
HO, ~'~ O ~CI
NH O s CI 1 ~H O
O~ ~ pgHN~O O
H
HzN NH O O O ~ ~' ~ N R~ O NH O H Oy' ~N R,
O a H ~ N N NH
3 N~NH ~ H O ~.,~ O
O ~ OH NHPg
OH NH2 PgHN O
HzN O
(2)
O
HO O
O OH HO
\ O OH
HO ~O NH H N OH O NH H N OH
/ NH O O~CI HO~,~ O CI
P HN O O (3) O
9 ~ .mH t P ".
O O H O~ N R p~ NHz
H N~-!'NH ~ O
OH ~NHPg OH NHPg
PgHN O PgHN U
1 ~4~
O
HO
p OH
HO O NH H N OH
~~ O~
O~CI
O
H
O H 0~~~~ N R
N 0~., N~ O
OH ' NH2
O
Scheme I
12
WO 01/05814 CA 02379851 2002-02-O1 PCT/US00/15017
The pendant amino groups at positions 2, 4 and 5 may be
protected using any standard means known to those skilled in
the art for amino protection. The exact genus and species
of amino protecting group employed is not critical so long
as the derivatized amino group is stable to the conditions
of subsequent reactions) on other positions of the
intermediate molecule and the protecting group can be
selectively removed at the appropriate point without
disrupting the remainder of the molecule including any other
amino protecting group(s). Suitable amino-protecting groups
include benzyloxycarbonyl, p-nitrobenzyloxycarbonyl,
p-bromobenzyloxycarbonyl, p-methoxybenxyloxycarbonyl,
p-methoxyphenylazobenzyloxycarbonyl,
p-phenylazobenzyloxycarbonyl, t-butyloxycarbonyl,
cyclopentyloxycarbonyl, and phthalimido. Preferred amino
protecting groups are t-butoxycarbonyl (t-Boc),
allyloxycarbonyl (Allot), phthalimido, and benzyloxycarbonyl
(CbZ or CBZ). Most preferred is allyloxycarbonyl and
benzyloxycarbonyl. Further examples of suitable protecting
groups are described in T.W. Greene, "Protective Groups in
Organic Synthesis," John Wiley and Sons, New York, N.Y.,
(2nd ed., 1991), at chapter 7.
The deacylation of a N-acyl group having a gamma or
delta hydroxylated side chain (e. g., 3,4-dihydroxytetra
deconoate) may be accomplished by treating the amino
13
WU 01/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
protected pseudomycin compound with a 5-20o aqueous acidic
solution. 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 run at about
40°C. A water soluble organic solvent may be used to assist
in solubilizing the pseudomycin compound. Suitable aqueous
solvent systems include acetonitrile, water, and mixtures
thereof. Acetonitrile was particularly useful when
deacylating a protected pseudomycin compound. A preferred
acidic solution for deacylating a protected pseudomycin
compound is 8% aqueous trifluoroacetic acid in acetonitrile.
Organic solvents accelerate the reaction; however, the
addition of an organic solvent may lead to other by-
products. Pseudomycin compounds lacking a delta 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 (see, e.g.,
U.S. Patent No. 5,573,936). 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
14
WO 01/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
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 or "PSN") 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-0-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).
Alternatively, a solid phase synthesis may be used
where a hydroxybenzotriazole-resin (HOBt-resin) serves as
the coupling agent for the acylation reaction.
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
W~ 01/05814 CA 02379851 2002-02-O1 PCT/US00/15017
groups; aliphatic acids containing nitrogen or oxygen within
the aliphatic chain; aromatic acids substituted with alkyl,
hydroxy, alkoxy and/or alkyl amino groups; and
heteroaromatic acids substituted with alkyl, hydroxy, alkoxy
and/or alkyl amino groups. The acylated product may be
useful as an active antifungal agent or as an intermediate
for the production of an active compound. Even though some
compounds were not as useful as others, the activity
profiles provide valuable insight into the design trends
needed to achieve optimum activity.
Once the amino group is acylated, then the amino
protecting groups (at positions 2, 4 and 5) may be 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),
16
W~ 01/05814 CA 02379851 2002-02-O1 PCT/jJS00/15017
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
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 (ATCC 67028) 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
17
WO 01/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
of Dutch elm disease," Proc. Natl. Acad. Sci. USA, 84, 6447-
6451 (1987).
A strain of P. syringae that is suitable for production
of one or more pseudomycins can be isolated from
environmental sources including plants (e. g., barley plants,
citrus plants, and lilac plants) as well as, sources such as
soil, water, air, and dust. A preferred stain is isolated
from plants. Strains of P. syringae that are isolated from
environmental sources can be referred to as wild type. As
used herein, "wild type" refers to a dominant genotype which
naturally occurs in the normal population of P. syringae
(e.g., strains or isolates of P. syringae that are found in
nature and not produced by laboratory manipulation). Like
most organisms, the characteristics of the pseudomycin-
producing cultures employed (P. syringae strains such as MSU
174, MSU 16H, MSU 206, 25-B1, 7H9-1) are subject to
variation. Hence, progeny of these strains (e. g.,
recombinants, mutants and variants) may be obtained by
methods known in the art.
P. syringae MSU 16H is publicly available from the
American Type Culture Collection, Parklawn Drive, Rockville,
MD, USA as Accession No. ATCC 67028. P. syringae strains
25-B1, 7H9-1, and 67 H1 were deposited with the American
Type Culture Collection on March 23, 2000 and were assigned
the following Accession Nos.:
18
WO ~l/05814 CA 02379851 2002-o2-O1 pCT/US00/15017
25-B1 Accession No. PTA-1622
7H9-1 Accession No. PTA-1623
67 H1 Accession No. PTA-1621
Mutant strains of P. syringae are also suitable for
production of one or more pseudomycins. As used herein,
"mutant" refers to a sudden heritable change in the
phenotype of a strain, which can be spontaneous or induced
by known mutagenic agents, such as radiation (e. g.,
ultraviolet radiation or x-rays), chemical mutagens (e. g.,
ethyl methanesulfonate (EMS), diepoxyoctane, N-methyl-N-
nitro-N'-nitrosoguanine (NTG), and nitrous acid), site-
specific mutagenesis, and transposon mediated mutagenesis.
Pseudomycin-producing mutants of P. syringae can be produced
by treating the bacteria with an amount of a mutagenic agent
effective to produce mutants that overproduce one or more
pseudomycins, that produce one pseudomycin (e. g.,
pseudomycin B) in excess over other pseudomycins, or that
produce one or more pseudomycins under advantageous growth
conditions. While the type and amount of mutagenic agent to
be used can vary, a preferred method is to serially dilute
NTG to levels ranging from 1 to 100 [ug/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
19
WO 01/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
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 [ug/ml. Preferred strains exhibit the
characteristic of producing one or more pseudomycins when
grown on a medium including three or fewer amino acids and
optionally, either a lipid, a potato product or combination
thereof.
Recombinant strains can be developed by transforming
the P. syringae strains, using procedures known in the art.
Through the use of recombinant DNA technology, the P.
syringae strains can be transformed to express a variety of
gene products in addition to the antibiotics these strains
produce. For example, one can modify the strains to
introduce multiple copies of the endogenous pseudomycin-
biosynthesis genes to achieve greater pseudomycin yield.
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.
WO 01/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
Alternatively, glycine is combined with one or more of a
potato product and a lipid. Culturing is conducted under
conditions effective for growth of P. syringae and
production of the desired pseudomycin or pseudomycins.
Effective conditions include temperatures from about 22°-C to
about 27°-C, and a duration of about 36 hours to about 96
hours. Controlling the concentration of oxygen in the
medium during culturing of P. syringae is advantageous for
production of a pseudomycin. Preferably, oxygen levels are
maintained at about 5 to 50% saturation, more preferably
about 30% saturation. Sparging with air, pure oxygen, or
gas mixtures including oxygen can regulate the concentration
of oxygen in the medium.
Controlling the pH of the medium during culturing of P.
syringae is also advantageous. Pseudomycins are labile at
basic pH, and significant degradation can occur if the pH of
the culture medium is above about 6 for more than about 12
hours. Preferably, the pH of the culture medium is
maintained between 6 and 4. P. syringae can product 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.
21
WO 01/05814 CA 02379851 2002-o2-O1 pCT/US00/15017
Choice of P. syringae strain can affect the amount and
distribution of pseudomycin or pseudomycins produced. For
example, strains MSU 16H and 67 H1 each produce
predominantly pseudomycin A, but also produce pseudomycin B
and C, typically in ratios of 4:2:1. Strain 67 H1 typically
produces levels of pseudomycins about three to five fold
larger than are produced by strain MSU 16H. Compared to
strains MSU 16H and 67 H1, strain 25-B1 produces more
pseudomycin B and less pseudomycin C. Strain 7H9-1 are
distinctive in producing predominantly pseudomycin B and
larger amount of pseudomycin B than other strains. For
example, this strain can produce pseudomycin B in at least a
ten fold excess over either pseudomycin A or C.
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.
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
22
WO 01/05814 CA 02379851 2002-02-O1 PCT/L1S00/15017
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,
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 prodrug 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 derivative) is
typically formulated into pharmaceutical dosage forms to
provide an easily controllable dosage of the drug and to
give the physician, patient, or veterinarian an elegant and
easily handleable product. Formulations may comprise from
23
WO 01/05814 CA 02379851 2002-02-O1 pCT/US00/15017
0.1% to 99.90 by weight of active ingredient, more generally
from about 10% to about 30% by weight.
As used herein, the term "unit dose" or "unit dosage"
refers to physically discrete units that contain a
predetermined quantity of active ingredient calculated to
produce a desired therapeutic effect. When a unit dose is
administered orally or parenterally, it is typically
provided in the form of a tablet, capsule,,pill, powder
packet, topical composition, suppository, wafer, measured
units in ampoules or in multidose containers, etc.
Alternatively, a unit dose may be administered in the form
of a dry or liquid aerosol which may be inhaled or sprayed.
The dosage to be administered may vary depending upon
the physical characteristics of the animal, the severity of
the animal's symptoms, and the means used to administer the
drug. 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,
24
WO 01/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
lubricating agents, surfactants, buffers, tonicity agents,
bulking agents, stabilizers, emulsifiers, suspending agents,
preservatives, sweeteners, perfuming agents, flavoring
agents and combinations thereof.
A pharmaceutical composition may be administered using
a variety of methods. Suitable methods include topical
(e. g., ointments or sprays), oral, injection and inhalation.
The particular treatment method used will depend upon the
type of infection being addressed.
In parenteral iv applications, the formulations are
typically diluted or reconstituted (if freeze-dried) and
further diluted if necessary, prior to administration. An
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. Iusitania); Torulopus spp.(i.e., T. glabrata);
Aspergillus spp. (i.e., A. fumigatus); Histoplasma spp.
WO 01/05814 CA 02379851 2002-o2-O1 pCT/US00/15017
(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 may be useful in the preparation of
medicaments for use in combating either systemic fungal
infections or fungal skin infections. Accordingly, a method
is provided for inhibiting fungal activity comprising
contacting Compound I of the present invention with a
fungus. A preferred method includes inhibiting Candida
albicans, Cryptococcus neoformans, or Aspergillus fumigatus
activity. The term "contacting" includes a union or
junction, or apparent touching or mutual tangency of a
compound of the invention with a fungus. The term does not
imply any further limitations to the process, such as by
mechanism of inhibition. The methods are defined to
encompass the inhibition of fungal activity by the action of
the compounds and their inherent antifungal properties.
A method for treating a fungal infection which
comprises administering an effective amount of a
pharmaceutical formulation of the present invention to a
host in need of such treatment is also provided. A
preferred method includes treating a Candida albicans,
Cryptococcus neoformans, or Aspergillus fumigatus infection.
26
WO ~l/~5814 CA 02379851 2002-02-O1 PCT/US00/15017
The term "effective amount" refers to an amount of active
compound which is capable of inhibiting fungal activity.
The dose administered will vary depending on such factors as
the nature and severity of the infection, the age and
general health of the host, the tolerance of the host to the
antifungal agent and the species of the host. The
particular dose regimen likewise may vary according to these
factors. The medicament may be given in a .single daily dose
or in multiple doses during the day. The 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 the like.
EXAMPLES
Unless indicated otherwise, all chemicals can be
acquired from Aldrich Chemical (Milwaukee, WI).
Biological Samples
P. syringae MSU 16H is publicly available from the
American Type Culture Collection, Parklawn Drive, Rockville,
27
WO 01/05814 CA 02379851 2002-o2-O1 pCT/US00/15017
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
Chemical Abbreviations
The following abbreviations are used through out the
examples to represent the respective listed materials:
ACN - acetonitrile
TFA - trifluoroacetic acid
DMF - dimethylformamide
DEAD - Diethylazodicarboxylate
EDCI - 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride
BOC = t-butoxycarbonyl, (CH3)3C-O-C(O)-
CBZ = benzyloxycarbonyl, C6HSCHz-O-C (O) -
FMOC = fluorenylmethyloxycarbonyl
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/0.1% aqueous TFA solvent
28
WO 01/05814 CA 02379851 2002-02-O1 PCT/US00/15017
system to 1000 acetonitrile over 20 minutes with a flow rate
of 1.5 ml/minute and using W 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.
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 5.0% dextrose and
sterile water for injection. The mice were monitored for 7
days following the first treatment and observed closely for
29
WO 01/05814 cA 02379851 2002-02-0l PCT/iJS00/15017
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
General procedures used to protect the pendant amino groups
at positions 2, 4 and 5 of Pseudomycin A, A', B, B', C or
C'.
Dissolve/suspend pseudomycin compound (R1=H) in DMF (20
mg/ml, Aldrich Sure Seal). While 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 ~,m, 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
HPLC yields an amorphous, white solid after lyophilization.
General procedures used for chemically deacylating the N-
acyl group of the L-serine unit.
Dissolve/suspend protected Pseudomycin A in
water/acetonitrile (2:1 H20:ACN, about 3.5 mg/ml) and add
WO 01/05814 CA 02379851 2002-02-O1 PCT/US00/15017
TFA (8o by volume) slowly at room temperature. Allow the
reaction to stir at room temperature until starting material
is consumed. Remove acetonitrile under vacuum at room
temperature and lyophilize the material. Dissolve resulting
solid in a small amount of DMF (add water and then an equal
amount of ACN if necessary). After preparative HPLC and
lyophilization, a white, amorphous solid (TFA salt,
presumably) is generally observed.
Solid phase acylation of the Pseudomycin nucleus using HOBt-
resin. The following example uses myristoyl acid; however,
the same general procedure may be used with other organic
acids.
In a 100 ml double-ended glass fritted reaction tube,
myristoyl acid (1.03 g, 3.62 mmol) was dissolved in 50 ml
1:1 DMF/THF. To this solution was added resin HOBt (1.94g,
2.9 mmol), and EDCI (0.556 g, 2.9 mmol) and was shaken
overnight. The solvent was drained and the resin was washed
with 2xDMF, 2xTHF, and 2x with 1:1 DMF/THF. CBZ-protected
Pseudomycin nucleus (1.0 grams, 0.723 mmol) was dissolved in
50 ml DMF/THF (20 mg/ml), and added to the resin bound
activated ester and mixed overnight on a rotator or shaker.
The product was drained away from the resin and the
remaining resin was washed with 2xDMF, 2xTHF, and 2x1:1
DMF/THF. The combined filtrates were isolated by reverse
31
WO 01/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
phase HPLC and lyophilized to yield (129 mg, 100) Myristoyl
acylated CBZ-protected Pseudomycin product.
Acylation of the Pseudomycin nucleus using an activated
ester (HOBt-mesylate). The following example uses glycine
myristoyl acid; however, the general procedures may be used
with other organic acids.
In a 500 ml round bottom flask, glycine myristoyl acid
(0.309 g, 1.1 mmol) was dissolved in 100 ml of DMF. To this
solution was added HOBt-mesylate (0.229 g, 1.1 mmol )and
triethylamine (0.0818, 0.8mmo1 )The solution was stirred
rapidly overnight under 1 atm N2. DMF and TEA were dried off
using the high vacuum. The residual oil was azeotroped 3x
with toluene till a white solid formed. To the solid was
added 100 ml of DMF and 1g of CBZ-protected Pseudomycin
nucleus. The solution was stirred overnight, and dried on
the high vacuum. The product was isolated by reverse-phase
HPLC and lyophilized to yield (233 mg, 20%) Myristoyl
acylated CBZ-protected Pseudomycin product.
General procedures used to deprotect the pendant amino
groups at position 2, 4 and 5 by hydrogenation.
Dissolve CBZ-protected acylated-derivative in a cold
13o acetic/methanol solution (5 mg/ml) and add an equivalent
amount of 10% Pd/C. Charge the reaction with hydrogen by
32
WO 01/05814 CA 02379851 2002-o2-O1 pCT/US00/15017
degassing reaction and replacing volume with H2 ,4-7 times.
Allow reaction to proceed at room temperature. Monitor the
reaction by HPLC and mass spectrometry every 15 minutes
until the starting material is consumed. When the reaction
is complete, remove balloon and filter reaction with 0.45 E.Lm
filter disk (Acrodisk GHP, GF by Gelman). Concentrate to
about 1/l0th volume and prep by HPLC. Lyophilize fractions
containing product.
Preparations
Preparation of Side-Chain Precursor (1c):
C,of'~zi
Cr12H25 ~ ~ I ~ C'12H25
1a 1b 1c
A 250 ml round bottom flask containing 100 ml of
degassed ACN was charged with m-bromobenzaldehyde (5.000 g,
27.02 mmol), triethylamine (5.490 g, 54.25 mmol) and 1-
dodecyne (5.000g, 30.06 mmol). To this mixture was added
PdCl2 (243.1 mg, 1.370 mmol), triphenylphosphine (718.8 mg,
2.740 mmol) and CuI (173.8 mg, 0.9120 mmol). The reaction
was then heated to reflux and allowed to react overnight.
The reaction was then cooled to room temperature and the
solvent was removed in vacuo. The resulting residue was
taken up in methylene chloride and washed 2 X 1N HCl and 1 X
33
W~ 01/05814 CA 02379851 2002-02-O1 PCT/US00/15017
brine. The organic layer was dried over MgS04. The drying
agent was then filtered off and the solvent was removed in
vacuo. Purification on a silica gel column eluting with 3%
EtOAc/hexanes yielded 3.73 grams of a the titled compound as
a brown oil. The spectral data was consistent with the
structure for m-(1-Dodecynyl)benzaldehyde (1a).
To 100 mL of EtOAc was added the above compound (1.00
g, 3.70 mmol) and 0.1 g of 5o Pd/A1203. The. reaction mixture
was subjected to 50psi of H2 at room temperature for 1 hour.
The reaction mixture was filtered over celite to remove the
catalyst and the celite was rinsed with copious amounts of
EtOAc. Removal of the EtOAc via a rotary evaporator yielded
882.1 mg of the product. This was used in the next step
without further purification. The spectral data was
consistent with m-Dodecylbenzaldehyde (1b).
An oven dried 50 ml round bottom flask was charged with
6.0 ml anhydrous THF under a nitrogen atmosphere at -78 °C
and lithium diisopropyl amine (1.2 mL of a 2M solution in
heptane/THF/ethylbenzene, 2.41 mmol) was then added. To
this was added t-butyl acetate (0.331 ml, 2.45 mmol) and the
resulting solution was raised to approx. -40°C and
maintained at this temperature for 1 hour. The above
compound (501.9mg, 1.83 mmol), dissolved in 4 mL anhydrous
THF and precooled to -40°C, was then added dropwise to the
anion. The reaction mixture was allowed to stir for 1 hour
34
WO 01/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
before quenching with 2 ml of saturated aqueous ammonium
chloride and 10 ml water. The reaction mixture was
partitioned between ether and water. The organic layer was
washed 1 X brine and dried over sodium sulfate. The drying
agent was then filtered off and the solvent was removed in
vacuo. Purification on a silica gel column eluting with 5%
EtOAc/hexanes yielded 238.1 mg of a yellow oil. The
spectral data was consistent with t-Butyl 3.-hydroxy-3-(m-
dodecylbenzyl) propionoate.
A solution of precooled (0°-C) of 4 ml TFA was added to
a 50 ml round bottom flask containing crude t-Butyl 3-
hydroxy-3-(m-dodecylbenzyl) propionoate. The reaction was
allowed to stir at this temperature for 25 min at which time
TLC (10% EtOAC/hexanes) indicated the consumption of the
starting ester. The TFA was removed in vacuo yielding an
oil (1c).
Preparation of Side-Chain Precursor (2c):
CsH~s
~ t-io
i
2a 2b 2c
Compound 2a is synthesized using the same procedures as
described above for Compound 1a using 1-octyne instead of 1-
WO 01/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
dodecyne. Compounds 2b and 2c are synthesized using the
same procedures described above for 1b and 1c, respectively.
Preparation of Side-Chain Precursor (3b):
H
H ~ OC» Hzs ~ Ho~~OC"I.'Izs
I ~ lvi I~~
3a 3b
A 500 mL round bottom flask containing 100 mL of
acetone was charged with m-hydroxybenzaldehyde (5.00g, 40.94
mmol), 1-bromoundecane (9.65g, 41.02 mmol) and K2C03 (8.488,
61.36 mmol). The reaction mixture was heated to reflux and
allowed to react for 10 h. The reaction was the cooled and
the acetone was removed in vacuo. The resulting residue was
partitioned between ether/water. The organic layer was
washed 2 X saturated aqueous NaHC03 and 1 X Brine. The
organic layer was dried over MgS04. The drying agent was
then filtered off and the solvent was removed in vacuo.
Purification on a silica gel column eluting with 30
EtOAc/hexanes yielded 1.6876 of a yellow oil. The spectral
data was consistent with Compound 3a. This aldehyde was
then carried through using the same procedures described in
the preparation of 1c to produce Compound 3b.
36
W~ 01/05814 CA 02379851 2002-02-O1 PCT/US00/15017
Preparation of Side-Chain Precursor (4c):
C,oH2, H / C,oH2,
C,zH2s
i
~i
i
1a 4a 4b
Compound 4a is synthesized using the same procedures as
described above for Compound 1c with the exception that
Compound 1a is not hydrogenated prior to condensation with
t-butyl acetate.
A 50 ml round bottom flask was charged with t-Butyl 3-
hydroxy-3-(m-dodecynylbenzyl) propionoate 4a(346.8 mg,
0.897) and dissolved in 10 ml MeOH /1 mL glacial AcOH.
Standard hydrogenolysis with 10% Pd/C (304.5 mg) for 24 h.
Removal of the catalyst via filtration and removal of the
solvent in vacuo led to 302.4 mg of t-Butyl 3-(m-
dodecylbenzyl) propionoate. The t-butyl ester was then
removed by treatment with TFA to produce Compound 4b.
Preparation of Side-Chain Precursor (5a):
H
HO ~ ~ C,2H2s
5a
To a 50 ml round bottom flask containing 5 mL THF at
-78°C was added sec-BuLi (1.56 mmol, 1.2 mL of a 1.3 M sol'n
in cyclohexane). To this mixture was added t-butyl
37
WO X1/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
bromoisobutyrate (in 2 ml THF at -78°C ) dropwise. This
reaction mixture was allowed to stir for 30 min at which
time Compound 1b (318.9 mg, 1.16 mmol in 2 ml THF at -78°C )
was then added to the reaction mixture dropwise. The
resulting reaction mixture was allowed to stir at -78°C for
30 min and then raised to 0°C over a period of 30 min and
allowed to stir at that temperature for 1.75 h. The
reaction was then quenched with 2 ml of saturated aqueous
ammonium chloride and allowed to warm to room temperature.
The reaction mixture was the partitioned between ether/water
and the organics were washed 1x brine and dried over MgS04.
The drying agent was then filtered off and the solvent was
removed in vacuo to yield 370.5 mg of crude material as a
racemic mixture. The t-butyl ester was then removed by
treatment with TFA to produce Compound 5a.
Preparation of Side-Chain Precursor (6a):
,CH3
HO ~ ~ C~aH2s
6a
To a 250 mL round bottom flask containing 100 mL MeOH and 1
mL of conc. H2S04 was added m-bromoanisic acid (5.00 g, 21.6
mmol). The resulting mixture was heated to reflux and
allowed to stir for 24 h. The reaction was cooled and the
38
WO ~l/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
solvent was removed in vacuo. The resulting solid was taken
up in ether and the organics were washed 2x water, 1x
saturated aqueous NaHC03 and 1x brine and dried over MgS04.
The drying agent was then filtered off and the solvent was
removed in vacuo to yield 4.728 of crude material which was
used without further purification. This was then coupled 1-
tetradecyne and underwent hygrogenolysis to the alkene
analogously to previous examples. The methyl ester was
converted to the carboxylic acid by suspending the methyl
ester (538.4 mg, 1.48 mmol) in 20 ml of a 30% HBr in AcOH
solution and heating to reflux. After 24 h at reflux the
solution was poured into 150 ml of water and extracted out
with 2x 200 ml CHZC12. The organics were washed with copious
amounts of water and dried over MgS04. The drying agent was
then filtered off and the solvent was removed in vacuo to
yield 398.78 of crude material 6a which was used without
further purification.
Preparation of Side-Chain Precursor (7a):
OH
HO ~ ~ C~aHzs
7a
Compound 6a (385.2 mg) was added to pyridinium
hydrochloride solid. The 2 solids were melted by heating to
39
WO 01/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
-- 220 °C and allowing the mixture to react for 3 h. The
reaction was then cooled and partitioned between CH2C12/1N
HCl. The organics were then washed 5x 1N HCl and dried over
MgS04. The drying agent was then filtered off and the
solvent was removed in vacuo to yield 158.8 mg of crude
material(7a) which was used without further purification.
Preparation of Side-Chain Precursor (8b):
O ~ I ~ I H ~ I ~ I
H Iw ~ - HO~
8a 8b
A 250 ml round bottom flask was charged with biphenyl
boronic acid (2.00 g, 10.1 mmol) and Pd(PPh)4 (980.0 mg,
0.848 mmol) in 60 mL toluene/30 mL 2 M aqueous Na2C03. To
this slurry was added m-bromobenzaldehyde (in 10 mL MeOH).
The reaction was heated to reflux and allowed to react for
h. The reaction was cooled and the organic layer was
washed 2x water, 1x brine and dried over MgS04. The drying
agent was then filtered off and the solvent was removed in
vacuo. The resulting solids were rinsed with cold hexanes
20 to remove any residual m-bromobenzaldehyde. The solid was
then slurried in hot hexanes and filtered hot to remove any
solids. The filtrate was then removed in vacuo to yield the
desired aldehyde 8a. Compound 8a was converted to 8b using
WO 01/05814 CA 02379851 2002-02-O1 PCT/US00/15017
the same procedures as described in the preparation of is to
yield a mixture of inseparable diastereomers.
Preparation of Side-Chain Precursor (9a):
O H3C OH
HO \(CH2)10CH9
9a
A solution of t-butyl acetate (2.02 ml, 0.015 mol) in
anhydrous THF (25 ml) was cooled to -78 °C and n-butyl
lithium (1.6 M in hexane)(9.35 ml, 0.015 mol) was added
dropwise. After 45 min a THF solution of 2-tridecanone (2.0
g, 0.01 mol) was added dropwise. The stirring was continued
at low temperature for 1 hr and then the reaction was
allowed to warm to room temperature over 15 min. Excess 1N
HC1 was added to quench the reaction and the aqueous
solution was extracted with ether (2x). The ether extracts
were dried over MgS04 and reduced in vacuo to give a crude
oil. Purification by column chromatography over silica (5%
ethyl acetate/hexane) gave 1.01 g (32o yield) of the t-butyl
ester. NMR was consistent with the desired product. The t-
butyl ester was then treated with trifluoroacetic acid to
cleave the t-butyl ester to produce Compound 9a with a
quantitative yield.
41
WO 01/05814 CA 02379851 2002-o2-O1 pCT/US00/15017
Preparation of Side-Chain Precursor (10e):
OHC / OH OHC / OR O ' / / OR
\I \
10a lOb
OH O OH
O , / I OR ~ I / OR's HO / OR
\~
lOc lOd 10e
R = n-C5H~ 1
To a THF solution (16 mL) of m-hydroxybenzaldehyde
(1.00 g, 8.20 mmol) was added at rt DEAD (1.29 mL, 8.20
mmol), PPh3 (2.15 g, 8.20 mmol) and n-pentanol (723 mg, 8.20
mmol). The reaction was stirred overnight at rt. After
silica gel purification, 1.02 g (650) of the desired product
10a was obtained.
To a THF solution of 10a (6.70 g, 34.90 mmol) was added
at 0°C Ph3P=CHO (10.6 g, 34.90 mmol). After stirring at rt
overnight, the reaction mixture was filtered and conc. in
vacuo to give a residue, which was purified by silica gel
chromatography to give 3.57 g (47%) of the desired
unsaturated aldehyde 10b.
An EtOAc solution (30 mL) of 10b (3.37 g, 15.5 mmol)
was subjected to hydrogenation (1.5 atm) using 10o Pd/C
(1.64 g, 1.55 mmol). The reaction was stirred overnight. At
this point, the reaction mixture was filtered through a pad
of Celite. The filtrates and rinses were conc. in vacuo. The
42
WO 01/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
residue thus obtained was purified by silica gel
chromatography (10% EtOAc/Hexanes) to afford 2.37 g (700) of
10c.
To a dichloromethane solution (23 mL) of aldehyde 10c
(1.26 g, 5.71 mmol) was added at 0°C allyltrimethylsilane
(0.91 mL, 5.71 mmol), followed by TiCl4 (0.63 mL, 5.71
mmol). After stirring for 1 hr at 0°C, the reaction was
quenched with saturated NaHC03 solution. The mixture was
diluted with dichloromethane (75 mL). The organic layer was
washed sequentially with saturated NaHC03, water and brine.
The organic layer thus obtained was dried, conc. and
purified via silica gel chromatography (10% EtOAc/Hexanes)
to afford 0.91 g (61%) of the desired allylic alcohol 10d.
Allylic alcohol 10d (0.91 g, 3.47 mmol) was dissolved
in aqueous acetone (7 mL each). To this solution was added
NMO (704 mg, 5.21 mmol) and a THF solution of Os04 (44 mg,
0.17 mmol). Atfer stirring at rt for 2 hr, the reaction was
quenched with NaHS03 (750 mg) to quench the excess oxidant.
The reaction mixture was diluted with brine (10 mL) and
extracted with EtOAc (3 x 50 mL). The combined organic
layers were dried and conc. in vacuo to provide 850 mg (82%)
of the desired triol intermediate. The triol (850 mg, 2.87
mmol) thus obtained was dissolved in MeOH (30 mL) and water
(6 mL). This solution was treated with NaI04 (1.38 g, 6.46
43
WO 01/05814 CA 02379851 2002-o2-O1 pCT/US00/15017
mmol) at rt. After 1 hr, the reaction mixture was filtered
through a pad of Celite. The filtrates were carefully conc.
in vacuo to yield the corresponding beta-hydroxy aldehyde.
This material was dissolved in a mixture of t-BuOH (14 mL)
and cyclohexene (2 mL). To the above solution was added an
aqueous solution (15 mL H20) containing KHzP04 (2.33 g, 17.7
mmol) and NaClOz (2.08 g, 23.0 mmol). The reaction was
stirred at rt for 5 hr and then acidified to pH =3 with 1N
HCl. The reaction mixture was extracted with EtOAc (3 x 50
mL). The combined extracts were washed with water and brine.
The organic layer was dried and conc. in vacuo to give the
crude acid 10e (1.5 g, ~3.4 mmol).
Compounds where R i s n-C6H13 , n-C7H15 , n-C8H17 , n-C9H19 , n-
CloHzl, and n-C14Hz9 were also made using the same procedures
as described above.
Preparation of Side-Chain Precursor (11d):
Me Me
R~~Me ( O~ ~
'OH
Me
R = (CH CH ~(CHZ)14CH3
2)14 3
11a lib
O OH
(CHz),4CHa ~ ~
HO~(CH2)~4CH3
11c 11d
44
W~ 01/05814 CA 02379851 2002-02-O1 PCT/US00/15017
To a dichloromethane solution (190 mL) of the chiral
acetal 11a (6.22 g, 19.1 mmol) was added at -78°C
trimethylallylsilane (10.9 mL, 68.69 mmol), followed by neat
TiCl4 (2.94 mL, 26.71 mmo1). The reaction was stirred at -
78°C for 1 hr and then at -40°C for 2 hr. At this point, the
reaction was quenched with methanol (15 mL) and diluted with
dichloromethane (200 mL). The resulting reaction mixture was
washed with 1N HCl (2 x 50 mL), water and brine. The organic
layer was dried and conc. in vacuo to give a residue, which
was purified by silica gel chromatography (100
EtOAc/Hexanes) to give 5.51 g (780) of the desired product
11b.
1H NMR of 11a (CDC13): 8 4.73 (m, 1H), 4.21 (m, 1H), 3.86 (m,
1H) , 1 . 75 (m, 1H) , 1 . 60-1.10 (m, 35H) , 0. 80 (m, 3H) . 1H NMR
of 11b (CDC13): 8 5.81 (m, 1H), 5.05 (m, 2H), 4.12 (m, 1H),
3.86 (m, 1H), 3.41 (m, 1H), 2.22 (m, 2H), 1.67-1.18 (m,
36H), 0.88 (m, 3H).
To a dichloromethane solution (155 mL) of 11b (8.56 g,
23.3 mmol) was added PCC (10.0 g, 46.5 mmol). The reaction
was stirred at rt for 18 hr, and then filtered through a pad
of Celite. The filtrates were concentrated in vacuo to give
a reddish residue, which was purified by silica gel
chromatography (10o EtOAc/Hexanes) to give 8.36 g (80%) of
the methyl ketone intermediate (structure not shown). The
WO Ul/~5814 CA 02379851 2002-02-O1 PCT/US00/15017
intermediate obtained herein (8.36 g, 22.8 mmol) was
dissolved in THF (60 mL) and MeOH (30 mL). To this solution
,, was added 7.5 M KOH (15 mL). After stirring 3 hr at rt, the
solvent was partially removed. The remaining reaction
mixtures were diluted with EtOAc/Et20 (3:1 ratio, 350 mL).
The organic layer was washed with water (3 x 50 mL) and
brine. The resulting organic layer was dried and conc. in
vacuo to give a residue, which was purified by silica gel
chromatography (10% EtOAc/Hexanes) to afford 6.22 g (960) of
the desired product 11c as white solids.
1H NMR of 11c (CDC13) : 85.75 (m, 1H) , 5.06 (m, 2H) , 3.56 (m,
1H), 2.23 (m, 1H), 2.07 (m, 1H), 1.75-1.17 (m, 28H), 0.80
(m, 3H).
Carbinol 11c (6.22 g, 22.0 mmol) was dissolved in an
aqueous THF solution (5.5 mL water and 55 mL THF). To this
solution was added NMO (4.42 g, 33.0 mmol), followed by Os04
(280 mg dissolved in THF, 1.10 mmol). The reaction stirred
at rt overnight. At this time, sodium bisulfide (4 g) was
added. The reaction was stirred for 2 hr, and then diluted
with EtOAc (300 mL). The whole mixture was washed with water
(2 x 40 mL) and brine. The resulting organic layer was dried
and conc. in vacuo to give the corresponding triol
intermediate. This material was dissolved in MeOH (200 mL)
and water (40 mL). To this solution was added NaI04 (10.6 g,
46
WO 01/05814 CA 02379851 2002-02-O1 pCT/jJS00/15017
49.5 mmol). After stirring at rt for lhr, the reaction was
filtered through Celite and purified by short column silica
gel chromatography (30% EtOAc/Hexanes) to afford ~10 g
(>1000) crude beta-hydroxyl aldehyde. The impuried aldehyde
thus obtain was dissolved in t-BuOH (100 mL) and cyclohexene
(14 mL). To this solution at rt was added an aqueous
solution (50 mL) of NaClOz (15.97 g, 176 mmol) and KHZP04
(17.8 g, 132 mmol). The reaction was stirred at rt for 6 hr
and then quenched at 0°C with 5N HCl to pH=4. The reaction
was extracted with 3:1 mix-solvent EtOAc/Et20 (3 x 250 mL).
The organic layer was washed with brine and dried and conc.
to provide 7.3 g (>100%) of the crude acid 11d, which was
used directly for the coupling reaction.
1H NMR of 11d (CDC13): 8 3.95 (m, 1H), 2.60-2.35 (m, 2H),
1.40-1.10 (m, 28H), 0.82 (m, 3H).
Preparation of Side-Chain Precursor (12c):
O
O O ~ O O~'0
~N. ---~ -- II
H3C-O C~3H27 E..~3C_O~N.C,sH27 H_O~N.C H
13 27
12a 12b 12c
A 1 liter round bottom flask was charged with
tridecanal (5.00 g, 25.2 mmol) and the HCl salt of Gly-Ome
(12.66 g, 100.8 mmol) in 600 ml anhydrous MeOH. To this
47
WO ~l/05814 CA 02379851 2002-02-O1 PCT/US00/15017
reaction mixture was added NaCNBH3 (1.787 g, 28.44 mmol) and
the reaction was allowed to stir overnight at room
temperature. The solids were filtered off and the solvent
was removed in vacuo. The resulting residue was partitioned
between CH2C12/saturated aqueous NaHC03. The organic layer
was washed 2x NaHC03 and dried over MgS04. The drying agent
was then filtered off and the solvent was removed in vacuo.
Purification on a silica gel column eluting.with 500
EtOAc/hexanes yielded 2.94 g of a white solid (12a).
A 50 ml round bottom flask was charged with the glycine
derivative 12a (504.6 mg, 1.86 mmol), triethylamine (224.6
mg, 2.22 mmol) in 10 ml anhydrous THF. To this was added
(BOC)20 (494.3 mg, 2.26 mmol) in one portion. The reaction
was allowed to stir for 18 h at which time the solvent was
removed in vacuo. The resulting oil was taken up in EtOAc
and washed 2x 1N HC1, 1x water, 1x brine and dried over
MgS04. The drying agent was then filtered off and the
solvent was removed in vacuo to yield 463.3 mg of a
colorless oil (12b) which was used without purificaton.
A 50 ml round bottom flask containing 5 ml THF was
charged with the methyl ester 12b (463.3 mg, 1.25 mmol). To
this was added 1.8 ml of a 1N LiOH solution. The resulting
reaction mixture was allowed to stir overnight. The
reaction was quenched by the addition of 1.8 ml of a 1N HCl
48
WO X1/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
solution. The THF was then removed in vacuo and the
resulting aqueous layer was extracted 2x CH2C12. The
organics were then dried over MgS04. The drying agent was
then filtered off and the solvent was removed in vacuo to
yield 246.2 mg of a colorless oil (12c) which was used
without purification.
Preparation of Side-Chain Precursor (23a):
O
~N.
HO C~3H2~
13a
The methyl ester 12b from above (499.7 mg/1.84 mmol)
was dissolved in a 1:1 mixture of acetic anhydride/pyridine
in a 50 mL round bottom flask. The reaction was allowed to
stir overnight at which time the solvent was removed in
vacuo. The resulting oil was taken up in CH2C12 and washed
2x 1N HCl, 1x water, 1x brine and dried over MgS04. The
drying agent was then filtered off and the solvent was
removed in vacuo to yield 319.9 mg of a colorless oil (13a)
which was used without purificaton.
General Preparation for Glycine side-chain precursors
(14-a)
49
WO 01/0$814 CA 02379851 2002-o2-O1 PCT/US00/15017
For example the following procedure describes the
synthesis of tridecanoyl-glycine-acid from Fmoc-glycine-wang
resin and tridecanoic acid.
In a 100mL double-ended glass fritted reaction tube,
Fmoc-glycine-wang resin (10g, 4.4 mmol) was added to 50 mL
of 30o Piperdine/DMF. The reaction was shaken for 20
minutes, and was washed 3x with DMF, 3x with isopropanol,
and 3x with DMF. To the resin was added a solution of
tridecanoic acid (4.7088, 22 mmol) in 50 mL DMF. To this
mixture was added HOBt (2.97 g, 22 mmol) and DIC (2.77 g,
22mmo1). The reaction vessel was put on a shaker overnight.
The resin was then washed 3x with DMF, 3x with
dichloromethane, 3x with MeOH, 3x with THF, and 3x with
dichloromethane. The resin beads were dried in a vacuum
oven for 1 hour. To the resin was added 100 mL of 950
TFA/H20. The reaction was shaken for 1.5 hours, and the
non-resin product was washed with TFA and collected. The
product was dried in a vacuum oven to a white residue, and
azeotroped with Toluene to yield tridecanoyl-glycine-acid
(1.14 g, 950) 1HNMR (THF) 0.82-0.92 (t, J= 7.2, 3H), 1.2-1.4
(s, 18H), 1.52-1.62 (m, 2H), 2.10-2.17 (t, J=7.2 , 2H),
3.83-3.89 (d, J=7.1 , 2H), 7.04-7.17 (s, 1H), 10.8-10.9 (s,
1H ) .
The following structure II will be used to describe the
products observed in Examples 1 through 17.
CA 02379851 2002-02-O1
WO 01/05814 PCT/US00/15017
O
HO
O
OOH
O H N OH
NH
H O ,,, O
~CI
O
O O
R'HN
O p ~~'NHR
H
N NH
O
O H '~N H R'
R HN p
II
Example 1
thesis of diastereomers 2-2 and 1-2:
H H
~ C~2H2s ~ C~2H2s
R= I~ R= I~
R' = CBZ R' = CBZ
1-1 1-2
Hydroxybenzotriazole (55.8 mg, 0.413 mmol) and 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride
(82.4 mg, 0.430 mmol) was added to Compound is in 4 ml
anhydrous DMF. This reaction mixture was allowed to stir
for 10 hours at room temperature at which time Z-PSN (375.2
mg, 0.271 mmol) was then added. HPLC indicated the
51
WO 01/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
consumption of CBZ-protected pseudomycin nucleus (Z-PSN)
after a period of 5 hours. The solvent was removed in vacuo
and the resulting residue was taken up in 1:1 ACN/H20 and
purified via preparatory HPLC. This yielded 2 major peaks
whose mass spectral data suggests that these peaks
correspond to the 2 diasteromers 1-1 (88.3 mg) and 1-2
(166.3mg).
tection of Diasteromer 1-1:
H
~ ~ Ci2Hzs
R=
R' = H
1-3
A 50 ml round bottom flask was charged with 10 ml MeOH
/1 ml glacial AcOH and diastereomer 1-1 (82.0 mg, 0.048
mmol). After degassing 89.1 mg of 10o Pd/C was added to the
reaction mixture and subjected to 1 atm H2 for 30 minutes.
Removal of the catalyst via filtration and purification via
preparatory HPLC and subsequent lyophilization provided 21.7
mg of Compound 1-3 (R1 - H). MS (Ionspray) calcd for
CS8H94C1N12O19 (M+H)+ 1297.64, found 1297.8.
Deprotection of Diasteromer 1-2:
52
W~ 01/05814 CA 02379851 2002-02-O1 PCT/US00/15017
OH
~ C,2H2s ~ ~ C,2H25
R= I~ R= I~
R'=H R'=H
1-4 1-5
Diastereomer 1-2 (152.8 mg, 0.089mmo1) was subjected to
hydrogenolyis as described for diasteromer 1-1 using 152.8
mg of 10% Pd/C for 30 min. HPLC indicated consumption of
starting material and formation of two product peaks which,
after preparatory HPLC and lyophilization, were found to be
Compound 1-4 (18.0 mg ) and Compound 1-5 (11.3 mg). Compound
1-4: MS (Ionspray) calcd for CS$H94C1N12O19 (M+H)+ 1297.89,
found 1297.8. Compound 1-5: MS (Ionspray) calcd for
CS$H9zC1N12018 (M+H)+ 1279.63, found 1281.7.
Each of the compounds listed in Table 1 below was
synthesized using the same acylation and deprotection
procedures as described above using the indicated side-chain
precursor. For each compound listed in Table 1, R1 is a
hydrogen.
Table 1
Example R = Side-Chaia
No. Precursor
H
2-1 I ~ CeH,~ 2c
H
2-2 I ~ CsH,~ 2c
2-3 ~ I ~ CBH~~ 2c
53
WO 01/0$814 CA 02379851 2002-02-O1 PCT/LTS00/15017
H
3-1 I ~ OCIIH~s 3b
H
3-2 I ~ OC"H23 3b
3-3 / I ~ OC,lH2s 3b
4-1 I ~ C12H2s 4b
H
5-1 ~ ~ C12H2s 5a
/
O .CH3
O 6a
6-1
I ~ C14H29
OH
7 -1 I w C14H2s 7 a
8-1 H , ~ I 8b
\ ~ I
I/
O H3C OH
9-1 9a
~CHz),oCHa
O OH
10-1 ~ OR 10e
O OH
11-1 ~~~HZ~~4cH3 11 d
12-1 12c
000
~N'C13H27
O
13-1 ~~ 13a
N.C1 sH2~
54
WO 01/05814 CA 02379851 2002-02-O1 pCT/[JS00/15017
O CH3 O CH
14 -1 * ~N~C" H23 H-O~N~C> > H2s
O O
(available from
Sigma)
*Although this compound and the compound where the N-methyl
group is absent shows little activity, Compounds where the
alkyl chain is increased progressively from C11 to C15 show
significant increased activity. Side chains of this class
may be prepared using the preparation described in
preparation 14-a.
Example Z5
Synthesis of Compound 15-2:
O
~N.
R = CisH2~
R' = H
15-1
To a 50 ml round bottom flask was added 31.3 mg (0.0237
mmol) of Compound 12-1 and 5 ml TFA (precooled to 0 °C).
The reaction mixture was allowed to stir at this temperature
for a period of 15 min at which time the TFA was removed in
vacuo. The residue was then taken up in water and
lyophilized to yield 24.3 mg of Compound 15-1. No further
purification was necessary.
Example 16
Example 16 illustrates the attachment of a Beta-amino
substituted side-chain.
Prepara ti on of Compounds 16a, 2 6b, 16c and 16d
WO U1/05814 CA 02379851 2002-02-O1 PCT/US00/15017
O / , H3C
~O~(CH ) CH -~ \ O N
2 10 3 ~ ~ ~
~O~(CHZ)~oCH3
16a
16b
O
O HN~O \ / ~..- O NHZ
HO (CHZ)~oCH3 / _O (CHZ)~oCH3
16d 16c
A solution of t-butoxycarbonylmethylenetriphenyl-
phosphorane (5.0 g, 13.3 mmol) and dodecyl aldehyde (2.2 ml,
10 mmol) in toluene (50 ml) was refluxed for 1 hr 20 min.
The solution was filtered through a plug of silica to remove
the phosphorus reagents and then reduced in vacuo to give a
crude oil. The oil was purified over a silica column by
elution with 2% ethyl acetate in hexane to give 2.36 g (840
yield) of compound 16a. MS- 283.4 (M+1) NMR consistent with
structure.
Butyl lithium (1.6 M in hexane)(1.19 ml, 1.9 mmol) was
added slowly to a solution of ( R )-benzylmethyl benzylamine
(0.42m1, 2.0 mmol) in THF (5 ml) cooled to -78 °C. A THF
solution of 16a (500 mg, 1.77 mmol) was then added dropwise.
The mixture was stirred at -78 °C for 1 hr. The reaction
mixture was then poured into sat. NH4C1 solution and
extracted with ether (2x). The ether solution was dried
over MgS04 and reduced in vacuo to give 0.95 g of 16b as a
56
WO 01/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
crude oil which was carried to the next step without
purification.
An ethanol (25 ml) solution of 16b (0.95 g) and
Pd(OH)2/C (0.47 g) was put under 60 psi of HZ at 55 °C for 18
hr. The suspension was filtered and then reduced in vacuo
to give 280 mg (53o yield) of crude 16c which was carried
directly to the next step.
Compound 16c (280 mg, 0.93 mmol) and N-benzyloxy-
carbonyloxysuccinimide (274 mg, 1.1 mmol) were mixed in THF
(10 ml) and stirred overnight. The solvent was removed in
vacuo and the product oil was purified by column
chromatography over silica using 5% ethyl acetate in hexane
as the eluant to give 268 mg (66o yield) of t-butyl ester of
16d. NMR was appropriate for the expected structure. The
ester (97 mg, 0.224 mmol) was dissolved in trifluoroacetic
acid (2 ml) at 0 °C for 0.5 hr to remove the t-butyl ester
(16d) with a quantitative yield.
57
WO ~l/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
Synthesis of Compound 16-1:
O NHZ
R = (CHZ)~oCH3
R'=H
Compound 16d was dissolved in DMF (2 ml).
Hydroxybenzotriazole (36.4 mg, 0.269 mmol) and 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride
(47.1 mg, 0.246 mmol) were added and the solution was
stirred 18 hr. The CBZ protected pseudomycin nucleus (Z-
PSN) (277 mg, 0.185 mmol) was added and the reaction was
stirred for an additional 18 hr. The reaction product was
purified by HPLC and lyophilization gave 136.3 mg (42%
yield) of the acylated CBZ protected pseudomycin derivative
as a white solid. MS- 1743 (M) and the NMR was consistent
with the proposed structure.
A methanol (10 ml) and acetic acid (1.5 ml) solution of
the acylated compound (130 mg, 0.0746 mmol) with 10% Pd/C
(120 mg) was put under a balloon of hydrogen for 20 min.
The solution was filtered and purified by preparative HPLC.
Lyophilization gave 23 mg (18% yield) of the trifluoroacetic
acid salt of 16-1 as a white solid. MS- 1206.8 (M) and the
NMR was consistent with the proposed structure. Compound
16-1 showed little or no activity against Candida Albicans
58
WO X1/05814 CA 02379851 2002-02-O1 PCT/US00/15017
and Cryptococcus neoformans which is a significant reduction
in activity as compared to the (3-hydroxy analog.
Example 27
Example 17 illustrates the attachment of a chiral side-
chain.
Preparation of side chain precursor 17d:
I
0
-O, o off
~n-Bu + O-Ti(OPr-i)2 r(R)
H (CH2 9 I ~ TMSO _ R' Me0 (CHz 9 n-Bu
Me0 uR' R R
17a 17b R' = CH3 17c
O OH
(R)-
HO~(CHz~n-Bu
R* R*
17d
To a THF solution of (S)-binaphthol (240 mg, 0.84 mmol)
was added 4A molecular sieves (4 g), followed by addition of
neat Ti(OPr-I)4 (0.25 mL, 0.84 mmol). The reaction mixture
turned red immediately and remained to be red. The chiral
catalyst (17b) thus prepared was used for the subsequent
reaction.
To a freshly prepared THF solution (4 mL) of (S)-
binaphthol-Ti catalyst 17b (0.42 mmol) was added at -78°C a
THF solution containing trimethylsilyldimethylketene acetal
(0.43 mL, 2.1 mmol) and the unsaturated aldehyde 17a (500
mg, 2.1 mmol) over 15 min. The reaction was stirred at -78°C
59
WO 01/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
for 1 hr and then at rt overnight. At this point, the
reaction was quenched with saturated NaHC03 solution and
extracted with EtOAc (100 mL). The organic layer was washed
with NaHC03, brine, and dried with anhydrous MgS04. Upon
filtration and conc. in vacuo. The residue was purified with
silica gel chromatography (10o EtOAc/Hexanes) to give 257 mg
(360) of the desired product 17c.
1H NMR of 17c (CDC13): 8 5.29 (m, 2H), 3.65 (s, 3H), 3.53 (t,
J - 7 . 3 Hz, 1H) , 2 . 33 (d, J - 6 . 3 Hz, 1H, 3' -OH) , 1 .97 (m,
4H), 1.65-1.22 (m, 20H), 1.13 (s, 3H), 1.12 (s, 3H), 0.84
(m, 3H) .
A THF solution (5 mL) of 17c (259 mg, 0.76 mmol) was
treated with an aqueous solution of NaOH (0.30 mL, 5N, 1.52
mmol). The reaction was heated overnight at 50°C. The
reaction mixture was cooled to 0°C and acidified to pH = 3
using 5N HC1. The reaction was then extracted with EtOAc (75
mL). The organic layer was washed with water and brine. The
organic layer thus obtained was dried and conc. in vacuo to
afford 222 mg (90%) of the crude acid 17d, which was used
directly for side chain coupling reaction.
1H NMR of 17d (CDC13) : b 5.28 (m, 2H) , 3 .56 (m, 1H) , 1.95 (m,
4H), 1.60-1.10 (m, 26H), 0.83 (m, 3H).
W~ 01/05814 CA 02379851 2002-02-O1 PCT/US00/15017
Preparation of 17-1 and 17-2:
O (R)OH
R = ~~(CH2~n'Bu R = (CH2~~'Bu
H3C CH3
R1 = H R1 = H
17-1 17-2
A THF solution (7 mL) of the crude acid 17d (240 mg,
0.74 mmol) was treated with HOBt (90.5 mg, 0.67 mmol) and
EDCI (128 mg, 0.67 mmol) at rt. After stirring for 1.5 hr,
DMAP (41 mg, 0.33 mmol) was added. After stirring for
another 2 hr, a DMF (4 mL) of CBZ-protected psuedomycin
nucleus (614 mg, 0.44 mmol) was added and the reaction was
stirred at rt overnight. The reaction mixture was purified
with preparative HPLC to give, after lyophilization, (160
mg, 210) of the desired product.
An acetonitrile solution (2 mL) of CBZ-protected
compound (53.4 mg, 0.032 mmol) was treated with TMSI (77 mg,
0.38 mmol) at 0°C. After 100 min, the reaction was quenched
with 1:1 CH3CN/H20. The resulting reaction mixture was
purified by reverse phase preparative HPLC to give 25.8 mg
(650) of the desired final product 17-1.
Compound 17-2 is made using the same procedures
described above using the appropriate starting materials
where R* is a hydrogen.
61
W~ 01/05814 CA 02379851 2002-02-O1 PCT/US00/15017
Example 18
Example 18 illustrates the attachment of a chiral
alkenyl side-chain.
Preparation of side chain precursor 18d:
Me Me
O j 1I
R~~Me ~ O OOH
v~ Me / /
~O
R = (CHZ)6CH=CH-CH=CHMe
18a 18b
Me Me
O O~O O OH
/ / / / 14
.a HO
18c lsd
To a dichloromethane solution (25 mL) of 18a (993 mg,
3.73 mmol) was added at -78°C methyl trimethylsilyl
dimethylketene acetal (2.27 mL, 11.2 mmol) and neat TiCl4
(0.49 mL, 4.48 mmol). After 2 hr, the reaction was quenched
at -78°C with MeOH (5 mL). The reaction mixture was
extracted with dichloromethane (3 x 40 mL). The combined
organic layers were washed with NaHC03 and brine. The
organic layer was dried and cons. in vacuo to yield a
residue, which was purified with chromatography (15-20o
EtOAc/Hexanes) to provide 837 mg (61%) of the desired
product 18b.
1H NMR of 18a (CDC13): 8 6.28-5.87 (m, 2H), 5.62-5.43 (m,
2H), 4.75 (m, 1H), 4.22 (m, 1H), 3.87 (m, 1H), 2.08-1.93 (m,
62
WO 01/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
2H), 1.80-1.60 (m, 3H), 1.60-1.40 (m, 3H), 1.38-1.10 (m,
15H). 1H NMR of 18b (CDC13): 8 5.98-5.90 (m, 2H), 5.55-5.47
(m, 2H) , 4. 08 (m, 1H) , 3 .86 (m, 1H) , 3.61 (s, 3H) , 3 .50 (m,
1H), 2.00-1.96 (m, 2H), 1.74-1.63 (m, 3H), 1.50-1.00 (m,
24H).
To a dichloromethane solution (22 mL) of 18b (837 mg,
2.27 mmol) was added PCC (0.98 g, 4.54 mmol). The reaction
was stirred at rt for 18 hr, and then filtered through a pad
of Celite. The filtrates were concentrated in vacuo to give
a reddish residue, which was purified by silica gel
chromatography (20o EtOAc/Hexanes) to give 441 mg (53%) of
the desired methyl ketone 18c.
1H NMR of 18c (CDC13): 8 5.92-5.87 (m, 2H), 5.48-5.43 (m,
2H), 3.88 (m, 1H), 3.56 (s, 3H),3.49 (m, 1H), 2.59 (dd, J
=
6.4, 14.7 Hz, 1H), 2.29 (dd, 5.9, 15.2 Hz, 1H), 2.06 (s,
J =
3H), 1.96 (m, 2H), 1.63 (m, 2H),1.40-0.90 (m, 20H).
To a THF (4 mL) and methane (2 mL) solution of 18c (440
mg, 1.20 mmol) was added at rt 7.5M KOH (1 mL). After
stirring at rt for 4 h, the reaction was acidified with 1N
HC1 to pH = 3. The reaction mixture was extracted with EtOAc
(3 x 30 mL). The combined extracts were washed with water (2
x 10 mL) and brine. The organic layer was dried and conc. in
vacuo to give 388 mg (>100%) of the crude acid 18d, which
was used directly for the side chain coupling reaction.
63
WO 01/05814 CA 02379851 2002-o2-O1 pCT/US00/15017
Preparation of Compound 18-2:
OH
R=
R'=H
~/~
18-1
To a THF solution (10 mL) of the crude acid 18d 01.66
mmol) was added HOBt (244 mg, 1.66 mmol) and EDCI (318 mg,
1.66 mmol). After stirring at rt for 5 hr, a DMF solution (5
mL) of Alloc-protected pseudomycin nucleus (614 mg, 0.50
mmol) was added. After stirring at rt for a few days, DMAP
(61 mg, 0.50 mmol) was added to the reaction mixture. After
stirring for additional 12 hr, the reaction mixture was
purified by reverse phase HPLC to afford, after
lyophilization, 225 mg (300) of alloc-protected acylated
derivative.
To a degassed THF (20 mL) and HOAc (1 mL) solution of
alloc-protected acylated derivative (240 mg, 0.16 mmol) was
added PdCl2(PPh3)2 (23 mg, 0.032 mmol) and Bu3SnH (0.87 mL,
3.23 mmol) at rt. After 1.5 hr, the reaction mixture was
purified by preparative HPLC to afford 33 mg (17%) of
Compound 18-1.
In addition to the compounds listed in the Examples
above, the following N-acyl derivatives were also made which
showed limited activity or non-significant activity.
64
WO 01/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
Even though these compounds may not be as useful as
antifungal agents, they provide valuable insight into the
design of compounds having optimal activity.
WO 01/05814 cA 02379851 2002-02-0l PCT/US00/15017
O -N O
O(CH2)9CH3
O ~ O
~~ N
_~ .. ~~3 _
O
O OH
O (CH2)2CH3 (CH2)SCH3
N
H ~I
~(CH2)2CH3
O
O OH
O O(CH2)4CH3
~N
t-butyl I
O O
i ~ (CH2)~CH3
~I
i o
0
O I ~ O CH CH
( 2)7 3
N\ /(CH2)8CH3
~O
O
~~N (CH2)~oCH3
O
O ~ ~ F
3
O(CH2)9CH3
O OH ~ O _
/ O(CH2OCH3
O(CH2)5CH3
O OH / I
66
WO 01/05814 CA 02379851 2002-o2-O1 PCT/US00/15017
Unless indicated otherwise, each of the compounds
listed in the Examples showed measurable activity against
Candida Alhicans, Cryptococcus neoformans, Aspergillus
Fumigatus, Candida Parapsilosis, or Histoplasma capsulatum.
However, the following basic trends in activity were
observed based on the compounds synthesized. The
stereochemistry of the (3-hydroxy group is preferably R.
Longer alkyl chain lengths (i.e., C1z-C2o) tend to have
higher activities than shorter alkyl chains (e. g., < C11)
regardless of stereochemistry or unsaturation levels.
Removal of the (3-hydroxy group, a,a-disubstitution, lower
alkyl chain lengths in both alkyl and alkoxy substituents,
extreme rigidity, and increased branching in the chain all
tended to have lower activity than the longer flexible
chains.
Consequently, alkyl side-chains represented by the
following structure are preferred for antifungal treatment:
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
67
WD 01/05814 CA 02379851 2002-02-O1 PCT/US00/15017
or Rb~ forms a six-membered cycloalkyl ring, a six-
membered aromatic ring or a double bond, or taken
together with R~ forms a six-membered aromatic ring;
Rb and Rb~ are independently hydrogen or methyl,
and either Rb or Rb~ is hydroxy provided that Rb~ is not
hydroxy when Ra, Rb, Rd, Re are hydrogen, R° is hydrogen
and Rf is n-hexyl, n-octyl or n-decyl, or Ra, Rb, Rd, Re
are hydrogen, R~ is hydroxy and Rf is n-octyl, n-nonyl,
or n-decyl;
R° is hydrogen, hydroxy, C1-C4 alkoxy,
hydroxyalkoxy, or taken together with Re forms a 6-
membered aromatic ring or CS-C6 cycloalkyl ring;
Re is hydrogen, or taken together with Rf is a
six-membered aromatic ring, CS-C14 alkoxy substituted
six-membered aromatic ring, or CS-C14 alkyl substituted
six-membered aromatic ring, and
Rf i s C8-C14 alkyl , or C5-C11 alkoxy .
68
